Protein Kinase C ? And CaMK? Involved In The Regulation Of PAkt1(Ser473) On The Release Of Mouse Oocytes From Diplotene Arrest

Objective:Up to the mid-two-cell stage about 27 hr postfertilization,the embryo appears to rely largely on protein and RNA synthesized during oogenesis.So the development of oocyte is crucial for the first cleavage of a fertilized egg.Mammalian oocytes undergo two complex meiotic processes during maturation,and are blocked in the premeiotic diploid phase of the first meiosis,when the nucleus is swollen,also known as vesicle germinal?GV?.Oocytes resume meiosis under the stimulus of pituitary hormone,acting as vesicle germinal breakdown?GVBD?.This restart is similar to the transition of mitosis from G₂ phase to M phase,which is the first important event in the maturation of oocytes and the key process to ensure the completion of meiosis and the normal development of embryos.Although it has been confirmed that Akt is involved in mouse oocyte maturation,its upstream and downstream molecular mechanisms still need to be further explored.Protein kinase B?PKB/Akt?,a Serine/Threonine protein kinase,is involved in variety cell activities.It is a key step for the activation of Akt that is PHD?pleckstrin homology domain?binds to phosphatidylinositol 3,4,5-trisphosphate?PIP₃?,a product of the PI3K signaling pathway catalyzes PIP₂ avtivated by growth factors.During the past25 years,the Akt signaling field has seen remarkable expansion and discoveries that have central relevance to health and human disease.Our previous reports have demonstrated that Akt may promote the cell division of fertilized mouse eggs by phosphorylating Cdc25B,which is an important upstream factor of Cdc2 and Cdc2 is one of the components of Maturation promoting factor?MPF?playing an important role in the G₂/M transition.Some studies have demonstrated that PKC,a Serine/Threonine protein kinase,may play a role in many cellular functions via regulating Akt,and it also participates in the processes of mammalian impregnation and zygote development.However,little information is available on mouse oocytes and relationship with Akt.Recent reports suggest that Calmodulin?CaM?and PIP₃ may synergistically bind with Akt?PHD?to promote efficient calcium signaling.Calmodulin-dependent protein kinase ??CaMK??is a serine/threonine specific protein kinase which is regulated by Ca²⁺/calmodulin complex.Studies have found that CaMK? is a target of calcium and calmodulin in the G₂/M transition in various cell types,but its mechanism in the meiosis process of oocytes from GV to GVBD is still not clear.In this study,qPCR,Western blotting and Immunofluorescence were used to verify the expression and localization of pAkt1?Ser473?in the oocyte from GV to GVBD;PAkt1?Ser473?specific inhibitor SH-6,PKC inhibitor Sotrastaurin and CaMK? inhibitor KN-93 were then used to treat the cells,and the expression and localization of pAkt1?Ser473?,pPKC?Thr505?,pCaMK??Thr286?,Cdc25Band pCdc2?Tyr15?were detected by Western blotting and Immunofluorescence.The role and relationship of pPKC?Thr505?and pCaMK??Thr286?in the first meiotic recovery of mouse oocytes.We preliminarily explored the roles and relationships of pAkt1?Ser473?,pPKC?Thr505?and pCaMK??Thr286?in the first meiotic recovery of mouse oocytes.Methods:1.Oocytes at GV,GVBD and M? stages were collected to detect the expressions of Akt subtypes by qPCR,and the protein expressions of pAkt1?Ser473?and Cdc25Bwere detected by Western blotting.To clarify the role of pAkt1?Ser473?in cells from GV phase to GVBD phase,GV cells were treated with SH-6,a pAkt1?Ser473?specific inhibitor,Western blotting and Immunofluorescence staining were used to detect the expression and localization of pAkt1?Ser473?,Cdc25Band pCdc2?Tyr15?in oocytes.2.After treatment with Sotrastaurin,GVBD percentage,pAkt1?Ser473?and Cdc25Blevels decreased,pCdc2?Tyr15?increased,while pPKC?Thr505?levels of SH-6cuitured cells showed no significant change.3.GVBD percentage and pAkt1?Ser473?levels decreased in the KN-93 treatment group,and pCaMK??Thr286?levels also decreased in the SH-6 treatment group.The results of Immunofluorescence showed that the localization of pAkt1?Ser473?,pCaMK??Thr286?,Cdc25Band pCdc2?Tyr15?was changed compared with the control group.Result:Down-regulation of pAkt1?Ser473?inhibits Cdc25B,reduces dephosphorylation of pCdc2?Tyr15?,and inhibits the release of mouse oocytes on the release of mouse oocytes from diplotene arrest.When pPKC?Thr505?inhibited,pAkt1?Ser473?is significantly decreased,suggesting that pPKC?Thr505?may be an upstream kinase of Akt1.pCaMK??Thr286?decreased when pAkt1?Ser473?was inhibited and inhibited pCaMK??Thr286?reduced pAkt1?Ser473?level,suggesting that pAkt1?Ser473?and pCaMK??Thr286?may interact in this process.