Effects Of Sphingosine-1-Phosphate On Granulosa Cells Derived From Porcine Early Antral Follicles

In mammalian ovaries,follicles at the initial stage of antral formation are called early antral follicles(EAFs).Most early antral follicles will undergo atresia or degeneration which results in the waste of abundant and high-quality oocyte resources.Apoptosis of granulosa cells is the major cause of follicle atresia.SIP,a product of intracellular sphingolipid metabolism,promotes survival and proliferation and inhibits apoptosis of various kinds of cells.However,there are few reports on the effects of SIP on follicular development.In this study,the effects of S1P on granulosa cells derived from early antral follicles(EAF-GCs)were studied to provide a theoretical basis for the optimization of in vitro culture system for follicles,and to better preserve and utilize the germplasm resources of precious and endangered species.Experiment 1:The concentration of SIP in GCs of healthy and atresia EAFs were detected by ELISA,and the localization of sphingosine-1-phosphate receptor 1(S1PR1)on porcine ovaries was detected by immunohistochemical staining.Results showed that the concentration of S1P in GCs of healthy EAFs was significantly higher than that of atresia EAFs(P<0.05).In addition,S1PR1 is expressed on oocytes,granulosa cells and theca cells of porcine primordial follicles,primary follicles,secondary follicles and antral follicles.Experiment 2:The effects of different concentrations of S1P(10 nmol/L,100 nmol/L,1?mol/L)on the proliferation of EAF-GCs were detected by MTT assay.It was found that different concentrations of SIP significantly promote the proliferation of EAF-GCs after 48 h(P<0.05),with 100 nmol/L S1P being the most significant group.Experiment 3:EAF-GCs were cultured in vitro,and treated with 100 nmol/L S1P,DMS and S1P+DMS for 48 h.DMS is the specific inhibitor of sphingosine kinase,which is the key of SIP synthesis.Flow cytometry,ELISA and RT-qPCR were used to detect the effects of SIP on apoptosis,steroid hormone synthesis and the angiogenesis gene expression of EAF-GCs.Results showed that SIP has no significant effect on apoptosis rate of EAF-GCs.SIP promote the generation of E2 and significantly up-regulate the ratio of E2 and P4 concentration(P<0.05),indicating that SIP is involved in the regulation of steroid hormone synthesis.The expression of VEGFA mRNA in SIP group was significantly lower than that in control group and DMS group(P<0.05).Conclusion:The concentration of SIP in granulosa cells of healthy EAFs is significantly higher than that of atresia EAFs.SIPRl is expressed on oocytes,granulosa cells and theca cells of porcine follicles at each phase.SIP significantly promote the proliferation of EAF-GCs and has no significant effect on the apoptosis of EAF-GCs.SIP regulate the synthesis of steroid hormones in EAF-GCs and inhibit angiogenesis within follicles.