| Objective To investigate the effects of dihydrotestosterone?DHT?on the expression of anti-Müllerian hormone?AMH?in follicular granulosa cells?GCs?of rat and growth of primordial follicles or AMH expression in mice.Methods 1.First part of experiment:GCs were extracted from the ovaries of 21d SD female rats for primary culture.Firstly,cell morphology was detected by HE staining and FSHR immunofluorescence detected cell purity after 48h of GCs culture.Then,cells were randomly divided into control group,10-5 mol/L DHT group,10-8 mol/L DHT group,2×10-5 mol/L MK-2206 2Hcl group and 10-8 mol/L DHT+2×10-5 MK-2206 2Hcl group,corresponding concentrations of regents were used 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 blot.2.Second part of experiment:The ovaries of 3d ICR mice were taken out and were randomly divided into control group?no drug intervention?,10-8mol/LDHT group,2×10-5 mol/LMK-2206 2Hcl group and 10-8 mol/L DHT+2×10-5 mol/LMK-2206 2Hcl group,corresponding concentration of reagents were used to intervene it 4d in vitro;HE staining counted the number of primordial follicles and early growing follicles in different groups;Immunohistochemistry was used to detect the expression of PCNA and AMH in ovarian tissue of different groups of mice;Western blot was used to detect the expression of AKT signaling pathway related major factors?AKT,FoxO3a,PTEN?,AMH and PCNA;HE staining,immunohistochemistry and Western blot were used to compare the changes of the number of follicles,protein expression of PCNA and AMH on day 3 and 7 in vivo.Results 1.First part of experiment:AMH was expressed in the membrane and cytoplasm of GCs in rats;Compared with the control group,both 10-8 mol/L DHT and 10-5 mol/L DHT can increase the expression of AMH in GCs?P<0.05?,there is no difference in the effect of these two concentrations of DHT on the expression of AMH in GCs?P>0.05?;Compared with the control group,10-8 mol/L DHT can increase p-AKT and AMH expression?P<0.05?,and2×10-5 mol/L MK-2206 2Hcl can decrease AMH expression?P<0.05?;Compared with 2×10-5mol/L MK-2206 2Hcl,AMH expression was increased in the 10-8 mol/L DHT+2×10-5 mol/L MK-2206 2Hcl-treated group?P<0.05?.2.Second part of experiment:The number of early growing follicles and the proportion of them to the total follicles in the DHT-treated group were higher than control group?P<0.05?;AMH mainly expressed in primary follicular granulosa cells,but less in primordial follicles;Expression of AMH,PCNA,p-AKT and p-FoxO3a all increased after DHT interfere with mouse ovaries in vitro?P<0.05?;However,when mice ovaries were treated with MK-2206 2Hcl which is the AKT signaling pathway inhibitors,not only number of early growing follicles as well as the proportion of them to the total follicles decreased,but also the protein expression of PCNA and AMH reduced?P<0.05?;In vivo experiments showed that expression of AMH in 7d group was higher than 3d group?P<0.05?,suggesting that AMH expression may increase with primordial follicles activation.Conclusion DHT not only can up-regulate the expression of AMH in GCs and mouse ovaries,but also promote the activation of mouse primordial follicles,which may be related to the activation of AKT signaling pathway. |
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