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June 1982, Volume 2, Number 1 Northwestern University

Suite 1525

875 North Michigan Avenue

Chicago, Illinois 60611

Editor: Gerald I. Zatuchni, M.D., M.Sc. Osborn Managing Editor: Kelley

RESEARCH FRONTIERS IN FERTILITY REGULATION

CURRENT STATUS OF PLANT PRODUCTS REPORTED TO INHIBIT SPERM

Norman R. Farnsworth, Ph.D. Professor of Pharmacognosy Department of Pharmacoqnosy and Pharmacology

College of Pharmacy, University of Illinois at the Medical Center

Chicago, Illinois

Donald P. Waller, Ph.D.

Associate Professor of Pharmacology Department of Pharmacognosy and Fharmacology

College of Pharmacy, University of Illinois at the Medical Center

Chicago, Illinois

The plant kingdom has abundant chemical compounds that elicit pronounced effects in animals and humans. A number of these compounds have become pharmacologic tools, and about one-hundred plant-derived compounds serve as drugs in the United States. According to the National Prescription Audit, 25% of all prescriptions dispensed from community pharmacies between 1959 and 1973 contained one or more active principles extracted from higher plants (63). In 1973, the American public spent an estimated $3 billion on plant-derived drugs (63), and in 1980 the expenditure was estimated to be $8 billion (58).

Although the prescription market for drugs made from plants is lucrative, plant-derived contraceptive agents have not been investigated extensively, and no such agents have found general acceptance for male fertility regulation (58,63). Djerassi suggests that the enormous development costs, and the considerable time required for approval by the U.S. Food and Drug Administration (FDA), limit the initiation of new contraceptive drug development programs in the United States, especially if continuous long-term dosing is required (55). Drug development programs involving plants are further discouraged because when a plant serving as a starting material for a drug is unavailable in the United States, controlling the source of supply becomes complicated. Other difficulties involve securing strong patent protection for natural products and eliminating biological variabiLty In plant material from batch to batch (59,177).

The scientific community in developing countries has greater interest than does the U.S. pharmaceutical in dustry in programs to develop new drugs from plants. In most developing countries, plant drugs are familiar and culturally acceptable to the general populace; they are already a part of many indigenous medical systems, and they represent an available and inexpensive starting material, should industrial development of a plant drug become feasible. One advantage in developing a male antifertility agent from a plant source, rather than through the complete synthesis of a new drug, is that a plant used as a contra ceptive agent in an indigenous medical system is likely to have a long folkloric history and an established low toxici ty potential. An impetus for research is that chemical templates to guide a chemist in the synthesis of active male contracep tives are rare, and even if contraceptive research on plants serves only to produce comp unds with novel structures, good activity, and some side effects, new molecular models will have been identified for further synthetic studies. There are no simple procedures to identify plants with biologically active principles (24,59). Most of the useful piant-derived drugs have been discovered as a result of follow-up studies of ethnomedical (folkloric) information, or when investigators have sought to identify the active principle in a plant extract reported to have biological ac-

Copyright PARFR 1982

tivity. Reliable ethnomedical data concerning the activity of plants for male contraception are few, and experimental data for plant extracts with effects on the male reproductive system are often unremarkable or irreproducible. A num ber of investigators have expressed negative opinions about the value of this type of research, but these opinions may represent personal biases rather than actual attempts to confirm the reported data. Although no plant has been thoroughly studied for its effects on male reproduction, serious scientific investigation of plant-derived agents having antisperm effects is underway in a number of laboratories, and has been for some years. In 1974, our group reviewed the potential value of plants as sources of new antifertility agents (60,61), and we subsequently assessed the problems associated with biological assays to determine the antifertility activity of plants and natural substances (24,62). In this report, we will consider the status of research on plant-derived agents that prevent sperm production if taken orally by the male, or that incapacitate or kill sperm on contact if used vaginally by the female. Problems involved in searching for plant-derived fertility regulating agnswill also be highlighted, agents wthen ANTIFERTILITY STUDIES USING PLANTS The male reproductive system is made up of tissues and structures involved in sperm production, storage, and transport to the female genital tract; interference with reproduction means interrupting any of these activities. Although hormones or hormone antagonists and steroids or steroid antagonists have all proven capable of regulating male fertility, they also exert their effects on nonreproductive tissues, as is the case with the steroid oral contraceptives in women. Therefore, it would be of great value to develop fertility inhibitors that are totally selective for reproductive systems and enzymes, and it is possible that a plant-derived drug may have this effect, Some of the plants that have been studied for their fertility inhibiting effects in the male, and some of the more promising and interesting investigations of those plants, are described arid briefly evaluated in the following section. Aristolochia indica L. (Arisolochlaceae). This species has been widely employed in ethnomedicine as a fertility regulating agent in women, and the fertility effects have been substantiated in various laboratories, To study the effects of A. indica in males, investigators administered a water soluble extract from a chloroform fraction of A. indica roots orally to male mice at a dose of 75 mg/kg every 3 days for seven doses. Varying degrees of degenerative change were found in the seminiferous germinal cell components, with prominent nuclear degeneration in all cell types. Reduction in the fluid c'-ntent 2

of accessory sex organs in A. indica-treated mice was prominent and seemed to indicate a decrease in en docrine function of the testis (133). After one of the constituents of A. indica responsible for ts ol oi th ctiv it ets o w to be ra ns iblo r its biological activity was shown to be trans-p-coumaric acid (PCA), Pakrashi and co-workers studied the action of PCA in prolactin-treated male mice (134) and in testosterone- or prolactin-treated orchiectomized rats as well (131). They concluded [in our opinion unconvinc ingly] that this substance was a prolactin inhibitor. Later, these workers reported that daily oral administration of PCA to male rats for 56 days produced complete loss of libido (132). The roots of A. indica are known to contain significant amounts of aristolochic acid and a variety of related com pounds. Aristolochic acid has antitumor and cytotoxic properties (106), which may explain at least some of the effects observed in the above experiments. Aadat ixdict Js A.M i a aes prepared water extract of crushed grien A. indica leaves was administered orally to male mice at a dose of 1.0 mI/animal daily for 1 month. Half of the animals were

sacrificed

and the testes were subjected to histo logical examination. The remaining treated animals were mated and the resulting pregnancies and litter sizes were noted. Following mating, the males were no longer given the test extract. Forty-five days later they were again mated with healthy females (49). The data from this study indicated an 80% reduction in pregnancies produced by A. indica-treated males, com pared with pregnancies in controls. The males regained 100% fertility after 45 days of no treatment. Histologic examination of the testes from treated animals revealed no evidence of an antispermatogenic effect (49). Although the data suggested that an antifertility effect was experienced by animals treated with the plant extract, too few details w-ere presented in the report to preclude the possibility i iat the effect was due either to male functional sterility or .o unsuccessful mating. In a later report, the same research group suggested that the activity could be due to a loss of libido in A. indica-treated animals (50). Balanites roxburghii Planch. (Zygophyllaceae). Pre liminary data suggested that oral administration of an ethanol extract of fresh, ripe, dried B. roxburghiifruitpulp induced infertility in male gerbils, with an associated 3- to 6-fold increase in blood glucose levels (53). Based on these findings, a study was initiated using healthy male dogs. An ethanol extract of B. roxburghii fruitpulp was given orally to five dogs at a dose of 35 mg/kg for 60 days. Another group of five dogs received only alloxan. After 60 days, all animals were sacrificed and the testes and epididymides from the animals were removed, weighed, anu examined histologically. A significant de

crease in testis and epididymis weight in both groups of animals was seen (53). The authors concluded that the atrophic changes found in the testes of B. roxburghiitreated dogs were secondary effects of hyperglycemia. Further study is warranted, to determine whether the hyperglycemic effect could be achieved separately from the effect on the testes of the treated dogs. Calotropisprocera (Ait.) R.Br. (Asclepladaceae). An ethanol extract prepared from C. procera flowers, and administered orally every other day for 30 days to Indian desert male gerbils (Meriones hurrianae Jerdon), at a dose of 20 mg/animal, produced marked testicular necrosis. Spermatogonia, spermatocytes, and Sertoli cells showed severe degenerative changes, and the lumen of the epididymis decreased in size and lacked spermatozoa. Protein, RNA, and sialic acid contents in tissues were also reduced. Acid and alkaline phosphatase, serum transaminases, cholesterol, and total lipids were increased. Liver damage was evidenced by tumorlike structures on the lobes, and the diameters of seminiferous tubules and Leydig cell nuclei were reduced (68). Calotropis procera contains cytotoxic and antitumor cardenolides (80). The results described above are similar to those expected when cytotoxic agents such as vinblastine, vincristine, and related agents are administered to laboratory animals (see Catharanthusroseus). A plant extact may have toxic effects as well as potentially useful iL t, Normally in such cases, the next step is to further iaciionate the active extract, in the hope of separating the useful from the toxic effect. This approach seems appropriate in investigating the antispermatogenic potential of C. procera. Carica papaya L. (Caricaceae). Sun-dried seeds from ripe papaya fruits (C. papaya)were ground and suspended in water, and the suspension was administered orally to fertile adult male rats at a dose of 20 mg/animal for 8 weeks. At the end of the dosing period, the treated males were mated with females of proven fertility and sacrificed, Also, t;.e motility pattern of sperm taken from the three segments of the epididymis and vas deferens was examined. The testes, epididymides, seminal vesicles, and ventral prostate were dissected from each animal and weighed. The papaya-treated male rats had a 40% reduced ability to fertilize the females. The weights of the genital organs and the motility pattern of spermatozoa of the treated animals were not significantly different from centrols (46). While these data are suggestive of a marginal antifertility effect, one must take into account that ground whole seeds were administered to the test animals at a fairly low dose level (20 mg/animal). In order to establish that these data represent a significant male antifertility effect, further testing is required, using concentrated solvent extracts from C. papaya seeds.

Catharanthus-oseus (L.) G. Don (Apocynaceae). The total alkaloid fraction prepared from C. roseus leaves, when administered to male rats by intraperitoneal injec tion, led to graded dcgenerative changes in the sper matogenic elements of the testis. Further, the testes of treated rats were flaccid and weighed less than those of control animals (88). These effects can be attributed to the vinblastine and vin cristine in C. roseus leaves; the former alkaloid is a major constituent of ihe total bases. As with most cytotoxic agents, vinblastine and vincristine are antispermatogenic in laboratory animals (32, 39, 108, 136). Vinblastine causes oligosperwc; in humans as well (183). Dieffenbachia seguine (Jacquin) Schott. (Araceae). The forced feeding of D. seguine to sterilize male and female prisoners in concentration camps during World War II was documented in 1949 in the accounts of the "Trials of War Criminals Before the Nuremberg Military Tribunals" (4). Apparently the active constituents of the plant lack selectivity, and D. seguine must be considered an irreversible sterilizant, nothi contraceptive agent. Both a mls ad fmls ls erdcie cpblt males and females lose their reproductive capability following ingestion of the plant. The plant's sterilizant effect was first reported in labora tory animals (123) following reports that it was used for sterilization purposes by natives in Brazil. Although the toxic effects of this plant have been well documented (20, 48, 56, 65, 103, 189, 190), the toxic or sterilizant elements have not been identified. There may be two types of compounds present in D. seguine, one causing the sterilizant effect and the other a toxic one. Further work appears warranted. Ecballiur elaterium A. Richard (Cucurbitaceae). The sole documentation that this plant may be useful as an antispermatogenic agent is a United States patent issued for this claim (130). The experimental evidence offered in support of male antifertility activity is confusing. For ex ample, the patent states, "Thus, the use of the flower in producing a new contraceptive is highly surprising" (36). But this statement follows: "In order to prepare the claim ed invention, the plant is collected, dried for two weeks, and the leaves and stems are ground up in a mortar and pestle until they become a powder" (130). The extract (1.0 mg) was administered by gastric intu bation to mature male rabbits, and 1 hour later, the animals were ejaculated by mechanical stimulation. Nor mal values for the control rabbit ejaculate were compared with those of the ejaculate collected 1 hour following dos ing of each rabbit with 1.0 mg of the plant extract (Table 1). A reduction in sperm motility and semen pH was reported. Addition of the extract to ox semen produced complete liquefaction, with a 90% drop in motility in vitro (130). 3

RABBIT PARAMETER VOLUME (ml) NUMBER OF SPERM/ml MOTILITY 1st hour 2nd hour

3rd hour

4th hour

MORPHOLOGY pH NORMAL VALUE 0.02-0.05t" 2-3,500!" 85-98% 75-55% 50-25% 25-5% 100% normal 6.2±0.05

I,

HUMAN

POST-DRUG VALUE* 0.02-0.5 2-35,0001 15-2% 0% 90-100% normal 1.4±0.01

$third to eighth hour §eighth to fourteenth hour

NORMAL VALUE 2-4 80-120 million 80-95% 80-95% 55-35% t 35-15% § 85-95% normal 7.2-7,6 ±0.05

POST-DRUG VALUE' 2-4 5-15 million 25-35% 25-5% 5-9% 85-95% normal 1.5-2.0±0.05

*data taken 1 hour after oral administration of the extract fmost likely these values represent type-setting errors

Table 1. Effect of Ecballium elaterium extract on rabbit and humar ejaculate 1 hour after oral administration (adapted from Nassar [130]). Administration of 1.0 mg of the plant extract orally to men ranging in age from 18 to 40 years resulted in a drop of pH in the ejaculate from a normal 7.2- 7.6 to a 1 hour post-treatment value of 1.5 - 2.0. Normal sperm counts were 80 to 120 million/ml, and 1 hour post-drug values were 5 to 15 million/ml. In 24 hours, all values in treated men returned to normal (130). Only time will tell whether these claims can be verified. Gossyplum species (Malvaceae). Gossypol is a yellow, phenolic, dimeric, sesquitr!rpene pigment derived primarily from various cotton (Gossypium) species. It presents an exciting lead for a potentially useful orally active male contraceptive agent. Human studies on gossypol were initiated in China in the early 1970s, but it was not generally known that this compound had a potential application as an orally effective male contraceptive until a paper written in English was published in 1978 (6). Since then, the published data based on human, animal, and in vitro studies have increased rapidly. Most studies have been published in Chinese, however, and thus have not been widely read. the status of gossypol Zatuchni and Osborn summarized as a male contraceptive as it was known in 1980 (214). A more recent review, in which unpublished data from China are included, has been published by Prasad and Diczfalusy (139). A World Health Organization (WHO)sponsored symposium in 1980 also summarized recent information on gossypol (64). Other reviews on gossypol are also available (8, 107, 117, 118, 208). Studies on various aspects of gossypol are listed in Table 2. There are several versions of the serendipitous discovery of the effects on spermatogenesis by gossypol (116, 141, 4 214). The earliest version we encountered was published in a Chinese journal in 1957. The following account is given (116): "In between 10-25 years before the war, there was a small village in Jiang-su called Wang Estate. A friend of mine and myself had visited that village in 1929.There were about 30 families living there. They were all wealthy farmers, but were very thrifty. They always ate the least expensive food. At that time, cot tonseed oil was far cheaper than bean oil so all of the families used cottonseed oil for cooking. During a period of more than 10 years when everyone ate cot tonseed oil, there was not a single child born. At that time, the fact that there were no children born in a wealthy family wa- regarded as a strange thing. In such a wealthy village where no family had any children born in such a long period, people just thought this was the deed of an evil spirit. The villagers tried everything, even marrying widows who had borne children. This produced no results; once the women a very the village, they never got pregnant. This wascame to frustrating thing in that village. "In that period of about 10 years, the news about that village had travelled very far. People just thought that this was a very strange thing About 5-6 years before the war, there was a large crop of soy bean in the Northeast provinces. Large amounts of soy bean oil were sold to the South. The price of soy bean oil be came much cheaper than cottonseed oil for cooking. A strange thing happened, every family in this village started to raise children. "My proposition is based on this observed fact. It seems that cottonseed oil can be used for birth control.

ability of gossypol to inhibit oocyte penetration t95,179) acrosin inhibition in vitro (95,179) antispermatogenic and/or antifertility effects on hamsters (34,186) male rats (9,34,42.44,75,76,86,92,115,164,194,196,204) mice (40) monkeys (160) rabbits (34) chronic toxicity in dogs and monkeys (151) rats (43,219) digestion with pancreatin of microcapsules containing gossypol (212) distribution in the subcellular fractions of, t testes (112) effects on adrenal cortex function (7) androgen-dependent organs in mice and rats (163) anterior lobe of pituitary of castrate hogs (218) autonomic nervous system of rodents (122) DNA content of spe-m from male subjects (202) exfoliated cells in human semen from subjects taking gossypol (162) fructose metabolism by sperm (138) growth and function of Leydig and Sertoli cells in culture (221) isolated bovine sperm plasma membrane + Ca +-ATPase (198) LH levels (113,192) + + Na K -ATPase of rat kidney (23,66,205,216) Na + K + -ATPase of guinea pig kidney (205)

rat liver (10,111,135)

rat liver enzymes (10) Sertoli cell junction of the guinea pig (137) serum prostaglandin levels (191) testes, cytological (209) testes, morphological (45)

testicular Isozymes (108) testosterone levels (113,114,150,192)

tumor promotion and/or initiation (79)

ultrastructure of human sperm in vitro (78)

ultrastructure of rat sperm in vitro (29,129) effects of sister chromatid exchange on CHO K-1 cells in vitro (110) human peripheral lymphocytes (67,203) mice (215) effects on the K + depleting effect on rabbit heart (144)

rats (140,143,166,210)

skeleta' muscle in vitro (207)

electron microscopy of

effects on cardiac muscle in monkeys (119)

gossypol toxic effects in rats (206)

histological effects on pituitary, adrenal cortex and hypothalamus of rabbits (150) rats (113,192,211) human studies in Brazil (41) in China (6,8,35,109,117,118,120,139,141,204,208) hypokalemia in humans (22,139,142) non-mutagenicity (38,193) pharmacokinetics and metabolism in rats

(101,165,167,173,195)

recovery of spermatogenesis in chronically dosed rats (75)

spermicidal effects in vitro (91,146,147,175,187,188)

in vivo (33,185,201)

synergism of gossypol antifertility effects in rats with

WIN-1844 (11)

Table 2. Varieties nf gossypol studies, with references.

The fact that when stopping eating cottonseed oil the birth control effect automatically disappears, shows that it is most economical, convenient and natural."

in Nanjing In 1971, Chinese investigators etermined that

ent time, the only evidence of an antifertility effect in humans is a marked decrease in sperm counts, usually to 4 million/ml or less after dosing for 2 to 3 months in 99%

of cases.

the active principle was gossypol, and toxicity ex, riments and preliminary dose evaluations were carried out. Semen analyses were carried out on five male subjects.

Concern has been expressed over the fertility recovery

rate of men who have been using gossypol. Preliminary data suggest that 74% of subjects receiving gossypol for

After administration of gossypol for 35 to 42 days, at a

dose of 60 to 70 mg daily, four of the males were

periods of from 6 months to 4.5 years, whose sperm

count was greater than 4 million/ml, recovered normal

azoospermic and one was necrospermic. These data led to the in 1972 testing of gossypol as a male antifertility clinical agent (141).

More than 8,000 men have now been treated with

fertility (139). Correlations of recovery rates with duration

of treatment have not as yet been published, but it Is ap

parent that chances of recovery are greater in subjects

who have not achieved azoospermic levels.

Side effects attributed to gossypol treatment in China are

gossypol in the 14 provinces of the People's Republic of China (120, 139). Some have received gossypol itself, some gossypol acetic acid (a more stable form), and a few

havie received gossypol formic acid. To date, there does

not appear to be any significant difference in the action of

fatigue (12%), gastrointestinal symptoms (7%), de-

creased libido (5%), dizziness (4%), dryness of mouth

(3%), minor occurrences of sleepiness, palpitation, eye

lid edema, decreased perspiration, skin rash, and hypo

kalemia (139).

these three materials. The usual dose administered is 20

mg daily for 60 to 70 days, followed by a maintenance dose of about 60 mg/week (107, 120, 139). At the pres-

Hypokalemia was reported in 66 of 8,806 men (0.75%)

using gossypol in clinical trials (120, 139). In China, the

5

incidence of hypokalemia has been associated with regional differences, which could be related to dietary intake. The familial thyrotoxicosis sometimes seen in Chinese males produces a periodic paralysis and other symptoms that are indistinguishable from those of hypokalemia due to gossypol (14, 126, 127). Treatment with potass:um chloride (1 gm/day) reduces the fatigue, lassitude, and electrocardiogram changes in subjects with hypokalemia, and also prevents hypokalemia paralysis (139, 140, 142). In animals, gossypol seems to have varying toxicity, based on species differences. Dogs appear to be more sensitive and monkeys least sensitive to its general toxic effects. Gossypol is not mutagenic in the Ames test (38, 193), but has been shown to be tumor-inducing or tumorproducing in mouse skin painting tests (79), a finding that requires further investigation, The loss of fertility in rats and hamsters due to gossypol administration has been repeated in severa! laboratories (34, 75, 76, 186). One study in non-human primates also demonstrated the ability of gossypol to decrease sperm concentration when administered to cynomolgous monkeys (Macaca fascicularis) (160). Species variability in response to gossypol has also been demonstrated in rats, hamsters, and mice (34). The effects of gossypol on serum testosterone, FSH, and L-i are inconsistent between species and in the same species. Hadley and co-workers reported a drop in serum testosterone and LH levels with no change in FSH in rats treated with gossypol, 30 mg/kg/day for 5 weeks oral"', (75). A slightly lower effective dose (20 mgg was (75).tA stly lowreaseerumetdosterne20 mg/kg) ws reported to decrease serum testosterone with no decrease in LH (163). Doses as low as 1 mg/kg daily for 1 week have also been reported to cause a lowering of serum testosterone (114). In non-human primates, however, no change in serum testosterone was observed, after oral dosing for 6 months with 10mg/kg/day (160). Lcydig cells isolated from gossypol-treated rats, as well as normal rat Leydig cells, in the presence of added gossypol, demonstrated a reduced production of testosterone when incubated in vitro with LH (75, 114). Although there is no question that gossypol inhibits fertility, the site of action is not clear; most evidence suggests that itdamages spermatids and spermatocytes. In semen sperma obtained from gossypol-treated animals, usually tozoa are decreased and some are abnormal (75, 160). Damage to the sperm frequently involves the mitochondrial sheath and degenerative changes in the early portions of the tail (75). Experiments with ligated epididymides suggest that the sperm are damaged before they reach the epididymis (42). Damage to the seminiferous tubules of hamsters and rats after treatment with gossypol is not evident (139, 164),

but close examination of sperm in the rat testes reveals damage primarily to spermatids and spermatocytes (43, 164). These findings concur with those of Chinese in vestigators, and suggest that gossypol produces its primary effect at the later stages of sperm maturation in the testes (6), probably at the late spermatocyte or spermatid stage. Testicular atrophy may also follow the direct effects on sperm after several months of exposure (219). However, effects on sperm occur much earlier than testicular effects, suggesting that testicular atrophy is secondary to accumulation of dead sperm in the tubules (43). Electron microscopic examination of rat testes after low doses (5 mg/rat for 4 weeks) of gossypol showed dilation of mitochondria of the mid-piece in spermatids (45). Animals dosed orally with 30 mg/kg/day experienced slight damage of the spermatids, with nuclear vacuola tion, wrinkly nuclear membrane, change in chromatin staining reaction, and swelling or displacement of the cap and acrosome. Spermatozoa obtained from the cauda epididym!dis of gossypol-treated rats have the same characterstics (29, 129). Continued treatment results in the appearance of exfoliated spermatids and sperma tocytes from germinal epithelium, as well as spermatozoa with detached heads in the lumen of the tubules (209). Spermatogonia appear unaffected morphologically by gossypol administration. A single dose of 100 mg/kg results in injury similar to that seen with low-dose, chronic administration. Nine to 11 days after treatment, only Ser toll cells and germ cells remain (209). Evaluation of the biochemical activity of developing sperm confirms the morphologically described site of damage. Inhibition of moplgiaydecbdstefdmg.Inbtonf incorporation of nucleic acid and protein precursors has been reported, with the greatest effect seen in spermatids and pachytene spermatocytes (209). Although gossypol appears to affect the late spermato cyte to spermatid stages, the biochemical mechanism for this action is unknown. Studies on the inhibition by gossypol of sperm enzymes, such as LDHX (108), Ca + +-ATPase (198) and acrosin (95, 179), as well as fructose metabolism (138), have not helped in the elucidation of the biochemical mechanisms of action. Many of the effects on biochemical assay systems are probably related to the antioxidant and chelating abilities of the chemically reactive gossypol molecule, resulting in non-specific inhibition of enzyme systems. Gossypol, as isolated from Gossypium species, is an op tically inactive molecule. Attempts have been made to separate the (+)-and (-)- forms of go:,sypol, but none have as yet been successful. (+)- Gossypol has been found in Thespesia populnea (Malvaceae) (100). A recent experiment with hamsters (184) confirmed the lack of effect of (+)-gossypol on male fertility initially identified in rats by Wang (194). This lack of male antlfe"

6

tility effect by the (+ )-form of gossypol may be a result of two possible mechanisms. First, it is possible that the physiological disposition of the (+)-isomer is different from that of the (-)-isomer. This is unlikely, since the major metabolite of gossypol is a glucuronide, and glucuronidation enzymes are non-specific. Second, the actions may be mediated by a stereospecific receptor site. The extremely small amount of gossypol that actually enters the testes to exert its action is good evidence that the antifertility effect is very specific. A review of these experiments clearly indicates that male infertility occurs before there is a decrease in sperm counts, the criterion utilized in assessing gossypol effects on humans and non-human primates. It is quite possible that the doses currently being utilized (Brazil, China) may be in excess of that required for infertility. More work is needed to accurately characterize the actions of gossypol. Doses may be substantially decreased, with an associated decrease in toxicity, if better criteria for gossypol-induced infertility are developed in animals and then extended to men. Future experiments on the actions of gossypol must distinguish between nonspecific toxic effects and the potential specific actions of gossypol. Hibiscus rosa-sinensis L. (Malvaceae) The first evidence that extracts of H. rosa-sinensis flowers produced an antispermatogenic effect in laboratory animals (97). pbihdin 1972 by Kholkuie and co-workers was

Hibiscus rosa-sinensis is a member of the Malvaceae, the same plant family as the cotton plant (Gossypium species) from which gossypol is obtained. To date, an analysis of H. rosa-sinensis for gossypol has not been reported in the literature. Hlupophae salcefolle D. Don (Eiaeagnaceae) A water soluble portion of a administered subcutaneously each salicifolia bark was ethanol extract prepared from H. day for 7 days to mature male rats. The animals were found to have degenerative changes in the seminiferous epithelium of the testes. Further, administration of the ex tract to another group of castrate rats receiving testos terone showed an inhibition of seminal vesicle develop ment (89). The authors concluded that the extract of H. salicifolia produced an antispermatogenic and an antian drogenic effect. [From the experimental data presented, we consider the latter effect questionable.] Leucaena glauca (L.) Benth, (Leguminosae). In Hawaii, where L. glauca grows abundantly as a weed, it is known as koa haole. Because the leaf meal of this plant has been found to contain more than 25% protein (dry weight), livestock producers and poultrymen use it for feed. However, some animals fed koa haole leaves have lowered fertility rates, and sows and rabbits develop alopecia (199). Ruminants do not seem to be affected in the latter respect (200). Male rats fed with a ration containing 15% koa haole leaf

published i192b

hkwancowres(7.

These investigators administered an ethanol extract of H. rosa-sinensis flowers to male rats orally for 30 days at dose levels of 50, 150, and 250 mg/animal. Definite histologic evidence for an effect on male reproduction was seen on day 14 of dosing. On day 30, the group of rats receiving the 250 mg/animal dose showed shrinkage of the seminiferous tubules, and complete disorganization of the testicular tissue and destruction of spermatogonial cells. Germinal cells were also affected and Leydig cells were absent. Sertoli cells were laast affected, and the seminal vesicles and prostate were also unchanged. There was no effect in rats dosed at 50 and 150 mg/ animal (97). The extract did not have androgenic or antiandrogenic activity, Later, the same investigators published data suggesting that the initial results found by them were caused by inhibition of gonadotropin release or synthesis (97,99). A benzene extract of flowers from this plant was similarly evaluated in male rats, with essentially the same results (168). A single 7.5 mg dose of H. rosa-sinensis flower extract was injected subcutaneously in a group of reproductively active male bats (Rhinopoma kinneari), and the six testicular LDH isozymes were measured daily. It was reported that LDH disappeared from the isozymograms on days 2 to 4, but reappeared on day 5 (98).

meal for 2 weeks prior to mating had a significant de

crease in pregnancies (121). Leucaenaglauca is known to contain significant amounts of the cytotoxic amino acid mimosine, about 3% in the leaves and 5% in the seeds. Mimosine may be responsi ble for the lowered male fertility in the previously men tioned study, since a mimosine-inactivating agent (sodium iron pyrophosphate) was added to the koa haole ration, which when fed to male rats did not impair fertility (121). This constitutes another example of the presence of a cytotoxic agent in plant material showing antifertility ef fects in the male. Lonicera clllosa Poir. (Caprlfollaceae). Male and female mice were dosed daily with 10 to 20 mg of a 50% ethanol-water extract prepared from L. ciliosa leaves. The extract was given 2 weeks prior to, and for 3 weeks during, cohabitation of the mice. This resulted in a pro nounced decrease in litters by the females in the study

(19).

From the design of the study, it was not possible to aEcer tain whether the effect of the plant extract was on the males or the females, or both. We believe that the plant should be reevaluated, using a more appropriate experi mental design. 7

Lupinus termis Forsk. (Leguminosae). Male rats were fed a ration containing 27% untreated or debittered (hot water treatment) L. termis seeds for 9 weeks. The animals were then sacrificed and the seminal fluid and testes were examined. Although the histologic evidence revealed a decreased sperm production in Lupin-treated animals, the biochemical evidence did not rule out the possibility of an indirect effect through decreased production of testosterone (174). This is another study suggesting the need for further experiments to determine if the antispermatogenic effect is worth pursuing. Malvaviscus conzattill Greenm. (Malvaceae). An ethanol extract prepared from the dried flowers of M. conzattii was administered orally for 25 days to male gerbils (Meriones hurrianae)at a daily dose of 25 mg/kg, and to maie rats at a dose of 50 mg/kg. On day 20, the animals were sacrificed and the testes, prostate, epididymides, and seminal vesicles were removed and weighed. The animals had no body weight loss relative to controls, but testicular weights decreased drastically in both species, as did the weights of the accessory sex glands. The weight of the adrenal glands increased significantly. The investigators suggest that both antispermatogenic and antiandrogenic effects were mediated by antigonadotropin activity (51). Similar results have been reported for an extract administered to bats (Rhinoporakinneari) (52) and mice (87, 182). PLANT FAMILY GENUS AND SPECIES

AMARANTHACEAE

DEERINGIA AMARANTHOIDES MERR.

Maluaviscus conzattii is closely related to Gossypium and Hibiscus species, and may contain gossypol. However, this species apparently has not been evaluated for the presence of gossypol, which would seem to be necessary before additional studies are carried out to substantiate the preliminary data. Momordica charantla L. irbltaceae). An ethanol extract of M. charantia fruits rJministered orally or sub cutaneously to adult male gerbils (Meriones hurrianae) for a period of 2 weeks reduced testicular weights and disrupted spermatogenesis without significantly affecting the sEminal vesicles or prostate. Doses ranged from 200 to 400 mg/kg (54). Dogs were dosed orally with the same extract at 1.75 mg/animal daily for 60 days, pro ducing similar results (54). Additional critical studies of this plant appear warranted. Ocimum sanctum L.(Lablatae). Oral daily dosing for 15 days with a benzene extract prepared from the dried leaves of 0. sanctum at levels of 100. 150, and 200 mg/kg decreased testes weight with no effect on the epididymides, seminal vesicles, prostate, or vas deferens of male rats (94, 156). A decreased sperm count in the rats receiving 150 mg/kg was also reported (94). These data should be confirmed in new studies using larger numbers of animals and dosing for more than 15 days. TYPE SPERM' RAT HUMAN

+ +

-

PLANT PART

Aerial parts Aerial paris

ACTIVE PRINCIPLE(S)

-

REFERFjNCE(S)

158 84,158

ARALIACEAE

SCHEFFLERA CAPITATA HARMS ASCLEPIADACEAE PYCNOSTELMA PANICULATUM K.SCHUM BALSAMINACEAE IMPATIENS DUTHIEI HOOK.F. CARYOPHYLLACEAE GYPSOPHILA CERASTOIDES D.DON GYPSOPHILA PANICULATA L, SAPONARIA OFFICINALIS L. VACCARIA SEGETALIS (NECK.) GARCKE COMBRETACEAE TERMINALIA BELLERICA ROXB. TERMINALIA HORRIDA STEUD. COMPOSITAE CALENDULA OFFICINALIS L. CENTRATHERUM ANTHELMINTICUM (WILLD.) KUNTZE SOLIDAGO VIRGAUREA L.

+

Scheffleroside (Triterpene saponin)

-

Not stated Whole plant Whole plant Roots Roots Not stated

+ t + + nt nt + t

nt

-

12 158

+ + + nt

-

+

-

158

I

1

Vacsegoside

(TrIterpene saponin)

Fruit Fruit Whole plant

Flowers

Seeds

Whole plant Whole plant Aerial parts

Whole plant Bark

Whole plant

Seed pod + nt + nt + + + + + nt + + nt + nt +

-

12

157,158

I

158

- - - - Acacic acid sapontn

-

1

158 157,158 158 157,158 158 16,17 157,158

157,158

+ + + nt + + +

GfLAMINEAE

DIMERIA GRACILIS NEES EX STEUD. LABIATAE

ANISOMELES MALABARICA R.BR. EX SIMS CALAMINTHA UMBROSA FISCH. & MEY. LEGUMINOSAE ACACIA CONCINNA DC. AESCHYNOMENE INDICA L. ALBIZIA LEBBEK BENTH.

Echinocystic and oleanolic acid saponins

Table 3. Plants reported to be spermicidal (in vitro), shown with their active principles. 8

Table 3 (continued) PLANT FAMILY GENUS AND SPECIES

ALBIZIA LEBBEK BENTH. ALBIZIA PROCERA BENTH. ALBIZIA PROCERA BENTH. ENTEROLOBIUM CYCLOCARPUM GRISEB. GLEDITSIA SINENSIS LAMK. GLYCYRRHIZA GLABRA L. MELILOTUS SICULA VITMAN

OUGENIA DALBERGIOIDES BENTH.

PLANT PART

Roots Roots Seeds Leaves Not stated Aerial parts Seeds

Stem bark

TYPE SPERM* RAT HUMAN

+ +

ACTIVE PRINCIPLE(S)

Lebbekanin E (Thterpene saponin) Oleanolic acid saponin Oleanolic acid saponin -

REFERENCE(S)

16,158,181 16-18,181 16-18 1 12 157,158 1

158

nt nt nt +t + nt

+

+ + + nt nt +

-

PITHECELLOBIUM DULCE (ROXB.) BENTH. PTEROLOBIUM INDICUM A. RICHARDS SAMANEA SAMAN (JACQUIN) MERR.

Seeds Aerial parts Aerial parts Flowers Seeds Whole plant Aerial parts Aerial parts Aerial parts Not stated Roots Fruit Aerial parts Not stated Whole plant Whole plant Fruit Aerial parts Fruit Not stated Seeds Seeds Seeds Seeds Seeds Whole plant Whole plant Whole plant Aerial parts

Whole plant

+ + + + + nt nt + + +t nt nt

+

+ + + + + + +

+

Oleanolic and echinocystc acid

saponins

-

18

157,158 158 180 158 217

1

158

158

12

159

171

83

5 16,1E

69,158

18,69,157 69,158

69,158

69 16,18 16,18,157 16,18 16,18 15,18 158

157

158 158

157

TRIGONELLA FOENUM.GRAECUM L. LILIACEAE PARIS POLYPHYLLA SMITH RUSCUS HYPOGLOSSUM L. MYRSINACEAE

ARDISIA NERIIFOLIA WALLICH MAESA INDICA VAR. ANGUSTIFOLIA HOOK.F. & THOMS.

PHYTOLACCACEAE

PHYTOLACCA ACINOSA ROXB. PHYTOLACCA AMERICANA L. PHYTOLACCA DODECANDRA L'HERIT. PITTOSPORACEAE PFITOSPORUMNEELGHERENSE WIGHT & ARNOTT POLEMONIACEAE POLEMONIUM CAERULEUM L. PRIMULACEAE

ANAGALLIS ARVENSIS L. RANUNCULACEAE

CALTHA PALUSTRIS L. SAPINDACEAE BLIGHIA SAPIDA KOENIG SAPINDUS MUKOROSSI GAERTN. SAPINDUS MUKOROSSI GAERTN. SAPINDUS TRIFOLIATUS L. SAPOTACEAE

MADHUCA BUTYRACEA (ROXB.) MACBRIDE MADHUCA LATIFOLIA (ROXB.) MACBRIDE MIMUSOPS ELENGI L. MIMUSOPS HEXANDRA ROXB. MIMUSOPS LFlTORALIS KURZ SCROPHULARIACEAE CELSIA COROMANDELINA VAHL HERPESTIS MONNIERA HBK. SOLANACEAE

SOLANUM XANTHOCARPUM SCHRAD. & WENDL. SYMPLOCACEAE

SYMPLOCOS GARDNERIANA WIGHT UMBELLIFERAE

CENTELLA ASIATICA URBAN

Samanin D (Triterpene saponin) -

nt + +

-

Oleanollc acid saponin

-

Oleanolic acid saponin Pitoside A; Pttoslde B (Triterpene saponin)

-

+

+ + + + + + + + + + + + nt + nt

-

nt + + + + nt + + nt nt nt + nt + +

+

Anagalligenone saponin

-

Oleanolic acid and hedera.qenin saponins

-

Hederagenin saponin

-

Bassic Bassic Bassic Bassic Bassc

-

acid saponin acid saponin acid saponin acid saponln acll saponin

Bacoside (Triterpene saponin)

- -

-

HYDROCOTYLE SAVANICA THUNB. Whole plant + 158

PIMPINELLA DIVERSIFOLIA DC. Whole plant + nt 158

VERBENACEAE

CLERODENDRUM SERRATUM SPRENG. Aerial parts + + 158

ZYGOPHYLLACEAE

BALANITES ROXBURGHII PLANCH. Fruit + + Diosgenin saponin 18

+ ,spermicldal; -, not spermicidal; nt, not tested (see original reference for concentrations used and test conditions) t bull sperm was used * reported used as a vaginal contraceptive in 100 women for up to 1 year at a concentration of 1:1,000, with no side effects and with complete protection.

