Functional Relationship Between Follicle Stimulation Hormone And Bone Morphogenetic Protein 9 In Regulating Proliferation By Human Granulosa Cells

Bone morphogenetic protein 9(BMP-9)has been recognized associated with cell proliferation,apoptosis and differentiation in various tissues.However the role of BMP-9 in human oocyte maturation regulatory networks and its relationship with follicle stimulation hormone(FSH)still been known a little.Here,we explored the effect of BMP-9 on the proliferation of human ovarian luteinized granulosa cells in vitro,and investigated the underlying mechanisms of the effect of BMP-9 on FSH-induced granulosa cell(GC)proliferation and steroid synthesis.The activity and proliferation of GCs treated by BMP-9 were observed through CCK-8 and EdU analysis.The level of associated mRNA,protein and cAMP synthetic induced by FSH under stimulation of BMP-9 of granulosa cell were measured by RT-PCR,Westernblot and cAMP kit,respectively.The results of CCK-8 showed that cultured with the addition of BMP-9 up-regulated the granulosa cell activity in dose and time-dependent,apporch the most significant increase at 100 ng / ml(3.52±0.17 folds,P <0.05)and at 48hours(0.20±0.11,0.46±0.10,P <0.05),respectively.The co-culture group of BMP-9 combined with FSH showed a larger proportion of EdU-labeled cells than control group(3±0.82,5±0.67,P<0.01).What's more,in consistent with observation of cell viability trend and EdU assay,BMP-9 increased the mRNA levels of PCNA in a dose-dependent manner(P<0.05),as well as increased the expression of FSH-induced PCNA mRNA.Compared with FSH group,the results of Westernblot showed that the level of FSHR protein was significantly heighten in BMP-9 extra added group(2±0.02,2.7±0.13 flods,P <0.01);the PKA protein level and the amount of cAMP production were decreased(6±0.17,4±0.06 flods,P <0.05);the Ras(2.2±0.11,3±0.10 flods,P <0.01)and Raf protein level(1.9±0.09,2.6±0.16 flods,P <0.01)and the phosphorylation of ERK1 / 2 were significantly increased(1.9±0.05,3±0.08 flods,P<0.05),as well.After inhibiting the expression of FSHR,PKA,ERK protein and cAMP synthesis in granulosa cells,respectively,the related change of PCNA,StAR,3?-HSD and P450 arom protein was observed in WB experiments.Results of this study indicated that BMP-9 promotes human ovarian granulosa cell proliferation related to the expression of PCNA protein.Furthermore,BMP-9 regulates the granulosa steroid synthesis by affecting FSH-induced FSHR / cAMP / PKA and Ras/Raf/ERK signaling pathways.