Zhang Jian, Zhang Sheng, Wang Yutian, Cheng Hui, Hao Linlin, Zhai Yanhui, Zhang Zhiren, An Xinglan, Ma Xiaoling, Zhang Xueming, Li Ziyi, Tang Bo
College of Veterinary Medicine, Jilin University, Changchun, Jilin, China.
Academy of Translational Medicine, First Hospital, Jilin University, Changchun, Jilin, China.
PLoS One. 2017 Dec 21;12(12):e0189542. doi: 10.1371/journal.pone.0189542. eCollection 2017.
DNA demethylation catalysed by the ten-eleven translocation (TET) protein is an important step during extensive global epigenetic reprogramming in mammals. However, whether TET proteins play a key role in DNA demethylation during the development of bovine pre-implanted embryos is still unclear. In this study, we utilized dimethyloxallyl glycine (DMOG), a small-molecule inhibitor of the TET protein, to impede the enzymatic activity of TET and explore subsequent effects on bovine parthenogenetic embryo development. We first detected the expression of the TET family, consisting of TET1, TET2 and TET3, in bovine MII stage oocytes and found that TET3 is more highly expressed than TET1 and TET2. Treatment with 1 mM DMOG increased 5mC levels (30.4% vs 79.8% at the 8-cell stage for satellite I, 25.3% vs 40.6% at the 8-cell stage for α-satellite, 20.5% vs 73.5% at the blastocyst stage for satellite I and 16.6% vs 30.0% at the blastocyst stage for α-satellite) at every bovine parthenogenetic embryo developmental stage. At the same time, DNA methylation level of satellite DNA and pluripotency gene promoters increased significantly. Real-time PCR analysis results indicated that the transcription levels of NANOG and OCT-4 decreased in the DMOG-treated group. Furthermore, TET inhibition negatively affected blastocyst formation, resulting in a decline in the blastocyst rate (17.1 ± 1.3% vs 24.1 ± 0.6%); however, the percentage of apoptotic cells was significantly increased according to the results of a TUNEL assay. Additionally, expression levels of the apoptosis-related gene BAX were up-regulated, while the expression of BCL-2 was down-regulated. In conclusion, these results support that TET plays important roles in bovine parthenogenetic embryo development by influencing DNA methylation reprogramming, gene expression and apoptosis.
由十一易位（TET）蛋白催化的DNA去甲基化是哺乳动物广泛的全基因组表观遗传重编程过程中的一个重要步骤。然而，TET蛋白在牛植入前胚胎发育过程中的DNA去甲基化中是否起关键作用仍不清楚。在本研究中，我们利用TET蛋白的小分子抑制剂二甲基乙二醛甘氨酸（DMOG）来阻碍TET的酶活性，并探索其对牛孤雌胚胎发育的后续影响。我们首先检测了由TET1、TET2和TET3组成的TET家族在牛MII期卵母细胞中的表达，发现TET3的表达高于TET1和TET2。用1 mM DMOG处理会增加牛孤雌胚胎每个发育阶段的5mC水平（卫星I在8细胞阶段为30.4%对79.8%，α卫星在8细胞阶段为25.3%对40.6%，卫星I在囊胚阶段为20.5%对73.5%，α卫星在囊胚阶段为16.6%对30.0%）。同时，卫星DNA和多能性基因启动子的DNA甲基化水平显著增加。实时PCR分析结果表明，DMOG处理组中NANOG和OCT-4的转录水平下降。此外，TET抑制对囊胚形成产生负面影响，导致囊胚率下降（17.1±1.3%对24.1±0.6%）；然而，根据TUNEL分析结果，凋亡细胞的百分比显著增加。此外，凋亡相关基因BAX的表达水平上调，而BCL-2的表达下调。总之，这些结果支持TET通过影响DNA甲基化重编程、基因表达和凋亡在牛孤雌胚胎发育中发挥重要作用。