9

Prunus emarginata Walp.(Rosaceae). This plant was tested for antifertility activity at the same time as was Lonicera ciliosa (19), but should be retested using more orthodox procedures to determine antifertility effects in the male. Withania somnifera (L.) Dunal (Solanaceae). Withania somnifera root powder was fed to male and female mice at a dose of 25 mg daily for 10 days. Males were then mated with the females and the feeding with W. somnifera root was continued during the mating period, until pregnant animals littered. Administration of this plant

seemed to delay mating, decrease pregnancies, and result in smaller litter sizes (70). However, since both males and females received the test drug, it isnot possible to determine whether the effect was on the male or the female, or both. Additional studies must be carried out using appropriate protocols to ascertain the effects on males. SPERMICIDES OF PLANT ORIGIN Most of the currently marketed spermicides, such as non oxynol-9, act by disruption of the spermatozoon plasma cell membranes. This action is only one of numerous

COMPOUND NAME

DL-ALANINE

BENZOQUINONE

BERBERINE

CATECHOL

CHIKUSETSUSAPONIN IV

3,4-DIHYDROXYCHALCONE EMETINE GLYCINE GOSSYPOL GUAIACOL

LEBBEKANIN E

LEMMATOXIN

LEMMATOXIN C

L-LYSINE HCL

2-METHOXY-5-METHYL-

BENZOQUINONE.

2-METHOXY-1,4-

NAPHTHOQUINONE

MYRICETIN

NIMBIN (NA SALT)

NUPHAR LUTEUM ALKALOID

OLEANOGLYCOTOXIN A

DL-ORNITHINE HCL

OUABAIN

PITTOSIDE A

PITT'OSIDE B

PAEONOL

PYROGALLOL

QUERCETIN

QUININE

CHEMICAL CLASS

Amino acid Benzoquinone Isoquinoline alkaloid Phenol Triterpene saponin Chalcone Isoquinoline alkaloid Amino acid Dimeric sesquiterpene Phenol Triterpene Triterpene Triterpene Amino acid Benzoquinone Naphthoquinone Flavonoid Triterpene Alkaloid Triterpene saponin Amino acid Cardiac glycoside Triterpene saponin Triterpene saponin Acetophenone Phenol Flavonold Quinoline alkaloid

EFFECTIVE CONC.

200 mMol 1-51,000 1-333

1-200

20-100g/ml 1-100

1-10,000

100 mMol

0.02-20 mg/ml

1-1,600 Not stated 80 ug/ml 20 Ag/ml 200 mMol 1,6,000 1,100 2 mg/ml 1-1,000

No: stated

10-100 pg/'ml 200 mMol 0.01-0.001 M 1.1,250 1-1,250 Not stated 1-50

1-1,000 1-100,00 1-2,000 1-1,000 1.1,000 1-1,000 1-100

1-50

1-25

1-500

1-500

1-100

1-1,000 1-1,000 1-3,200 1-12,800 1-2,000 1-16,667 1-4

Not stated

1-6,400

SPERM SOURCE

Rabbit Guinea pig Bull Guinea pig Human Rat Human Rabbit Human Boar Guinea pig Human Human Human Rabbit Guinea pig Rat Human Rat Human Human Rabbit Boar Human Human Bull Guinea pig Rat -luman Guinea pig Rat Dog Rat Rat Human Rat Human Rat Human Rat Rat Guinea pig Guinea pig Human Rat Human Bull Guinea pig

REF.

154

73

149

73

171

172

30,36 154

91,146,147,187,188 175

73

181

171

171

154

73

171

172

161

2

171

154

25-27

83

83

12

73

172

30,36,159

28

28

28

172

172

180

180

84

84

159

172

172

73

73

81

47

178

12

73

RUFIGALLIC ACID

RUTIN

SAMANIN D

SCHEFFLEROSIDE TANNIC ACID

TAXIFOLIN

3,3',4,4'-TETRA-

HYDROXYCHALCONE

THYMOL THYMOQUINONE TOLUQUINONE UREA VACSEGOSIDE O-VANILLIN

Phenol Flavonoid Triterpene saponin Triterpene saponin Polyphenol Flavanone Chalcone Monoterpene Monoterpene Quinone Simple amide Triterpene saponin Benzenoid derivative

Table 4. Plant principles reported to have spermicidal properties (in vitro). 10

mechanisms to kill spermatozoa. In the past, mercuric compounds were used as spermicides and acted as general protoplasmic poisons, but the marketing and use of mercury-containing preparations has been discontinued. Numerous enzyme systems in viable sperm are susceptible to inhibition, for example, those involving glycolysis, energy production, and myosin contraction. The local application of spermicides allows for less specificity of action, when compared with the specificity required for oral or injectable contraceptive agents. Although development of specific inhibitors of sperm metabolic pathways may be difficult, sperm-specific isozymes, such as LDHx, are being identified. It is quite possible that future investigations into the biochemistry of spermatozoa will provide new and more specific sites for their destruction, A large number of plants have been randomly selected and screened for spermicidal activity in vitro, and several appear promising. Those species found to be active, and the nature of the active principle(s), when known, are presented in Table 3. A list of plant-derived chemical substances of known or partially known structure, reported to be spermicidal in vitro, appears in Table 4. The active compounds represent a variety of chemical structures, many of which appear to be too toxic for consideration as spermicidal agents in humans. A majority of plant-derived spermicides are triterpene saponins of several structure types; steroid s~ionins are less frequently encountered. Combinations of sugars, attached in glycosidic linkage to the aglycones, include arabinose, fructose, fucose, galactose, glucose, glucuronic acid, rhamnose, and xylose. At the present time, just which structural features within this class of spermicides are required for optimal activity is open to speculation. A study comparing the spermicidal effects of various triterpene saponins of known structure could reveal information that might be helpful in this respect. Mechanistic studies have not been reported relative to the spermicidal action of the triterpene saponins. Since all saponins exhibit surface-active properties, the most likely mechanism for their action is a disruption of the sperm plasma cell membrane, in a manner similar to that of nonoxynol-9. Plants are an untapped resource for inexpensive and effective spermicide preparations. The initial screening of plant extracts for spermicidal activity is simple and does not require elaborate facilities. In countries where re search costs limit the type of experimental studies that can be conducted, and where flora is abundant, programs to develop inexpensive vaginal spermicides should prove productive,

SEMEN COAGULATION AND LIQUEFACTION Human semen coagulates immediately following ejacula tion, trapping and immobilizing spermatozoa in a tight fibrin matrix. Under normal circumstances, liquefaction occurs within several minutes and is usually complete after 5 to 20 minutes. The semen of some species (guinea pig and rat) coagulates but does not liquefy; rather, a plug is formed from the ejaculate. Semen from other species (dog and bull) does not coagulate at all; the coagulum temporarily immobilizes spermatozoa and al lows a resting phase before the sperm become motile and travel through the female reproductive tract. Components of the coagulation system are added to the ejaculate via secretions from the seminal vesicles. The Ii quefaction factors, primarily plasminogn activators, originate from the secretions of the Cowper and prostate glands. Modification of Cowper and prostate gland fluids could eliminate or greatly reduce the rate of liquefaction of semen and thereby decrease fertility. This effect on the liquefaction rate has been observed in men with patho k ical conditions that result in delayed liquefaction of semen. A long-lasting coagulum could cause a significant reduction in fertility in the human, but whether increased coagulation time alone would result in consistent com plete loss of fertility is doubtful. Agents that extend the coagulation period could be utilized in conjunction with agents acting on other spermatozoon properties, and might result in a synergistic effect to reduce fertility. Currently, the coagulation system in semen has not been carefully examined biochemically to identify differences from other coagulation systems. Until specific differences are discovered, the use of coagulation-inducing sub stances will be restricted to vaginal contraceptives. With the exception of a few plants with lectins that coagulate semen, plants have not been studied for this ef fect until recent years; in 1977, about 1600 Indian plants were screened, and 90 showed positive semen coagu lating properties (158). Initial testing involv;.d preparation of ethanol/water (1:1) extracts of dry plant material, and testing of these (after removal of solvent) at a 2% concen tration on human semen and rat vasal or epididymal con tents (196). All 90 positive extrats coagulated rat vasal or epididymal contents, whereas only 49 coagulated human semen. In ten active extracts, further study sug gested that the active principles were tannins. Most likely, these extracts had a denaturing effect on one or more proteins involved in the semen coagulation-liquefaction process. SPERM AGGLUTINATION Spermatozoa normally bind several substances to their outer membrane; these substances, called sperm-coating antigens, are derived from the male genital tract fluid and are usually associated with sperm fertility inhibition. Ex 11

amples of sperm-coating antigens are decapacitation factor and proteinase inhibitors. These binding substances normally block sperm fertility and are removed from the sperm head or are deactivated during the process of capacitation in the female genital tract. Several agglutinins, such as piant lectins, have been shown to adhere to the plasma membrane of human spermatozoa and inhibit the normal processing of sperm. Tight binding of exogenous substances to spermatozoa may result in perturbations in the plasma membrane, which then prevent capacitation. Andie haga semspecyinhified antigetyn sumase LDH , have successfully inhibited fertility in female animals. Although the results are inconclusive, the aggluti on of sperm by means of sperm-specific antibodies may be possible. Although investigators must exercise caution in evaluating the sperm-agglutinating properties of agents capable of interacting with the immune system, agents that cause sperm agglutination through direct interactions with the sperm membrane may be valuable adjuncts in vaginal contraception. As with the coagulation system, this mechanism may reduce fertility, but it is not likely to be 100% effective.

Plants warrant systematic study as a potential source of sperm-agglutinating compounds.

SPERM-SPECIFIC ENZYME INHIBITORS Investigations into the biochemistry of fertilization have revealed some of the enzymes required for fertilization; of major importance are acrosin, hyaluronidase, and corona-penetrating enzyme (CPE). These enzymes are attached to the acrosomal membrane located on the head of the spermatozoon. Each of the enzymes has an important role in the fertilization process. Corona-penetrating enzyme is important for the penetra tion of the spermatozoon through the corona radiata. Hyaluronidase attacks the intercellular matrix of the cumulus oophorus and clears a path to the zona pellucida, where the enzyme acrosin, a protease, assists in the penetration. Binding of the spermatozoon to the zona is necessary for fertility, but the exact mechanism is unknown. Endogenous inhibitors of CPE (decapacitation factor) and acrosin (proteases) are found attached to the sperm head and are normally removed during capacitation. Substances that inhibit acrosin and hyaluronidase have been identified and are being studied as possible fertility-

COMPOUND NAME

CHEMICAL CLASS

REFERENCES

regulating agents, primarily as vaginal contraceptives.

Highly specific inhibitors of acrosin, hyaluronidase, and CPE may be developed, not only for vaginal application, but for oral use as well. Finding substances that specifically inhibit these enzymes may be difficult, however, because of their similarity to enzymes in non-reproductive tissues.

ASCORBIC ACID (+) - CATECHiN CIRANTIN DIHYDROQUERCETIN

ECHINACIN ERIODICTYLOL

Vitamin Flaonoid Flavonoid Flavanone

Unknown Flavanone Organic acid

145

145

71 148

102,104,105 148

FUMARIC ACID GENTISIC ACID HESPERIDIN

HESPERIDIN,

Phenolic acid Fiavonoid giycoside Favonoid derivative

Phenolic acids Flavanone

13 128 82 90,104,169

77,155 148

Acrosin inhibitors. No effort has been made to broadly

screen plants for acrosin inhibitors. Plant-derived acrosin inhibitors are few, and include soybean and other trypsin inhibitors as well as a large number of monosaccharides. D-hexoses are generally more effective acrosin inhibitors than are L-hexoses (3). Gossypol has recently been reported to inhibit acrosin (95,179). The area has been

ACID DERIVATIVES NARINGENIN

PHOSPHORYLATED HYDROXYBENZOIC

well reviewed by Zaneveld (213).

QUERCETIN QUERCETRIN RESERPINE RHAMNETIN RUTIN TECTORIDIN

TECTORIGENIN

Flavonol Flavonol Indole alkaloid Flavonol Flavonol glycoside Isofavone

Isofiavone

148 148 21,124 148 148 5L.

57

Hyaluronidase inhibitors. Very few plant extracts have been tested 'or hyaluy.nidase inhibitory effects. Only three appear to produce significant activity: the seeds of Aescuius hippocastanum L.(Hippocastanaceae)(72); Echinacea purpurea Moench (Compositae), whole plants (105); and the root essential oil of Curcuma longa (Zingiberaceae) (74).

Compounds of known structure in higher plants that have antihyaltronidase activity represent a hetero geneous group, and are listed in Table 5,

XANTHORHAMNOL

Flavonold glycoside

57

Table 5. Plant-derIved hyaluronidase Inhibitors determined by in vitro and/or in vivo evidence. 12

,

.,

S Initially, we noted that plants are definitely a source of many useful and widely-employed drugs, and that practical fertility-regulating agents are likely eventually to be discovered from this source. There is little folkloric information about the use of plants to regulate fertility in the male. Some published reports exist on the testing of crude plant extracts for male antifertility activity, but these represent only a fraction of the number of reports suggesting effects of plant extracts on ftmale fertility. There seems to be a lack of correlation between experimental results obtained by one group of investigators and another, in data obtained in vitro and in vivo, and in experimental results and information found in folklore. Fac tors complicating the adequate assessment of plants af fecting male fertility are inadequate numbers of vehicletreated controls, poor experimental design, problems related to insolubility of crude plant extracts, variation in routes of administration, and the diversity in reproductive function and control among various laboratory species. Another significant problem involves the manner in which plant names are cited in scientific articles. Botanical nomenclature can be confusing when Latin binomials of plants are either so grossly misspelled as to be unrecognizable, or the misspelled name resembles another taxon. Two identical Latin binomials can represent entirely dif-

ferent species. Such problems can be resolved only ifthe authority for each taxon is given. Thus, if a plant being studied is represented by an inadequately documented Latin binomial, the data may be irreproduclble. Drug development programs that involve the synthesis of useful compounds are complex, requiring intensive effort by organized teams of highly qualified investigators. Even so, it might take 10 years or more to develop an effective agent that may be safely evaluated in the human. Fur thermore, not all compounds that enter clinical trials emerge as markeiable drugs. It thus seems logical to ex pect that similar high intensity programs, well organized and appropriately budgeted, would have to be imple mented ifuseful fertility regulating agents for the male are to be isolated from plants and identified. The plant kingdom remains a virtually untapped source of new fer tility regulating agents.

ACKNOWLEDGEMENTS Collection, compilation, and computerization of the liter ature cited has been supported, in parl, through funds from the World Health Organization, Special Programme of Research, Development, and Research Training in Human Reproduction (Project 78135), and from the Na tional Cancer Institute (U.S.A.) contract CM-97259). The authors wish to thank Mr. Chun-Tao Che for translating into English much of the work published in Chinese that has been cited herein.

REFERENCES

1. Abd Elbary A, Nour SA: Correlation between the spermilcidal activity and the hemolytic ;ndex of certain plant saponins. Pharmazie 34:560,1979. 2. Aleshkina YA, ll'inskaya TN, Rubinchik MA, Vichkanova SA: Alkaloid from Njmphaeaceae. British Patent 968,042. Chemical Abstracts 61:15939c. 3. Anderson RA Jr, Oswald C, Leto S, Zaneveld LJD: Inhibition of human acrosin by fructose and other monosaccharides. Biol Reprod 22:1079, 1980. 4. Anonymous: Experiments for rrass sterilization. In Trials of War Criminals before the Nuremberg Military Tribunals 1:694, 1949. 5. Anonymous: Annual report, Central Drug Research Institute, Lucknow, India, 1978, p 8. 6. Anonymous: Gossypol - a new antifertility agent for males. Chinese Med J (New Series) 4:417, 1978. 7. Anonymous: Effect of gossypol on adrenal cortex function. Shan-hsi Hsin I Yao 8(7):50, 1979. 8. Anonymous: Gossypol - a new antifertility agent for males. Gynecol Obstet Invest 10:163, 1979. 9. Anonymous: Gossypol - a new contraceptive for males. Wu-han I Hsueh Yuan Hsueh Pao 8:102, 1979. 10. Anonymous: Effects of gossypol on serum transaminases of rats. Shan-hs Hn I Yao 98:46, 1980. 11. Anonymous: Antifertility synergism of gossypol and WIN-1844. Yao Hsueh T'ung Pao 15(9):42, 1980. 12. Anonymous: Study on spermicidal agents originating from Chinese medicinal plants. Abstract of a paper obtained from the Pharmaceutical Factory, Nanjing College Pharmacy, Nanjing, People's Republic of China,of1981. 13. Arai T, Suehiro S: Influence of fumaric acid on organisms, with special reference to the atrophy of gonads. Wakakama Med Rept 1:35-42. Chemnical Abstracts 48:46 8 MR. 14. Au KS, Yeung R: Thyrotoxic periodic paralysis: Periodic variations in the muscle calcium pump activity. Arch Neural 26:543, 1972. 15. Banerji R, Masera G, Nigam SK: Constituents of Mimusops littoralis seeds. Fitoterapia 50:53, 1979. 16. Banerji R, Misra G, Nigam SK, Singh S, Saxena RC: Steroid and triterpenold saponins: Possible spermicidal agents. J Steroid Biochem 9:864, 1978. 17. Banerji R, Nigam SK: Chemistry of Acacia conm- cinno and A. caesia bark. J Indian Chem Soc 56:1043, 1980. 18. Banerjl R, Srivastava AK, Sra GM, Nlgan SK, Singh S, Nigam SC, Saxena RC: Steroid and triterpenold saponins as spermicidal agents. Indian Drugs 17:6, 1979. 19. Barfknecht CF, Peng HC: Antifertility factors from plants. I.Preliminary extraction and screening. J Pharm Scl 57:1607, 1968. 20. Barnes A, Fox LE: Poisoning with Diefen. bochia. J Hist Med Allied SO. 10:173, 1955. 21. Bertelli A, Cogni G, Testi G: The antipermeabllty effect of reserpine. Boll Soc Ital Biol Sper 32:832, 1956. 22. B XF, Ye YX, Yang HF, Zhang ZR: Preliminary study on gossypol In causing hypokalemia. Chung kuo I Hsueh Ko Hsueh Yuan Hsueh Pao 3:175, 1981. 23. BI XF, Zheng YJ, Yang HF, Zhang ZR: The elfect of gossypol on ATPase activity of the kidney. Chung-kuo K'o Hsueh 24: 573, 1981. 24. Bingel AS, Farnsworth NR: Botanical sources of fertility regulating agents: Chemistry and phar macology. In Corbin M, Comn A (eds): Progress in Hormone Biochemistry and Pharmacology, vol. 1, St. Alban, Vt, Eden Press, 1980, p 149. 25. Boender J: Effect of ouabain on the motility of boar spermatozoa. Proc Kon Ned Akad8 We!ensch, 3 Ser C 77:125, 1974. Chemical Abstracts :53930y. 26. Boender J: The relation between sperm quality and the effect of Diergeneeskd 101:1134,of 1976. sper matozoa. Tijdschr ouabain on the motility Chemical Abstracts 86:65395 . 27. Boender J: The effect of chelators, calcium and magnesium on the ouabain sensitivity of boar sper matozoa. Proc Kon Ned Wetensch, Ser C 80:35, 1977. Chemical Abstracts 86:1838991. 28. Booysen C: Quinine as a contraceptive. Mar riage & Hygiene 2:54, 1935. 29. Bozek SA, Jensen DR, Tone JN: Scanning elec tron microscopic study of spermatozoa from gossypol-treatedrats. Cel Tssue Res 219:659, 1981 30. Brown-Woodman PD, White IG: Investigation of the spermicidal action of quinine and emetine. Therlogenology 8:199, 1977.

13

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206. Wu YW, Zong SD, Shleh SP: liectron microscopic observation of the toxic effect of gossypol on rats. Chleh P'oy Hsueh Par) 10:92, 1980. potassium content In Isolated91 skeletal muscle. Yao Hsuh Tn 62:4 207. Xu Y, Qlan SZ: Effect of gossypol on sue Tung Pao 16(2):14, 1981. 208. Xue SP: Studies on the antifertility effect of gossypol, anew contraceptive for males. In Fen CC, Griffin D (eds): Recent Advances in Fertility Regula ton. Being, September 2-5, 1980. Geneva, Atar SA, p 122. 209. Xue SP, Zog SD, Su SY, Wu YW, Liu Y, Zhou ZG, Ma XX: Antispermatogenic effect of gossypol on the germinal epithelium of the rat testes. A cytological autoradiographical and ultrastructural observation. Chung-kuo K'o Hsueh 23:642, 1980. 210. Yu TH, Zhang XD, Wang ZH: Ineffectiveness of gossypol on deoxycorticosterone acetate (DOCA)-induced hypokalemia in rats. Sheng Chih Yi Bi Yun 1(2):39, 1981. 211. Yuan DX, Liu XY, Cao YC, Wang JY, Fu ZLHistochemical observation of the effect of gossypol on pituitary, adrenal cortex and hypothalamus in rats. Chieh P'ou Hsueh Pao 11:331, 1980. 212. Yuan XB: The digestion of capsule film with pancreatin for the determination of gossypol in microcapsules. Yao Hsueh Tung Pao 16(3):15, 1981. 213. Zaneveld LJD: Sperm enzyme inhibitors as an tifertility agents. In Hafez ESE fed): Human Sc,,nen and Fertility Regulation in Men. St. Louis, CV Mosby Co., 1976, p 570. male antifertility agent. Report of a workshop. Research Frontiers in Fertility Regulation 1(4):1, 1981. 215. Zhang ZS, Wang MM, Lu Q, Yiao YL, Jiang XR, Wan ; RL, Zheng HZ, Tan YB: Genetic studies of gossypol. I. The testing of gossypol acetic acid on sister chromatid exchanges In vitro In the mouse. Sheng Chih Yu Bi Yun 1:42, 1981. 216. Zhao RS, Fu YF: Effects of gossypol acetic acid

on NA + -K+ -ATPase activity in the kidney of rats.

Chung-hua I Hsueh Tsa Chih 61:12, 1981.

217. Zheng YK, Lu FY: Spermicidal activity of Paris polyphyla Smith. Chung Ts'ao Yao 12(2):40, 1981.

218. Zhou JY, Zhag Y, Zhang SX, Bo AH, Han 214. Zatuchni GI, Osborn CK: Gossypol: A possible

tions of extracts and compounds from Phytolacca dodecandra. Contraception 10:135, 1974. 172. Swayne VR, Beller JM, Martin GJ: Sper-

micidal effect of certain physiologically active

substances. Proc Soc Exp Biol Med 80:384, 1952.

173. Tang XC, Zhu MK, Shi QX: Comparative

studies on the absorption, distribution an"d excretion

14 of C- ossypol in four species of animals. Yao

Hsueh Cset Pao 15:212, 1980. 174. Tannous RI, Nayfeh SN: Effect of feeding lupine seeds on spermatgenesis in the rat. Austo2 ,193. 175. Tso WW, Lee CS: Effect of gossypol on boar

spermatozoa in vitro. Arch Androl 7:85, 1981.

176. Tsubura Y, Shimomura H, Yamamoto H, Imai

S, Seklgawa S: Toxicity test of keracyanin on rats

given orally for three months. Nara Igaku Zasshi

30:13, 1979. 177. Tyler VE: Plight of plant-drug research in the

United States today. Econ Bat 33:377, 1979.

178. Umekaz C, Fordney-Settlage DS: In vitro studies on cervical contraception: Use of urea as a spermicidal agent. Contraception 12:465, 1975. 179. Van der Ven HH, Kennedy WP, Bhattacharyya AK, Waller DP, Polakoski KL, Zaneveld LJD: Gossypol inhibition of human publication. and oocyte penetration. Submitted for sperm acrosin 180. Varshney IP,Khanna NK: Partial structure of a new saponin samanin-D from the flowers of Pithecellobium saman Benth. Indian J Pharm Sci 40:60, 1978. 181. Varshney IP, Vyas P, Srivastava HC, Singh PP: Study of Albizzia lebbek Benth. wood saponin, lebbekanin E.Natl Acad Sd Lett (India) 2:135, 1979. 182. Verma OP, Joshl BC, Kumar S, ChatterJee SN: Antifertility effects of Malvauiscus conzatti Greenm. flower extract (sc) on male albino mice. Indian J Exp Biol 18:561, 1980. 183. Vilar 0: Effect of cytostatic drugs on human testicular function. Sterility. New Martini L (eds): Male Fertilityp and In Mancini RE, York, Academic Pr 1974, 423. tly, 184. Waller DP, Bunyapraphatsara N, Martin A, Vournazos CJ, Ahmed MM, Soejarto DD, Cordell GA, Fong HHS: Effect of (+)-gossypol on fertility in male hamsters. Submitted for publication. 185. Waller DP, Cameron SM, Zaneveld LJD: Spermicidal effect of gossypol in an in vivo animal model for vaginal contraceptives. Abstr Amer Soc Androl Meet, New Orleans, March 11-14, 1981. Soejarto 186. Waller DP, Fang HHS. Cordell GA, Sojro DD: Antifertility effects of gossypol and its Impurities on male hamsters. Contraception 23:653, 1981. 187. WaIler DP, Fang HHS, Zaneveld LJD: Spermicidal composition. U.S. Patent 4,297,431, 1981.

SQ, Chen NZ: A histological study of the effect of gossypol on the anterior lobe of pituitary of castrated hog. Chieh P'ou Hsueh Pao 12:101, 1981. 219. Zhou LF, Chen CC, Wang NK, Lei HP: Ex perimental observations on long-term administration of gossypol acetic acid. Chung-hua I Hsueh Tsa Chih 60:343, 1980. 220. Zhou LF, Lei HP: Recovery of fertility in rats after gossypol treatment. In Fen CC, Griffin D (eds): Recent Advances in Fertility Regulation. Beijing, September 2-5, 1980. Geneva, Atar SA, 1981, pp 147-151. 221. Zhuang LZ: Effect of gossypol on the growth and function of the Leydig cell and Sertol cell in culture. Abstr 14th Annual Meet Soc Study Reproduction, August 10-13, 1981, p 137.

This publication was supported by the United States Agency for International Development (USAID). The contents do not necessarily reflect USAID policy.

16

September 1982, Volume 2, Number 2

Northwestern University

875 North Michigan Avenue Chicago, Illinois 60611 Editor: Gerald I. Zatuchni, M.D., M.Sc.

II

f

Managing Editor: Kelley Osborn

RESEARCH FRONTIERS IN FERTILITY REGULATION

NOVEL PEPTIDES IN OVARIAN FOLLICULAR FLUID:

IMPLICATIONS FOR CONTRACEPTIVE DEVELOPMENT

Neena B. Schwartz, Ph.D.

Deering Professor

Department of Neurobiology and Physiology

Northwestern University

Evanston, Illinois

With the demonstration and isolation of hypothalamic peptides that control the secretion rates of anterior pituitary hormones in the 1970s, a new era of identifiable physiologic control signals began (47). During the past ten years, hypothalamic peptides such as thyrotropin releasing hormone (TRH), somatostatin, and gonadotropin-releasing hormone (GnRH) have also been iden tified in extrahypothalamic brain tissue and in the gut (29, 36), and GnRH-specific receptors have been found in the gonads themselves (31). The placenta has been found to be quite rich in unexpected peptides, such as GnRH, TRH, somatostatin, adrenocorticotropic hormone (ACTH), thyroid-stimulating hormone (TSH), and prolactin (35). Ovarian follicular fluid has also been examined for peptides exerting a variety of activities. For many years, physiologists have known that follicular fluid (FF) of human, bovine, porcine, and equine origin is a rich source of ovarian steroids (18), and more recently, the fluid has also been shown to contain three pituitary hormones: follicle-stimulating hormone (FSH), luteinizing hormone (LH), and prolactin (42). Within the past five years, numerous other peptides have been identified in FF: oocyte maturation inhibitor (OMI), FSH receptorbinding inhibitor (FSHRBI), luteinization stimulator and inhibitor (LS and LI),adovarian goadosati. folliculostatin (FST, goadorini an inhibin or Th later terms are generic names for factors that stimulate or inhibit secretion of both FSH and LH from the pituitary gland. The discovery of these novel peptides has engendered excitement and controversy, and a number of books have been written about them (24,32,53). This article is a progress report on this rapidly moving field, including a

dE scription ofa new "morphology" of the ovarian follicle, and a review of the interrelationships of ovarian micro scopic morphology and gonadotropin receptors, and ovarian secretion of steroids (34, 52). LIFE HISTORY OF "HE FOLLICLE The morphologic changes that occur during primordial, primary, and early secondary follicle growth are illus trated in Figure 1. When a primodial follicle begins to grow, a series of ultrastructural and biochemicalchanges is initiated by the dictyate oocyte, culminating in the formation of a fully grown and physiologically differen tiated ovum, enclosed in a glycoprotein coat called the zona pellucida, and single layer of cuboidal granulosa cells (20). This follicular unit, called a primary follicle, is approximately 150 p in diameter, and steadily increasing granulosa cell mitosis causes the primary follicle to grow to a diameter of approximately 200 . Meanwhile, the granulosa cells develop FSH, estrogen, and testosterone receptors and become physiologically coupled by gap junctions, and the fully differentiated primary follicle migrates into the medulla, where it acquires a theca tissue. In response to FSH, the development of the secondary follicle begins (20). The follicles that contain oocytes become atretic, for the most part, and fewer than 1%of all such oocyte-follicle complexes ovulate (20, 34, 52). The morphologic se quence, which culminates in ovulation, is coordinated with gonadotropin secretion rates, and steroid secretion, as well as with specific hormonal receptors in the follicle cells. Information about specific receptors, by which circulating hormones act on ovarian cells, explains why some follicles fail to grow to maturity until near the time of

@Copyright PARFR 1982

PRIMORDIAL

FOLLICLE

FOLLICLE

Bt-ENT LAMINAE

GRANULOSA CELLS

TYATE OOCYTE

menopause, while others grow and ovulate at the time of puberty, even though all follicles within the ovary are

exposed simultaneously to the same hormones in the

LAMINAE BASEMENT

GRANULOSA CELLS

capillary blood (46).

During each menstrual cycle, some follicles increase in size by proliferation of granulosa cells, and begin to

PRIMARY FOLLICLE

GROWN FULLY OOCYTE

ZONA PELLUCIDA

secrete small amounts of estrogen (15) (P"gure 2). It

appears that the granulosa cells in prolifei ing follicles start with a small complement of FSH mem :'ane recep

BASEMENT LAMINAE

GRANULOSA CELLS

tors and estradiol cytosol receptors, and that progressive

development changes occur in the response of granulosa cells to hormones (Figure3).

SECONDARY

PELLUCIDA ZONA

0

FOLLICLE

FULLY GROWN OOCYTE

The current hypothesis proposed to explain the "two cell

theory" of follicle estrogen production is that in response to Lli ;timulation, the steroid secreting cells in the theca interna secrete androstenedione. As shown in Figure 4, the androstenedione diffuses across the basement lamina

BASEMENT LAMINAE STEROID SECRET_N

of the follicle and enters the granulosa cells; here it is

aromatized into estrogen by the aromatase enzymes in the granulosa cells by FSH, and the newly synthesized estrogen is released into the follicular fluid

ANTRUM_

BO Vinduced

EARLY-

TERTIARY

T ERTIR

00 .

ZONA PELLUCIDA '

and peripheral circulation (20). Thus, estradiol synthesis

and secretion probably require LH-acting on the LH in theca cells, which make androstenedione and testosterone. These precursors are converted to estradiol under the influence of FSH acting on granulosa cells (17). Thus, as each small follicle begins to synthesize and secrete very small amounts of estradiol, the local estradiol and FSH, acting on receptors, cause granulosa cell division, with enhanced numbers of FSH receptors within the follicle; each granulosa cell appears to have an unvarying number of FSH receptors. A given follicle will start to enlarge, and if its rate of enlargement coincides with the follicular phase of the menstrual cycle, it may enlarge to a great enough size that estradiol secretion, and estrogen and FSH acting together, will increase the

FULLY GROWN

_ULTIPLE LAYERS OF

GRANULOSA CELLS

-THECA INTERNA

P

Lreceptors

Figure 1. Life history of the follicle (Reproduced, with permission, from Erickson GF: Normal ovarian function. Clin Obstet Gynecol 21:31, 1978).

Androgen PITUITARY

Theca cells

Hi

FPTIAYto

(+oestrogens)

RLFS RLH

F a

Oestrogen

blood

FS.LH

l~ln

~

s~

R, Ho

Basement membrane

Granulosa

cel

bloodsAoB diiso obo dAdiffeetanBdiffe Oestrogens

diiso

retato differentiatio

Follicular flud

Figure 3. Illustration of hypothetical changes in response Figure 2. Part of follicle showing sites of action of gonadotropins, and production and action of steroids. (Reproduced, with permission, from Dorrington JH: Pituitary and placental hormones. In Austin CR, Short RV (eds): Mechanisms of Hormone Action. Cambridge, Cambridge University Press, 1979, pp 53-80).

ofgranulosa cells to hormones. RFsH, RLH,RE: Receptors

for FSH, LH, and estradiol; T: Testosterone; E: Estradiol. (Reproduced, with permission, from Richards'chapter in Jones RE (ed): The Vertebrate Ovary: Comparative Biology and Evaluation. New York, Plenum Press, 1978, p 336).

2

THECA INTERNA

Lutlinizing Hormone I

CL

.......

Cholesterol

f f

l iu

l (

EI I M I F 1

)g es of w,

L F

i VLF ,f

f

Andro(tenedionReprdua, wmn

menstrual cyclei

Rese

\

Basmen Lfollicle.

rAnodartestnedi

ewomen. f komaliation Gyneco nd2 1:3,.in,

ATP

FAmture 5. Sequence of hormone present in follicular fluid growth of follicle: Increasing concentration (e) and durinon npeak oncrtration (E) of estradiol Follicular proges o terone (P) is at very high concentrations in preovulatory f Early (EF), middle (M late (), and very late follicular (VLF) growth phases of menstrual cycle in (Reproduced, with permission, from: McNatty

KP: Follicular Fluid. In Jones RE (ed): The Vertebrate Ovary. New York, Plenum Press, 1978, p 215). )ogn

junctions first appear between the granulosa cells shortly after the ocyte completes its growth in the primary follicle. They facilitate cellular interchange

Fo c id

nuL

rAor

Isuth n

tos Enzyme

of small

gen

Follicle StimultingHrmone

molecules the size of Lyclic adenosine monophosphate and steroid hormones, and are thought to play an

GRANULOSA CELLS

Figure 4. Chart showing hypothetical process of follicle estrogen production. (Reproduced, wi tehrough from Erickson GF: Normal ovarian function. ClinObstet Gyneco1 21:31, 1978).

hormonal stimulation of important cell function during follicle development (Figure granulosa role in coordinating

6). There is also easy access to the interior of the oocyte its surrounding C muLs of granulosa cells, which connects to the interior of the oocyte (26) (Figure 7). The adaptive significance of the fluid micro-environ ment of the cells within the follice is clear: the fluid comartment provides access for FSH and LH molecules cell and other while providing a local area contiguous steroidreceptors, signals. Thus, in the ovary, of amplified follicles may be at very different growth stages, but all within a given follicle are bathed in the same environment.

oto (3.Tis grnubrof Lrcell s

i utherenhsance estro-

eces Ti re lls c gen Load e ticells By this time, the follicle we have followed becomes the dominant follicle in the cycle; it secretes enough estrogen to trigger the release of the LH surge. This LH, acting on the LH receptors scattered throughout the follicle, sets into play the steps leading to the resumption of reiosis in the oocyte (4) and otslation within the dominant follicle, butgrow, but (48). not reach critical ovary thatat the time to no other did Any follicle in the maturity had begun when LH and FSH levels were optimal, now becomes atreticansaearse pced and granulosa cells of show gap juncthe ovulated follicle producing progesterone ase the dominant steroid of the luteal phase. At the point when a follicle exceeds 0.2 mm in diameter, fluid begins to accumulate within the follicle (Figure5). The contents suggest that follicular fluid is derived partly from the plasma and partly from secretory products of3

the granulosa cells themselves (18). Morphologically, granulosa cells are loosely packed and show gap junctions, which permit free passage of materials throughout the interior of the follicle. The specialized membrane

.. ,.ol

l

N

.' 6 E two 4( GF: Nn

'/ 1

21: 1,..9

B puteinize,

,"

Figure 6. Electron micrograph of gap junction between

two adjoining granulosa cells (A, 13) in early secondary

follicle. (Reproduced, with permission, from Erickson GF: Normal ovarian function. Clin Obstet Gyencol 21:31, 1978). 3

"Plasma proteins" Steroid binding protein Enzymes Intracellular Extracellular "Ovulating"

-

plasminogen; proteases (proteoglycans)

4G .Mucopolysaccharides

"Chrondroitin "Z

9*,

Hyaluronic acid sulfate acid Steroids

Estrogens Androgens Pituitary protein hormones FSH LH Prolactin Non-steroidal ovarian factors Oocyte meiosis inhibitor (OMI) FSH secretion suppressing factor or folliculostatin)

Luteinization stimulator (LS)

Luteinization inhibitor (LI)

FSH-receptor-binding-inhibitor (FSHBI)

Gonadocrinin

Gonadostatin

Table 1. Interesting substances found in follicular fluid.

ing of the largest follicle. Activation of a protease in follicular fluid is usually assumed to be necessary for reduction of wall strength, with subsequent rupture. Under the influence of FSH, plasminogen is converted to plasmin, a protease that Beers, Strickland, and associates believe may cause ovulation (6,55). Espey has suggested other candidates as the critical protease (22). Follicular fluid is a very viscous liquid, and the stickiness that plagues everyone who has worked with it is due to mucopolysaccharides, or proteoglycan molecules, composed of sugars attached in chains of polypeptides (3). These molecules have a high viscosity, and exert a kind of matrix structure in solvents. Proteoglycans, which have been identified in FF, include chondroitin sulfate, hyaluronic acid, dermatan sulfate, and heparin. FSH appears to enhance the synthesis of these materials by granulosa cells (2). These substances are involved in cumulus expansion (19) and are correlated with antrum formation (41). The concentrationofchondroitin sulfate and heparin sulfates decreases as follicular maturation occurs, being more concentrated in fluid from small pig follicles thin from large ones (3).

.

-Progestins

f ~,. ~q,*I~

."

*

.:: .

.

1

..

_

_

_(inhibin

zona pellucida (ZP)

Figure 7.

Structure of fully formed around an oocyte in Graafian follicle. Microvilli arising from oocyte interdigitate with processes from granulosa cells (G). These processes penetrate into cytoplasm of oocyte (C) and may provide nutrients and maternal

protein. (N: Oocyte nucleus.) (Reproduced, with permission, from Baker TG: Oogenesis and ovulation. In Austin

CR, Short RV (eds): Germ Cells and Fertilization. Cambridge, Cambridge University Press, 1972, p 25).

