Study On The Relationship Between Donor Cell Chromatin Histone Acetylation Level And Reconstructed Buffalo Embryos Developmental Potential

Effect of donor cell chromatin histone acetylation level on nuclear transferefficiency was investigated, and the relationship between acetylation andembryos developmental potential was also analyzed by comparing histoneacetylation level among buffalo embryos derived from nuclear transfer (NT),parthenogenetic activation (PA) and in vitro fertilization (IVF).A method for detecting mRNA expression in buffalo fibroblasts was set up byusing real. time fluorescence quantitative PCR. Gene ,fragments of buffalohistone2A, histone acetyltransferasel (HAT1), histone deacetylase 1 (HDAC1)were cloned, and sequencing result showed that these cDNA got about 98%homologous with bovine's. The primers were sensitive enough and fulfilledSYBR GreenⅠreal-time fluorescence quantitative PCR. Amplificationefficiency of target genes (HAT1, HDAC1) was equal to reference gene (H2A).The best efficiency of SYBR Green real-time fluorescence quantitative PCR wasgiven when 0.5μL primers at 1μM and 0.5μL cDNA templates were containedin onereaction system.Expression patterns of HAT1, HDAC1 in donor cells were detected afterTSA-treated. Results showed that gene expression levels changed correspondingto different TSA concentration and treated time. The level of mRNA for HAT1was enhanced significantly after treating by 0.15μM TSA for 72h (P＜0.05).Alternatively, expression of HDAC1 was restrained after TSA-treated for 48h(P＜0.05).Effect of chromatin histone acetyiation level on nuciear transfer efficiency was investigated through transferring TSA-treated somatic cells into enucleatedoocytes. More oocytes cleaved and developed to blastocyst after donor cellswere treated by higher concentration of TSA. Developmental potential ofreconstructed embryos was improved significantly when donor cells weretreated by 0.15μM TSA for 72h, 0.3μM TSA for 48h,0.6μM TSA for 24h(P＜0.05, blastocyst ratios were 30%, 30.8%,30.3% respectively vs. 19%).Acetylation levels of buffalo embryos derived from NT, IVF and PA wereinvestigated. Different kinds of embryos in different development stages showeddynamic change. NT embryos from TSA-treated donor cells showedsignificantly higher acetylation levels than other kinds of embryos, and NTembryos from untreated cells showed similar acetylation dynamics, even thelevels were lower. Acetylation levels of IVF embryos were increasing stably tothe blastocyst stage. PA embryos showed a steady histone acetylation patterneven it was lower in four cell or eight cell stage.In conclusion, (1) the proper primers~ designed for H2A, HAT1, HDAC1 inreal-time PCR can be guided by the homology sequence of bovine, (2)theproper primers, top-quality cDNA and optimized reaction system are keys forreal-time fluorescence quantitative PCR, (3) effect of TSA on somatic cellsdepends on treating time and TSA concentration, proper concentration andtreating time can change gene expression levels significantly, (4) nucleartransfer efficiency may be relevant to donor cell Chromatin histone acetylationlevel, donor cells undergo TSA, treated properly can improve developmentalpotential of reconstructed embryos, (5) embryo developmental potentialcorrelate closely to its histone acetylation, IVF embryos show higher histoneacetylation level than PA embyos.