Suppression of sperm function by depot medroxyprogesterone acetate and testosterone enanthate in steroid male contraception*t

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1 FERTILITY AND STERILITY Copyright" 8 The America Fertility Society Vol., No., April8 Prited i U.S.A. Suppressio of sperm fuctio by depot medroxyprogesteroe acetate ad testosteroe eathate i steroid male cotraceptio*t Frederick C. W. Wu, M.D.:j: R. Joh Aitke, Ph.D. Medical Research Coucil (MRC) Reproductive Biology Uit, Cetre For Reproductive Biology, Ediburgh, Scotlad Te ormal me were give three mothly itramuscular ijectios of mg of depot medroxyprogesteroe acetate (MPA) ad mg of testosteroe (T) eathate. Six me became azoospermic, while four remaied oligozoospermic, with a mea sperm desity of. ±. stadard error of the mea millio/mi. Zoa-free hamster oocyte peetratio was abolished i all oligozoospermic samples at the ed of treatmet. Twety of the oligozoospermic samples yieldig at least. to. millio motile spermatozoa showed a complete absece of oocyte peetratio. Seme parameters retured to ormal, although some took up to moths. These fidigs demostrated a atifertility actio of MP A ad T eathate o the fuctioal capacity of residual spermatozoa ad support the view that extreme oligozoospermia may be a teable target for reversible steroid male cotraceptio. Fertil Steril:, 8 The choice of reliable cotraceptio curretly available to me is limited to two methods, amely codoms ad vasectomy. The former is the most widely used reversible method, although its efficacy is user-depedet ad its acceptability had bee o the declie util the advet of the huma immuodeficiecy virus epidemic recetly. Vasectomy, though icreasigly i demad, is essetially a irreversible meas of male sterilizatio. Hece, there is a geuie eed ad demad for ew methods of male fertility regulatio equivalet i terms of efficacy, safety, ad acceptability to those beig employed i female cotraceptio. Received July, 88; revised ad accepted December, 88. * Supported by the World Health Orgaizatio Special Programme of Research, Developmet, ad Research Traiig i Huma Reproductio. t Preseted i part at the Fifth Europea Workshop o Molecular ad Cellular Edocriology of the Testis, Brighto, Uited Kigdom, April to, 88. :j: Reprit requests: Frederick C. W. Wu, M.D., MRC Reproductive Biology Uit, Chalmers Street, Ediburgh EH EW, Scotlad. Although the female cotraceptive pill has bee pheomeally successful i the past years, sex steroid supp,ressiori of spermatogeesis has ot, to date, bee vigorously pursued as a potetial male hormoal cotraceptive. This is because, eve with the most effective steroid regimes, azoospermia ca oly be reliably iduced i % to % of subjects, with the remaider beig severely oligozoospermic. Pregacies have bee reported i parters of me beig treated with depot medroxyprogesteroe acetate (MP A) ad testosteroe (T) eathate whe sperm couts were < X /ml, with half of the pregacies occurrig i those with sperm couts < X /ml. Furthermore, hypogoadotropic patiets, whe treated by goadotropis, may become fertile with sperm desities < X I ml. This is i cotrast to aother report i which oly seve uplaed pregacies occurred i almost voluteers usig various steroid suppressive regimes over a period of years. Sigificatly, oe of the pregacies occurred whe sperm desities were < X /ml. Give this ucertaity regardig the fertility of me who remai oligo- Vol., No., April8 Wu ad Aitke Sperm fuctio i male cotraceptio

2 zoospermic, potetial cotraceptive agets that produce a sigificat proportio of oazoospermic me are ulikely to be cosidered for cliical efficacy trials. The availability of i vitro tests of sperm fertilizatio potetial has provided a reliable laboratory method of assessig the fertility of oligozoospermic ifertile me. Such techiques would appear to be particularly suited to examiig the effectiveess of potetial cotraceptive regimes that do ot cosistetly iduce azoospermia. The cocept of evaluatig cotraceptive actio i terms of suppressio of sperm fuctio is oe that so far has ot bee seriously explored. The aim of this study was to assess the i vitro fertilizig capacity of residual spermatozoa from me redered oligozoospermic by a combiatio of depot MP A ad T eathate usig the iterspecific hamster oocyte peetratio (HOP) assay. Subjects MATERIALS AND METHODS Te healthy ormal fertile