If one accepts this argument for the adaptive significance of follicular fluid, one can understand why the discovery of a number of putative peptide signals or factors in easily obtainable follicular fluid has aroused excitement and has suggested that these factors may take part in the process of follicular individualization,

FOLLICULAR FLUID Follicular fluid provides an easy sample of intrafollicular interstitial or extra-cellular fluid, and is a rich source of many substances. A partial list of the fluid contents is provided in Table 1 (8, 18, 41). Since follicular fluid is at capillary pressure, the follicular basement membrane is not a barrier to water movement (18, 41). Concentration of serum proteins is somewhat i (18, 1). folicularenlutha n in bloo. rotnsoet lower in follicular fluid than in blood. Serum proteins Preovulatory gonadotropin surges initiate rupture of the follicular wall, which occurs after the preovulatory swell-

4

The exact role of these molecules in oocyte-cumulus changes and ovulation remains to be elucidated, but they do FSH to enhance synthesis of granulosa cells by se. appearcanbe secretion products ofthese moleculesper granulosa cells, and apparently can cause cumulus exansion (ellarnd he arentcyte, caur w acis exp a ns io n (aro un d th e oo c yte ), frr wh ich h ya fo synthesis isnecessary. While LH isusually considered to be the "ovulating hormone" (48), FSH undoubtedly

be te "vulainghormne"(48) FS undubtdly

OOCYTE MATURATION INHIBITOR (OMI) After a variable number of years of rested meiosis, the Afte within the follicle destined o ovulate iis, th e oocyte withinabenumbe des to ate iri

u of t e va y b or th Hs

that cycle

lu ron ic a c ido

resumes meiosis within an hour or so of appearance in the blood of the LH surge (4). If an entire follicle isshelled out of the ovary before theunless the has taken place, the oocyte remains arrested, LH surge LH is placed within

the explant medium. If an oocyte (w~th cumulus) is teme mem f *tumulus removed from a follicle an oocyte and explanted, "it undergoes

ge as ak n p c ,t e

participates importantly as the follicular morphology, and volume of FF as wellin determining the composition Follicular fluid is rich in steroids, and steroid concentration in follicular fluid greatly exceeds that in blood. Not surprisingly, more estradioi is seen in follicles with fluid that also contains FSH since, as we saw above, FSH plus estradiol enhances granulo~a cell mitosis and thus enlarges follicles (46) (Figure 5). When follicles of different sizes and at different stages of the menstrual cycle are compared (41), the highest levels of estradiol are found in the very late follicular phase large follicles, which are preovulatory. Progesterone isfound in fairly low levels, except inthe late follicle. Follicles also contain measureable amounts of FSH, LH, and prolactin, always, of course, at concentrations lower than are found in the blood (41). FSH is found in all follicles that have started to form an antrum, while LH is seen only in follicles of very late phase. LH and progesterone appear together in these preovulatory follicles. Prolactin levels tend to fall with follicular maturation. Thus, follicles have their own hormonal environment (Figure 5), and amechanism appears to restrict diffusion of steroids from the follicular fluid into the blood, as can be seen from the extraordinarily high estrogen levels achieved. Movement of steroids into follicular fluid from nearby follicu'ar structures is also restricted, as can be seen by the Lw progesterone levels detected in follicles during the luteal phase, in the presence of an ipsilateral corpus luteum (41). Prostaglandins, which may be involved in ovulation, are also found in follicular fluid from preovulatory follicles, after the LH surge (22, 41). Table 1 lists the non-steroid "novel" factors found in follicular fluid. Investigators seeking steroids and gonadotropic hormones in FF are also seeking local factors to account for the heterogeneity of morphology, secretory potential, and destiny of contiguous follicles within each ovary. To understand the process by which a follicle becomes "chosen" for agiven cycle, one must see what isdifferent among follicles, rather than what is the same. While preferential vascularity has been one explanation of differences, local factors within the follicles probably play nivotal roles in differentiation,

meiosis without addition of hormones to the culture medium (56). This suggests that within the protected environment of the follicle, completion of meiosis is prevented until the LH preovulatory surge removes an inhibitor or renders it inactive. Tsafriri and associates have shown that FF from several species contains afactor that can prevent oocyte matura tion in uitro (56, 57, 58). The investigators named the factor Oocyte Maturation Inhibitor, or OMI. Since mat uration is inhibited when oocytes aie co-cultured with granulosa cells, granulosa cells appear to secrete OMI into the medium or into follicular fluid in situ. Small follicles seem to secrete more OMI than large follicles, and FF from small and medium-sized follicles contains more OMI activity than FF from large follicles. FF also inhibits progesterone secretion by cumulus-enclosed oocytes (9), presumably via OMI. We have already seen that the oocyte is connected by processes to granulosa cells in the cumulus layer (26) (Figure 7). OMI may be delivered to such oocytes from the mass of granulosa cells in situ, so that when the oocyte-cumulus mass is cultured alone, lack of OMI delivery accounts for "spontaneous" maturation. The LH surge causes a break in connections between cu mulus and oocyte, and thus may initiate meiosis by preventing OMI from reaching the oocyte (26). Purification of OMI has been difficult, possibly because of the uncertainties of a bioassay system that depends on assessment of oocyte germinal vesicle breakdown. The OMI, a protein not a steroid, with a molecular weight between 1,000 and 10,000, has been identified in bovine, porcine, and human FF. LUTEINIZATION INHIBITOR AND

STIMULATOR

Luteinization is a process that converts the granulosa cell-theca unit remaining after ovulation to cells with 1)a large cytoplasm/nucleus ratio, 2) a profusion of LH receptors, and 3) the capacity to secrete high rates of progesterone. Among mammalian species, dependence of the corpus luteum on aparticular luteotropic hormone to "rescue" it at the onset of pregn;ncy varies consider ably, but all species appear to depend on progesterone from the corpus luteum to sustain pregnancy, at least at 5

the beginning. Whether a local signal or more than one local signal prevents luteinization of cells within the preovulatory folicle and then permits these cells to luteinize to form a corpus luteum is not known. While luteinization depends on LH, FSH, and/or prolactin, or on estrogen (this varies with the species), it also may depend on intrinsic factors. If granulosa cells from large preovulatory follicles are cultured in vitro, they undergo "spontaneous" luteinization in the absence of added luteotropic signals like LH; on the other hand, granulosa cells from small follicles will not luteinize unless gonadotropins are added to the culture medium. These observations suggest that small follicles might contain a "luteinization hiliibition" factor, whereas large follic!es might

contain a "luteinization stimulation" factor (, 24, 37). Figure 8.

80 70

0

60

In Vitro

In Vivo

o 40

40

20

0 ,o 0

0 L pFF-D, jtg

(Chorcool

Extrocted)

If FF from small antral follicles or from atretic large follicles isadded in vitro to granulosa cells harvested from

large follicles, it can prevent luteinization. If this luteinization inhibitor (LI) is added to granulosa cells from small

Effect of porcine follicular fluid (charcoal on secretio i of FSH by rat pituitary. In vitro extracted) study utilized a dispersed monolayer of pituitary cells, exposed to pFF for 48 hours. In vivo study utilized an acutely ovariectomized rat, injected intravenously with pFF and autopsied 5.5 hours post-injection. FSH was

follicles, it will prevent the induction of LH receptors by FSH that must precede luteinization. On the other hand, follicular fluid from large follicles can enhance luteinization and progesterone secretion in granulosa cells har vested from small follicles and tested in vitro. The fluid can also enhance the induction of LH receptors by FSH in these granulosa cells. The factor from large follicles has been named luteinization stimulator (LS). The immature follicles appear to contain afactor that inhibits luteinization (LI) and the accumulation of LH receptors. Follicles destined to become responsive to gonadotropins, and eventually to ovulate, acquire a higher ratio of LH receptors to LI; follicles destined to become atretic do not acquire LS in sufficient quantity. What controls the acquisition of these factors isnot known at present, nor is their mechanism of action understood. Their identification has provided yet another set of local factors distinguishing contiguous follicles, These factors have not been isolated. They appear not to be steroids, since charcoal-extraction does not remove activity. They both appear to have a molecular weight greater than 10,000.

measured by RIA either in culture medium (in vitro) or in serum (in vivo). Porcine serum was used as control for

pFF.

ferential negative effects on FSH, since we had shown that estradiol negative feedback is much more effective on LH than on FSH (51). Porcine follicular fluid that has been charcoal-extracted to remove steroids exerts suppression of FSH secretion into the plasma of rats in vivo (50), and also suppresses FSH secretion when applied to pituitary cells in vitro (12) (Figure 8). LH secretion is not inhibited in vivo (50) nor in vitro under basal conditions (with no added GnRH) (12, 28). The FSH-inhibiting or suppressing follicular factor has been found in FF from bovine, sheep, porcine, equine, and human sources, and it suppresses FSH in rats, mice, horses, hamsters, and monkeys (49). About 2hours are needed for it to suppress FSH significantly in vivo, and the effect of a single intravenous injection disappears after about 10 hours. It cannot block ovulation if given before the ovulatory surges of LH or FSH, but it can prevent growth of smaller follicles dependent on elevated FSH (30, 50). FF taken from small and medium porcine follicles contains ahigher concentration of folliculostatin activity than FF from large follicles (39). FF from human follicles, taken during the follicular phase, has more folliculostatin activity than follicles sampled during the (10). ase luta luteal phase (10). Folliculostatin appears to be aprotein, and isassociated with a molecule (or complex) with a molecular weight over 70,000 (28, 33, 39). All its known characteristics suggest thjt it may be identical to testicular inhibin but more potent in FF than in testicular sources.

FOLLICULOSTATIN (OVARIAN INHIBIN) Folliculostatin is a factor in follicular fluid that suppresses the rate of FSH secretion by the anterior pituitary gland, It was discovered simultaneously by investigators in two laboratories searching for such afactor for two different reasons. DeJong and co-workers (13) were searching for a new source of inhibin, and reasoned that granulosa cells were analogous to Sertoli cells, and thus might secrete an inhibin (24). At Northwestern University, we were looking for an ovarian feedback signal with pre6

Unlike the other factors discussed (OMI, LS, and LI), folliculostatin must act via the bloodstream to suppress pituitary function, i.e.. it must be a hormone. It is secreted by granulosa cells in culture (21); it may be found in ovarian vein blood of rats and monkeys (1,9,38). Until a more sensitive assay for its measurement is achieved, the verification of its hormonal status will be unclear. Whether folliculostatin also has an effect on the follicle itself is not known, since the FSH-suppressing activity isfound in FF not only containing OMI, LS, and LI, but also showing some activities that alter receptorbinding of LH and FSH. Until these factors are isolated, their individual characteristics cannot be assessed. high potency and specificity of folliculostatin in suppressing FSH but not pituitary LH secretion has focused much attention on its possible hormonal status.

'

GnRH AND THE OVARY Perhaps the most unexpected findings relative to intra ovarian peptides concern GnRH-related factors. The discovery that GnRH in humans and other species eventually caused ovarian and testicular failure (25) was at first ascribed to "down-regulation" of GnRH receptors shown to act locally on the ovary, preventing FSH associated estradiol secretion and induction of LH re c at e n plaedi secro and ut n c on o ce ceptors when placed in itro with cultured granulosa cells (31). This action of GnRH can be blocked by specific antagonists of that peptide. Simultaneous with the above observations was the discovery of specific receptors for GnRH in ovaries (and testes), which fits with the observation that GnRH can act on the ovary (31). Yet, the levels of GnRH in peripheral blood (outside the pituitary portal system) are not high enough to activate the gonadal GnRH receptors. This suggests that GnRH or a related peptide might be produced locally, and thus could modulate the effects of gonadotropins locally. Guillemin and co-workers have claimed that a GnRH-like peptide (called gonadocrinin) can be found in FF, and can stimulate LH and FSH secretion when applied to isolated pituitary cells (the basic bioassay for hypothalamic GnRH itself) (62). Whether gonadocrinin really exists or merely represents

GnRH contamination isnot clear. Gonadocrinin has not in the anterior pituitary. However, GnRH was then

GONADOTROPIN BINDING INHIBITORS As indicated earlier, ovarian cell receptors are of central importance to LH and FSH in determining the ability of blood LH and FSH levels to influence selective follicles. Also, the induction of such receptors is an important method for controlling ovarian function. There appears to be another level of control of receptor function; substances have been identified inovarian tissu xtracts

subtanes avebee varan iss~ ;trats idntiiedin

and FF that interfere with the binding of FSH and LH to their respective ovarian receptors. Reichert and co-workers have identified afactor they call FSH binding inhibitor (FSHBI) in bovine FF and in human serum (11, 45). The material isassessed by its ability to inhibit binding of radio-labeled FSH to granulosa cells in uitro. The concentration of FSHBI increases with increasing size of the bovine follicles from which FF is harvested. The inhibitor appears to be a peptide with a molecular weight of less than 5000, and may exist in several component molecular weight fractions. Ward and associates have discovered a factor that inhibits the binding of LH to its ovarian receptors, and have designated the activity as LH receptor binding inhibitor (LHRBI) (59,60). The factor has been identified in corpora lutea from rat and porcine ovaries. Not only does it inhibit LH binding, but it also prevents LHstimulated ovarian steroidogenesis. It is a peptide present in anon-dializable fraction (MW less than 10,000) and adializable fraction (MW less than 3800). It appears not to bind to the same site on the LH receptor as LH itself (i.e., it isnot acompetitive binder). This factor has not been identified in FF, and unlike inhibin and GnRH, it has not been identified in testicular preparations. Reichert has identified a factor in bovine FF that stimulates LH binding (LHSA) (45).

bnhonto inas n mediu whn grnascll been shown to increase in medium when granulosa cells are incubated, as folliculostatin has been shown to be secreted. Ying and Guillemin have also found a substance in FF that they have called "gonadostatin" (61). This factor inhibits the secretion of both LH and FSH by pituitary cell cultures that have been challenged with exogenous GnRH. This material appears to have amolecular weight below 10,000. In contrast, the activity we have identified as folliculostatin, or ovarian inhibin (Figure 8), has a molecular weight greater than 10,000 and specifically inhibits FSH secretion in basal pituitary cell culture. Since all of these activities are being detected within a common source, follicular fluid, various claims of different factors will not be resolved until some purification has been achieved. A SPECULATIVE VIEW OF THE ROLE OF

PEPTIDE FACTORS IN THE LIFE HISTORY

OF THE FOLLICLE

Each follicle probably is initially provided with granulosa cells that have FSH receptors. These follicles may acquire circulating FSH and start granulosa cell mitosis. With mitosis and increasing numbers of cells, the follicle develops the capacity to synthesize estradiol. Increasing the estradiol enhances FSH effects, and the follicle grows further and antral fluid begins to collect. 7

At this stage, locally produced OMI continues to hold the oocyte, contained within the follicle, in the dictyate stage of meiosis. Local LI prevents FSH from inducing LH receptors prematurely until a critical follicle size is reached. The critical size may be signaled locally by the concentration within the FF of an unknown factor. The thecal cells have acquired LH rec'.ptors under the influence of estrogen and FSH. Follicular secretion of estrogen increases in proportion to numbers of granulosa cells, and folliculostatin secretion also increases. The folliculostatin is responsible for falling FSH values during the late follicular phase. Gradually, LI is lost and LS concentrations rise, permitting induction of LH receptors within the granulosa cells. When estrogen levels are high enough to trigger the precipitous pre-ovulatory LH and FSH surges, these surges set into play a cascade of local events in the dominant follicle. LH immediately causes breaking of the cumulus cell-oocyte connections and meiosis resumes, since OMI is not delivered to the oocyte. FSH causes beginning dispersal of the cumulus cells around the oocyte. Intra-follicular proteases begin to decrease the strength of the follicle wall. The size of the antrum increases, perhaps because osmotic pressure increases, leading to enhanced water transfer from the blood. Blood vessels begin to invade the granulosa cell layers. Eventually, the follicle ruptures at the narrowest point, and the cumulus is delivered to the fimbriae, along with the oocyte, which by now has thrown off a polar body. that has been left The corpus luteum a enzyatieclet behind luteinizes bnd cretespromhorpslical morphologically and enzymatically, and secretes progesterone under the influence of LH. If pregnancy occurs, it is maintained under the continuing influence of Ascribing a role for the LH and FSH receptor binding inhibitors is difficult. They may represent artifactual pieces of membrane receptors that impede binding, or they may represent fail-safe mechanisms that are necessary to block hormone binding to receptors, if something goes wrong in the chosen follicle. One possibly adaptive contigency for the latter mechanism might be the occurrence of oocyte damage. The aborting of such a follicle by atresia would be highly adaptive. The local production of GnRH-like molecules and of GnRH receptors may represent accidental and unused nuclear translation and transcription of a genetic message, or it may involve a local neural mechanism (43). There are suggestive reports that neural representation occurs for the ovary. Recent evidence of noradrenergic participation in gonadotropin transduction and steroidogenesis (52) suggests that the ovary may be subject to the influence of efferent neural input and may influence CNS function by a neural pathway back to the nervous system. The whole issue of peptidergic neurons and

neural tissue as targets for pituitary hormones as well as for steroids suggests that more surprises could develop concerning ovarian regulation.

CONTRACEPTIVE AND CLINICAL

IMPLICATIONS

The specificity of the factors discussed, and their pre surned localization of action, suggest a number of new targets for contraception. If the LH surge were pre vented from inducing withdrawal of OMI from the oocyte in the dominant follicle, all of the endocrine events of the cycle would occur, but the oocyte would not be viable. Injection of OMI in such a way as to reach the oocyte during the time when the surge is occurring could prevent resumption of meiosis. The ovarian GnRH receptors are a prime target for contraception; they appear to be the conduit through which GnRH and its agonists can suppress steroido genesis, ovulation, and corpus luteum function (25). Such agonists working via the pituitary gland can also lead to indirect suppression of ovarian function, through down-regulation of GnRH receptors within the pituitary and subsequent failure of LH and FSH secretion. An tagonistic analogues t- GnRH can also block pituitary gonadotropin secretion, leading to suppression of LH and FSH. Obviously, timed administration of GnRH agonists or antagonists can block ovulation, follicular development, or corpus luteum function, depending on when during the cycle administration occurs. The promising data showing that luteolysis can be induced by GnRH agonists, resulting in contraception, have been modulated by demonstrations that hCG from an inter vening implantation may be able to rescue the corpus luteum (24). While at the present time, gonadal receptors have proven interesting possible targets for contracep tion, we do not know as yet what these receptors normally recognize as ligands in the in situ ovary. If there is a local release of GnRH-like peptides, exerting some hitherto unsuspected local regulation, there will un doubtedly be other contraceptive targets. At present, the GnRH receptors in the ovary have been indistin guishable from those in the pituitary, except in the numbers of receptors per cell. If there are differences in recognition sites, analogues might be synthesized that could selectively act as agonists or antagonists at the gonadal level but not at the pituitary level, or vice versa. While GnRH agonists can cause luteolysis via LH withdrawal from normal corpora, it might be possible to prevent the induction of LH receptors in the first place. Luteinization inhibitor, if it could be isolated and purified, might enter the dominant follicle via the circulation, and permit an ovulatory response to the LH surge, but no luteinization. Conversely, an antibody to luteinization

8

stimulator might prevent LH receptors from being induced in growing follicles, blocking ovulation in response to an LH surge, or preventing corpus luteum maturation. If, as has been proposed, luteinization occurs with adequate LS/LI ratios, then atresia of the dominant follicle might be achieved by treatment altering the ratio of these putative follicular factors. Clearly, a complex cascade of events lies between secretion of GnRH and action of LH and FSH on the ovary. Just as antagonists to the decapeptide can compete with GnRH on its pituitary or ovarian receptors, the receptor-binding inhibitors to LH and FSH could provide local interfering agents that would prevent gonadotropin regulation of ovarian function. If the events of the cycle were permitted to proceed normally, and then timed injections of such binding inhibition factors were carried out, LH or FSH could be prevented from exerting target effects.

steroids as a pituitary inhibitor lies in the possiblility that such proteinaceous materials may have fewer targets. However, the unexpected distribution of many peptide receptors in gut and brain should prevent indiscriminate use of"specific" peptides in the expectation that no other targets exist.

The factors under discussion could offer important

insights in cases of ovarian failure as well. Women with secondary amenorrhea caused by follicular loss or absence show higher serum FSH values than do women with follicles; while serum LH values overlap, serum FSH levels are inevitably higher (27). Inadequate formation or maintenance of corpora lutea might result from local over-production of LI. At present, the factor influencing the control of synthesis and release of the local peptides is unknown, but such information is being sought.

Folliculostatin is the most specific suppressing agent for FSH that has been used. Injected into rats or cattle, itCONC.. .. . . can prevent or delay follicular recruitmaent after ovulation CONCLUSION, (50). Inmonkeys itcan suppress FSH secretion, and this material, if given during the follicular phase, can cause a Seldom has a field grown as fast as has the investigation short Theoretically,which can beduring luteal phase, if injected overcome with hMG (14, of local ovarian peptides. This research interest can be 54). the elevated FSH o oa vra etds hsrsac neetcnb viewed as a part of the broader hunt for peptide peak that occurs in primates at the time of menses, the in neural tissue, the GI tract, and the placenta. secretion The new folliculostatin should prevent growth by inhibiting FSH. class of regulators has revolutionized our views of cell to The rodent data suggest, however, that itmight just delay cell communication in the nervous system, and may have the cycle, and after cessation of treatment, a rebound of the same effect on our concepts of ovarian physiology. FSH would occur (50). Like the nervous system, the ovary has contiguous Folliculostatin can prevent FSH secretion in male recipstructures that must be kept separated from each other ients (40), and could be used as a block for spermatoin function, and peptides may provide an important part genesis. The possible advantage of folliculostatin over of the localizing mechanism for both organs.

9

REFERENCES

16. Dorrington JH: Pituitary and placental hormones. In Austin CR, Short RV (eds): Mechanisms of Hormone Action. Cambridge, Cambridge University Press, 1979, pp 53-80. 17. Dorrington JH, Armstrong DT: Effects of FSH on gonadal functions. Rec Prog Horm Res 39:301, 1979. 18. Edwards RG: Follicular fluid. J Reprod Fertil 37:189, 1974. 19. Eppig JJ: Gonadotropin stimulation of the expansion of cumulus oophori isolated from mice: general conditions for expansion in vitro. J Exp Zool 208:111, 1979. 20. Erickson GF: Normal ovarian function. Clin Obstet Gynecol 21:31, 1978. 21. Erickson GF, Hsueh AJW: Secretion of 'inhibin' by rat granulosa cells in vitro. Endocrinology 103:1960, 1978. 22. Espey LI: Ovulation. In Jones RE (ed):

34. Jones RE (ed): The Vertebrate Ovary: Comparative Biology and Evolution. New York, Plenum Press, 1978. DT: Placenta as a source of "brain" and "pituitary" hormones. Biol Reprod 26:55, 1982. 36. Krieger DT, Martin JB: Brain peptides. N Engl J Med 304:876, 944, 1981. 37. Ledwitz-Rigby F, Rigby BW, Gay VL, Stetson M, Young J, Channing CP: Inhibitory action of porcine follicular fluid upon granu losa cell luteinization in vitro: assay and influence of follicular maturation. J Endocrinol 74:175, 1977. 38. Lee VWK, McMaster J, Quigg H, Findlay J, Leverska L: Ovarian and peripheral blood inhibin concentrations increase with gonado tropin treatment in immature rats. Endocrin ology 108:2403, 1981. 39. Lorenzen JR, Channing CP, Schwartz NB: Partial characterization of FSH sup pressing activity (folliculostatin) in porcine follicular fluid using the metestrous rat as an in viuo bioassay model. Biol Reprod 19:635, 1978. 40. Lorenzen JR, Dworkin GH, Schwartz NB: Specific FSH suppression in the male rat by porcine follicular fluid. Am J Physiol 240:E209, 1981. 41. McNatty KP: Follicular fluid. In Jones RE (ed): The Vertebrate Ovary. New York, Plenum Press, 1978, pp 215-259. 42. McNalty KP, Hunter WM, McNeilly AS, Sawers RS: Changes in the concentration of pituitary and steroid hormones in the follicular fluid of human Graafian follicles throughout the menstrual cycle. J Endocrinol 64:555, 1975. 43. Niall HD: The evolution of peptide hor mones. Ann Rev Physiol 44:615, 1982. 44. Niswender GD: Mechanisms controlling luteolysis. In Schwartz NB, Hunzicker-Dunn M (eds): Dynamics of Ovarian Function. New York, Raven Press, 1981, pp 153-160. 45. Reicher LE Jr, Sanzo MA, Dias JA: Studies on purification and characterization of gonadotropin binding inhibitors and stimula tors from human serum and seminal plasma. In Franchimont P, Channing CP (eds): Intra gonadal Regulation of Reproduction. London, Academic Press, 1981, pp 61-80. 46. Richards JS: Hormonal control ofovarian follicular development: a 1978 perspective. Rec Prog Horm Res 35:343, 1978. 47. Schally AV: Aspects of hypothalamic Science regulation of the pituitary gland. 202:18, 1978.

1. Anderson LD, DePaoio LV: Control of inhibin secretion from ovary. In Franchimont P, Channing CP (eds): Intragonadal Regulation of Reproduction. London, Academic Press, 1981, pp 343-364. 2. Ax RL, Ryan RJ: FSH stimulation of 3Hglucosamine-incorporation into proteoglycans by porcine granulosa cells in vitro. J Clin Endo Metab 49:646, 1979. 3. Ax RL, Ryan RJ: The porcine ovarian follicle. IV. Mucopolysaccharides at different stages of development. Biol Reprod 20:1123, 1979. 4. Ayalon D, Tsafriri A, Lindner HR, Cordova T, Harell HR: Serum gonadotropin levels in proestrous rats in relation to the resumption of meiosis by the oocytes. J Reprod Fertil 31:51, 1972. 5. Baker TG: Oogenesis and ovulation In Austin CR, Short RV (eds): Germ Cells and Fertilization. Cambridge, Cambridge University Press, 1972, pp 15-45. 6. Beers WH, Dekel N: Intercellular communication and the control of oocyte maturation. In Schwartz NB, Hunzicker-Dunn M (eds): Dynamics of Ovarian Function. New York, Raven Press, 1981, pp 95-104. 7. Chang SCS, Anderson W, Lewis JC, Ryan RJ, Kang YH: The porcine ovarian follicle. I. Electron microscopic study of surface features of granulosa cells at different stages of development. Biol Reprod 16:349, 1977. 8. Chang SCS, Jones SD, Ellefson RD, Ryan RJ: The porcine ovarian follicle: I. Selected chemical analysis of follicular fluid at different developmental stages. Biol Reprod 15:321, 1976. 9. Channing CP, Anderson LD, Hoover DJ, Kolena J. Osteen KG, Pomerantz SH, Tanabee K: The role of non-steroidal regulators in control of oocyte and follicular maturation. Rec Prog Horm Res 38:331, 1982. 10. Chappel C, Holt JA, Spies HG: Inhibin: differences in bioactivity within human follicular fluid in the follicular and luteal stages of the menstrual cycle. Proc Soc Exp Biol Med 163:310, 1980. 11. Darga NC, Reichert LD Jr: Some properties of the interaction of follicle stimulating hormone with bovine granulosa cells and its inhibition by follicular fluid. Biol Reprod 19:235, 1978. 12. DeJong FH, Smith SD, van der Molen HJ: Bioassay of inhibin-like activity using pituitary cell in vitro. J Endocrino] 80:91, 1979. 13. DeJong, FH, Sharpe RM: Evidence for inhibin-like activity in bovine follicular fluid. Nature 263:71, 1976. 14. DiZerega GS, Hodgen GD: Follicular phase treatment of luteal phase dysfunction. Fertil Steril 35:428, 1981. 15. DiZerega GS, Hodgen GD: Folliculogenesis in the primate ovarian cycle. Endocrine Reviews 2:27, 1981.

The Vertebrate Ovary. New York, Plenum Press, 1978, pp 503-532. 23. Farookhi R: Atresia: a hypothesis. In Schwartz NB, Hunzicker-Dunn M (eds): Dynamics of Ovarian Function. New York, Raven Press, 1981, pp 13.23. 24. Franchimont P, Channing CP (eds): Intragonadal Regulation of Reproduction. London, Academic Press, 1981. 25. Fraser HM: Luteinizing hormone-releasing hormone and fertility control. In Finn CA (ed): Oxford Reviews of Reproductive Biology. Oxford, Clarendon Press, 1981, pp 1-48. 26. Gilula NE', Epstein ML, Beers WH: Cellto-cell communication and ovulation. A study of the cumulus-oocyte complex. J Cell Biol 78:58, 1978. 27. Goldenberg RL, Grodin JM, Rodbard D, Ross GT: Gonadotropins in women with amenorrhea. The use of plasma follicle-stimulating hormone to differentiate women with and without ovarian follicles. Am J Obstet Gynecol 116:1003, 1973. 28. Grady RR, Charlesworth MC, Schwartz NB: Characterization of the FSH-suppressing activity in follicular fluid. Rec Prog Horm Res 38:409, 1982. 29. Guillemin R: P'ptides in the brain: The new endocrinology of the neuron. Science 202:390, 1978.

. 30. Hoak DC, Schwartz NB: Blockad of recruitment of ovarian follicles by suppre.sion of the secondary surge of follicle-stimulating hormone with porcine follicular fiuid. Proc Nat Acad Sci (USA) 77:4953, 1980. 31. Hsueh AJW, Jones PBC: Extrapituitary

actions of gonadotropin-releasing hormone. Endocrine Reviews 2:437, 1981. 32. Jagiello G, Vogel HJ (eds): Bioregulators of Reproduction. New York, Academic Press, 1981. 33. Jansen EHJM, Steenbergen J, DeJong FH, van der Molen HJ: The use of affinity matrices in the purification of inhibin from bovine follicular fluid. Mol Cell Endocrinol 21:109, 1981.

48. Schwartz NB: The role of FSH and LH and of their antibodies on follicle growth and on ovulation. Biol Reprod 10:236, 1974. 49. Schwartz NB: Inhib.n (folliculostatin) in the female. In Frajese G, Hafez r SE, Conti C, Fabbrini A (eds): Oligozoospermia: Recent Progress in Andrology. New York, Raven Press, 1981, pp 139-146.

10

50. Schwartz NB: Role of ovarian inhibin (folliculostatin) in regulating FSH secretion in the female rat. InChanning CP, Segal S (eds): Intra-Ovarian Control Mechanisms. New York, Plenum Press, 1982. 51. Schwartz NB, Channing CP: Evidence for ovarian "inhibin": suppression of the secondary rise in serum follicle stimulating hormone levels in proestrous rats by injection of porcine follicular fluid. Proc Nat Acad Sci (USA) 74:5721, 1977. 52. Schwartz NB, Hunzicker-Dunn M (eds): Dynamics of Ovarian Function. New York, Raven Press, 1981. 53. Spilman CH, Wilks JW (eds): Novel Aspects of Reproductive Physiology. New York, Spectrum Publications, 1978. 54. Stouffer RL, Coensgen JL, DiZerega GS, Hodgen GD: Induction of defective corpus luteum function by administration of follicular fluid to monkeys during the follicular phase of the menstrual cycle. In Schwartz NB, Hunzicker-Dunn M (eds): Dynamics of

Ovarian Function. New York, Raven Press, 1981, pp 185-190. 55. Strickland S, Beers WH: Studies on the role of plasminogen activator in ovulation. J Biol Chem 251:5694, 1976 56. Tsafriri A: Oocyte maturation in mai mals. In Jones RA (ed): The Vertebrate Ovary. New York, Plenum Press, p 409. 57. Tsafriri A, Channing CP, Pomerantz SH Lindner HR: Inhibition of maturation of isolated rat oocytes by porcine follicular fluid. J Endocrinol 75:285, 1977. 58. TsafririA, Pomerantz SH, Channing CP: Inhibition of maturation by porcine follicular fluid: partial characterization of the inhibitor. Biol Reprod 14:511, 1976. 59. Ward DN, Nahm HS, Shalek RJ, SchwartzNB, LorenzenJR, MooreRB, Grady RR, Channing CP, Glenn SD, Sugino H, Yang K-P: In pursuit of physiological inhibitors of and from the ovary. InJagiello G, Vogel HJ: Bioregulators of Reproduction. Now York, Academic Press, 1981, pp 371-387.

60. Yang KP, Neira ES, Yen HN, Samaan NA, Wong TS, Ward DN, Channing CP: Corpus luteum LH-receptor binding inhibitor (LH-RBI). In Franchimont P, Channing CP (eds): Intragonadal Regulation of Reproduc tion. London, Academic Press, 1981, pp 133-156. 61. Ying SY, Guillemin RG: Gonadostatine et gonadocrinine polypeptide d'origine ovar ienne 6 activites hypophysiotropes. CR Acad Sci Series D 289:943, 1979. 62. Ying S, Ling N, Bohlen P,Guillemin R: Gonadocrinins: Peptides in ovarian follicular fluid stimulating the secretion of pituitary gonadotropins. Endocrinology 108:1206, 1981.

11

December, 1982, Volume 2, Number 3 Northwestern University

Suite 152

r

875 North Michigan Avenue

Chicago, Illinois 60611

Editor: Gerald I. Zatuchni, M.D., M.Sc. Managing Editor: Kelley Osborn

RESEARCH FRONTIERS IN FERTILITY REGULATION

SPERM ENZYME INHIBITORS FOR VAGINAL AND OTHER CONTRACEPTION

Lourens J.D. Zaneveld, D.V.M., Ph.D. Professor Department of Physiology and Biophysics, and

Department of Obstetrics and Gynecology

College of Medicine, University of Illinois, Chicago, Illinois

Ever since scientists identified the ejaculate, and specifically spermatozoa, as the male factor that causes pregnancy, attempts have been made to prevent the fusion of the male and female gametes. At present, physical methods to prevent sperm migration include permanent or reversible obstruction of the vas deferens or the fallopian tube, covering the external cervical os with a cap or diaphragm, and the penile condom. Chemicals are used to interrupt the functional activity of the spermatozoa; vaginal contraceptives presently on the market employ spermicidal agents (primarily nonoxynol-9) that immobilize the spermatozoa by their surfactant activity. Immunologic research is aimed at developing systemic or local antibodies against spermatozoa, resulting in their agglutination and immobilization, Today, the only nonsurgical sperm-directed techniques in clinical use are the spermicidal agents, the cervical diaphragm and cap, and the condom. Used in combination with each other, the diaphragm and spermicide have a mean use-effectiveness of 13 unwanted pregnancies per 100 woman-years, whereas spermicides alone have a mean use-effectiveness of 20 unwanted pregnancies per 100 woman-years (59). Vaginal contraceptives have a variable success rate, which is due in part to the human factor, that is, the need for the spermicidal agent to be deposited directly into the vagina shortly before each intercourse, and for the diaphragm (or cap) to be placed correctly over the cervix. Today's vaginal contraceptives also have physiologic limitations, particularly ifspermicides are used without a diaphragm. Spermatozoa can enter cervical mucus within 30 to 60 seconds after being deposited vaginally, which may not be enough time for the spermicide to contact the gametes. Additionally, human semen coagulates im-

mediately upon ejaculation, trapping the spermatozoa. Surfactants, the most frequently used spermicides, may have difficulty passing through the coagulum and con tacting spermatozoa. The coagulum is often located very close to the cervical os, so that as liquefaction occurs, the spermatozoa enter the cervical mucus immediately, with out having contacted the spermicide. The physiologic limitations of presently-marketed sper micides were shown by a primate study performed with one of the more effective vaginal contraceptive formula tions on the market (76). Although the formulation was placed vaginally immediately before each coital act, half of the primates became pregnant within three to four menstrual cycles. Less than desirable contraceptive activity was also obtained in the rabbit with presently marketed vaginal contraceptives (9, 27, 46, 77). Thus, the human factor alone apparently accounts only partially for the failure rate of vaginal coitraceptives, and agents are needed that are more effective, either because they are more active spermicides, or because they prevent the functional activity of the spermatozoa by a different mechanism. Additionally, it would be beneficial if these agents could 1) readily pass into the seminal coagulum, and 2) enter cervical mucus, thereby hindering cervical passage of the spermatozoa. During the last three decades, much has been learned

about the biochemistry of the spermatozoon and the role of some of its chemical entities in the fertilization process. For instance, we know that certain sperm enzymes, several of which are specific to tile spermatozoon, have a key role in the maintenance of sperm motility and in the ability of the spermatozoon to penetrate into and fuse with the egg. Inhibition of these enzymes will lead to infertility.

'

@Copyright PARFR 1982

Utilizing this inhibition is an attractive approach to the development of new contraceptive agents, particularly if specific inhibitors can be developed. Such sperm enzyme inhibitors may be useful not only as vaginal contracep tives, but also for enhancing the effectiveness of the intrauterine device (IUD), or even as systemic contraceptive agents. Although the idea of using sperm enzyme inhibitors is by no means novel, and was already suggested in the 1940s, recent data show that this approach is feasible in developing a new class of compounds for contraceptive purposes. FUNCTIONAL ACTIVITY OF THE SPERMATOZOON IN FERTILIZATION MorpholoSically, the spermatozoon consists of a head and a tail. The human sperm head measures approximately 2 x 3 x 5 p, and the tail is about 45 p in length. Most of the sperm head comprises a large nucleus that contains tightly packed chromosomal material (Figure1).

Plasma Membrane

A lysosome-like organelle, the acrosome, surrounds the anterior portion of the nucleus. An inner and outer acrosomal membrane limit the acrosome proper. The first portion of the sperm tail is the midpiece, which contains a mitochondrial sheath surrounded by a small amount ofcytoplasm. The mitochondrial sheath encircles the tail fibers (axonemal complex). The entire sper matozoon, including the midpiece cytoplasm and acro some, is surrounded by a plasma membrane. Basically, the spermatozoon possesses two enzyme sys tems that are essential for its activity. The first system is associated with the midpiece and tail, and includes the enzymes involved in the membrane transport of com pounds, glycolysis, citric acid cycle, oxidative phosphorylation, and other metabolic activities. These processes serve primarily to generate the energy required for the sliding of the axonemal microtubules, that is, for sperm motility. Interference with the midpiece enzyme system results in sperm immobilization.

The second enzyme system is associated with the acro

Acrosome

Vacuole

uci .ar Envelope

------. \

some, and includes enzymes such as hyaluronidase and acrosin. The acrosomal enzymes have an essential role in the ability of a spermatozoon to fertilize the egg (38, 44).

