Studies on the Male Antifertility Constituents of Tripterygum hypoglaucum(Lévl.)Hutch.

昆明山海棠茎皮乙醇提取物中雄性抗生育活性成分的分离,显示氯仿部分及正丁醇部分为活性部分。用硅胶柱对这两部分进一步分离后,得到雷藤素乙(Ⅰ)、雷藤素甲(Ⅱ)、雷酚二萜酸(Ⅲ)、雷酚萜醇(Ⅳ)、3-oxo-olean-12-en-29-oicacid(Ⅴ)、齐墩果酸(Ⅵ)、3β,22α-dihydroxy-△^(12)-oleanen-29-oic acid(Ⅶ)、3-乙酰氧基齐墩果酸(Ⅷ)、雷公藤内酯甲(Ⅸ)、雷公藤次硷(Ⅹ)、胡萝卜甙(Ⅺ)、β-谷甾醇(Ⅻ)、1-表儿茶素(ⅩⅢ)及富马酸(ⅩⅣ)。其中3-oxo-olean-2-en-29-oic acid,齐墩果酸及富马酸为首次从雷公藤属植物中发现。雷藤素乙,雷酚萜醇,3-β,22α-dihydroxy-△^(12)-oleanen-29-oic acid 及胡萝卜甙为首次从昆明山海棠中分离得到。药理筛选显示雷藤素乙及雷藤素甲对于雄性大鼠具有可逆性雄性抗生育活性,其有效剂量分别为每天200及30μg/kg。

Antifertility effects of Azadirachta indica methanol seed extract on canine spermatozoa in vitro

Objective:To investigate the effects of Azadirachta(A.)indica methanol seed extract on canine male fertility in vitro.Methods:The effect of the A.indica methanol seed extract(2.5,5.0,10.0 and 20.0 mg/mL,respectively)on canine spermatozoa was evaluated in vitro at 1.0,2.5,5.0 and 10.0 min post-treatment.The two controls were comprised of sperm sample in phosphate-buffered saline,and sperm sample in phosphate-buffered saline and 1%dimethyl sulfoxide,respectively.The experiment was repeated(n=3)in duplicates with semen collected from 3 adult Basenjis.The effect of A.indica methanol seed extract treatments on canine spermatozoal total and progressive motility(phase-contrast microscopy),sperm vitality(eosin-nigrosin vital staining method)and total abnormalities(phase contrast microscopy,eosin-nigrosin staining and Papanicolaou staining methods)were determined.Results:A.indica methanol seed extract treatment caused a concentration and time-dependent decrease in sperm percentage total motility,progressive motility and vitality.The absence of sperm revival post-treatment provided evidence of a permanent or irreversible incapacitation of sperm by A.indica methanol seed extract.Conclusions:The inhibition of sperm motility and significant spermicidal effect observed post-treatment suggest that A.indica methanol seed extract may produce a potent antifertility or contraceptive effect in dogs if applied as an intra-vaginal cream pre-coitus.

Evaluation of Antifertility Effect and Recovery of the Seed Oil Constituents of Iranian Species of Melia Azadrach L. in Male Rats

Objective To evaluate the fertility effects of seed extract of Melia azadarach L. treatment effects on fertility indices in male Wistar rats. Methods The seed oil extract had been prepared according to conventional methods. The rats were randomly divided into three study groups. Groups A and B received graded doses of 50 mg/kg and 100 mg/kg body weight of the extract oil, respectively on daily basis for 60 d. Animals in control group received I ml of maize oil. At the end of 60 d and 3 months treatment period, 6 animals per group were randomly selected and fertility was evaluated with mating test. GSI （gonadosomatic index） sperm motility, sperm viability, ESR （epididymal sperm reserves）, DSP（daily sperm production） and testosterone concentration were also assessed.Results In the first stage, a significant reduction infertility indices especially in higher dose was observed compared with the control. During the next stage, the significant increase in fertility indices are the indication of reasonable recovery and reversibility of extract activity. Conclusion The seed oil of Melia azadarach L. has antifertility activity, but its effects is reversible.

