Martinez-Diaz Mario A, Che Limei, Albornoz Marcelo, Seneda Marcelo M, Collis Daryn, Coutinho Ana Rita S, El-Beirouthi Nayala, Laurin Denyse, Zhao Xin, Bordignon Vilceu
Department of Animal Science, McGill University, Ste-Anne-de-Bellevue, Québec, Canada.
Cell Reprogram. 2010 Feb;12(1):85-94. doi: 10.1089/cell.2009.0047.
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.
本研究评估了用组蛋白脱乙酰酶抑制剂（HDACi）曲古抑菌素A（TSA）处理后，体细胞核移植（SCNT）猪胚胎中表观遗传标记的变化以及植入前和植入后的发育情况。使用体外成熟的卵母细胞和来自雄性胎儿成纤维细胞（FF）细胞系或三只成年母猪的骨髓细胞（BMC）的细胞核来生成胚胎。核移植后，细胞融合1小时后，卵母细胞要么暴露于10 ng/mL TSA中10小时，要么不暴露于TSA中。单细胞期和分裂期（二至四细胞期）胚胎样本在细胞融合后15至18小时或46至48小时固定，并进行免疫细胞化学处理，使用特异性一抗检测赖氨酸14处的组蛋白H3乙酰化（H3K14ac）或赖氨酸9处的组蛋白H3二甲基化（H3K9m2）。TSA处理增加了来自FF和BMC的分裂期胚胎中H3K14ac的免疫荧光信号，但不影响H3K9m2。TSA处理使FF细胞产生的胚胎发育到囊胚期的比例增加（45.2%对23.9%），但对BMC产生的胚胎没有影响（30.6%对27.4%）。当将来自FF细胞的第5至6天囊胚期胚胎移植到受体雌性子宫中时，两种处理都产生了克隆仔猪。在移植成年母猪BMC来源的经TSA处理的囊胚后也产生了克隆仔猪，但对照胚胎未产生克隆仔猪。这些发现表明，组蛋白脱乙酰酶的抑制对增强由不同细胞类型重构的SCNT胚胎中的H3K14ac具有相似的作用，但对体外和体内发育的影响似乎因核供体细胞类型而异。