At the time of fertilization, the egg is surrounded by

Nucleus

-

Equatorial Segment

Postnuclear Cap Redundant Nuclear

Envelope... Connecting Piece

several investments: 1) the follicle cell layer, consisting of the cumulus oophorus only, or the cumulus oophorus and corona radiata; and 2) the zona pellucida, a mucopolyzona Pellucida z-

\\ AOuter Dense FibersMtonr~Axoneme

Egg

. 11

Cumulus Oophorus

"

A

"Oviduct

Uterus

i /

/

Level of Section A.

FImbri

CooaCdit

co

Rsda

patcita

NonCspaclted

A Centra Pair

MSpermatozoa

Outer Pair

1

.I

praoo

EjnCacuated

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ItII Spermatozoa

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Vaglna

Figure 1. Schematic representation of the human spermatozoon. (From Zaneveld LJD: The biology of human spermatozoa. In Wynn RM (ed): Obstetrics and Gynecology Annual, Vol 7. New York, Appleton-CenturyCrofts, 1978, p 17) 2

Figure 2. Schematic representation of the fertilization process. The two round bodies between the zona pel lucida and the egg are the polar bodies. (From Zaneveld LJD: The biology of human spermatozoa. In Wynn RM (ed): Obstetrics and Gynecology Annual, Vol 7. New York, Appleton-Century-Crofts, 1978, p 33)

saccharide layer (Figure 2). In some textbooks, one reads that many spermatozoa reach the egg and release their acrosomal enzymes, which lyse the investments away from the egg so that one spermatozoon can fuse with the oocyte. This is not the case; rather, fertilization occurs with the oocyte investments intact, requiring th2 fertilizing spermatozoon to penetrate each of these layers. The acrosomal enzymes aid the spermatozoon in contacting and penetrating the oocyte investments. When spermatozoa enter the uterus, they begin to undergo an activation process called capacitation (56). This process is biochemical in nature and prepares the spermatozoon for the morphologic changes that the acrosome undergoes (the acrosome reaction) when the spermatozoon contacts and penetrates the egg. Without having undergone the acrosome reaction, the spermatozoon cannot pass through the zona pellucida (40, 56). The acrosome reaction may result in the exposure of one or more of the acrosomal enzymes that are essential for the penetration of the spermatozoon through the zona. A number of naturally occurring and synthetic sperm enzyme inhibitors have been shown to prevent fertilization, but only a few, among them inhibitors of hyaluronidase and acrosin, have been investigated in some detail. Seminal plasma itself contains enzyme inhibitors that, when added to spermatozoa, can prevent fertilization, Finally, antibodies have been prepared against certain sperm enzymes that inhibit these enzymes and can prevent thc fertilizing capacity of the spermatozoa. The following is a briet discussion of these inhibitory agents and the work done so far to establish their potential as contraceptives. li is important to keep in mind that the terms spermicide and sperm-directed antifertility agent are not synonymous. Clinically, these terms are sometimes used interchangeably, but such use is incorrect. A spermicide causes the death of the sperm cell. Antifertility agents can prevent fertilization by many other mechanisms besides immobilizing and killing spermatozoa; for example, by inhibiting sperm transport, preventing capacitation, disallowing the acrosome reaction, hindering penetration through oocyte investments, etc. The following discussion deals only with those agents known to be sperm enzyme inhibitors.

gametes (28, 53). When the follicle cell layer is removed from mouse eggs, the hyaluronidase inhibitors no longer prevent fertilization. This is not the case with hamster gametes, since the one hyaluronidase inhibitor that was tested also blocked the penetration of spermatozoa through the zona pellucida (48). The inhibitor did not prevent sperm passage through the vitelline membrane, however. Table I lists, in chronologic order, various studies showing the in vivo and antifertility activity of synthetic hyaluroni dase inhibitors. Already in the late 1940s and the 1950s, investigators were testing hyaluronidase inhibitors as contraceptives. Those inhibitors receiving the greatest attention were derivatives of hyaluronic acid and certain flavonoids, particularly phosphorylated hesperidin. In laboratory animals, the hyaluronidase inhibitors decreased the conception rate, either when added to spermatozoa before artificial insemination, or when placed vaginally before coitus. A controversial study performed at that time with humans is of particular interest (62). Phosphorylated hesperidin was administered orally to both men and women of 300 married couples over a period of 3 to 30 months. Only two pregnancies occurred, owing to a failure to take the hyaluronidase inhibitor. No side effects were noted, and the contraceptive effect was completely reversed within 48 hours of halting inhibitor administration. However, other investigators were unable to confirm the antifertility activity of this hyaluronidase inhibitor when they admin istered it orally or intraperitoneally to rodents (Table 1). Even though no argument was raised regarding the vaginal contraceptive potency of the inhibitors, further research was halted at that time, in part because of the controversy that had developed, and in part because of the advent of the steroidal contraceptive techniques. During the past decade, two types of antihyaluronidases have been evaluated for their contraceptive activity: hyaluronidase antibodies, and chemical inhibitors. Anti bodies prepared against sperm hyaluronidase inhibit the lytic activity of this enzyme toward synthetic substances and the follicle cell layer (41, 78), and can prevent the in vitro fertilization of spermatozoa (14). Since the hyalur onidases of the spermatozoa and testis are identical, and sperm hyaluronidase appears to be specific to the male genital tract, that is, it differs from that of other tissues and organs (41, 78), these antibodies should not crossreact with other tissues. Thus, an immunologic approach with sperm hyaluronidase as antigen could be applied to both men and women to prevent conception, without causing unwarranted side effects. In vivo antifertility studies need to be performed after immunization with pure hyaluronidase to ensure that hyaluronidase antibodies can enter the genital tract in

HYALURONIDASE INHIBITORS One of the first enzymes found to be associated with the sperm acrosome is hyaluronidase. The enzyme causes the dispersion of the follicle cell layer of the egg, and the spermatozoon appears to employ this enzyme to lyse a passage through this investment (4). Indeed, inhibitors of hyaluronidase specifically prevent the penetration of spermatozoa through the follicle cell layer of mouse

3

high enough concentractions to form a complex with the hyaluronidase of the spermatozoa and prevent fertilizat4ion. Such studies appear to have been attempted by only one group of investigators so far (7). Using purified bovine testicular hyaluronidase as antigen, infertility could not be induced in female rabbits, but a significant decrease in sperm ccunt occurred in male rabbits on immunization. The effect was completely reversible, and the males became fertile 9 to 15 weeks after the last antigenic stimulus. The effectiveness of the antibodies in the male rabbit is somewhat- surprising, since bull and rabbit hyaluronidase crossreact poorly, if at all (78). It is of

INHIBITOR

Nitrated hyaluronic acid derivatives

interest that a number of women with unexplained infertility possess antibodies against sperm hyaluronidase, in addition to the usual sperm agglutinating and sperm immobilizing antibodies (41). The ability of chemical hyaluronidase inhibitors to prevent fertilization was confirmed recently (29, 30). Phosphorylated hesperidin and PSs&both prevented conception on vaginal placement in rabbits before coitus (Table 1). These results, as well as t: ose reported in the 1940s and 1950s, encouraged us to perform some detailed studies evalu ating the practical use of the hyaluronidase inhibitors as

SIGNIFICANT OBSERVATIONS

At concentrations of 0.5 to 2.3 mg/ml, inhibitors actively prevented fertilization when mixed with ejaculated spermatozoa; at 8 to 40 mg/ml, inhibitors significantly reduced conception when placed vaginally Intraperitoneally (20 mg/kg/day) or orally (100 mg/kg/day) administered inhibitors prevented fertiliza tion in about 80% of animals. The cycle was not affected and the effect was reversible Fertility was decreased at a concentration of 20 mg/kg/day Inhibitor was administered in tablets at a dose of 100 mg/kg/day, and completely prevented conception in 300 married couples with only 2 exceptions over test periods from 3 to 30 months. The two failures were due to not taking the medication. No side effects were noted and the contraceptive activity was completely reversible after withdrawal of the inhibitor within a 48.hour period Inhibitor did not affect fertility when injected intraperi. toneally (20 mg/kg/day), administered orally (100 mg/kg/day), or deposited into oviducts (0.03 ml; 1%in saline). Fertility was significantly depressed on addition of inhibitor (0.1 to 1% saline) to ejaculated spermatozoa before vaginal insemination Fertility was not affected by inhibitors at concentra tions of approximately 150 mg/kg/day

STUDY

Pincus et al., 1948 (49)

TEST METHOD

Inhibitors added to ejaculated rabbit spermatozoa before artificial insemination; vaginal application of inhibitors to rabbits before coitus Intraperitoneal or oral administration to female rats before coitus

Phosphorylated hesperidin

Martin & Beiler, 1952, (36)

Phosphorylated hesperidin

Sieve, 1952 (62)

Inhibitor injected intraperitoneally into mice before coitus Inhibitor administered orally to men and women

Phosphorylated hesperidin

Chang & Pincus,

1953 (10)

Inhibitor administered intraperitoneally or orally to female rats before coitus; deposited into oviducts of rabbits 10.5 to 11 hours after coitus; or added to ejaculated rabbit spermatozoa before vaginal insemination

P.hosphorylated hesperidin, dehydroquercitin, hesperidin

methylchalcone

PSs3 (53 D/K)*

Thompson et al.,

Inhibitors administered orally to 1953 (68)

male and female mice before coitus Parkes et al., 1954 (47) Joyce et al.,

1979 (29)

Inhibitor added to ejaculated rabbit spermatozoa before vaginal insemination Rabbit spermatozoa treated before vaginal insemination; or vaginal application of inhibitors in rabbits before coitus Inhibitor added to epididymal hamster spermatozoa before insemination into the uterus Inhibitors placed vaginally in rabbits before coitus

Fertilization was prevented

Phosphorylated hesperidin, PSs.*

Hesperidin (1%) and PSs3 (10 to 14 mg/ml) reduced fertilization in both test systems. Contraceptivc activity was greatly enhanced by adding acrosin inhibitors Contraceptive effect at 10 mg/mI but not at 5 mg/ml

Sodium aurothiomalate (myocrisin) Sodium aurothiomalate (myocrisin), phosphorylated hesperidin, fenoprofen Ca++,

phenylbutazone,

oxyphenbutazone, penicillamine, PSs3*

Perreault et al.,

1980 (48)

Joyce &

Zaneveld, 1981

(30)

Phenylbutazone (0.02 mg/ml), oxyphenbutazone (0.1 mg/ml), phosphorylated hesperidin (10 mg/ml) and PSs5 mg/ml) were highly contraceptive; myocrisin (2.5 (75 mg/ml) was slightly active; and fenoprofen Ca++ (0.65 mg/ml) and penicillamine (1 mg/ml) were inactive (see Table 2 for further detail)

*Hydroquinone-suifonic acid formaldehyde polymer.

Table 1. In vivo antifertility activity of synthetic hyaluronidase inhibitors.

4

contraceptives. Little was known about the toxicity of the compounds that had been used so far, but we felt that agents could be found that were inhibitory toward hyaluronidase, and were already used clinically as noncontraceptive pharmaceuticals. Since such compounds are already on the market, their side effects should be minimal and, if they are contraceptive, they should be rapidly available for clinical use. This approach appeared realistic when we found that myocrisin (disodium aurothiomalate), an anti-inflammatory agent, prevented the penetration of mouse and hamster spermatozoa through the layers surrounding the egg (48, 53). A number of marketed compounds were screened for their ability to inhibit hyaluronidase. Of these, five were effective (Table 2). Their enzyme inhibitory activity toward some typical acrosomal enzymes was established and, with one exception, all were specific for hyaluronidase. The antifertility activity of these compounds was tested by mixing them with capacitated mouse spermatozoa before adding the treated spermatozoa to oocytes in vitro. All the compounds, except for one, prevented fertilization (Table 2). No effect on sperm motility or forward progression was noted at the concentration of the inhibitors used. Several of the inhibitors, again at concentrations that did not affect sperm motility, were also contraceptive when placed vaginally in rabbits before coitus (Table2). Phenylbutazone and oxyphenbutazone, which are presently both used clinically as anti-inflammatory agents, were particularly effective even when used at concentrations as low as 0.02 mg/ml and 0.1 mg/ml, respectively,

These results are encouraging, especially considering that under exactly the same conditions in rabbits, presently marketed vaginal contraceptives were reported to reduce the conception rate only partially, usually by only 50% to 70% or less (9, 27, 46, 72, 77). Several of these vaginal contraceptives contain nonoxynol-9 at concentrations of approximately 50 mg/ml. Thus, at least in the rabbit, phenylbutazone and oxyphenbutazone appear to be several orders of magnitude more potent than non oxynol-9, the spermicide most frequently used in present day vaginal contraceptives. ACROSIN INHIBITORS Vaginal Contraceptive Action. Acrosin, a sperm proteinase, is probably involved in several aspects of the fertilization process. Inhibitors of this enzyme were reported to prevent the sperm acrosome reaction, sperm binding to the zona pellucida, and sperm passage through the zona pellucida and the vitelline membrane (40,56, 57, 64, 73). Thus, treatment of spermatozoa with acrosin inhibitors prevents their fertilizing capacity. Acrosin has received a significant amount of attention during the past decade, and its properties have been fairly well characterized (6,38, 45). As with other proteinases, the enzyme can be inhibited by both naturally occurring and synthetic agents. One naturally occurring acrosin inhibitor is present in seminal plasma and can, on addition to capacitated rabbit spermatozoa, prevent their in uiuo fertilization when the treated spermatozoa are insemi nated into the oviduct (80). Since this inhibitor is naturally

ENZYME

N.acetyl

COMPOUND (COMPANY) Hyaluronidase + + + + +

Acrosin

-

fi-glucuronidase +

-

glucosaminidase

P-galactosidase

ANTIANTIFERTILITY FERTILITY ACTIVITY ACTIVITY ON VAGINAL INVITRO (MOUSE PLACEMENT GAMETES) IN RABBITS + +

(MERCK, SHARP &

DOHME)

FENOPROFEN Ca++ (LILLY) PENICILLAMINE OXYPHEN-

MYOCRISIN

(MERCK, SHARP & DOHME)

-

+

-

+

BUTAZONE (CIBA-GEIGY)

PHENYL-

+

+

BUTAZONE (CIBA-GEIGY) Table 2. Enzyme inhibitory spectrum and antifertility activity of certain pharmaceutical agents that inhibit

hyaluronidase. 5

present, it must be of low toxicity, and would be attractive contraceptive agent. However, the seminal inhibitor is mostly removed from the spermatozoa during capacitation and/or the acrosome reaction (24, 81). Therefore, it cannot be used as a vaginal contraceptive, but it may be potentially useful as an agent that can be released from an IUD to enhance the effectiveness of this device (see next section). The same type of reversible binding to spermatozoa probably occurs with other naturally occuring proteinase inhibitors. A number of these inhibitors, particularly the Kunitz pancreatic trypsin inhibitor (Trasylol), were shown to be cortraceptive in in vitro fertilization systems, or when mixed with spermatozoa before the treated gametes were inseminated into oviducts (74). However, they do not appear practical as contraceptive agents, unless they are released directly into the uterus or fallopian tubes, or are administered systemically at high concentrations (see further). For e: ample, Schumacher and associates (58), treating rabbit spermatozoa with soybean trypsin inhibitor or Trasylol (4 to 22.5 mg/ml) prior to insemination into the vagina, noted no effect on conception. Similarly, 0.9 mg/ml of antipain, a bacterial proteinase inhibitor, placed in the vagina of primates before coitus, did not cause a significant reduction in conception (75). In contrast to the naturally occurring inhibitors, certain synthetic agents can bind irreversibly or pseudo-irreversibly to proteinases, and should have more immediate clinical potential as contraceptives. A number of synthetic acrosin inhibitors have been tested for their ability to prevent conception in uiuo (Table 3). Of these, N-a-p-tosyl-L-lysine chloromethyl ketone (TLCK) and p-nitrophenyl-p' -guanidinobenzoate (NPGB) were particularly potent when tested in rodents and rabbits. After vaginal application to primates, NPGB (at 0.09 mg/ml) was much more effective than TLCK (10 mg/ml), and equally or slightly more effective than Delfen Vaginal Cream (Ortho Pharmaceutical Co.) in preventing pregnancy (75). Although these results showed clearly that the vaginal placement of acrosin inhibitors prevents or decreases conception, a problem was foreseen with their clinical applicability. For instance, TLCK is an alkylating agent (potentially carcinogenic), and NPGB releases nitrophenol when it reacts with acrosin. For the acrosin inhibitors to be practical, the compounds must be of low toxicity. Hall and co-workers synthesized N-carbobenzoxy amino acid esters that showed contraceptive activity when placed vaginally in mice (26). The N-carbobenzoxyglycine vinyl ester was particularly potent and had a low acute toxicity with an LDs0> 500 mg/kg (the LDso is the dose at which half of the animals die immediately when acompound is administered). Subsequently, these investigators pre-

pared a number of N-protected glycine activated esters, all of which decreased fertilization when placed vaginally at 10 mg/kg/day for 4 weeks in mice (13). N-carbobenz oxyglycine vinyl ester, and N-carbobenzoxyglycine 1,2 dibromoethyl ester, were most effective, resulting in 0% fertilization rates. The acrosin inhibitory activity of the compounds correlated positively with the vaginal con traceptive potency. A different approach was used by our laboratory. Since NPGB was an effective contraceptive agent with an LDso of 180 mg/kg in mice (75), but it released nitrophenol, we replaced the nitrophenol by a phenol already on the market and approved by the U.S. Food and Drug Administration for human use. The guanidinobenzoic acid portion of the molecule is probably of low toxicity, and similar compounds have been used clinically. Nine such phenol derivatives of guanidinobenzoic acid (aryl guanidinobenzoates) were synthesized (31) (Table 4). All of them were found to be potent inhibitors of huma acrosin with 150 values (the concentration at which half of the enzyme is inhibited) of 10- 7 to 10-10 M. The arylguanidinobenzoates blocked fertilization at con centrations that did not affect sperm motility when added to capacitated mouse spermatozoa before the treated gametes were mixed with eggs in uitro. Subsequently, the in viuo contraceptive efficacy of the inhibitors was screened by placing them at 0.1 mg/ml concentrations in the vagina of rabbits before coitus (Table 4). All com pounds except two inhibited fertilization. The arylguani dinobenzoates containing the phenols acetoininophen, ethylparaben, and methylsalicylate were most active. Similar to the hyaluronidase inhibitors, these arylguani dinobenzoates appear to be orders of magnitude more potent than nonoxynol-9 (see section on Hyaluronidase Inhibitors). Since none of the acrosin inhibitors had ever been tested to determine if they could prevent the fertilization of human spermatozoa, we mixed the arylguanidinobenzo ates (at 10- 4 to 10- 6M) with human spermatozoa before the gametes were capacitated and added to denuded hamster oocytes, to assess their fertilizing capacity (69). No effect on sperm motility was observed, and all compounds tested decreased the fertilizing capacity of the human spermatozoa. Thus, inhibitors of acrosin are also contraceptive toward human gametes. Toxicologic studies with the arylguanidinobenzoates are presently in progress in our laboratory. Acute studies in mice and rats showed that the compounds are of very low toxicity in regard to the amount needed for contraception. The LDso of the compounds, on intraperitoneal injection, varied in most cases from 500 to 1000 mg/kg and was occasionally even higher. By comparison, nonoxynol-9, when tested for its acute toxicity in our laboratory under

6

INHIBITOR*

TLCK, TPCK, NPGB, EPGB

STUDY

Zaneveld et al., 1970 (79)

TEST METHOD

Capacitation assay; inhibitors added to ejaculated rabbit spermatozoa before uterine insemination; or vaginal application of inhibitors to rabbits before coitus

SIGNIFICANT OBSERVATIONS

5 TLCK but not TPCK (5 to 15/ug/10 sperm), blocked of capacitated spermatozoa; TLCK fertilizing ability and NPGB but not EPGB (10 pg/l10 sperm) decreased fertilization when added to ejaculated spermatozoa before uterine insemination; TLCK (3 mg/mI in K-Y jelly) inhibited conception when deposited vaginally before coitus

TLCK

Zaneveld et of., 1971 (77) Newell et al., 1972 (46)

Vaginal application to rabbits before coitus Inhibitors applied vaginally to rabbits before coitus

Mixing TLCK (5 mg/ml) with Delfen Vaginal Cream (Ortho Pharm. Co., containing 50 mg/mI nonoxynol-9) greatly increased the contraceptive properties of Delfen TLCK (3mg/ml in K-Y jelly) depressed fertilization to a larger extent than GPB, ABP, or L-arginine; a mixture of TLCK, GPB, APB, NPGB, poly-L-lysine, benzami dine, and L-arginine (each 2 to 3 mg/ml in K-Y jelly) possessed greatest antifertility activity; addition of TLCK (3mg/ml) and GPB (2 mg/ml) to Delfen Vaginal Cream enhanced the contraceptive activity of Delfen Both inhibitors significantly decreased the fertilizing capacity of spermatozoa at concentrations of 0.003% to 0.005% with only slight impairment of motility. No effect on fetal weight or other anatomic aspect of the fetus was noted Both inhibitors (3 mg/ml) prevented blastocyst implantation when added to uteri; combined capsule and injection was the most effective mode of administration In K-Y jelly, NPGB (0.09 mg/ml) possessed significantly higher contraceptive effect than TLCK (10 mg/ml) and was equally as effective as Delfen Vaginal Cream Most compounds (10 mg/kg/day) decreased or com pletely inhibited fertilization. The carbobenzoxy-glycine, -leucine, and -proline esters were most effective. TLCK and TPCK also inhibited conception The inhibitors effectively prevented fertilization in both systems (NPGB at 100 pg/ml; TLCK at 200 pg/ml)

TLCK, GPB, APB, NPGB, L-arginine, polyL-lysine, benzamidine

TLCK, TPCK

Miyamoto & Chang, 1973 (42)

Uterine insemination of inhibitortreated ejaculated hamster spermatozoa

TLCK, TPCK

Dabich & Andary, 1974 (11) Zaneveld et of., 1979 (75) Hall et of., 1979 (26)

Uterine injections or slow release of inhibitor from capsules placed in utero in mice 2 to 4 days after coitus Vaginal application of inhibitors to .Aump-tailed macaques (Mococa arctoides) before coitus Vaginal application or intraperi. toneal injection into mice

NPGB, TLCK

Activated N-carbobenzoxy amino acid esters, TLCK, TPCK TLCK, NPGB

Joyce et of., 1979 (29)

Rabbit spermatozoa treated with inhibitors before vaginal insemination, or the vaginal application of inhibitors to rabbits before coitus Vaginal application or intraperitoneal injection into mice

N-protected glycine activated esters, TLCK, TPCK

Drew et al., 1981 (13)

At 10 mg/kg/day, N-carbobenzoxy-vinyl ester and N-carbobenzoxy-glycine 1,2-dibromoethyl ester com pletely prevented pregnancy when applied vaginally. All other esters also showed contraceptive activity. The compounds were less active when applied intraperi toneally. Acrosin inhibitory activity of the esters cor related with their vaginal contraceptive potency. TLCK and TPCK prevented fertilization when placed vaginally Aminobenzamidine (97.1 mg/lg/day), NPGB (0.8 mg/kg/day) and MUGB (1.0 mg/kg/day) caused a 50% decrease in the fertilization rate. Benzamidine had no effect A number of compounds possessed high contraceptive activity at 0.1 mg/ml. (see Table 4)

Benzamidine, aminobenzamidine, NPGB, MUGB Phenol derivatives of guanidino benzoic acid (arylguanidinobenzoates)

Beyler & Zaneveld, 1982 (5) Kaminski et of., 1981 (31)

Release from subcutaneously placed minipumps in mice before blastocyst implantation Vaginal insemination into rabbits

*

TLCK =N-a-p-tosyl-L-lysine chloromethyl ketone TPCK =L-1-tosylamide-2-phenylethyl-chloromethy ketone =p-nitrophenyl-p'-guanidinobenzoate NPGB = EPGB ethyl-p-guanidinobenzoate

MUGB = p-methyluthibelliferyl-p -guanidinobenzoate APB = p-amidophenacyl bromide GPB = p-guanidinophenacyl bromide

Table 3. In viuo antifertility activity of synthetic acrosin inhibitors.

exactly the same conditions, had an LDso of 180 mg/kg, worse than that of any of the arylguanidinobenzoates. In regard to acrosin inhibition and vaginal contraception, it isalso worthwhile to consider certain ions, particularly zic. Most of the acrosin present on ejaculated sperr iatozoa isinazymogen form, called proacrosin. Proacrosin conversion to acrosin isprobably anecessary step for the fertilization process. At least in some species, acrosin activity and proacrosin conversion can be inhibited by zinc ions. Zinc ions also inhibit the acrosome reaction of hamster spermatozoa (40). Deposition of certain zinc salts inthe vagina of rabbits before coitus decreases their fertilization rate (72). Such zinc salts also enhance the effectiveness of presently marketed vaginal contracep tives, at least in the rabbit (72).

Action in Association with an Intrauterine Device

The use of acrosin inhibitors to enhance the contraceptive potency and to decrease the side effects of the IUD is particularly attractive for three reasons. First, the agents are potent inhibitors of the sperm acrosome reaction and fertilization. Second, certain acrosin inhibitors can pre vent blastocyst implantation, further assuring the con traceptive activity of the inhibitors when released inutero (11) (implantation appears to involve a blastocyst pro teinase that has a number of properties in common with acrosin, and can be inhibited by similar agents) (12). Third, some side effects of the IUD can be minimized if certain antifibrinolytic agents (inhibitors of plasminogen activation, and/or plasmin) are deposited into the uterus (61, 67). The IUD appears to cause an activation of fibrinolytic activity in the uterus (60), which in turn results in

decreased clot formation and enhanced menstrual flow

(IUD). Besides being useful for vaginal contraception, acrosin inhibitors may also be employed in conjunction with the IUD. The IUD causes several undesirable side effects, among them menorrhagia, dysmenorrhea, and pelvic inflammatory disease (PID). These side effects are due at least in part to the shape of the IUD, which makes the device irritating to the endometrium. Howeve, such irritation ispresently necessary for the IUD to be effective, since the device probably acts primarily by inducing a chronic inflammatory response of the endometrium, so that blastocyst implantation is prevented. One could decrease the irritating properties of the IUD by altering its design so it fits the uterus better, while retaining its contraceptive activity by releasing antifertility agents. For instance, the release of copper from the IUD enhances its contraceptive effectiveness. Other agents, including the enzyme inhibitors, are likely to be much more potent than copper in preventing conception.

and pain. When injected into the uterus, the Kunitz pan creatic trypsin inhibitor (Trasylol) isparticularly effective in reducing such pain and bleeding (67). Release of antifibrinolytic agents from IUDs in women reduces the excessive menstrual blood flow to approximately phys iologic levels (51, 66). Thus, release of certain proteinase inhibitors from the IUD may not only increase the effectiveness of this device, but should also prevent some of the side effects commonly associated with IUD use, particularly if the shape of the IUD can be altered so that it is less irritating to the uterus. Since the three enzymes (acrosin, the blastocyst pro teinase, and plasmin) have a number of properties in common, and can be inhibited by similar agents, it isvery likely that inhibitors of acrosin and the blastocyst pro teinase can be used that also have antifibrinolytic activity. For instance, Trasylol isnot only an antifibrinolytic agent

CONTRACEPTIVE POTENCY OF PHENOL WHEN PLACED VAGINALLY IN RABBITS BEFORE COITUS (0.1 mg/ml)t

PHENOL MOIETY OF THE ARYLGUANIDINOBENZOATE* ACETOMINOPHEN ETHYLPARABEN METHYLPARABEN SALICYLAMIDE 8-HYDROXYQUINOLINE METHYLSALICYLATE

PHARMACOLOGIC ACTIVITY OF PHENOL Analgesic, antipyretic Preservative

Preservative

Analgesic

+-40

++

Antimicrobial

Counter-irritant, flavoring agent

++H+i-

EUGENOL

HEXYLRESORCINOL THYMOL

Dental obtundant, topical anesthetic Antiseptic

Antibacterial, antifungal

.. +0

*All the arylguanidinobenzoates are potent inhibitors of human acrosin (31).

t

+

+--

0

- highly contraceptive (0-10% fertilization) =contraceptive (10-30% fertilization) = not contraceptive (80-100% fertilization)

Table 4. Phenol derivatives of guanidinobenzoic acid (arylguanidinobenzoates) as vaginal contraceptives in rabbits.

8

but isalso a potent inhibitor of tre blastocyst proteinase and acrosin, and prevents fertilization when mixed with spermatozoa before oviductal insemination (80). Since NPGB inhibits acrosin, plasmin, and the blastocyst proteinase, it is likely that the arylguanidinobenzoates synthesized by our laboratory will be useful in conjunction with the IUD. Several patents were recently awarded to Eli Lilly Co. (U.S.) for the controlled relase of certain acrosin in(U.S. patents hibitors from silicone into the uterusThe compounds 4264-575, 4264-576,4264-577, 4264-578). covered included sulfonate salts of the form ula RSO 3M (M is a cation), sulfoalkyl alkanoate salts of the formula RCOO(C-.),SO3M, salts of the formula ROSOM, and sterol sulfates derived from cholesterol, campestrol, Psitosterol, lanosteral, erosterol, dehydrocholesterol or /3-hydroxycholenic acid. Systemic Action. Acrosin inhibitors may also be applied systemically to induce infertility. Long-term intraperitoneal injection of N-carbobenzoxy amino acids, particularly N-protected glycine activated esters (10 mg/kg/ day), into female mice decreases the' fertility (13,26). The intraperitonc-il administration of leupeptin and antipain (5 mg/day) over long periods to mice also decreases conception (32), but no antifertility activity was observed when the Kunitz pancreatic trypsin inhibitor (Trasylol) was administered systemically to rabbits and mice (6to 20 mg/kg/day) (58). When agents are administered for prolonged periods of time, one cannot differentiate whether the inhibitors prevent fertilization, implantation, or fetal development, In order to determine if systemically administered acrosin inhibitors can specifically prevent the processes that occur before implantation, we released benzamidine (88.6 mg/kg/day), aminobenzamidine (97.1 mg/kg/day), NPGB (0.8 mg/kg/day) and the arylguanidinobenzoate containing methylumbelliferone as phenol (MUGB) (1.0 mg/kg/day) from subcutaneously implanted minipumps in mice during the preimplantation period only (5). Except for benzamidine, all three inhibitors decreased the fertilization rate by about 50%. This finding was especially encouraging because NPGB and MUGB were applied at low concentrations. The systemic application of inhibitcrs needs to be studied in greater detail, however, before it can be used clinically. Preferably, the inhibitors should be specific for acrosin i Since acrosin is biochemictly and immunologically dift fm acrosin ferent from trypsmn, the enzyme most similar totnally (6, 45), this should be possible. However, until now, only certain monosaccharides have been found to selectively 2).semen inhiit arosn raherthantrysin Antibodies prepared against acrosin inhibit the activity of this enzyme toward high molecular weight substrates, and

do not crossreact with trypsin, that is, they appear to be specific (6, 45). Such antibodies may be useful from a contraceptive standpoint. Antibody studies have been complicated, becausc of difficulty in preparing enough pure acrosin as antigE nso that high antibody titers can be produced. Some da-a are available, however, showing that rabbits immunized with acrosin and possessing high antibody titers are not likely to become pregnant when mated, whereas fertility was not prevented when the rabbits showed lower acrosin antibody titers (65). Also, treatment of capacitated rabbit spermatozoa withofanti acrosin immunoglobulins before tubal insemination the f5) in e . ndersore a mes re gametes renders the rabbits infertile (15). GOSSYPOL Gossypol, adisesquiterpene aldehyde, obtained primarily from the cotton plant, isan antispermatogenic agent when given orally to men and to several other animal species. This was first established in China (3), and has since been confirmed by others. The clinical use of gossypol has been argued, however, because of the possible toxicity of this compound. One of the first effects noted after administration of gossypol isadecrease in sperm motility. For this reason, our laboratory and other research groups investigated the spermicidal activity of gossypol. When it ismixed with polyvinylpyrrolidone (PVP), which solubilizes gossypol, or even in the absence of PVP, gossypol has in vitro spermicidal activity approaching that of existing vaginal contraceptive formulations (50, 54, 70). When placed vaginally in primates before coitus, gossypol-PVP causes a dose-dependent decrease in the motility of the spermatozoa (8). The antimotility effect of gossypol is caused possibly by its ability to inhibit a number of midpiece metabolic enzymes, such as lactate dehydro genase and several enzymes involved in oxidative phos phorylation (1, 37, 43, 50). Its spermicidal activity makes gossypol potentially useful as a vaginal contraceptive; however, its potency may not be high enough to compete with presently marketed vaginal contraceptives. Thus, based strictly on its sper micidal properties, gossypol would probably be useful only in those parts of the world that do not have ready access to marketed formulations. We therefore initiated a study to see if low, nonspermicidal dose levels of gossypol could prevent fertilization. Preliminary exper iments by Williams in rabbits had shown that such low wnthach low n raits h ens by Woss when placed vagi levels of gossypol were contraceptive before coitus (72). The effect of gossypol on the fertilizing capacity of human spermatozoa was evaluated by mixing low amounts of the compound with whole ov the smina plsm and fo minutes, semnfor 55mntsremoving the seminal plasma and excess gossypol by washing the spermatozoa, capacitating the gametes in medium, and adding them to hamster oocytes from which the outer investments had been

9

removed. At concentrations ranging from 5.5 to 16.5 pg/ml semen, gossypol showed no effect on sperm motility, but a dose-dependent inhibition of fertilization took place (33). Additionally, there was a parallel, dosedependent decrease in the ability of proacrosin (the zymogen form of acrosin that is prevalent in ejaculated spermatozoa) to convert to acrosin. Apparently, the antifertility mechanism ofgossypol at low, non-spermicidal dose levels isdue inpart to its ability to prevent proacrosin activation. Vaginal contraceptive experiments in rabbits, using low gossypol doses, are presently in progress, and appear to confiim the results of Williams (72). Thus, gossypol may have potential from avaginal contraceptive standpoint. If its vaginal absorption isalso low, toxic effects should be minimal or nonexistent. LACTATE DEHYDROGENASE-X ANTIBODIES Lactate dehydrogenase (LDH) is one of the metabolic enzymes of the sperm midpiece. The testis and spermatozoa possess a form of this enzyme (LDH-X or LDH-C4) that isspecific to the male genital tract. In the female, LDH-X is absent from all tissues, including those of the reproductive tract (34). LDH-X anibodies do not bind to LDH-1 or LDH-5, and do not inhibit these isozymes, but they do inhibit the activity of LDH-X (19). Immunologically, LDH-X is attractive for the development of a contraceptive method that is specifically directed toward spermatozoa, without affecting any other tissues. The reality of this approach was shown by Goldberg and coworkers, who immunized female mice, rabbits, and primates with LDH-X and noted asignificant decrease in their conception rate (20, 23, 34). However, the passive immunization with LDH-X had no effect on the fertility of female mice (17). Immunization of males with LDH-X also had contraceptive effects, at least in the rabbit (22) and the guinea pig (71). As with acrosin and hyaluronidase, use of LDH-X as an antigen for contraceptive purposes poses problems of both supply and homogeneity. These limitations can be overcome if one or more peptides can be obtained that are small enough for chemical synthesis and have antibodies that cross-react with the native antigens. Such peptides have been prepared from LDH-X (21) and their antibodies were shown not only to cross-react with LDH-X but also to prevent the fertilization of mouse gametes (18). OTHER ENZYME INHIBITORS For successful fertilization, spermatozoa require the activity of anumber of other enzymes not yet discussed (6,38,40, 44, 56). Inhibition of these enzymes would lead to 10

infertility. For instance, numerous compounds can inhibit the glycolytic and respiratory enzymes of the midpiece, thus inducing sperm immobilization. Many respiratory enzyme inhibitors are available, and a number of these have been shown to prevent sperm motility, including hydrazine, hydroxyl amine, sodium azide, ;,yu, uquinone, cyanide, carbon monoxide, and malonate. Inhibitors of glycolytic enzymes that were shown to prevent sperm motility include iodoacetate, iodosobenzoate, iodoaceta mide, p-chloromercurobenzoate, hydrogen peroxide, phenyl mercuric acetate, p-carboxyphenylarsine and the metal ions copper, cadmium, mercury, lead, sodium arsenite, and selenite. These compounds interact with the sulf hydryl groups of the enzymes through oxidation, mercaptide formation, or alkylation. Other glycolytic enzyme inhibitors that were shown to cause sperm immobilization are D,L-glyceraldehyde, quinones, quinols, and fluoride. Several of these enzyme inhibitors are already used in presently marketed vaginal contracep tives; however, they are rarely the primary ingredient. Most are too toxic for clinical use, because they affect the metabolic processes of other tissue cells, as well as those of spermatozoa. The response of spermatozoa to these midpiece enzyme inhibitors varies tremendously among different species, and differs also according to the genital tract origin of the spermatozoa tested, that is, from the epididymis or the ejaculate. If a compound inhibits the motility of sper matozoa of one species we cannot assume that it will inhibit the motility of spermatozoa of another. Before we can conclude that such compounds may be of clinical use, their immobilizing activity toward human spermatozoa needs to be investigated. The inhibition of several acrosomal enzynnes besides acrosin and hyaluronidase can prevent capacitatic nand/or the acrosome reaction, and thus fertilization. For instance, glycosidase inhibitors were reported to inhibit the capaci tation of hamster spermatozoa (25) although in our laboratory, the addition of/3-glucuronidase and N-acetyl glucosaminidase inhibitors to capacitated mouse sper matozoa did not prevent their in vitro fertilizing capacity (28). A phospholipase inhibitor, p-bromophenacylbro mide, inhibits the acrosome reaction of hamster sper matozoa (35). The acrosome reaction isalso prevented by inhibitors of enzymes involved in protein phospholipid transmethylation (39). So far, no in vivo contraceptive studies have been performed with these inhibitors. Certain seminal plasma components that have enzyme inhibitory properties can prevent the fertilizing capacity of spermatozoa. One of these is an acrosin (proteinase) inhibitor (see Acrosin Inhibitors). Another is a glyco protein with a molecular weight varying from 200,000 to 370,000, depending on the species (16, 52). This glyco protein isoften called "decapacitation factor," because it

renders capacitated spermatozoa infertile. The rabbit glycoprotein can be broken down to low molecular weight peptides (containing about 10 amino acids) that retain their antifertility activity (38,55). The exact mechanism of action of the glycoprotein is not known, but the glycoprotein prevents penetration of spermatozoa through the investments surrounding the egg (52) either by inhibiting the acrosome reactior (16), by inhibiting an acrosomal hydrolase called "corona penetrating enzyme" (CPE) (82), or by an as yet undiscovered mechanism. Another glycoprotein (fetuin) present in serum also inhibits CPE and prevents fertilization when added to capacitated rabbit spermatozoa (63). Such naturally occurring factors, particularly ifactive in a low molecular weight, synthesizable form, are interesting, because they should be of low toxicity. However, at least the glycoprotein from seminal plasma appears to be removed from th' spermatozoa during capacitation, so that the compound will probably not be useful as a vaginal contraceptive, unless an irreversibly binding form can be produced. .also

"Suicide" inhibitors, which become inactive after they react with a specific sperm enzyme,can also be developed. The phenol derivatives of guanidinobenzoic acid (aryl guanidinobenzoates) that inhibit acrosin are an example. As vaginal contraceptives, the enzyme inhibitors can be used either alone or in combination with spermicides, so that the benefit of both activities can be obtained. Some acrosomal enzyme inhibitors are also spermicidal, and may be dually active as spermicides and antifertilization agents. Since several of the hyaluronidase and acrosin inhibitors have already been shown to be more effective and less toxic than nonoxynol-9, one can expect that enzyme inhibitors will appear on the market in vaginal contraceptive preparations in due time. Fnzyme inhibitors can also be used to enhance IUD effectiveness. Although the IUD already has a very low failure rate, it causes menorrhagia, dysmenorrhea, and pelvic inflammatory disease. Some of these side effects can be minimized by designing !UDs with shapes that are less irritating to the endometrium, but retain their contra ceptive potency by releasing enzyme inhibitors. Addi tionally, certain acrosin and/or fibrinolytic inhibitors are capable of decreasing the menorrhagia and possibly

the dysmenorrhea associated with IUD uF. .

rCO.CIA.J

iU.

