双氢睾酮对大鼠原代卵泡颗粒细胞抗苗勒管激素表达的影响

Effects of dihydrotestosterone on the expression of anti-Müllerian hormone in primary follicular granulosa cells in rats

目的探讨双氢睾酮(DHT)对大鼠原代卵泡颗粒细胞抗苗勒管激素(AMH)表达的影响。方法从95只21 d SD雌性大鼠中提取颗粒细胞原代培养48 h后,先用HE染色检测细胞形态,卵泡刺激素受体(FSHR)细胞免疫荧光检测细胞纯度;然后根据干预方法和实验的不同,随机将细胞分为对照组(无药物干预)、10-8mol/L DHT组、10^-5mol/L DHT组,2×10^-5mol/L蛋白激酶B(Akt)抑制剂(MK-22062Hcl)组和10-8mol/L DHT+2×10^-5 mol/L MK-22062Hcl组,分别使用相应浓度的试剂干预细胞3 h。干预完成后,细胞免疫荧光染色观察AMH表达的部位和变化;Western blotting测定不同组的AMH、Akt、p-Akt蛋白表达量。结果大鼠原代卵泡颗粒细胞的纯度为93.33%±3.09%,AMH表达在大鼠原代卵泡颗粒细胞膜和细胞质中;与对照组相比,10-8 mol/L DHT和10^-5 mol/L DHT均可使颗粒细胞AMH表达增加(P<0.05),但这两个浓度的DHT对颗粒细胞AMH的表达影响差异无显著性(P>0.05);与对照组相比,10-8mol/L DHT可以使p-Akt、AMH表达升高,2×10^-5 MK-22062Hcl使AMH表达降低(P<0.05);与2×10^-5MK-22062Hcl相比,10-8mol/L DHT+2×10^-5 MK-22062Hcl处理组AMH表达升高(P<0.05)。结论DHT可以上调大鼠原代卵泡颗粒细胞AMH的表达,这种作用可能与Akt信号通路有关。

Objective To explore how dihydrotestosterone(DHT)affects the expression of anti-Müllerian hormone(AMH)in primary rat follicular granulosa cells,and then provides a new experimental basis for the role of DHT and AMH in follicular development.Methods Granulosa cells was extracted from the ovaries of 9521 days SD female rats for primary culture.Firstly,cell morphology were detected by HE staining and follicke stimulating hormone receptor(FSHR)immunofluorescence detected cell purity after 48 hours of cell culture.Then,cells were randomly divided into control group(without drug intervention),10-8mol/L DHT group,10^-5mol/L DHT group,2×10^-5 protein kinase B(Akt)inhibitor(MK-22062Hcl)group and 10-8 mol/L DHT+2×10^-5 mol/L MK-22062Hcl group according to different intervention method and experiments,corresponding concentrations were used in each group to intervene cells for 3 hours.After intervention,sites and expression changes of AMH were detected by immunofluorescence.Changes of AMH,Akt and p-Akt were detected by Western blotting.Results Immunofluorescence showed that the purity of primary follicular granule cells in rats was 93.33%±3.09%,and AMH was expressed in the membrane and cytoplasm of primary follicular granule cells in rats;Compared with the control group,both 10-8mol/L DHT and 10^-5mol/L DHT increased the expression of AMH in granulosa cells(P<0.05),but there was no difference in the effect of these two concentrations of DHT on the expression of AMH in granulosa cells(P>0.05);Compared with the control group,10-8 mol/L DHT increased p-Akt and AMH expression,and 2×10^-5 MK-22062Hcl decreased AMH expression(P<0.05);Compared with the 2×10^-5 MK-22062Hcl,AMH expression was increased in the 10-8mol/L DHT+2×10^-5 MK-22062Hcl treatment group(P<0.05).Conclusion DHT can up-regulate the expression of AMH in primary follicular granulosa cells in rats,and this effect may be related to the Akt signaling pathway.