Effect Of Kisspeptin On The Secretion Of Steroid Hormone In Tan Sheep Ovary Granulosa Cells And Its Regulation Mechanism

In this study,the primary ovarian granulosa cells of Tan sheep were isolated,cultured,and identified;secondly,ELISA,CCK-8,flow cytometry and q-PCR were used to study the effects of kisspeptin on the secretion of steroid hormones in Tan sheep ovarian granulosa cells.,Granulosa cell proliferation,cell cycle,cell apoptosis and related gene expression,and further explored the signal pathway of kisspeptin regulating the synthesis of steroid hormones in Tan sheep ovarian granulosa cells.The aim is to clarify the specific regulatory effects of kisspeptin on primary ovarian cells of Tan sheep and reveal its mechanism of action and signaling pathways,which will help to improve and enrich the theoretical knowledge of kisspeptin’s regulation of the entire reproductive axis,for the development of kisspeptin-mediated The new technology of animal reproduction regulation lays the foundation for the early stage.The main results are as follows:1.Primary ovarian granulosa cells of Tan sheep were correctly isolated and cultured,then the property of ovarian granulosa cells was confirmed by FSHR immunofluorescence;meanwhile,it was proved that ovarian granulosa cells could express kiss1mRNA and GPR54 mRNA;2.Kisspeptin alone could promote the secretion of steroid hormones in Tan sheep ovarian granulosa cells,which had a synergistic effect with hCG co-treatment;Kisspeptin alone could significantly up-regulate the gene expression of key enzyme genes and related genes regulating steroid hormone snythesis,such as STAR,CYP19,HSD17B2,FSHR,ERβ,PGR,LHR and INHBA(P<0.05);3.Kisspeptin could promote the proliferation and inhibit apoptosis of ovarian granulosa cells in Tan sheep;kisspeptin significantly up-regulates genes expression related cell proliferation and cell cycle,which included CDK1,CDK2,CDK4,CCND1,CCNB2,CCNE2,and PCNA(P<0.05);meanwhile,kisspeptin also remarkably down-regulated the expression of apoptosis-promoting gene including p53,Bax and Caspase-3(P<0.05)and up-regulated the expression of apoptosis-inhibiting gene Bcl-2(P<0.05);4.Kisspeptin significantly increased the expression of FOS,MAP2K2,RAF1,PRKCE,and PLG2 in the PLC/PKA/Raf/MAPK signaling pathway(P<0.05),and significantly increased the expression of phosphorylated ERK1/2 protein(P<0.05).In conclusion,the ovarian granulosa cells of Tan sheep cultured in vitro express kiss1 and GPR54 genes;Kisspeptin promotes Tan goat ovarian granulosa cell proliferation,inhibits apoptosis,or up-regulates the expression of key enzymes for steroid hormone synthesis in Tan sheep.Ovarian granulosa cells secrete steroid hormones;in Tan sheep ovarian granulosa cells kisspepin regulates the synthesis of steroid hormones in granulosa cells through PLC/PKC/MAPK intracellular signaling pathways.