Involvement Of SGK1 In The Development Of One-cell Mouse Fertilized Eggs

Objective Serum and glucocorticoid-induced protein kinase（SGK）is one of the members of the AGC protein kinase family.Currently,three SGK genes have been found in human and mouse.SGK1,SGK2,and SGK3 proteins were encoded,of which SGK1 and SGK3 are expressed in all tissues.As an important participant in the PI3K/AKT/m TOR signaling pathway,SGK1 is involved in the regulation of cell division,growth,migration,apoptosis and other life processes.It has been established that SGK1 plays an important regulatory role in tumor,inflammation and diabetes.These functions are dependent on the phosphorylation of SGK1 at Ser422 and Thr256 downstream of the PI3K signaling pathway.In addition,as a multifunctional protein,phosphorylation of SGK1 is involved in the regulation of CCNB/CDK1（Cdc2）complex activity,which promotes cell cycle progression in starfish and mouse oocytes.However,the role of SGK1 in the early development of mammalian fertilized eggs has not been reported so far.Our previous study showed that SGK1 was expressed in G₁、S、G₂ and M phases of the mouse fertilized egg cell cycle,and the expression level of SGK1 gradually increased from G₁ phase to G₂ phase,and reached the peak in G₂ phase,while the expression level of SGK1 decreased in M phase.This implies that the alteration of SGK1 expression level may be related to the progression of cell cycle.Therefore,this study will take the mouse one-cell stage fertilized eggs as the research object,to further explore the role of SGK1 in the development of mouse one-cell stage fertilized eggs,and preliminarily explore the mechanism of SGK1,which will provide experimental basis for further research on the molecular regulatory mechanism of SGK1 in the early development of mouse fertilized eggs.Methods1.Full G₁ stage mouse fertilized eggs were collected under solid microscope after superovulation.2.The pc DNA3.1-MYC-SGK1-mcherry eukaryotic expression vector was constructed.3.The constructed expression plasmid was transcribed into m RNA in vitro for microinjection.4.SGK1-m RNA was microinjected to observe the effect of SGK1 overexpression on the early development of fertilized eggs,and the phosphorylation level of Cdc2-Tyr15 was detected at the corresponding time points.5.G₁ phase fertilized eggs were cultured with different concentrations of SGK1 antibody.The development of fertilized eggs was observed under phase contrast microscope,and the phosphorylation level of Cdc2-Tyr15 was detected at the corresponding time points.6.Western blotting was used to detect the phosphorylation expression level of SGK1-Thr256 and Cdc2-Tyr15 in mouse fertilized eggs at different time points.Results1.In the SGK1-m RNA injection group,cleavage began at 27～27.5h after HCG injection,the cleavage rate of fertilized eggs was 98.0%at 31h after HCG injection and 100.0%at 33h after HCG injection.The cleavage of fertilized eggs in the no-injection group and the TE buffer injection group began 28～28.5h after HCG injection.The cleavage rates in the no-injection group and TE buffer injection group were 50.0%、48.0%at 31h after HCG injection,and 91.0%、92.0%at 33h after HCG injection,respectively.Compared with the control group（no injection group and TE buffer injection group）,the cleavage rate of SGK1-m RNA injection group was significantly increased（P<0.05）.2.The Cdc2-Tyr15 phosphorylation signal in the SGK1-m RNA injection group was attenuated at 27～28h after HCG injection,and completely disappeared at 29h after HCG injection.The Cdc2-Tyr15 phosphorylation signal was attenuated at 28～29h after HCG injection and disappeared completely at 30h after HCG injection in both non-injected and TE buffer injection groups.3.The mouse fertilized eggs in G₁ phase were cultured in diluted SGK1 antibody medium of 0,1:200,1:100,1:50,and 1:25,respectively.The initiation time of cleavage of the five groups was as follows:28～28.5h,28～28.5h,28.5～29h,29～30h,30～31h after HCG injection;The cleavage rates of fertilized eggs in the five groups at 31 h after HCG injection were 52.0%、50.0%、35.0%、21.0%、10.0%,respectively.Compared with the control group,the cleavage rates of fertilized eggs in 1:100,1:50 and 1:25 concentration groups were significantly decreased（P<0.05）.The cleavage rate at 33h after HCG injection was 93.0%、95.0%、80.0%、33.0%、16.0%,respectively.Compared with the control group,the cleavage rate of fertilized eggs in 1:100,1:50 and 1:25 concentration groups were significantly decreased（P<0.05）.4.Cdc2-Tyr15 phosphorylation signal of mouse fertilized eggs was detected in five antibody concentration groups:0,1:200,1:100,1:50,1:25.The time when Cdc2-Tyr15phosphorylation signal began to decrease was 28～29h,28～29h,29～30h,29～30h,30～31h after HCG injection.The time when Cdc2-Tyr15 phosphorylation signal disappeared in the first four groups was 30h,30h,31h,32h after HCG injection,the weak Cdc2-Tyr15 phosphorylation signal was still detected in the 1:25 antibody concentration group at 32h after HCG injection.5.Phosphorylation of SGK1-Thr256 and Cdc2-Tyr15 in mouse fertilized eggs was detected at 27h,28h,29h,30h and 31h after HCG injection.The results showed as follows:The weak SGK1-p Thr256 could be detected at 27h after HCG injection,and the phosphorylation signal of SGK1-Thr256 was gradually enhanced at 27h,28h,29h,30h and 31h after HCG injection.The Cdc2-Tyr15 phosphorylation signal was significantly weakened at 28～29h after HCG injection,and the phosphorylation signal of Cdc2-Tyr15 could not be detected at 30h after HCG injection.Conclusion1.Overexpression of SGK1 can advance the one-cell mouse fertilized eggs enter M stage.2.Inhibition of SGK1 can arrest the one-cell mouse fertilized eggs in G₂/M phase.3.The activation time of SGK1 is earlier than that of Cdc2.SGK1 may be the upstream regulator of Cdc2,and SGK1 regulates the development of the one-cell mouse fertilized eggs through Cdc2.4.SGK1 is a positive regulator on the development of one-cell mouse fertilized eggs.