Involvement Of Cdc14B In The Development Of One-cell Mouse Fertilized Eggs

Objective Cell division cycle protein 14B(Cdc14B)belongs to the serine / threonine protein phosphatase family and plays an important role in cell cycle regulation,growth and development,and signal transduction.Studies have shown that human Cdc14 B is localized in the nucleolus during interphase of mitosis,and its biological functions mainly include regulating mitotic exit,assembly of nuclear tissue and mitotic spindle,centrosome replication,and regulating the effectiveness of DNA damage checkpoint in G2 phase.Studies on mouse oocytes have shown that overexpression of Cdc14 B can delay the recovery of meiosis in oocytes,and down-regulation of Cdc14 B expression can cause early recovery of meiosis.Studies on mammalian cells indicate that a small amount of Cdc14 B is located in the mitotic centrosome and maintains the nuclear structure together with polo-like kinase Plk1.The study of U2 OS cell line believes that Cdc14 B has the function of binding and stabilizing microtubules.It is located in the nucleus in the interphase and gathers in the central region of the spindle at a later stage.Cdc14 B regulates the functions of spindle and microtubules through its subcellular localization,thereby regulates the mitotic process of cells.Our previous research has confirmed the expression of Cdc14 B in one-cell-stage mouse fertilized eggs.In one-cell mouse fertilized eggs,Cdc14 B and ?-tubulin are co-localized in the cortex of fertilized mouse eggs in G1 phase and S phase.In the early G2 phase,Cdc14 B and ?-tubulin are co-localized in the cytoplasm.In late G2 phase,part of Cdc14 B entered nucleus.In the M phase,Cdc14 B and ?-tubulin colocalized to the spindle.In our study,with the one-cell fertilized mouse eggs as the research object,through microscopic injection of the m RNA of Cdc14 B,the author observed their influence on the cleavage rate,death rate and G2/M transition of one-cell-stage mouse fertilized eggs;The cleavage rate,mortality,cell morphology,and spindle changes of one-cell mouse fertilized eggs are observed by microinjection of Cdc14B-MO.The subcellular localization in mousefertilized eggs is observed by microscopic injection of p CMV3-GFPSpark-Cdc14 B.This provides a theoretical basis for further studying the molecular regulatory mechanism of Cdc14 B during one-cell mouse fertilized eggs development.Methods 1.After mouse are superovulation then we collect one-cell fertilized mouse eggs.2.Construction of fluorescent expression plasmid p CMV3-GFPSpark-Cdc14 B.3.Transcribe the constructed fluorescent expression plasmid into m RNA in vitro for microinjection.4.Observe the effects on the development of mouse fertilized eggs after the microinjection of m RNA.5.Observe the effects on the development of mouse fertilized eggs after microinjecting Cdc14B-MO.6.Verify the interference efficiency after the microinjection of Cdc14B-MO by RT-PCR and Western blotting.7.Observe the co-localization of Cdc14 B and ?-tubulin after silencing Cdc14 B by indirect immunofluorescence.8.Observe the subcellular localization of the Cdc14 B by direct immunofluorescence.Results 1.In the Cdc14 B m RNA injection group,cleavage occurred at 32-32.5h after h CG injection,and the cleavage rate of mouse fertilized eggs was 45.7% at 33 h after h CG injection;In the control group,including the no-injection group and the TE buffer injection group,cleavage of mouse fertilized eggs began to occur at 28.5-29 h after the h CG injection,and the cleavage rate were 63%?61.7% respectively at 31 h after the h CG injection,at 33 h after h CG injection,the cleavage rate were 94.7%?93.6%respectively.2.RT-PCR results showed that the Cdc14 B gene was expressed in one-cell mouse fertilized eggs in each group,and the expression level of the Cdc14 B m RNA injection group was significantly higher than that of the control group.Western blotting results showed that 12 hours after the injection of Cdc14 B m RNA,m RNA has been translated into protein.3.In the Cdc14B-MO injection group,cleavage began to occur at 27-27.5h after h CG injection.At 31 h after h CG injection,the cleavage rate of mouse fertilized eggs was85.9%,and the cleavage rate was up to 97.8% at 33 h after h CG injection.4.RT-PCR results showed that the Cdc14 B gene was expressed in one-cell mouse fertilized eggs in each group,and the expression level of the Cdc14B-MO injection group was significantly lower than that of the control group.Western blotting detected Cdc14 B expression in the Cdc14B-MO injection group,which was about 80% lower than that in the control group.It shows that Cdc14B-MO has better interference effect.5.The fluorescence signal of Cdc14 B was significantly weakened after interference by indirect immunofluorescence.In the Cdc14B-MO injection group,the spindle constructed by ?-tubulin in the M phase of the fertilized eggs collapsed,and no bipolar formation was observed.6.After microinjection of p CMV3-GFPSpark-Cdc14 B,the fluorescence signal was concentrated in the spindle in metaphase,indicating that Cdc14 B is located in the spindle in metaphase;In anaphase,Cdc14 B was located in the spindle and the cortex.Conclusion 1.Overexpression of Cdc14 B can delay the entry of M phase of one-cell mouse fertilized eggs.2.Knockdown of Cdc14 B accelerates the cleavage of one-cell fertilized mouse and the transition of G2/M phase.3.Down-regulation of Cdc14 B leads to abnormal spindle formation in one-cell mouse fertilized eggs.4.The localization of Cdc14 B in the spindle during mitotic division regulates spindle assembly and the mitosis of one-cell mouse fertilized eggs.