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The present study assessed changes in epigenetic markers and pre- and postimplantation development in somatic cell nuclear transfer (SCNT) porcine embryos after treatment with the inhibitor of histone deacetylases (HDACi), Trichostatin A (TSA). Embryos were generated using in vitro matured oocytes and nuclei from either a male fetal fibroblast (FF) cell line or bone marrow cells (BMC) from three adult sows. After nuclear transfer, oocytes were either exposed or not to 10 ng/mL TSA for 10 h starting 1 h after cell fusion. Samples of one-cell stage and cleaved (two- to four-cell stage) embryos were fixed at 15 to 18 h or 46 to 48 h after cell fusion and immunocytochemically processed to detect histone H3 acetylation at lysine 14 (H3K14ac) or histone H3 dimethylation at lysine 9 (H3K9m2) using specific primary antibodies. TSA treatment increased the immunofluorescent signal for H3K14ac in cleaved embryos derived from both FF and BMC but did not affect H3K9m2. Development to the blastocyst stage was increased by TSA treatment (45.2 vs. 23.9%) in embryos produced from FF cells but not in those produced from BMC (30.6 vs. 27.4%). Cloned piglets were produced from both treatments when day 5 to 6 blastocyst-stage embryos derived from FF cells were transferred into the uterus of recipient females. Cloned piglets were also produced after the transfer of TSA-treated blastocysts derived from BMC of adult sows but not from control embryos. These findings suggest that the inhibition of histone deacetylases have similar effects on enhancing H3K14ac in SCNT embryos reconstructed from different cell types but the effect on in vitro and in vivo development seems to differ according to the nuclear donor cell type.

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Phosphorylated histone H2A.x (H2AX139ph) is a key factor for the repair of DNA double-strand breaks (DSBs) and the presence of H2AX139ph foci indicates the sites of DSBs. In this study, we characterized the presence of H2AX139ph during in vitro development of porcine embryos produced by IVF and somatic cell nuclear transfer (SCNT). Pronuclear stage embryos produced by IVF had, on average, 9.2 H2AX139ph foci per pronucleus. The number of H2AX139ph foci was higher in the 2-cell-stage embryos than in the 4-cell-stage embryos fixed at 48 h post-fertilization. The percentage of H2AX139ph-positive nuclei was higher in SCNT embryos that were activated with ionomycin (ION) alone than in those activated with ION and strontium chloride (IONCSr 2C ). A negative correlation was found between the percentage of H2AX139ph-positive cells and the total number of cells per embryo in day 7 blastocysts produced by IVF or SCNT. Based on the detection of H2AX139ph foci, the findings of this study indicate that DSBs occur in a high proportion of porcine embryos produced by either IVF or SCNT; fast-cleaving embryos have fewer DSBs than slow-cleaving embryos; the oocyte activation protocol can affect DNA integrity in SCNT embryos; and better-quality blastocysts have fewer DSBs. We propose that the presence of H2AX139ph foci can be a useful marker of embryo quality.