雷帕霉素对小鼠始基卵泡激活及生长发育的作用研究  

Effects of Rapamycin on the activation and growth of primordial follicles in mice

作　　者：陈秀英[1] 管海云 李斌[1] 张炜[1] Chen Xiuying;Guan Haiyun;Li Bin;Zhang Wei(Obstetrics and Gynecology Hospital of Fudan University,Shanghai 200011,China)

机构地区：[1]复旦大学附属妇产科医院,上海200011

出　　处：《中华生殖与避孕杂志》2020年第1期24-29,共6页Chinese Journal of Reproduction and Contraception

基　　金：国家自然科学基金面上项目(81771587)。

摘　　要：目的 研究哺乳动物雷帕霉素靶蛋白(mTOR)特异性抑制剂雷帕霉素对小鼠始基卵泡激活及生长发育的作用.方法 选取C57BL/6雌性小鼠40只,随机分为对照组(n=20,等体积生理盐水)和雷帕霉素处理组(n=20,雷帕霉素5 mg/kg),持续给药30 d后每组分别取7只小鼠,取卵巢组织行石蜡切片,HE染色观察组织形态学变化并进行卵泡分类计数,免疫组织化学法检测DDx4/MVH和Ki-67分子的表达水平,另提取卵巢组织蛋白,Western blotting法检测mTOR及其下游信号分子的表达水平.停用雷帕霉素8周后,对照组和雷帕霉素处理组分别取6只,行始基卵泡计数,免疫组织化学法检测抗苗勒管激素(AMH)和Ki-67分子的表达水平,另取7只,予孕马血清促性腺激素(PMSG)和人绒毛膜促性腺激素(hCG)促排卵,24 h后取材行HE染色观察组织形态学变化.结果 雷帕霉素处理组小鼠始基卵泡数量较对照组显著增多(P=0.040),可见大量始基卵泡聚集,DDx4/MVH分子的表达增加;生长卵泡数量则明显减少(P=0.002),且Ki-67表达水平降低.停用雷帕霉素8周后,雷帕霉素处理组小鼠始基卵泡数量仍明显多于对照组,小鼠卵巢中可见各级生长卵泡,正常表达Ki-67和AMH.促排卵后,雷帕霉素处理组和对照组小鼠卵巢均可见大量成熟卵泡或黄体.Western blotting结果显示雷帕霉素处理组小鼠卵巢mTOR及其下游信号分子p70S6K、rpS6和eIF4B磷酸化水平显著低于对照组(P均<0.05),而总的分子水平未发生明显变化(P>0.05).结论 雷帕霉素通过抑制mTOR信号通路分子磷酸化水平,抑制始基卵泡的激活,减缓始基卵泡向生长卵泡的转化速率,并抑制卵泡生长发育,从而保护卵泡储备功能.Objective To investigate the effects of Rapamycin,a mammalian target of rapamycin(mTOR)specific inhibitor,on the activation and growth of primordial follicles in mice from the signal transduction of follicular development.Methods A total of 40 C57BL/6 female mice were randomly divided into control group(n=20,same volume of saline)and Rapamycin treatment group(n=20,5 mg/kg Rapamycin).Seven mice in each group were treated after 30 d,and ovarian histomorphological changes were observed by HE staining,the expressions of DDx4/MVH and Ki-67 were detected by immunohistochemistry,the level of mTOR and its downstream molecules were detected by Western blotting.Eight weeks after administration,6 mice in control group and Rapamycin treatment group were selected for follicle count,and the expressions of anti-Müllerian hormone(AMH)and Ki-67 were detected by immunohistochemistry.Another 7 mice of both groups were treated with pregnant mare serum gonadotropin(PMSG)and human chorionic gonadotropin(hCG)for ovulation induction.The ovaries were obtained for histomorphological observation by HE staining.Results The number of primordial follicle in Rapamycin treatment group was significantly increased compared with control group(P=0.040),and a large number of primordial follicles were clustered,while the number of growing follicles was significantly decreased(P=0.002).The number of primordial follicle,expressing DDx4/MVH molecule,was increased,while the expression of Ki-67 was decreased compared with control group.Eight weeks after Rapamycin treatment,the number of primordial follicles was still more than that in control group,and the expressions of Ki-67 and AMH in growing follicles were normal.After ovulation induction treatment,large numbers of mature follicles or corpus luteum could be observed in both two groups.Western blotting showed that the phosphorylation levels of mTOR and its downstream molecules p70S6K,rpS6 and eIF4B in Rapamycin treated group were significantly lower than those in control group,while the total molecula

关 键 词：雷帕霉素 哺乳动物雷帕霉素靶蛋白 始基卵泡 卵巢储备 

分 类 号：R965[医药卫生—药理学]