Indian J Physiol Ph~rmacol 2001; 45 (I): 7-21

REVIEW ARTICLE

RESEARCH IN REPRODUCTION: THE INDIAN SCENARIO IN THE LAST DECADE [II]*

JAYASREE SENGUPTA

Department of Physiology, All India Institute o.f Medical Sciences, New Delhi - 110 029

(Received OD August 10, 2000 )

Abstract: Developing new, improved and totally safe, effective and acceptable contraceptives based on the recent advances in cellular and molecular biology of reproduction is a new challenge to biomedical scientists involved in research in reproductive biology. The present article reviews some of the major contributions made during the last decade by scientists working in lndia in developing new strategies and technologies for better human reproductive health and fertility regulation. Key words: antHmplantation contraception fertility regulation hormonal antagonists hormonal contraceptives immuno-contraception natural contraceptives reproductive health

INTRODUCTION to unprotected and which will decrease the resort to abortion; In a UNICEF report it has been expanded male contraceptive choices, stated, "Family planning could bring more participation and responsibility (3). Now the benefits to more people than any other challenge for scientists in the related single technology now available to the areas is to translate this concept into human race" (1). The necessity of developing developing new, improved and totally safe, a strategy for woman centred agenda effective and acceptable contraceptives had been highlighted at the Cairo b~sed on the recent advances in cellular International Conference on Population and molecular biology of reproduction. and Development (2) and it emphasized on In this review 1 have attempted to tell the three specific needs which were previously story of the contributions made during unmet by the currently available methods: the last decade by scientists working women~controlled methods that provide in India in developing new strategies additional protection against sexual and technologies for better human transmitted infections; methods which a reproductive health and fertility woman can use as a back-up when exposed regulation.

-For part I of the article, see Indian Jour/lal of PhysiQla/JY and Pharmacalagy 2000; 44(3); 237-254 SeOIUpu lodiao J Pbysiol Pbarmacol 2001: 45(1)

Phy..ieo·chemica' met-hod. for fert.i1ity replaUoo suggest that exfoliation of epithelium due ia the male to vu occlusion by SMA is reversed after 150 days of noninvasive reversal (8). while At. the Centre for Biomedical necrospermic status of the spermatozoa Engineering of the All Indian Instit.ut.e of during initial ejaculations have been Medical Sciences, Guha and colleagues suggested to offer instant: sterility after vas developed an injectable method for inducing occlusion with SMA (9). occlusion using maleic anhydride with dimethyl sulphoxide (DMSOl UR or plaat produeu ror the developme.at- of Dove.l as its solvent (4). A special feature of this contraceptive .t-rat-e.rie. treatment was that of spontaneous re· establishment of passage with time, As an alternate approach to . and the restoration period could be long-term contraception in the male was regulated from three months to five years. tested at the National Institute of Phase I clinical trials on male volunteers Immunology, New Delhi, by injecting a showed that the treatment was well single dose of oil extract.ed from a traditional tolerated with only minimal side effects and Indian plant, Neem (Azodirachta indica) with no long·term adverse effects (5). These into the vos deferens of the rat (0). The results paved the way for the phase 11 authors reported an antifertility response clinical trials and the drug, styrene maleic throughout the 8 month observation period anhydride (SMA) in DMSO (now named as without any inOammatory or obstructive R,sug) was successfully administered to changes in and vas deferens, and sexually active healthy male volunteers with no change in testosterone levels, whose wives were without any form of however spermatogenic block was observed contraceptive support. The results obtained which was presumably mediated by from this study were encouraging as one local immune mechanism. though anti· year treatment led to and sperm antibody could not be detected. provided protection (6). At the Subsequently, polyherbal neem as a cream University of Rajasthan, Jaipur., Lohiya and preparation was developed by this group and colleagues examined the feasibility of a was tested in rabbits and monkeys; it spacing method for contraception using SMA showed contraceptive efficacy after 8S a vas occlusive agent in male langur intravaginal application and was found to monkeys (7). The results suggest that the be safe in subacute toxicity st.udies SMA·based spacing technique for mole performed in monkeys (1). The spermicidal contraception could be extrapolated to the activity of neem oil was first report.ed by human by use of no·scalpel injection and Riar and colleagues of the Defense Institute non·invasive reversal. Ultrastructural of Physiology and Allied Scienees, Delhi (l2). studies revealed that after non-invasive Subsequent studies revealed that the reversal, the vos epithelium regained a state spermicidal action with loss of motility of normalcy as evidenced by prominent was due Lo the formation of pores and plasma membrane, nucleus, cytoplasmic vesicles on sperm head indieating damage organelles, and stereocilia. The results to cell membrane; supplementation of lIldian J Ph,..iol PharmacoI2001; 45(1) Re!leart.b in ReproductIon in India 9 pentoxifyJline which i9 known to enhance attachment. and proliferation and loss of motilit.y could not reverse the spermicidal fertility (20). At the Indian Institute of action of neem oil (13). In addition, the use Chemical Biology, Calcutta, Pakrashi and of neem as a spermicidal agent would coUeagues investigated the anti-ovulatory provide protect.ion to women against. a (21l and anti-spermatogenic (22) potential variety of microorganisms and sexually of Molviscus conzotti nower in the rat. transmitted diseases (14). Lohiya and colleagues at the University The long-term contraceptive potential of of Rajasthan, Jaipur have performed neem for women was proposed by Upadhyay experiments with several plant products et a!. 05), using an intraut.erine mode of using small laboratory animrds and non­ delivery The numbers of MHC II human primate species. Using purified positive cells were found to be high in gossypol acetic acid Blone and in uterine endometrium of bonnet monkeys combination with potassium chloride, the following such neem treatment. Oral antifertility action and possible administrat.ion of purified neem extract, hypokalcmia were tested in adult male Pronl!l!m, to pregnant rats led to resorption Iangurs fOT 120 days. The treatment of embryos with elevated levels of interferon resulted in severe oligospermia, with gamma (IFN-y) and tumour necrosis factor­ impairment of sperm motilit.y. The functions alpha (TNF-a) (16). A post.-implantation of accessory glands and , however, aoortifecient response to orally administered remained unimpaired. Complete reversal of Deem was similarly reported for the rabbit these changes were noted after 90 to lOS and the baboon, and the effect was shown days of withdrawnl of treatment leading the to be reversible (17). The partially investigators to suggest that, while fractionated active principle of neem has oligospermia achieved was reversible, the been suggested to function as an hypokalemic response of langun~ is similar immunomodulator in causing pregnancy to human and not related to impurit.y of failure with decline in chorionic gossypol (23, 24). Monkey'" rcceiving gonadotropin (CG) and progcsterone levels gossypol plus potassium salt showed normal in thc bllboon, and increasc in CD4+ and serum potassium level. Scanning electron CDB+ cells in spleen and mcsenteric lymph microscopy of spermato2.oa rcvealed nodes ClB). The active principal in neem deleterious abnormalities in hend and responsible for reversible anti-fertility midpieccj testicular morphology following response in rodent, lagomorph and primate gossypol exposure resulted in a decrease in species was shown to be a mixture of six seminiferous tubule diameter nnd arrest of components which include saturated mono­ (2Sl. The contraceptive and di-uDsnturated free fatty acids and their efficacy of a chloroform extract. of papaya methyl esters (9). A post-coital action of seeds was investigated. and It was shown Deem oil has been suggested based on the to induce a defect which was reversible and ob ervation that. in utl.ro exposure of two was observed to be mamly post-testicular cell mouse embryo.9 to neem oil led to failure in nature without IDnu(>ncing toxicologic:lJ of blastocyst development. trophoblast profile and libido in rats :lnd rabbits 10 Senguptn Indian J Physiol Pharmacol 200J; 45(1)

