Studies On Improving Development Of Cloned Sheep Embryos By Regulation Of H3K9 Methylation And DNA Hydroxymethylation

Although animal cloning by somatic cell nuclear transfer(SCNT)has been succeeded in many species,the low efficiency has hindered the utility in agricultural and biomedical applications of the SCNT technology.Although researchers have tried a lot to improve the cloning efficiency,the effectiveness is relatively limited.The efficiency of SCNT still needs to be improved.The developmental defects of SCNT embryos has been largely attributed to incomplete epigenetic reprogramming of donor cell genome,which includes DNA methylation,histone acetylation,histone methylation and so on.Histone 3 lysine 9 trimethylation(H3K9me3)was recently identified as a key barrier to efficient reprogramming by SCNT in mouse.In this study,we compared the H3K9 trimethylation and demethylation(me2)levels between sheep embryos produced by in vitro fertilization(IVF)and SCNT.The results showed that compared to stage-matched IVF embryos,SCNT embryos exhibited higher levels of H3K9me3/2 at the 1-cell,2-cell and blastocyst stages.To regulate the H3K9me3/2 levels in SCNT embryos,this study used histone methyltransferase SUV39 inhibitor,chaetocin,or histone lysine demethylase(KDM)protein to treat the donor cells or SCNT embryos.We found that chaetocin treatment could efficiently down-regulate the H3K9me2 and H3K9me3 levels in cultured donor cells and the resulting SCNT embryos,but it failed to promote the development of SCNT embryos.Direct treatment of the SCNT embryos with chaetocin could also significantly reduce the H3K9me2 and H3K9me3 levels,but the development of embryos was severely impaired by chaetocin treatment.When sheep fetal fibroblast(SFF)cells were treated with recombinant human KDM4D protein(rhKDM4D),the levels of H3K9me3 and H3K9me2 were both significantly decreased.After SCNT,rhKDM4D-treated donor cells supported significantly higher percentage of cloned embryos developing into blastocysts as compared to non-treated control cells.Moreover,the blastocyst quality was also improved by rhKDM4D treatment of donor cells,as assessed by the total cell numbers in blastocysts and the expression of developmental genes including SOX2,NANOG and CDX2.In conclusion,KDM4D protein treatment of sheep donor cells can effectively reduce the levels of H3K9me3 and H3K9me2 in cells and promote the development of SCNT embryos.The abnormal DNA methylation in cloned embryos is also an important factor hindering the development of SCNT embryos.There are higher levels of 5-methylcytosine(5mC)during the early development of SCNT embryos.5-hydroxymethylcytosine(5hmC)is the first oxidation production of 5mC.It is still unclear whether it will promote the development of cloned embryos by increasing the level of 5hmC.Recently studies have identified that vitamin C is a cofactor of Fe2+ and a-ketoglutarate-dependent dioxygenases to promote the activity of ten-eleven translocation(TET).In this study,we investigate the effect of vitamin C on the early development of mouse and sheep embryos.When sheep donor cells were treated with vitamin C,the 5hmC levels were increased but the 5mC levels were not affected.Further,vitamin C treatment of serum-starved donor cells could induce the decrease of 5mC levels in parall with the increase 5hmC levels.However,any vitamin C treatment of donor cells could not improve the blastocyst development of resulting SCNT embryo although the expression of some pluripotency related genes like KLF4 and SOX2 were upregulated.We found that addition of 200?M vitamin C into in vitro maturation(IVM)medium or in vitro culture(IVC)medium could significantly improve the blastocyst development of mouse embryos.Similarly,supplementation of(10 and 50 ?M)IVC medium with vitamin C could also improve the preimplantation development of sheep SCNT and IVF embryos.Immunostaining indicated that the 5hmC levels were elevated in vitamin C-treated blastocyst.In summary,sheep cloned embryos in 1-cell,2-cell and blastocyst stage have higher levels of H3K9me3/2.Chaetocin treatment of cultured donor cells can reduce the levels of H3K9me3/2 in cells,but it can not promote the in vitro development of cloned embryos.rhKDM4D treatment can decrease the levels of H3K9me3/2 in donor cells and can promote the in vitro development of resulting SCNT embryos.Vitamin C can increase the levels of 5hmC and decrease the levels of 5mC in serum-starved donor cells,but it has not beneficial effect on the development of resulting SCNT embryos.Addition of vitamin C in IVC medium can significantly promote the development of IVF and SCNT embryos.This study provides important information for improving the procedure and efficiency of animal cloning by SCNT.