male voluteers, mea age. ±.8 (stadard deviatio) years were recruited from the geeral commuity, postatal wards of the Simpso Memorial Materity Pavilio ad the Family Plaig Cliic, Ediburgh. All subjects had previously fathered childre ad had ormal sperm desity (> X / ml), motility (>%), morphology (>% ormal) ad HOP rates (>%) i seme samples, i compariso with a fertile populatio of me evaluated i our laboratory. All but oe subject was married at the time of study. They were advised to cotiue with a effective alterate form of cotraceptio for the duratio of the study. Approval for the project was grated by the local Ethical Committees ad writte iformed coset was obtaied. Treatmet Each subject received itramuscular (IM) ijectios of mg of depot MPA (Upjoh, Kalamazoo, MI) ad mg of T eathate (Scherig, Berli, West Germay) at -weekly itervals o three occasios durig a -week period of treatmet. I additio, two placebo (salie) ijectios were admiistered i a sigle-blid fashio at - weekly itervals for 8 weeks before commecig the steroid treatmet regime i five subjects. Assessmet ad Follow-up Each subject was reviewed ad examied at itervals of weeks durig treatmet ad recovery, which cotiued for moths after the last ijectio. Sexual activity was recorded i daily logs to moitor ay variatios i sexual fuctio. At each review, seme aalyses ad HOP tests were performed. Blood samples were obtaied for estimatio of plasma cocetratios of luteiizig hormoe (LH), follicle-stimulatig hormoe (FSH), T, MPA, estradiol (E), ad sex hormoe-bidig globuli (SHBG) immediately before ad,, ad days after each ijectio durig the placebo ad treatmet periods. Durig recovery, blood samples for hormoal estimatios were take at -weekly itervals i the first moths ad at -weekly itervals i the last moths. Routie hematology, biochemistry ad liver fuctio tests were carried out at -weekly itervals throughout the study. Aalyses of Seme Seme samples were obtaied by masturbatio after a period of to days of ejaculatory abstiece. Seme volume, sperm desity, ad motility were aalyzed by stadard techiques. Zoa-free hamster oocyte peetratio tests were performed o all seme samples cotaiig at least. millio motile sperm accordig to the method of Aitke et al. 8 Sufficiet umbers of spermatozoa ad oocytes were icorporated ito the assay i order to provide a valid assessmet of huma sperm fuctio, ad the results were expressed i terms of the percetage of oocytes peetrated at a cocetratio of millio motile spermatozoa/ml. Hormoe Assays Luteiizig hormoe, FSH, T, MPA, ad E were measured by radioimmuoassay (RIA).U- Sex hormoe-bidig globuli was measured by the method of Roser. Durig the placebo phase, oly LH, FSH, ad T measuremets were carried out o samples obtaied o day 8. Durig the treatmet ad after treatmet periods, LH, FSH, ad T were estimated i all blood samples, but MP A, E, ad SHBG were measured o selected samples oly, as idicated i the results. Statistical Aalyses Results are expressed as mea (± stadard error of the mea [SEM]). Data were subjected to aaly- Wu ad Aitke Sperm fuctio i male cotraceptio Fertility ad Sterility

3 Table Distributio of Semial Parameters i Te Subjects Treated by Depot MP A ad T Eathate Treatmet moth Post- Post- Post- Post- Post- Post Pretreat- Pretreat- Placebo Placebo Treat- Treat- Treat- treat- treat- treat- treat- treat- treatmet met met met met met met met met met met Sperm desity (millio/ml) > - < Azoospermia 8 Total sperm cout (millio) > - < Azoospermia Motile sperm cout (millio) > - < Sperm motility(%) > - < Azoospermia 8 Hamster oocyte peetratio (%) > < Isufficiet/ Techical problems 8 Uliquefied seme. Hamster oocytes uavailable. ses of variace for repeated measures with post hoc comparisos usig Newma-Keuls test. The twotailed t-test was used to compare results betwee azoospermic ad oligozoospermic subjects. Statistical sigificace was set at P <.. RESULTS All te subjects completed the study. Four of the plaed seme samples were ot collected, while the HOP assay was uable to be performed o 8 collected samples because of oliquefied seme (), isufficiet eggs available (), ad iadequate umber of motile sperm () (Table ). The two placebo ijectios i five me durig the first 8 weeks produced o sigificat chages i mea sperm couts, HOP rates (Fig. ), or plasma T, LH, ad FSH cocetratios (Fig. ). Throughout the -moth study, a sigificat (P <.) correlatio was observed betwee the HOP ad sperm desity (r =.), total sperm cout (r =.), motile sperm cout (r =.), ad percetage motility (r =.) i the samples i which all of these parameters were measured. Durig treatmet (Fig. ), mea sperm desity, total sperm cout, ad motile sperm cout declied sigificatly from the ed of the secod moth reachig the adir (. ±. millio/ml,. ±. ad. ±. millio, respectively) at the ed of the third moth. Mea HOP rate decreased i parallel with sperm couts from over % i pretreatmet samples to ±.