Effect And Mechanism Of Kisspeptin-10 On Ovarian Granulosa Cells Of Qinghai Yak

Kisspeptin,a key factor in mammalian reproductive initiation,is a short peptide encoded by Kiss-1 gene.In the study of various mammals.Kisspeptin is found to be distributed in hypothalamus,nerve center,ovary,etc,while its smallest active fragment,Kisspeptin-10?kp-10?,which is highly conservative among species,has a promoting effect on the proliferation of granulosa cells and progesterone secretion in the ovary of animals.Whether kp-10 can also regulate the proliferation and hormone secretion of ovarian granulosa cells in Qinghai yak is unclear.In this experiment,Qinghai yak ovarian granulosa cells were extracted by suction method,and immunofluorescence technology was used to test whether the cultured cells were ovarian granulosa cells.HE staining was used to observe the morphology of granulosa cells.CCK and ELISA methods were used to detect the proliferation and progesterone secretion of Kp-10 on ovarian granulosa cells of Qinghai yak.Flow cytometry was used to detect the effect of Kp-10 on apoptosis of granulosa cells.In further research,combined with ELISA and flow cytometry,the possible mechanism of Kp-10 on progesterone secretion from ovarian granulosa cells of Qinghai yak was preliminarily discussed.This research mainly includes the following three parts:?1?Ovarian granulosa cells of Qinghai yak were cultured in vitro.After selecting an appropriate ovary,the cells were extracted by suction method,and the cultured cells were examined by immunofluorescence?FSHR?to see if they were granulosa cells.Through HE staining and CCK method,the changes of cell morphology and cell activity of granulosa cells in vitro culture.The test results showed that the positive rate of FSHR was>97%,which verified that the cells were ovarian granulosa cells.Yak ovarian granulosa cells with good growth can be obtained.Granulosa cells are polygonal or spindle-shaped and extend out pseudopodia and grow radially.After 24 hours of culture,granulosa cells entered logarithmic phase and plateau phase 72 hours later.?2?Kp-10 affects the proliferation,apoptosis and progesterone secretion of ovarian granulosa cells of Qinghai yak cultured in vitro.Qinghai yak ovarian granulosa cells cultured in vitro were cultured in a medium containing fetal bovine serum for 6 hours and then gradually changed to a serum-free medium.Kp-10 with different concentrations was added.CCK was used to observe the proliferation of cells within 72 h,and the progesterone content in the culture medium was detected by ELISA.The results showed that Kp-10 treatment could significantly increase the activity of granulosa cells?p<0.05?,and 100nmol·L^-1 could significantly promote the secretion of progesterone?p<0.05?.PI staining was used to detect cell apoptosis.It was found that when 100nmol·L^-1 was cultured for 24 hours,the cell apoptosis occurred.?3?The mechanism of kp-10 on progesterone secretion in ovarian granulosa cells of Qinghai yak may be affected by intracellular calcium signaling pathway.After adding Kp-10 and Verapamil with different concentrations to treat the cells individually or jointly for 24 hours,the progesterone content in the supernatant was measured by ELISA,and the fluorescence intensity of intracellular calcium ions was detected by flow cytometry.Verapamil,a calcium ion blocker,can reduce the secretion of progesterone,reaching a significant reduction level at 20 and 50 nmol·L^-1?p<0.05?.When 100nmol·L^-1Kp-10 was treated with Verapamil of different concentrations,the progesterone content increased,but its inhibitory effect on progesterone secretion could not be reversed?p>0.05?.The intracellular Ca²⁺ concentration was detected by flow cytometry,and the results were consistent with the progesterone secretion level of granulosa cells.In conclusion,Kp-10 can promote progesterone secretion of yak ovarian granulosa cells cultured in vitro,and its mechanism may be related to intracellular Ca²⁺ concentration.