Expression And Function Of ASH1L Methyltransferase In Bovine Cumulus Cells And Embryos

In mammals,cell proliferation and embryo development are regulated by epigenetic modification.Histone methylation and acetylation can change chromosome conformation,regulate gene expression and silencing,even further control the animal development.Absent small or homeotic 1(ASH1L)is a histone methyltransferase,which can promote the development and growth of individuals by methylating H3K36 and antagonizing the expression of polycomb proteins.This research studied the expression and function of ASH1 L in bovine cumulus cells and embryos,which provides the basis for clarification of regulatory mechanism of ASH1 L methyltransferase.Experiment 1: To study the expression and function of ASH1 L in bovine cumulus cells,the expression of ASH1 L in bovine cumulus cells was detected.Results showed that ASH1 L was located in the nucleus of bovine cumulus cells and distributed in a dotted pattern.We successful screened out the effective siRNA(siRNA-2)targeting the bovine Ash1 L gene and the interfering efficiency reached 60-70%.The methylation levels of H3K36me1/2/3 in cumulus cells transfected with siRNA-2 were significantly lower than those in the control group(P<0.05).Additionally,Ash1 L gene interference could increased the number of apoptotic cells,significantly up-regulated the expression of apoptosis-related genes Bax,Bcl-2 and caspase-3.However the levels of apoptosis genes Bax and caspase-3 were higher than that of anti-apoptosis gene Bcl-2(1.311 and 1.179 vs 1.146).At the same time,the mRNA expression of PRC2 protein subunit was significantly increased in cells in which Ash1 L was knock-down with siRNA-2(P<0.05).Experiment 2: To study the expression and function of ASH1 L in bovine embryos fertilized in vitro,the expression of ASH1 L in embryos at different developmental stages was detected.Results showed that ASH1 L was located in the nucleus of bovine oocytes and embryos at 2,4,8 and 16-cell stages,while ASH1 L was spotted in the nucleus during morula and blastocyst stage.H3K36me2/3 was found in oocyte nuclei and IVF embeyos at all developmental stages,and it was weak in 4-cell embryo,and the signal is enhances from 8-cell to blastocyst.The expression of Ash1 L gene in 16-cell and morula stage was significantly higher than that in other stages(P<0.05).The blastocyst development rate and quality were significantly decreased after injected siAsh1L(P<0.05).The mRNA expression of pluripotent genes was significantly decreased(P<0.05).The expression of PRC2-related subunit EZH2 was significantly increased from 2-cell to morula stage(P<0.05).The expression of EED and Suz12 was significantly increased in 16-cell and morula stage(P<0.05).Experiment 3: To predict the target genes of Ash1 L,the differential expression profiles of genes were measured using RNA_sequencing.The results showed that a total of 828 differentially expressed genes(DEGs)were screened,including 514 up-regulated genes and 314 down-regulated genes.A total of 1723 GO items were selected by GO annotation,58 signal pathways were obtained by KEGG analysis.ASH1 L protein is mainly involved in lysine degradation pathway and tight junction pathway.Among them,the expression levels of ACAT2 and ZAK genes were relatively different,both of them were related to cell growth and embryonic development.The target genes of ASH1 L enzyme may be ACAT2 and ZAK.However,the interaction mechanism between those genes and ASH1 L,and their regulation on embryo development required further study.This paper concluded that ASH1 L protein was located in the nucleus of cumulus cells,oocytes and early embryos.Interfering with Ash1 L gene expression,the apoptosis rates of cumulus cells and the expression levels of apoptosis-related genes were significantly increased,and H3K36me1/2/3 methylation level decreased significantly.Similarly,embryo developmental rate blastocyst quality,and mRNA levels of pluripotent genes were decreased significantly,and the expression levels of PRC2 protein subunit genes were significantly increased in cells and embryos.The results of transcriptome sequencing showed that the target groups of Ash1 L methytransferase were ACAT2 and ZAK genes.Therefore this study provides a theory basis to reveal the regulation mechanism of ASH1 L in animal embryos development.