The Study On Zygotic Gene Activation And Epigenetic Modification During Porcine Somatic Cell Nuclear Transfer Early Embryo Development

In mammals,the early stage of embryonic development is regulated by maternal factors derived from oocytes,and the zygotic genome is in a state of transcriptional silencing at this time.Maternal factors are gradually consumed with the continuous cleavage,the zygote genome is activated in a timely manner,and the dominance of regulating embryo development changes from maternal to zygote.The developmental arrest occurs in almost all mammalian early embryos cultured in vitro,and the major developmental arrest period coincides with the time of zygotic activation,which fully indicates that the correct activation of zygotic genes is of great significance for early embryo development.People have been working on somatic cell nuclear transfer(SCNT)for more than 50 years,and the low cloning efficiency has been the main reason that restricts the application of this technology.Previous studies have found that there are abnormal zygotic gene activation in mouse and human SCNT early embryos which lead to the incomplete reprogramming and development arrest.Abnormal epigenetic modifications are one of the key reasons responsible for zygotic activation barriers.The pig is not only a kind of livestock with important economic value,but also can be used as bioreactors for producing humanized proteins and ideal animal models for heterologous organ donors.Therefore,it is of great scientific significance and application value to improve the porcine SCNT efficiency.Previous studies have seldom studied the zygotic activation and epigenetic motification of porcine SCNT embryos at the transcriptome level.Therefore,in our study we compared gene expression pattern of in vivo fertilization(IVV)embryos and SCNT embryos at transcriptome level and the expression patterns of various epigenetic modification enzymes by PCR array.Our study was designed to elucidate the underlying mechanisms by which epigenetic modifications affect zygotic gene activation and embryonic development during porcine SCNT.The main results of ourstudy are as follows:1.The transcriptome analysis of IVV embryos at different stages during porcine preimplantation development revealed that:(1)The transcriptome patterns during porcine early embryonic development exhibited dynamic changes,and the zygotic gene activation occured at 4-cell stage.Combined expression profiles analysis of humans,mice,and cattle showed that,the time of mouse zygotic activation was the earliest,but it experienced two activations at 2-cell and 8-cell stage respectively;human zygotic activation occurred in 8-cell embryos;bovine zygotic activation occurred at the latest which appeared at 16-cell stage;(2)Gene function of differentially expressed genes during the zygotic activation of four mammalian were generally the same as each other,but pigs and cattle had the highest similarities in each aspect;(3)The core KEGG pathway in which differentially expressed genes during the zygotic activation of four mammalian enriched were basically the same,however they also had their own species-specific pathway enrichment.2.The comparison of transcriptomes and the expression pattern of multiple histone H3K4 methyltransferases between porcine IVV embryos and SCNT embryos revealed that(1)Compared with IVV embryos,porcine SCNT early embryos existed abnormal zygotic gene activation at 4-cell stage.Most zygotic genes failed to transcription activation at this time,and mainly of them were related to ribosome synthesis and translation;(2)Compared with IVV embryos,almost all H3K4 methyltransferases were expressed at abnormally higher levels in porcine SCNT embryos at the transcriptome level,and SCNT embryos showed abnormally higher H3K4me3 level at protein levels.3.The study about the effects of MM-102,a small-molecule inhibitor of H3K4 methyltransferase MLL1,on early SCNT embryos showed that:(1)MM-102 treatment under the conditon of 75 ?M and 72 hours significantly increased the development rates of porcine SCNT embryos at each stage;(2)MM-102 treatment reduced the overall H3K4,H3K9 methylation and 5m C levels of porcine early SCNT embryos,especially at zygotic activation stage and blastocyst stage,making them more similar to IVF embryos;(3)MM-102 treatment resuced the expression patterns of various epigenetic modification enzymes in SCNT embryos at 4 cell stage and blastocyst stage,and mainly down-regulated a series of DNA and histone methyltransferases,and up-regulated some histone acetyltransferases andtranscriptional activators,making their overall expression patterns more similar to that of IVV embryos;(4)MM-102 treatment increased m RNA expression levels of pluripotent genes POU5F1,NANOG and anti-apoptosis gene BCL2 in porcine early SCNT embryos,but reduced the expression of apoptotic gene BAX.The proportion of apoptotic cells in blastocysts was reduced,so the quality of the blastocysts were improved.From the above results,we concluded that the zygotic gene activation occurring in mammalian early embryos was abnormal in porcine SCNT embryos,and abnormally high H3K4 methylation might be one of the key reason for zygotic gene activation barrier.MM-102 could rescued epigenetic reprogramming levels in porcine SCNT embryos thereby improving the and the efficiency of SCNT.