Effect Of PsA On Early Development Of Bovine Cloned Embryos

Somatic cell nuclear transfer(SCNT)is a technology that makes use of cytoplasmic reprogramming material in the oocyte to make reprogramming of highly differentiated somatic cell nuclear to restore the totipotency and develop into a new member.So far,more than 10 kinds of animals have been cloned by the SCNT technology,including cow,mice,pig,rabbit,etc,but the low cloning efficiency limits its application.Epigenetic modifications,such as histone deacetylase inhibitors,can correct the abnormal epigenetic modifications and promote the reprogramming of SCNT embryos,so as to improve development of early embryos.Psammaplin A(PsA)is a novel histone deacetylase inhibitor,so there is little report available on its effect on early development of bovine cloned embryos.Therefore,the effects of PsA on cleavage rate,blastocyst rate,expression of key genes and expression of histone markers in bovine parthenogenetic embryos were analyzed by immunohistochemistry and fluorescence quantitative PCR,and the experimental concentration and time of PsA were determined in the research.Ultimately,the effects of PsA on the early development bovine cloned embryos were studied.The results are as follows:1.The effect of PsA on cleavage rate and blastocyst rate in bovine parthenogenetic embryos: PsA treatment of 6 h 5 ?M and 10 ?M as well as 12 h 5 ?M and 10 ?M had no significant effect on the cleavage rate of bovine parthenogenetic embryos,which ranged from 62% to 68%.The blastocyst rate of bovine parthenogenetic embryos was significantly reduced by PsA treatment at 12 h and 6 h 10 ?M,while the blastocyst rate was increased at 6 h 5?M,but the difference was not significant.The results showed that the cleavage rate and blastocyst rate of bovine parthenogenetic embryo decreased with the increasing concentrations and prolonged treatment of PsA.2.The effect of PsA on gene expression in bovine parthenogenetic embryos: The relative expression levels of HDAC1 and DNMT1 were significantly decreased by PsA treatment.For the pluripotent gene Nanog,compared with control group,the relative expression levels of 6 h 10 ?M was increased,6 h 5 ?M and 12 h 5 ?M were reduced but the differences was not significant,12 h 10 ?M was significantly reduced.The expression level of SOD was significantly decreased and BCL was reduced compared with control group.The results showed that PsA inhibited the expression of HDAC1 and DNMT1,and increased oxidation and apoptosis.Based on the results of development rate and gene expression,we selected the the concentration and time of PsA which could be conducive to the development of embryo,10 ?M 6 h.3.The effect of PsA on expression of histone H3K9me3 and H3K9 ac in bovine parthenogenetic embryos: In the PsA treatment group,the relative expression of H3K9 ac was significantly increased,while H3K9me3 was significantly decreased in 4 cell and blastocyst of bovine parthenogenetic embryo.4.The effect of PsA on cleavage rate and blastocyst rate in bovine SCNT embryos: The PsA treatment increased the embryo cleavage rate and blastocyst rate,the cleavage rate increased by 6.47%,and the blastocyst rate increased by 1.15%.The results indicated that PsA treatment could promote the development of SCNT embryo.5.The effect of PsA on gene expression in bovine SCNT embryos: Compared with the SCNT group,PsA treatment inhibited the expression of HDAC1,and increased the expression of DNMTI but the difference was not significant.Compared with the SCNT group,PsA treatment reduced the expression of SOD,and increased the expression of Nanog as well as improved the expression of BCL,but the difference was not significant.Compared with the IVF group,the expression of HDAC1 and DNMTI were increased,SOD and BCL was improved,Nanog was decreased in the SCNT group.The results indicated that PsA significantly inhibited the expression of HDAC1 in bovine SCNT embryos,promoted the expression of pluripotent gene Nanog and inhibited apoptosis.6.The effect of PsA on expression of histone H3K9me3 and H3K9 ac in bovine SCNT embryos: The expression of H3K9 ac was up-regulated and the expression of H3K9me3 was down-regulated by PsA treatment,and there was no significant difference between the SCNT and IVF groups.The results suggested that PsA treatment could induce chromatin remodeling in bovine SCNT embryos,alleviate transcriptional inhibition,and contribute to reprogramming of reconstructed embryos.In conclusion,PsA treatment inhibited the expression of HDAC1,promoted the expression of pluripotent gene Nanog,up-regulated the expression of H3K9 ac,down-regulated the expression of H3K9me3,promoted the development of bovine SCNT embryos,and contributed to nuclear reprogramming,which laid a theoretical foundation for improving the efficiency of cloning.