Kisspeptin Participates In The Regulation Of Progesterone Secretion In Granulosa Cells Derived From Polycystic Ovarian Syndrome Patients

Background Polycystic ovarian syndrome(PCOS) is a endocrinologic disease which is characterized by hyperandrogenism,chronic anovulation and multiple follicular in ovary, and its morbidity may include hyperinsulinemia, insulin resistance, early onset of type 2 diabetes mellitus(DM), dyslipidemia, cardiovascular disease and infertility.By far,the pathological changes of PCOS include gonadotropin-releasing hormone,hyperpulsatile gonadotropin secretion, increased ovarian,adrenal steroidogenesis and menstrual irregularity.GPR54(G-protein-coupled receptor54)is a number of rhodopsin family which is coded by KISS-1R. GPR54 was found to have a key regulatory position in the hypothalamic–pituitary–gonadal axis by Roux and Seminara respectively in 2003. Kisspeptin is a ligand of GPR54,and the polypeptide is encoded by KISS-1 in humans. Kisspeptin combining with GPR54 in Hypothalamus as a vital signal trigger the peak of Gn RH,which is considered the most effective stimulation in mammalian. Young etfound that plasma kisspeptin levels were significantly higher in the PCOS patients than normal women,which is considered as a clue to one conjecture:kisspeptin may participate in the pathological process of PCOS.S.A. Yilmaz et reported kisspeptin levels correlated significantly positively with luteinizing hormone(LH), total testosterone(T), modified Ferriman–Gallwey(m FG) scores and free androgen index(FAI).About the participation of kisspeptin in the pathological process of PCOS,most researches focused on the central level and its expression in hypothalamus.Researches about the expression of kisspeption in gonads were less and insufficient.In this study,we compared the level of kisspeptin in follicular fluids between pcos patients and nomal women,and research its effects on the hormones which were secretion by granulosa cells and related genes.It is speculate that kisspeptin may participate in the microenvironment of follicular by autocrine and paracrine in the pathological process of PCOS.Purpose 1.Compared the protein level and gene level of kisspeptin in follicular fluids between pcos patients and nomal women to explore the changes of kisspeptin and its recepter in PCOS. 2.Explore the effect of kisspeptin and its agonist p234 on the hormones which were secretion by granulosa cells and related genes.Method 1.Detect the protein level of kisspeptin in follicular by ELISA. 2.Detect the expression of KISS-1/KISS1 R m RNA in granulosa cells abtained from the folicular fluids of patients who receive oocyte retrieval treatment with RT-PCR. 3.Detect the cell proliferation after the stimulation of kisspeptin through CCK-8. 4.Detect the influence of kisspeptin on the secretion of estrogen and progesterone in granulosa cells. 5.Detect the influence of kisspeptin on the change of the expression of related genes in granulosa cells with RT-PCR. 6.Detect the expresion of protein GPR54 by immunofluorescent staining.Result1.The level of kisspeption in follicular was higher in PCOS patients than normal women with statistical significance. 2.The expression of KISS-1 in granolosa cells was higher in PCOS patients than normal women(BMI<25kg/m2)with statistical significance.In non-PCOS patients,the expression of KISS-1 had positive correlation with BMI(r=0.743,P<0.01),the correlation did not exist in pcos patients. 3.Different concentrations of kisspeptin has different effect towards KGN( granulosa cell line):High concentration inhibited the growth of KGN,while low concentration promoted the cell proliferation 4.After treated with kisspeptin,there was no statistical change about the secretion of E2 and progestin from KGN.In luteinized granulosa cells,the secretion of E2 and progestin both increased significantly(P<0.01). 5.After treatment with 600ng/ml and 1600ng/ml kisspeptin, the expression of St AR m RNA、KISS-1R、β-HSD、LDLR、Adponectin m RNA in luteinized granulosa cells did not change significantly. 6.After treatment with p234 at 1600ng/ml, the expression of St AR、β-HSD、P450scc、P450arom �'� LHCG m RNA decreased in luteinized granulosa cells, the difference was NOT significant.The expression of Adponectin、Adipo-R and FSHR m RNA increased,and the change of Adipo-R m RNA is significant. 7.GPR54 expressed in granulosa cells.The protein located in the cell membrane.Conclusion Kisspeptin may participate in the microenvironment of follicular by autocrine and paracrine in the pathological process of PCOS.