Involvement Of SGK3 In The First Meiotic Resumption Of Mouse Oocytes

Objective Serum and glucocorticoid-induced protein kinase(SGK)3 is a serine/threonine kinase containing a PX domain.SGK3 is a multifunctional protein and plays an important regulatory role in sea star oocyte meiotic recovery and tumor cell cycle transition.After the activation of the PI3K-PDK1/TORC2 signaling pathway,PDK1 and TORC2 phosphorylate Thr320 on the A-loop and Ser486 on the HM of SGK3,respectively,to activate SGK3.However,the role of SGK3 in mouse oocyte meiotic recovery has not been reported in the literature.Our previous study explored the protein expression and subcellular localization of SGK3 in the development of mouse oocytes and discussed the mechanism of SGK3 in meiotic division.Based on the above information,we speculate that SGK3 may play an important role in the first meiotic division recovery of mouse oocytes.In this study,we used mouse GV-stage oocytes as the research object,observed the GVBD rate and death rate of mouse oocytes by microinjection of SGK3 mRNA,and observed the GVBD rate,death rate,cell morphology,and CDC2-Tyr15 phosphorylation level of mouse oocytes in each experimental group by SGK3 antibody inhibition experiment.Western blotting was used to detect the phosphorylation levels of SGK3-Ser486 and CDC2-Tyr15.This provides a theoretical basis for further research on the molecular regulatory mechanism of SGK3 in the first meiotic division recovery of mouse oocytes.Methods:1.GV-stage mouse oocytes were obtained using superovulation method.2.The eukaryotic expression plasmid pc DNA3.1-Myc-SGK3 was constructed.3.pc DNA3.1-Myc-SGK3 was transcribed into mRNA in vitro for subsequent microinjection.4.Microinjection of SGK3 mRNA was performed to observe the effect on oocyte development.5.SGK3 antibody inhibition experiment was performed to observe the effect of different concentrations of antibody on oocyte GVBD rate and CDC2-Tyr15 phosphorylation level.6.Western blotting was used to detect the phosphorylation status of SGK3-Ser486 and CDC2-Tyr15.Result:1.Compared with the control group(uninjected group and TE-injected group),the GVBD rates of the SGK3-mRNA injection group were 78% at 1 h,94% at 2 h,96% at 3 h,and100% at 4 h after microinjection.In contrast,the GVBD rates of the uninjected group and TE-injected group(both control groups)were 53% and 51%,76% and 73%,90% and91%,and 95% and 96% at 1 h,2 h,3 h,and 4 h after microinjection,respectively.Compared with the control group,the GVBD rate in the SGK3-mRNA injection group was significantly increased(P < 0.01).2.Detect the phosphorylation level of CDC2-Tyr15 in the control group and the SGK3-mRNA injection group.The phosphorylation signal of CDC2-Tyr15 in the control group completely disappeared 2 hours after culture,and the phosphorylation signal in the SGK3-mRNA injection group was no longer detectable 1 hour after microinjection.Compared with the control group,the complete dephosphorylation time of CDC2-Tyr15 was at least 1 hour earlier.3.In detection 1,GVBD of GV stage oocytes was assessed after treatment with SGK3 antibody dilutions of 1:200,1:100,1:50,and 1:25.The results showed that after 1 h of in vitro culture,the GVBD rates of oocytes in the control group,1:200,1:100,1:50,and1:25 treatment groups were 53%,50%,38%,19%,and 8%,respectively.The GVBD rates of the 1:100,1:50,and 1:25 treatment groups were significantly lower than that of the control group(P < 0.01).After 2 h of in vitro culture,the GVBD rates were 76%,74%,49%,26%,and 11%,respectively.The GVBD rates of the 1:100,1:50,and 1:25treatment groups were significantly lower than that of the control group(P < 0.01).After3 h of in vitro culture,the GVBD rates were 90%,88%,63%,35%,and 15%,respectively.The GVBD rates of the 1:100,1:50,and 1:25 treatment groups were significantly lower than that of the control group(P < 0.01).After 4 h of in vitro culture,the GVBD rates were 95%,93%,75%,44%,and 21%,respectively.The GVBD rates of the 1:100,1:50,and 1:25 treatment groups were significantly lower than that of the control group(P < 0.01).4.In the antibody dilution experiment,the phosphorylation level of CDC2-Tyr15 was detected.The phosphorylation signals of CDC2-Tyr15 in the control group and the 1:200antibody treatment group disappeared completely after 2 h of culture.There was no difference in the time required for complete dephosphorylation of CDC2-Tyr15 between the 1:200 antibody treatment group and the control group.The phosphorylation signals of CDC2-Tyr15 in the 1:100 antibody treatment group began to weaken after 2 h of culture and disappeared completely at 4 h.The phosphorylation signals of CDC2-Tyr15 were detectable in the 1:50 and 1:25 antibody treatment groups at 1 h,2 h,3 h,and 4 h of culture.5.The phosphorylation of SGK3-Ser486 and the phosphorylation of CDC2-Tyr15 were detected at 0 min,30 min,60 min,90 min,and 120 min in vitro culture of GV stage oocytes.The results showed that the relative expression levels of SGK3-p Ser486 at the five time points of oocyte development were 0.156 ± 0.003,0.775 ± 0.002,0.858 ± 0.004,0.959 ± 0.005,and 0.981 ± 0.007,respectively.The phosphorylation signal of SGK3-Ser486 was significantly enhanced when GV phase oocytes were cultured in vitro for 30 minutes,while the phosphorylation signal of SGK3-Ser486 remained at a high level when cultured for 60,90,and 120 minutes.The relative expression levels of CDC2-p Tyr15 at five time points during oocyte development were 0.956 ± 0.008,0.941 ±0.015,0.567 ± 0.011,0.477 ± 0.011,and 0.030 ± 0.020.The phosphorylation signal of CDC2-Tyr15 was relatively strong at 0 and 30 min of culture in vitro,and the phosphorylation signal of CDC2-Tyr15 was significantly weakened at 60 min,but no phosphorylation signal of CDC2-Tyr15 was detected at 120 min.Conclusion:1.Overexpression of SGK3 promotes the recovery of the first meiotic division in mouse oocytes.2.Inhibition of SGK3 kinase delays the recovery of the first meiotic division in mouse oocytes.3.The activation time of SGK3 is earlier than that of CDC2,and SGK3 kinase may be an upstream regulatory enzyme of CDC2.4.SGK3 can regulate the recovery of the first meiotic division in mouse oocytes.SGK3 is a positive regulatory factor for the recovery of the first meiotic division in mouse oocytes.