Study On The Expression And Subcellular Localization Of SGK3 In Mouse Oocytes

Objective:Serum and glucocorticoid-induced protein kinase(SGK)is a Ser/Thr double specific protein kinase,which belongs to AGC(PKA/PKG/PKC)kinase family.SGK has high homology with second messenger proteins such as protein kinase B(AKT/PKB),and both are regulated by phospho lnositide-3-kinase/phosphoinositol-dependent protein kinase 1(PI3K/PDK1)signaling pathway.SGK is widely involved in gene rapid transcription regulation and intracellular localization regulation in eukaryotes.SGK mediates DNA synthesis,glucose transport,cell survival,cytoskeleton rearrangement and other physiological processes,and regulates cell proliferation,differentiation,or apoptosis through its shuttling between cytoplasm and nucleus.Mammalian cells express three isoforms of SGK1,SGK2 and SGK3.Recently,more and more reports have revealed the role of SGK3 in human tumors,such as regulating tumor growth,survival and metastasis,affecting tumor stem cells(CSCs)and participating in tumor therapeutic resistance,and also proved that SGK3 is related to the occurrence and development of a variety of diseases.SGK directly phosphorylates cell cycle factor 25(CDC25)and myelin transcription factor 1(MYT1)and triggers the activation of cyclinb-CDK1,leading to the G2/M phase of meiosis.Mouse oocyte is one of the ideal models for mammalian cell cycle research,but the specific mechanism which SGK3may involve the regulation of cell cycle process has not been thoroughly investigated.However,whether SGK3 is expressed in mouse oocytes and whether it has a regulatory effect on the resumption of the first meiosis during oocyte maturation has not been reported at home and abroad so far.Therefore,in order to verify the role and influence of SGK3 in the resumption of the first meiosis of mouse oocytes,real-time PCR,Western blotting and indirect immunofluorescence techniques were used to detect the expression and subcellular localization of SGK3 in mouse oocytes.This study provided experimental basis for studying the role of SGK3 in regulating the first meiosis of mouse oocytes,and laid a solid foundation for further studying the mechanism of early development of mouse oocytes in the future.Methods:1.Mouse oocytes of GV,GVBD,MⅠ and MⅡ phase were collected by superovulation technique.2.Real-time PCR was used to detect m RNA relative expression levels of SGK1-3 in four phases of mouse oocytes.3.The protein expression levels of SGK1-3 in mouse oocytes were detected by Western-blotting method.4.The subcellular localization of SGK3 in GV,GVBD,MⅠ and MⅡ of mouse oocytes were observed by indirect immunofluorescence.Results:1.Real-time PCR showed that SGK1-3 m RNA were expressed in GV,GVBD,MⅠ and MⅡ phases of mouse oocytes,but the relative expression levels were different.SGK1 and SGK2 were mainly expressed in GV,and both showed a decreasing trend from GV to MⅡ,but the expression level of SGK1 was significantly higher than that of SGK2 in the same period,and the expression level of SGK3 in GVBD and MⅡ was significantly higher than that of GV and MI.2.Western blotting results showed that the expression trends of SGK1-3 in GV,GVBD,MⅠ and MⅡ were basically consistent with their m RNA expression trends.The expression levels of SGK1 and SGK2 decreased gradually from GV to MⅡ,while the expression levels of SGK3 increased significantly from GV to GVBD,and decreased significantly in MⅠ,then increased again and reached the peak in MⅡ.3.Cellular immunofluorescence experiments indicated that SGK3 signals which coupled with red fluorescent were localized in the nuclear membrane in GV-stage oocytes;translocated to the nucleus before GVBD and localized in the nucleus during GVBD;SGK3 signal(red fluorescent)were distributed in the whole cell in the MⅠ phase and translocated to nucleus in the MⅡphase.Conclusion:1.SGK1-3 was expressed in the four phases GV,GVBD,MⅠ and MⅡ of mouse oocytes,and the expression levels were different.SGK1 and SGK2 were mainly expressed in GV,but SGK2 was relatively less,SGK3 was mainly expressed in GVBD and MⅡ.2.SGK3 protein kinase was expressed in all four phases,and the expression level increased from GV to GVBD,decreased significantly in MⅠ,and increased sharply in MⅡ and reached the peak.3.In mouse ooctyte,SGK3 locates in cytoplasm in GV,translocated to the nucleus before GVBD,and distribute in the whole cell in GVBD and MⅠ,then translocated to the nucleus from cytoplasm in MⅡ.The nucleo-cytoplasmic shuttling of SGK3 may participate in the activation of Cyclinb-CDC2.