Preliminary study on antifertility activity of Enicostemma axillare leaves and Urena lobata root used in Indian traditional folk medicine

Objective:To evaluate the possible antifertility activity of Enicostemma axillare(E.axillare) leaves and Urena lobata(U.lobata) root in adult male Wistar albino rats.Methods:Six groups of rats were treated with ethanolic(70%v/v) extracts of E.axillare(375 and 750 mg/kg body weight) and U.lobata root(300 and 600 mg/kg body weight) once daily for 55 days.Control groups received the distilled water and vehicle.All the treated rats had corresponding recovery groups.At the end of each treatment periods,animals were killed and organ weights,sperm characteristics,testicular and epididymal biochemicals as well as testicular enzymes were assessed.Results:The E. axillare and U.lobata at tested doses did not decrease body weight,whereas the weight of testes, epididymides and seminal vesicles were significantly(P【0.01) reduced.Significantly(P【0.01) more reductions in the sperm motility,viability and counts,epididymal and testicular protein contents were noted in the rats treated with higher dose of both the plants.Both the plants at the higher dose caused a marked increase(P【0.01) in sperm morphological abnormalities,testicular cholesterol and ascorbic acid contents were remarkably increased(P【0.01),while,the activities of testicular glucose-6-phosphate dehydrogenase(G-6-PDH) andΔ~5-3β-hydroxy steroid dehydrogenase(Δ~5-3β-HSD) were significantly reduced(P【0.01).However,reversal of these changes occurred after 55 days of treatment withdrawal.Conclusions:This study suggests that the ? axillare leaves and U.lobata root reversibly inhibited spermatogenesis and steroidogenesis indicating reversible antifertility activity which could partially support the traditional of these plants as male contraceptives.

Antifertility effects of 60-day oral gavage of ethanol extract of Spondias mombin leaves in guinea pigs: A biochemical, reproductive and histological study

Objective:To investigate the impact of ethanol extract of Spondias(S.)mombin leaves on the biochemical parameters of testicular function,hormonal profile,sperm profile and reproductive outcomes of guinea pigs.Methods:Sexually matured male[(1000.40±8.12)g]and female[(810.00±7.09)g]guinea pigs were used.In testicular function study,a total of 24 male guinea pigs were randomised into 4 groups of 6 guinea pigs each.Group A(control)was orally administered 1 mL of physiological saline,once daily for 60 days while groups B,C and D were treated like the control group except that they were orally administered 100,250 and 500 mg/kg body weight of ethanol extract of S.mombin leaves.For the fertility study,the same animal groupings and treatments in the testicular function study were adopted.The male guinea pigs were paired with the females(1:1)and afterwards examined for pregnancy outcomes.Results:The ethanol extract of S.mombin leaves contained saponins,alkaloids,flavonoids,tannins,steroids,phenolics,phlobatannins,cardiac glycosides,cardenolides and dienolides with saponins(4.80 mg/mL)occurring the most whilst cardenolides and dienolides(0.08 mg/mL)were the least.The ethanol extract of S.mombin leaves significantly and dose dependently reduced the activities of alkaline phosphatase,glutamate dehydrogenase,3-hydroxy-3-methylglutaryl coenzyme A reductase,malic enzyme,17-β-hydroxysteroid dehydrogenase,lactate dehydrogenase,catalase and superoxide dismutase as well as levels of testosterone,glycogen,total protein and ascorbic acid in the testes when compared with the control group(P<0.05).All the doses of ethanol extract of S.mombin leaves also reduced the levels of sorbitol dehydrogenase,3-β-hydroxysteroid dehydrogenase,glucose-6-phosphate dehydrogenase and sialic acid whereas the levels of testicular acid phosphatase,gamma glutamyl transferase and cholesterol increased dose dependently(P<0.05).The serum luteinising hormone,testosterone and estradiol were reduced after the administration of ethanol extract of S.mombin leaves whereas levels of serum follicle stimulating hormone increased significantly.The 100 and 200 mg/kg body weight of ethanol extract of S.mombin leaves increased the testosterone/estradiol ratios whilst the 500 mg/kg body weight of ethanol extract of S.mombin leaves decreased it.The sperm motility,sperm count,normal sperm morphology,sperm density,sperm viability and semen viscosity were significantly reduced in the ethanol extract of S.mombin leaves-treated guinea pigs(P<0.05)whereas the head-,tail-and neck-defects increased significantly when compared with the control group(P<0.05).In contrast,the semen volume and pH were not significantly altered by the ethanol extract of S.mombin leaves(P>0.05).The ethanol extract of S.mombin leaves at both 100 and 250 mg/kg body weight significantly reduced the total number,circumference,weight and length of the pups whereas the 500 mg/kg body weight of ethanol extract of S.mombin leaves-treated rats did not produce any pup.The 250 and 500 mg/kg body weight of ethanol extract of S.mombin leaves induced degenerative and necrotic changes in the seminiferous tubules with vacuoles in the germinal epithelium and a few to complete absence of spermatozoa.In all of these,the 500 mg/kg body weight of ethanol extract of S.mombin leaves produced the most pronounced alteration on the parameters.Conclusions:S.mombin leaves have induced infertility in the male guinea pigs via endocrine dysregulation,anti-spermatogenic activity,testicular dysfunction and oxidative stress and made possible by the presence of saponins,alkaloids,flavonoids,tannins,steroids,phenolics,and cardiac glycosides.

