【作者】 张才田；

【导师】 崔毓桂；

【作者基本信息】 南京医科大学 ， 临床检验， 2008， 硕士

【摘要】 精子发生是在内分泌系统的调控下完成的。下丘脑分泌促性腺激素释放激素（GnRH）刺激垂体分泌卵泡刺激素（FSH）和黄体生成素（LH）,LH作用于睾丸间质细胞,使其分泌睾酮（T）。一般认为精子发生需FSH和T的双重作用。如果给与外源性雄激素（TE、TU）,反馈性抑制FSH和LH,使睾丸局部T浓度下降,可抑制精子发生。男性激素避孕的主要方法有单用雄激素、雄激素与孕激素合用、雄激素与其他制剂及物理方法合用。目前国际上广泛采用的男性避孕药是雄激素类和孕激素类甾体激素的药物组合,如醋酸赛普特隆（cyproteroneacetate）和十一酸睾丸酮（testosterone undecanoate,TU）、DMPA+TU,目的是达到最大程度的FSH和LH的反馈抑制,同时减少雄激素剂量,以避免因单独使用雄激素而导致其血液浓度的巨大波动。近年,国际上提出了男性抗生育的“双重打击”学说,即在激素作用（及激素机制）之外,合并使用物理的、化学的、或者其他分子机制途径的方法。雷公藤多甙具有明显的抑精作用,早在上个世纪80年代初,我国学者已发现口服雷公藤多甙可致男性精子计数下降,停药后可恢复正常。后来动物实验研究表明雷公藤多甙主要影响附睾精子,表现为损伤精子微管、微丝和胞膜头尾分离和顶体弯曲,在附睾管腔可见脱落的精子细胞,组织学观察显示曲细精管和附睾上皮损伤轻微。但睾丸生精细胞也受影响。然而雷公藤多甙对睾丸生殖细胞作用的深层次的分子机制仍然不清楚。定性研究一些雷公藤多甙抑制精子发生、诱导生精细胞凋亡分子信号通路,为将来与男性激素合并应用作为男性避孕药奠定基础。研究目的本项研究中,探讨雷公藤多甙抑制大鼠精子发生、诱导生精细胞凋亡的作用,同时观察对精子活率、畸形率的影响;用免疫组化的方法研究雷公藤多甙促进生殖细胞凋亡的多个分子信号通路上的关键分子。深入的细胞与分子机制的研究,验证雄激素合并雷公藤多甙组合方式可能的“双重打击”机制,为将来雷公藤多甙与男性激素合并应用作为男性避孕药奠定基础。研究方法本研究设计中,40只成年雄性SD大鼠被分为4组,正常对照组,灌胃给予生理盐水;3个雷公藤多甙处理组,灌胃给予雷公藤多甙16mg/kg,给药时间分别为2周、4周、6周。每日一次,在2及6周用化学发光法检测血清睾酮、FSH、LH和可的松水平;在2、4及6周取一侧附睾进行精子计数和活动率检测。在2、4及6周分别用Bouin’s和5%二甲砷酸钠灌流并固定睾丸二甲砷酸钠灌流并固定的睾丸用于原位末端标记法（TUNEL）观察睾丸生精细胞凋亡;Bouin’s灌流并固定的睾丸,HE染色后光镜观察睾丸组织的形态学变化,免疫组织化学法观察凋亡通路的相关蛋白Bax/Bcl-2、Fas/FasL和Caspase-3的表达。研究结果给药2周后大鼠血清FSH、LH、T、E2和可的松平均值分别为3.85IU/L、3.98 IU/L、3.91 nmol/L、201.3×10^-15mol/L和113.4×10^-9mol/L,6周后分别为4.1 IU/L、3.88IU/L、3.82nmol/L、226.5×10^-15mol/L和102.1×10^-9mol/L,与对照组相比(3.9IU/L、4.21IU/L、3.71 nmol/L、207.6×10^-15mol/L和105.3×10^-9mol/L)都无显著差异（P均＞0.05）;给药2周后平均精子数下降到52×10⁶/ml,活精子率下降到60%,和畸形率上升到1.2%,与对照组（平均精子数:62×10⁶/ml,活精子率:79%,畸形率:1.2%）相比有显著差异（P均＜0.05）;给药4周（平均精子数:15×10⁶/ml,活精子率:24%,畸形率:56%）和6周（平均精子数:9×10⁶/ml,活精子率:8%,畸形率:66%）平均精子计数下降、活精子率下降和精子畸形率升高更显著（P＜0.001）;组织学检查,正常对照组可见大鼠睾丸曲细精管生精细胞数目较多及层次清晰,管壁较厚,生精细胞排整齐,给药组可见大鼠睾丸曲细精管生精细胞数目减少及层次减少,管壁变薄,生精细胞排列紊乱,原始精原细胞和支持细胞未见明显改变。TUNEL显示给药2周后生精小管内的生精细胞凋亡增加到42.2%,与正常对照组（21.1%）相比,有显著差异（P＜0.05）,4周（81.1%）和6周（96.5%）生精细胞凋亡增加更明显（4和6周P＜0.001）。免疫组织化学显示,凋亡相关蛋白Bax、Fas、FasL和Caspase-3的表达明显上调,Bcl-2的表达无显著差异。研究结论雷公藤对大鼠精子发生的抑制作用,其细胞学机制表现为诱导生精细胞凋亡增加,同时精子的畸形率升高,活率下降,表明雷公藤对精子的发生和附睾内的精子都有明显的作用。雷公藤多甙使睾丸内Bax、Fas、FasL和Caspase-3表达增加,与诱导生精细胞凋亡相关,是相关的信号通路之一。雷公藤多甙抗生育作用的睾丸细胞学机制,与单纯雄激素抗生育的机制一致;但分子信号机制两者不完全相同。进一步研究雷公藤多甙作用的分子信号机制有助于今后降低剂量、减少毒副作用、探讨其作为男性避孕研究与开发的可能性。更多还原

【Abstract】 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.更多还原