Tsuji Yuta, Kato Yoko, Tsunoda Yukio
Laboratory of Animal Reproduction, College of Agriculture, Kinki University, Nara, Japan.
Zygote. 2009 May;17(2):109-15. doi: 10.1017/S0967199408005133. Epub 2009 Jan 28.
To facilitate nuclear reprogramming, somatic cells or somatic cell nuclear-transferred (SCNT) oocytes have been treated with the histone deacetylase inhibitor trichostatin A (TSA), or the DNA methyltransferase inhibitor, 5-aza-2'-deoxycytidine (5-aza-dC), to relax epigenetic marks of differentiated somatic cells. TSA-treated SCNT oocytes have increased developmental potential, but the optimal treatment period is unknown. Reduced methylation levels in somatic cells have no positive effect on SCNT oocytes, but the treatment of SCNT embryos with 5-aza-dC has not been investigated. We examined the effect of TSA treatment duration on the developmental potential of mouse SCNT oocytes and the effect of 5-aza-dC treatment on their in vitro and in vivo developmental potential. To determine the effects of TSA treatment duration, nuclear-transferred (NT) oocytes were cultured for 0 to 26 h with 100 nM TSA. SCNT oocytes treated with TSA for 8 to 12 h had the higher rate of development to blastocysts and full-term fetuses were obtained after treatment for 8 to 12 h. When oocytes were treated for 14 h and 26 h, blastocyst rates were significantly decreased and fetuses were not obtained. To examine the effect of 5-aza-dC, 2-cell stage SCNT embryos were cultured with 10 or 100 nM 5-aza-dC for 48 h to the morula stage and transferred. The potential of embryos treated with 5-aza-dC to develop into blastocysts was decreased and no fetuses were obtained after transfer. The findings demonstrated that long-term TSA treatment of SCNT mouse oocytes and treatment with 5-aza-dC inhibit the potential to develop into blastocysts and to fetuses after transfer.
为促进细胞核重编程,体细胞或体细胞经核移植(SCNT)的卵母细胞已用组蛋白脱乙酰酶抑制剂曲古抑菌素A(TSA)或DNA甲基转移酶抑制剂5-氮杂-2'-脱氧胞苷(5-aza-dC)处理,以放松分化体细胞的表观遗传标记。经TSA处理的SCNT卵母细胞发育潜力增加,但最佳处理时期尚不清楚。体细胞中甲基化水平降低对SCNT卵母细胞没有积极影响,但5-aza-dC对SCNT胚胎的处理尚未进行研究。我们研究了TSA处理持续时间对小鼠SCNT卵母细胞发育潜力的影响以及5-aza-dC处理对其体外和体内发育潜力的影响。为确定TSA处理持续时间的影响,将核移植(NT)卵母细胞与100 nM TSA培养0至26小时。用TSA处理8至12小时的SCNT卵母细胞发育至囊胚的比率更高,处理8至12小时后获得了足月胎儿。当卵母细胞处理14小时和26小时时,囊胚率显著降低且未获得胎儿。为研究5-aza-dC的影响,将2细胞期的SCNT胚胎与10或100 nM 5-aza-dC培养48小时至桑椹胚期并进行移植。经5-aza-dC处理的胚胎发育成囊胚的潜力降低,移植后未获得胎儿。研究结果表明,对SCNT小鼠卵母细胞进行长期TSA处理以及用5-aza-dC处理会抑制移植后发育成囊胚和胎儿的潜力。