Effects Of PI3K/Akt Signaling Pathway On Porcine Oocytes In Vitro Maturation

Oocytes are the basis of animal embryo engineering technology research and development,its mature quality directly affects the efficiency of embryo engineering technology.The quality of porcine oocytes in vitro maturation is significantly lower than that of in vivo,and the technology system of porcine oocytes in vitro maturation needs to be further improved.Phosphatidylinositol-3-kinase/Protein kinase B（PI3K/Akt）signaling pathway is related to ovarian function,mainly including the proliferation of primary follicle recruitment、follicle granulosa cells luteal formation and oocyte maturation.But there are few studies on the maturation about porcine oocytes.In this study,the relationship between PI3K/Akt signaling pathway and porcine oocytes in vitro maturation was preliminarily explored,so as to improve the quality of porcine oocytes in vitro maturation by regulating the PI3K/Akt signaling pathway.1.The effects of EGF-activated the PI3K/Akt signaling pathway on porcine oocytes in vitro maturation were investigated.The results showed that addition of 10 ng/ml EGF in the mature medium significantly increased the expansion of cumulus cells（P<0.05）,it significantly increased the mature rate of porcine oocytes（P<0.05）,what’s more,it significantly increased the 2-cell rate,4-cell rate and blastocyst rate of embryos from parthenogenetic activation（P<0.05）.The analysis of RT-q PCR showed that activation of the PI3K/Akt signaling pathway significantly up-regulated the expression levels of GDF9、Akt、CDC2、Cyclin B1 and MOS genes（P<0.05）,significantly up-regulated the expression levels of P34（P<0.01）,and up-regulated the expression levels of BMP15（P>0.05）.Western blot detection showed that 10 ng/ml EGF significantly increased the phosphorylation level of p-Akt ^Thr308（P<0.05）.2.The effects of PI3K/Akt signaling pathway activator PS48 on porcine oocytes in vitro maturation and proliferation of cumulus cells were studied.After Hoechst33342 staining of oocytes cultured at different concentrations,it was found that PS48 of 10μM could significantly improve oocytes develop to MII stage（P<0.05）.RT-q PCR analysis that the relative expression of GDF9 and BMP15 were significantly reduced,compared with the control group（P<0.05）.The relative expressions of MOS and Cyclin B1 were significantly increased compared with the control group（P<0.01）.Relative expressions of Akt and m TOR were significantly increased compared with the control group（P<0.05）.The relative expression of CDC2 was not significantly different from control group（P>0.05）.Western blot detection showed that PS48 of 10μM significantly increased phosphorylation level of p-Akt ^Thr308 and Akt protein（P<0.05）.The analyzed of proliferation and RT-PCR about cumulus cells,the results showed that 10μM PS48 could promote the proliferation and increase the mitochondrial membrane potential of cumulus cells,BCL2 and Caspase3 were significantly down-regulated（P<0.01）,BAX was significantly down-regulated（P<0.05）,the expression of CDK4 and P27 was down-regulated with no significant difference（P>0.05）.3.The effects of PI3K/Akt signaling pathway inhibitor LY294002 on porcine oocytes in vitro maturation were investigated.The results showed that 50μM of LY294002 had a significant inhibitory effect on cumulus cells expansion（P<0.05）,and significantly inhibited the mature rate and2-cell rate of porcine oocytes（P<0.05）.RT-q PCR detection showed that the expression of GDF9、BMP15、Akt、CDC2、Cyclin B1 and P34 genes were significantly down-regulated（P<0.01）.Western blot results showed that both Akt and p-Akt ^Thr308 were expressed in porcine oocytes,while LY294002 of 50μM inhibited both Akt and p-Akt ^Thr308 in porcine oocytes,was significantly lower than control group（P<0.01）.In summary,EGF of 10 ng/ml added to the medium of porcine oocytes in vitro maturation,it significantly increased the phosphorylation level of p-Akt ^Thr308 and further improved the mature rate of oocytes and embryonic development after parthenogenetic activation.Adding 10μM of PS48 to the mature medium could also improve the mature rate of oocytes.On the contrary,Adding 50μM of PI3K/Akt signaling pathway inhibitor LY294002 to the mature medium can inhibit oocyte maturation,and reduce the number of blastocyst cells of parthenogenetic activation.