双氢睾酮对小鼠卵泡颗粒细胞增殖与抗苗勒管激素表达的影响

Effects of Dihydrotestosterone on Proliferation and Expression of Anti-Müllerian Hormone of Mouse Granulosa Cells

【目的】探讨双氢睾酮(DHT)对小鼠颗粒细胞增殖与抗苗勒管激素(AMH)表达的影响。【方法】给3周龄的昆明小鼠注射孕马血清促性腺激素(PMSG,10 IU/只)以获取颗粒细胞,颗粒细胞传代后48 h HE染色鉴定形态,绘制颗粒细胞的生长曲线,免疫荧光法鉴定促卵泡素受体(FSHR)的表达。当第2代颗粒细胞汇合度达到50%时,先用无血清的DMEM/F12培养基饥饿处理12 h,然后在培养基中添加不同浓度的DHT(0、10^(-9)、10^(-8)、10^(-7)、10^(-6)、10^(-5)mol/L),培养48 h后检测颗粒细胞增殖情况,实时荧光定量PCR法及ELISA法分别测定AMH基因及蛋白的表达。在颗粒细胞培养液中分别添加10^(-6)mol/L Flutamide(雄激素受体(AR)特异性抑制剂)、10^(-7)mol/L DHT、10^(-7)mol/L DHT+10^(-6)mol/L Flutamide、10^(-5)mol/L DHT、10^(-5)mol/L DHT+10^(-8)mol/L 11-ketodihydrotestosterone(AR特异性激动剂),分别记为F、D7、DF、D5、DK组,以不添加药物为对照组。培养48 h后,检测各组颗粒细胞增殖及AMH蛋白含量。【结果】体外培养的小鼠颗粒细胞呈梭形或铺路石状,生长曲线呈S形,细胞普遍表达FSHR;与0 mol/L DHT组相比,10^(-8)、10^(-7)mol/L DHT组细胞增殖分别显著和极显著增加(P<0.05;P<0.01),10^(-5)mol/L DHT组细胞增殖显著降低(P<0.05);10^(-7)mol/L DHT组AMH基因的相对表达量和AMH蛋白含量均极显著增加(P<0.01)。与D7组相比,DF组颗粒细胞增殖和AMH蛋白含量均极显著降低(P<0.01);与D5组相比,DK组颗粒细胞增殖和AMH蛋白含量均极显著升高(P<0.01)。【结论】10^(-7)mol/L DHT能够显著促进颗粒细胞增殖和AMH表达,而10^(-5)mol/L显著降低了颗粒细胞增殖以及AMH表达,且AR介导了DHT调控颗粒细胞增殖和AMH表达。

【Objective】The aim of this study was to investigate the mechanism of dihydrotestosterone(DHT)involved in the regulation proliferation and the expression of anti-Müllerian hormone(AMH)of mouse granulosa cells.【Method】Kunming mice(3 weeks)were injected with pregnant horse serum gonadotropin(PMSG,10 IU/mouse)to obtain granulosa cells.After granulosa cells was passaged for 48 h,the morphology of granulosa cells was identified by HE staining,the growth curve of granulosa cells was drawn,and the expression of follicle stimulating hormone receptor(FSHR)was identified by immunofluorescence.When the confluence of the second generation granulosa cells reached 50%,they were starved with serum-free DMEM/F12 medium for 12 h,and different concentrations of DHT(0,10^(-9),10^(-8),10^(-7),10^(-6),10^(-5)mol/L)were added to the medium.After 48 h of culture,the proliferation of granulosa cells was detected.The expression of AMH gene and AMH protein were measured by Real-time quantitative PCR and ELISA,respectively.10^(-6)mol/L flutamide(AR specific inhibitor),10^(-7)mol/L DHT,10^(-7)mol/L DHT and 10^(-6)mol/L flutamide,10^(-5)mol/L DHT,10^(-5)mol/L DHT and 10^(-8)mol/L 11 ketodihydroprosterone(AR specific agonist)were added to the medium of granulosa cells,which were recorded as F,D7,DF,D5 and DK groups respectively,and the control group was not added with drugs.After 48 h of culture,the proliferation of granulosa cells and the content of AMH protein were detected.【Result】Mouse granulosa cells cultured in vitro were spindle or paving stone shaped,and the growth curve was S-shaped.The cells were generally expressed FSHR.Compared with 0 mol/L DHT group,the cell proliferation of 10^(-8)and 10^(-7)mol/L DHT group were significantly and extremely significantly increased(P<0.05;P<0.01),and that of 10^(-5)mol/L DHT group was decreased significantly(P<0.05).The relative expression of AMH gene and the content of AMH protein in 10^(-7)mol/L DHT group were extremely significantly increased(P<0.01).Compared with D7 group,the proliferation of granulosa cells and the content of AMH protein in DF group were extremely significantly decreased(P<0.01).Compared with D5 group,the proliferation of granulosa cells and AMH protein in DK group were extremely significantly increased(P<0.01).【Conclusion】10^(-7)mol/L DHT could significantly promote the proliferation and AMH expression of granulosa cells,while 10^(-5)mol/L DHT significantly reduced the proliferation and AMH expression.Moreover,AR could mediate DHT to regulate proliferation and AMH expression of granulosa cells.