Study On The Expression And Subcellular Localization Of CDC14B Protein In 1-cell Fertilized Mouse Eggs

Objective cell division cycle protein 14B(CDC14B)is a member of the CDC14 phosphatase family,which is widely expressed in eukaryotic cells.It is a highly conserved serine/threonine protein phosphatase,which can dephosphorylate the threonine and serine of the target protein and further regulate the biological activity of the target protein.Studies have shown that CDC14 B is widely involved in mitosis,meiosis,cytokinesis,G2 damage checkpoint activation,centrosome replication,spindle stability maintenance,DNA damage repair,carcinogenesis,cilia formation and other physiological and pathological processes.Because CDC14 B has many biological functions,it has become a research hotspot.Another important cell cycle protein,Cyclin B1,is the regulatory subunit of CDC2-dependent kinase,which combines with CDC2 to form the CDC2/Cyclin B1 complex,mitosis promoting factor(MPF),promoting the G2/M transition.Studies have shown that CDC14 B is a negative regulator in cell division,because it is expressed and located differently in different subcells and regulates cell cycle progression through its expression and location.Overexpression of CDC14 B blocks the activation of mitotic and zygotic genomes in mouse preimplantation embryos,while deletion of CDC14 B results in abnormal spindle and cytoplasmic division defects,indicating that CDC14 B is necessary in the process of cell division.Studies on human osteosarcoma cell line U2 OS showed that CDC14 B was located in the nucleus in the interphase and aggregated in the central region of the spindle in the later phase,with the function of binding and stabilizing microtubules,thus regulating the mitotic process of cells.In mouse oocytes,CDC14 B is located in the spindle during MI-MII phase.By regulating cadherin 1(CDH1),CDC14 B can degrade Cyclin B1(CCNB1),reduce the binding of CCNB1 to cyclindependent kinase CDK1,antagonism on CDC2 activity,inhibit the activity of MPF(CDC2/Cyclin B1),and prevent meiotic resumption.However,it is not clear whether CDC14 B,a phosphatase,is present in one-cell fertilized mouse eggs and its regulatory function in mitotic division.Whether CDC14 B is also located in the spindle and inhibits MPF activity by degrading Cyclin B1,thus preventing G2/M transition of fertilized mouse eggs.So far,it has not been reported at home or abroad.Therefore,this project mainly studies the expression and localization of CDC14 B in G1,S,G2 and M phases of one-cell fertilizedmouse eggs and the expression of Cyclin B1,providing a theoretical basis for the study on the mechanism of CDC14 B regulating G2/M transition in one-cell fertilized mouse eggs.Methods 1.Mouse superovulation technique was used to collect G1,S,G2 and M in one-cell fertilized mouse eggs.2.The relative expression levels of CDC14 B mRNA and Cyclin B1 mRNA in each phase of one-cell fertilized mouse eggs were detected by real-time PCR.3.The expression of CDC14 B protein in each phase of one-cell fertilized mouse eggs was detected by Western blotting.4.Co-localization of CDC14 B and ?-tubulin in one-cell fertilized mouse eggs in G1,S,G2 and M was observed by cell immunofluorescence.Results 1.Real-time PCR showed that the relative mRNA expression levels of CDC14 B mRNA were different in the four phases of one-cell fertilized mouse eggs,and gradually increased from G1 to S phase,reached a peak at G2 phase,and then decreased at M phase.The expression of Cyclin B1 in G1 phase was higher than that of in S phase,and the highest expression was found in G2 phase.There was no significant difference between G2 phase and M phase.2.Western blotting showed that CDC14 B protein was expressed in G1,S,G2 and M of one-cell fertilized mouse eggs,and the expression was increased in G1 to G2,and was highest in G2 phase,but decreased in M phase.3.Cellular immunofluorescence assay showed that CDC14B(red fluorescence)and ?-tubulin(green fluorescence)were co-located in the cortex of fertilized mouse eggs in G1 phase and S phase.In the early G2 phase,CDC14B(red fluorescence)and ?-tubulin(green fluorescence)co-localized in the cytoplasm.In late G2 phase,part of CDC14 B entered nucleus.In the M phase,CDC14B(red fluorescence)and?-tubulin(green fluorescence)co-localized to the spindle.Conclusion 1.CDC14 B protein is expressed in all four phases of one-cell fertilized mouse eggs,with low expression in G1 and S phases,highest expression in G2 phase,and decreased expression in M phase.2.The expression of Cyclin B1 mRNA is relatively low in G1 and S phases,but significantly increased in G2 and M phases,and there is no significant difference in G2 phase and M phase.Therefore,CDC14 B can not regulate G2/M transition by degradation of Cyclin B1 in one-cell fertilized mouse eggs.3.CDC14 B is located in the cortex and cytoplasm of one-cell fertilized mouse eggs during the interphase of mitosis,and in the spindle during the mitotic phase,and may has the function of regulating the spindle.