The Regulatory Effects Of RFRP-3 On Proliferation,apoptosis And Steroidogenesis Of Ovarian Granulosa Cells In Tan Sheep

In order to clarify the regulatory role of RFRP-3 on the function of primary ovarian granulosa cells in Tan sheep,this study firstly isolated and cultured primary ovarian granulosa cells from Tan sheep in vitro.The expression of RFRP-3/GPR147 was identified by RT-PCR and Western blot.Secondly,RT-qPCR,ELISA,CCK-8,flow cytometry and Western blot were used to analyze the effects of RFRP-3 on steroid hormone production,proliferation,cell cycle,apoptosis and expression of related genes and proteins in Tan sheep ovarian granulosa cells,and further investigated the signaling pathway of RFRP-3 regulating steroid hormone production and proliferation in Tan sheep ovarian granulosa cells.The main results are as follows:1.The RFRP-3 gene was found to be expressed on Tan sheep ovarian granulosa cells,and the GPR147 gene and protein were also expressed in granulosa cells;2.RFRP-3 treatment inhibited steroid hormone secretion in Tan sheep ovarian granulosa cells,and RFRP-3 alone significantly down-regulated the expression of key enzyme genes and proteins HSD17B2,STAR,CYP19A1,as well as genes LHR,FSHR,INHBA,PGR and ERβ that regulate steroid hormone synthesis(P<0.05);3.RFRP-3 inhibited granulosa cell proliferation and promoted granulosa cell apoptosis in Tan sheep ovaries;significantly down-regulated the expression of granulosa cell proliferation,cyclerelated proteins(PCNA,CDK1,CCND1)and cycle-related genes PCNA,CDK4,CCND1,CCNB2,CCNE2,CDK1,CDK2(P<0.05);meanwhile,RFRP-3 significantly down-regulated the expression of apoptosis-inhibiting Bcl-2 genes and proteins(P<0.05)and significantly up-regulated the expression of pro-apoptosis-related proteins(p53,Bax)and genes(p53,Box,Caspase-3)(P<0.05).4.RFRP-3 significantly down-regulated the expression of FOS,MAP2K2,RAF1,PRKCE,and PLCG2 genes in the ERK1/2 signaling pathway that regulates granulocyte steroid hormone production(P<0.05)and significantly decreased the expression of P-ERK1/2 protein(P<0.05).5.RFRP-3 significantly downregulated the expression of PIP2,Bad,Bid,GSK-3β genes in the PI3K/AKT/mTOR signaling pathway that regulates granulosa cell proliferation(P<0.05),and significantly decreased the expression of P-PI3K,P-AKT and P-mTOR proteins(P<0.05).