% (ot sigificat) ad. ±.8% (P <.) at the ed of the first ad secod treatmet moth, respectively. After the third treatmet, six subjects had become azoospermic, while i the four oligozoospermic me, mea sperm desity was. ±. (. to.) millio/ml; total ad motile sperm couts were. ±.8 (.8 to.) ad. ±. ( to.8) millio, respectively. Oe of the oligozoospermic samples was devoid of motile sperm, while the remaiig showed zero oocyte peetratio capacity (Table ). Betwee ad oazoospermic ejaculates from each of the te subjects showed abset oocyte peetratio durig Vol., No., April8 Wu ad Aitke Sperm fuctio i male cotraceptio

4 :.. 8 ~~~ N.. I ~~'~P Moths N. 8 II 8 8 Figure The effects of IM depot MP A mg ad T eathate mg ('f') mothly for moths o mea ± SEM sperm desity, total ad motile sperm couts, sperm motility ad (HOP) rate i te ormal me. Five received placebo (\) for moths. Values bracketed idicate sigificat differece from pretreatmet-for details, see Results. the treatmet ad the first post-treatmet moths. I these samples, the mea sperm desity was. ±.8 millio/ml (< to.); total ad motile sperm couts were. ±. (. to.) ad. ±. (O to.) millio, respectively. Ofthe oligozoospermic samples with sperm desities < X /ml durig treatmet ad recovery, showed abset oocyte peetratio, yielded <. X /ml of motile sperm, which was the miimal cocetratio required for the oocyte peetra~ tio assay, oe was ot tested due to oavailability of hamster oocytes, ad oly oe sample with sperm desity of X /ml (Subject post-treatmet moth ) showed % oocyte peetratio. Sigificatly (P <.) decreased sperm motility of.8 ±. (O to ) ad. ± 8. ( to %) was observed oly i the third treatmet ad the first post-treatmet moth, respectively, compared with pretreatmet motilities of over %. Durig recovery, mea total sperm cout, sperm desity, ad the cocetratio of motile spermatozoa retured to withi the pretreatmet rage i the sixth post-treatmet moth. By the fourth post-treatmet moth, mea HOP rates were ot sigificatly differet from pretreatmet levels (Fig. ). Oly oe subject failed to produce at least oe seme sample with sperm desity > X / ml i either the fifth or sixth post-treatmet moth (Table ). This ma's recovery coicided with aperiod of uusually high sexual activity prompted by a ewly formed relatioship. His seme aalyses evetually retured to ormal pretreatmet values, while his parter coceived at moths after the last treatmet; a ormal female ifat was delivered at full term. The parter of subject coceived ad moths after his last treatmet. Both pregacies eded i miscarriage at weeks. Subsequetly, a third pregacy wet to term, deliverig a ormal female ifat. I three subjects, HOP capacity remaied uder % throughout the -moth recovery period (Table ). Follow-up studies i the esuig moths cofirmed the retur to ormal HOP rates i two me, but the last cotiued to show abormal results of % to %. I this subject, the HOP rate had already declied ito the subormal rage durig the placebo phase. Subject (who received additioal T ijectios durig treatmet) reestablished ormal HOP capacity as early as the first moth after his last treatmet, whereas the majority oly regaied sperm fuctio i the third or fourth moth (Table ). Mea plasma T ad MPA cocetratios icreased sigificatly (P <.) o day after each ijectio oft eathate ad depot MPA (Fig. ). Testosteroe the declied to the lower limit of the ormal rage (. ±., 8. ±., ad. ±. moljl for first, secod, ad third treatmet moth, respectively) immediately before the ext ijectio. Medroxyprogesteroe acetate showed evidece of accumulatio with repeated doses, the plasma level o day 8 of the third treatmet cycle beig sigificatly higher tha the pretreatmet level (P <.) ad that of day 8 of the first treatmet cycle. Plasma MP A was still detectable i three me at the ed of the th post-treatmet moth. Both LH ad FSH were suppressed to the limit of detectio by day followig the first treatmet. Luteiizig hormoe icreased sigificatly o day 8 from the adir of the first ad secod moth (P <. ad., respectively), but FSH rose sigificatly o day 8 oly after the first ijectio. Testosteroe, LH, ad FSH remaied suppressed for at least moths after treatmet, oly Wu ad Aitke Sperm fuctio i male cotraceptio Fertility ad Sterility

