| It had been reported that there were lots of problems in SCNT embryos,including epigenetic modification,maternal-to-zygote transition(MTZ),maintenance of pluripotency,which were different with normal in vivo and in vitro fertilization(IVF)embryos.H3K9me3 histone methylation plays an important role in somatic cell reprogramming and embryonic development.Chaetocin is a specific inhibitor of histone methyltransferase SUV39H1/2 and G9a.It can reduce the expression of H3K9me3 histone methylation in cells.To?nderstand the impact of reducing the expression level of H3K9me3 on Debao black pig SCNT embryo development potential,in this study,we treated Guangxi Debao black pig ear fibroblast and SCNT embryos with chaetocin,then investigated the effect of chaetocin treatment on in vitro developmental potential of somatic cell nuclear transfer embryos.1.Optimum chaetocin concentration for treatment of porcine parthenogenetic embryos.The results showed that?sing different concentrations of chaetocin treatment pig parthenogenetic embryos 24 h,2 nM significantly increased the blastocyst rate of porcine PA(46%vs 37%,P<0.05).2 nM treatment of porcine PA embryos in different times,24 h significantly increased blastocyst rate(46%vs 37%,P<0.05).The qRT-PCR results showed that compared with the control group,2 nM chaetocin for 24 h decreased expression of SUV39H1/2 and G9a in the embryos and were significantly reduced in SCNT 2-and 4-cell embryos(P<0.05).?nder this processing condition,chaetocin could significantly increase the expression of eIF3A,TFIIA in SCNT 2-and 4-cell embryos(P<0.05),and also significantly increased the expression of Oct4,Nanog in SCNT blastocyst.The histone H3K9me3 acetylation level was studied by immunohistochemical technology,the results showed the levels of histone H3K9me3 acetylation of PA embyos at various stages(4-cell and Blastocyst)were significantly lower than the control group(P<0.05).2.Effects of chaetocin treatment on development of Debao black porcine SCNT embryos.In this study,compared with the control group,2 nM chaetocin treated SCNT embryos 24 h would significantly increase the porcine somatic cell nuclear embryo 2-cell embyos and blastocyst rate(79%vs 68%,21%vs13%,P<0.05).The qRT-PCR results showed that compared with the control group,treatment of SCNT embryos with chaetocin could decrease expression of SUV39H1/2 and G9a.The expression of SUV39H1,G9a and SUV39H2 genes in 2-cell and 4-cell embryos was significantly decreased(P<0.05).In SCNT 2-and 4-cell embryos,the expression of eIF3A,TFIIA was significantly increased(P<0.05),and the expression of Nanog,Oct-4 in blastocyst was improved(P<0.05).Finally,the H3K9me3 level in Debao black porcine SCNT embyos was analyzed by immunohistochemistry.The result showed that,in comparison with non-treated group,the chaetocin could significantly decrease H3K9me3 level in SCNT 2-and 4-cell embryos(P<0.05).3.The effects of donor cells treated with chaetocin on in vitro developmental potency of Debao black pig SCNT embryos were investigated.In this study,the effect of Debao pig ear fibroblasts treated with different concentration chaetocin 24 h was investigated.The results showed that cells treated with 20 nM chaetocin had similar growth curve with that of the control group,and all treated cells had "S" shape of growth curve.With increasing of chaetocin concentrations,cells showed distortion,growth inhibition in varying degrees,and even death.DeBao black pig fibroblasts treated with chaetocin from 20nM didn't show significantly alteration on the chromosome ploidy.The cleavage and morula rates of SCNT embryos produced with donor cell treated with chaetocin had a significant difference with that of the control group.The blastocyst rate of 20 nM chaetocin the treatment groups of was higher than that of other groups(19%vs 13%,P<0.05).The qRT-PCR results showed that compared with the control group,20nM chaetocin treatment for 24 h reduced the expression of SUV39H1/2 and G9a.Significantly increased the expression of eIF3A,TFIIA in SCNT 2-and 4-cell embryos(P<0.05),and the expression of Nanog and Oct-4 in blastocyst was improved.The expression of H3K9me3 was detected by immunofluorescence,20 nM chaetocin treated donor cells for 24 h,reduced H3K9me3 expression in embryos,significantly decreased in SCNT 2-and 4-cell embryos(P<0.05)In conclusion,the above results demonstrated that?nder suitable chaetocin processing conditions could reduce the levels of H3K9me3 histone methylation in SCNT embyos,and improve the development ability of early nuclear transfer embryos... |
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