Antifertility properties ofCentella asiatica ethanolic extract as a contraceptive agent: Preliminary study of sperm proteomic

Objective:To investigate the antifertility properties ofCentella asiatica L. ethanolic extract and identify the sperm proteomic changes in treated male rats.Methods: A total of 32 male Sprague-Dawley rats were divided into two groups: control group (n=16) received distilled water and treatment group (n=16) which received 300 mg/kg ofCentella asiatica L. extract for 42 d. After the treatment period, the number of implantation sites was recorded and the sperm proteomic changes were analysed by 2D gel electrophoresis. In addition, the expression of protein spots was quantified by MALDI-TOF analysis.Results:Centella asiatica L. extract resulted in low number of implantation sites in the treatment group (100.00±2.82) compared to the control group (183.00±2.14). The percentage of infertile male rats in the treatment group was higher (43.75%) compared to the control group (18.75%). Proteomic analysis showed the expression of protein spots identified in the treatment group decreased with 234 spots compared to the control group with 282 spots.Conclusions:The results revealed that there was antifertility activity in the male rats with the administration ofCentella asiatica L. ethanol extract and the identified proteins could provide understanding on the adverse effect ofCentella asiaticain male reproductive system.

Tripterygium Wilfordii： Structure，Chemistry and Activityof Male Antifertility Constituents

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Analysis of Oleanolic Acid and Ursolic Acid, Potential Antifertility Agents in Moringa （Moringa oleifera） Seed

Oleanolic acid （OA） and ursolic acid （UA） are isomeric tritcrpenes that are difficult to separate chromatographically. Studies have indicated that both OA and UA have antifertility properties in several livestock species, suppressing attainment of puberty, gonad function and gamete production. Being able to determine the presence and quantity of the OA and UA in moringa and pawpaw seeds will allow for calculation of optimal inclusion levels of these acids in animal diets for antifertility-activity. The purpose of the study was thus to quantify the levels of OA and UA in Moringa and pawpaw seed powder by means of ultra-high performance liquid chromatography with electrospray ionization tandem mass spectrometry （UHPLC-ESI-MS/MS）. Only OA was detected in moringa seeds at a concentration level of 0.508 p.g/g ＋ 0.032 pg/g dry weight. UA and OA could not be detected in pawpaw seeds used in this study. The presence of OA in moringa seeds suggest that the seeds can be included in tilapia diets as a potential antifertility treatment to control precocious breeding in tilapia culture in small scale farming systems in Sub-Saharan Africa. Further studies are warranted to determine the levels of OA and UA composition in moringa seeds from other parts of the Sub-Saharan Africa （other than Zambia）.

Im m unoregulatory Effects of Triptergium W ilfordii Hook at Antifertility Dose on Splenic NK Cells Activityin Male Mice

Effects of Tripterypium Wilfordii Hook f (TWH) on sperm atozoa in the epi- didym is and splenic NK cells activity in m ale m ice w ere observed using MTT assay and silver impregnation m ethods. The results show ed that the density, viability and m otility of the epididym alsperm atozoa in the experim entalgroupstreated w ith TWH w ere m ore significantly reduced than those in the controlgroup (P< 0.01). The head sw elling, head separation from tailin the groups treated w ith TWH w ere observed. The inhibition of splenicNK cellsactivity in m iceby TWH w asdose-dependent. Inhi- bition by TⅡand TWH athigh dose on the NK cells activity w as significant (P< 0.01 and P< 0.05), w hileinhibitory effectsof TWH atinterm ediateand low doseson the NK cells activity w ere notobserved (P> 0.05). Itw as concluded thatTWH at low er antifertility dose did not significantly inhibit the splenic NK cells activity. It m ightbe usefulforevaluating thetherapeuticeffectsof TWH in futureclinicalprac- tice.