5 Prortr atmort PlacebO Pra-troratmorl Placebo,, E E ~ ' ~ ~ z ~ ~ ' ~ o '' r , , r ~ f Q ~ 8 ~ l i I' I < ' ~ " ~ ' MONTHS Figure Chages i MP A, T, E, LH, FSH, ad SHBG before, durig, ad after IM depot MP A mg ad T eathate mg ('f') or placebo (\) at mothly itervals for moths i te ormal me. Values bracketed idicate a sigificat differece from pretreatmet-for details, see Results. * deotes a sigificat icrease from pretreatmet or adir. regaiig pretreatmet cocetratios at the fifth or sixth post-treatmet moth. Estradiol followed the same patter as that oft ad MPA, while SHBG was sigificatly (P <.) suppressed compared with pretreatmet durig the secod ad third moths of treatmet (Fig. ). The four subjects who remaied oligozoospermic after three doses of depot MP A ad T eathate compared with the six azoospermic subjects were ot sigificatly differet i terms of age, pretreatmet sperm desities, or HOP rates. The oligozoospermic subjects had lower LH, FSH, ad T cocetratios tha the azoospermic me durig treatmet; but the differeces did ot reach statistical sigificace. Oe of the first five patiets, who did ot receive placebo, reported a declie i sexual fuctio i the first moth that was immediately alleviated by booster T eathate ijectios i the secod ad third moths. However, the lack of adequate baselie iformatio ad placebo-cotrolled data i these five subjects precluded comparative statistical aalyses to be carried out, eve though there was a suggestio that frequecy of coitus ad morig erectios were higher durig tha after treatmet. I the other five subjects, the first placebo ijectio produced a trasiet icrease i frequecy of morig erectio ad sexual thoughts, while subsequet treatmet also was associated with a sigificat icrease i the same measures, which retured to pretreatmet levels o withdrawl. Aother subject oticed a trasiet fall i his performace i competitive ruig durig treatmet. A mior ad trasiet rise i gammaglutamyltraspeptidase was recorded i oe subject whose level was at the upper limit of ormal before treatmet. DISCUSSION The preset study has demostrated for the first time a suppressio of i vitro fertilizig potetial i me with oligozoospermia iduced by shortterm treatmet with depot MP A ad T eathate. After three mothly admiistratios of the steroid combiatio, six out of te subjects became azoospermic, while over % of oazoospermic ejaculates with sperm desities of < X /ml failed to exhibit ay capacity for fertilizatio i vitro. The zoa-free HOP assay assesses the global ability of the spermatozoa to capacitate, acrosomereact, ad geerate a fusogeic equatorial segmet capable of fusio with the vitellie membrae of the oocyte. The demostratio of defective sperm fuctio by the HOP assay i steroid-iduced oligozoospermia therefore does ot idetify which of the above processes have bee compromised. Furthermore, other aspects of sperm fuctio such as Vol., No., April8 Wu ad Aitke Sperm fuctio i male cotraceptio

6 sperm movemet characteristics ad iteractio with zoa pellucida were ot examied i these subjects. Our results did reveal, however, that a major compoet of the fertilizatio process, amely sperm-oocyte fusio, was abset or severely impaired i all oligozoospermic subjects durig or immediately after the cessatio of depot MP A ad T eathate treatmet. Although the predictive value of positive HOP results for i vivo fertility i oligozoospermic me remais somewhat cotroversial, there is a cosesus that a zero HOP score is highly correlated with a egative fertility outcome, provided that appropriate measures have bee take to miimise the false egative rate. It is kow that the cocetratio of motile spermatozoa ca ifluece the oocyte peetratio rates, ad i severely oligoastheozoospermic samples, the miimal cocetratio of motile sperm to esure positive oocyte peetratio may ot be attaied ad false egative results thus occur. It might therefore be argued that the low or egative fertilizatio rates ecoutered i the severely oligozoospermic subjects i this study