Huan Yan Jun, Zhu Jiang, Xie Bing Teng, Wang Jian Yu, Liu Shi Chao, Zhou Yang, Kong Qing Ran, He Hong Bin, Liu Zhong Hua
College of Life Science, Northeast Agricultural University, Haerbin 150030, China.
J Reprod Dev. 2013 Oct;59(5):442-9. doi: 10.1262/jrd.2013-026. Epub 2013 Jun 10.
The efficiency of cloning by somatic cell nuclear transfer (SCNT) has remained low. In most cloned embryos, epigenetic reprogramming is incomplete, and usually the genome is hypermethylated. The DNA methylation inhibitor 5-aza-2'-deoxycytidine (5-aza-dC) could improve the developmental competence of cow, pig, cat and human SCNT embryos in previous studies. However, the parameters of 5-aza-dC treatment among species are different, and whether 5-aza-dC could enhance the developmental competence of porcine cloned embryos has still not been well studied. Therefore, in this study, we treated porcine fetal fibroblasts (PFF) that then were used as donor nuclei for nuclear transfer or fibroblast-derived reconstructed embryos with 5-aza-dC, and the concentration- and time-dependent effects of 5-aza-dC on porcine cloned embryos were investigated by assessing pseudo-pronucleus formation, developmental potential and pluripotent gene expression of these reconstructed embryos. Our results showed that 5-aza-dC significantly reduced the DNA methylation level in PFF (0 nM vs. 10 nM vs. 25 nM vs. 50 nM, 58.70% vs. 37.37% vs. 45.43% vs. 39.53%, P<0.05), but did not improve the blastocyst rate of cloned embryos derived from these cells. Treating cloned embryos with 25 nM 5-aza-dC for 24 h significantly enhanced the blastocyst rate compared with that of the untreated group. Furthermore, treating cloned embryos, but not donor cells, significantly promoted pseudo-pronucleus formation at 4 h post activation (51% for cloned embryos treated, 34% for donor cells treated and 36% for control, respectively, P<0.05) and enhanced the expression levels of pluripotent genes (Oct4, Nanog and Sox2) up to those of in vitro fertilized embryos during embryo development. In conclusion, treating cloned embryos, but not donor cells, with 5-aza-dC enhanced the developmental competence of porcine cloned embryos by promotion of pseudo-pronucleus formation and improvement of pluripotent gene expression.
通过体细胞核移植（SCNT）进行克隆的效率一直很低。在大多数克隆胚胎中，表观遗传重编程并不完全，而且基因组通常处于高甲基化状态。在先前的研究中，DNA甲基化抑制剂5-氮杂-2'-脱氧胞苷（5-aza-dC）可以提高牛、猪、猫和人类SCNT胚胎的发育能力。然而，不同物种间5-aza-dC处理的参数有所不同，并且5-aza-dC是否能提高猪克隆胚胎的发育能力仍未得到充分研究。因此，在本研究中，我们用5-aza-dC处理猪胎儿成纤维细胞（PFF），然后将其用作核移植的供体细胞核或用于成纤维细胞来源的重构胚胎，并通过评估这些重构胚胎的假原核形成、发育潜力和多能基因表达，研究5-aza-dC对猪克隆胚胎的浓度和时间依赖性影响。我们的结果表明，5-aza-dC显著降低了PFF中的DNA甲基化水平（0 nM对10 nM对25 nM对50 nM，分别为58.70%对37.37%对45.43%对39.53%，P<0.05），但并未提高源自这些细胞的克隆胚胎的囊胚率。与未处理组相比，用25 nM 5-aza-dC处理克隆胚胎24小时显著提高了囊胚率。此外，处理克隆胚胎而非供体细胞，在激活后4小时显著促进了假原核形成（处理的克隆胚胎为51%，处理的供体细胞为34%，对照组为36%，P<0.05），并在胚胎发育过程中将多能基因（Oct4、Nanog和Sox2）的表达水平提高到体外受精胚胎的水平。总之，用5-aza-dC处理克隆胚胎而非供体细胞，通过促进假原核形成和改善多能基因表达，提高了猪克隆胚胎的发育能力。
