Kisspeptin-10 modulates hormone secretion and proliferation of follicular granulosa cells by upregulating expressions of the kiss-1r gene

The present study aimed to determine the subcellular localization of KISS-1 protein, and also to investigate the effects of Kisspeptin-10 (KP-10) on the proliferation viability and hormone secretion of follicular granulosa cells (FGCs), and its molecular mechanisms. FGCs were cultured in vitro in DMEM media supplied with the different doses of KP-10 (0,10,100 and 1000 nmol/L) which were allocated into a control group (CG), experiment group 1 (EP1), experiment group 2 (EP-2) and experiment group 3 (EP-3), respectively, CG, EP-1, EP-2 and EP-3. Subcellular localization of FSHR and KISS-1 proteins in FGCs were assayed using immunofluorescence assay. A Cell Counting Kit 8 (CCK8), real time quantitative PCR (qPCR), and western blotting were used to detect the proliferation activities and expression levels of genes and proteins in the FGCs. Elisa kits were applied to measure the contents of estrogen (E2) and progesterone (P4). Immunofluorescence assay showed that KISS-1 protein was positively expressed in sheep FGCs and mainly distributed in the cytoplasm. At 48h and 72h following KP-10 treatment, the cell viability of the FGCs in groups EP-2 and EP-3 were significantly enhanced compared to CG (P<0.05 or P<0.01). The estrogen (E2) and progesterone (P4) contents of group EP-2 were increased in comparison with CG (P<0.01). Additionally, expression levels of Kiss-1R, StAR, 3β-HSD and CYP11 mRNAs in group EP-2 were remarkably increased compared to CG (P<0.05 or P<0.01) with increments of 65.9%, 30.7%, 73.4% and 71.4%, respectively. In conclusion, KISS-1 proteins were located mainly in the cytoplasm of FGCs. KP-10 could enhance FGC vitality, and promote progesterone and estrogen secretion, which is possibly achieved by upregulation of expression levels of Kiss-1R, StAR, 3-β-HSD and CYP11 genes. KP-10 significantly upregulated mRNA and protein levels of Bcl-2 and PI3K (P<0.01), and downregulated expressions of mRNA and protein levels of Bcl-2 and PI3K (P<0.05). Our outcomes provide a solid basis for searching after novel improvements of reproduction of animals.