The Role Of WNT5A In Regulating The Expression Of HAS2 And Insulin Reactivity In Ovarian Granulosa Cells

BACKGROUNDThe effects of gonadotropins,including follicle-stimulating hormone(FSH)and luteinizing hormone(LH),on follicular development and ovulation are coordinated by local signal molecules produced by various cells in the ovary.WNT signaling molecules are an important type of ovarian local factors.WNT signaling pathway is highly conserved among many species.WNT signaling regulates cell proliferation,cell fate determination,and cell polarity and other important processes in cell growth and differentiation.According to the different signaling cascades of WNT proteins,the WNT pathways can be divided into:the canonical WNT signaling pathway(?-catenin-dependent)and the non-canonical WNT signaling pathway(?-catenin-independent).Among them,the non-canonical WNT signaling pathway participates in the regulation of the cytoskeleton formation process and the establishment of cell polarity by activating downstream signaling pathways(WNT-Ca2+and WNT-PCP).The various components of WNT and its signaling pathways are specifically expressed at different stages of follicular development,and are essential for the normal development and maturation of ovaries and follicles.Polycystic ovary syndrome(PCOS)is a complex endocrine metabolic disease determined by genes and environment.It is a common ovarian organic disease that causes ovulation disorders and infertility in women.The incidence of insulin resistance(IR)in patients with polycystic ovary syndrome(PCOS)can reach 70%.Insulin resistance is critical in the pathophysiological changes of PCOS.When the body's sensitivity and responsiveness to insulin decrease,and glucose uptake and utilization by insulin target organs or tissues cannot be promoted by normal levels of insulin,systemic or local insulin resistance will occur.In addition,PCOS patients are often accompanied by abnormal ovulation or ovulation disorders,and insulin resistance can also exacerbate ovulation abnormalities or disorders,which is also a common cause of infertility in PCOS patients.Studies have shown that the expression of WNT5A,a gene that encodes the non-canonical WNT signal,is increased in the ovarian granulosa cells of patients with polycystic ovary syndrome,and WNT5A mainly regulates inflammation by activating the PI3K/AKT/NFKB signaling pathway.WNT5A is involved in the follicular response of gonadotropins and is necessary for normal follicular development.However,the mechanism of WNT5A in follicular development and ovulation,especially the effect of WNT5A on granulosa cell ovulation response and insulin responsiveness needs further study.Chapter I Gonadotropins regulate the expression of Wnt5a in mouse ovarian granulosa cellsOBJECTIVE:The expression of Wnt related genes in ovarian tissue was detected to clarify the cell specificity of Wnt protein expression during ovarian development,and the role of Wnt related genes in ovarian follicle development was discussed.MATERIALS AND METHODS:Immunohistochemistry and qRT-PCR were used to detect the expression of genes in ovaries of mice at 3 and 6 weeks,during estrous cycles and after ovulation induction.Western blotting was used to detect the expression of the target protein in ovulation induced mice ovaries and GC cells supplemented with recombinant IL6 protein.RESULTS:Immunohistochemical results showed that the specific expression of Wntless and ?-catenin in small follicles was stronger than large follicles in 3-week or 6-week mice.Wntless and ?-catenin were expressed in all follicles.With the development and maturation of follicles,the expression of Wnt5a was gradually increased.The qRT-PCR results showed that the expression of Wnt5a in the ovaries of 6-week mice was significantly higher than that of 3-week mice.The expression of Wntless and Axin2 increased during the diestrus and proestrus,but decreased during the estrus and metestrus.The expression of Wnt5a was relatively increased during estrus and metestrus.Compared with the control group,the expression of Fshr,Lhcgr,Areg,Has2,Ptgs2,Wnt5a,Wntless,Axin2 and IL-6 were significantly increased after ovulation induction treatment.WB results showed that the expression of Wnt5a was increased after ovulation induction treatment or GC cells added recombinant IL6 protein,and the expression of p-NFkB/NFkB was similar to Wnt5a.The expression of p-Akt/Akt decreased after ovulation induction treatment,but increased after the addition of IL6 protein.CONCLUSION:The related genes in the Wnt pathway:Wntless,?-catenin and Wnt5a,are expressed in various stages of ovarian follicle development.Wntless and?