Inhibition of Acrosomal Enzymes by Gossypol Is Related to Its Antifertility Action

To investigate into the mechanisms underlying the irreversible sterility induced by gossypol, we studied the relationship between its inhibitory action on acrosomal enzymes and its antifertility effect.As shown by our result, after exposure to gossypol (l.25-60 μg/ml) for 15 min. in vitro,the sperms' ability to penetrate bovine cervical mucus and the fertility rate were significantly reduced. Also, following administration of gossypol (12.5 mg/kg/day) for six weeks, the rate of fertilization in vitro by hamster sperm was significantly decreased. In the gossypol-treated group, extracts of testis sperm delayed dispersion of cumulus cells, suggesting inhibition of hyaluronidase and other acrosomal enzymes. Furthermore, the acrosin and arylsulfatase activities were shown to be markedly inhibited. Thus, a parallelism was displayed between the reduction of fertility and the decreasc in acrosin and arylsulfatase activities in epididymis sperms.Besides, the inhibition was reversible and was dosage-and durationdependent. In conclusion, the assay of acrosin activity might serve as a useful tool for monitoring the irreversible sterility induced by gossypol,

Antifertility effects of quinestrol in male lesser bandicoot rat, Bandicota bengalensis, and potential in managing rodent population under field conditions

Integrating fertility control techniques using steroid hormones after lethal control can help reduce post control rebuildup of rodent populations.The current study is thefirst to assess the antifertility effects of quinestrol in male lesser bandicoot rat,Bandicota bengalensis which is the predominant rodent pest species in Southeast Asia.Rats in different groups were fed bait containing 0.00%,0.01%,0.02%,and 0.03%quinestrol for 10 days in laboratory and evaluated immediately,and 15,30,and 60 days after treatment discontinuation for effect on reproduction and other antifertility parameters.Effect of 0.03%quinestrol treatment for 15 days was also observed in managing rodent populations in groundnut cropfields.Treatment resulted in average consumption of 19.53±1.80,67.63±5.50,and 246.67±1.78 mg/kg bwt active ingredient by three treated groups of rats,respectively.No reproduction was observed in female rats mated with male rats treated with 0.03%quinestrol,even 30 days after cessation of treatment.Post-mortem examination showed a significant(P<0.0001)effect of treatment on organ weights(testis,cauda epididymis,seminal vesicles,and prostate gland)and different sperm parameters(sperm motility,sperm viability,sperm count,and sperm abnormality)in the cauda epididymalfluid with partial reversibility after 60 days.A significant(P<0.0001)effect of quinestrol on the histomorphology of testis and cauda epididymis was observed,suggesting its effect on spermatogenesis.Affected cell association and cell count in seminiferous tubules did not fully recover within 60 days of stopping treatment.Evaluation of the effects of quinestrol treatment in groundnutfields showed greater reductions in rodent activity infields treated with 2%zinc phosphide followed by 0.03%quinestrol treatment as compared tofields treated with 2%zinc phosphide alone.Research concludes that quinestrol has the potential to reduce fecundity and post control rebuildup of B.bengalensis populations,but long-term studies of the effectiveness of quinestrol under large-scalefield conditions are needed to use it as part of an integrated pest control program for rodents.

Drug synergistic antifertility effect of combined administration of low-dose gossypol with steroid hormones in rats

Background Our previous studies suggested that low-dose gossypol combined with steroid hormones has a reversible antifertility role in adult male rats, and the course of treatment was shorter than that of either gossypol or steroid hormones alone. This result suggested that low-dose gossypol and steroid hormones have a drug synergistic effect on antifertility. The aim of the study was to find the target organs of the antifertility synergistic effect of the combined regimen. Methods Thirty-two adult male rats were divided into four groups randomly： group GH,

Antifertility Effects of Orally Administration of Low Dose Gossypol Acetic Acid Combined with Methyltestosterone Plus Ethinyl Estradiol on Male Rat