(26-28). Reversible sterility could also be Hormoual contraception for tbe male induced in male rats by using an aqueous extract of papaya seeds without any adverse There is a need for more participation effects on libido and on toxicological profile by men in fertility regulation, however, a (29). significant degree of gap exists between the need and the demand for novel male The fields of family planning and the contraceptives and the state of prevention and cure of sexually transmitted understanding of the basic knowledge of the disease (STDs), including HIV/AIDS have functions of the male reproductive system. traditionally been operated independently Sustained research efforts are therefore with the underlying operating assumption needed to provide broader contraceptive that the STDs are not of concern to married choict:!5 and to make men share the couples creating families; for them the risk responsibility and the burden for fertility of sexually transmitted diseases are regulation with their partners. generally considered trivial. However, epidemiological and sociologic facts now Taking a lead from the report of Ready indicate that an increasing size of global and Rao (33) that daily application of population is simultaneously vulnerable to testosterone (T) to men resulted in unwanted conception and unwanted suppression of spermatogenesis and infection (30). Thus, targeted drug delivery azoospermia, and normal spermatogenesis via the vaginal route may pave the way for regained with the withdrawal of the the development of new methods for treatment, Rajalakshmi and coworkers at protection against pregnancy and STDs, the All India Institute of Medical Sciences. thereby allowing improved reproductive New Delhi, studied the pharmacokinetics of health care. The hypothesis that potent anti­ a long acting , testosterone microbial, anti-tumour agents like magainin enanthate (TE) in monkeys (34). They peptides, and antibiotic and anti-angiogenic agents like fumagillin can inhibit blastocyst observed a spurt of supra·physioiogical implantation has been tested in the rhesus levels of testosterone within 3 days of monkey model at the All India Institute of treatment which was followed by a gradual Medical Sciences. These agents were decline; dihydrotestosterone (DHT) and intravaginally administered through levels were also found to be tampons to proven-fertile, mated rhesus elevated (34). Since similar data were also monkeys and were observed to cause "one available from human studies with TE, a hundred per cent pregnancy protection concern was voiced whether exposing the without any marked change in menstrual male to intermittent spurts of supra­ cycle length of treatment cycles and of physiological levels of and subsequent cycles. Thus, intravaginal could induce patho-physiological delivery of anti·microbiai agents like changes in the target organs. To this effect, magainins and fumagillol may lead to the the actions of long-term use of TE on development of potential anti-implantation prostate structure and function using the strategy for interception of pregnancy rhesus monkey as a primate model was (31, 32). critically evaluated (35). Long·term Indian J Physiol Pharmaeol 2001; 450) Research in Reproduct.ion in India 11 exposure to TE (once in two weeks for 33 (37). In monkeys subjected to controlled months) resulted in an increase in weight dietary conditions, long-term exposure to TE of cranial and caudal lobes of prostate, failed to show any changes in glucose cellular hypertrophy and increased secretory tolerance test, however, serum insulin levels function of cells. cellular hyperplasia, decreased significanUy (38). The effects of increased levels of estradiol and prostate­ a long-acting androgen ester testosterone specific acid phosphatase and marked bucic1ate (TB) administered on days 1 and increase in fibromuscular stroma in central 91 of a 360 day study period to bonnet and peripheral zones of both lobes of monkeys revealed suppression of testicular prostate in adult male monkeys. In an and epididymal functions suggesting that attempt to understand the direct actions of th.is long-acting androgen may have the steroid hormones and growth factors on potentiality to induce and maintain prostate physiology, prostate epithelial cells reversible sterility (39). The rise in T levels were collected from rhesus monkeys, following TB injection was gradual and peak isolated and cultured in flasks coated with values were attained by day 14 after either collagen IV or laminin; the influence injection and the levels were found to be of growth factors such 8S IGF I and IGF II within the normal range. Similarly, the in combination with androgens such as levels of dihydrotestosterone was within the dihydrotestosterone and androstenedione on normal range and though estradiol level was cell number, thymidine incorporation and elevated, it was lower compared to the rise secretory activity were assessed Of the two observed following TE injection (40). At the IGFs, IGF I was more effective than IGF II. National Institute of Health and Family DHT with IGFs was more potent in inducing Welfare, New Delhi, Das and colleagues proliferation, differentiation and secretion observed that STS·557 07-alpha­ than androstenedione. It is considered that cyanomethyl-17-beta-hydroxy-estra-4, 9 (19)­ using such a primary culture system the dione-3-one) exhibited, contraceptive physiology of prostatic epithelial cells can potential when tested in the bonner monkey. be monitored vis-a-vis the actions of drugs Daily treatment for 12 weeks resulted in that can inhibit cell proliferation (36). significant decline in count, motility, acrosin and hyaluronidase activities and in the Though there was suppression of the fertilizing ability of spermatozoa by sixth nocturnal rise in T level, TE injection to week of treatment. The blood testosterone rhesus monkeys led to increased prMile declined by the second week bioavailability ofT which favoured a change coinciding with rising level of STS-557 in in its metabolism by the liver and instead circulation (41, 42). of androstenedione, androsterone became the major metabolite as spermatogenesis HormonaJ aot.ronbts (or pregDIIDcy interception became arrested. Liver function tests revealed that long-term TE exposure led to It is established that embryo increased liver transominases (SGOT and implantation occurs in a favourable SGPT) and these enzymes returned to uterine milieu which exists in ­ baseline values during the recovery period primed, -dominated state of 12 SellgupLl1 Indian J Physiol Pharmacol 2001; 45(I) endometrium (44). It is therefore likely that has