The Effect Of Cdc14A On G2/M Transition Of One-cell Fertilized Mouse Eggs

Objective: Cell division cycle 14A(Cdc14A)is a highly conserved dual-specific phosphatase and widely expressed in eukaryotic cells.It plays a conservative role in eukaryotes,which involved in mitosis,cytokinesis,meiosis,and DNA damage repair.The cell division cycle 25(Cdc25)protein family participates the regulation of cell cycle.The Cdc25 family consists of three phosphatases,Cdc25 A,Cdc25B,and Cdc25 C,and Cdc25 B has been identified as the primary promoter of mitosis.In Saccharomyces cerevae,Cdc14 is sequestered in the nucleoli in the interphase and is inactived.At the mitotic phase,Slk19 promotes part of Cdc14 release from the nucleoli to the nucleus,Cdc14 dephosphorylates the substrate of Cdc2 phosphorylation,and then Dbf2 directly phosphorylates Cdc14 to facilitate the release of Cdc14 into the cytoplasm.The study of Saccharomyces pombe showed that Cdc14 could rapidly degrade Cdc25 of mitotic inductor after cell division,and Cdc25 was proved to be the substrate of Cdc14 in vitro.Cdc25 activity was down-regulated by Cdc14 to ensure the rapid inactivation of mitotic Cdk complex and exit mitosis.Our previous studies have confirmed that Cdc14 A is expressed in one cell stage fertilized mouse eggs,and the expression of Cdc14 A is the highest in G2 phase,and decreased in M phase.The localization of Cdc14 A showed that Cdc14 A was located in the nucleoli of cells in G1,S and G2 phases of one-cell stage fertilized mouse eggs,and Cdc14 A was released into cytoplasm in M-phase of one-cell stage fertilized mouse eggs.In addition,our previous study found that Cdc25 B was located in cytoplasm in G1 and S phases and entered nucleus in G2 phase.Then,we constructed the Cdc14 A mutants(WT,active in the wild type and C278 S,inactive in the mutant),and found that overexpression of Cdc14A-WT delayed the entry M phase of one-cell fertilized mouse eggs;Cdc14A-C278 S had no effect on G2/M transition of one-cell fertilized mouse eggs.And there may be interaction between Cdc14 A and Cdc25 B in fertilizedmouse eggs,and the specific site of interaction may be Cdc25B-S15,rather than Cdc25B-S186.Based on the above data,we hypothesized that Cdc14 A and Cdc25 B were co-located in the nucleus during the G2/M phase conversion of the one-cell stage fertilized mouse eggs.The combination of Cdc14 A and Cdc25 B caused the dephosphorylation of Cdc25 B and thus unable to activate MPF,so the fertilized eggs were blocked in the G2 phase.In order to verify the above hypothesis,we use one-cell fertilized mouse eggs as the research object,and by knocking down the expression of Cdc14 A,we observed their influences on the G2/M transition of one-cell stage fertilized mouse eggs.The interaction between Cdc14 A and Cdc25 B and the specific sites of interaction were determined by immunoprecipitation experiments and indirect immunofluorescence co-localization of Cdc14 A and Cdc25 B,this stuyd provided theoretical basis for further research on the molecular regulatory mechanism of Cdc14 A and Cdc25 B during the development of one-cell stage fertilized mouse eggs.Method: 1.After mouse were superovulation then we collect one-cell fertilized mouse eggs at G1,S,G2,M phases;2.Observe the effects on the development of fertilized mouse eggs after the microinjection of Cdc14A-MO,using RT-PCR and Western Blotting experiments to verify the effectiveness of interference;3.Construct eukaryotic expression fluorescent plasmids :p CDNA3.1-MYC-Cdc14A-mcherry,p EGFP-Cdc25B-S15 A,p EGFP-Cdc25B-S15D;4.Observe the subcellular localization of the Cdc14 A and Cdc25 B by direct immunofluorescence;5.Immunoprecipitation was used to determine whether Cdc14 A interacted with Cdc25 B.Results(1)In the control groups(without injection group and TE buffer injection group),cleavage occurred at 28h-28.5h after hCG injection,and the cleavage rates were 21.7 % and 23.4 %,29 h after hCG injection,the cleavage rates were 53.6 % and55.3 %,and 31 h after hCG injection,the cleavage rates were 93.5% and 91.4 %,the cleavage rate between the control group had no difference(P > 0.05);Compared with the control groups,Cdc14A-MO group cleavage occurred at 26h-26.5h afterhCG injection.,29 h after hCG injection,with cleavage rates of 58.3 % and 31 h after hCG injection the cleavage rates were 92.4 %.(2)Indirect immunofluorescence showed that Cdc14 A was located in the nucleus of G1,S and G2 phases,while Cdc25 B was located in the cytoplasm of G1,S and early G2 phases,and both of Cdc14 A and Cdc25 B were located in the nucleus of late G2 phase of one-cell stage fertilized mouse eggs.(3)Immunoprecipitation experiments showed that Cdc25 B could bind to Cdc14A-C278S;Cdc14A could bind to Cdc25B-WT and Cdc25B-S15 D,but Cdc14 A could not bind to Cdc25B-S15 A when the Ser15 of Cdc25 B mutated to alanine(Ala).Conclusions(1)After Cdc14 A was knocked down,one-cell fertilized mouse eggs was accelerated to enter M stage.Cdc14 A is a negative regulator of the one-cell fertilized mouse eggs.(2)Cdc4A could bind with Cdc25 B,namely,Cdc4 A can interact with Cdc25 B.(3)The Ser15 of Cdc25 B is the binding site of Cdc14 A and Cdc25 B.Cdc14A can bind with Cdc25B-WT and phosphomimic Cdc25B-S15 D but not with unphosphorylated Cdc25 B,When Ser15 of Cdc25 B is mutated to an unphosphorylated residue Ala,the Cdc14 A binding is abrogated.