Study On The Expression And Subcellular Localization Of Cdc14A Protein In 1-cell Fertilized Mouse Eggs

Objective:Cell division cycle 14A（Cdc14A）phosphatase is a protein phosphatase,which was originally discovered and named by L.Hartwell.He accidentally found it to be a serine/threonine bispecific phosphatase family when screening genes regulating cell cycle of budding yeast^[1].It plays a stable conservative role in eukaryotes（from yeast in single-celled fungi to humans）G2 phase^[2].Cdc14 can down-regulate the activity of Cdk1 in G2/M transition and its function involves the regulation of anaphase of mitosis and the exit of mitosis and cytokinesis^[1].According to previous studies on yeast and human osteosarcoma cells,Cdc14 is an important regulator of cell cycle progression,and its lack of expression not only inhibits DNA synthesis,but also hinders cell division.Mammalian cells express two isoforms of Cdc14,hCdc14A and hCdc14B,which are homologous,similar to their functions in yeast^[3].HCdc14A is located in the centrosome during the interphase and has the function of regulating the centrosome replication cycle.The realization of this function depends on its interaction with the centrosome^[4,5].However,hCdc14B is different from hCdc14A in both localization and function.hCdc14B is mainly located in the nucleoli of cells during interphase and partially located in the centrosomes and spindles.Due to its different localization,hCdc14B usually directly binds to microtubules to stabilize the spindle^[6].Different expression levels of hCdc14A in cells also have different effects on cell division.When hCdc14A is overexpressed,the S-phase centromere will be separated early,and the multi-pole spindle will be formed.When its expression level drops,mitosis will produce a variety of defects,including cytoplasmic non-division^[1].Cdc14A phosphatase in mouse fertilized eggs localizated in cell nucleolus,nucleoli Cdc14A gathered around the chromosomes after mouse fertilized eggs broke.And in the first meiotic prophase（MⅠ）and in the middle of the second meiosis（MⅡ）Cdc14A scattered throughout the cytoplasm^[1].The mouse fertilized eggs is the most simple and natural cell cycle model in vertebrate which is close to human.The research of mouse zygote will be helpful to reveal the reproductive and developmental regulation mechanism of mammals.In this study,we investigated the expression of Cdc14A protein and the localization of Cdc14A in 1-cell fertilized mouse egg by molecular biology techniques and methods.Our results will provide useful information on the machanisms which Cdc14A protein control early development of mouse fertilized eggs.Methods:1.Mice were intraperitoneally injected with PMSG and HCG,and the fertilized eggs of G1、S、G2 and M phase were collected.2.The relative expression levels of Cdc14A mRNA in four phases（G1、S、G2、M）of mouse one-cell fertilized eggs were detected by real-time PCR.3.Western blot was used to detect the expression of Cdc14A protein in each phase;4.The localization and nucleic acid staining of Cdc14A in one-cell fertilized eggs of mice were observed by Immunofluorescent staining.Results:1.Cdc14A protein was expressed in G1、S、G2 and M phase in 1-cell fertilized eggs of mouse,with lower expression in G1 and S phase,higher expression in G2 phase and lower expression in M phase 2.Western blot was performed to detect the expression of Cdc14A in the one-cell phase（G1、S、G2 and M）of mouse,the expression in G1 and S phases was lower,while the expression in G2 phase was higher and lower in M phase.3.The localization of Cdc14A in mouse one-cell phase fertilized eggs was observed by Immunofluorescent staining.Finally,it was found that Cdc14A was localized in the nucleus in G1、S and G2 phases,and released from the nucleus and spread to the cytoplasm in M phase.Conclusion:1.Cdc14A protein was expressed in G1、S、G2 and M phases in 1-cell fertilized eggs of mouse,with lower expression in G1 and S phase,higher expression in G2 phase and lower expression in M phase.2.Cdc14A was located in the nucleus of G1、S and G2 of mouse1-cell fertilized eggs,that was released from the nucleus and spread to the cytoplasm in M phase.