outlined the earlier studies of these two methods to intercept the process of embryo fertility regula,ting drugs, clomiphene citrate implantation in a potential conception cycle and centchroman and their current uses without compromising normal menstrual (48). cyclicity could provide potential new tools for developing ul:ler·acceptable, safe and The development of novel anti-progestins need·driven contraceptive strategies for which bind to progesterone receptor at the women. cellular level to inhibit progesterone action in target cells has allowed for the Ccntchroman, a non-steroidal antif­ elucidation of the hormonal basis of the ertility agent with weak estrogenic and cellular and molecular events leading to potent anti-estrogenic activities was embryo implantation. Anti·progestins are alJP('oved for its use as 'once-a-weak' pill in now being widely used to develop retro­ India since 1991. The drug discovered by active contraception which include the Central Drug Research Institute of , luteal Lucknow is thought to exert its contraception and mense·inducers to be used contraceptive action by disrupting the at the expected time of menstruation. It is balance of estrogen and progesterone anticipated that the development of such IlE-CeSB8ry to prepare the uterus for contraceptive technology will help to implantation. Centchroman is also reported minimise the increasing incidence of unsafe to accelerate ovum transport in animal abortion. models. The resulting asynchrony between uterine preparation and ovum transport Antiprogestins such as lilopristone prevents implantation of the fertilized egg (ZK98734), onapristone (ZK98299) and (41»). The contraceptive efficacy and safety (RU486) have potent anti­ of 30 mg centchroman administered bi­ progestational as well as variable degree of weekly for the first three months followed anti-glucocorticoid activities as judged from thereafter at weekly intervals was studied irt vivo and in vitro animal experiments (49). in a Olulticentre trial. A total of 377 women At the Institute for Research in volunteers were covered for 3932 months of Reproduction. Mumbai, Puri and colleagues use with pregnancy protection and Pearl have used onapristone (ZK98299) to study index of 1.83. About 90% menstrual cycles its effects on pregnancy and fetal outcome were within the normal range and about in bonnet monkeys (50). Limited data on 8 4(',4 were prolonged. The contraceptive effect animals demonstrated that onapristone was readily reversible and subsequent given for four consecutive days between were normal (46). Roy has days 20 to 30 post-ovulation terminated chronicled the unfolding saga of an anti­ pregnancy in 62% animals and in cases estrogenic compound, clomiphene citrate in where treatment failed the endocrine Its failure to be used as a fertility regulating function of placenta was affected and agent, while it obtained approval, on the fetal growth retarded. Usc of another other hand, as a drug now widely used for antiprogestin, Jilopristone at different time­ t.hl:' treutmllnt of ovulatory failure (47), and points after ovulation resulted in luteolysis Indian J Physiol Phannacol 2001; 45{l) Research in Reproduction in India 13 and pregnancy failure due to its actions on Research in Reproduction, Mumbai have gonadotrophin release in the bonnet monkey also studied the effects of daily interanasal (51). Administration of onapristone at administration of (NET) on weekly intervals to adult bonnet monkeys folliculogensis, cervical mucus, vaginal for 10 weeks resulted in inhibition of cytology and endomertial morphology in the ovulation in all monkeys studied with the human, which indicated its potential use as arrest of typical follicular phase rise .of an antifertility agent (58). estradiol and of the midcycle surge in the levels of bioactive LH along with very low Understanding the cellular and level of progesterone throughout the cycle molecular basis of blastocyst implantation, causing atrophy of endometrium (52). and the development of novel anti­ Lilopristone administered on a weekly implantation strategies using the rhesus regime to bonnet monkeys inhibited monkey as a primate model is the focus of folliculogenesis which was reversed by attention of Sengupta and colleagues at the exogenous FSH and LH administration (52). At the All India Institute of Medical In a study using cyclic bonnet monkeys, Sciences. In the pre·ovulatory phase, Ishwad et al. (53) observed that weekly, low progesterone has been suggested to play a dose administration of onapristone did not critical role in the regulation of mid-cycle block ovulation but had an inhibitory effect gonadotrophin surge leading to delayed on endometrial development (54). Low dose ovulation and altered profiles of ovarian onapristooe administered throughout the hormones. Thus it is possible that menstrual cycle prevented pregnancy administration of mifepristone, an anti­ without disturbing menstrual cycle and progestin during the peri-ovulatory stage ovulalion in majority of cycles studied may render hostility to pregnancy in bonnet monkeys (55). The binding establishment by acting at the ovarian level characteristics of onapristone to and at the endometrial level. Using the progesterone receptors in human rhesm' monkey as a primate model, Ghosh myometrial cytosol was also examined (56). et 81. (59) aimed to test two hypotheses: The results of this study revealed that (i) inhibition of prevulatory progesterone although progesterone and onapristone are action can delay or inhibit ovulation, and mutually competitive for binding to (ii) inhibition of preovulatory progesterone progesterone receptor, onapristone also has action can inhibit post-ovulatory phase distinctive binding sites. Further studies to 'tndometrial receptivity for implantation. characterize such binding sites for The results of this study supported earlier onapristone to progesterone receptor in reports that follicular phase mifepristone human myometrial cytosol revealed that the can inhibit or disrupt follicular maturation antiprogestin effects of onaprisone may and delay ovulation, however, it failed to result from suboptimal nuclear binding! inhibit implantation, because gonadal retention of antiprogestin4receptor hormones including progesterone, resume complexes (57). The relative binding affinity normal functions once ovulation takes place. of onnpristone for nuclear progesterone It appears that peri-ovulatory stage receptor was 33% of that for progesterone. mifepristone treatment shall not provide Kumar and colleagues at the Institute for pregnancy protection to women. 14 Sengupta IndiaD J Physiol Pharmacol 2001: 45(1)