are the cosequece of iadequate sperm umbers rather tha of ay idepedet defects i the fuctioal competece of the spermatozoa. To circumvet these practical ad iterpretative difficulties, we have used a calcium ioophore, A8, for the iductio of the acrosome reactio i order to icrease the level of sperm-oocyte iteractio. We also have used the Poisso-Gamma distributio model to provide thresholds of gamete cocetratios that must be achieved i order for ay give level of oocyte peetratio (icludig zero) to idicate a sigificat declie i sperm fuctio relative to the ormal fertile populatio. To maitai a adequate gamete cocetratio i the assays for this study, oocyte umbers were icreased i order to compesate for ay deficits i the desity of motile spermatozoa. To further stadardize the HOP assay for oligozoospermic samples yieldig variable motile sperm cocetratios, all results i this study were corrected to a costat motile sperm cocetratio of millio/ml usig formulas based o the Poisso model. Together, these safeguards esured as far as possible that the observed declie i fertilizig potetial i our subjects with steroididuced oligozoospermia was idicative of a geuie disruptio of fuctioal itegrity i the residual spermatozoa, idepedet of the reductio i sperm umbers. The repeatedly egative oocyte peetratio results i oligozoospermic subjects would led further support to the cotetio that the state of steroid-iduced oligozoospermia may costitute a sufficietly safe target for male cotraceptio. It is ow appropriate to coduct Phase cliical trials to demostrate the cotraceptive efficacy of sex steroid regimes ad to ascertai the upper limit of sperm desity compatible with effective suppressio of fertility. Whether other steroid regimes ca reproduce the same effects o sperm fuctio as depot MP A ad T eathate also remais to be elucidated. The oly oligozoospermic ( < X /ml) sample that showed ay oocyte peetratio (.%), cotaiig X spermatozoa/ml, was from Subject durig the secod post-treatmet moth. His uusually rapid ad efficiet recovery of sperm fuctio was quite ulike the rest of the group ad may be associated with the fact that this was the oly subject to have received booster ijectios oft eathate. This result also uderlies the ability of our HOP assay to assess sperm fuctio i the presece of low sperm umbers. Sex steroid suppressio of spermatogee.sis higes o the ihibitio of pituitary goadotrophi ad Leydig cell testosteroe productio. 8 This deprives the semiiferous tubules of the high itratesticular testosteroe cocetratio required for quatitatively ormal spermatogeesis. 8 Our results suggest that this type of edocrie maipulatio of the testis ca also produce defects i sperm fuctio, although the precise mechaisms ivolved are ukow. A similar suppressio of hamster oocyte peetratio ability was recetly reported i mokeys redered oligozoospermic by goadotropi-releasig hormoe atagoist (GRHa)/ suggestig that T deprivatio may be the key. Itratesticular deficiecy of T may impair Sertoli cell fuctio or prevet ormal codesatio of sperm heads, thus leadig to defective sperm maturatio ad failure i the subsequet expressio of sperm fuctio. The time course of the effect of depot MP A ad T eathate o sperm fuctio would be i keepig with actios o the testis (spermatogeesis ad/or spermiogeesis) rather tha o the epididymis. The fidigs i Subject also suggest that high T cocetratios as a result of booster ijectios may have had certai protective effects that hasteed the retur of ormal sperm fuctio. This may be similar to the protective effect of testosteroe observed durig GRH-a suppressio of spermatogeesis. Medroxyprogesteroe acetate, i additio to goado- Wu ad Aitke Sperm fuctio i male cotraceptio Fertility ad Sterility

