Effects Of GDF9 And BMP15 On Chicken Follicular Granulosa Cell Proliferation And Progesterone Production

Chicken ovarian development is a complicated and dynamic process,which is regulated by multi-factors.Female laying performance is directly dependent on the follicular development.Researches in chicken follicular development will lay the theoretical foundation for the understanding of avian reproductive biology and the improvement of chicken laying performance.Growth differentiation factor 9(GDF9) and bone morphogenetic protein 15(BMP15) both belong to transforming growth factor-?(TGF-?) superfamily and have been reported to play important regulating roles in mammalian ovarian development but remain unclear in avian ovaries.The current study aims to investigate the effects of GDF9 and BMP15 on follicular granulosa cells(GCs).We first determined the concentrations of sex hormones,and the expression levels of both receptors of GDF9 and BMP15 in the chicken developmental follicles.Then in vitro cultured GC models were constructed and their abilities to proliferate and produce progesterone were confirmed.In the end,the effects of GDF9 and BMP15 on GCs were investigated using in vitro GC models.The main results of our study are detailed below:1.The dynamic production of progesterone(P4) and estradiol(E2) were showed during the development of ovarian follicles.P4 production began in the hierarchical follicles,increased dramatically at the preovulatory stages(especially F1-F2),declined after the ovulation.E2 was produced predominantly in the prehierarchical follicles,declined gradually in the hierarchical follicles and terminated after the ovulation.The dynamic and opposite relationship between P4 and E2 levels indicated that follicular cells had divergent functions at different developmental stages.2.Around follicle selection,the mRNA expression levels of FSH receptor(FSHR),GDF9 and BMP15 receptors(BMPR2 and ALK6) were upregulated in the GC layer,indicating their potential roles during follicle selection.Remarkably the unique expression pattern of FSHR in the GC layers of SYFs suggested that FSHR and FSH may participated in the follicle selection and granulosa cell differentiation.Higher expression levels of BMPR2,ALK5 and ALK6 in the GC layers than that in the TC layers implied that GDF9 and BMP15 may regulated GC functions.3.GC models in vitro could mimic the true states of GCs in developmental follicles in vivo and thus were suitable for the researches on GC proliferation and P4 production.The differences of cell proliferation and the responses to forskolin between GCs from prehierarchical follicles(phGCs) and GCs from preovulatory follicles(poGCs) indicated that GC differentiated upon follicle selection.GCs cultured in vitro proliferated better in response to EGF stimulus and poGCs produced more P4 and upregulated the expression levels of steroidogenesis genes STAR,CYP11A1 and HSD3 B in response to FSH stimulus.All these observations indicated that endocrine FSH and paracrine EGF both participated in the follicular development.4.GDF9 regulated GC functions in many aspects.GDF9 inhibited GC proliferation in a dose-dependent manner,potentiated cell cycle progression(G1/S transition) and regulated related gene expressions.GDF9 also promoted DNA replication and decreased cell apoptosis in phGCs but not in poGCs.5.In the poGC assays,GDF9 alone did not affect the basal P4 production,while in the cotreatment with FSH,GDF9 promoted FSH-induced P4 production and mRNA expression level of STAR gene,probably by mediating the regulation of FSHR expression.6.BMP15 decreased GC proliferation,which could be blocked by inhibitors LY2109761 and dorsomorphin instead of SB505124,which may suggest that BMP15 signals via BMPR2/ALK6 instead of ALK5.7.BMP15 showed an anti-proliferating and pro-differentiating effect on GCs in follicular development.In prehierarchical follicles,BMP15 downregulated EGF-induced phGC proliferation;in preovulatory follicles,BMP15 enhanced FSH-induced P4 production.