Study On The Expression And Subcellular Localization Of Wee1A In One-cell Fertilized Mouse Eggs

Objective:Wee1A protein kinase was firstly isolated from Schizosaccharomycescells by Nurse et al.Wee1A protein kinase can inhibit cell mitosis by inhibiting the activity of cell division cyclin 2（Cdc2,human homolog Cdk1,also known as Cdc28 in budding yeast）,so it is named"wee"family.Wee1A protein kinase is a member of serine/threonine bispecific kinase family,which is involved in cell mitosis,meiosis,DNA damage response,regulation of replication forks and genome stability.At present,the identified Wee1 protein kinase family includes Wee1A,Wee1B and PKMYT1.Wee1A is also called G₂checkpoint kinase,PKMYT1 is called membrane associated tyrosine and threonine specific Cdc2 inhibitory kinase,and Wee1B is called oocyte meiosis inhibitory kinase.As one of the ideal models for studying mammalian cell cycle,the mechanism of early development of mouse fertilized egg has not been thoroughly studied.Among them,Wee1A protein kinase family is involved in the regulation of cell cycle progression.At present,only the localization and expression of Wee1A in oocytes,Schizosaccharomyces cells and somatic cells have been reported.In this study,we used RT-PCR to detect the relative expression of Wee1A m RNA and Wee1B m RNA in G₁,S,G₂and M stages of mouse one-cell fertilized eggs,and determined the main expression subtypes.Western blotting was used to detect whether the expression of Wee1A and Wee1B proteins in G_1,S,G₂and M stages of mouse one-cell fertilized eggs changed with the progress of cell cycle.The localization of Wee1A,Wee1A-p S123 in mouse one-cell fertilized eggs was observed by indirect immunofluorescence technique,which provided experimental basis for further study on the early development mechanism of Wee1protein kinase family members in mouse one-cell fertilized eggs basis.Methods:1.Mice were intraperitoneally injectedwith PMSG and HCG by superovulation.According to the time of HCG injection and the morphology of fertilized eggs,the fertilized eggs of G_1,S,G₂and M phase were collected.2.RT-PCR was used to detect the relative expression of Wee1Am RNA and Wee1Bm RNA in G_1,S,G₂and M phase of mouse one-cell fertilized eggs.3.Western blotting was used to detect the expression of Wee1A protein and Wee1B protein in G_1,S,G₂and Mphase of mouse one-cell fertilized eggs.4.Indirect immunofluorescence technique was used to observe the localization of Wee1A,Wee1A-p S123 in four stages of mouse one-cell fertilized eggs.Results:1.RT-PCR results showed that Wee1A m RNA and wee1B m RNA were expressed in the four stages of mouse one-cell fertilized eggs,and the m RNA expression level of wee1A was significantly higher than that of wee1B at the same stage.2.Western-blotting results showed that the protein expression levels of Wee1A and Wee1B were consistent with their m RNA expression levels in the four stages of mouse one-cell fertilized eggs,which increased gradually from G₁stage to G₂stage,and decreased gradually to M stage.The expression level of Wee1A protein was significantly higher than that of Wee1B protein at the same time.3.Indirect immunofluorescence results showed that Wee1A protein coupled red fluorescence signal was located in the cytoplasm in G₁and S phases of mouse one-cell fertilized eggs,some Wee1A protein began to enter the nucleus in the early G₂phase,and in late G₂and M phases,the red fluorescence signal was significantly enhanced,at the same time,the number of wee1A-p S123 in nucleus increased with the progress of cell cycle.Conclusion:1.Wee1A and wee1B proteins were expressed in G₁,S,G₂and M phases of mouse one-cell fertilized eggs.The expression of Wee1A and Wee1B proteins was low in G₁and S phases,the highest in G₂phase,and decreased in M phase.The expression level of Wee1A protein was significantly higher than that of Wee1B protein at the same stage.2.Wee1A was localized in cytoplasm at G₁and S phases.In early G₂phase,part of Wee1A entered the nucleus,and in late G₂and M phases,the number of Wee1A entered the nucleus increased significantly,at the same time,the number of wee1A-p S123 in nucleus increased with the progress of cell cycle,which may regulate the development of mouse one-cell fertilized eggs through the phosphorylation and nonphosphorylation of Wee1A or the subcellular localization of Wee1A.