BMP6 Regulates The Function Of Goat Ovarian Granulosa Cell And The Mechanism Of AMH Expression

Anti-Mullerian hormone(AMH)is produced by the granulosa cell of the female mammalian ovary and inhibits the recruitment of primordial follicles,thereby avoiding premature depletion of ovarian reserve follicles.Therefore,as an important regulatory hormone for early ovarian follicle development,the role of AMH in mammalian reproduction has gradually attracted the attention of researchers.Relevant in vitro studies have confirmed that bone morphogenetic protein 6(BMP6),an oocyte exocrine protein,has the characteristics of temporal and spatial expression similarity with AMH during follicle development,it indicates that BMP6 plays a potential role in the regulation of AMH expression,but the specific mechanism of action has not been elucidated.Based on this,in this study,by culturing Dazu black goat ovarian granulosa cell in vitro,using cell immunostaining,exogenous BMP6 protein addition,flow cytometry,q RT-PCR,Western blot,small molecule interference and other techniques and methods to reveal the effect of BMP6 on the granulosa cell proliferation,cycle,apoptosis and steroid hormone secretion in ovarian,to clarify the regulatory mechanism of BMP6 on AMH expression,provide a basis for in-depth study of AMH function in ovarian granulosa cell.The main findings and conclusions of this study are as follows:1.The effect of BMP6 on the proliferation,cycle,apoptosis and steroid hormone secretion of granulosa cells.Exogenous addition of 50 ng/m L BMP6 to goat granulosa cell did not affect the proportion of Ed U positive cells and had no significant effect on the m RNA and protein expression level of the proliferation-related gene PCNA(P>0.05).BMP6 had no significant effect on the cycle phase distribution of granulosa cells(P>0.05),but increased the m RNA expression of CDK4(P<0.05)and CCND1(P<0.01),and decreased the m RNA expression of CCNE1(P<0.01);BMP6 had no significant effect on granulosa cell apoptosis and also had no significant effect on the m RNA expression levels of apoptosis-related genes BAX,BCL 2 and Caspase3(P>0.05).BMP6 could up-regulate the levels of E2 and P4 secreted by granulosa cell(P<0.01),The m RNA expressions of the rate-limiting enzymes CYP11A1 and CYP19A1 were significantly increased(P<0.01),and the m RNA expressions of 3β-HSD and St AR were significantly decreased(P<0.01).The above results indicated that exogenous BMP6 treatment did not affect the proliferation,cycle phase distribution and apoptosis of granulosa cells,but promoted the secretion of E2 and P4 in granulosa cells,and up-regulated the m RNA expressions of CYP11A1 and CYP19A1.2.The effect of BMP6 on AMH secretion in granulosa cell.BMP6 can promote the secretion of AMH hormone in granulosa cell(P<0.01),and up-regulate the m RNA and protein expression levels of AMH(P<0.01);BMP6 can also up-regulate the m RNA expression levels of genes SOX9 and GATA4 related to AMH transcription initiation(P<0.01).),and significantly increased the protein expression level of GATA4(P<0.01);after knockdown of GATA4 gene,the m RNA expression level of AMH was significantly decreased(P<0.05),while the protein expression level of AMH had no significant change(P>0.05).These results indicate that BMP6 promotes the secretion,m RNA and protein expression levels of AMH,and upregulates the expression of genes SOX9 and GATA4 related to AMH transcription initiation.3.Study on the pathway of BMP6 regulating AMH expression.Exogenous addition of BMP6 treatment promoted the phosphorylation levels of SMAD1/5/8(P<0.01)and ERK1/2(P<0.05).It was further found that inhibiting the SMAD1/5/8 signaling pathway significantly abolished the effect of BMP6 on AMH m RNA and protein.Up-regulation effect(P<0.05),and also eliminated the up-regulation effect of BMP6 on GATA4;further verified by simply activating the SMAD1/5/8 signaling pathway,it was found that compared with the BMP6 treatment group,the activation pathway did not affect AMH and GATA4 m RNA and protein expression levels(P>0.05).The results showed that BMP6 promotes AMH expression through the SMAD1/5/8 signaling pathway.