The use of low dose, single method to detect ovulation needs to be administration of mifepristone, an anti­ developed. Few leads have emerged from the progestin during the early luteal phase, use of mifepristone as a tool to induce however, leads to one hundred per cent endometrial de-sychronization, loss of pregnancy protection in the rhesus monkey endometrial receptivity and failure of without affecting menstrual cyclicity (60). implantation in the primate. The studies Similar results were observed in women by revealing the distribution, and regulation a Swedish group of investigators (61). of local factors such as vascular endothelial Mifepristone action during the early luteal growth factor (VEGF) and placental protein phase led to endometrial contraception with 14 (PPI4) in receptive and non-receptive significant degree of desynchronization and stage endometria suggest that they may loss of receptivity for blastocyst serve as specific targets to modulate implantation (62). Furthermore, as has been endometrial differentiation towards discussed earlier, the absence of dialogue receptivity for blastocyst implantation between the developing preimplantation (68, 69). The close association of VEGF and embryo and receptive stage endometrium another angiogenic factor, placental growth was associated with failure of factor (PIGF) in influencing vascular and preimplantation embryo growth and endometrial functions at the time of viability and lack of gestational stage implantation have been examined based on endometrial glandular hyperplasia and their localization and expression in the vascularity (63, 64). Anti-nidatory action of primate uterus (68). luteal phase mifepristone was found to be associated with changes in endometrial lmmuno-coDtraceptioD prostaglandin concentration during the implantation window (65). The hypothesis Vaccines for the control of fertility are that prostaglandins are involved in the likely to have an important impact in anti-nidatory action of mifepristone was improving the currently available further tested and it was observed that the reproductive health options. Basic research anti-gestation activity of mifepristone and in the field of gonadotropins and their associated endometrial changes could not actions on target cells led to the testing of be accentuated or attenuated with the long·term contraceptive efficacy of ovine co-administration of prostaglandin E or FSH vaccine by Moudgal and coworkers in cyclo-oxygenase inhibitor (diclofenac), o·or healthy, fertile bonnet monkeys using pure could these be mimicked by these agents FSH from sheep pituitaries at the Indian alone (66, 67). The authors concluded that Institute of Science, Bangalore (70). The the anti-nidatory action of mifepristone is vaccine elicited an immunogenic response most likely mediated through a in all ten monkeys studied. Immunization multifactorial mechanism and the for 4.7-5.7 years did not affect the health prostaglandin milieu is one of the modules and libido of the animals. Concentration of in the complex mechanism. However, for T in serum remained normal, but within mifepristone to be used as an early luteal 150 days of immunization there was a phase, once-a-month contraceptive, a simple marked decrease (75-100%) in the number Indian J Physiol Pharmacol 2001; 45(1) Research in Reproduction in India 15