A number of data have recently been accrued which clearly indicate that inhibitors of sperm enzymes, whether chemical agents or antibodies, are potent contraceptives. Their use is attractive because the enzymes are often sperm-specific, so that development of inhibitory agents directed only toward spermatozoa, and thus, having minimal side effects when used clinically, becomes feasible. A sperm-directed contraceptive method could be employed in both men and women. However, it will probably be more readily applicable to the woman, because only 0.1% to 1%of the ejaculated spermatozoa reach the uterus, and an even smaller number of spermatozoa (thousands) reach the egg. Thus, the concentration of the inhibitors required to induce contraception in the female is much less than in the male. Additionally, the inhibitors car. be applied iocally in the woman. Use of the chemical enzyme inhibitors is most immediately apparent from a vaginal cot traceptive standpoint, Since most of the inhibitors are of iew molecular weight, they should be able to enter the coagulum and inactivate spermatozoa. Additionally, they may pass into the cervical mucus, so that any spermatozoa not contacted in the vagina by the inhibitors can be inactivated during cervical transport and storage. The surfactants (such as nonoxynol-9) used as spermicides in present-day vaginal contraceptives are much less likely to enter the coagulum and cervical mucus.

Enzyme inhibitors can be employed systemically to prevent conception. At present, no good chemical inhibitors are available for this purpose, because none :o as yet specific enough toward spermatozoa; thus, tlhey also inhibit certain enzymes from other tissues. However, the immunologic approach seems feasible. In the past, immunizing animals with testis, testicular extracts, sper matozoa, and sperm extracts, has resulted in some degree of infertility. However, such a heterogeneous antigen preparation ismuch less attractive from a clinical standpoint than a homogeneous preparation consisting of a sperm specific er zyme, such as lactate dehydrogenase, acrosin, or hyaluronidase. Inthis regard, the results with LDH-X have been very encouraging. The research so far has focused on only a very small number of the possible enzymes that can be inhibited to prevent fertilization, primarily because we know too little about the biochemical properties of the sperm enzymes and their role in the fertilization process. Investigators should give considerable attention to this important area of contraceptive research during the coming decades. The use of gamete-directed agents holds great promise in leading to generally acceptable, non-toxic, highly effective contraceptive methodology.

Most of the research on this topic done in our laboratory was supported by the Program for Applied Research on Fertility Regulation (PARFR-204) and by the National Institutes of Health (NIH HD 09868). 11

REFERENCES

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14. Dunbar S, Munoz MG, Cordle CT, Metz CB: Inhibition of fertilization in vitro by treatment of rabbit spermatozoa with univalent isoantibodies to rabbit sperm hyaluronidase. J Reprod Fertil 47:381, 1976. 15. Dudkiewicz AB: The effect of antiacrosin antibodies on fertilization in vivo. Biol Reprod 26, Suppl 1:145a, 1982. 16. Eng LA, Oliphant G: Rabbit sperm reversible decapacitation by membrane stabilization with a highly purified glycoprotein from seminal plasma. Biol Reprod 19:1083, 1978. 17 Erickson, RP Hoppe PC, Tennenbaum D, Spielmann H, Epstein CJ: Lactate dehydrogenase-X: effects of antibodies on mouse gametes but not on early development. Science 188:261, 1975. 18. Goldberg E,Beyler S: Personal com munication, 1982. 19. Goldberg E: Immunochemical spe cificity of lactate dehydrogenase-X. Proc Natl Acad Sci 68:349, 1971. 20. Goldberg E: Infertility in female rabbits immunized with lactate dehydrogenase-X. Science 181:458, 1973. 21. Goldberg E, Gonzales-Prevatt V, Wheat TE: Immunosuppression of fertility in females by injection of sperm specific LDH-C4 (LDH-X): prospects for development of acontraceptive vaccine. InSemm K, Mettler L (eds): Proc III World Con gress on Human Reproduction. Amsterdam, Excerpta Medica, 1981, p 360. 22. Goldberg E,Wheat TE: Induction of infertility in male rabbits for immunization with LDH-X. In Spilman CH, Lobl TJ, Kirton KT (eds): Regulatory Mechanisms of Male Reproductive Physiology. New York, American Elsevier Publ Co, 1976, p 133. 23. Goldberg E, Wheat TE, Powell JE, Stevens VC: Reduction of fertility in female baboons immunized with lactate dehydrogenase-C4. Fertil Steril 35:214, 1981. 24. Goodpasture JC, Reddy JM, Zaneveld LJD: Acrosin, proacrosin and acrosin inhibitor of guinea pig spermatozoa capacitated and acrosome reacted in vitro. Biol Reprod 25:44, 1981.

25.glycosidaseRBL, Anderson OF: Effect of Gwatkin inhibitors on the capacita tion of hamster spermatozoa by cumulus cells in vitro. J Reprod Fertil 35:565, 1973. 26. Hall IH, Drew JH, Sajadi Z, Loeffler LJ: Antifertility and antiproteolytic ac tivity of activated N-carbobenzoxy amino acid esters. J Pharm Sci 68:696, 1979. 27. Homm RE, Doscher GE, Hummel 27. Gomm RE, R el activity of three vaginal contraceptive products in the rabbit: relationship to in vitro data. Contraception 13:479, 1976. 28. Joyce C: The antifertility activity of sperm hyaluronidase inhibitors. Biol Reprod 26, Suppl 1:117a, 1982. 29. Joyce C, Freund M, Peterson RN: Contraceptive effects of intravaginal application of acrosin and hyaluronidase inhibitors in rabbit. Contraception 19:95, 1979. 30. Joyce C, ZaneveldLJD:Inhibitorsof sperm hyaluronidase as vaginal contra ceptive agents. J Androl 2:17, 1981. 31. Kaminski J, Bauer L, Zaneveld LJD, Bhattacharyya AK, Nuzzo N, Van der Ven HH: Synthesis and evaluation of enzyme inhibitors with contraceptive potency. XIXth Annual Meeting of Medicinal Chemistry, Iowa City, Iowa, 1981. 32. Katz J, Troll W, Adler SW, Levitz M: Antipain and leupeptin restrict uterine DNA synthesis and function in mice. Proc Nat Acad Sci, U.S.A. 74:3754, 1977. 33. Kennedy WP, Van der Ven HH, Waller DP, Polakoski KL, Zaneveld LJD: Gossypol inhibition of oocyte penetra tion and sperm acrosin. Biol Reprod 26, Suppl 1:118a, 1982. 34. Lerum JE, Goldberg E: Immu.nologi cal impairment of pregnancy in mice by lactate dehydrogenase. Biol Reprod 11:108, 1974. 35. Lui CW, Meizel S: Further evidence in support of a role for hamster sperm hydrolytic enzymes in the acrosome re action. J Exp Zool 207:173, 1979. 36. Martin GJ, Beiler JM: Effect of phos phorylated hesperidin, a hyaluronidase inhibitor on fertility in rat. Science 115:402, 1952. 37. Maugh TH: Male "pill" blocks sperm enzyme. Science 212:314, 1981.

9. Chang MC: Effect ofcommercial contraceptive jellies placed into the rabbit vagina before mating. Fertil Steril 11:109, 1960. 10. Chang MC, Pincus G: Does phosphorylated hesperidin affect fertility? Science 117:274, 1953. 11. Dabich D, Andary TJ: Prevention of blastocyst implantation in mice with proteinase inhibitors. Fertil Steril 25:954, 1974. 12. DenkerH-W:Theroleoftrophoblastdependent ane of uterine proteases in initiation of implantation. In Ludwig H, Tauber PF (eds): Human Fertilization. Stuttgart, George Thieme Publ, 1978, p 204. 13. Drew JH, Loeffler L, Hall IH: Antifertility activity of N-protected glycine activated esters. J Pharm Sci 70:60, 1981.

12

38. McRorie RA, Williams WL: Biochemistry of mammalian fertilization. Ann Rev Biochem 43:777, 1974. 39. Meizel S: Inhibition of the hamster sperm acrosome reaction by transmethylation inhibitors. J Exp Zool 217:443, 1981. 40. Meizel S: The mammalian sperm acrosome reaction, a biochemical approach. In Johnson MH (ed): Development in Mammals. Amsterdam, NorthHolland Publ Co, 1978, Vol 3, p 1. 41. Metz CB: Role of specific sperm antigens in fertilization. Fed Proc 32:2057, 1973.

42.Miymot H Chng G:Effctsof 42. Miyamoto H, Chang MC: Effects of

51. Ragab MI, Thomas MN: The use of tranexamic acid (AMCA) in IUDs as an antibleedingagent. Int J GynaecolObstet 14:137, 1976. 52. Reddy JM, Audhya TK, Goodpasture JC, Zaneveld LJD: Properties of a highly purified antifertility factor from human seminal plasma. Biol Reprod. In Review. 53. Reddy JM, Joyce C, Zaneveld LJD: Role of hyaluronidase in fertilization: the antifertility activity of myocrisin, a nontoxic hyaluronidase inhibitor. J Androl 1:16, 1980. 54. Ridley AJ, Blasco L: Testosterone

and gossypol effects36:638, 1981. sperm motility. Fertil Steril on human

63. Srivastava PN, Gould KG: Inhibition of fertilizing capacity of rabbit sperma tozoa by sialoproteins. Contraception 7:65, 1973. 64. Stambaugh R, Smith M: Sperm pro teinase release during fertilization of rabbit ova. J Exp Zool 197:121, 1976. 65. Syner FS, Kuras R, Moghissi KM: Active immunization of female rabbits with purified rabbit acrosin and effect on fertility. Fertil Steril 32:468, 1979. 66. Tauber PF, Kloppel A, Goodpasture JC, Burns J, Ludwig H, Zaneveld LJD: Reduced menstrual blood loss by intra of an antifibrinolytic agent fromrelease

o natfbioyi gn rmita

proteinase inhibitors on the fertilizing capacity of hamster spermatozoa. Biol Reprod 9:533, 1973. 43. Myers BD, Throneberry GO: Effect ofgossypol on some oxidative respiratory enzymes. Plant Phvsiol 41:787, 1966. 44. Morton DB: Lysosomal enzymes in mammalian permatozoa. In Dingle JT, Dean RT (eds): Lysosomes in Biology and Pathology. Amsterdam, North-Holland PubI Go, 1976, Vol 5, p 203. 45. Morton DB: The occurrence and function of proteolytic enzymes in the reproductive tract of mammals. InBarrett AJ (ed): Proteinases in Mammalian Cell and Tissues. New York, North-Holland Publ Co, 1977, p 445. 46. Newell SD, Polakoski KL, Williams WL: Inhibition of fertilization by proteinase inhibitors. Proc 7th International Congress on Animal Reproduction and Artificial Insemination, Munich, Germany, 1972, Vol 3, p 2117. AS, iocemcalaspctsof he 47. Parkes Bioogial HJ, Spensley PC: nd Rogers Biological andbiochemical aspects ofthe prevention of fertilization by enzyme inhibitors. Proc Soc Study Fertil 6:65, 1954.

55. Robertson RT, Bhalla VK, Williams WL: Purification and peptide nature of decapacitation factor. Biochem Biophys Res Commun 45:1331, 1971. 56. Rogers BJ, Brentwood BJ: Capacitation, acrosome reaction and fertilization. In Zaneveld LJD, Chatterton RT (eds): Biochemistry of Mammalian Reproduction. New York, J Wiley, 1982, p 203. 57. Saling PM: Involvement of a trypsinlike activity in binding of mouse spermatozoa to zonae pellucidae. Proc Nat Acad Sci 78:623 1, 1982. 58. Schumacher GFB, Swartwout JR, Zuspan FP: Fertility experiments in mice and rabbits with the trypsin-kallikrein inhibitor from bovine lung. In Fritz M, Tschesche M(eds): Proteinase Inhibitors. New York, Walter de Gruyter, 1971, p 247. 59. Sciarra JJ: Vaginal contraception: historical perspective. In Zatuchni GI, (eds): Vaginal Sobrero AJ, Speidel Contraception: NewJJDevelopments, Cotaeto:NwDvlpet. Hagerstown, Harper & Row, 1979, p 2. 60. Shaw ST, Cihak RW, Moyer DL: Fibrin proteolysis in the monkey uterine cavity. Variation with and without an intrauterine contraceptive device. Nature 226:1097, 1970. 61. Shaw ST, Moyer DL, Aaronson DE, Underwood J, Forino RV: Intrauterine medication with epsilon aminocaproic acid. Effect on rhesus monkeys wearing intrauterine devices. Contraception 11:395, 1975. 62. Sieve BF: A new antifertility factor. Science 116:373, 1952.

uterine devices. Am J Obstet Gynecol 140:322, 1981. 67. Tauber PF, Wolf AS, Herting W, Zaneveld LJD: IUD induced hemorrhage: control by local proteinase inhibition. Fertil Steril 28:1375, 1977. 68. Thompson RQ, Sturtevant M, Bird OD: Effect of phosphorylated hesperidin in mice. Science 118:657, 1953. 69. Van der Ven HH, Bhattacharyya AK, Kaminski J, Binor H, Bauer L, Zaneveld LJD: Inhibition of human sperm capacitation by proteinase inhibitors and a high molecular weight factor from human seminal plasma. Biol Reprod 24, Suppl 1:38a, 1981. 70. Waller DP, Zaneveld LJD, Fong HHS: In uitro spermicidal activity of gossypol. Contraception 22:183, 1980. 71. Wellerson R, Wagstaff P, Asculai S, 7 Weson , Wagstaff PBAscti 5, Hudson M,Kupferberg AB: Induction of aspermatogenesis in guinea pig through immunization with lactate dehydrogenaseX isozyme. Int J Fertil 19:65, 1974. 72. Williams WL: New antifertility agents active in the rabbit vaginal contraception (RVC) method. Contraception 22:659, 1980. 73. Wolf DP: Involvement of a trypsin like activity in sperm penetration of zona free mouse ova. J Exp Zool 199:149, 1977. 74. Zaneveld LJD: Sperm enzyme in hibitors as antifertility agents. In Hafez ESE (ed): Human Semen and Fertility Regulation in Men. St Louis, CV Mosby, 1976, p 570.

48. Perreault S, Zaneveld LJD, Rogers BJ: Inhibition of fertilization by myocrisin, a hyaluronida- -:,hibitor. J Reprod Fertil 60:461, 1980. 49. Pincus G, Pirie NW, Chang MC: The effects of hyaluronidase inhibitors on fertilization in the rabbit. Arch Biochem 19:388, 1948. 50. P6s6H, WichmannK, JanneJ, Luukkainen T: Gossypol, a powerful inhibitor of human spermatozoal metabolism. Lancet 1:885, 1980.

13

75. Zaneveld LJD, Beyler SA, Kim DS, Bhattacharyya AK: Acrosin inhibitors as vaginal contraceptives in the primate and their acute toxicity. Biol Reprod 20:1045, 1979. 76. Zaneveld LJD, Bhattacharyya AK, Primate model for the evaluation of vaginal contraceptives. Am J Obstet Gynecol 129:368, 1977. 77. Zaneveld LJD, Polakoski KL, Robert son RT, Williams WL: Trypsin inhibitors and fertilization. In Fritz H, Tschesche H (eds): Proceedings of the First International Resealch Conference on Proteinase Inhibitors. New York, Walter de Gruyter, 1971, p 236.

78. Zaneveld LJD, Polakoski KL, Schumacher GFB: Properties of acrosomal hyaluronidase from bull spermatozoa. J Biol Chem 248:564, 1973. 79. Zaneveld LJD, Robertson RT, Williams WL: Synthetic enzyne inhibitors as antifertility agents. FEBS Letters 11:345, 1970.

81. Zaneveld LJD, Srivastava PN, Williams WL: Relationship of a trypsin like enzyme in rabbit spermatozoa to capacitation. J Reprod Fertil 20:337, 1969. 82. Zaneveld LJD, Williams WL: A sperm enzyme that disperses the corona radiata and its inhibition by decapacita tion factor. Biol Reprod 2:363, 1970.

80. Zaneveld LJD, Robertson RT, Kes sler M, Wi,iams WL: Inhibition of fertili zation in uiuo by pancreatic and seminal plasma trypsin inhibitors. J Reprod Fertil 25:387, 1971.

14

May. 1983. Volume 2. Number 4 Suite 1525 Northwes'kern University 875 North Michigan Avenue Chicago, Illinois 60611 Editor: Gerald I. Zatuchni, M.D., M.Sc. Managing Editor: Kelley Osborn

r

r

RESEARCH FRONTIERS IN FERTILITY REGULATION

CURRENT STATUS OF RESEARCH AND DEVELOPMENT

OF VAGINAL CONTRACEPTIVE RINGS AS A

FERTILITY CONTROL METHOD IN THE FEMALE

Subir Roy, M.D.

AssociateProfessor and

Daniel R. Mishell, Jr., M.D.

Professorand Chairman Department of Obstetrics and Gynecology University of Southern California School of Medicine Los Angeles, Caliornia

The contraceptive vaginal ring (CVR) is a clinically tested contraceptive steroid delivery system that provides 1) more constant circulating drug levels than oral for-

Size Determination of the optimum size of the toroidal CVRs

rmulations; 2) minimal ,ubject-physician contact; and 3) acceptable use-effectiveness and continuation rates. The rationale for this mode of drug delivery combines the knowledge that steroids (as well as other agents) can be rapidly absorbed into the circulation from the vagina, and the observation that the steroids contained in dimethylpolysiloxane (Silastic) devices can be released for long periods of time without producing local discomfort or significant systemic or local side effects, This article reviews the development of the CVR,with

Thisartcle eviws te dvelomen of he VR, ith

special emphasis on the device developed by the Population Council, and summarizes the clinical and metabolic studies that have been performed to date. The device being developed by the World Health Organization, designed to release levonorgestrel (LNG) at a rate of approximately 20 /g/day, does not consistently inhibit ovulation, and will not be discussed here because no publications about this device are as yet available (4). CHARACTERISTICS OF SILASTIC DEVICES Devices made of dimethylpolysiloxane are non-toxic (26), they release the steroids they contain at rates proportional to their surface area (7) and inversely proportional to the thickness of the outer wall of the

devices (11), and the amount of steroid contained within

must take into account subject comfort as well as the possibility of spontaneous expulsion of the device with activities that increase intra-abdominal pressure. The initial studies were performed with devices 70 andi 80 mm in outer diameter and 10 mm thick (14). Because of concern about possible expulsion, devices 75 mm in diameter contained a flat spring much like that in a conventional diaphragm. These rings were abndoned because, although none of the subjects COroplled of slippage, vaginal erosions were producd in over half Ihe subjects (15). A study of rings with on outer diatieter of 65 mm and a thickness ef 7 ton denmotnstrat('d that these, tha t

ion m c e d eve7 n d thinn r i thinner rings produced even more erosion than did the thicker rings (16). Subsequently, nnqS With Ile diameter of 61 mi (18,19), 60 titl (3, 13, 17, 3033), 58 mm (2729), 55 trn (11), and 50 tutu (2729), and I thickness of 9 to 9.5 nin have been well tolerated. From the results of these early proottype stu(lies, it ou inter (hida'ter of ,bout 5(to appears that CVIN -with

58 un antd a thickness (if 7

tolerated by most women. women, and unlike the diaphrqlti, the devic, do', not have to be fitled or l)]aced in, certaitin psititi, ilthou,1lu it geaerally becomes oriertitd oround h t vix As lootni as the outer surface of the CVlI is iii contnI with th Ntroids vaginal epit helium, systemic absi O tiolt of .h1

will occur. Vaginal mucosal vrolot occuts vely itl

to to (Mmin ate well 1)5 Onelting is suil, bl Iin all

the device determines the duration of action (6).

frequently, and heals spontaeously folli winq sitiple

'-Copyright PARFR 1983

removal of the CVR (15, 16, 19). Spontaneous expulsion of the device can be easily corrected by having the user reinsert the device. Upon colposcopic examination of women following 2 years of use, no changes of the vaginal epithelium adjacent to the CVR have been noted (25). Ring design Homogeneous. The initial design of the CVR was a ring made of a homogeneous mixture of steroid and Silastic (14) (Figure 1A). This device was found to release high initial dosages of steroid resulting in high circulating blood levels, followed by a rapid reduction of steroid levels (13, 30) coinciding with episodes of breakthrough bleeding or bruakthrough spotting (BTB/BTS). Core. In order to eliminate the initial high levels followed by the rapid decline, the core r:ng was developed (Figure 1B). Rings 9 mm thick, with steroids contained within a 3.5 mm central core, were tested and produced fairly uniform serum levels while in situ, except for an initial period, and had sufficient steroid capacity to allow for several cycles (3).

Shell. In an effort to develop devices with more uniform release rates, rings with a shell design were developed (Figure IC). These shell rings have a steroid and Silastic layer applied around an inner core of inert Silastic (16, 19, 31). The active layer is covered with another layer of inert Silastic tubing, thus providing an almost uniform distance through which the steroid must travel in order to be absorbed (11, 12, 18, 19, 31, 32). Collagen band. In an effort to develop a device that would potentially use less steroid and would be easier to fabricate, the collagen band device was developed (Figure I D). Collagen bands containing steroid were designed to lie in a groove on an inert inner core of Silastic. These devices were abandoned, because the bands broke or were displaced and unpredictable blood levels of steroid occurred (33). Drugs tested Studies have been performed utilizing the following progestogens alone or in combination with estrogens: medroxyprogesterone acetate (MPA), chlormadinone acetate (CMA), norethindrone (NET), R2323, di-nor gestrel (dl-Ng), levonorgestrel (LNG), LNG plus estradiol benzoate (EB), and LNG plus estradiol (E-). The 21 carbon compounds, e.g., MPA, which suppressed ovula

tion while being released in doses of 520 to 1219 pg per

day from a homogeneous ring, and CMA released from a shell ring, produced blood levels similar to those achieved after oral administration; however, following reports of mammary tumors in beaS!e dogs (14-16, 17), these compounds were no longer studied. Homogeneous NET rings that release 850 or 1529 /g of

the drug per day produced unacceptable episodes of

breakthrough bleeding and spotting associated with an offensive odor, and c.ulation occurred in one-quarter of the cycles studied (17). R2323 administered in a core device releasing 150 to 400 jg per day produced no BTB/BTS and blocked ovulation. Following removal of the device, withdrawal bleeding occurred promptly (3). Further tests with this promising agent were abandoned when men ingesting 100 mg of R2323 per week developed increased transaminase levels (3). In tests of rings releasing dl-Ng at rates of 120 to 350 pg per day, the major problem was BTB/BTS, which occurred in 63% of cycles studied. Ovulation also occurred in approximately 15% of cycles with this device in place (31). In an effort to reduce the incidence of BTB/BTS, a collagen band device was developed, into which LNG plus EB was incorporated. However, BTB/BTS was observed in 6 4 % of cycles studied, suggesting that no

Figure 1. Diagrammatic cross-sectional representation of the contraceptive vaginal ring. Figure A. Vaginal ring, made of a homogeneous mixture of contraceptive steroid and Silastic. Figure lB. Core vaginal ring, with a total thickness of 9 mm and central core of 3.5 mm, surrounded by 5 mm Silastic. Figure IC. Shell vaginal ring, fabricated around central core of Silastic, surrounded by layer of homogeneous mixture of contra ceptive steroid and Silastic, covered by a tube of Silastic. Figure 1D. Collagen band vaginal ring, containing contraceptive steroid in a band of collagen held in place by placement in groove on an inert Silastic ring. (Reproduced, with permission, from Hafez ESE, Van Os WAA (eds): Biodegradables and Delivery Systems for Con traception, p 169. Lancaster, England, MTP Press, Limited, 1980.) 2

benefit accrued from the addition of EB to the band, since the amounts (50 to 100/g)of estradiolbenzoate available for vaginal absorption were very small, and once absorbed, the drug did not alter circulating serum estradiol levels (33). Shell rings releasing LNG alone demonstrated higher sustained circulating serum LNG levels (1.6 to 2.4 ng/ml) than those shell rings releasing dl-Ng (1.2 to 1.7 ng./ml, over a 6-month period (18, 31).

Thesc LNG shell rings were associated with BTB/BTS in 33%, lack of withdrawal bleeding in 5%, and ovulation in 3% of cycles studied. Investigators considered the possibility that adding E2 to the LNG ring would more effectively control bleeding and suppress ovulation. In the first trial with rings releasing LNG plus E2, significantly improved bleeding control was observed. BTB/BTS occurred in only 7 % of cycles studied. Withdrawal bleeding after ring removal occurred within I to 5 days, with a mean of 2.6 days, and lasted from 3 to 7 days, with a mean of 4.5 days. There was no failure of withdrawal bleeding, and no ovulation occurred inanyofthe cycles studied (19). These rings released an average of 289 pg of LNG per day, and 212 pig of E2 per day, and produced fairly constant serum LNG levels of 1 to 3 ng/ml and an initial peak level of E, of about 100 pg/mil, which rapidly declined but presumably stimulated the endometrium sufficiently to provide improved bleeding control. Use schedule The original use schedule was similar to that recommended for oral contraceptive use. The CVR was inserted on day 5 of the cycle and left in place for 21 days.

occurred during this study, the vaginal rugae were

flattened. The fact that the patient did not need to

remember when the ring had to be removed was thought

to counterbalance the unpredictable occurrence of bleed ing episodes with this schedule. When questioned,

however, subjects preferred the fixed time schedule to

this erratic schedule.

Thus, the protocol now recommended for the use of the

vaginal ring is to have the woman insert it herself on day 5

of the cycle and wear it for 3 weeks, then remove it for a

7-day period to allow withdrawal bleeding. Should the

CVR interfere with coitus, it may be removed for up to 3

hours and then reinserted postcoitally without reducing

the contraceptive effect. Pharmacodynamics When the self-administered CVR containing both levonorgestrel and estradiol is placed in the vagina, the steroids are released from the surface and absorbed through the vaginal epithelium into the circulation at a fairly constant rate (Figure2). Patients using the rings had mean levonorgestrel levels of 2.5 /g/ml for the first cycle and 1.3 ug/ml during the sixth cycle (19). These levels are sufficient to inhibit ovulation, but are several tmes less than the 4 to 8 pg/ml peak levels obtained with daily ingestion of tablets containing 500 ug norgestrel (250 ug levonorgestrel) (5). Ovulation continues to be inhibited during the week that the rings are not in place. Although midcycle gonadotropin peaks are abolished,

CYCLE

V.

4

During the first studies with medroxyprogesterone

acetate rings in women following this schedule, a high incidence of BTB/BTS occurred, suggesting that in

,0,000

-

2

3

5

6

i

I

B

I

I

a

·

sufficient amounts of endogenous estrogen were being

secreted during use of this progestogen-only containing CVR (14, 15). Therefore, a trial was performed in which the CVR was inserted on day 10, when more endogenous estradiol would be secreted by the ovary. Since the incidence of BTB/BTS was not reduced with this schedule, the approach was abandoned (16). With another regimen, ableeding signal was tested; each subject was instructed to leave the ring in place and to

1000

, ;, °

10

., ,:

:

C:

remove it for 5 days only if she experienced BTB/BTS

(32). In studies with this regimen, the incidence of bleeding and spotting was usually lower than that expected during a comparable period of untreated cycles, The bleeding was also :;cantier than that during the subjects's ordinary menstruations, but prolonged amen-

1

14

1

14

1 14

1

14

1

14

1

14

1

DAY

orrheic episodes occurred. In almost all instances, the

ring was re-inserted within 5 days after removal, and no ovulations were observed. Although no vaginal erosions

Figure 2. Serum estradiol and levonorgestrel levels on log-scale and progesterone levels during six treatment cyclcs with vaginal rings. Rings were inserted on day 1 and removed on day 21 during each cycle. Open bars at top of graph indicate 3-week treatment cycles with rings inplace. Black bars indicate bleeding days (full height for bleeding and half height for spotting). (Reproduced, with permission, from Am J Obstet Gynecol 130:58, 1978.)

3

d-Ng ESTRADIOL

E-

contraceptive steroids: first, a natural estrogen, estradiol,

is released from the CVR instead of the more potent (in

CV

CI40-

CV-

CV5

C

=3!

" E-

terms of hepatic effects) synthetic estrogen, ethinyl estradiol, found in the oral tablets. Second, a smaller

of estrogen is absorbed from the CVR, and this

30

,amount

20-

=absorption " 0 otreatment

Figure 3. Three times a week assay of serum LH (solid line) and FSH (dotted line) during six treatment cycles. Open bars at top of graph represent the 3 weeks rings were in place. Values for LH and FSH are for same subject as in Figure 2. (Reproduced, with permission, from Hafez ESE, Van Os WAA (eds): Biodegradables and Deliory Systems for Contraception, p 165. Lancaster, England, MTP Press, Limited, 1980.)

occurs only during the first few days of each cycle (Figure2), because of the relatively lower solubility and diffusion of estradiol in comparison to levonorgestrel in dimethylpolysiloxane. Third, the CVR route of administration initially bypasses the liver, while

ro

isaonintally passes the liver whie

the steroids absorbed orally pass directly to the liver after absorption in the gut. For these reasons, the CVR has

lessofaneffectonhepaticalterationsthandocombination

oral contraceptives, which may increase angiotensinogen and steroid binding globulins while decreasing anti thrombin III (9, 21).

gonadotropin peaks have been observed during the 1week interval when the ring is removed, or soon after its re-insertion (Figure 3). There are three differences accounting for the variation in estrogenic effect produced by the CVR and oral NET RATES

50 mm 58 mm

Shell rings used for six or seven consecutive 21-day

cycles, with a 7-day non-use interval between cycles, have been analyzed for average steroid loss (12). In uivo, rings 58 mm in diameter were found to release levonor gestrel and estradiol at mean rates of 293 + 54 pg per day and 183 + 34/pg per day, respectively. Rings of 50 mm GROSS RATES

50 mm 58 mm

CVR RATE PREGNANCY MEDICAL TERMINATION USE-RELATED TERMINATION PERSONAL TERMINATION MOVING LOST TO FOLLOW-UP (LFU) CONTINUATION, LFU A TERMINATION CONTINUATION, LFU NOT A TERMINATION TYPE OF TERMJ N'ATION PREGNANCY MEDICAL REASONS USE-RELATED REASONS PERSONAL REASONS MOVING LOST TO FOLLOW-UP ALL NO. WOMEN ENROLLED NO. WOMEN AT RISK, 12 MO.

* P<0.01

CVR 1.0 22.5 4.4 9.7 2.1 9.9 50.4 56.8 EVENTS (NO.)

NORDETTE 2.0 18.7 2.0 11.2 2.0 25.9 38.2 55.4

CVR 2.4 29.2 8.4 12.2 2.8 9.5 48.8 54.0

CVR 1.4 27.0 5.4 13.7 3.2 11.3 50.4 56.8

NORDETTE 3.3 24.7 3.6* 18.5 3.2 31.Ot 39.2t 55.4

1.8 23.5 6.6 8.8 2.0 8.5 48.8 54.0

STANDARD ERRORS 10 95 10 55 10 133 313 553 193 0.8 2.3 1.4 1.8 0.9 1.4 2.3 0.7 2.2 1.1 1.9 1.0 1.5 2.3 1.1 2.3 1.2 2.4 1.1

2.3

2.2

9 119 35 45 10 46 264 547 239

5 115 23 47 10 53 253 556 220

f P<0.001

(From Sivin I, Mishell DR Jr, Victor A et al: A multicenter study of levonorgestrel-estradiol contraceptive vaginal rings. I. Use effectiveness. An international comparative trial. Contraception 24:341, 1981).

Table 1. One-year termination and continuation rates per 100 acceptors by regimen and reason for termination (aJl segments of use). 4

diameter had mean levonorgestrel and estradiol release rates of 252 + 34 pg per day and 152 + 21 pg per day, respectively. Sufficient steroid was present in each of these rings so that they could be used for at least six treatment cycles before the drug was depleted. CLINICAL STUDIES In order to assess the contraceptive effectiveness and acceptability of the shell C conang LN n o devices of two sizes - 50 mm and 58 mm in outer diameter and a thickness of 9.5 mm - were fabricated and users' experience was compared to experience of users of an oral contraceptive containing 150 pg LNG users o r (Nordetteremoval r pg v0 LNG with 30 pg EE2 (Nordette). These rings were compared to Nordette in a multicenter study involving data from eight clinics - in Brazil, Chile, Denmark, Dominican Republic, Finland, United States (Los Angeles), Nigeria, and Sweden (27-29). A total of 547 women using the 50 mm CVR, 556 women using the 58 mm CVR, and 553 women using the Nordette oral contraceptive participated in the study. Among the CVR users in all segments of use, 1-ye.r net pregnancy rates were less than 3 per 100, approximately the same as the pregnancy rates observed among users of Nordette (Table 1). Continuation rates at 1year were 50 per 100 users of the rings (all segments of use). This rate was significantly higner than, or equal to, the rate observed among the users of Nordette - 38.2 or 55.4 per 100 users, depending upon whether the lost to follow-up rates of these subjects were considered a termination or 50 mm

PROBLEM VAGINAL CVR 34.1

not, respectively. The prcfile of women terminating was similar for the users of the two sizes of rings, but it differed significantly from that of the Nordette acceptors. Gross 1-year rates of termination for medical reasons ranged from 25 to 29 per 100 for the three regimens, without a significant difference. However, ring users were more likely to terminate use for vaginal problems and pill users for headaches, nausea, and other systemic symptoms. Problems relating to use of the CVR regimen accounted for a significantly higher discontinuation rate among CVR than among Nordette users. Terminations for use-related reasons were coded into seven categories: frequent ring expulsion, interference of the ring with rin or o th rs' disiof orffic coit cotsuer'dlieforifcuiwthnetonr of the ring, unpleasant ring odor, difficulties in storage of the ring or pill, ring loss, and problems associated with use of the pill. Lost rings, insertion and removal difficulties, or users' dislike of insertion or removal, accounted for the majority of CVR use-related terminations. These trials indicate that CVRs of this design are as effective in use and have continuation rates at least equal to, and possibly superior to, Nordette under the same study conditions. In addition, side effects of the rings and Nordette were evaluated by noting spontaneous complaints, by re cording additional medications taken by users, and by physical examination (28). Inquiries about changes in the frequency of specific conditions were made at the end of the subjects' participation in the first year of the study (Table 2). The incidence of spontaneous complaints was similar among users of the two different-sized rings and of Nordette. 58 mrnm

CVR 37.8 NORDETTE 9.6

SIGNIFICANCE

(3 REGIMENS)

*

LOWER ABDOMINAL PAIN HEADACHE

NERVOUSNESS DEPRESSION

10.5 7.7

2.8 1.7

14.6 7.1

3.6 2.1

9.6 19.4

5.9 0.7

NS

*

NS NS

*

MENSTRU 4L ACNE OR OTHER SKIN PROBLEMS MASTALGIA OTHER ALL FIRST PROBLEMS WOMEN-YEARS

NS = not significant * P<0.001

34.7 7.7 0.3 21.0 120.5 351.8

22.0 8.0 0.9 21.4 117.6 335.8

21.7 5.3 1.6 31.6 105.4 335.8

NS NS t NS

t P<0.01

(From Sivin I,Mishell DR Jr, Victor A et al: A multicenter study of levonorgestrel-estradiol contraceptive vaginal rings. 11.Subjective and objective measures of effects. An international comparative trial. Contraception 24:359, 1981).

Table 2. Fit st problems mentioned in response to question, "How have you been feeling since last visit?" By regimen; all segments of use; incidence per 100 women-years during first 12 months.

5

Vaginal complaints obviously were far more frequent among ring users, while only users of the 50 mm ring had significantly greater menstrual complaints than users of either the 58 mrn ring or Nordette. Headaches, dizziness, and nausea were reported more frequently by users of Nordctte. All the regimens were associated with weight gain of about 1 kg and increased hemoglobin levels of about 0.5 gm/dl. Nordette, but not the CVR, was associated with small but significant increases in both mean diastolic (0.9 mm Hg) and systolic (1.5 mm Hg) blood pressure. A more detailed examination of the menstrual events, based on diaries, demonstrated that the CVRs, used continuously for 3 weeks and then removed for 1 week, and Nordette produced approximately the same total number of bleeding and spotting days during six cycles of use (27-29) but the small (50 mm outer diameter) ring was associated with somewhat more spotting (Table 3). This ring was also associated with somewhat more prolonged bleeding and spotting runs and with more prolonged nonbleeding intervals than reported by users of the larger

(58 mm outer diameter) ring or Nordette. On the average, CVR users experienced about 1day per month of bleeding or spotting with the ring in place. Evidence from menstrual diaries indicates that the 58 mm ring provides control over the menstrual cycle comparable to that of Nordette (29). In an effort to determine acceptability of these rings in a rural setting, an investigation was undertaken in rural, small town, and urban slum clinics in four locations, two in Brazil and two in the Dominican Republic (8). The CVR was offered as a new method in the clinics and described as similar to the pill but placed in the vagina for 3 weeks each month followed by a 1-week rest interval. Follow-up surveys were carried out in the four locations at the end of the experimental period. Of the total contraceptive acceptors in each of the four locations, 3%, 8 %, 9 %,and 12.5% chose the CVR. The acceptance rate was higher in three rural clinics, where the nurses themselves used the ring. The follow-up surveys showed that the user's ability to place and keep the ring in the vagina without removal for a prolonged period was the 50 mm CVR 58 mm CVR

DAYS

NORDETTE

MEAN NUMBER OF DAYS OF BLEEDING AND SPOTTING PER 30-DAY REFERENCE PERIODS 1-30 (from acceptance) 31-60 61-90 91-120 121-150 151-180 N = days 1-30 N =days 151-180 5.90 4.78 4.80 4.60 4.76 4.66 500 356 5.57 4.75 4.54 4.28 4.30 4.41 493 335 6.42

4.51

4.54

4.30

4.37

4.43

422

270

PERCENTAGE DISTRIBUTION OF BLEEDING AND SPOTTING, DAYS 1-168

(6x28 DAY REFERENCE PERIOD)

<12 13-24 25-36 37-48 49-60 >60 No. women Mean ± S.D. 4.0 39.0 35.8 14.4 5.3 1.3 374 28.67 ± 12.01 3.9 41.7 42.0 7.6 3.9 0.8 357 26.80 ± 10.12 0.7

43.5

42.4

11.2

1.4

0.7

278

27.29 ± 9.05

MEAN NUMBER OF BLEEDING AND SPOTTING DAYS EXPERIENCED IN DAYS 169-336 No. women Mean ± S.D.

international comparative trial. Contraception 24:377, 1981).

