Study On The Mechanism Of PKC?/Cdc25b Pathway In Early Development Of Fertilized Mouse Eggs

Objective: Since its discovery in 1977,Protein kinase C(PKC)an important kinase in the intracellular signal transduction system,widely involed in many process of the life span in cells.Such as cell growth,differentiation,cell cycle control,participation in cell apoptosis may be related to tumorigenesis,plays an important role in the development of many diseases.PKC participates in multiple signal transimission because of various subtypes of PKC execute different cellular functions,expression and subcellular locations.Its molecular mechanism involved in growth regulation is also complicated by the fact that cells themselves can synthesize several PKC isoenzymes.Cell cycle is controlled by the interaction of a variety of phosphatase and kinase.As a member of the PKC family,PKC? is the major intracellular kinase implicated in a large number of signal transduction processes transducing extracellular signals into intracellular events.It is of great significance to explore the role of PKC? signaling pathway in cell cycle regulation.Cdks are cyclin-dependent kinases,firstly binding with cyclin,after the kinases are acativated,then they together participate in cell cycle control.cdks participate in the regulation of G2/M transition.Cyclin as the regulatory subunit of cdks,combined with it,the Cyclin/cdk complex formed,when cdks are in the state of activation,the cdks play their regulatory role in the cell cycle.The heterodimer mitosis promoting factor(MPF)composed of Cdc2 and cyclin B,which is crucial for G2/M transition.Prior to mitosis,Cdc2 is inactivated due to phosphorylation of the Tyr15 and Thr14 residues of Cdc2,forming the inactive pre-MPF,when the inactive pre-MPF phosphatase upon dephosphorylation on Thr14 and Tyr15 is by the dual specificity Cdc25 in mistios,it is activated.G2/M transition is regulated by cdc2,the Cdc25 B phosphatase plays an essential role during G2/M transition,and is the promoter in mitosis,is the essential phosphatase for meiotic resumption.In the early development of fertilized mouse eggs,little is known about the mechanism especially that of 1-cell to 2-cell cycle transition.PKC? could be detected both at M? and 2-cell cycle of fertilized mouse eggs,but the mechanism of PKC? in early development of fertilized mouse eggs.Our previous research foud that PKC could speed up the development of fertilized mouse eggs through activating MPF,and activity of PKC enhanced at M phase.Also,other studies have shown that PKC? constitutes the majority of PKC's activity,and the increase of PKC? expression at M phase may be related to the enhancement of PKC activity.In this research,we mainly study on 1-cell cycle of fertilized mouse eggs,focused on the expression at m RNA and protein level,subcellular localization of PKC? at G1,S,G2,M phases in 1-cell cycle of mouse embryos,in existence of the inhibtor and small inhibitor RNA(si RNA)against PKC? to study the PKC? function,discussing the role and mechanism of PKC? in early development of fertilized mouse eggs.With the help of Scansite soft ware,Ser96 of Cdc25 B maybe a phosphorylation site of PKC?,in mammalian cells Cdc25 B was a direct substrate of PKC? or not,the regulation of Cdc25B-Ser96 in the cell cycle of the fertilized mouse egg has not been reported.Hence,study of PKC? and the PKC?/Cdc25 B pathway in 1-cell phase fertilized mouse eggs especially in the G2/M transition will extend the mechsiam of PKC? in cell cycle control.Methods: Kunming genealogy-specific pathogen-free mice(females at 4-6 weeks and 20-24g;males at 8 weeks and 30-35g)were supplied by Experimental Animal Center of China Medical University.Female mice were firstly injected with PMSG and then with human chorionic h CG in the abdominal cavity,embryos were cultured in M16 medium to different cell cycle requirements.Set the experimental group and the control group separately,and conduct the following studies:(1)Real time-PCR was used to detect expression of PKC? in 1-cell cyclle of mouse embryos.(2)Western-blot was used to detect expression of PKC? in 1-cell cyclle of mouse embryos.(3)Indirect immunofluorescence analysis subcellular localization of PKC? and Cdc25 B.(4)Inhibtor and si RNA against PKC? were used to analyze the cleavage rate of mouse embryos and activity of MPF,phosphorylation status of Cdc2-Tyr15.(5)The specific antibody of Cdc25BSer96 was used to detect the phosphorylation status of Cdc25 B at G1,S,G2,M sages.(6)Predict the phosphorylation site in potential substrate of PKC?,construction mimicking phosphorylated and dephosphorylated mutants of Cdc25 B,transcribed in vitro into m RNA.(7)Cdc25B-m RNAs were microinjected into G1,S,G2,M phase mouse embryos,analyze expression of Cdc25B,cleavage rate of mouse embryos and activity of MPF,phosphorylation status of Cdc2-Tyr15.(8)When the activity of PKC? was inhibited,microinjecting with Cdc25B-m RNAs,analyze expression of Cdc25 B,cleavage rate of mouse embryos and activity of MPF,phosphorylation status of Cdc2-Tyr15.(9)PKC?-si RNA and Cdc25B-m RNAs were microinjected into different phases of mouse embryos,analyze expression of Cdc25 B,cleavage rate of mouse embryos and activity of MPF.Results: 1,PKC? could be detected at m RNA and protein levels during the 1-cell Phase embryos,the G1,S,G2 phase had no difference,with a significantly increase at the M-phase,activity of PKC? increased at M phase,subcellular localization showed that PKC? and Cdc25 B distributed in all the 4 phases.2,When activity of PKC? was inhibited,the cleavage rate and MPF activity decreased,development of mouse embryos were supressed.3,Higher expression levels of phosphorylated Cdc25B-Ser96 were observed during the G2 and M phases compared with expression in G1 and S phases.4,Microinjecting Cdc25B-S96D(Asp)-m RNA could activate MPF in advance,obviously promote the cleavage of fertilized mouse eggs.5,Microinjection of Cdc25B-S96 D mutants speed mitotic resumption in rottlerin or si RNA arrested eggs.Conclusion: 1,PKC? expressed in different phases of 1-cell fertilized mouse eggs,and the expression and activity increased significantly at M phsae.2,The selective inhibitor of PKC? and si RNA delayed the active of MPF,So the G2/M transion was ihibited,the ratio of cleavage,all these results demonstrated the positive role of PKC? in regulating early development of mouse embryos.3,Cdc25BSer96 was phosphorylated at G2,M phase,microinjection of mimic phosphorylated Cdc25BSer96-m RNA can significantly promote the development of fertilized mouse eggs,the PKC?/Cdc25 B pathway plays an important role in early development of mouse embryos.Cdc25 B was a potential substrate of PKC?,may through the phosphorylation of Cdc25BSer96,then activate MPF promotes G2/M transion.