Regulation Of LRH-1 On Apoptosis And Secretion Of Steroid Hormones In Bovineovarian Granulosa Cells

LRH-1?Liver receptor homolog-1,NR5A2?,also known as NR5A2 is a member of the nuclear receptor family.It acts as a transcriptional activator to regulate the ex pression of steroid-related genes,and regulates the secretion of steroid hormones and the regulation of breast cancer factors.Important role.Most follicles in female mam mals are blocked.Hormone regulation is also one of the factors of follicular atresia.Apoptosis of granulosa cells is considered to be the main mechanism of follicular atr esia,but whether LRH-1 is involved in the proliferation of bovine ovarian granulosa cells.The regulation of death still needs to be confirmed.More and more studies hav e shown that the progesterone receptor PGR meAdiates the anti-apoptotic signaling pa thway of cells,and the effect of progesterone?P4?on the reproductive tissues of fem ale animals is also mediated by PGR.Progesterone receptor signaling pathway is inv olved in the ovarian estrous cycle and luteinization,and regulates the body's ovarian development,pregnancy and other important activities.It is also closely related to so me diseases,including Alzheimer's disease,Parkinsons and so on.However,there is no report about LRH-1 mediating apoptosis of bovine ovarian granulosa cells,and its regulatory mechanism is unknown.Activation of LRH-1 expression and expression of progesterone receptor signaling pathway-related receptors by specific small molecule activators?DLPC and P₄?using immunohistochemistry,RT-qPCR,Western blot,ELISA,immunofluorescence cell assay,Annexin V-FITC/PI and CCK-8 and other techniques,to explore the LRH-1 synthesis of bovine ovarian granulosa cell steroid hormone and its physiological regulation of b ovine ovarian granulosa cells,trying to elucidate LRH-1 regulation of follicular devel opment The new mechanism provides preliminary verification and reference for the s ubsequent study of ovarian function.The test mainly obtained the following results:1.This experiment uses bovine ovarian granulosa cells in vitro serum-free cultur e system.The isolated and cultured bovine ovary granulosa cells had high purity and the isolation rate reached 95.5%.Immunohistochemistry revealed that LRH-1 was hig hly expressed in bovine ovarian granulosa cells.2.The bovine granulosa cells were treated with different concentrations of DLP C?50?M,100?M,125?M,150?M?for 24h.The content of progesterone,estradiol a nd testosterone secreted by granulosa cells was detected by ELISA.The results show ed that different concentrations of DLPC inhibited the secretion of progesterone?P₄?and estradiol?E₂?.The inhibition of 50?M and 100?M DLPC was more obvious.Int erestingly,150?M DLPC could induce the secretion of testosterone?T?.This indicates that LRH-1 activation inhibits the secretion of P₄ and E₂ hormones in bovine ovarian granulosa cells.3.Addition of 50?M DLPC significantly promoted the expression of LRH-1 gen e and protein?P<0.05?;Adding 50?M DLPC up-regulated the expression of AR and StAR,and 100?M DLPC promoted the expression of CYP19,HSD3?1,HSD17?1,ES R1 and ESR2 genes,and down-regulated the expression of CYP17?CYP11.This indi cates that upregulation of LRH-1 expression regulates the expression of steroid genic pathway-related genes?StAR,CYP11,CYP19,HSD3?1,HSD17?1,CYP17?and recept ors?AR,ESR1,ESR2?.4.PPAR?protein expression was detected by Western blot.Cell proliferation wa sdetected by CCK-8,and apoptosis was detected by Annexin V-FITC/PI.The results revealed that different concentrations of DLPC inhibited the expression of downstream PPAR?protein of LRH-1?P<0.05?.Different concentrations of DLPC inhibited the pr oliferation of bovine ovarian granulosa cells,and 50?M DLPC significantly promotedt he apoptosis of bovine ovarian granulosa cells?P<0.05?.This indicates that up-regulat ion of LRH-1 inhibits the expression of downstream protein PPAR?,promotes apopto sis,and inhibits cell proliferation.5.Up-regulation of LRH-1 expression significantly inhibited the expression of pr ogesterone receptors?P<0.05?.Immunofluorescence showed that 50?M DLPC+10?M P₄ significantly increased the expression of PGR?P<0.05?,but had no significant effe ct on the expression of progesterone receptor membrane protein?PGRMC1??P<0.05?.CCK-8 showed that 50?M DLPC+?5?M P₄ or 10?M P₄?significantly increased cell proliferation in bovine granulosa cells.Annexin V-FITC/PI showed that 50?M DLPC+?5?M P₄ or 10?M P₄?significantly inhibited apoptosis of bovine ovarian granulosa cells?P<0.05?.Western blot was used to detect the expression of apoptotic proteins.50?M DLPC+?5?M P₄,10?M P₄ or 100?M P₄?significantly inhibited the expression of Cleaved-Capase3 protein?P<0.05?;50?M DLPC+?10?M P₄ or 100?M P₄?signific antly promoted Bcl-2 Protein expression?P<0.05?.The results of this experiment indi cate that the progesterone receptor activator reverses the inhibition of LRH-1 on the apoptosis of bovine ovarian granulosa cells and the inhibition of cell proliferation.In summary,LRH-1 inhibits the biosynthesis of P₄ by regulating the steroidogeni c synthesis pathway.In addition,LRH-1 promotes apoptosis of ovarian granulosa cell s by inhibiting P₄/PGR signaling,thereby mediating follicular atresia.The results of t his study provide a new theoretical basis for the study of subsequent follicular devel opment mechanisms.