Study On Effect Of Kiss1 On Secretion Of E2 By Granulosa Cells In Porcine Ovaries

The state of growth and development of ovarian follicles directly determines the reproductive performance of sows.Granular cell function affects the growth and atresia of follicles.It was found that KISS1 can promote the development of follicles and affect the function of granulosa cells in ovarian tissues,but the expression regulation mechanism and cell function of KISS1 in porcine ovarian granulosa cells remains unclear.This study mainly focused on landrace × large white sows,using the porcine ovarian granulosa cells as a cell model.First of all,we studied the expression and regulation mechanism of KISS1 itself,and then studied the effect of this mechanism on the function of granulosa cells.To provide molecular basis for improving the expression regulation of Kiss1 in porcine granulosa cells,and to provide a molecular basis for the analysis of the factors affecting the growth,development,and maturation of ovarian follicles in the growing binary sows.In this study,we detected the expression level change of mRNA in ovarian tissues during the onset of puberty in landrace × large white sows by qRT-PCR.Clone the KISS1 promoter region of the sow,construct a dual luciferase reporter gene recombinant containing the deletion fragment of KISS1 promoter,and analyze the activity of the deletion fragment in the promoter region of KISS1 by dual luciferase reporter system and determine its core promoter region.Bioinformatics website predicts potential transcription factor binding sites in the KISS1 promoter region and validates using ChIP.The eukaryotic expression vector and siRNA containing the CDS region of transcription factor were constructed.The activity of KISS1 promoter and the expression of KISS1 mRNA and protein were analyzed by dual luciferase reporter system,qRT-PCR and Western Blot.Construction of recombinant vector and siRNA containing the CDS region sequence of KISS1 and Transfection into ovarian granulosa cells in pigs.Detection of the concentration of E2 in the supernatant of ovarian granulosa cells by ELASA Kit,analysis of the effect of KISS1 on estrogen receptor gene ESR1 and ESR2 by qRT-PCR technique and analyze the changes of the key gene mRNA level of PI3 K signaling after overexpression or interference with KISS1,thus confirming the function of Kiss1 gene in porcine ovarian granulosa cells.The main results are as follows:(1)The expression of KISS1 mRNA in pig ovary was significantly higher than P1 and P3 at P2.(2)The full-length 2482 bp of the KISS1 gene promoter region-2261 ~ +221 was amplified by PCR,and the dual luciferase system analysis revealed that there was a negative regulatory element binding site in the-850 ~-289 region.(3)Transcription factor prediction website forecast and ChIP validation indicated that transcription factors STAT4 and CEBP? can bind to the promoter region of KISS1 through corresponding DNA action elements,Respectively,specifically bound to the KISS1-309 ~-297 and-744 ~-733 regions.(4)After overexpression of STAT4 and CEBP?,the activity of KISS1 promoter was decreased and the mRNA and protein expression levels were significantly decreased.The expression of KISS1 mRNA and protein were significantly increased after interfering the transcription factors STAT4 and CEBP?.(5)After overexpression of KISS1,the apoptotic rate of granulosa cells decreased,the concentration of E2 in the supernatant of cells increased significantly,and the mRNA expression levels of ESR1 and ESR2 increased significantly;after interference with Kiss1 expression,the apoptosis rate of granulosa cells increased,E2 The concentration of Star,CYP17,3B-HSD,17B-HSD,CYP19 A,ESR1,and ESR2 mRNA levels decreased significantly.(6)After overexpression of KISS1,the mRNA expression levels of PIK3 CG,PI3CI,and PDK increased significantly,and the mRNA expression levels of FOXO3,TSC2,and BAD decreased significantly.After the interference of Kiss1 expression,the mRNA expression levels of PIK3 CG,PI3CI,PDK,FOXO3,TSC2,and BAD decreased.Decline,but the difference is not significant.Based on the above results,the main conclusions are as follows: Transcription factors STAT4 and CEBP? can inhibit KISS1's promotion of granulosa cells secreting E2 by inhibiting the expression of KISS1.