双氢睾酮对绵羊小腔卵泡颗粒细胞的抗凋亡作用

Anti-apoptotic effects of dihydrotestosterone on small antral follicle granulosa cells of sheep

为了探讨双氢睾酮(dihydrotestosterone,DHT)对绵羊小腔卵泡颗粒细胞凋亡的调控作用,试验选择直径为2~5 mm的绵羊卵泡颗粒细胞,通过Western-blot检测卵泡刺激素受体(follicle stimulating hormone receptor,FSHR)与胆固醇侧链裂解酶(cholesterol side chain cleavage enzyme,CYP11A1)蛋白的表达变化,来验证颗粒细胞在体外培养过程中是否发生黄体化;通过添加黄体生成素受体(luteinizing hormone receptor,LHR)颉颃剂BAY-899建立非黄体化颗粒细胞培养体系,利用细胞计数法检测不同培养体系下颗粒细胞增殖活力;利用实时荧光定量PCR方法与Western-blot技术检测不同浓度DHT对凋亡相关基因与蛋白[B细胞淋巴瘤-2(B-cell lymphoma 2,Bcl-2)和Bcl-2相关蛋白X(Bcl-2 associated X,Bax)]表达量的影响;采用ELISA法检测不同浓度DHT对颗粒细胞分泌抗缪勒氏管激素(antimüllerian hormone,AMH)含量的影响。结果表明:与新鲜分离的颗粒细胞比较,体外培养48 h后颗粒细胞的FSHR蛋白相对表达量极显著下降(P<0.01)、CYP11A1蛋白相对表达量显著升高(P<0.05)。与体外培养的颗粒细胞比较,在含BAY-899培养基中培养的颗粒细胞FSHR蛋白相对表达量极显著升高(P<0.01),CYP11A1蛋白相对表达量极显著降低(P<0.01),且通过生长曲线比较,其增殖活力无明显差异。用不同生理浓度DHT处理颗粒细胞后,抗凋亡基因与蛋白Bcl-2呈浓度依赖性升高(P<0.05或P<0.01),而促凋亡基因与蛋白Bax表达则未发生显著变化(P>0.05);AMH蛋白表达量随DHT浓度升高呈浓度依赖性升高趋势(P<0.05或P<0.01)。说明颗粒细胞在体外培养过程中发生了黄体化,BAY-899能够抑制颗粒细胞转变为黄体化颗粒细胞,DHT可能通过激活AMH促进Bcl-2的表达来抑制颗粒细胞的凋亡,从而对小腔卵泡发育具有正向调控作用。

To investigate the role of dihydrotestosterone(DHT)in regulating apoptosis of ovine follicular granulosa cells,sheep follicular granulosa cells with a diameter of 2-5 mm were selected for the experiment,and the protein expression changes of follicle-stimulating hormone receptor(FSHR)and cholesterol side chain lyase(CYP11A1)were detected by Western-blot to verify the luteinization of granulosa cells during in vitro culture.Non-luteinizing hormone receptor(LHR)antagonist BAY-899 was used to establish a non-luteinizing granulocyte culture system,and cell counting method was used to detect the proliferation activity of granulocyte cells in different culture systems.Real-time fluorescence quantitative PCR and Western-blot were used to detect the effects of different concentrations of DHT on the expression of apoptosisrelated genes and proteins B lymphoma-2(Bcl-2)and Bcl-2 related protein X(Bax).The effects of different concentrations of DHT on the secretion of anti-mullerian hormone(AMH)in granulosa cells were detected by ELISA.The results showed that compared with freshly isolated granulosa cells,the relative expression level of FSHR protein was significantly decreased(P<0.05),and the relative expression level of CYP11A1 protein was significantly increased 48 h after in vitro culture(P<0.01).Compared with granulosa cells cultured in vitro,the relative expression level of FSHR protein in granulosa cells cultured in BAY-899 medium was extremely significantly increased(P<0.01),and the relative expression level of CYP11A1 protein was extremely significantly decreased(P<0.01),and there was no significant difference in proliferation activity through growth curve comparison.The expression of anti-apoptotic gene and protein Bcl-2 increased in a dose-dependent manner after DHT treatment(P<0.05 or P<0.01),while the expression of pro-apoptotic gene and protein Bax had no significant changes(P>0.05).The expression of AMH protein increased with DHT concentration(P<0.05 or P<0.01).It indicated that BAY-899 could inhibit the transformation of granulosa cells into luteinized granulosa cells,and DHT might inhibit the apoptosis of granulosa cells by activating AMH protein to promote the expression of Bcl-2,thus playing a positive role in regulating the development of small coelomic follicles.