7 tropi suppressio, also may act as a atiadroge, thus coferrig a additioal atifertility effect either at the testicular or epididymal level, although a direct effect o spermatozoa caot be excluded. With or without T, MP A ca also reduce the testicular ad epididymal cotet of adrogebidig protei via a direct actio o Sertoli cells. The reversibility of short-term suppressio of testicular fuctio has bee cofirmed i this study. All subjects attaied ormal sperm couts, although full recovery required moths i oe subject. Oocyte peetratio ormalized i ie out of te subjects by 8 moths after treatmet. The remaiig subject, although ormal before treatmet, first showed low egg peetratio durig placebo admiistratio, which did ot retur to the ormal rage at moths after treatmet. This should ot, therefore, be regarded as orecovery. Two subjects have sice fathered ormal ifats. Despite the low ormal plasma T i the last weeks of the treatmet cycle, oly oe subject (who had marital difficulties durig the study ad subsequet separated from his wife) reported a trasiet declie i sexual fuctio. Iterestigly, a icrease i some reported measures of sexual fuctio was observed durig treatmet i the placebocotrolled group. This may be related to the suppressed SHBG makig more free T available. Although the absece of major side effects over the course of this study is oteworthy, it must be emphasized, however, that the duratio of treatmet was short ad the effects of loger-term exposure of me to exogeous sex steroids remai to be determied. Of particular cocer is the lowerig of high-desity lipoprotei cholesterol observed i healthy me followig withdrawal of depot MP A ad T eathate treatmet, presumably related to the effects of uopposed progestage. I summary, this study has demostrated a suppressio of sperm fuctio idepedet of the reductio i sperm umbers i me redered oligozoospermic by sex steroids. This additioal atifertility actio should lead to a reexamiatio of the cotraceptive efficacy ad targets achievable by steroid suppressio of spermatogeesis. Ackowledgmets. We thak Ms. Vivie Turbull for efficiet maagemet of the study, Ms. Nacy Loudo ad staff of the Ediburgh FPA cliic for help i recruitmet, Ms. Jae Clarkso for performig the HOP assay, Ms. Vicky Sweetig for goadotrophi ad testosteroe RIAs, Ms. Jea Wickigs for estradiol RIA, Dr. Ke Fotherby for advice o MPA RIA, ad Ms. Pam Warer ad Dr. Mark Watso for help with statistics. REFERENCES. Me-ew focus for family plaig programmes. Popul Rep [J]:8, 8. Shearer SB, Alvarez-Sachez F, Aselmo J, Breer P, Coutiho E, Latheo-Faudes A, Frick J, Heiild B, Johasso EDB: Hormoal cotraceptio for me. It J Adrol(Suppl):8, 8. Barfield A, Melo J, Coutiho E, Alvarez-Sachez F, Faudes A, Brache V, Leo P, Frick J, Bartsch G, W eiske W, Breer P, Mishell D, Berstei G, Oritz A: Pregacies associated with sperm cocetratios below millio/ml i cliical studies of a potetial male cotraceptive method, mothly depot medroxyprogesteroe acetate ad testosteroe esters. Cotraceptio :,. Sheris RJ: Hypogoadotrophic hypogoadism i me. I Curret Therapy i Edocriology. Edited by DT Krieger, CW Bardi. Philadelphia, Decker, 8, p. Paulse CA, Bremmer WJ, Leoard JM: Male cotraceptio: cliical trials. I Advaces i Fertility Research. Edited bydrmishell,jr. New York, Rave Press, 8,p. Paulse CA: Aother look at the sperm peetratio assay. Fertil Steril :, 8. Belsey MA, Eliasso R, Gallegos AJ, Moghissi KS, Paulse CA, Prasad MRN: Laboratory Maual for the Examiatio of Huma Seme-Cervical Mucus Iteractio. Sigapore, Press Cocer, 8 8. Aitke RJ, Ross A, Hargreave T, Richardso D, Best F: Aalysis of huma sperm fuctio followig exposure to the ioophore A8. J Adrol:8, 8. Aitke RJ, Elto RA: Applicatio of a Poisso-Gamma model to study the ifluece of gamete cocetratio o sperm-oocyte fusio i the zoa free hamster egg peetratio test. J Reprod Fertil 8:, 8. Aitke RJ, Elto.RA: Applicatio of Poisso distributio theory to the zoa-free hamster oocyte peetratio test to assess sperm fuctio of me with astheozoospermia. J Reprod Fertil :, 8. Huter WM, Beie JG: Reductio of o-specific serum resposes i huma pituitary goadotrophi radioimmuoassay. J Edocriol8:,. Corker CS, Davidso DW: Radioimmuoassay of testosteroe i various biological fluids without chromatography. J Steroid Biochem :, 8. Shrimaker K, Sazea BN, Fotherby K: A radioimmuoassay for serum medroxyprogesteroe acetate. J Steroid Biochem :, 8. Hillier SG, Tsois CG, Wickigs EJ, Eide KA: Ihibitio of FSH stimulated graulosa cell fuctio by a sythetic fragmet of the porcie ihibi a-subuit: evidece for ivolvemet of GRH receptors. J Edocriol :R, 8. Roser W: A simplified method for the quatitative determiatio of testosteroe oesradiol bidig globuli activity i huma plasma. J Cli Edocriol Metab :8,. Y aagimachi R: Zoa free hamster eggs: their use i assessig fertilizig capacity ad examiig chromosomes of huma spermatozoa. Gamete Res :8, 8 Vol., No., April8 Wu ad Aitke Sperm fuctio i male cotraceptio

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