| BackgroundSpermatogenes were completed under the regulation of the endocrine system.Gonadotropin-releasing hormone secreted from hypothalamus can stimulate pituitary to secret follicle stimulating hormone and luteinizing hormone.Under the action of LH,the testis leydig cells secreted testosterone.Generally Spermatogenes need diphasic action of LH and Fsh.FSH and LH feedback inhibited by exogenous androgen lowered testosterone local in testis,spermatogenesis were inhibted in the end.There were Androgen alone,the combination of androgen and progestogen,the combination of androgen and GnRH about main androtin contraceptive method.The steroid hormone unions of androgens and progestogens were extentive adopted in the international.For instance,the union of cyproterone acetate and testosterone undecanoate inorder to FSH and LH be feedback inhibited extently,reduce the dose of androgen and avoid the tremendous fluctuation of serum testosterone for its use alone.The "two hit"theory about male antifertility were proposed recently in the international.The method were to combine physical, chemical or other molecule mechanism besides hormonal action.In the 80s of the last century,the scholar in our country found the Sperm count were lowered when Tripterygium wilfordii for oral use,it could recovery to normal after drug withdrawal.The animal experiment manifested tripterygium wilfordii main effected sperm in epididymis,showing that microtubule,microfilament of sperm were injuried,head-tail separation, and acrosome bending.The deciduous sperms were found in the duct of epididymis.The histology manifested seminiferous tubules and epididymis epithelium were injuried little.But spermatogenic cell and Leydig cell in testis were effected too.It was unclear about its cellular and molecular mechanisms of the suppression of the spermatogenesis.Some protein of qualitative investigation on the way of tripterygium wilfordi inhibited spermatogenesis were conbined with androtin to establish a foundation for male contraceptive.ObjectiveThis study was designated to explore the suppression of spermatogenesis induced by Tripterygium wilfordiis,at the same time obsersating its effect on the sperm count,the rate of vivi-sperm,the rate of deformity.The Immunohistochemistry revealed many important molecules involved in germ cell apoptosis induced by GTW.The further research of cell and molecule mechanisms were used to verificate the possible "two hit"mechanisms about the union of androgen and tripterygium wilfordii.This could make progress of GTW as a potential and safe contraceptive for the male.MethodIn present study,the forty SD adult rats were divided four groups.The normal control were treated with normal sodium;The thirty SD adult rats were treated with Tripterygium wilfordiis(16 mg/kg)by intragastric administration four 2 weeks,4 weeks,and 6 weeks,one time per day.The serum T,FSH,LH and Cortisone were measured in duplicate by chemiluminescence.The distal cauda epididymis was removed and minced with scissors to release the epididymal content into a 35-mm Petri dish containing 2mL warm normal sodium(pH 7.4)to epididymal sperm counts and motility at 2,4 and 6weeks.The glutaraldehyde-fixed testes were used to determine germ cell apoptosis with TUNEL.The expression of Bax/Bcl-2,Fas/FasL and Caspase-3 were analyzed by Immunohistochemistry.ResultsThe rat serum average value of FSH,LH,T,E2 and Cortisone were 3.85 IU/L,3.98IU/L,3.91 nmol/L,201.3×10-15mol/L and 113.4×10-9mol/L after the rats were treated with tripterygium wilfordiis for two weeks,4.1 IU/L,3.88 IU/L,3.82 nmol/L,226.5×10-15mol/L and 102.1×10-9mol/L after the rats were treated for 6 weeks,compared to the control group, were not changed(all P＞0.05).The mean sperm count decreased to 52×106 /ml,the rate of vivi-sperm decreased to 60%and the rate of deformity upgrade to 1.2%after the rats were treated with GTW for 2 weeks,compared to normal control(mean sperm count:62×106/ml,the rate of vivi-sperm:24%,the rate of deformity:1.2%)were significantly changed(all p＜0.005),4 weeks(mean sperm count:15×106/ml,the rate of vivi-sperm:24%,the rate of deformity:56%)and 6 weeks(mean sperm count:62×106/ml,the rate of vivi-sperm:8%,the rate of deformity: 66%),copared to normal control,the mean sperm count,the rate of vivi-sperm and the rate of deformity were signifigantly changed (P＜0.001).The histology revealed that each spermatocytes and spermids obviously reduced,spermatogenic cells arranged chaoticly.The TUNEL revealed the apoptosis of spermatogenic cells in the seminiferous tubule were 21.1%in the normal control,upgrading to 42.2%for 2 weeks, 81.1%for 4 weeks,96.5%for 6 weeks,increasing significantly(P＜0.05). he Immunohistochemistry revealed that the expression of Bax,Fas/FasL and Caspase-3 were up-regulated.ConclusionsGTW suppressed spermatogenesis by induced- apoptosis of germ cells in the testis associated with reduction of germ activity and an increase of the deformed sperms through its action on both epididymis and testis. GTW did not affected hormone production in Leydig cells and the adrenal function.Fas/FasL system and Bax pathway could be part of mechanisms involved in germ cell apoptosis induced by GTW.The results suggest it is important for considering a lower dose of GTW for reducing adverse effects,this could make progress of GTW as a potential and safe contraceptive for the male. |
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