Hua Song, Lu Chenglong, Song Yakun, Li Ruizhe, Liu Xu, Quan Fusheng, Wang Yongsheng, Liu Jun, Su Feng, Zhang Yong
College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi Province 712100, People's Republic of China.
Reprod Fertil Dev. 2012;24(3):501-9. doi: 10.1071/RD11091.
To investigate the effect of mitochondrial heteroplasmy on embryo development, cloned embryos produced using bovine oocytes as the recipient cytoplasm and ovine granulosa cells as the donor nuclei were complemented with 2pL mitochondrial suspension isolated from ovine (BOOMT embryos) or bovine (BOBMT embryos) granulosa cells; cloned embryos without mitochondrial injection served as the control group (BO embryos). Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and sodium bisulfite genomic sequencing were used to analyse mRNA and methylation levels of pluripotency genes (OCT4, SOX2) and mitochondrial genes (TFAM, POLRMT) in the early developmental stages of cloned embryos. The number of mitochondrial DNA copies in 2pL ovine-derived and bovine-derived mitochondrial suspensions was 960±110 and 1000±120, respectively. The blastocyst formation rates were similar in BOBMT and BO embryos (P>0.05), but significantly higher than in BOOMT embryos (P<0.01). Expression of OCT4 and SOX2, as detected by RT-qPCR, decreased significantly in BOOMT embryos (P<0.05), whereas the expression of TFAM and POLRMT increased significantly, compared with expression in BOOMT and BO embryos (P<0.05). In addition, methylation levels of OCT4 and SOX2 were significantly greater (P<0.05), whereas those of TFAM and POLRMT were significantly lower (P<0.01), in BOOMT embryos compared with BOBMT and BO embryos. Together, the results of the present study suggest that the degree of mitochondrial heteroplasmy may affect embryonic development.
为研究线粒体异质性对胚胎发育的影响，以牛卵母细胞作为受体细胞质、绵羊颗粒细胞作为供体细胞核生产的克隆胚胎，分别用从绵羊颗粒细胞（BOOMT胚胎）或牛颗粒细胞（BOBMT胚胎）中分离出的2μL线粒体悬液进行补充；未进行线粒体注射的克隆胚胎作为对照组（BO胚胎）。采用逆转录定量聚合酶链反应（RT-qPCR）和亚硫酸氢盐基因组测序分析克隆胚胎早期发育阶段多能性基因（OCT4、SOX2）和线粒体基因（TFAM、POLRMT）的mRNA及甲基化水平。2μL绵羊来源和牛来源的线粒体悬液中线粒体DNA拷贝数分别为960±110和1000±120。BOBMT胚胎和BO胚胎的囊胚形成率相似（P>0.05），但显著高于BOOMT胚胎（P<0.01）。RT-qPCR检测显示，与BOOMT胚胎和BO胚胎相比，BOOMT胚胎中OCT4和SOX2的表达显著降低（P<0.05），而TFAM和POLRMT的表达显著升高（P<0.05）。此外，与BOBMT胚胎和BO胚胎相比，BOOMT胚胎中OCT4和SOX2的甲基化水平显著更高（P<0.05），而TFAM和POLRMT的甲基化水平显著更低（P<0.01）。本研究结果表明，线粒体异质性程度可能影响胚胎发育。
