The Effect Of Histone Deacetylase Inhibitor Oxamflatin On In Vitro Developmental Efficiency Of Porcine Somatic Cell Nuclear Transfer Embryos

Somatic cell nuclear transfer(SCNT)refers to the technique of fusing a differentiated somatic cell with enucleated oocyte to form a reconstructed embryo and the genetic background of the cloned offspring is identical to the donor cell.Since the birth of Dolly sheep in 1996,numerous species have been successfully cloned using SCNT technology,including mouse,dog,cat,cow and pig.Although this technology had been developed in the past 20 years,the efficiency of SCNT remains low(about 1%-5% in mammals).The cloned animals often have some abnormal phenotypes,such as low implantation rate,high abortion rate,and too large body accompanied by organ developmental defects,also known as large fetal syndrome.However,the cloned animals with abnormal phenotype can reproduce the normal offspring,indicating that the abnormal phenotype of cloned animals is caused by the abnormal epigenetic modification rather than genetic material changes.Studies have shown that low SCNT efficiency is due to incomplete or abnormal epigenetic reprogramming of somatic nucleus in enucleated oocyte.SCNT is a promising technology,which can help us understand the mechanism of somatic cell reprogramming process,also can be used for human organ transplantation and other basic medical research.The combination of SCNT and gene editing technology can be used to produce transgenic pigs,including disease-resistant,excellent meat quality traits,and environment-friendly pigs.Considering that the important application value and the low cloning efficiency of SCNT,in this study,we focused on how to improve the efficiency of pig cloning,meanwhile,we seek to elucidate the molecular mechanism of SCNT reprogramming,and the main results are as follows:1.We performed a preliminary experiment and found that supplemented with a certain amount of histone deacetylase inhibitor Oxamflatin to porcine zygotic medium 3(PZM-3)significantly increased the rate of in vitro blastocyst formation of porcine SCNT embryos.We then optimized the treatment conditions of Oxamflatin with different treatment concentrations and durations,and we found that by treating the reconstructed embryos with 1?M histone deacetylase inhibitor Oxamflatin for 15 h significantly improved the in vitro blastocyst development rate(treated vs.non-treated;10.3% vs.25.5%;p<0.05).2.Treatment of pig cloned embryos with 1?M Oxamflatin significantly reduced the total deacetylase activity at the pronuclear stage of the reconstructed embryos and enhanced the total histone acetylation levels of H3K9 and H4K5 at pronuclear,2-cell and 4-cell stages.We then examined the mRNA expression levels of four types of histone deacetylases(including HDAC1-11,and Sirt1,2)in MII oocytes and porcine fetal fibroblasts(PFF).The results indicated that the relative mRNA expression of HDAC1,2 and 3 in MII oocytes was higher than that of in PFF,and also higher than other types of histone deacetylases genes.Oxamflatin treatment significantly reduced the expression of HDAC1 in the pronuclear stage of porcine reconstructed embryos and this can,at least,partly explained the hyperacetylated histone H3K9 and H4K5 levels mediated by treatment of Oxamflatin.3.We also detected the acetylation level of non-histone ?-tubulin protein and found that treatment with 1 ?M of Oxamflatin for 15 h postactivation significantly increased the level of acetylated ?-tubulin in the midbody and spindle of porcine reconstructed embryos during the first two mitotic cell cycle progression,which may be regulated by inhibition of HDAC6 expression.4.RT-qPCR result showed that the mRNA level of DNA methyltransferase 1(DNMT1)is dominant in porcine MII oocytes,and its expression level is higher than that of its homologous DNMT2,DNMT3 a,and 3b(more than 50 fold).We detected the total 5-mC and 5-hmC levels of porcine cloned embryos at 2 cell and 4 cell stages using immunofluorescence co-staining,and the result suggests that the total 5-mC and 5-hmC levels of porcine SCNT embryos were decreased from 2 to 4 cell stages.Treatment of porcine reconstructed embryos with 1 ?M oxamflatin for 15 h significantly reduced total DNA methylation in 2 cell stage of porcine cloned embryos and also decreased the expression of DNMT1 at 2-cell stage.5.The expression of the POU5F1(POU Class 5 Homeobox 1)gene in the blastocyst stage of pig SCNT embryos was significantly improved by treatment with 1 ?M Oxamflatin for 15 h.This may be due to a decrease of the DNA methylation level of the POU5F1 promoter region,but Oxamflatin treatment did not alter the methylation level of porcine satellite DNA sequence.6.Low-dose Oxamflatin treatment did not inhibit the growth of porcine fetal fibroblasts.Oxamflatin treatment also resulted in higher acetylation levels of histone H3K9,H4K5 and non-histone ?-tubulin in porcine fetal fibroblasts and,to some extent,Oxamflatin treatment reduced the total DNA methylation level of porcine fetal fibroblasts.However,pretreatment of porcine fetal fibroblasts with Oxamflatin did not improve the rate of in vitro blastocyst formation of porcine reconstructed embryos.These results indicated that Oxamflatin treatment resulted in a higher efficiency of somatic cell cloning by inhibiting the activity of histone deacetylase in oocytes,rather than by decreasing the activity of histone deacetylase in donor cells.In this study,we had a better understanding of the molecular reprogramming mechanism of the effect of histone deacetylase inhibitor Oxamflatin on porcine cloning efficiency.This will be useful to better understand the reprogramming mechanism of somatic cell nuclear transfer in the future.