-catenin are more affected by FSH promotion in primary and secondary follicles,and their expression is relatively high.However,their expression is relatively low when the follicle maturation or ovulation is regulated by LH.The expression of Wnt5a is relatively low in primary and secondary follicles due to FSH,while the promoted expression of LH is significantly increased in mature follicles or ovulatory follicles.Be consistent with studies,LH is likely to directly or indirectly affect the expression of Wnt5a through the regulation of IL6 and NFkB,which requires further verification.Chapter ?.The role of WNT5A in LH-mediated ovulation response of granulosa cellsOBJECTIVE:To explore the mechanism of LH-mediated elevated Wnt5a expression in ovulation occurrence and development.MATERIALS AND METHODS:qRT-PCR was used to detect gene expression in KGN cells treated with Forskolin+PMA and BAY-117082(NFkB inhibitor),and Western Blotting(WB)was used to detect protein expression.WB was used to detect the expression of the target protein in KGN cells treated with Forskolin+PMA and BAY-117082.Forskolin+PMA treated KGN cells for different time(0,1,6,24,48h),WB was used to detect the protein expression level.KGN cells was treated with WNT5A recombinant protein,LY-294002(PI3K inhibitor)and SP600125(JNK inhibitor),the expression level of target gene was detected by qRT-PCR,and the level of target protein was detected by WB.siRNA-Wnt5a was successfully transferred into KGN cells,WB detect the protein expression level.RESULTS:qRT-PCR results showed that the expressions of WNT5A,AREG,IL6 and HAS2 were increased in KGN cells treated with Forskolin+PMA compared with the control group,while the expressions of WNT5A,HAS2 and PTGS2 were significantly decreased in KGN cells treated with BAY-117082(NFkB inhibitor)compared with the treatment with Forskolin+PMA.The expression level of HAS2 in KGN cells treated with WNT5A recombinant protein and inhibitors SP600125 and LY294002 was significantly lower than the group treated with WNT5A recombinant protein.WB results showed that the expressions of WNT5A and p-NFkB/NFkB were significantly increased in KGN cells treated with Forskolin and PMA for 24 hours compared with the control group,and the Akt/GSK3B pathway was significantly inhibited by LH.When KGN cells were treated with Forskolin and PMA for 24h and 48h,WNT5A expression was further increased,HAS2 was also increased,and PI3K/Akt/GSK3b pathway was activated again.The addition of WNT5A recombinant protein,PI3K inhibitor and JNK inhibitor in KGN cell culture significantly reduced the expression of HAS2 and inhibited the PI3K/AKT/GSK3B pathway.siRNA-WNT5A was successfully transferred into KGN cells,and the expression of WNT5A was significantly reduced,the expression of HAS2,and the PI3K/AKT/GSK3b pathway was inhibited.CONCLUSION:KGN cells were treated with PMA+Forskolin to simulate the peak of LH.LH has an inhibitory effect on PI3K-AKT-GSK3B under physiological conditions.With the occurrence of ovulation,LH may regulate the expression of downstream WNT5A through IL-6 regulation of NFKB pathway,and WNT5A regulates the expression of HAS2 through PI3K-AKT-GSK3B,and then regulates the occurrence of ovulation of ovarian follicles.Chapter ?.Correlation between WNT5A overexpression and insulin resistance in ovarian granulosa cellsOBJECTIVE:To further explore the role of WNT5A in insulin sensitivity and insulin resistance in granulosa cells.MATERIALS AND METHODS:Add different concentrations of insulin to KGN cells for different time,and add Forskolin+PMA or WNT5A recombinant protein treatment,WB detects the protein expression level.Plasmid p-EGFP-WNT5A-N1 was constructed and transferred into KGN cells,and p-EGFP-N1 was used as the control,insulin was added,protein expression was detected by WB,and metabolism level of lactic acid in KGN cells was detected by lactate detection kit.RESULTS:When KGN cells were treated with insulin,WB results showed that the best treatment time and concentration of insulin was 100ng/ml for 30min.Adding Forskolin and PMA to simulate LH ovulation induction,the effects of insulin(100ng/ml,30min)treatment measured by WB showed that p-AKT/AKT and p-GSK3?/GSK3? phosphorylation decreased.After WNT5A recombinant protein was added for 3 hours,the WB results showed that the phosphorylation of p-AKT/AKT and p-GSK3?/GSK3?increased.When KGN cells were transfected with p-EGFP-N1-WNT5A,the expression of WNT5A was significantly increased,and the effects of insulin(100ng/ml,30min)examined by WB showed that the expression of p-AKT/AKT,p-GSK3?/GSK3? was significantly lower than that of the control group.The determination of lactate showed that,compared with the p-EGFP-N1 treatment group,the production of lactic acid in the cells was gradually decreased by overexpression of WNT5A in KGN cells.CONCLUSION:When WNT5A is successfully overexpressed in KGN cells,it is no longer a promoter but an inhibitor of AKT-GSK3?.WNT5A overexpression will cause disorder of insulin sensitivity through the AKT-GSK3? pathway,and then lead to the occurrence of insulin resistance in granulosa cells.