猪卵母细胞线粒体分布及线粒体DNA拷贝数变化

摘要

To observe mitochondrial distribution and mitochondrial DNA copy number changes in porcine oocytes at different periods during in vitro maturation. Mito-Tracker Green and real-time Quantitative PCR were used to respectively detect the mitochondrial distribution and mitochondrial DNA copy number in porcine oocytes. The results showed that the mitochondrial distribution was gradually translocated from the cortex to the perinuclear area of oocytes, and then spread in the cytoplasm thoroughly, and mitochondrial clusters became larger, stain became deeper. Mitochondrial DNA copy number of 0, 11, 22 h were (2519. 52±940. 39), (3421. 47±345. 71) ,(9747. 58±1928. 24), respectively, and there was no significant difference (P＞0. 05) among them. Mitochondrial DNA copy number of 33 h was (39913. 61±1180. 26), which was significantly higher than those of 0,11,22 h (P＜0. 05). Mitochondrial DNA copy number of 44 h was (130074. 30±78119. 45), which was significantly higher than that of 33 h (P＜0. 05). In conclusion, as the oocyte maturation process forward, mitochondrial activity enhanced and mitochondrial DNA copy number increased.%本研究旨在观察猪卵母细胞线粒体分布及线粒体DNA拷贝数变化,以期作为判定哺乳动物卵母细胞胞质成熟的指标,同时也为今后克隆技术的发展和相关基因表达调控的研究提供基础.运用线粒体分子探针标记技术检测体外成熟不同时期卵母细胞中线粒体的分布变化,运用实时荧光定量PCR技术检测其线粒体DNA拷贝数的变化趋势,揭示线粒体分布、线粒体DNA拷贝数变化与卵母细胞发育潜能的关系.结果表明,猪卵母细胞成熟前后,线粒体分布由未成熟的周边分布变为成熟后的均匀分布,并且线粒体簇变大,着色变深.卵母细胞成熟0、11、22 h的mtDNA拷贝数分别为(2 519.52士940.39)、(3 421.47士345.71)和(9 747.58士1 928.24),他们之间无显著性差异(P＞0.05).卵母细胞成熟33 h的mtDNA拷贝数为(39 913.61±1 180.26),显著高于成熟0、11和22 h的mtDNA拷贝数(P＜0.05).卵母细胞成熟44 h的mtDNA拷贝数为(130 074.30±78 119.45),显著高于成熟33 h的mtDNA拷贝数(P＜0.05).由此可见,随着卵母细胞成熟进程的推进,线粒体活性增强,线粒体DNA拷贝数明显增加.