| Wnt/?-catenin signaling regulates numerous physiological activities,including cell fate decision,embryo development,nervous system development and tumorigenesis,et al.,and plays a crucial role in follicle development,granulosa cells growth and oocyte maturation.Cell proliferation,apoptosis and steroidogenesis of granulosa cells dependent on regulation of Wnt/?-catenin signaling.Melatonin is a vital indole hormone,and performs a significant function on nervous system development and anti-tumor.Melatonin plays a vital regulatory role on development of granulosa cells.There are more and more researches which demonstr-ate that Wnt/?-catenin signaling participates in the regulation of melatonin in multiple physio-logical activities.However,it remains to know whether Wnt/?-catenin signaling participate in regulating development of granulosa cells.Phosphatidylinositol-3-kinase(PI3K)signaling acts as a regulator on cell fate determination,nervous system development and ovary develop-ment in high organism.Moreover,PI3 K signaling relates to some human diseases,such as diabetes and Alzheimer diseases and so on.There are more and more researches on tumorigen-esis and physiological activities that prove the tight relationship between Wnt/?-catenin signa-ling and PI3 K signaling.However,it is still unknown that the interaction between Wnt/?-catenin signaling and PI3 K signaling on the regulation of ovary development.In this study,the regulatory role of Wnt/?-catenin on development of porcine granulosa cells was investigated.To demonstrate the interaction between Wnt/?-catenin and melatonin and the crosstalk between Wnt/?-catenin and PI3 K signaling involving cell proliferation,cell apoptosis and steroidogenesis of porcine granulosa cells and so on,Wnt/?-catenin and PI3 K signaling were modulated using small-molecule inhibitor,and cell viability,gene expression and protein level of ?-catenin were detected by CCK-8 kit,qRT-PCR and Western Blot,respectively.This study would expound a new modulation of development of porcine granulosa cells and provided initial exploration and theoretical reference for the advanced investigation of regulatory mechanism of Wnt/?-catenin signaling.This study had acquired the results presented as followed:1.Effect of Wnt/?-catenin signaling on cell proliferation and cell apoptosisPorcine granulosa cells were cultured with Wnt/?-catenin inhibitor IWR-1-endo(DMSO,1,5,10,20,30 ?M)for 48 h.The cell viability of porcine granulosa cells was detected by CCK-8 kit.The protein level of ?-catenin was determined by Western Blot and the genes expression of granulosa cells was detected using qRT-PCR.The results indicated that the cell viability of granulosa cells cultured with 5 ?M IWR-1-endo was reduced signifi-cantly,and degraded protein level of ?-catenin.Expression of cell cycle-related genes CCNA2?CCNB1 and p34 was decreased brightly.And the higher expression of inflammatory –related and apoptosis-related genes TNFAIP6?PTSG2 and Caspase3 was identified in granulosa cells treated with 5 ?M IWR-1-endo.It proved that inhibition of Wnt/?-catenin signaling was likely to suppress cell cycle and enhanced cell apoptosis.2.FSH involved in regulation of Wnt/?-catenin on genes expressionPorcine granulosa cells were cultivated with 5 ?M IWR-1-endo in culture system with FSH(0.1 IU/mL)and FSH-free system.It was indicated that inhibition of Wnt/?-catenin induced upregulation of steroidogenic genes CYP19A1 and StAR and upregulation of FSHR and LHCGR,however,enhanced the expression of ESR2 in granulosa cells cultured with 0.1 IU/mL FSH.While the inhibition of Wnt/?-catenin by IWR-1-endo reduced expression of CYP19A1?StAR and CCND2,and increased expression of FSHR.It was revealed that Wnt/?-catenin signaling regulated steroidogenesis and expression of gonadotropin receptors,and FSH modulated the FSH target genes via ?-catenin/TCF,the downstream transfer factor of Wnt/?-catenin.3.Regulation of melatonin on Wnt/?-catenin signaling induced cell proliferation and genes expressionPorcine granulosa cells cultured with melatonin(DMSO,0.01,0.1,1,10,100 ng/mL)for 48 h acquired higher cell viability,and the highest cell viability was detected in granulosa cells treated with 0.1 ng/mL melatonin.Moreover,upregulation of Wnt/?-catenin was determi-ned treated with 0.1 ng/mL melatonin.Compared to the control group,increase of cell cycle-related genes CCNA2 and CCNB1 while reduction of CCND2 appeared in granulosa cells with 0.1 ng/mL melatonin.Melatonin also increased expression of CYP19 A and St AR,and decreased the expression of TNFAIP6?PTSG2 and Caspase3 to suppress cell apoptosis.It was indicated that Wnt/?-catenin signaling was activated by melatonin and probably to enhance cell cycle,inhibited cell apoptosis and promote syntheses of oestrogen and other steroid hormone.4.Effect of inhibition of PI3 K signaling by Wortmannin on Wnt/?-catenin signalingPorcine granulosa cell cultured with Wortmannin(DMSO,0.1,1,1.5,3,5 ?M)for 48 h.The result showed that ?-catenin was decreased and correspondingly,the cell viability of granulosa cells was declined significantly.In addition,the remarkable inhibition appeared in granulosa cells treated with 5 ?M Wortmannin.Porcine granulosa cells were cultured with 5 ?M Wortmannin for 48 h,and the genes expression was determined by qRT-PCR.The results showed that expression of CYP11A1 and CYP19A1 was reduced vividly induced by Wortmannin.Decrease of mRNA of LHCGR and FSHR and increase of mRNA of ESR2 were reveal in granulosa cells incubated with 5 ?M Wortmannin.The elevated expression of PTSG2 and Caspase3 was tested after the induction of Wortmannin for 48 h.We summarized that Wortmannin blocked PI3 K signaling and resulted in inhibiting Wnt/?-catenin signaling,suppressing cell viability,accelerating cell apoptosis,and decreasing expression of gonado-tropin receptors.It suggested that the crosstalk between PI3 K signaling and Wnt/?-catenin signaling was likely to modulate granulosa cells development.In Conclusion,Wnt/?-catenin signaling played a crucial role on regulating physiological activities during the development of porcine granulosa cells.Melatonin and PI3 K signaling maintained close coordination with Wnt/?-catenin signaling to modulate development of granulosa cells and to ensure the development of porcine proceeded normally.This study proved a reliable theoretical foundation for the further research of regulatory mechanism of Wnt/?-catenin. |
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