of spermatozoa in seminal ejaculates. All ten immunization on days 1.20,40 and 70 (73). animals proved infertile in repeated matings The kinetics of antibody production for both with females of proven fertility and after the immunogen (oFSH) and the cross stopping booster injections, nine out of ten reactive antigen (hFSH) were essentially animals regained fertility which closely similar. The volunteers responded to the correlated with the rate of decline of first two immunizations with production of antibody titres. Srivastava and Daa (71) antibody which was specific for FSH and reported that male bonnet monkeys were had no significant change in the values of rendered oligospermic, but not azoospermic related glycoprotein hormones (LH, TSH). following active immunization with ovine Seminal plasma protein, transferrin, a FSH. The percentage of having good marker of and of seminiferous motility was reduced with concomitant tubular function showed reduction (30-90%) increase in sperm ATPase activity. Eight out following immunization with oFSH with of ten monkeys failed to impregnate females reduction in sperm counts (30-74%). Thus, of proven fertility after mating for three long·term blockade of endogenous FSH consecutive cycles while the other males action in men using oFSH as an immunogen impregnated after cohabitation with females can potentially be used in reducing the at a time when the antibody titre was quantity and the quality of spermatozoa reduced. Thus active immunization with resulting in infertility. Moudgal et al. (74) ovine FSH may not produce azoopsermia but have commented that male contraceptive renders infertility to male monkeys provided vaccines nre well tolerated by the primate sufficient antibody titre is maintained. including the human and do not give rise Immunization of proven fertile male to any known t.oxic symtoms or immediate adult monkeys with recombinant FSH health hazards. Contraceptive vaccines for receptor protein preparation representing the male thus appear to be a feasible 1-134 amino acids of the extracellular strategy. Close attention is however needed domain of the receptor results in production to evaluate carefully the bioefficacy of of receptor blocking antibodies. Serum T and antibodies raised to recombinant ovine FSI-I LH levels remained unchanged following beta or FSH receptor over the LHRH/LH immunization but there was 50% reduction based vaccine since the former do in transformation of spermatogonia (2C) to not require exogenous testosterone primary spermatocytes (4C) and the 4C: 2C supplementation to maintain accessory ratio showed a correlative change with gland function and libido. While the LHRHI reduction in fertility index (sperm counts x LH vaccine results in azoospermia. the FSH motility score) and monkeys became vaccine causes the production of low infertile between 242-368 days. The numbers of poor quality sperms which fail observed actions were near indentical to to impregnate cyclic females. that seen following immunization with FSH (72). At the National Institute of Immunology, Gupta and coworkers have identified the Human male volunteers were examined peptide epitopes of porcine zona to test their response to ovine FSH after glycoproteins (pZPC) as an initial step in 16 Sengupta Indian J Physiol Pharmacol 2001; 45(1) an immuno·contraceptive vaccine based on acrosomal surface of mature spermatozoa synthetic peptides corresponding to pZPC from different mammalian species (83). The or its homologues in other species (75). authors suggest that testicular RCP may Immunization of female bonnet monkeys playa role in celI·cell communication. with pig zona pellucida glycoprotein ZP3 Similar to RCP, thiamin carrier protein using adjuvants permissible for human use (TCP) has also been shown to be synthesized led to infertility; fifty per cent of animals and secreted by rat Leydig cells (84) and regained fertility with declining antibody Sertoli cells in vitro (85). The immuno­ titre (76). The zona pellucida glycoprotein 3 contraceptive potential of RCP has been alpha (ZP3alpha) is the primary sperm highlighted by Adiga et aJ. (86) and it has receptor ligand in porcine been suggested that RCP or its defined interaction, and its epitopes have been framenta could be a novel, fi.rst generation mapped by using monoclonal antibodies. The vaccine for regulating fertility in both sexes. authors suggest that such studies will help Immunohistochemically Rep has been in designing synthetic peptide~based localized on ovulated oocytcs and early immuno-contTaceptive vaccine (77, 78). embryos, and active immunization in rats and bonet monkeys with avian RCP prevents The immuno-contraceptive potential of pregnancy without causing adverse vitamin carrier proteins in innuencing physiological effects on the mother in pregnancy has been the focus of attention terms of her vitamin status, reproductive of Adiga and colleagues at the Indian cycles and reproductive endocrine profiles. lnstitute of Science, Bangalore. Studies in Active immunization of male rats the rat revealed that bioneutralization of and monkeys with denatured Rep which is endogenous maternal ribonavin carrier more effective in eliciting neutralizing protein (RCP) by antibody against denatured antibodies markedly reduces fertility by avian vitamin carrier resulted in higher impairing the fertilizing potential of rates of fetal resorption as against animals spermatozoa (86). immunized with the native protein (79). Passive immunization of pregnant rats on At the National Institute of Immunology days 10 to 12 of gestation with monoclonal and at the International Centre for Genetic antibody to chicken thiamin carrier protein Engineering and Biotechnology, New Delhi, resulted in resorption (80). Leydig cells of Talwar and colleagues have been developing adult rat testis have been shown to a vaccine to prevent pregnancy. Talwar et synthesize and secrete estrogen-inducible a1. (87) reported the development of a RCP under positive regulation of LH (81). vaccine for inducing antibodies against Sertoli cells werl! shown to synthesize and human chorionic gonadotrophin (hCG). The secrete in vitro an estogen-inducible RCP vaccine which had earlier been shown to be in culture suggesting a functional role of reversible, and without any notable side this protein as a carrier of riboflavin to effects on endocrine, cardiovascular and developing germ cells in rodents (82). other body functions was observed to require Riboflavin carrier protein (RCP) has also an antibody titre> or = 50 ng of hCG been localized on germ cells, and also on bioneutralization capacity per ml. Talwar Indian J Physiol Pharmacal 2001; 45(1) Research in Reproduction in India 17 and colleagues (88) now report that 148 adequate antibody titre for a sufficient women of proven fertility who were period of time, high degree of individual volunteers for the phase II efficacy trial for variation and induction of incomplete the HSD-hCG vaccine showed high efficacy abortion (92). Thus the future considerations with 1 pregnancy in 1224 cycles, at or above in the development of safe and effective 50 ng/mt led to conceptions indicating regain vaccine for fertility regulation will need to of fertility (89). The antibody response was take into account the mechanisms that found to be predominantly against an exclu5ively cause cell-mediated immune epitope in the core part of the beta heG responses and the definition of antibody molecule. Beside~ its use in the reversible titres to assess the role that each plays in control of fertility, hCG vaccine also has the infertility and ccll and tissue damage (93). potential to inhibit tumour growth as various types of cancers including lung ACKNOWLEDGMENTS carcinoma cells produce heG (90). To minimize multiple injections for antibody The studies reported in the present article titre maintenance, Singh et al. (91) have and the previous article (IJPP 2000; 44(3): rcported the development of a biodegradable 237-254) from the author's laboratory delivery system for a were generously funded over the years vaccine as tested in the rat and monkey by the Indian Council of Medical Research, models. A single injection of the immunogen the Council of Scientific and Industrial entrapped in microspheres generated a Research, the Department of Science response comparable to that obtained by and Technology, the Department of the same immunogen on alum injected at Biotechnology. Government of India, the a monthly interval and antibodies generated Special Programmc of Research, Department had good bioneutralization capacity and Training in Human Reproduction, World indicating immunogen integrity. Health Organization, the India-held US Rupee Fund of the National Institute of The potential pitfalls in any immuno­ Health, USA, and the Rockefeller contraceptive strategy must be seriously Foundation. The author takes this considered; these include cross-reactivity of opportunity to thank Dr. D Ghosh for his antibody with non-target tissues, failure to comments and suggestions for these two produce antibody titre, failure to sustain articles.

