S Effects Of Dihydrotestosterone On Expression Of Anti-mullerian Hormone And Proliferation Of Mouse Granulosa Cells

In female mammals,follicular development plays an important role in oocyte maturation.Follicular granulosa cells are one of the important components of follicles,and their fate is closely related to the development of follicles.Studies have shown that the proliferation of granulosa cells is involved in the process of follicular growth,but the apoptosis of granulosa cells can participate in the process of follicular atresia.Other studies have shown that dihydrotestosterone（DHT）,its receptor（AR）and anti mullerianhorone（AMH）are highly expressed in the early development of antral follicles,but low in the late development of antral follicles.DHT,AR and AMH are the key factors of follicular development.The correlation between them and granulosa cell proliferation has not been reported.In this study,the cell purity,morphology and growth activity of mouse follicular granulosa cells in vitro culture system were identified by cell immunofluorescence technology,he staining technology and drawing growth curve.The number of mouse follicular granulosa cells cultured in vitro after 48 hours of treatment with different concentrations of DHT was detected by cell counting;Then,combined with enzyme-linked immunosorbent assay and real-time quantitative RT q PCR,the effects of different concentrations of DHT on the protein and gene expression of AMH secreted by mouse follicular granulosa cells were discussed;Finally,in the culture system without other hormones,different concentrations of DHT and AR regulators（11 ketodihydroprosterone,AR agonist;flutamide,AR inhibitor）were added alone or in combination to explore their effects on the proliferation and AMH protein expression of mouse follicular granulosa cells.The results showed that（1）mouse granulosa cells cultured in vitro had high purity,the morphology was classic spindle or polygon,and the growth curve was classic"s"curve;（2）In the range of 10^-9-10^-7mol/LDHT concentration,the proliferative activity of granulosa cells increased gradually with the increase of DHT concentration;In the range of 10^-7-10^-5mol/LDHT concentration,the proliferative activity of granulosa cells decreased gradually with the increase of DHT concentration;Compared with the control group,the proliferation activity of granulosa cells in 10-⁷mol/LDHT treatment group was the highest（P<0.05）,and the proliferation activity of granulosa cells in 10^-5mol/LDHT treatment group was weaker（P<0.05）.（3）In the range of 10^-9-10^-7mol/LDHT concentration,the expression of AMH in granulosa cells increased gradually with the increase of DHT concentration;In the concentration range of 10^-7-10^-5mol/LDHT,the expression of AMH in granulosa cells decreased gradually with the increase of DHT concentration;The protein and gene expression of AMH in 10^-7mol/LDHT treatment group were significantly up-regulated compared with the control group（P<0.05）,but there was no significant difference between10^-5mol/LDHT treatment group and the control group（P>0.05）.（4）In the culture system without other hormones,compared with 10-7mol/L DHT treatment group,the combined addition of 10^-7mol/L DHT and 10-6m flutamide could significantly reduce the number of granulosa cell proliferation（P<0.05）;Compared with 10^-5mol/L DHT treatment group,the combined addition of 10^-5mol/L DHT and 10^-8m 11 ketodihydroprosterone could significantly increase the number of granulosa cell proliferation（P<0.05）.（5）In the hormone free culture system,after treatment with DHT,flutamide and 11ketodihydroprosterone alone or in combination for 48 hours,the 10^-7mol/L DHT treatment group could significantly increase the expression of AMH protein,and the promotion effect could be offset by flutamide（P<0.05）.The 10^-5mol/L DHT treatment group significantly reduced the expression of AMH protein,This effect was also counteracted by 11ketodihydroprosterone（P<0.05）.The above results show that the purity,morphology and growth activity of mouse follicular granulosa cells cultured in vitro were good,which could provide experimental materials for the next experiment.In the process of vitro culture,10^-7mol/L DHT promoted the proliferation and AMH expression of granulosa cells by up regulating the expression of AR in granulosa cells.The results of this study reveal that DHT has a dual effect on the proliferation of mouse follicular granulosa cells,which was helpful to deeply understand the mechanism of mouse follicular development.