Abstract:

Objective To discuss the regulatory effect of serum and glucocorticoid-induced protein kinase 1 （SGK1） in the early development of fertilized eggs at G₁ phase of the mice， and to clarify the related mechanism. Methods Some female mice aged 4-6 weeks and weighed about 20 g， and several male mice aged over 8 weeks and weighed about 30 g were selected. The female mice were intraperitoneally injected with 10 IU of pregnant mare serum gonadotropin （PMSG）， followed by 10 IU of human chorionic gonadotropin （HCG） after 48 h. After HCG injection， the female mice were caged overnight with the male mice at a ratio of 1∶1.The fertilized eggs at G₁， S， G₂， and M phases were collected at 12-21 h， 21-26 h， 26-28 h， and 28-30 h after injected with HCG， and their cellular morphology at different cell cycles were observed under light microscope. The mouse fertilized eggs at G₁ phase after superovulation were collected， the mRNA was synthesized in vitro， and divided into no injection group， Tris-EDTA buffer injection group （TE injection group）， and SGK1-mRNA injection group.The SGK1 antibodies were mixed with KSOM culture medium with the concentrations of 1∶25， 1∶50， 1∶100， 1∶200， and 0 to culture the mouse fertilized eggs at G₁ phase. Western blotting method was used to detect the expression levels of SGK1 protein in fertilized eggs of the mice in various groups and the dephosphorylation for phosphorylated SGK1-Threonine 256 site tyrosine15 site of cell diusion cyclin 2（Cdc2）（Cdc2-pTyr15） in the fertilized eggs of the mice in various groups and different concentrations of SGK1 antibody groups and the developmental states of the fertilized eggs in the fertilized eggs of the mice in various groups and different concentrations of SGK1 antibody groups were observed under phase contrast microscope； the expression levels of phosphorylated SGK1-Thr256 （SGK1-pThr256） and Cdc2-pTyr15 proteins in fertilized eggs at different post-HCG injection times were detected by Western blotting method. Results Compared with no injection and TE injection groups， the expression level of SGK1 protein in the cells in SGK1-mRNA injection group was significantly increased （P<0.01）. 27-28 h after injected with HCG， the phosphorylation signaling of Cdc2-pTyr15 in fertilized eggs of the mice in SGK1-mRNA injection group was gradually disappeared， and there was no phosphorylation signaling 29 h after injected with HCG. At 28-29 h after injected with HCG，the phosphorylation signaling of Cdc2-pTyr15 in fertilized eggs of the mice in no injection and TE injection groups gradually disappeared， completely disappeared at 30 h after injected with HCG. With the increasing of the concentration of SGK1 antibody， the disappearing time of the Cdc2-pTyr15 phosphorylation signaling was increased. At 27 h after injected with HCG，the fertilized eggs of the mice in SGK1-mRNA injection group was initiated cleavage； at 31 h after injected with HCG， nearly all the fertilized eggs turned into G₂ phase； at 33 h after injected with HCG， all the fertilized eggs in 0 and 1∶200 SGK1 antibody groups underwent cleavage. However， with the increasing of SGK1 antibody concentration， the cleavage of the fertilized eggs in 1∶25， 1∶50， and 1∶100 SGK1 antibody groups was gradually decreased， particularly at 1∶25 SGK1 antibody group. Compared with no injection and TE injection groups， the death rate of the fertilized eggs of the mice in SGK1-mRNA injection group was significantly decreased at 31 h after injected with HCG （P<0.05）， and the cleavage rate was increased（P<0.05）. With the increasing of the SGK1 antibody concentration， the death rates of the fertilized eggs in different concentrations of SGK1 antibody group were increased （P<0.05）， with the extending of cleavage time was increased， and the cleavage rate of the fertilized eggs was decreased in a dose-dependent manner， and the cleavage rate of fertilized eggs in 1∶25 SGK1 antibody group was the lowest.The expression level of SGK1-pThr256 protein in fertilized eggs of the mice was gradually increased from 27 h after injected with HCG （P<0.05 or P<0.01） in a time-dependent manner； at 28 to 29 h after injected with HCG， the expression levels of Cdc2-Tyr15 protein were gradually decreased （P<0.05） in a time-dependent manner， and had completely disappeared at 30 h after injected with HCG. Conclusion Both the over-expression and inhibition of SGK1 can affect the time for the fertilized eggs at G₁ phase to entry into M phase， suggesting that SGK1 protein may be one of the regulatory factors in the early development of fertilized eggs at G₁ phase of the mice， and it may regulate the development of the fertilized eggs at G₁ phase through regulation of Cdc2.

Key words: Serum and glucocorticoid-regulated protein kinases 1, Fertilized egg, Cell division, Phosphatidylinositol 3-kinase, Cleavage