Objective To investigate the feasibility and optimal regimen of orally administration of low dose gossypol acetic acid （GA） combined with methyltestosterone （MT） plus ethinyl estradiol （EE） for contraception in males.Methods Wistar male rats were randomly assigned into four groups, 20 in each group. Animals in group A or B were administered daily with 1% methyl cellulose or GA （12 mg/kg） suspended in 1% methyl cellulose, respectively. Rats in group C or D took firstly GA 12 mg/kg＋MT 20 mg/kg＋EE 0.1 mg/kg or MT 20 mg/kg＋EE 0.1 mg/kg, in asuspension with 1% methyl cellulose, via gastric intubation. After the infertilities were initiated（6 weeks for group C, 8 weeks for group D）, GA was served alone while MT＋EE were withdrawn in rats of groups C and D. The treatment was ceased after 18 weeks and some males from group C were permitted to recover. Fertility testing, 10 males per group, was served for determining infertility or restoration of fertility in treated rats. Examinations of histology and biochemistry in treated rats were used to examine the morphologic influences on sperm, testis, epididymides and viscera, and biochemical changes in blood. The growth and development of F1 generation of the rats would also be tested in a series of behavioral tests.Results Ten rats from group C were infertile at week 6 after treatment, and the fulfilled infertility was maintained with low-dose GA （12 mg/kg） only daily. Six weeks after cessation of treatment, all of treated males recovered their fertility. However, 8 of 10 rats from group D were in sterility at 6th week of treatment and all at 8th week of treatment, but the infertility could not be kept with the similar dose GA alone later on. Moreover, no adverse effects were found in our present experiments.Conclusion Administration of oral low dose GA combined with MT and EE as loading dose could successfully induce infertility in short term, whereafter the efficacy could completely be maintained by similar low dose of GA alone for long term. The combined regimen should be safe, effective, reversible and long term, and then optimal and promised contraception for male.

Antifertility activity of Artemisia vulgaris leaves on female Wistar rats

AIM： To evaluate the antifertility activity of Artemisia vulgar＆ leaves on female Wistar rats. METHOD： The plant extract was tested for its effect on implant formation at two dose levels, 300 and 600 mg.kg 1, respectively. The effective methanolic plant extract was further studied for estrogenic potency on ovariectomised immature female Wistar rats. RESULTS： The data presented in this study demonstrate the antifertility potential of Artemisia vulgar＆ methanolic leaf extract, which shows a strong and significant decrease in implant formation （100%）, and a strong estrogenic effect resulting in a significant increase in uterine weight in immature ovariectomised rats. These observations suggest that the methanolic extract of Artemisia vulgaris leaves has strong anti-implantation activity and estrogenic activity. CONCLUSION： The methanolic plant extract ofA. vulgaris has antifertility activity.

Phytochemical analysis and antifertility potential of Cynodon dactylon in female Wistar rats:A herbal approach towards contraception

Objective:The objective of the present study was to evaluate the antifertility activity of ether(ErCD),chloroform(CeCD)and ethyl alcohol(EyCD)extracts of the whole plant of Cynodon dactylon in female Wistar albino rats.Methods:Acute oral toxicity and an antifertility study were performed in female Wistar rats with two dose levels(200 and 400 mg/kg,orally)of EyCD.The estrogenic and progestogenic effects of EyCD were further observed by administering it to immature Wistar rats by investigations of vaginal cornification,hormonal level,uterus weight,biochemical parameters,histopathology of the uterus and deciduoma formation,respectively.Isolation of EyCD was carried out by Flash Chromatography and isolated fraction was estimated by HPLC.Results:No toxicity with any of the extract was found up to the dose of 2000 mg/kg body weight.EyCD treated rats exhibited maximum reduction in pregnancy(83.33%).Estimation of EyCD on vaginal cornification,estrogen-induced uterotrophic assay and deciduoma model demonstrated vaginal cornification,significant(P<0.01)increase in uterine weight and uterine proliferation in histopathology and reduced deciduoma formation respectively.Hormonal and biochemical parameters confirmed the above findings indicating estrogenic potential and antiprogestogenic potential of EyCD that might be attributed to the presence of phytoestrogen(apigenin)in EyCD.Conclusion:The results suggested that extracts of C.dactylon possess significant antifertility activity,which is consistent with the literature reported in folk medicine of this plant in fertility regulation.

Approaches to post-testicular contraception

The induction of infertility in males of several species through epididymal interference is more difficult to achieveby reduction of the mounts of epididymal secretions (eg α-glucosidase, L-carnitine) or immunological interferencewith secreted proteins ( eg D/E, P34H, P26h) than by direct actions of drugs on sperm function (eg inhibition ofglyceraldehyde 3-phosphate dehydrogenase by chloro-compounds). The latter approach holds promise for mankind ashuman sperm are susceptible to glycolytic inhibition. Future contraceptive developments may arise from production oftargeted inhibitors, research on the displacement of sperm proteins in the epididymis and interference with sperm plasmamembrane ion channels.