246 25.30 ± 12.26

220 24.55 ± 10.60

169 24.42 ± 6.95

(From Sivin I,Mishell DR Jr, Victor A et al: A multicenter study of levonorgestrel-estradiol contraceptive vaginal rings. I11.Menstrual patterns. An

Table 3. Bleeding and spotting days by regimen (data from menstrual diaries). 6

most important attribute of the method and played a large role in the woman's reaction to it. Anticipated use-related problems were the most prominent reason given by pill acceptors for not choosing the ring; however, ease of use was named as the "most liked" characteristic by 55% of the ring i iers. Women tended to remove the ring for intercourse and to wash it frequently, often with detergents, indicating their concern with "cleanliness" of an object kept within a body cavity for long periods.

With the oral contraceptive, there was no effect on the fasting glucose concentation or on glucose tolerance. The insulin concentrations, particularly in fasting states and at 60 minutes of the IV-GTT, showed a tendency to higher values. This is in accordance with earlier findings of peripheral insulin resistance when estrogens of the synthetic type are used in combination with levonor gestrel (24). All liver function values remained within normal range in all subjects. There was a small significant decrease in alkaline phosphatase in both groups, which is in contrast to the eievation noted with higher dosage combined oral contraceptives. It was concluded that neither of these two contraceptive methods, the effects of which are predominantly gesta genic, seems to cause impairment of glucose tolerance or hepatic function. Globulins. A study comparing the combination CVR to various combination oral contraceptives has demon strated that the CVR produces no changes in cor ticosteroid binding globulin-binding capacity (CBG-BC), angiotensinogen, or antithrombin Ill, in contrast to oral contraceptives, which produce significant increases of the first two and asignificant reduction in the last. These globulins respond to estrogen-dominant preparations and suggest that the combination CVR-releasing levonor gestrel and estradiol is a relative progestogen-dominant preparation(21). Indeed, the CVR produces asignificant reduction of sex hormone binding globulin-binding capacity (SHBG-BC) and no change in the total serum norgestrel, but significantly greater non-SHBG-bound norgestrel (both per cent and mass,imn) when cor pared to an oral contraceptive containing 300pg nor gestrel and 30p g ethinyl estradiol (9). The overall effect of such a preparation is to produce no estrogen-mediated effects, for reasons already described (see Pharmaco dynamics section), while producing relative progestogen dominance (see Lipids and lipoproteins section, following). Lipids and lipoproteins. Because of the concern that gestagen-dominant contraceptive steroids may adversely affect lipids and lipoproteins, several studies investigating the effect of the CVR on lipids, lipoproteins, serum lipoproteins, and apolipoproteins have been undertaken. In a study in the United States, lipids and lipoproteins, as determined by analytic ultra-centrifugation, were studied in 5 controls and 10 women using 58 mm CVRs releasing 290 pg per day of LNG and 180 pg per day of E2. The groups were comparable for race, age, parity, and obesity

METABOLIC STUDIES Carbohydrate and liver function. A prospective, long-term study undertaken to compare the metabolic effects of the contraceptive vaginal ring and Nordette in two groups of women (n = 22 and 20, respectively) has also been reported (1). An intravenous glucose tolerance test (IV-GTT), including determination of the insulin response to glucose, and liver function tests (bilirubin, alanine amino transferase, asparagine amino transferase, and alkaline phosphatase) were performed pretreatment and after 2,6, and 12 months of treatment, and at about 1 month post-treatment (Table 4). Both the glucose tolerance and fasting values of glucose were unaltered, The early insulin response to glucose increased by 50k in the CVR group after 1 year of treatment, but not in the oral contraceptive group. All other insulin values were unchanged. The effect on the peak insulin is not related to or indicative of any change of peripheral insulin sensitivity, but shows, rather, the sensitivity of the pancreas to the glucose stimulus. Since there is evidence of progesterone receptors in the beta cells (10), at least the initial insulin response to a glucose load might be', irectly influenced by progestogens. The unchanged fasting and 60 minute insulin levels in this study, as well as the normal glucose tolerance, indicate that levonorgestrel, on its own, does not cause either impairment of glucose tolerance or peripheral insulin resistance.

CVR 3 months 6 months 12 months * P<0.05

t P<0.01

Calculated on paired data.

Oc 11

17

5

26* 32t 50t

(From Ahren T, Victor A,Lithell Het al: Comparison of the metabolic

effects of two hormonal contraceptive methods: An oral formulation and a vaginal ring. Contraception 24:415, 1981).

indices, alcohol ingestion, smoking, diet, and exercise.

Table 4. Mean increase of peak insulin in per cent of pretreatment values,

Fasting blood samples were obtained twice before CVR treatment, after 2 and 7 weeks of treatment and 1 week

7

thereafter (Table 5). The women using the CVR had a significant incremental reduction of cholebterol hum baseline to treatment (15%), which was distributed among all the lipoprotein classes - especially HDL-C (2 7%). The cholesterol/HDLC ratio was significantly increased with treatment. All mean changes were within the reference range. The reduction in HDL (21%), especially in the subclasses HDL 2a (48%) and HDL 2b (71%), was significant, and for the subclasses HDL 2a and HDL 2b, reduction was outside the reference range. The HDL/HDL ratio increased significantly (44%), while the LDL/HDL 2a + 2b ratio increased significantly outside the reference range ( 13 1%) with treatment. Of the lipid and lipoprotein measurements that changed significantly with treatment, HDLC, HDL, HDL 2a, LDL/HDL, and LDL/HDL 2a +2b changed significantly toward baseline in the 1week when the subjects were off treatment (23). A 1-year, prospective Swedish study compared the effects on lipoproteins, lipids, and apolipoproteins of a combined oral contraceptive (30 pg ethinyi estradiol and (abut15pg 0 perday)and a CVR levonorgestrel) andout i ehigher.

(about 180 pug per day) and levonorgestrel (about 290 pg

LDL and HDL particles, with an altered lipid/protein ratio, during both contraceptive treatments. Despite the impressive relative increase in the LDL/HDL ratio in the contraceptive ring group, the average absolute value of this ratio did not reach the mean for healthy men (2). The effects of the 50 mm combination levonorgestrel and estradiol ring, which release 250 pg per day and 150 pg per day, respectively, of these steroids, were studied in 10 healthy, normally menstruating women attending afamily planning clinic in Santo Domingo (20). A schedule of 21 days of use followed by 7days of non-use was followed for 6 cycles (Table 7). During the first two cycles of use, concentrations of cholesterol, HDL cholesterol, triglyc erides, and LDL cholesterol declined significantly from control levels - as much as 2 0% for cholesterol, 18% for HDL cholesterol, 25% for triglycerides, and 13% for LDL cholesterol. There were no subsequent changes with continued use. These declines are similar in direction but of lesser magnitude than those reported from the frete re Unite t an Swee clse

chesroHDcoltrlraidungramn.Te

ment plasma levels of the same lipids were considerably There was no significant change in the total

per day) (Table 6). The two treatments induced significantly different effects. Inthe OC group, the lipoproteinlipid concentrations showed only minor changes, but apolipoprotein B and A-1 increased by about 15%a. In contrast, during treatment with the CVR, there was a 25% decrement of cholesterol in high density lipoprotein and a 10% decrement in low density lipoprotein cholesterol, with only minor effects of apolipoprotein Band A-I. The ratio of LDL and HDL cholesterol increased in the CVR group, but not in the oral contraceptive group. The results also indicate a change in the composition of the

TEST

REFERENCE

cholesterol/HDL cholesterol ratio during treatment. The differences observed among ihese studies may be due to the different ethnic groups, their dietary practices, or to the different (lower) release rate of steroids in the latter study. A possible potential reduction of predicted incidence of myocardial infarction with use of the CVR suggested by a reduction in total cholesterol appears to be counter balanced by a reduction in HDLC and increases in the cholesterol/HDL-C and LDL/HDL ratios. The potential clinical implications of these findings, if any, remain to

be determined. Vaginal flora. The CVR is a foreign body that is placed into the vaginal vault for an extended period of time. Therefore, concern has been expressed whether any TEST HDL-C LDL-C LDL-C/HDL-C Apo Bt Apo A-If PRETREATMENT 1.43 2.81 2.00 88 97 12 MOS 1.06 2.69 2.70 103 98 A%* -24t -10 +2k5f +17t NC

RANGE

TREATMENT TREATMENT A'Ot

PRE

Cholesterol HDL-C Cholesterol/ HDL-C HDL HDL2a HDL 2b LDL/HDL LDL/HDL 2a + 2b

*

127 37

-218 63

183 52 3.5 318 115 48 0.9 1.6

.

155 38 4.0 250 60t

14f

-15 -27 +14 -21 -48 -71 +44 +131

2.5- 5.0 224 - 380 79 - 165 15 - 139 0.51.4 0.9 - 2.9

1.3

3.7t

Significant changes

* Percentage change from pretreatment t Significan changes

T Arbitrary units (Adapted from Ahren T, Lithell H, Vicior A ei al: Comparison of the metabolic effects of two hormonal contraceptive methods: An oral formulation and a vaginal ring. II. Serum lipoproteins and apolipo. proteins. Contraception 24:451, 1981).

t Percentage change from pretreatment

t Outside the reference range

(Adapted from Roy S, Krauss RM, Mishell DR Jr ei al: The effect on lipids and lipoproteins of a contraceptive vaginal ring containing levonorgestrel and estradiol. Contraception 24:429, 1981).

Table 5. Average values of selected lipids and lipoproteins (mg/dl) in 10 women using 58 mm LNG + E2 CVRs. 8

Table 6. Average values of selected lipids and lipopro teins (mmol/I) in 22 women using 58 mm LNG + E2 CVRs.

changes in the flora of the vagina occur with CVR usage. A variety of studies with different agents (MPA, CMA, dl-NG, LNG-E2) have shown that although vaginal secretions are increased with CVR usage, which in some instances were the result of pathogenic organisms such as Candida, the resulting vaginitis could be treated while the woman continued to use the CVR (11, 16, 19, 31). To more carefully study this issue, a prospective study was undertaken in which premenopausal women seeking a steroid contraceptive method were allowed to choose between a CVR containing levonorgestrel and estradiol used in a 3 week in, 1 week out regimen (n = 20) and an oral contraceptive containing levonorgestrel (150 pg) and ethinyl estradiol (30 pg) in a 28-day regimen (n = 10). Cultures from the posterior vaginal fornix were obtained prior to therapy in both groups, and monthly for 6 months for the CVR group, and after 1,3, and 6 months for the OC group. These cultures were streaked on specific media to provide quantitative aerobic and anaerobic organisms, Lactobacillus sp., Candida sp., Gardnerellauaginalis and Neisseria gonorrhoeae counts in micro-organisms per ml. A comparison of the number and types of organisms isolated from vaginal cultures obtained initially and after 6 months of use demonstrated no statistically significant differences in colony counts between CVR and OC users. The results of this study suggest that the use of the CVR is not associated with a greater growth of pathogens than isoral administration of a progestin and estrogen combination (24).

CONCLUSION

The shell ring with an outer diameter of 58 mm and thickness of 9 to 9.5 mm, releasing LNG (280 jg per day) and estradiol (180pg per day), which is self-administered removed for 1 and used for 3continuous weeks and thenrtes o e th w wek s aocied with acceptable rates of break week, isassociated through bleeding and spotting, almost complete inhibi tion of ovulation, and acceptable rates of withdrawal bleeding upon ring removal, and it may be re-used for at least 6 months. These rings are as effective as, and have continuation rates at least equal to, oral contraceptives. Additionally, since headache and nausea are less common w!ih ring use, this method of contraception may be preferred by women who complain of these or other symptoms while taking the oral steroid. The ring produces few, if any, of the metabolic effects that are associated with oral estrogen administration, such as an increase in angiotensinogen and steroid binding globulins, and a decrease in antithrombin Ill. Thus, the side effects of hypertension and thrombosis, which can occasionally occur with oral contraceptives, would most likely not be increased with CVR usage. Studies from some, but not all, centers indicate that women wearing vaginal rings have a decrease in total cholesterol, LDL cholesterol, and HDL cholesterol. The changes in the commonly used "risk ratios" (total cholesterol/HDL or LDL/HDL) are small in most women and the values usual ly do not approach those found in normal male or femaie patients with cardiovascular disease. The relevance of these observations is currently being investigated. A concern that the ring would not be used by women in developing countries has not materialized. About 10% of women who were offered the ring as a new method without having received any previous information re garding the possible advantages of the method demon strated that they would choose the method and use the

TEST

PRETREATMENT

TREATMENT 2 CYCLES 134 38 32 93 4.38

A%* -20t -25t -18 -13t NC

167 Cholesterol 51 Triglycerides 39 HDL-C 118 LDL-C Cholesterol4.44 HDL-C * Percentage change from pretreatment t Significant changes

(Adapted from Robertson DN, Alvarez F,Sivin I et al: Lipoprotein patterns in women in Santo Domingo using alevonorgestrel/estradiol contraceptive ring. Contraception 24:469, 1981).

rings with relatively high continuation rates. Thus, this

new method of steroid contraception is likely to be an

effective alternative for women who cannot or will not use

the other currently available contraceptive methods.

Table 7. Average values of selected lipids (mg/dl) in women using 50 mm LNG -t E2 CVRs.

I0

9

REFERENCES

1. Ahren T, Victor A, Lithell H et al: Comparison of the metabolic effects of two hormonal contraceptive methods: An oral formulation and a vaginal ring. Contraception 24:415, 1981. 2. Ahren T, Lithell H, Victor A et al: Comparison of the metabolic effects of two hormonal contraceptive methods: An oral formulation and a vaginal ring. 11. Serum lipoproteins and apolipoproteins. Contraception 24:451, 1981. 3 3. Akinla 0, Lahteenmaki P, Jackanicz T: Inlravaginal contraception with a synthetic progestin, R2323. Contraception 14:671, 1976. 4. Benagiano G: Sumnary of WHO program. In Gabelnick HL (ed): Drug Delivery Systems. Washington DC, USDHEW Pub] No (NIH) 77-1238, US Department of Health, Education and Welfare, 1977. 5. Brenner PF, Mishell DR Jr, Stanczyk FZ et al: Serum levels of d-norgestrel luteinizing hormone, follicle-stimulating hormone, estradiol and progesterone in women during and following ingestion of combination oral contraceptives containing dl-norgestrel. Am J Obstet Gynecol 129:133, 1977. 6. Crank J, Park GS: Methods of measurement. In Crank J, Park GS (eds): Diffusion in Polymers. New York, Academic Press, 1968. 7. Dzuik PJ, Cook B: Passage of steroids through silicone rubber. Endocrinology 78:208, 1966. 8. Faundes A, Hardy E, Reyes Q et al: Acceptabilityandthe contraceptive vaginal ring by rural of urban population in two Latin American countries. Contraception 24:393, 1981. 9. Granger LR, Roy S, Mishell DR Jr: Changes in unbound sex steroids and sex hormone binding globulins-binding capacity during oral and Ainl Obstet gestogen administration. vaginal pro-

J gynecol 144:578, 1982. 10. Green IC, Howell SL, ElSeifi S et al: Binding of H-progesterone by isolated rat islets of Langerhans. Diabetologia 15:349, 1978. 11. Henzl MR, Mishell DR Jr, Velasquez JC et al: Basic studies for prolonged progestogen administration by vaginal devices. Am J Obstet Gynecol 117:101, 1973. 10

12. Jackanicz TM: Levonorgestrel and estradiol release from an improved contraceptive vaginal ring. Contraception 24:323, 1981. 13. Johansson EDB, Luukkainen T, Var tiainen E, Victor A: The effect of progestin R2323 released from vaginal rings on ovarian function. Contraception 12:229, 1975. 14. Mishell DR Jr, Talas M, Parlow AF et al: Contraception by means of a Silastic vaginal ring impregnated with medroxyprogesterone acetate. Am J Obstet Gynecol 07:100, 1970. 15. Mishell DR Jr, Lumkin ME: Contraceptive effect of varying dosages of progestogen in Silastic vaginal rings. Fertil Steril 21:99, 1970. 16. Mishell DR Jr, Lumkin M, Stone S: Inhibition of ovulation with cyclic use of progestogen-impregnated intravaginal devices. Am J Obstet Gynecol 113:927,

1972.

17. Mishell DR Jr, Lumkin M, Jackanicz T: Initial clinical studies of intravaginal rings containing norethindrone and nor gestrel. Contraception 12:253, 1975. 18. Mishell DR Jr, Roy S, Moore DF, Brenner PF, Page MA: Clinical perfor. mances and endocrine profiles with conIraceptive vaginal rings containing d-norgestrel. Contraception 16:625, 1977. 19. Mishell DR Jr, Moore DE, Roy Set al: Clinical performance of endocrine profiles with contraceptive vaginal rings containing a combination of estradiol and d-norgestrel. Am J Obstet Gynecol 130:55, 1978. 20. Robertson DN, Alvarez F, Sivin I et al: Lipoprolein patterns in women in Santo Domingo using a levonorgestrel/ estradiol contraceptive ring. Contracep tion 24:469, 1981.

23. Roy S, Krauss RM, Mishell DR Jr et al: The effect on lipids and lipoproteins of a contraceptive vaginal ring containing levonorgestrel and estradiol. Contracep tion 24:429, 1981. 24. Roy S, Wilkins J, Mishell DR Jr: The effect of a contraceptive vaginal ring and contraceptives on the vaginal flora. Con traception 24:481, 1981. 25. Roy S: Personal communication, 1983. 26. Selmanowitz VJ, Orentreich N: Medicalgadflid s J r atoh Medical grade fluid silicone. J Dermatol

Surg Oncol 3:597, 1977.

27. Sivin I, Mishell DR Jr,Victor A et al: A multicenter study of levonorgestrel estradiol contraceptive vaginal rings. I. Use effectiveness. An international corn parative trial. Contraception 24:341, 1981. 28. Sivin I, Mishell DR Jr, Victor A et al: A multicenter study of levonorgestrel estradiol contraceptive vaginal rings. II. Subjective and objective measures of effects. An international comparative trial. Contraception 24:359, 1981. 29. Sivin 1, Mishell DR Jr, Victor A et al: A multicenter study of levonorgestrel estradiol contraceptive vaginal rings. II1. Menstrual patterns. An international comparative trial. Contraception 24:377, 1981. 30. Victor A, Edqvist LE, Lindberg P et al: Peripheral plasma levels of d-norges trel in women after oral administration of d-norgestrel and when using intravaginal rings impregnated with di-norgestrel.

Contraception 12:261, 1975.

31. Victor A Jla 3.VtoA, Johansson EDB: Plasma levels of d-norgestrel and ovarian function in women using intravaginal rings im pregnated with di-norgestrel for several cycles. Contraception 14:215, 1976.

32. Victor A, Johansson EDB: Contra

21 Roy S,Mishell DR Jr, Gray C et a]: ceptive rings: Set-admniiistered treat ment governed by bleeding. Contracep Comparison of metabolic and clinical tion 16:137, 1977. effects of four oral contraceptive formu lations and a contraceptive vaginal ring. Am J Obstet Cynecol 136:920, 1980. 22. Roy S,Mishell DR Jr: Contraceptive vaginal rings: Mechanisms of action and historical development. In Hafez ESE, Van Os WAA (eds): Biodegradables and Delivery Systems for Contraception, pp 163-174. Lancaster, England, MTP Press, Limited, 1980. 33. Victor A, Nash HA, Jackanicz RM et al: Collagen bands: A new vaginal delivery system for contraceptive ster oids. Contraception 16:125, 1977. 34. Wynn V, Godsland I, Niththyanan than R et al: Comparison of effects of different combined oral contraceptive formulations on carbohydrate and lipid metabolism. Lancet i:1045, 1979.

July 1983, Volume 2,Number 5 Northwestern University Suite 1525 875 North Michigan Avenue Chicago, Illinois 60611 Editor: Gerald I. Zatuchni, M.D., M.Sc.

Managing Editor: Kelley Osborn

p r

RESEARCH FRONTIERS IN FERTILITY REGULATION

CURRENT STATUS OF ANTAGONISTIC ANALOGS OF LH-RH

AS A CONTRACEPTIVE METHOD IN THE FEMALE

Andrew V. Schally, Ph.D., D.Sc.H.C., M.D.H.C.

Section of Experimental Medicine, Department of Medicine,

Tulane University School of Medicine,

and Endocrine and Polypeptide Laboratories,

Veterans Administration Medical Center, New Orleans, Louisiana

We are fast approaching an era in which "zero population growth" will be a vital necessity rather than merely a desirable alternative. Uncontrolled population growth in many parts of manypars oftheworl the world ha conribted to gave has contributed o grave economic, political, and social problems. faster rate the poorer, less developed nations have a Ironically, of poorr, ess eveopednatonshavea fsterrat of population extent. Reducing the millionscontrol it to a increase and are unable to of unplanned significant ainiunantexe .Reacis wthdwde wiioud olvanyd economic and health problems and would enable developing countries, particularly, to improve their standard of living (54). This need for regulating human fertility in order to slow the rate of population increase has been recognized for decades by various sociologists, but only in recent years, and to a lesser degree, by political leaders and governments. Presently available methods of contraception are either inadequate, inconvenient, or unacceptable to some groups and individuals (79). Better control of human fertility will require the development and testing of new methods of contraception that will be effective and, at the same time, will be safer, simpler and more convenient than the methods now available (79). This task can be achieved through a greater commitment to contraceptive research. A series of complex processes is involved in human reproduction. In the human female, a delicate qualitative and quantitative interplay between the hypothalamus, pituitary, and ovary, and luteinizing hormone (LH), follicle-stimulating hormone (FSH), and ovarian steroids is essential for follicular maturation, ovulation, implantation, nidation, and maintenance of gestation (70). Development of contraceptive methods has been and can continue to be based on interference with some of these processes. Many agents, including the hormones themselves, depending on the time they are administered,

interfere with these mechanisms and thus produce antifertility effects. Among several contraceptive approaches being con sd r d a d d v l p d i n a e n a ao s o h sidered and developed controlling secretion of both the is one based on analogs of LH hypothalamic hormone and FSH from the anterior pituitary gland (Figure1). anis fromch piitay gln igureti). This approach was ae made possible by the isolation, determination of structure, and synthesis of this hor mone in 1971 (55, 56, 69, 71, 75, 77). The hypothalamic hormone is called the LH- and FSH-releasing hormone, abbreviated as LH-RH/FSH-RH, or simply gonadotropin releasing hormone (Gn-RH) (70, 73). While LH-RH is also accepted as the main FSH-releasing hormone, for reasons of convenience and historical continuity the abbreviation LH-RH is generally used for naming its analogs. LH-RH is the main link between the brain and the pituitary so far as reproductive function is concerned (Figure2). Much work indicates that this link can be disrupted by various means. Consequently, approaches based on the use of suitable LH-RH analogs should, in the foreseeable future, lead to the development of a method of contraception at the central level that might be easy to apply and free of undesirable side effects (Figure2). Some selected background information, as well as the very latest findings on the topic of antagonistic analogs of LH-RH, will be reviewed here.

THEORETICAL CONSIDERATIONS FOR

ANTAONNS FOR

ANTAGONISTS OF LH-RH

The design of modified structures that might compete with a biologically active compound for the same receptor sites, and yet exhibit little intrinsic activity, is a classical concept that has been used to develop a number of

@ Copyright PARFR 1983

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Figure 1. The molecular structure of luteinizing hormone-releasing hormone (LH-RH). drugs. For example, certain micro-organisms require p-aminobenzoic acid to form folic acid. Sulfanilamide, a structural analog of p-aminobenzoate, blocks folic acid synthesis by substrate competition, and is thus an effective inhibitor of growth of susceptible bacteria (41). Sulfanilamide is used for treatment of various infections. These findings on sulfanilamide were a milestone in the history of chemotherapy and initiated a new era in medicine (41). Another classical example of an antago nist is 5-fluorouracil, a pyrimidine antirnetabolite used for cancer chemotherapy. competitive inhibition occurs at the substrate binding or receptor site, the receptor being defined as a cellular transducer of hormone action that binds the in a reversible interaction. Receptors for peptide hormones are found in the cell membrane. The chemical structure and stereochemistry of an inhibitory analog of a peptide must resemble that of the substrate or the hormone.

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success in the peptide field. When we proposed the development of inhibitory analogs of LH-RH in 1971 (74), the concept of a peptide endowed with anti-LH-RH activity was based purely on theoretical considerations. Knowledge of the structural features that constitute the active site in a peptide is the primary step for the synthetic development of specific antagonists. We cor rectly forecast that replacement or deletion of some

2

LH-RH RECEPTOR INTERACTION WITH

LH-RH ANTAGONISTS

Irelatively

1

F[L-H1 R- -7]

gcertain TTARY

Competitive LH-RH antagonists appear to be ideal candidates for the development of a new contraceptive method, since they should disrupt the reproductive cycle safely.

RELATIONSHIP BETWEEN STRUCTURE AND BIOLOGICAL ACTIVITY OF LH-RH Various studies with many LH-RH analogs indicate that amino acids can be replaced in the LH-RH molecule without a major loss of activity (70, 73). It appears that amino acids in positions Iand 4 to 10 may be involved only in binding to the receptors and/or in exerting conformational effects. However, histidine and tryptoph, are likely to exert a functional effect in addition to providing receptor-binding capacity, since simple substitutions or deletions in posi tions 2 or 3 greatly decrease or abolish LH-RH activity (70). Indeed, the His-Trp sequence appears to constitute an "active center" in LH-RH. Thus, the imidazole group of histidine in position 2 possesses features, such as acid-based character and hydrogen bonding capacity, that render it necessary for expression of activity; and replacement or deletion of histidine drastically reduces agonist activity. Similarly, the deletion or replacement of tryptophan in position 3, or even a change in its configuration, also results in nearly complete loss of activity. This crucial role of Trp may be linked in some way to the electron-donating capacity of the indole nucleus of tryptophan. The functional character of histidine and L-tryptophan in positions 2 and 3 of LH-RH has been further confirmed by synthesis of many inhibi tors of LH-RH with changes in these positions (see section below). It is interesting that while most alter ations in the 2 and/or 3 position(s) of LH-RH destroy the gonadotropin-releasing effect, some of these modifica tions may preserve or even increase the binding affinity of the analog to the receptors. In the synthesis of LH-RH antagonists, virtually all investigative groups have taken advantage of the informa tion gained from work with the superactive agonists that replacement of glycine in the 6 position of LH-RH by D-Ala, D-Leu, D-Phe, D-Trp, or other D-amino acids leads to a major increase in biological activity (21, 58). This phenomenon is attributed to better binding confor mation than in LH-RH and is also indicated by empirical energy calculations ofMomany (57). The activities of the D-6-amino acid analogs appear to increase with the size of the side chain, which suggests that lipophilicity may also be a factor. The most active analog seems to be D-Trp 6-LH-RH, which also shows prolonged activity (28). Clinical results indicate that the potency of D-Trp 6-LHRH in human beings is 50 to 100 times greater than that of LH-RH.

CELL

MEMBRANE

EFFECTS AND EVENTS

INTHE CELL Figure 3. Highly simplified schematic representation of how LH-RH antagonists prevent LH-RH from binding to the receptors on the cell membrane and activating the events in the cell. amino acids in LH-RH might result in an analog possessing the structural features requisite for effective binding with the receptor, but lacking those necessary to elicit a physiological response, i.e., the stimulation of LH and FSH release (74). Such analogs could be competitive inhibitors of LH-RH; that is, they would be devoid of intrinsic LH-RH activity, but by competing for attachment to the receptor site with endogenous LH-RH, they might reduce LH and FSH secretion (Figure 3). Synthesis of non-competitive inhibitors of LH-RH, such as chlorambucil-l-LH-RH, which might react irreversibly with the pituitary receptor by alkylating it, has also been attempted (10), but these compounds appear to be less potent than the modern inhibitors. It must be emphasized that competitive LH-RH antagonists were and are intended primarily for female contra ception. It is important to realize that abolishing the basal secretion of LH and FSHI is not necessary. On the contrary, complete inhibition of LH and FSH release would be harmful, since it would interfere with ovarian steroidogenesis, which in turn might have undesirable effects on women. Instead, the task of LH-RH antagonists would be to block the midcycle surge of LH and FSH necessary for ovulation. To be more practical, a contraceptive polypeptide would probably have to be given by a route of administration other than parenteral. Results from clinical experiences with LH-RH and its stimulatory analogs indicate that intranasal administration may be the most efficacious. An inhibitor could also be conveniently administered every 1 to 3 months in the form of injectable biocompatible microcapsules of a biodegradable polymer that would allow its slow release.

3

The incorporation of ethylamide (EA) in the 10 position and a D-amino acid in the 6 position produces some analogs 30 to 100 times more potent than LH-RH, and they cause prolonged release of LH and FSH (22, 35). D-Leu 6,desGly 10-LH-RH EA and D-Ser-(Bu t )6-LH-RH EA (H766) were reported to be 60 and 100 times -nore potent, respectively, than LH-RH (51, 70). Thus, the changes in the 6 and 10 positions reinforce each other and produce superactive, long-acting analogs. These superactive analogs of LH-RH are active when administered intravenously, subcutaneously, intramuscularly, orally, intravaginally, intranasally, or even rectally (70, 73). DEVELOPMENT OF LH-RH ANTAGONISTS One of the early analogs, desHis 2-LH-RH, was reported to competitively antagonize LH-RH inmonolayer cultures of rat anterior pituitary cells when present in concentrations 10,000 times greater than LH-RH (85). However, in our hands, desHis 2-LH-RH did not show significant antiLH-RH activity in various in vivo tests (76). We tried to increase the weak antagonist activity of this analog by adding superactive modifications in positions 10 and 6 of the LH-RH sequence. We synthesized desHis2 ,desGly 0 LH-RH EA and showed that in ovariectomized, steroidtreated rats, it inhibited LH-RH-induced LH release (21, 89). This was the first peptide that significantly reduced LH secretion in response to LH-RH in vivo (89). Tryptophan in position 3 could also be replaced to give peptides with some inhibitory activity, such as Leu 3-LHRH (70). A whole series of potential antagonists was then synthesized and assayed in immature male rats. This assay permitted us to accurately compare the effectiveness of LH-RH antagonists by measuring the inhibition of response to LH-RH. Among the analogs tested, desHis,Leu,desGly -LH-RH EA; desHis,D-Ala 6, tested, desly s2,O-LH-RH EA;desisD-Aa-LH-R desHis 2 , d desGly'-LH-RHEA;desHiS 2,D-Ala 6-LH-RH and desHiS2, Leu 3,D-Ala 6,desGly 0 -LH-RH EA caused a significant inhibition of LH-RH-induced release of LH and FSH. However, desHis 2,D-Ala 6,desGly 10-LH-RH EA and desHis 2,Leu 3 ,D-Ala 6,desGly 0 -LH-RH EA also showed some intrinsic LH- and FSH-releasing activities (90). It appeared that, for a given antagonist, inhibitory effect was improved by incorporating either the D-amino acid in the 6 position, or a C-terminal ethylamide group. However, incorporation of both these modifications within the same analog often increased the inherent agonist activity (90). The substitution of D-leucine or D-phenylalanine in position 6 further increased antagonist activity. For instance, in immature male rats, desHis 2,D-Phe 6-LH-RH caused inhibition of response to LH-RH for 4 hours (70). Another important discovery was made by Rees and associates, that replacement of His in position 2 by DPhe, rather than its deletion, produces a better antagonist 4

(63). D-Phe 2-LH-RH was a far more effective inhibitor than desHis2-LH-RH. D-Phe 2,D-Leu 6-LH-RH and D Phe 2,D-Phe 6-LH-RH inhibited 50% of LH release induced by LH-RH at a molar ratio (MRwo) of 150 and 25, respectively. In general, the D-Phe 2-analogs were about three times more active than the corresponding desHis 2 analogs in vitro and in uiuo (70). D-Phe 2,D-Phe 6-LH-RH produced significant inhibition of LH and FSH for 6 to 8 hours after injection in immature male rats. Subsequently, the assay based on blockade of ovulation in4-day cycling rats became the most reliable method for testing antago nists (20, 30). At first, multiple doses of antagonist were given, since their duration of action was not sufficient to produce complete inhibition of the LH surge that takes place over an 8-hour period in the rat. Analogs such as D-Phe 2 ,D-Ala 6-LH-RH and D-Phe 2,D-Leu 6-LH-RH were able to block ovulation and the preovulatory gonado tropin surge in cycling rats when multiple doses of 12 mg/kg of body weight were given during the afternoon proestrus (20, 30). In 4-day cycling rats, D-Phe 2,D-Leu 6LH-RH caused 83% blockade of c',ulation after three injections of 2 mg on the proestrous day (30). D-Phe 2,DPhe6 -LH-RH was effective in single doses of about 6 mg/kg in the rat. The antagonist D-Phe 2,D-Phe6 ,Az Gly I-LH-RH containing an Aza-Glycine residue in position 10 was reported to block ovulation induced by LH-RH in androgen-sterilized constant estrus rats (33). Attempts were then made by modifying position 3 to synthesize peptides with lower inherent LH-RH activity thatretained thedesirableinhibitory properties ofD-Phe2 D-Phe 6-LH-RH. D-Phe2 ,Phe 3,D-Phe6-LH-RH, administer ed in a dose of 1.5 mg at noon on the proestrous day, caused a 95% reduction of preovulatory LH surge (30). Three subcutaneous injections of mg each of D-Phe 2, Thesuctnosijtosof1meahfD-e, Phe 3,D-Phe 6-LH-RH into proestrous rats at noon, 2:30 pm, and 5 pm completely suppressed spontaneous ovulation (30). Later, we determined that a single injec tion of 1.5 mg of this peptide at noon was sufficient to block spontaneous ovulation completely (70). The replacement of Trp by D-Trp in position 3 appeared to increase the potency of inhibitory peptides significantly (29). In tests for inhibition of ovulation, D-Phe 2,D Trp3 ,D-Phe 6-LH-RH (Figure 4) was about twice as effec tive as D-Phe 2,Phe3 ,D-Phe 6-LH-RH and longer acting (29). D-Phe 2,D-Trp 3,D-Phe 6-LH-RH was the first inhibi tory analog found to be active in human beings (36). Other replacements of positions 3 and 6 of the LH-RH sequence also produced potent inhibitory analogs. DPhe 2,Pro3 ,D-Trp 6-LH-RH, infused by a minipump at the rate of 375 pg/day for 4 days to cycling rats, inhibited ovulation (49). An improvement in antagonistic activity resulted from the replacement of L-pyroGlu by the D-pyroGlu group (66). Another approach that has been used for increasing

p-GLU-D-PHE-D-TRP-SER-TYR-D-PHE-LEU-ARG-PRO-GLY-NH

2

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2

3

4

5

6

7

8

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D-p-C1-Phe 2,D-Phe6 -LH-RH was also very active (26, he 34). When D-Ala was incorporated into one of 1 more active sequences in position 10 to give [Ac-D-Phel,D-p Cl-Phe2 ,D-Trp 3,6,D-AlaOI]-LH-RH, another two-fold in crease in activity was found (26). [Ac-D-p-Cl-Phe1, 2,

Trp 3,D-Phe 6,D-Ala1°J-LH-RH was effective in blocking

D-PHE ,D-TRP D-PHE -LH-RH Figure 4. The molecular structure of an early [H-RH 3 2 antagonist, D-Phe ,D-Trp ,D-Phe6-LH-.RH, active in hu

ovulation in rats when given in 10-15 pg doses at 12 noon on proestrus (34). which occurred whenProlonged blockade of ovulation, 150/pg doses of this analog were given, was associated with a 90% reduction in pituitary binding sites for LH-RH (59, 72). A similar improvement in activity occurred in N-Ac-D-Trpl analogs containing D-Ala'0(34). N.Ac.D.Trp, 3 ,D-p.Cl.Phe2,D.Phe6.D.Ala'o was active at G-)se. ,A 10 pg in blocking ovulation inrats (26, 34, 72). Other groups used different structural substitutions in position 1. Spatola and Agarwal reported that Ac-Gly-l D-p-Cl-Phe 2,D-Trp 3,6-LH-RH inhibited ovulation in rats in doses of 25 pg/rat (81). Rivier and co-workers studied several analogs containing Ac-dehydro-Prol(A3Pro) substitution in position 1,com bined with D-p-Cl-Phe in position 2 (65). Ac-dehydro Pro-D-p-CI-Phe 2,D-Trp 3 '-LH-RH andAc-dehydro-Pro1 D-p-F-Phe2,D-Trp3,-LH-RH inhibited ovulation in doses of 7.5 pg and 5pg respectively. Rivier's group also made some analogs with /3-(2-Naphthyl-D-Ala) substitutions in positions 3 and 6 (65). Ac-dehydro-Pro-D-p-F-Phe./3 (2-Naphthyl)-D-Ala3 , 6-' H-RH was highly active at 2.5 pg/rat in the ovulation test. Both N-Acetyl-dehydro-Pro and /3-(2-Naphthyl-)D-Ala appeared to be good substitu tions. Bowers and Folkers' group synthesized various series of LH-RH antagonists with D-amino acid substitutions in positions 2,3,6 as well as 4,5,8 (9). Of these analogs, N-A c-Thr',D-Phe2,D-Trp 3 ,6,D-Ser'I,D-TyrS,D-Arg8 was active at doses of 12.5 pg/rat and showed no changes in conformation. It has recently been demonstrated in our laboratory that insertion of D-lysine, or better still, D-arginine, in the

man beings.

the inhibitory activity of antagonists has been the substitution of D-Lys for Gly in position 6, and the use of its E-,mino as apeptide branching or dimerization point (27, 80). These analogs with increased antiovulatory activity could be more effective as aresult of their ability to bind to two receptor sites simultaneously. Branched chain analog called the Y peptide [D-Phe 2,D-Trp3,N-(pyroGluD-Ph2. D-Trp-Ser-Tyr).D-Lys6]-LH-RH had greater inhibitory activity than D-Phe 2,D-Trp 3,D-Lys 6-LH-RH on a weight basis, despite its higher molecular weight. This increased activity was probably due to the presence of two N termini that could interact with two receptor sites simultaneously. However, apeptide with three N-termini had decreased activity, presumably because of steric

problems between chains.

