| The purpose of present study was to investigate the expression patterns and the roles of histone H3S10phosphorylation during oocytes maturation and embryonic development of nuclear transfer, and the relationship between histone phosphorylation and acetylation. It would lay foundation to improve embryo developmental quality by using epigenetic regulation method.First, to understand the phosphorylation status of histone H3S10during oocyte meiotic maturation, spatial and temporal distribution of phosphorylated serine10of histone H3during in vitro matured rabbit and porcine oocytes were examined by immunofluorescence staining method, the effects of ZM447439treatment on expression of histone H3serine10phosphorylation were also detected. The results showed that:At GV stage, few oocyte was stained by H3S10phosphorylation specific antibody. While oocytes grew to GVBD stage, the different expression patterns were observed between rabbit and porcine oocytes. Higher phosphorylation level of H3S10was clearly observed in rabbit oocytes, and this high expression trend maintained until MⅡ stage. Phosphorylation of H3S10was reduced at M I stage, followed by gradually increased from anaphase/telophase(A I) to metaphase(M II) stage. Among all of the maturation process, the highest phosphorylation level of H3S10of rabbit oocytes was at GVBD stage. For porcine oocytes, the phosphorylation of H3S10was gradually increased from GVBD to A I stage, the highest phosphorylation level was at M Ⅰ/A Ⅰ stage, followed by reduced level at MⅡ stage. The influence of low concentration of ZM447439on in vitro-matured rabbit and porcine oocytes meiotic maturation was limited, oocytes treated with30μM ZM447439stoped at GVBD stage. Few of rabbit and porcine oocytes treated with5μM ZM447439was dephosphorylated at H3S10site(20.7%vs10.9%), the remained oocytes had similar H3S10phosphorylation level with the control groups. When treated with10μM ZM447439,45.8%of rabbit and35.2%of porcine oocytes were phosphorylated at histone H3S10. When treated with30μM ZM447439, the phosphorylation of H3S10in most of oocytes disappeared. When treated with lOnM TSA,89.5%of porcine oocytes was phosphorylated at histone H3S10, similar with the control group(p>0.05).When treated with50nM TSA,2.3%of pig oocytes were phosphorylated at H3S10.Secondly, the phosphorylation distribution and expression level of histone H3S10during pig early embryonic development were examined. For4-cell stage embryos, the effect of TSA concentration on histone H3S10phosphorylation of pig nuclear transfer embryos during mitosis was explored. The results showed that:Histone H3S10is phosphorylated at the prophase stage of the cell division. The phosphorylation level at the metaphase stage was the highest, following with gradually reduced from the telophase stage. The phosphorylation level of nuclear transfer embryo during mitotic in all stages was significantly higher than that of the PA and TSA treatment groups(p<0.05). The expression status of the histone H3S10phosphorylation at mitotic metaphases in different stages of porcine preimplantation embryos was also examined. The results showed that, the phosphorylated level of pig nuclear transfer embryo was firstly reduced and then gradually increased from2-cell to blastocyst. And the phosphorylation level of histone H3S10at mitotic metaphase during pig nuclear transfer embryo was significantly higher than that of the PA and the TSA treated groups(p<0.05). Meanwhile, the expression of AURKB and HDAC2genes in porcine nuclear transfer preimplantation embryos treated with different concentration TSA was analyzed. The results showed that, the expression of AURKB and HDAC2genes were significantly lower than that of control groups from2-cell to blastocyst stages, the expression of the two genes was gradually reduced with the development of embryos(p<0.05). These results suggested that TSA treatment could significantly reduce the expression of AURKB and HDAC2gene during embryonic development.In conclusion:(1) The dynamic changes of histone H3S10phosphorylation during mammalian oocytes meiosis could impact the oocyte maturation.(2) The AuroraB kinase inhibitors-ZM447439, which is one of target antitumor drugs, had damage effect on the germ cell development.(3) There was a close positive corrrlation among AuroraB, H3S10phosphorylation and dynamic chromosome in in vitro-maturated oocytes.(4) The histone H3acetylation could be essential for H3phosphorylation of in vitro-maturated oocytes.(5) TSA could significantly reduce the phosphorylation level of histone H3, as well as the expression of AURKB and HDAC2genes during embryonic development. |
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