Effect Of P53on Bovine Oocytes Maturation In Vitro

p53is a kind of tumor suppressor genes, it plays a role in the process of controlling cellcycle arrest, senescence, autophagy and apoptosis. It expressed widely in various tissues oforganism. In male germ cells, p53take part in repairing DNA in the process ofspermatogenesis, and controlling the cell cycle checkpoint before meiosis and participating inthe male germ cell apoptosis. In the female germ cells, p53paly an important role in theoogenesis in utero female mice and the selection of oocyte. Thus detect whether p53wasinvolved in the repair of DNA damage and its potential of regulation cell cycle checkpoint inoocyte maturation process is very necessary. The purpose of this study is to clone the bovinep53gene and construct a eukaryotic expression vector pp53-venus, and then discuss its effecton oocyte maturation in vitro.Firstly, we cloned the total CDS sequence of p53gene from bovine cumulus cells andconstructed eukaryotic expression vector pp53-venus; Secondly, we collected bovine oocytesin different developmental stages in vitro culture, and detected the changes of p53mRNAexpression in different developmental stages in vitro culture via the real-time PCR; Finally,we transcribed p53-venus into mRNA using in vitro transcription kit and over expression andinterference p53gene in bovine oocytes by microinjection technique, then statistics of the firstpolar body extrosion rate, studying the effect of p53gene on maturation of oocyte in vitro.Research results are as follows:1. We cloned the total CDS sequence of p53gene from bovine cumulus cells and linkedit into pMD19-T. After identified pMD19-T-P53by double restriction enzyme digestion, wecloned the p53into expression vector pvenus, and then we constructed eukaryotic expressionvector of pp53-venus. After pp53-venus recombinant plasmid transfected into Hela cellsmediated by Lipofectamine2000, we confirmed the expression and position of therecombinant plasmid pp53-venus in Hela cells. It mainly located in the nucleus of the Helacells.2. We collected bovine oocytes in different developmental stages in vitro culture andextract RNA in oocytes with trace method, then detected the changes of p53mRNAexpression in different developmental stages in vitro culture via the real-time PCR. Theresults showed that the expression of p53mRNA lifted quickly from GV stage to MI period and the expression quantity is the highest in MI period, and then it gradually decreased. Thismay suggest the role of p53in control of metaphase checkpoint.3. We transcribed p53-venus into mRNA using in vitro transcription kit and observed theexpression and localization of p53-venus mRNA in bovine oocyte after microinjection. Wefound that it expressed not only in cell nucleus, but also in cytoplasm, but the expressionamount is much smaller than in nucleus. Over expression of p53by microinjection p53-venusmRNA has no siginificent effect on the first polar body extrosion rate of bovine oocyte and itcan cleavaged and developed into embryos after parthenogenetic activation. Then we observedthe spindle morphology of the oocytes which injected in p53-venus mRNA by immuno-fluorescence. The morphology of spindle is normal and the immature oocytes was most rest inmeiosis I metaphase. Interference the expression of p53by microinjection of bovine p53siRNA also has no siginificent effect on the first polar body extrosion rate of bovine oocyteand it can developed into embryos after parthenogenetic activation.Conclusion: we cloned the total CDS sequence of p53gene and constructed eukaryoticexpression vector of pp53-venus; transfected pp53-venus recombinant plasmid into Hela cells,it can correctly expression and located in the nucleus; the changes of p53mRNA expression indifferent developmental stages in vitro culture lifted quickly from GV stage to MI period andthe expression quantity is the highest in MI period, and then it gradually decreased; overexpression and interference p53gene in bovine oocytes by microinjection has no siginificenteffect on the first polar body extrosion rate of bovine oocyte.