Dimers of D-Phe 2,D-Trp 3,D-Lys6.LH-RH were also potent inhibitors, the best of the series being the isophthaloyl and succinoyl dimers (80, 82). One of the most active compounds in this series was the isophthaloyl dimer of D-pyroGlu,D-Phe 2 ,D-Trp 3,D-Lys6-LH-RH, which gave significant blockade of ovulation in the rat at adose of 0.5 mg (27, 80). This dimer was also found to be active in human beings (72). Some modifications in the peptide backbone, e.g., by introduction of the OJCH-CH3-S group, have also led to active antagonists, probably because of resistance to enzymatic degradation (82).

Subsequent work was continued on positions 1 and 2, particularly on the effects of various substituents on the benzene ring of the D-Phe residue, and it was found that the p-CI-peptides had improved inhibitory activities (14, 25,26, 34). Channabasavaiah and Stewart (14) reported that substitution of acylated D-amino acids, including D-Phe, in position 1led to good inhibitors. In our hands N-Ac-D-PheI,D-p-CI-Phe2,D-Trp3,D-Trp6-LH-RH produced good blockade of ovulation at a low dose of 62 pg when injected in propylene-glycol-saline solution and at 31 pg in corn oil (25, 26). Replacement of D-Phe by D-p-CI-Phe in position 1 improved the inhibitory activity about two-fold (26). p-FPhe- reptides were about equally effective. Ac-L)-Trpl,3,

position of LH-RH antagonists results in greater anti ovulatory activity as compared to that found in the corresponding D-Phe 6 or D-Trp 6 analogs (23). For instance, [Ac-D-p-CI-Phe' 2,D-Trp3,D-Arg 6,D-Ala'0 ].LHRH (Figure 5)exhibited antiovulatory activity at adose of 3pg in 40% propylene glycol/saline and at 750 ng in corn oil per rat (23, 72). This was the first competitive LH-RH antagonist with activity in the range below I pg (23, 72). In addition, this antagonist issoluble in saline and active when given orally (61). (N-Ac-D-P-CI-Phe1'

2 ,D-Trp 3 ,D-Arg 6 ,D-Alo 1

) -LH-RH

Figure 5. Structure of Arg6 antagonist.

5

The antiovulatory activity of various antagonists of LHRH isillustrated inTable 1. It can be seen that during the past decade at least a 1000-fold increase in inhibitory activity has been achieved, LH-RH Analog D-Phe 2.D-Leu 6 D-Phe-D-Phe3-D-Phe6 D-Phe2-D-Trp 3-D-Phe6 D.Phe-D-pCI-Phe 2_ D-Trp 3.D.Phe 6 Ac-D-Phe1-D-pC-Phe 2 ,D-Trp Ac-D-pCI-Phe1, 2,D-Trp 3 6 Ac'D-pCI-Phe1' 2,D-Trp 3, D-Phe 6-D-Ala' 0

Ac'D'p'CI'Phe1' 2D-Trp 3, D-LyS6,D-Ala 0

Dose* (mgi) 2.0 (x 3) 1.5 1.0

,6

Blockade of Ovulation 82 100 90 82 100 70 100

50

tests extensively. We used in vitro tests occasionally to guide early attempts to synthesize LH-RH antagonists, to obtain evidence for an effect on the pituitary gland, and to shed light on the mechanisms of action. More recently, various groups have carried out measurements of the binding of antagonists to receptors in order to obtain information for designing new antagonists. IN VITRO ASSAYS Since LH-RH and its analogs stimulate the release of LH and FSH from cultured rat pituitary cells (84, 86), methods have been developed to measure suppression of response to LH-RH by inhibitory analogs in this system. After 4 or 5 days in culture, cells were washed and incubated for 5 hours at 37°C in Dulbecco-modified Eagle's medium in the presence o- absence of 3 x 10- 9 M

LH-RH. Antagonistic LH-RH aialogs added 10 minutes

0.25 0.062 0.015 0.010

0.003

Ac-D-p-CI.Phe 1,2,D-Trp 3, D.Arg 6.D.Ala' 0

0.003

78

prior to LH-RH inhibited LH and FSH release (24, 52). In

our hands, in vitro activities cf some LH-RH antagonists

*Most analogs were injected in40(kpropylene glycol/saline. Modified from Schally AV, A:imura A, Coy DH: Vitamins and Hormones 38:257-323, 1980; de ]a Cruz A, Coy DH, Vilchez-Martinez

JA et al: Science 191:195.197, 1976; Coy DH, Nekola MV, Erchegyi J, Coy EJ et al: In Zatuchni GI, Shelton JD, Sciarra JJ (eds): LHRH Peptides as Female and Male Contraceptives. Philadelphia, Harper & Row, 1981, pp 37-45; Coy DH, Horvath A, Nekola MV et a]: Endo cr'inology 110:1445-1447, 1982.

were sometimes different from in vitropotencies (24, 52). However, Bowers and co-workers fc. md that for most of the antagonistic analogs, in vitro and in vivo potencies closely paralleled each other (9). STUDIES WITH PITUITARY

MEMBRANE RECEPTORS

The investigation of binding affinity of antagonists of LHRH with adenohypophysial plasma membranes should help in designing better inhibitory analogs. Various groups have studied the interaction of some early inhibitory analogs with pituitary receptors for LH-RH. 10 2 SponashowedthattheearlyantagonistdesHis desGly LH-RH EA competed with LH-RH for LH-RH binding sites on the pituitary plasma membrane (83). In our initial studies, rat pituitary homogenate was incubated with ([125l]LH-RH) in the presence of different concentrations of either cold LH-RH or D-Phe 2,DTrp3 ,D-Phe 6-LH-RH. but the antagonist displaced the sites with the tracer, LH-RH Lompeted for receptor t a ategreest did the sites tracer from receptor sites to agreater degree than did the

parent hormone (70, 72). Similarly, the displacement of [125]LH-RH by cold LH-RH in adenohypophysial plasma membrane preparations was also smaller than that lr n mrae perations was a produced by D-Phe 2 ,D-Trp 3,D-Phe 6 -LH-RH (70, 72). There is now excellent evidence that LH-RH and agonistic analogs bind to specific receptors in the anterior pituitary gland. These receptors are located on the surface of the gonadotrophs. Several groups worked out the optimized conditions for in vitro receptor binding studies (17, 43, 44, and 45,47,53). Both low (KA = 106 M -1) high (KA = 108-109 M-t ) affinity sites for LH-RH have been demonstrated in pituitary membrane preparations (12, 17, 53). Modifica tions in the 6 and 10 positions, which produce super

Table 1.Artiovulatory activity of various LH-RH antagonists in 4-day cycling rats.

In conclusion, although additional improvements in inhibitory activity are likely to be achieved, several recently synthesized antagonists are already potent enough to be practical for clinical tests. Plans have been made to subject these antagonists of LH-RH to systematic clinical trials as soon as necessary pharmacological and toxicological tests are completed.

BIOLOGICAL EVALUATION OF LH-RH ANTAGONISTS Bothin ivoandin

used by nearly all itr mehodswer Both in vivo and in vitro methods were usd b nerlyall

groups to test inhibitory activity of various investigative analogs of LH-RH made by synthetic peptide chemists (9, 29, 65, 70, 72). Although scien.tists in our laboratory pses considr2).Aleoexperciencin possessed considerable experience in in virlaorstdi in vitro studies with isolated pituitary tissue dating back more than two decades (67), we decided to base the evauation of antagonists on the results of in vivo tests. This was prompted by the realization that in order fer these antagonists to form the basis of an effective contra ception method, they would have to perform under in vivo conditions where various effects, such as enzymatic degradation in the blood stream, would have to be taken into account. A high in vitro activity of an antagonist might be of little relevance if it is rapidly degraded in the blood stream. Nevertheless, some groups used in vitro 6

active agonists, also increase binding affinity to LH-RH receptors (17). In the rat, significant variations in the LH-RH receptors occur under several physiological conditions, including estrus cycle and pregnancy (12, 18, 68). The affinity of modern antagonists for LH-RH receptors is as high as that of some of the most potent agonists, the equilibrium association constants (KA) being 4-6 x 109 M-1, i.e., 8 to 10 times higher than that of LH-RH (KA = 6.6 x 108 M- 1 ) (15, 17, 59). The postcastration rise in pituitary LH-RH receptors can be prevented by treatment with LH-RH antagonist N-AcetylAla ',D-p-CI-Phe2,D-Trp 3 ,6-LH-RH (16). When measured in the unprocessed homogenate, this reduction in receptors could be accounted for in part by the receptor occupied by the antagonist. The dissociation rate of this antagonist from pituitary membrane preparations was also four times slower than the dissociation rate of a potent agonist (46). The inhibition of ovulation by LHRH antagonist is also accompanied by a reduction in pituitary LH-RH binding sites (59). Conn and co-workers demonstrated recently that a typical antagonist, D-pyro-Glu-D-Phe 2 -D-Trp3,D-Lys 6LH-RH, can becnme an LH-RH agonist if it is cross-linked to form a dimer and conjugated to an antibody to D-Lys 6-

propylene glycol/saline solution or corn oil were injected subcutaneously, usually at 12 noon on the proestrus day. Control groups were given vehicle alone. On the follow ing morning, the oviducts of thc animals were inspected for ova under a dissecting microscope (20, 30, 88). The most convcnient way to express the potency was to compare the percentage of blockade of ovulation in response to a certain dose of inhibitory analog. Earlier studies in proestrous rats showed only a partial blockade of ovulation after repeated administration (6 times) of large doses of antagonist D-Phe2,D Ala6-LH-RH (20). It was found later that a single administration of better antagonists could reduce or depress the preovulatory surge of LH and FSH and block ovulation (30, 70, 72) (Figure 7). A single subcutaneous injection of 1.5 mg of D-Phe 2,Phe 3, D-Phe6-LI-I-RH given at noon on the proestrus day resulted in 95% reduction of preovulatory LH surge and 84% reduction of FSH surge (30). Modern antagonists such as N-Ac-D-p-Cl-Phel, 2,D-Trp3,D-Arg 6,D-Ala'0 -LHRH can block ovulation in rats in much smaller doses- 3 pg in propylene glycol/saline or 0.75 pg in oil (Table 1).

LH-RH (19). The stimulatory effect might be caused by receptor microaggregation, i.e., bringing two receptor

molecules within a critical distance.

-uen, 1D5 Diluent

I 10

(Group 1) LH-RH (Group 2)

-Phe -D-Phe -LH-RH * LH-RH (Group 4)

3 6

D-p2-pe3-D-Pne6-H-RH + Diluent (Group 3) D-ph,

2

o

·

IN VIVO EVALUATION OF LI--RH ANTAGONISTS

inhibition of LH and FSH release and blockade of ovulation in rats. In early studies, various in ulivo methods, for instance inhibition of LH-RH induced LH release in ovariectomized rats treated with steroids, were used to follow the anti-LH-RH activity of the LH-RH antagonists (89). Only the most significant studies will be discussed here. One of the i nost convenient assays consisted of blockade of LH and FSH release in immature male rats (88, 90). Animals were injected subcutaneously with antagonists or with vehicle alone. Two hundred nanograms of synthetic L I-RH or saline solution were injected subcutaneously at the same time or at different times thereafter. Blood was collected 30 minutes after LH-RH or saline administration. Active antagonist had the

'.

67/, 6

5

92.6% 0

, 94.4% z 2 E '. 30Conol

Time of

.

ability to reduce LH and FSH release. Typical results are illustrated in Figure 6. The anti-LH-RH activity was

calculated as a percentage of inhibition of gonadotropin release over a 1 to 6 hour period. However, the most meaningful data were based on blockade of ovulation in proestrous rats. Antiovulatory tests were performed with adult female rats weighing approximately 200 g. Daily vaginal smears were taken and only rats that showed at least two successive regular 4-day cycles were used. Inhibitory analogs dissolved in

0

30

60

120

i

88% 88%

240

$cinjection of LH-RH or Diluent (min

Figure 6. Time course in immature male rats of blockade of LH and FSH release in response to LH-RH by D-Phe2, Phe 3,D-Phe 6-LH-RH(500pg/rat)injectedsubcutaneously. The analog was injected at time 0. The rats were decapitated 30 minutes after administration of synthetic LH-RH (200 ng/rat) or diluent. The percentage blockade of response to LH-RH for 60 and 240 minutes is indicated. (From de la Cruz A, Coy DH, Vilchez-Martinez JA et al: Science 191:195, 1976).

7

100

Injection

+ 75

11

=: 50 -

\

A--

3 6 D-Phe 2 KJgsc.) (1, -Phe -D-Phe -LH-RH 500

e--o

Diluent ( 20 %Propylene Glycol / Saline)

EI

12

2

4

6

8 ( PM)I

10 and

12

2 Estrus IAM

4

6

Hours of Proestrus

(1.5 Figure 7. Effect of single subcutaneous administration of D-Phe 2 -Phe3 ,D3-Phe 6-I -RH1111mg) on the preovulatory, surge Vilcbez-Martinez JA et al: Science 191:195, 1976). of LH in proestrus rats. (From de la Cruz A, Coy 131-11,

The LH-RH antagonist Ac-D-p-CI-Phe 1 ,2,D-Trp 3 ,D-Arg6, D-Ala' 0 -LH-RH is also active when given orally (61). Administration b gavage of 2 mg antagonist at 2 pmn on proestrus day blocked the preovulatory surge of LH and ovulation (61). Ovulation was also delayed for at least one day in some animals given 1 or 0.5 mg antagonist orally (61). The demonstration that antagonists of LHRH can exert contraceptive effects when given orally enchances their therapeutic potential. Pharmacologic doses (1000 pg) of this LH-RH antagonist N-Ac-D-p-CI-Phe 1 '2,D.Trp3,D-Arg 6 ,D.AlaIO.LH-RH were also reported to inhibit ovulation induced by hCG in pregnant mare serum (PMS)-pretreated hypophysectomnized rats (60). Although these doses were about 300 times larger than those needed to block ovulation at the pituitary level in proestrus rats, these studies; suggest that some of the effects of LH-RH antagonists, in analogy to agonists, can be exerted in part at the ovarian level. Previous studies by Hseu and associates (48,50) support the idea that antagonists of LI--RH may exert some direct action on the gonads.

Disruption of the estrus cycle and mating behavior

of female rats when given indaily doses of 200pg. Cycles resumed 6 to 9 days after cessation of treatment. Another potent antagonist, Ac-dehydro-Pro1 ,D-p-CI,Phe 2 , . D-Trp3 6 -H.RH-(2), produced a maiked decrease in lordosis behavior when infused into the third ventricle (32). These results suggest that antagonist analogs of LH-RH reduce sexual receptivity in the female rat (32). D-Phe 2,D-Ala-LH-RH has been rigorously tested by Corbin and Beattie (7,20). They found that high doses (6 mg) of this peptide could prevent pregnancy when administered precoitally, during proestrus, to rats which were then subjected to mating (7, 20). Rivier and associates '64) also repo! ted recently that Ac-dehydroPro ',D-p-Cl-?he 2 ,D-Trp3 ,6,Me-LeU7-LH-RH administered daily on days 7 to 12 of pregnancy indoses of 1mg caused a decrease in plasma progesterone levels. Golden Hamsters. In golden hamsters, ovulation was moedfiutoblcwthniioraaogtantws mreaiiuts tobok7ihiniior0nlostani.a In cycling hamsters, repeated administration of D-Phe 2, D-L.eu 6 -LH-RH on the afternoon of proestrus decreased LH surge by 83% (70) but this suppressed ovulation by only 30%. The reason for this was discovered later, when it was shown that hamsters can ovulate fully with only about 10% of the gonadotropins normally released during proestrus (70). We also examined various early analogs for their ability to suppress LH-RH-induced ovulation in

in rats. Various investigators reported previously that larger doses of early antagonists of LH-RH could disrupt the estrus cycle in rats (20, 70). Recently, Rivier and associates (64) reported that Ac-dehydro-Pro 1 ,D-p-CI. Phe 2,D-Trp3 ' 6,MeLeu 7-LH-RH disrupted the estrus cycle 8

phenobarbital-blocked hamsters. Only D-Phe2 -D-Leu 6LH-RH blocked ovulation when itwas injected 60 minutes before 75 ng LH-RH. For reasons explained above, subsequent studies in hamsters were abandoned (70). Rabbits. LH-RH antagonist can also inhibit ovulation caused by mating orinducedbyL-Rinmaturefemale3 2 hen multiple large 3to D-Phe ,Phe , adinit,rd rabbits (62). WD-Ph6-L-RHwer doses of ties t hlfD-Phe6.LH-RH were administered 3 to 5 times at halfhour intervals beginning 30 mirnutes prior to mating, the rise in plasma LH was reduced, and three out of five treated rabbits showed partial or complete blockade of ovulation. Similar results were obtained with DPhe2,D-Trp 3,D-Phe 6-LH-RH. Corbin and Beattie were the first to report that 25 mg of D-Phe 2,D-Ala 6-LH-RH could block LH-RH-induced ovulation in rabbits (20). We showed that a peak in the plasma LH level and full ovulation, routinely induced in all does by intravenous injection of LH-RH, could be suppressed by pretreatment with D-Phe 2,Phe 3,D-Phe 6LH-RH (62). A STUDIES WITH

elevation of gonadotropins 3 to 4 days after castration. After discontinuation of treatment with the antagonist, gonadotropin levels rose to elevated levels observed in control animals (3). Acute intramuscular administration of I mg of this antagonist to ovariectomized rhesus monkeys decreased LH and FSH levels withir 2 to 4

reduced for at hours24 hours. gonadotropin levels were in response to least (4). The The rise in LH and FSH te ntl inca a st ratno f 150 rg LH as s

LH-RH was significantly inhibited administration of 150 /ig

ir.variectomized animals by pretreatment 18 hours o

before with 1 mg antagonist (5). In another study, daily

administration of 1-mg doses of the same antagonist to 7 normally cycling rhesus monkeys from days 10 to 14 of the cycle significantly delayed the preovulatory LH surge and ovulation in 5 animals (6). The length of the cycle was proportionately increased in these animals, ovulation occurring 6 to 10 days later. In the two remaining animals, LH and estrogen peaks occurred while they were receiving LH-RH antagonist, but ini both cases, estrogen ,alues were already ascending when treatment was started (6). One of these animals did not ovulate, as determined by laparoscopy, and did not form a visible corpus luteum (6). This and another study showed that a marked decrease in postovulatory LH levels induced by antagonist does not alter the length of the luteal phase (6). It was also demonstrated that 1-mg doses of N-Ac-D-Trpl, 3,D-p-CI-Phe 2,D-Phe 6,D-Ala 0 -LH-RH de creased the amplitude of LH-pulses in rhesus monkeys, while 2 mg of this antagonist abolished both the frequency and amplitude of LH-pulses. Other investigations indi cated that LH-RH antagonists can suppress positive feedback and potentiate negative feedback of estradiol benzoate on gonadotropin secretion in ovariectomized rhesus monkeys (1, 2). Animals that received N-Ac-DTrpl,3,D-p.Cl-Phe2,D-Phe6,D-Ala'0-LH-RH in doses of I H mg, or Ac-D-p-CI-Phel , 2,D-Trp 3,D-Arg 6,D-Ala ° -L -RH in doses of 0.2 mg, in addition to 40 pg of estradiol benzoate, showed a more pronounced and prolonged suppression of gonadotropin levels during the first 24 hours than animals treated with estradiol benzoate alone. Animals injected with the antagonist also showed a suppression of LH and FSH peaks typically observed 26 to 48 hours after estradiol benzoate administration (1, 2). The results of this study indicate that LH-RH is an essential prerequisite for the mechanism of positive feedback of estrogen on gonadotropin secretion. Work in cynomolgus monkeys (Macaca fascicularis) also showed, in analogy to results in rhesus monkeys, that antagonist N-Ac-D-Trp',3,D-p-Cl-Phe2,D-Phe6,D -

Alaio-LH-RH,given subcutaneously, in doses of Img, for 25 days from the first day of the menstrual cycle, suppressed ovulation (8). The animals remained anovu latory during administration of the antagonist. Four animals ovulated 7to 15 days following discontinuation of the drug and 1 monkey ovulated 125 days later. These

LH A

AGS ILH-RH

Various problems were encountered in initial studies with antagonistic analogs in primates until suitable models were found (70). Initial tests demonstrated that large doses of early LH-RH antagonists, D-Phe 2,D-Phe 6-LH-RH and D-Phe 2,D-Trp 3,D-Phe 6-LH-RH, inhibited responses to LH-RH in baboons, rhesus monkeys, marmosets, and chimpanzees (40, 42, 70). Chimpanzees respond to LHRH in amanner closely similar towomen. D-Phe 2,D-Trp 3, D-Phe 6-LH-RH, in doses of 35 mg, inhibited the LH response of chimpanzees given 10/pg of LH-RH without altering basal gonadotropin levels (40). Wilks and associates reported modification of preovulatory gonadotropin ;, estrogen peaks in cycling rhesus monkeys id treatcd with 300 mg doses of D-Phe 2,Pro,D-Phe 6-LH-RH (92). However, even at these large doses, the analog did not completely inhibit gonadotropin surges and luteinization (92). Recent investigations, particularly by Asch and his associates (1-6,8) provided proof that LH-RH antagonists, developed primarily on the basis of antiovulatory tests in rats, are active in female primates (1-8). In a series of studies, Asch and co-workers demonstrated inhibitory effects of modern LH-RH antagonists on gonadotropin production and ovulation in rhesus monkeys and cynomolgous monkeys (1-6, 8). In the first study, it was shown that daily administration of 1 mg of Ac-D-Trp',a,D-p-CI-Phe2,D-Phe6,D-Ala'°-LH-RH prevented the castration-induced rise of LH and FSH in female rhesus monkeys (3). Controls showed significant

9

studies in rhesus and cynomolgous monkeys showed

clearly that chronic administration of LH-RH antagonist

can abolish ovulation in primates (6,8).

These investigations also demonstrated that a nonhuman primate model can be used to test the efficacy of LH-RH antagonists as potential contraceptive agents in the human female. LH-RH antagonists were found to be potent, long-acting inhibitors of LH and FSH secretion in nonhuman primates, with potential clinical application in fertility control. CLINICAL TRIALS WITH ANTAGONISTS CLRL WITH AN N IT In the course of development of LH-RH antagonists, as inhibitory activity improved, we felt it would be of critical importance to show that these analogs were active in women. The demonstration that large doses of D-Phe2 ,D-Trp3,DThedemonstraiminishd thtargdosoD-PhesDpnse t LH-RH innormal men led us to investigate whether this inhibitory analog had the same effect innormal ovulatory women (36) (Figure8). Eight woman physicians aged 20 to 28 years, who were normally menstruating, cyclic, and ovulatory, volunteered for the investigation (11). Intra muscular administration of 90 mg of D-Phe 2,D-Trp 3,DPhe 6-LX-RH on days 12 to 14 significantly reduced LH and FSH response to 50 jug LH-RH given 3 or 6 hours later. Basal levels of gonadotropins were not affected. The menstrual cycle was disrupted in all eight women and ovulation did not occur (11). Six women experienced earlier menstrual bleeding and two had delayed menses

15 DAY I DAY 2

(11). This study demonstrated that inhibitory analogs of LH-RH are active inwomen and that the analogs can be considered for the development of new methods for

female contraception.

The next step was to investigate the influence of this inhibitory analog dlone on elevated gonadotropin levels in oophorectomized or postmenopausal patients. We found that the administration of 90 mg of D-Phe2,D-Trp 3,D-Phe 6 LH-RH to a45-year-old woman 12 years after oophorec tomy decreased LH values up to 8 hours after the injection. In another woman who had undergone oopho rectomy, resting FSH levels also decreased up to 24 hours after injection in response to the inhibitor. A simultaneous suppression of LH and FSH serum levels occurred in a 35-year-old woman with ovarian amenor rhea (38,39). It was subsequently determined that more modern antagonists were active in much smaller doses. The isophthaloyl dimer of D-p-Glu1 ,D-Phe2 ,D-Trp 3,DLys 6-LH-RH, administered in doses of 50 mg to climacteric women and to women with ovarian amenorrhea lowered high LH and FSH levels for a period of 6to 8 hours (72).

2 N-Ac-D-Phe ,D-p-C-Phe2,D-Trp3.6-LH-RH and N-Ac-D 10 mg and 5 mg respectively, lowered LH and FSH in climacteric women for more than 24 hours (72).

Both of these analogs were then tested in normal women with the aim of inhibiting ovulation. N-Ac-D-Phe,D-pCI-Phe 2,D-Trp 3,6-LH-RH blocked ovulatory LH peak and ovulation in 8out of 14 normal women when given on day 12 of the cycle in doses of 10 mg. Six of these women menstruated 12 to 20 days later and two experienced earlier uterine bleeding (72, 93). The other six women

DAY 3 -15

0

C/)

4

10

5.

-5

TIME LH-RH 25p g

4

0

h

L

2r

LH-RH 25 pg

LH-RH 25 Pg

LH-RH 2 5 pg

LH-RH 25 pg

Figure 8. Mean response in serum LH levels of 4 men to 25 /g LH-RH before (control) and after intramuscular administration of 90 mg D-Phe 2,D-Trp3,D-Phe6-LH-RH. Asterisks indicate values significantly (p < 0.01) different from the value at that time duringthe control period. (From Gonzalez-Barcena D, Kastin AJ, Coy DH et al: Lancet ii:997-998, 1977).

10

showed some attenuation of both LH and FSH surges but ovulated. All 14 patients showed normal ovulation and resumed regular menses in the next cycle. This proved that the effect of the antagonist are reversible (93). In another study, a still more powerful antagonist, N-Ac10 6 D-p-Cl-Phe 2 ,D-Trp3,D-Phe ,D-Ala -L-RHmature ed on day 12 in adose of 5 mg, blocked ovulation based on LH, FSH and progesterone levels in 6out of 9normal women. No breakthrough bleeding occurred. In some wome, period ws dlayd up to 10 ays women, the menstrual te mnstualperod u to10 days was delayed but in others, menstruation occurred within the normal limits of the cycle (72, 93). N-Ac-D-p-CI-Phel, 2,D-Trp 3,D-Phe 6,D-Ala'O-LH-RH was then tested in smaller doses in ten normally menstruating ovulatory women aged 20 to 30 years (94). Intramuscular administration of the antagonist in doses of 2 mg on day 12 of the menstrual cycle inhibited midcycle surge of LH and FSH and ovulation in 6out of 10 women (Figure9). Serum progesterone levels and urinary pregnanediol

SLHRH

values in these 6 women were consistently low, and corresponded to anovulatory cycles (94). These women menstruated 12 to 26 days after injectionof the antagonist. Two patients in whom the analog did not abolish the LH mid-cycle surge showed a short luteal phase and pre menstruation. An early decline inprogesterone levels suggested that luteolysis took place. Two women ovulated, but no pregnancies occurred. Endometrial biopbies revealed moderate proliferative endometrium in era ed o untoward o mes rnewhom o deat i blocked. No untoward women in whom ovulation waswere recorded, but the side effects of this antagonist injection was reported to be painful by all patients because of 40% propylene glycol/saline solution used as a carrier. All women resumed regular menses and ovula tion in the subsequent 2 to 4 cycles. No episodes of breakthrough bleeding occurred in treated and post control cycles (94). So far only one other group has reported clinical studies on LH-RH antagonist (13). A powerful LH-RH antagonist, Ac-dehydro-Pro1 ,D-p-F-Phe 2,D-Trp, 6-LH-RH, adminis

tered intravenously in doses of 80 pg/kg, lowered serum

ng/

0

ANALOG

CONTROL

levels of LH and FSH by 43% and 18% respectively within

4 hours in 5 hypergonadotropic women, and decreased

500400-

o--o TREATED

400 30020

-intramuscular

0.o oo 0

the pulses of both gonadotropins (13). Subcutaneous or injection of this antagonist suppressed LH levels by 39% and FSH by 13% to 27% at 6 to 8 hours,

gonadotropin levels returning to normal within 24 hours.

,0o0F SHgonadotropin

When this antagonist was given during an intravenous infusion of LH-RH (0.2 pg/min) at a time of maximal

stimulation, it reduced LH levels by 49%

o o00,

0

400300

,oo/ ° 0\

-'o"

and FSH by 13% within 2 hours. These studies confirmed our previous findings (11, 36, 38, 39, 93, 94) that LH-RH antagonists can suppress endogenous or LH-RH-induced gonadotropin release.

No data are available sorA on the clinical efficacy of the far roL2TrA

200100

E STRADIOLanaoit Pg/,,,t

400]

powerful antagonist N-Ac-D-p-CI-Phel,',D-Trp3,D-Arg 0

o.

6,

400

3 2001

1

0

0

D-Ala' -LH-RH, which is soluble in water or saline. Evaluation of effects of chronic administration of this antagonist and other powerful modern antagonists on

101

0 1

PROGESTERONE

Ag/mI

ovulatory patterns in women will have to await the completion of long-term toxicity studies. THE STATUS OF FEMALE CONTRACEPTION

1'

6

4

A

1

BASED ON LH-RH ANTAGONISTS

-10

,

,

-

-

-2

, ,

0 +2 4 DYO'CYCLE

6

+

I 0

T

+12

Results of our studies described above indicate that a single administration of LH-RH antagonists can abolish

the mid-cycle surge of LH and FSH and inhibit ovulation

in most women, or induce luteolysis in others. Con

Figure 9. Inhibitory effect of N-Ac-D-p-CI-Phe' 2,D-Trp3, D-Phe 6,D-Ala10-LH-RH on the midcycle peak of LH and

sequently, exerted through effect of LH-RH antagonists could be the contraceptive two possible mechanisms. These findings confirm the view that LH-RH antagonists can be used for the development of anew contraceptive method.

11

FSH in comparison with the control cycle in a normal woman (From Zarate A, Canales ES, Sthory I et al:

Contraception 24:315-320, 1981).

The use of antagonists of LH-RH may, however, be connected with some problems. The irregular bleeding after a single administration of the antagonists seems to be at present one of the inconveniences (93, 94). Itwill be necessary to administer the antagonists for several cycles or even a more prolonged period of time in order to evaluate the anovulatory action and chronic effects, as well as the morphological changes in the endometrium. Since the antagonists were designed for inhibition of LH and FSH secretion, they should not cause any transient stimulatory "flare-ups" and should lead to a better ovarian suppression than the agonists, which vpre originally intended for treatment cf infertility. uoppsed incienc ofendoetral ypeplaia rd Thus, the incidence of endometrial hyperplasa aid unopposed estrogen secretion is expected to be low after treatment with the antagonists. It is also hoped that antagonists will offer other advantages over the agonistic analogs of LHRH in fertility control. For example, itmay be possible to administer the antagonist from day 5 to day 25, as in the case of the oral contraceptive pills, or only a few times near mid-cycle, to inhibit ovulation, in contrast to daily administration of agonists. It would be desirable to preserve a normal menstrual cycle, if possible, during artagonist administration. Some minor problems that became apparent during the acute trials with the antagonists can be solved without major difficulties. The discomfort associated with the injection of 40% propylene glycol as vehicle could be easily eliminated by the use of suitable carriers such as gelatin-mannitol solution for the more lipophilic antagonists. In addition, the powerful antagonist N-Ac-D-p-CIPhe1' 2,D-Trp 3,D-Arg 6,D-A ia ° -LH-RH is soluble in water and saline solution. Evaluation of various routes of administration of these antagonists will also have to be carried out and suitable routes devised. Frequent parenteral administration would be inconvenient and, hence, impractical. Oral administration of LH-RH agonists in rats or humans required doses at least 1000 times larger than those causing a comparable elevation of gonadotropins by the parenteral route (31, 37, 70, 72, 73). The same appears to be true for the antagonists on the basis of oral administration tests in rats (61). Thus, in human beings, oral administration of LH-RH antagonists would probably have a very low degree of effectiveness, of the order of about 1/1000, as compared with the parenteral route. Hence, a practical route of administration for the antagonists appears to be intranasal. Intranasal application has been found to be a convenient and practical way of administering LH-RH and its agonists, H766 and DTrp 6-LH-RH (91), and the same should be possible for more lipophilic LH-RH antagonists. In addition to intranasal sprays, recent studies indicate that injectaule microcapsules with LH-RH agonists can be prepared from biodegradable biocompatible polymer poly (d,l. lactide-co-glycolide) for once-a-month administration (78, 87). These microcapsules with D-Trp 6-LH-RH or D 12

Nal(2) 6-LH-RH were designed for constant controlled release of the agonists over a30-day period. The efficacy of these release systems in rhesus monkeys and rats has been demonstrated (87). Similar or related formulations of polymers for microen capsulation of LH-RH antagonist are certain to be tried and developed. Since such microcapsules need to be administered only every 30 to 90 days, this method would further facilitate the development of contraceptive approaches based on LH-RH antagonists. Periodic intramuscular administration of microcapsules with the antagonistwouldavoidthepregnancies that wouldreullt antailue byavome pren follow ther regimens from the failure a method wouldto be other result

properly. Such some women also appropriate

for

poper Scmethodouldlso e women in developing countries. In case the analogs already developed should not have the efficacy required for clinical trials, success in this project may be ensured by synthesis of additional LH-RH antagonists by peptide chemists and subsequent testing by reproductive biologists and clinicians.

CONCLUSION

The feasibility of creating, by synthetic approaches, LHRH analogs that inhibit LH and FSH release and ovulation, which was forecast in 1971, has been clearly proved. LH-RH antagonists already have sufficient potency to permit extensive trials on blockade of ovulation innormal women. In these trials, the analogs could be injected intramuscularly in a special vehicle or as microcapsules for controlled release over a longer period. More convenient routes of administration, such as nasal spray, will also have to be investigated. It is probable that antagonists of LH-RH could form the basis of new contraceptive methods. These methods would be based on blockade of the ovulatory mid-cycle peak of LH by antagonists of LH-RH or on induction of luteolysis. A peptide contraceptive that is free of the side effects of antifertility steroids would be a welcome addition to presently available methods for the control of human fertility. ACKNOWLEDGMENT It is a pleasure to acknowledge the advice of Ricardo Asch, M.D., Manuel Neves eCastro, M.D., and especially Ana Maria Comaru-Schally, M.D., and the editorial assistance of Judy Gernsbacher in the preparation of this manuscript.

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1

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13

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4.HzmE htafnt aeigo

50. Jones PBC, Hsueh AJW: Direct effects of gonadotropin releasing hor mone and its antagonist upon ovarian functions stimulated by FSH, prolactin, and LH. Biol Reprod 24:747-759, 1981. 51. Konig W, Sandow J, Geiger R: Struc ture-function relationships of LH-RH/ FSH-RH. InWalter R and Meienhofer J (eds): Peptides: Chemistry Structure and Biology. Ann Arbor, Ann Arbor Science Pub, Inc, 1975, pp 883-888. 52. Labrie F, Savary M, Coy DH, Coy EJ, Schally AV: Inhibition of luteinizing hormone release by analogs of luteinizing hormone-releasing hormone (LHRH) in Vitro. Endocrinology 98:289-294, 1976. 53. Marian J, Conn PM: Subcellular localization of the receptor for gonado tropindreleasing hormone inpituitary and ovarian tissue. Endocrinology 112:104 112, 1983. 54. Mastroianni L: Symposium on con traceptive technology. Fed Proc, Fed Am Soc Exp Biol 37:2664-2665, 1978.

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43. Hazum E: Photoaffinity labeling of luteinizing hormone releasing hormone receptor Endocrinology rations. of rat pituitary membrane prepa109:1281-1283, 1981. 44. Hazum E: Some characteristics of GnRH receptors in rat-pituitary mem branes: Differences between an agonist and an antagonist. Mol Cell Endocr 23:275-281, 1981.

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55. Matsuo H, Arimura A, Nair RMG, Schally AV: Synthesis of the porcine LH and FSH releasing hormone by the solidphase method. Biochem Biophys Res Commun 45:822-827, 1971. 56. Matsuo H, Baba Y, Nair RMG, Arimura A, Schally AV: Structure of the porcine LH- and FSH-releasing hormone, Part 1. The proposed amino acid sequence. Biochem Biophys Res Commun 43:1334-1339, 1971. 57. Momany FA: Conformational energy analysis of the molecule luteinizing hormone-releasing hormone tetrapeptide and decapeptide analogues. JAm Chem Soc 98:2996-3000, 1976. 58. Monahan MW, Amoss MS, Anderson HA, Vale W: Synthetic analogs of the hypothalamic luteinizing hormone releasing factor with increased agonist or antagonist propertes. Biochemistry 12: 4616-4620, 1973. 59. Nekola MV, Ge L-J, Pedroza E, Erchegyi J, Coy DH, Schally AV: Luteinizing hormone-releasing hormone associated with reduced pituitary binding sites. Endocrinology (In press, 1983). 60. Nekola MV, Horvath A, Coy DH: Direct inhibition of ovulation by an antagonist of LHRH. Fed Proc 42:Abstract No 4015, March 1983.