REFERENCE

1. Grant JP. The State of the World's Children 1992 deferens. Contraception 1990; 41: 323-331. Oxford University PreBs, Oxford. 1992; JIll 58 .. 5. Guha SK, Singh G, Anand S, Kumar S, Kaul S, 2. Van Look PFA. Perelo-Palacios G. COfllraceptille Kaul V. Phase 1 clinical trial of an injectable Research and Development 1984 to 1994. Odord contraceptive {or the male. COIl/raception 1993; 48: University Press, New Delhi, 1994. 367-375. 3. Fathalia MF. Contraeeption-21. Internal J OY/lecof 6. Guhll SK, Singh S, Ansari S, Kumar S, Srivastava Ohstflr 1999; 67; 55-812. A, Koul V, Das HC, Malhotra RL. Das SK. Phllse II clinical trial of II vas deferens injectAble 4 Guhll SK, Anand 5, Ansari S, Farooq A, Sharma contraceptive for the male. COlltroceptioll 1997; 56: ON. Time-controlled injectable occlusion of the vas 245-250. 18 Sengupta Indian J Physiol Pharmacal 2001: 45(1)

7. Lohiya NK, Manivannan B, Miahra PK_ Repeated 19 Garg S, Talwar GP. Upadhyay SN. vas occlusion and non-invasive revonal with Immunocontraceptive activity guided fractionation styrene maleic anhydride for male contraception and characterization of active constituenls of neem in langur monkeys. lilt J Alldrol 2000; 23: 36-42. (A:r.adirachta indica) seed ertrftcts. J Ethl/ophQrmocol 1998; 60: 235-246. 8. Manivannan B, Miahra PK, Lohiya NK. Ultrastructural changell in the vas deferens 20. JuneJa SC, Pfeifer T, Williams RS, Chegini N. Neem of langur monkeYII Presbytis entellus entellus oil inhibits two-cell embryo development and after vas occlusion with styrene maleic trophectoderm altachment and proliferation in anhydride and after ils reversaL Contraception vitro. J ASlist Reprod Genet 1994; II: 419-427. 1999: 59; 137-144. 21. Bannerjee R, Pal, AK, Kabir SN Pakraahi A. 9. Lohiya NK, Manivannan B, Mishra PK. Alltiovulatory faculty of the nower of Ma{uoviscus Ultrastructural changes in the spermatozoa of conzottii Phytother Reff 1999; 13: ] 69-171. Inngur monkeys Presbyti/! elltellus entellua after 22. Chakraborty S. Pakrashi A. Antifertility efftlct of vas occlusion wilh styrene maleic anhydride. chronically administered Malvaviscus conzattii COlltraeepllQn 1998; 58: 125-132. flower extract on fertility of male rats, 10. Upndhyay SN. Dhawan S, Talwar GP. Antifertility Contraceptioll 1991; 43: 273-285, errects of neem (Azadirachta indica) oil in male 23. Lohiya NK, Sharma K, Kumar M. Sharma S. rats by single intra-vas adminilllration: an Limltationll in developing gossypol acetic acid 8S a alternate approach to vasectomy. J Androl 1993; male contraceptive. Contrclception 1990; 41: 14: 275-281. 519-532. 11. Garg S, Taluja V, Uplldhyay SN. Talwar GP. Studies 24. KUDlar M, Sharma S, Lohiya NK GOS!lypol·induced on the contracelltive efficacy of Praneem polyherbal hypokalemia and role of exogenous potassium salt cream. ContNlception 1993; 48: 591-596. supplementation when used as an 12. Riar 5S. Devakumar C, Havazhagan G, Bardhan J, antispermatogenic agent in male langur monkey. Kain AK, ThomalJ P, Singh R, Singh B. Volatile Contraception 1997; 56: 251-256. fraction of neem oil as a . COl/lraceplioll 25. Sharma S. Kumar M, Goyal RB, Mnnivannam B, 1990: 42: 479-487. Lohiya Nk. Reverillble antillpermatogenic effect of 13. Sharma SK, Sai Ram M, llava:r.hangan G, Devendra gossypol in langur monkeys (Pre$byti$ entelillS elltellu& Ado Conlracept 1999; 15: 15-27. K. Shivaji 55, Selvamurthy W. Mechanism of actiou ofNIM-76: It novel vaginal contraceptive from neem 26. Lohiya NK. Goyal HB. Antifertility invelltigations oil. Contraceplio/l 1996; 54: 373-378. on the crude chloroform extract of Carica papaya Linn. lIeeds in Inale albino fIltS. Illd J Erp BioI 14. Talwar GP. Raghuvanashi P, Mishra R, Mukherjee 1992; 30: 1051-1055. S, Shah S. Immuliol Cell Bioi 1997; 75: 190-192. 27. Lohiya NK, Pathak N, Millhra PK, Manivnnnan B. 15. Upadhyay S, Dhawan S, Sharma MG, Talwar GP. Reversible contraception with chloroform extract Long-term copntraceptive effects or intrauterine of Carica. papaya Linn. Seeds in male rabbits. need treatment. (JUNT) in bonnet. monkeYII: an Reprod Toxicol 1999; 13: 59-66. alternate to intrauterine contraceptive devicell. Contrnception 1994; 49: 161-169. 28. Lohiys NK, Pathak N, Mishra PK, Manivannan B. Cortraceptive evalution and toxicological study of 16. Mukherjee S, Talwar GP. Teemination of pregnancy aqueoull extract of the aeedll of Carica papaya in in rodents by oral administration of praneem, a male rabbits. J Elhnophormacol 2000; 70: 17-27. purified need used seed extract. Am J Reprod Immunol1996; 35: 51-56. 29. Lohiya NK, Goyal RB, Jaynprakash 0, Ansari AS. Sharma S. Antifertility effects of aqueous extract 17. Mukeherjee S, Lohiya NK, PaJ R. Sharma MG. of Carica papaya seeds in male rats. Pltlt1!/J Med Talwar GP. Purified neem (Azadirachta indica) lIeed 1994; 60: 400-404. extracts (Praneem) abrogate pregnancy in primates. Contraception 1996; 53: 375-378. 30. Harrison PF, Rosenfield A. Contraceptive Research and Development Looking to the Future, National 18. Talwar GP, Shah 5. Mukeberjee S, ChabraR. Academy Press, Washington DC, 1996, pp 26-48. Induced termination of pregnancy by purified extracts of Azadirachta indica (Neem): mechanisms 31. Dhawan L, Ghosh D, Lalitkumar POL, Sharma D, involved. Am J Reprod lmmu"ol 1997; 37: 485-491. Lailley B, Overstreet JW. Sengupta J. Anti-nidatory Iltdiaa J Pbyaiol Pbannacol 2001, "5(1) Research in ReprodudioQ UI India 19

