| Maturation of mammalian oocytes involves a series of complex physiological events,such as chromatin condensation and separation,which may be closely related to histone methylation modification.Histone H3K4 trimethylation(H3K4me3)is associated with transcriptional activation,Histone H3K9(H3K9me3)trimethylation inhibits gene expression,both methylation modifications are regulated by histone methyltransferases and demethyltransferases.Chaetocin can specifically inhibit the expression of histone methyltransferase SUV39H1/2 and G9A,and reduce the level of H3K9me3.CPI-455 is a specific inhibitor of histone demethyltransferase KDM5A and KDM5B,CPI-455 treatment can increase the expression of H3K4me3 in cells.Our study used Chaetocin and CPI-455 to treat porcine oocytes in vitro maturation,analyze the changes of gene expression,understand the effects of histone trimethyl modification on porcine oocytes maturation and early embryo development.This will lay a foundation for further understanding the mechanism of histone methylation modification in porcine oocytes in vitro maturation and early embryo development.1、Effects of regulation of H3K9me3 expression on porcine oocytes maturation and early embryo development.Adding different concentrations of Chaetocin into in vitro maturation culture medium during 0-22 h,the results showed that the levels of in vitro maturation rate,4-cell rate and blastocyst rate were significantly increased in 2 nM group compared with that of non-treated group(81.5%VS 72.62%,74.43%VS 68.69%,40.43%VS 27.48%,P<cell 0.05).There were no significant differences in cleavage rate,4-rate,blastocyst rate and total number of blastocysts among different groups treated with Chaetocin at 22-44 h and 0-44 h,respectively.Compared with untreated group,the rate of MIIoocytes significantly increased in 0-44 h treat with 5 nM group(87.39%VS 71.95%,P<0.05).Immunofluorescence results showed that there had H3K9me3 signals at GV stage,no expression was found in oocyte after 22 hours of culture.Compared with untreated group,the H3K9me3 levels in blastocysts were significantly decreased in 2 nM group.The results of qRT-PCR showed that,compared with untreated group,the expression of SUV39H1/2 and G9A in oocyte decreased as a whole,the expression of Nanog,Oct4 and CDX2 in blastocyst significantly increased in 2 nM group(P<0.05),and the total number of blastocyst had no difference(P<0.05).2、Effects of regulation of H3K4me3 expression on porcine oocytes maturation and early embryo development.Adding different concentrations of CPI-455 into in vitro maturation culture medium during 0-22 h,the results showed that the levels of in vitro maturation rate and blastocyst rate were significantly increased in 5 μM group compared with that of untreated group(88.82%VS 78.20%,41.84%VS 28.4%,P<0.05).When porcine oocytes were treated with CPI-455 at different stages of maturation in vitro,it was found that,compared with untreated and 1 μM groups,the 2-cell rate of 5p.M treatment group decreased significantly after treated at 22-44 hours of maturation in vitro.(84.94%,86.34%VS 74.15%,P<0.05);When CPI-455 was added at 0-44 h during in vitro maturation,no significant differences were found in vitro maturation rate,2-cell rate,4-cell rate,blastocyst rate and total blastocyst number bettwen the groups(P>0.05).Immunofluorescence results showed that the expression of H3K4me3 in blastocysts was significantly increased in 5μM group compared with untreated group(P<0.05).The results of qRT-PCR showed that compared with untreated group,the expression of KDM5A and KDM5B in oocyte was decreased and the expression of Nanog,Oct4,CDX2 in blastocyst was significantly increased in 5μM group(P>0.05),but the total number of blastocysts was not different(P>0.05).Chaetocin and CPI-455 combined treatment during 0-22 h had no significant effect on oocyte maturation in vitro and early embryo development(P>0.05).In conclusion:Suitable concentrations of Chaetocin and CPI-455 could reduce the expression of SUV39H1/2 and G9A,KDM5A and KDM5B genes in porcine oocytes,respectively.The expression of pluripotent genes in blastocysts was upregulated,and the in vitro maturation rate and early embryonic development rate of were increased. |
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