Chen Fan, Li Ming-Guo, Hua Zai-Dong, Ren Hong-Yan, Gu Hao, Luo An-Feng, Zhou Chang-Fan, Zhu Zhe, Huang Tao, Bi Yan-Zhen
Hubei Key Laboratory of Animal Embryo Engineering and Molecular Breeding, Institute of Animal Husbandry and Veterinary, Hubei Academy of Agricultural Sciences, Wuhan 430070, China.
College of Animal Science and Technology, Shihezi University, Shihezi 832061, China.
Int J Mol Sci. 2023 Aug 5;24(15):12455. doi: 10.3390/ijms241512455.
The ten-eleven translocation (TET) enzyme family, which includes TET1/2/3, participates in active DNA demethylation in the eukaryotic genome; moreover, TET1/2/3 are functionally redundant in mice embryos. However, the combined effect of TET1/2/3 triple-gene knockdown or knockout on the porcine oocytes or embryos is still unclear. In this study, using Bobcat339, a specific small-molecule inhibitor of the TET family, we explored the effects of TET enzymes on oocyte maturation and early embryogenesis in pigs. Our results revealed that Bobcat339 treatment blocked porcine oocyte maturation and triggered early apoptosis. Furthermore, in the Bobcat339-treated oocytes, spindle architecture and chromosome alignment were disrupted, probably due to the huge loss of 5-hydroxymethylcytosine (5hmC)and concurrent increase in 5-methylcytosine (5mC). After Bobcat339 treatment, early parthenogenetic embryos exhibited abnormal 5mC and 5hmC levels, which resulted in compromised cleavage and blastocyst rate. The mRNA levels of EIF1A and DPPA2 (ZGA marker genes) were significantly decreased, which may explain why the embryos were arrested at the 4-cell stage after Bobcat339 treatment. In addition, the mRNA levels of pluripotency-related genes OCT4 and NANOG were declined after Bobcat339 treatment. RNA sequencing analysis revealed differentially expressed genes in Bobcat339-treated embryos at the 4-cell stage, which were significantly enriched in cell proliferation, cell component related to mitochondrion, and cell adhesion molecule binding. Our results indicated that TET proteins are essential for porcine oocyte maturation and early embryogenesis, and they act by mediating 5mC/5hmC levels and gene transcription.
包括TET1/2/3的10-11易位（TET）酶家族参与真核基因组中的主动DNA去甲基化；此外，TET1/2/3在小鼠胚胎中功能冗余。然而，TET1/2/3三基因敲低或敲除对猪卵母细胞或胚胎的联合作用仍不清楚。在本研究中，我们使用TET家族的特异性小分子抑制剂Bobcat339，探索了TET酶对猪卵母细胞成熟和早期胚胎发育的影响。我们的结果表明，Bobcat339处理可阻断猪卵母细胞成熟并引发早期凋亡。此外，在经Bobcat339处理的卵母细胞中，纺锤体结构和染色体排列被破坏，这可能是由于5-羟甲基胞嘧啶（5hmC）大量丢失以及5-甲基胞嘧啶（5mC）同时增加所致。Bobcat339处理后，早期孤雌胚胎表现出异常的5mC和5hmC水平，这导致卵裂率和囊胚率受损。EIF1A和DPPA2（合子基因组激活标记基因）的mRNA水平显著降低，这可能解释了为什么Bobcat339处理后的胚胎停滞在4细胞阶段。此外，多能性相关基因OCT4和NANOG的mRNA水平在Bobcat339处理后下降。RNA测序分析揭示了在4细胞阶段经Bobcat339处理的胚胎中差异表达的基因，这些基因在细胞增殖、与线粒体相关的细胞成分以及细胞粘附分子结合方面显著富集。我们的结果表明，TET蛋白对猪卵母细胞成熟和早期胚胎发育至关重要，它们通过介导5mC/5hmC水平和基因转录发挥作用。