erred of ..acinally admUUltend (Ala' U.IO)_ "2. Shanal RK. D.. RP Etrecta oC STS·557 and 20 .acaiDID II amide in tbe rbelul monkey. Act·l OD sperm (uncholUl and aerum le.ela of ColitroccphOIi 2000; 10 Pre... Wlollterone io bonnet IDOllIr.ey rMacoeo ra4u"a). (Amtroupt14a 1992; "5: ....3-191 52 L.htkulDu PGL, Seo(upta J. DbawaD L, h.ar•• 0, Luley 8, Oventreet Jw. Gho.b D. Anti­ 43. Gh..h 0, De P, Se:OCupta J Luteal pba.e _.nan DJd.tory .fftld .fnCloally ad_uw:tered fu.m.aplliu "alroCeD i. 1I0t ....aliaJ for ImplallLatJolI and ID the rba,uI monke,. CO"'f'CUtphO,. 2000; mlUDtenance oC prepa.ntJ' (rollll lu..rrocate embryo 11:1 pf"ft6. trail.fer iQ lbe rhe.us monk., H"m Rt:prod 199",9.629-637, 33. IWddy PRK, Rao JM Ravanlble aatifertilit,. action of telleolterone propionate In buman males...... Ghoah 0, Sengupta J. ReceDt de.elopmenls in C(J,.troccptto,. 1972; 5: 295-301 endocrinoiocy and paracrinology of blutocyst Implantation in tbe primAte. /lum Reprod Update 9.. RaJalahhmi M, Ramahilhnan PRo 1998; lli1J-168. PharmacokineliQ and pharmacodynamiQ of a Dew 10nC·acrin, andrOll:eD luter malotenance of "5. KlImboj VP, Ray S, Dhllwlln 8N. Centchroman. phYliolo,ical androll:eD levea. (or" months .ner a Dru,. 0( T0d.4y 1992; 28: 227-232. aincle l.qjectIOD. Co,.,ra«p'IO,. 1989; "0: 399--412. ..6 Nhylnaod S, Kamboj VP. Chandrav.li, Das K, 55 Udaykumar TS, T)'ac1 A. a.,jalabmi 101, Des SN, Gupta K, Rohat(i R, Baveja R, Jina R, Matra R. Hashim S, Sajaj J5. Chalices In atructure and Shar",a U Conll'lIIcepli..e .mcac, and lafety of 'UDttIOIUl of prolLate by lon,-term administration CIlntc:hromall with biwHkl,-cuID.w...kl,. schedule. of aD JUKlroceo. tMloIIterone enanlhaleul rhe.us 10, CP Puri PFA Van Look (Ed.). C.rrent Collcepu _ookl1" 'MoeGUI "..lo'o). Anat Rec: 1998; 252; III Fertl1tty Rqulatloa Gad Reprodllc',on, Wile,. 137-6..5 E8lotern Lillllited • New Ace loternatioo.l Limited. New O•.lhi, 199..; pp 61-68 S6 Uda,aku.at" TS, Jl1yara,J DA, a.,jdakshmi M., Sbarw.a RS Culture of proaLate epltheUal cells of "7. Ro, SEarl, duelopm.atat atudiea witb tba rb.,uI monke,. on eslneellular matris clomlpheol eitr.te: Their lubaeqllent ...lidatioll lubatr.te" loOuence of ltenllda and IDSulin-lii. and utelllion. /,.d J &p BIoi 1992, 30: 931-946. 162~ crowth (acton. J End/Xr'lflol 1999; "'3--450. "8. Roy S. E.xperierl.ee in earl, developmental Itudies 37 Tyaci A, Rlijalauhmi M, Jeyaraj DA Sharma RS, with two fertility fIlculatlO;- druCI Health S"lIj JS Effects of lonc-term UN of tutoll.erone .-d Populotion: Pe"pedHJe. G,.d l ..uu enlnthel.e II Effect on lipida, high and loW' denaity 1998,21 170-182, lipoprotein cholelhrol And liver function .e9. Van Look PFA, von Hert'leD II. POlt-ovulatory parAmetera. Int J Androi 1999; 22: 3"'-355. methodl of fertility regulation: The emergence of 38. Tya,l A., Rnjalaltlhmi M, Jeyaraj DA, Sharma RS, antiprogc1t0ll:enl. In, PFA VnnLook PFA, G Perez­ Bl\Illj JS. Effecll of long.lerm adminiltration of Palaciol (Edl), ContraCflptiue Reuarch and lOltOlterollU enRnthate on glucolll mehlbolilm in DCI!Jelopment 1984 to 1994, Oxrord Univcrtlity Prell8, rhuua monkeYI. Contrace/llion 1999; 59: 333-337. New DeIhl, 1994; pp 151-201. 39. Kinll:er S, Pal PC. Rajalakalhmi M, Kumar Sharma 50. Purl CP, Patl! RK, Elger WA, Pongubaja JM. Effects of pro'l!Ilterone antall:0Qlll ZK 98.299 on early DNA, 8-.iiV JS. Effects of teltollerone buciclate precnlllncy and foetal outcome in bonnet monkeys. on tellicular and epididymal lperm functionl in ContNJtltp',orl 1990; 41; 197-205 bonnet monkeYI. ContNJ«ptlOn 1995; 52: 21-27. 51 Puri CP Kalkam RR. D'SGuu A. Eigu WA, Palil ..0 Ra,Jalaksbmi M. Illductian of aaoolpermia by KK. .ffcd of proceal.eronl antaconia", Iilopriatone hormollftl methodl in Don-human prim.to In, CP (ZK 98 13.. ) an induction of ",eoltrualion, Puri, PFA Van Look (I~(b). C"rrent CaNup" III iahibitiorl. of aldation and tumiDatioo of prepau.cy Tertility Rt:It"tatio,. oNd Reprod.c:ho'l. Wiley 1ft boonet monkeys. Bu,/. Rtprot/ 1992; "3: "37-443. Eutem Limned·New A,e lalernatiooaJ Limited, New Deih" 199", pp 117-132 52.. Puri CP, KatkaDl RR Vadl,oppula AD, Gop.lakriahnao K. Elpr WA, SiIlDloria Fa. PatH ... a"Jnmd., SS, Chaudhuzy J, Sharma RK, Das RP. KK. Contrac:epti..e potential of 8JI anhpf"OCel!Itin ZK Conlr.cepll.e poll!ntial .f S1'S-557·. feasibility 98.7S4-lIIduoed blacbde of follicu1occnuis with Itud, In male bonnet malllr.e, (Macaca radlaW, PSH and LH aod their differeotial ..ffec:t.I in haODet Colltroupllo,. 1990;"1 641-663, Dlanb,a. lild J Brp BIOI 1992, 30 987-995. 20 Sengupta Indian J PhYlliol Pharmacol 2001; 45(1)