The antibody against a nuclear autoantigenic sperm protein can result in reproductive failure

To study whether the antibody against the testis form of the nuclear autoantigenic sperm protein(tNASP)could result in reproductive failure,we successfully cloned and expressed a 339-bp cDNA fragment of mouse tNASP(mtNASP).Using mouse as a model,recombinant mtNASP(rmtNASP)and a synthetic peptide,human tNASP393-408(htNASP393-408),were investigated for their antifertility effect.Active immunization with rmtNASP or the synthesized peptide raised high antibody titers in the immunized mice.Sperm-egg binding and fusion assay were carried out in 8-10-week-old BALB/c mice.Sperm-egg binding and in vitro fertilization of mouse oocytes were inhibited by co-incubation of zona-free mouse oocytes with capacitated mouse spermatozoa in the presence of varying concentrations of the antisera against rmtNASP.There was a significant antifertility effect in animals immunized with rmtNASP or the synthesized peptide.The effect on fertility in the mice immunized with the synthesized peptide was reversible.Our data indicate that active immunization with rmtNASP antigen may induce a strong antibody response that causes an inhibition of fertility.

Effect of Solanum surattense seed on the oxidative potential of cauda epididymal spermatozoa

Objective:To evaluate the effect of aqueous seed extract of Solarium suratlense(S.suraltense)on the oxidative potential of cauda cpididymal spermatozoa.Methods:S.surattense seed extract was orally administered at the dosage of 10 mg/kg b.w.for 15 days,after which aspartate transferase(AST),alanine transferase(ALT),glutamate dehydrogenase(GDH),citric acid and isocitrate dehydrogenase(ICDH)were assayed.Results:The activity levels of the enzymes AST and ALT,which are considered to be the androgenicity in the sperm suspension,were depleted in the extract fed rats.The activity level of the enzyme ICDH,was reduced significantly in the treated group(P<0.00l).Conclusions:It can be concluded that the oral administration of the aqueous seed extract of S.surattense can deplete the oxidative stress of cauda epididymal spermatozoa in albino rats.

ANTISPERMATOGENIC EFFECT OF TRIPTERYGIUM WILFORDII AND TRIPCHLOROLIDE（T4） ON RAT GAMETOGENESIS AND SPERMATOZOA

The multiglycosides of Tripterygium uilfordii(TII), a ready-made Chinese herbal medicine used for the treatment of rheumatoid arthritis , have been shown to cause oligospermia in patients. In the present study, the antifertility effects of TII and tripchlorolide (T4, isolated from TII) were observed in male rats. In rats fed with TII at a dose of 10mg·kg·d for 7 weeks, the seminiferous tubules were essentially not influenced. However, most of the sperm heads along the surface of the tubular lumen were transformed from the normal sickle-shaped to round shaped. suggesting a possible mutagenic action. There was minimal testicular change but prominent epididymal spermatozoa damage in all rats treated with T4 (0. 05 mg·kg·d) for 7 weeks. The epididymal spermatozoa showed various structural abnormalities, including disrupted connecting pieces and cracked midpieces. and more than 80% of the spermatozoa were decapitated. No significant changes were seen in the main visceral organs. The data suggest that T4 may have good prospects as a male contraceptive.

GENERATION AND PARTIAL CHARACTERIZATION OF MONOCLONAL ANTIBODIES AGAINST SOLUBILIZED MEMBRANE FRACTION OF HUMAN SPERMATOZOA

Human sperm membrane antigens extracted by deoxycholate (DOC) were used to immunizeBALB/c mice.Hybrid cell lines secreting sperm-specific monoclonal antibodies were generatedby cell fusion in a semi-solid medium and screened by indirect immunofluorescent assay usinglive and methanol-fixed sperm.Out of 850 hybrid clones from cell fusion,28 were shownto secrete sperm-specific antibodies which reacted with the acrosome,equatorial segment,whole surface plasma membrane or tail of spermatozoa.Finally,seven hybrid cell lineswere established and shown to secrete monoclonal antibodies which had no cross-reactivitywith arty human tissues other than testis and sperm.The majority were also shown toinhibit fertilization of mouse oocytes in vitro and human sperm penetration of zona-freehamster ova.Western blot analysis revealed that some of these antibodies reacted withsperm membrane antigens of distinct molecular size.