Department of Theriogenology and Biotechnology, College of Veterinary Medicine, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul 08826, Korea.
Division of Animal & Dairy Science, College of Agriculture and Life Science, Chungnam National University, Daejeon 34134, Korea.
Int J Mol Sci. 2021 Jul 20;22(14):7757. doi: 10.3390/ijms22147757.
Artificial activation of oocytes is an important step for successful parthenogenesis and somatic cell nuclear transfer (SCNT). Here, we investigated the initiation of DNA synthesis and in vivo development of canine PA embryos and cloned embryos produced by treatment with 1.9 mM 6-dimethylaminopurine (6-DMAP) for different lengths of time. For experiments, oocytes for parthenogenesis and SCNT oocytes were cultured for 4 min in 10 μM calcium ionophore, and then divided into 2 groups: (1) culture for 2 h in 6-DMAP (DMAP-2h group); (2) culture for 4 h in DMAP (DMAP-4h group). DNA synthesis was clearly detected in all parthenogenetic (PA) embryos and cloned embryos incorporated BrdU 4 h after activation in DMAP-2h and DMAP-4h groups. In vivo development of canine parthenogenetic fetuses was observed after embryo transfer and the implantation rates of PA embryos in DMAP-2h were 34%, which was significantly higher than those in DMAP-4h (6.5%, < 0.05). However, in SCNT, there was no significant difference in pregnancy rate (DMAP-2h: 41.6% vs. DMAP-4h: 33.3%) and implantation rates (DMAP-2h: 4.94% vs. DMAP-4h: 3.19%) between DMAP-2h and DMAP-4h. In conclusion, the use of DMAP-2h for canine oocyte activation may be ideal for the in vivo development of PA zygotes, but it was not more effective in in vivo development of canine reconstructed SCNT oocytes. The present study demonstrated that DMAP-2h treatment on activation of canine parthenogenesis and SCNT could effectively induce the onset of DNA synthesis during the first cell cycle.
卵母细胞的人工激活是孤雌激活和体细胞核移植（SCNT）成功的重要步骤。在这里，我们研究了不同时间用 1.9mM6-二甲氨基嘌呤（6-DMAP）处理犬 PA 胚胎和克隆胚胎的 DNA 合成起始和体内发育。对于实验，孤雌激活和 SCNT 的卵母细胞分别在 10μM钙离子载体中培养 4 分钟，然后分为 2 组：（1）在 6-DMAP 中培养 2 小时（DMAP-2h 组）；（2）在 DMAP 中培养 4 小时（DMAP-4h 组）。在 DMAP-2h 和 DMAP-4h 组中，激活后 4 小时所有孤雌（PA）胚胎和克隆胚胎均明显检测到 DNA 合成。在胚胎移植后观察到犬孤雌胚胎的体内发育，PA 胚胎在 DMAP-2h 中的植入率为 34%，明显高于 DMAP-4h（6.5%，<0.05）。然而，在 SCNT 中，DMAP-2h 和 DMAP-4h 之间的妊娠率（DMAP-2h：41.6%对 DMAP-4h：33.3%）和植入率（DMAP-2h：4.94%对 DMAP-4h：3.19%）均无显著差异。总之，DMAP-2h 用于犬卵母细胞激活可能是 PA 合子体内发育的理想选择，但在犬重构 SCNT 卵母细胞的体内发育中效果并不理想。本研究表明，DMAP-2h 处理可有效诱导犬孤雌激活和 SCNT 的 DNA 合成起始，启动第一个细胞周期。