53. Ishwad PC, Katkam RR, Hinduja IN, Chwalis:e K, in the rhesus monkey. Hum Reprod 1997; 12: Eiger W, Puri CPo Treatmen~ with a progesterone 575-582. antagonist ZK 98.299 delays endometrial development without blocking ovulation in bonnet 64. Ghush 0, Lalitkumar PGL, Wong VJ, Hendrickx AG, Sengupta J. Preimplantation embryo monkeys. Contraception 1993; 48: 57-70. morphology following early luteal phase anti­ 54. bhaw PC, Garde SV, Sheth AR, Puri CPo Expression nidatory treatment with mifeprilltone (RU486) of a 10.5 kDa inhibin in the endometrium of bonnet in the rhesus monkey. Hu.m Reprod 2000; 15: monkey on treatment with an antiprogestin Zk 180-188. 98.299: an immunochemical study. Contraception 1993; 49: 593-599. 65. Nayak NR, Ghosh 0, Sengupta J. Effects of luteal phase administration of mifepristone (RU486) and 55. Katkam RR, Gopalkrisan K, Chwalis:e K, Schilinger prostaglandin analogue or inhibitor on E., Puri cpo Onapristone(ZK 98. 299): a potential endometrium in the rhesus monkey. Hum Reprod antiprogestin for endometrial contraception. Am J 1998; 13: 047-1056. Obstet Gynecol 1995; 173: 779-787. 66. Nayak NR, Sengupta J, Ghosh D. Antinidatory 56. D'Sou:ea A, Hunduja IN, Puri CPo Antiprogestion effect of luteal phase administration of ZK 98. 299 and progesterone display differential mifepristone (RU485) is associated with changes binding characteristics in the human myometrial in endometrial prostaglandins during the cytosol. Biochem Biophys Acta 1992; 1125: 73-80. implantation window. Contraception ]998; 58: 111­ 57. 0' Souza A, Hinduja IN, Kodali S, Moudgi VK, Puri 117. CPo Interaction of newly synthesi:eed 67. Nayak NR, Ghosh 0, Lasley BL, Sengupta j. Anti­ antiprofesterone ZK 98. 299 with progesterone implantation activity of luteal phase mifepristone receptor from human myometrium. Arol Cdl administration is not mimicked by prostaglandin Biochem 1994; 139: 83-90. synthesis inhibitor or prostaglandin analogue in 58. Kumar Te, Shah KS, Chitlange SM, Hauri KT, the rhesus monkey. Contraception 1997; 55; 103­ Gopalkrishnan K, Vadigoppula AD, Vernkar VJ, 114. Borknr OM, Puri Cpo Effects of intranasal 68. Ghosh 0, Sharkey A, Charnock-Jones 5, Dhawan administration of norethisterone on L, Ohara 5, Smith SK, Sengupta J. Expression of foUiculogenesis, cervical music, vaginal cytology, vascular endothelial growth factor (VEGF) and endometrial morphology and reproductive placenta growth factor (PIGF) in concepts and endocrine profile in women. Con/raception 1991; endometrium during implantation in the rhesus 44; 245-257. monkey. Mol Hljtll Reprod 2000; 6: 935-941. 59. Ghosh 0, Nayak NR, Sengupta J. Effect of follicular 69. Lalitkumar PGL. Sengupta J. Ghosh D. Placental phase administration of mifepristone (RU486) on protein 14 in elldometrium during menstrual cycle blastocyst implnntation in the rhesus monkey. and effect of esrly luteal phase mifepristone Controception 1997; 56: 117-122. administration on its expression in implantation 50. Ghosh 0, Sengupta J. Anti·nidatory effect of stage endometrium in the rhesus monkey. Hum a single early post-ovulatory administration of Reprod 1998; 13: 3478-3486. mifepristone (RU486) in the rhesus monkey. Hu.m 70. Moudgal NR, Ravindranath N, Murthy as, Reprod 1993; 8: 552-558. Dighe RR, Aravindan GR, Martin F. Long­ 61. Gemzell·Daniellson K, Swahn ML, Svalander P, term contraceptive effie8cy of vaccine of ovine Bygdeman M. Early luteal treatment ""'ith follicle stimulation hormone in male bonnet mifepristone (RU486) for fertility regulation. Hum monkeys (Macaea radiata). J Reprod Fertil 1992; Rcprod 1993; 3: 870-873. 96: 91-102. 62. Ghosh D. Sengupta J, Hendriclot AG. Effect of 71. Srivastava A, Das RP. Sperm production and single-dose early luteal phase administration of fertility of bonnet monkeYII (Mucaea radiala) mifepristone (RU486) on implantation stage following immunization with ovine follicle endometrium in the rhesus monkey. Hum Reprod stimull\ting hormone. Ind J Exp Bio1 1992; 30: 1993; 9: 2026-2035. 574-577. 63. Ghosh 0, Kulaf PGLI, Sengupta J. Early luteal 72. Moudgal NR, Sairam MR, Krishnamurthy H. Khan phase administration of mifepristone inhibits H. Immunization of male bonnet monkeys (M. preimplantaion embryo development and viability radiata) with a recombinan~ FSH receptor Indian J Physiol Pharmacol 2001; 45(1) Research in Reproduction in India 21

preparation affects testicular function and fertility. rat Sertoli cells in culture. Mol Cell Elldorcrinol Endocrinotogy 1997; 138: 3065-3068. 1996; 18: 41-50. 73. Moudglll NR. Murthy GS, Prasanna Kumar KM. 83. Bhat KG, Malhotra P, Karande AA, Adiga PRo Martin F, Suresh R. Medhaffiurthy R. Pati S. Sehgal Immunohistochemical locali:tation of riboflavin S, Sazen:l. BN. Responaiveness of human male carrier protein in testicular cells of mammals.lnd volunteers to immunization with ovina follicle J Exp BioI 1995; 33: 12-16. stimulating hormone vaccine: results of a pilot study. Hum Reprod 1997; 12: 457-463. 84. Subramanian S, Adiga PRo Characterization and hormonal modulation of immunoreactive thiamin 74. Moudgal Jllyakumar M. Krishnamurthy HN, car~ier protein secreted by adult rat leyding cells Sridhar S, Krillhnamurthy H, Martin F. in vitro. J Endocrinol 1999; 162: 49-56. Development of male contraceptive vaccine-a perspective. Hum Reprud Update 1997; 3: 335-346. 85. Subramanian S, Adiga, PRo Characterization and hormonal modulation of immunoreactive thiamin 75. Gupta SK. Yurewiez EO, Af:talpurkar A. R:l.o KV. carrier protein secreted in immature rat Sertoli Gage DA, Wu FI, Sacco AG. Localization of epitopes cells in culture. J Steroid Bicx;hem Mol Bioi 1999; for monoclonal antibodies at the N-terminus of the 68: 23-30. porcine zona pellucide glycoprotein pZPC. Mol Reprod Dcu 1995; 42: 220-225. 86. Adiga PR, Subramanian S, Ran J. Kumar M. Prospects of riboflavin carrier protein (RCP) as an 76. Kaul R, Afzalpurkar A, Gupta SK. Strategies for antifertility vaccine in male and female mammals. designing an immunocontraceptive vaccine bused Hum Reprod Update 1997; 3: 325-334. on zona pellucida synthetic peptides and recombinant antigen. J Reprod Fcrtil (Suppf) 1996; 87. Talwar GP, Singh O. Pal R, Chatterjee N, Ulladhyay 50; 127-134. SN, Kaushie C, Garg S, Kaur R, Singh M. Chadmsekhllr S et a!. A birth control vaccine is on 77. Gupta SK, Chadha K, Harris JD, Yurewicz EO, the horizon for family planning. Anll Med 1993; 25: Sacco AG. Kolluri SK, Afzalpurker A. Mapping of 207-212. epitopes on porcille zona pellucida-3 al·pha by monoclonal antibodies inhibiting oocyte-sperm 88. Talwar GP, Singh O. Gupta SK, Hasnain SE, Pal interaction. Biol Reprpd 1996; 55: 410-415. R, Majumdar 5S, Vrati S, l'o'lukhopadhay A, Srinivall J, Deshmukh U et at The HSD·hCG vaccine 78. Gupta SK. Sharma M, Behera AK. Bisht R