The Effects And Mechanism Of Kisspeptin-10 On Hormone Secretion And Cell Proliferation Of Sheep FGCs

Objective: To investigate the effect and mechanism of Kisspeptin-10(KP-10)on steroid hormone synthesis,cell proliferation and apoptosis in sheep primary follicular granulosa cells(FGCs).Method: Immunofluorescence detection technology was used to isolate,culture,and identify sheep FGCs.Different concentrations of KP-10 were added to the complete culture medium,and the secretion of estrogen and progesterone in FGCs was measured using ELISA.Steroid hormone synthesis and gene expression of cell proliferation and apoptosis were measured using qPCR.Cell viability was measured using CCK-8,and protein expression related to cell proliferation and apoptosis was measured using Western blot.The main results showed that:1.The primary FGCs of sheep were successfully isolated and cultured,and further confirmed to be FGCs using FSHR immunofluorescence identification method;And it was demonstrated that the KISS-1 gene is expressed in the cytoplasm of FGCs;2.KP-10 alone can promote the synthesis and secretion of steroid hormones in sheep FGCs,and KP-10 alone can significantly upregulate the key enzyme genes St AR,CYP11A1,and 3 for steroid hormone synthesis β-The expression of HSD and KISS-1 receptor genes;3.RNA seq sequencing technology was used to screen and compare different differentially expressed genes between the normal FGCs cell group and the FGCs cell group treated with KP-10 at a concentration of 100 nmol/L.The results showed a total of 202 differentially expressed genes,including 119 up-regulated genes and 83down-regulated genes.Six differentially expressed genes related to cell proliferation and cycle were screened and validated using qPCR technology.The results obtained were basically the same as those of RNA seq sequencing.4.KP-10 promotes the proliferation of FGCs cells,inhibits cell apoptosis,and significantly upregulates the expression of Bcl-2,PI3 K,and AKT m RNA(p<0.01);Significantly downregulated the expression of Bax and Caspase-3 m RNA(p<0.05).100 nmol/L KP-10 treatment of FGCs significantly upregulated the expression level of Bcl-2 protein(p<0.01),and significantly downregulated the expression levels of Bax and AKT protein(p<0.01).In summary,the KISS-1 gene is expressed in the cytoplasm of sheep FGCs cultured in vitro;KP-10 can significantly promote the synthesis and secretion of steroid hormones in sheep FGCs by promoting their proliferation,inhibiting cell apoptosis,or upregulating the expression of key enzymes involved in steroid hormone synthesis;KP-10 can regulate FGC cell proliferation and inhibit cell apoptosis in sheep through the PI3K/AKT signaling pathway.