61. NekolaMV, HorvathA, GeL-J, Coy

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77. SchallyAV, NairRMG,ReddingTW, Arimura A: Isolation of the luteinizing hormone and follicle-stimulating hor mone-releasing hormone from porcine hypothalami. J Biol Chem 246:7230 7236, 1971. 78. Schally AV, Redding TW, ComaruSchallyAM: Inhibition of prostate tumors by agonistic and antagonistic analogs of LH-RH. Proceedings of the Prostate LHcr Proce othe Prostae Cancer Project Workshop, Buffalo, New York, March 21-23, 1983.The Prostate (In press). 79. Sciarra JJ: The continuing need for contraceptive research. Fertil Steril 36:697-698, 1981. 80. SeprodiJ, Coy DH, Vilchez-Martinez JA, Pedroza E, Schally AV: Branched chain analogs of luteinizing hormone releasing hormone. J Med Chem 21:276 280, 1978. 81. Spatola AF, Agarwal NS: A highly potent antiovulatory LH-RH analogue with no. I-position side chain. Biochem Biophys Res Commun 97:1571-1574, 1980. 82. Spatola AF, Bettag AL, Agarwal NS, Saneii H, Vale WW, Bowers CY: The role of peptide backbone modifications in increasing biological stability of LH-RH analogs. In Zatuchni GI, Shelton JD, auhi(,SetnJD Sciarra JJ (eds): LHRH Peptides as Fe male and Male Contraceptives. Phila delphia, Harper & Row, 1981, pp 24-36. 83. SponaJ: LH-RH-recoptor interaction is inhibited by des-His', des-GlyO-LH RH-ethylamide. FEBS Lett 48:88-92, 1974. 84. Vale W, Grant G, Amoss M, Black well R, Guillemin R: Culture of enzy matically dispersed anterior pituitary cells: functional validation of a method. Endocrinology 91:562-572, 1972. 85. Vale W, Grant G, Rivier J, Monahan M, Amoss M, Blackwell R, Gurgus R, Guillemin R: Synthetic polypeptide antag onists of the hypothalamic luteinizing hormone releasing factor. Science 176: 933-934, 1972. 86. Vale W, Rivier C,Brown M, Rivier J: Pharmacology of thyrotropin releasing factor (TRF), luteinizing hormone releas ing factor (LRF), and somatostatin. In Porter JC (ed): Hypothalamic Peptide HormonesandPituitaryRegulation. New York, Plenum Press, 1977, pp 123-156.

DH, Schally AV: Suppression ofovulation in the rat by an orally active antagonist of luteinizing hormone-releasing hormone. Science 218: ;60-162, 1982. 62. Phelps CP, Coy DH, Schally AV, Sawyer CH: Blockade of LH release and ovulation in the rabbit with inhibitory analogues of LH-RH. Endocrinology 100:1526-1532, 1977. 63. Rees RWA, FoelITJ, ChaiS-Y, Grant N: Synthesis and biological activities of analogs of the luteinizing hormone-releasing hormone (LH-RH) modified in position 2. J Med Chem 17:1016-1019, 1974. 64. Rivier C, Rivier J, Vale W: Antireproductive effects of apotent GnRH antagonist in the female rat. Endocrinology 108:1425-1430, 1981. 65. Rivier, J, Rivier C,Perrin M, Porter J, Vale WW: GnRH analogs: Structureactivity relationships. In Zatuchni GI, Shelton JD, Sciarra JJ (eds): LHRH Peptides as Female and Male Contraceptives. Philadelphia, Harper & Row, 1981, pp 13-23.

Steri1 22:703-721, 1971.

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87. Vickery B, McRae G, Tallentire D: Disparate effects of daily versus continuous administration of a potent LHRH agonist on plasma LH levels in female rhesus monkeys. Fertil Steril 39:417418, 1983. 88. Vilchez-Martinez JA, Coy DH, Coy EJ, Arimura A, Schally AV: Comparison ofthe anti-LH/FSH-RH and anti-ovulatory activities of [D-Phe 2,D-Leu6]-LH-RH and [D-Phe2 ,D-Ala"I-LH-RH. Endocr Res Commun 3:231-241, 1976. 89. Vilchez-Martinez JA, Schally AV, CoyDH, CoyEJ, DebeljukL, ArimuraA: In uivo inhibition of LH-release by a synthetic antagonist of LH-releasing hormone (LH-RH). Endocrinology 95:213218, 1974.

90. Vilchez-Martinez JA, Schally AV, Coy DH, Coy EJ, Miller CM Ill, Arimura A: An in uivo assay for anti-LH-RH and anti-FSH-RH activity of inhibitory analogs of LH-RH. Endocrinology 96:1130-1134, 1975. 91. Wass JAH, Besser GM, Gomez-Pan A, Scanlon MF, Hall R, Kastin AJ, Coy DH, Schally AV: Comparison of longacting analogues of luteinizing hormonereleasing hormone in man. Clin Endocrinol 10:419-430, 1979. 92. Wilks JW, Folkers K, Humphries J, Bowers CY: Effect of [D-Phe 2,Pro3 ,DPhe 6]-luteinizing hormone releasing hor mone, an antagonist, on preovulatory gonadotropin secretion in the rhesus monkey. Biol Reprod 23:1-9, 1980.

93. Zarate A, Canales ES, Schally AV, Coy DH, Comaru-Schally AM: The use of LHRH agonists and antagonists as antifertility agents in the human female. In Zatuchni GI, Shelton JD, Sciarra JJ (eds): LHRH Peptides as Female and Male Contraceptives. Philadelphia, Har per & Row, 1981, pp 227-236. 94. Zarate A, Canales ES, Sthory I, Coy DH, Comaru-Schally AM, Schally AV: Anovulatory effect ofaLHRH antagonist in women. Contraception 24:315-320, 1981.

This publication was supported by the United States Agency for International Development (USAID). The contents do not necessarily reflect USAID policy.

December 1983, Volume 2,Number 6

Northwestern University 875 North Michigan Avenue

PQ(

Chicago, Illinois 60611

Editor: Gerald I. Zatuchni, M.D., M.Sc.

Managing Editor: Kelley Osborn

RESEARCH FRONTIERS IN FERTILITY REGULATION

CURRENT STATUS OF RESEARCH ON SPERM ANTIGENS: POTENTIAL APPLICATIONS AS CONTRACEPTIVE VACCINES

Erwin Goldberg, Ph.D. Professor Department of Biochemistry, Molecular Biology and Cell Biology

Northwestern University

Evanston, Illinois

The immunogenicity of spermatozoa manifests itself in a variety of clinical conditions. Autoimmune orchitis is the result of an immune response to either spermatozoa or constituents of the testes. Idiopathic infertility of both males and females is often associated with antibodies to spermatozoa. In addition, antisperm immunoglobulins are a consequence of vasectomy that may compromise restoration of fertility. These pathologic conditions are consistent with the observations that testes and spermatozoa are antigenic in both males and females. Analysis of the nature of the antigenic stimulus is the basis for treatment and prevention of disease, as well as for restoration of fertility. If these uncontrolled pathologic infertilities were understood, the underlying mechanism could be manipulated to produce controlled infertility, Indeed, the fertility in females of several species is reduced following immunization with sperm or testes extracts. A well-defined sperm antigen could be applicable in immunization of both sexes. Thus, it may be possible to exploit the antigenicity of spermatozoa in the development of a new contraceptive technology. The antigenicity of spermatozoa and testes has been known since the early 1900s; nevertheless, a precise description of the immune response to male reproductive tissues requires the isolation and characterization of specific antigens. There have been many attempts to identify the antigenic constituents of testes and spermatozoa, and to detect immune reactions and infertility following immunization with a variety of sperm "fractions" (27). More recently, there have been attempts to isolate and characterize specific sperm antigens (40). Considerable progress has been made in understanding the structure, localization, and antigenic properties of one enzyme, lactate dehydrogenase C 4 (LDH-C 4; LDH-X), which serves as a useful model for devJoping a contraceptive vaccine (63).

ANTIGENICITY OF TESTIS CONSTITUENTS The mammalian testis is a bifunctional organ specialized for hormone release as well as spermatogenesis. This latter function is a highly structured sequence of cell differentiation to produce the exquisitely specialized spermatozoon. This process takes place in the rigorously controlled environment of the germinal epithelium, which is sequestered from the general body circulation by the blood-testis barrier. This isolation of spermatogenesis from the circulatory system and the production of unique, specialized proteins required for the construction and function of a sperma tozoon underlies testicular antigenicity. The immune system is fully functional at the time spermatogenesis commences. Therefore, new protein species synthesized during spermatogenesis would be recognized as foreign. Generally, the blood-testis permeability barrier restricts exposure of these potential antigens to the immune system. The compartmentation of the germinal epithelium has been described by Fawcett (4). Specialized junctional complexes between adjacent Sertoli cells are the principal structural basis of the blood-testis permeability barrier. The epithelium is partitioned into a basal compartment occupied by the spermatogonia.The more advanced germ cells are contained in an adluminal compartment (4). Presumably, the Sertoli cell junctions and their special ized permeability properties lead to maintenance of a suitable environment for germ cell differentiation. From an immunologic standpoint, antigens of germ cells are prevented from reaching the blood and inducing an auto immune reaction (16). Similarly, circulating antibodies are blocked from access to the germinal epithelium by this same barrier. Disruption of the barrier can provoke antisperm antibodies as well as autoimmune orchitis.

@ Copyright PARFR 1983

AUTOIMMUNE DISEASE The pathogenesis of experimental allergic orchitis (EAO)

xpermenal The athgensisof llegicvasectomized

immunoA was reviewed revealed by Tungdetailed complexof EAO in pathology is recently by the (54). studies the guinea pig. Furthermore, there are examples of pig.curthrmor e, thdrfimmne resaple o ntuial

naturally occurring orchitis and of immune response to

system as a result of the surgical procedure. Auto antibody responses to sperm-specific antigens appear in animals of all speckcs studied. Also, post adieA pigs vasectomy ocitis op in raits and guinea pis

vasectomy orchitis develops in rabbits (1.Aprnlteatimn

(57). However, only 50% to 60% of the vasectomized subjects, regardless of species, develop such antibodies

epnei tan

testis-specific antigens in some vasectomized men (54). An interesting model of naturally occurring orchitis has been discovered in acolony of dark mink in which 20% to 30% of the males were infertile (56). Orchitis and/or spermatogenesis with testicular sperm antigen-antibody complexes were unequivocally demonstrated in many of these animals. Primary infertility was observed in some mink soon after puberty. These animals had low levels of antisperm antibodies. Aspermatogenesis, but rarely orchitis, was the main testicular abnormality. Tung observed that mink with primary infertility were defective in gonadotropin releasing hormone (GnRH) secretion, presumably due to abnormal hypothalamic function or its control mechanisms (55). Secondary infertility occurred in animals after aperiod of proven fertility. In these mink, there were significantly higher levels of antisperm antibodies, many testes with severe orchitis and/or aspermatogenesis, and presumptive evidence of immune complex deposition. The symptoms of secondary infertility increased during the breeding season. Ultrastructural studies revealed breakdown of tight junctional complexes between adjacent Sertoli cells (55). These findings suggest that the blood-testis barrier becomes defective during seasonal regression of the testis. Autoimmunization may lead to the development of orchitis, although the exact role of immune complexes in pathogenesis of secondary infertility remains to be defined, Obviously, precise analysis of the immune response and of immune pathogenesis in males would be greatly facilitated by definition of the aspermatogenesis antigen(s). Previous studies on isolation and characterization are cited by Tung (54). Teuscher and associates have isolated and partially characterized a polypeptide from guinea pig testis that induces EAO (53). This peptide, designated AP3, has an apparent molecular weight of 12,500 and induced severe EAO in 100% of the guinea pigs tested. Neither the subcellular location nor the functional role of AP3 is known at present. IMMUNOLOGIC CONSEQUENCES OF VASECTOMY Tung has summarized the immunopathologic complications of vasectomy and evidence of postvasectomy orchitis (54). Essentially, vasectomy creates a pool of spermatozoa that becomes accessible to the immune 2

(51). Apparently, the autoimmune response is strain dependent and controlled by asingle gene in the guinea pig model. Takami and associates demonstrated that in vasectomized rats, autoantibody production to sperm is not influenced by genes in the major histocompatibility complex or by genes on the Y chromosome (50). After vasectomy, some serum antibody reacts with cell surface antigens, while other antibody is directed to internal antigens of testicular cells and sperm (57). Presumably, the latter reflects release of sperm deg radation products. The cell surface antigens recognized postvasectomy include at least three major polypeptides, however the contribution of each to antigenicity remains unclear. Autoantibodies isolated from the sera of vasectomized guinea pigs react with three major sperm plasma membrane antigens of 69,000,42,000 and 20,000 daltons, respectively (51). These peptides are signif icantly larger than the AP3 antigen of EAO. Identification and characterization of the antigenic stimulus isessential andachieveran erstanigenic stis isessei to achieve an understanding of orchitis and postvasec tomy immunopathology. IMMUNOLOGIC INFERTILITY Serum antisperm antibodies have been implicated in cases of unexplained infertility. In fact, such observations have been the basis of attempts to manipulate the immune response to spermatozoa, both to alleviate undesired infertility and to develop acontraceptive. A variety of assays for spermagglutination, immobiliza tion, and cytotoxicity have been used in attempts to establish a quantitative relationship between infertility and antisperm antibodies. This work was recently reviewed by Menge (28). The incidence of antibodies detected by sperm agglutination and immobilization methods varies respectively from 5%to 30% and 2%to 16% for infertile women, and from 3%to 20% and3%to 7% for infertile men (31). Moghissi and Wallach also implicate sperm antibodies either in sera, reproductive tract fluids, or both in couples with persistent unexplained infertility (34). These authors caution, however, that many pub lished reports of an immunologic basis are inadequate in their evaluation of infertility. Reduced penetration of cervical mucus by sperm is associated with serum titers of antisperm antibodies in both sexes and also with immobilizing antibody activity in cervical mucus (31). The incidence of subsequent

pregnancy in 376 infertile couples was reduced significantly ifthe man or the woman had antisperm antibodies in serum or in genital tract secretions. Agglutinating antibodies in both sexes occurred about three times more frequently than immobilizing antibodies (31). There have been no reports to date ot i,.h-mobilizing antibody activity in the sera of fertile individuals (31). In general, infertility in human couples may be caused by immunologic reactions in which sperm penetration of cervical mucus is impeded, transport and viability in the female reproductive tract is decreased, penetration of the ovum by the sperm the fertilized development of is inhibited, or normal postfertilization ovum is impaired (31). Serious problems exist with most of the assay procedures used to detect antisperm antibo,'es in sera and reproductive tract fluids. These assays are complicated by Fc receptors on spermatozoa that cause a nonspecific binding of immunoglobulins in uitro (64). At the very least, both interpretation and quantitation of the various assays of spermagglutination are difficult, so there is great demand for improved methods. Wolf and associates have recently des. -ibed solid phase assays utilizing radiolabeled or enzyme-linked second antibody that may provide increased sensitivity and specificity to detect antisperm antibodies in infertile couples or vasectomized males (65). Little is known about the specific antigens underlying these immunologic infertilities. When human serum samples containing antisperm antibodies were analyzed by a radioimmunoassay (24), unique sperm antigens that elicit these antibodies could be identified. Thus, sera from four patients reacted with a protein of about 30,000 daltons. Sera from two patients reacted with a protein band having a molecular weight of about 60,000. Sera from an additional nine patients did not react with unique peptides of sperm.

Millette and Bellv6 immunized rabbits with type B sperma togonia, 82% to 88% pure, from the mouse, and detected antigenic membrane components on germ cells at all stages of differentiation, ranqing from primitive Type A spermatogonia to mature spermatozoa (32). Further more, certain antigenic components of the plasma membrane of early germ cells were partitioned selectively during spermiogenesis into that portion of the plasma membrane destined for the residual body. The complexity of the plasma membrane components of developing mammalian spermatogenic cells is described d ee n M ulinspeAm a to 30 poly by Millette and Moulding (33). At least 25 to 30 poly peptides were detected from pachytene primary sper matocytes and round spermatids, with no peptide unique to either stage. Kramer and Erickson described stage-specific protein synthesis patterns during spermatogenesis (21). Pachy tene spermatocytes were more active in protein synthesis than either early or late spermatids. Approximately 85% of the total extractable supernatant proteins were present in all three cell types. Twenty proteins were detected in both early and late spermatid fractions but not in other stages. Four proteins were found only in pachytene spermatocytes, three in spermatocytes and early sper matids, ten in early spermatids, and only one protein was found in late spermatids. Solubilized particulate proteins also showed stage specificity, although 75% to 80% were in all cell types (21). Protein synthetic patterns in mouse spermatocytes and spermatids were examined in cultures of seminiferous tubules or isolated germ cells (3). Soluble polypeptides synthesized by middle to late pachytene spermatocytes and round spermatids were resolved as about 250 radiolabeled spots, compared to only 100 spots from intermediate spermatids. There :s a drastic reduction in the number of newly synthesized polypeptides during spermiogenesis, with few new molecular species that are translated postmeiotically. Stage-specific synthesis of peptides is clearly reflected in gene expression during munne spermatogenesis (7, 48,

49). Pachytene spermatocytes and round spermatids

synthesized approximately peptides, with less synthesis equivalent numbers of poly in elongating spermatids and residual bodies (48). Stage specificity was detected in mNppltosbtitrsfeetvpouto mRNA populations, both in terms of selective production and selective utilization of mRNA (7). It appears that much of the protein synthesis in the elongating spermatid is directed by mRNAs that had been stored in round spermatids (49). ISOLATION OF SPECIFIC PROTEINS Of the many peptides synthesized during spermato

ANALYSIS OF SPERM ANTIGENS The antigenic complexity of the male gamete undoubtedly confuses the analysis of these clinically significant conditions. The same pathology can reflect a variety of antigens, perhaps to different extents and with variation amntiniphas to4). dIffet ietetsan, wthrv ,to among individuals (24). It is necessary, therefore, to consider the total immunogenic inventory of the male reproductive systemm As noted above, many of the proteins that are synthesized only during spermatogenesis represent potential autoantigens, since sexual maturation begins long after immunologic maturity. In addition to various testisspecific enzymes, plasma membrane components of developing germ cells could be antigenic.

3

genesis, not all are antigenic. In most cases, the functional or metabolic role of these sperm-specific proteins has not been 'lentified. Such identification would represent a major accomplishment in the study of autoantigenicity of testicular constituents by facilitating isolation procedures. Proteins that have been purified from test.s or spermatozoa include some of defined function, e.g., enzymes, and some that play an as yet undetermined structural role, e.g., plasma membrane constituents. Purification strategies for enzymic proteins can be relatively straightn forward, and readily yield homogeneous products. In general, measurements of enzyme activity monitor the isolation protocol, and the steps are relatively mild, particularly in the case of soluble proteins. The primary limiting factor is the obtaining of sufficient starting material to yield a useful amount of product for further has been isolated, analysis. Once ahomogeneous protein its auto- or isoantigenicity can be readily establishd. Thus, LDH-C,,, PGK-2, cytochrome c, and possibly acrosin and hyaluronidase are testis-specific and autoantigenic (10). Purification of testes or sperm peptide6 without known biological activity presents a much more formidable be employed problem. Immunochemical techniques may to monitor the isolation of antigenic peptides. The simplest experiment involves immunization with intact sperm and subsequent measurement of antibody binding to these cells. Lopo and Vacquier showed that rabbit antiserum to sperm of the sea urchin Strongyocentrotus purpuratus reacts with sperm from 28 species and from 7 phyla of the animal kingdom, including mammals (26). Antibody binding is not due to H-Y or Fc receptors or to cell surface tubulin. This report suggests the possible eistence of common antigenic determinants on the surface of all animal sperm (26) It would be exceedingly useful to have a species from which sperm are abundantly available, to isolate a determinant common to human spermatozoa. A number of investigators have immunized animals with sperm or testes extracts, or extracts of isolated populations of germ cells aL various stages of spermatogenesis. Antisera obtained by these procedures may be used to purify specific antigenic peptides, to localize them on the sperm surface, or to follow their appearance and distribution during spermatogenesis. In the case of the guinea pig, EAG induction has been used to follow the isolation of the autoantigens AP2 (52) and AP3 (53). AP2 is a soluble antigen of 9000 daltons M.W. This protein is releasc. by the in vitro-induced acrosome reaction ofguinea pig cauda epididymal sperm. Thus, this strategy defines an activity (EAO.) and subjects it to a purification protocol.

O'Rand and Porter used an autoantisera immunoad sorbent column and preparative gel electrophoresis to purify small quaintities of a rabbit sperm membrane autoantigen, designated RSA-1 (42). Subsequently, they described a procedure that greatly increased the yield of RSA-l and also uncovered RSA-2 (43). Testis membrane pellets were fractionated by an SDS-7% to 15% poly acrylamide gradient prep-disc gel column. The investi gators recovered RSA-1 in a yield sufficient to determine carbohydrate and amino acid content. From its average hydrophilicity, RSA-1 appears to be a relative nonpolar, asymmetric protein in the general category of fibrous or tropomyosin-like proteins (43). It is a sialoglycoprotein of 13,000 ± J200 daltons intrinsic to the plasma mem brane of spermatozoa and similar in several respects to the guinea pig testis autoantigens (4). However, neither AP3 (53), which is almost the same size as RSA-1, nor AP2 (52) appears to be a glycopeptide. Lee and co-workers reported that antisera from rabbits immunized with homologous epididymal sperm reacted with three major protein bands on SDS gels, with correspondir' - molecular weights of about 70,000, 14,000 :s, respectively (24). Whether one of and 13,0( is homologous to RSA-Iremains reptides these sma: , ie toebe det erm RSA-I vias localized on the pla-rnamembraneof sperma tozoa 3nd spermatogenic cells by fluorescein- and peroxidase-labeled antibodies. It appears first on the surface of pachytene spermatocytes and is present throughout spermatogenesis (42). On the mature sper matozoon, RSA-1 is concentrated in the postacrosomal and middle-piece regions, but may occur in isolated iiatches over the acrosome and tail regions. It would be interesting to demonstrate this antigen in other species. Romrell and associates identified rabbit sperm surface autoantigens that appeared during spermatogenesis (44). Two distinct subclasses of autoantigens were detected: an early class appearing on pachytene spermatocytes, but not present on spermatozoa, and a late class first appearing on spermatids. The authors suggest that each stage of spermatogenesis contains a few so-called differ entiation antigens whose function is of limited duration. Specific surface proteins may appear during formation of Sertoli-germ cell junctions and disappear during dis ruption of the junction occurring with sperm release. Autoantibodies to guinea piq sperm autoantigens have been localized in plasma membrane over the entire sperm head, acrosomal contents, fibrous sheath, and outer dense fibers of the tail filament and the inner acrosomalmembraneofsomeacrosome-reactedsperma tozoa (58). All these studies employed polyclonal sera against

4

complex immunogens. Thus, many antigens are detected, and the concentration of the specific antibodies and the affinities are variable. Restriction of the assay to a single antigen will greatly facilitate purification. Monoclonal antibodies add both sensitivity and specificity to probe the antigenic peptides of spermatozoa.

MONOCLONAL ANTIBODY PROBES OF

SPERM ANTIGENS

Hybridoma technology allows the production of mono specific antibodies without the need for immunization with a purified antigen (20). Each clone from the fusion of a myeloma cell with a lymphocyte produces an antibody of unique specificity. Not only does this permit more precise discrimination in the analysis of sperm antigens, but such antibodies are also potentially useful in establishing a functional role for these peptides. Feuchrer and associates developed four monoclonal antibodies by hyperimmunization of male mice with murine sperm from the cauda epididymis (6). Each of the monoclonal antibodies bound to a specific region of the sperm surface. Three of these antibodies recognized topographically restricted antigenic determinants on the sperm head and also bound to sperm from rat, rabbit, hamster, and guinea pig. The fourth monoclonal antibody detected a determinant on the sperm tail, and appe .red to be specific. None of these antibodies reacted with ejaculated human sperm. The antigenic determinants were first detectable on sperm from the corpus epididymis, suggesting that maturation in the epididymis involved unmasking or modificatio,. of preexisting surface components, release of molecules from the sperm cytoplasm, orattachmentofnewcomponents to the sperm surface (6). The monoclonalan aibody that binds to the sperm tail also binds to epithelial cells of the epididymis (59). This antigen is, therefore, probably secreted by principal cells occupying a short segment of the distal caput epididymis. Similar results were obtained with antisera raised against purified epididymal-specific proteins from hamster and rabbit (35) and rat (5, 19, 22). Apparently, mammalian spermatozoa are coated with antigenic glycoproteins dtuiy epididymal transit (22). There is evidence to show, in the ram, a highly specific uptake of components from cauda epididymal fluid by testicular sperm (60). It shouldantibodies to dissect this process with mono clonal be possible to define more precisely the nature and and role of epididymal constituents in sperm maturation, Monoclonal antibodies also provide a powerful tool to unravel the role of sperm-specific proteins in fertilization, Alexander (1) has developed several monoclonal antibodies by immunization of mice with human spermatozoa.

Many of these are directed against evolutionarily con served antigens that are especially valuable, since the effect of a particular antibody on fertilization can be tested in species other than man. Some of Alexander's monoclonals react with fresh but not capacitated sperm, an indication that those antibodies are to antigens associated with the acrosome reaction. One of these is specific to an antigen in the 20,000 dalton range. At least two monoclonals are directed against sperm-specific surface antigens that may be glycoproteins ranging from approximately 18,000 to 40,000 daltons. Monoclonal antibodies prepared against mouse epidid ymal sperm by Schmell & associates indicated patterns of spatial differentiation of target antigens in localized regions of the sperm surface (46). Furthermore, the anti-tail binding monoclonal diltibodies also appeared to be species-specific. These investigators combined radio labeling, immunoprecipitation, and gel electrophoresis to detect 200,000, 68,000 and 40,000 dalton antigens of the sperm surface (47). Monoclonal antibodies to syngeneic testis cells were prepared to define differentiation antigens of sperma togenesis in mouse testes (2). One such antigen appears at about the time meiosis begins and persists at least to the early spermatid stage. It may be present in relatively low concentration in epididymal sperm. A second mono clonal antibody recognizes a testis antigen that may first appear in spermatogonia or early prophase spermato cytes and that cannot be detected on epididymal sperm. The monoclonal antibodies described above are directed to antigens whose function has not been identified. Hybridomas have also been generated following immuni zation with LDH-C4 . These monoclonal antibodies, described in detail below, have proven useful in assessing the evolutionary history of this sperm-specific enzyme and in relating protein structure to antigenicity (14). Specific antigens identified by these chemical and immu nological techniques will be useful in dissecting the various immunopathologies of sperm, as well as in describing the stages of gene expression in sperma tol, nesis. They may also serve as the basis for a contraceptive vaccine. EXPERIMENTALLY INDUCED

IMMUNOLOGIC INFERTILITY

A contraceptive vaccine is suggested by the observed association between infertility and anti-sperm antibodies in both male and female human beings. Suppression of fertility is well documented in female laboratory animals immunized with sperm. The goal of present-day research remains that of identifyirng the responsible antigen(s) from extracts of sperm and testes.

5

Menge and associates extracted rabbit sperm with lithium diiodo., :cylate (LIS) (30). Rabbits isoimmunized with LIS-soluble extracts of sperm and testis showed reduced fertility. They produced sera containing sperm immobilizing and agglutinating antibodies. Treatment of sperm with the sera prior to artificial insemination (AI) of nonimmunized does abolished fertility. The antisera also blocked attachment of rabbit sperm to rat ova in vitro, Rabbits immunized with the LIS-extracted sperm pellet showed a normal frequency of fertilization but a reduced rate of implantation. Antibodies present in these sera did not inhibit attachment of rabbit sperm to rat ova in vitro, or fertilization by Al in nonimmune rabbits. There was a significant reduction in implantation of fertilized ova transf erred from nonimmunized does to rabbits immu nized against the LIS-extracted sperm pellet. Saling and O'Rand prepared rabbit anti-mouse sperm Fab fragments that were capable of inhibiting sperm45 zona interaction and sperm-egg fusion invitro ( ). This serum contained antibodies to four major and three minor antigens. As noted below, it may be that these antigens play an essential role in fertilization, but itis not possible to rule out mechanical blockage of sperm function by antibody binding, Menge and Black described the effects of rabbit or rhesus monkey of zona-free sperm sera on humanTreatment of tration anti-human hamster ova (29). sperm penetraton amser oa (9).Treamen of f zna-fee sperm with normal sera tended to increase the percentage of ova penetrated, compared with basic medium alone. Antisera against sperm, sperm extract, and testes, as well as Fab preparations of these antisera, caused significant decreases in the number of ova pe.etrated. Pretreatment of zona-free ova vith antisperm Fab preparations had no effect on the sperm penetration rate but artisperm serum dd depress the rate of penetration. These authors suggest that antibodies against integral surface antigens of sperm membranes may either be covering receptor molecules for the egg membrane or causing a steric hindrance of the membrane interaction between the gametes. Unfortunately, the egg pretreatment results are consistent with a nonspecific blocking reaction.

solubilized rabbit epididymal sperm, and developed two hybridomas secreting monoclonal antibodies against sperm (36). Inseminalion of female rabbits with sperm treated with either of the monoclonal antibodies resulted in significant reduction in fertility, as seen by the percent age of 9-day embryo implants recovered. Although these antibodies inhibited in vitro binding of rabbit sperm with the zona pellucida of rat ova, fertilization in vivo was not significantly affected. Both of these monoclonal anti bodies appear to recognize the same antigen of testicular origin and with an approximate molecular weight of 63,000 daltons. Naz and Menge used an immunoaffinity column to isolate the ant gen (37). The antigen was identified as a glyco protein containing about 20% carbohydrate and was immunogenic in mice but not in rabbits (38). Neverthe less, the monoclonal antibodies reacted with rabbit, human, and murine sperm (37). The immunoreproduc tive significance of this antigen requires further study. However, the postfertilization effects of these antibodies and the self-recognition of the glycoprotein by the immune system of the rabbit suggest a somatic origin of the antigen: stimulus. This could lead to a cross reactivity with tissues that would compromise the con traceptive potential of this antigen. Naturally occurring antibody-mediated infertility should tive del fody-met ine N a ul o provideausefulmodelfordevelopmentofacontraceptive

vaccine. It seems reasonable to propose, for example, that enhancement of an immune response to male t is resse gat nheme ould ine gametes inthe female could indeed suppress fertility. It is obvious from the foregoing discussion that sperm antigens do existan anre immunogenic. In some cases, they also can provoke an immune response or development of antibody that renders sperm incapable of fertilizing the egg, of reaching the site of fertilization, or of inhibiting postimplantation development of the embryo. The most important requirement for development of a contraceptive vaccine is identification of a highly specific and immunogenic sperm antigen. Inaddition, the antigen must be available in relatively large quantities, and this is unlikely if it is to be obtained from natural sources (i.e., testes or sperm). Therefore, the antigen must be amenable to chemical syithesis. The major problem with the various antigens so far described is that useful Fab fragments derived from antisera to RSA- 1were used wihtevrosaignsofrdcibdstatufl There was a 68% prior to Altuses spermatozoathenumbero Al (41). to treat srt necessary for precise structural and immuno ered. aquantities waSpe (4) ttreaucton chemical characterization are not available, and may not reduction in the number of fetuses recovered. Sperm be, without application of heroic isolation and purification binding tc the zona was reduced in vitro, and there was r rocedures. At the present time, the most viable sperm complete inhibition of egg penetration. Since RSA-1 specific antigen for vaccine development is lactate de cannot be detected on acrosome-r"eacted sperm by hydrogenase C4 (LDH-C 4; LDe--X). immunofluorescence (41), it islikely that the specific Fab enzymes required antibody blocks release of acrosomal DEVELOPMENT OF A CONTRACEPTIVE

for zona penetration. Alternatively, RSA-1 may play an VACCINE; SPERM-SPECIFIC LDH-C4

important role during penetration through the zona (41). Naz and co-workers immunized mice with detergentAll vertebrate tissues contain LDH. This tetrameric

6

Figure 1. Cross-section of mature mouse testis showing immunofluorescent localization of LDH--C 4. Note the absence of fluorescence staini:,g in spermatogonia, interstitial, and Sertoli cells. There isa marked increase in the LDH-C 4 content of the more advanced germinal elements. enzyme occurs in multiple molecular forms, or isozymes, which are randomly assembled from two types of subunits, A and B. A third subunit type, C, issynthesized only in testes during active spermatogenesis, and is distinct from the A and B peptides of somatic origin, LDH-C, 1 first appears in the primary spermatocyte during mid-pachytene ofthe firstmeiotic division.The restriction of LDH-C, to the germinal epithelium is apparent in Figure 1. LDH-C,; isdemonstrated by immunofluorescent localization in primary spermatocytes and spermatids, while the spermatogonia and the non-germinal elements, such as interstitial and Sertoli cells, are cle rly devoid of this isozyme. The appearance of LDH-C 4 first in midpachytene primary spermatocytes is illustrated in the immature testes section in Figure 2. Synthesis of this isozyme continues, so that it becomes the most abundant form of LDH in spermatozoa. The unique developmental and biochemical properties of LDH-C, 1 are of considerable interest and significance to testes and sperm metabolism. They were reviewed recently in detail by Wheat and Goldberg (63).

Figure 2. Cross-section of immature mouse testes. The most advanced stage of spermatogenesis is the mid pachytene primary, spermatocyte, the only cell type that contains LDH-C4.

POW

Since LDH-C 4 isnot found inprepubertal testes or inany other tissue of t.,e male, and is also completely absent from the female, it is antigenic in both sexes of homologous andheterologousspecies. Rabbit antiserum to mouse LDH-C. reacts with this isozyme from all mammalian species, but does not react with isozymes composed of A and B subunits (8). The availability of relatively large quantities (hundreds of milligrams) of mouse LDH-C 4 (23, 61) facilitates rigorous analysis of the effect of immunization with LDH-C 4 on fertility. Female laboratory animals were immunized with LDH-C 4 and then mated. Fertility was significantly reduced in bothmice and rabbits(9, 25). Thestudy was extendedto nonhuman primates (1 ', and nine adult female baboons (Papio anubis) of proven fertility were immunized with mouse LDH-C4. Circulating antibody was produced in all animals and reacted with mcuse, baboon, and human LDH-C. All animals showed normal ovulatory men strual cycles during the immunization protocol. In a series of breeding experiments, 22 of 30 mnatings, or 74%,

SCHEMATIC

VIEW OF ONE

LDH SUBUNIT

LOOKING ALONG THE MOLECULAR Q AXIS TOWARDS THE CENTER OF THE MOLECULE

IP

Figure 4. Model depicting the location of the antigenic peptides of LDH-C 4, and seen as shaded regions in this photograph. (Modified from Adams et al: In Jaenicke R, Helmreich E (eds): Protein-Protein Iteractions. Berlin and New York, Springer-Verlag, 1972, p 131).

Figure 3. Immunofluorescent localization of LDH-.C4 spermatozoa.

to

these immunoglobulins would impair energy metabolism arid functional motility of the sperm. Antibodies to LDH-C 4 do inhibit enzyr.,e activity in uitro (8). Alter natively, antibody binding sites provided by LDH-C 4 on the sperm surface would mediate spermagglutination, as

well as complement dependent cytotoxicity (11) (Figure 3). Thus, a useful contraceptive effect of immunization

the spermatozoon principal piece of the tail. The pattern seen here for murine sperm is the same in human

were infertile, as compared to 28% in control matings. The contraceptive effect of the vaccine containing LDHC4 was associated with high antibody titer. Normal pregnancies ensued in baboons in which titer declined after termination of booster injections. The conceptions that did occur during the experiment were restricted to animals with poorly developed immune responses and did not yield normal fetuses. Thus, this treatment is effective, it does not impair embryonic development or damage the female reproductive system, and is reversible, For LDH-C 4 to provide the basis for a practical immunological contraceptive, two major problems must be solved. First, a synthetic substitute for the natural product antigen must be ident,fied. Second, the contraceptive effect must be made more nearly complete. Knowledge of the mechanisms of immunocontraception can lead to improved effectiveness. Antibodies to LDH-C 4 may suppress fertility in at least two ways. Inhibition of intracellular enzyme activity by 8

with LDH-C 4 can be achieved by the action of specific antibodies in the female reproductive tract at a sufficiently high concentration to provide an immunologic barrier preventing sperm from reaching and fertilizing the egg. Anti-LDH-C 4 serum is transferred into the female repro ductive tract (17) and sperm transport is markedly inhibited in the genital tract of immunized females (18). These data suggest that immunization with LDH-C 4 results in immunoglobulins that entei the female re productive tract and prevent sperm from fertilizing the egg. Ensuring a high concentration of anti-LDH-C 4 by stimulation of the secretory immune system in the female reproductive tract would make the contraceptive effect more complete. Female mice immunized with LDH-C 4 placed directly into the uterus exhibited elevated levels of LDH-C4-specific IgA in their uterine fluids. In animals receiving a systemic immunization 7 to 10 days prior to intrauterine inoculation, LDH-C 4-specific IgA and lgG could be detected in both uterine fluids and in serum (Shelton and Goldberg, unpublished). These results suggest that manipulation of the secretory immune system of the female reproductive tract could enhance the immunocontraceptive effects of LDH-C 4.

Problems of both supply and homogeneity preclude use of a natural product antigen as a contraceptive vaccine, Our strategy in development of such a vaccine, ti! erefore, has been to map antigenic determinants of LDH-C 4. The procedure essentially involves chemical or enzymic digestion of LDH-C4 and isolation of the fragments. Those peptides that bind anti-LDH-C, are chemically characterized. Appropriate peptides are synthesized and conjugated to carrier molecules for immunization. The anti-peptide antibodies are tested for binding to LDH-C 4. Finally, the immunocontraceptive properties of these synthetic determinants are assessed. Although these studies are still in progress, we have demonstrated that our strategy is sound. Thirteen antibody-binding peptides have been identified. A structural model of the LDH-C subunit indicates the positions of these peptides (Figure 4). Immunoglobulins that react with LDH-C4 have developeL. in rabbits immunized with four different synthetic peptide fragments of the native protein (13, 15, 62). Furthermore, these antibodies agglutinate sperm and significantly inhibit in uitro fertilization (Beyler et al, unpublished).

support this suggestion. The use of a well-defined, synthetic antigen is essential to progress in developing a vaccine for fertility control and to establish with certainty the utility of such technology. These studies with LDH-C4 are consistent with the feasibilty of a synthetic antigen for use in a vaccine to control fertility. LDH-C 4 remains the best-developed candidate for future studies. As noted above, immunization with LDH-C 4 does suppress fer tility, and the complete biochemical characterization of this isozyme is well underway. These studies will provide a wealth of potential synthetic antigens to substitute for the natural product in experiments to determine whether immunologic contraception is appropriate and desirable for wide-scale application in human beings. Although there are certainly a number of practical problems to be resolved, prospects are good that during the next decade, a vaccine to prevent pregnancy will become available. ACKNOWLEDGEMENT Research from this laboratory was supported by a grant from the National Institutes of Health (NIH) and by contracts with the Contraceptive Development Branch of NIH, the World Health Organization, and the Program for Applied Research on Fertility Regulation (USAID/ DSPE-C-0035). I thank Dr. T. E. Wheat and Dr. J. Shelton for their critical reading of this manuscript, and gratefully acknowledge the outstanding secretarial assist ance of K. Clisscld and Mary Smith.

CONCLUSION Immunocontraception has been suggested as a solution to the need for alternative and reversible contraceptives, Both pathologic and experimentally-induced infertility

9

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