Amoushahi Mahboobeh, Salehnia Mojdeh, Mowla Seyed Javad
Department of Anatomy, Tarbiat Modares University, Tehran, Iran.
Department of Biotechnology, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran.
J Reprod Infertil. 2017 Oct-Dec;18(4):343-351.
The objective of this study was determination of the changes in the reactive oxygen species (ROS) level, mitochondrial DNA (mtDNA) copy number and enzyme activity and transcription factor A (TFAM) gene expression in oocytes after vitrification.
The oocytes at metaphase II (MII) stage (n=320) were collected from super-ovulated adult female mice (n=40). These oocytes were divided into vitrified and non-vitrified groups (n=160 in each group). After vitrification of oocytes, ROS level, mtDNA copy number; TFAM gene expression and mitochondrial enzymes activity (cytochrome C oxidase and succinate dehydrogenase) were assessed and compared with non-vitrified group. Visualization of the mitochondria was done using Mitotracker green staining under confocal microscope. Data were compared by independent T-test. Values of p<0.05 were considered as statistically significant.
The survival rate of oocytes after vitrification and warming was 96.05%. The intensity of cytochrome C oxidase activity, mtDNA copy number and TFAM gene expression in non-vitrified oocytes were significantly lower and the level of ROS was higher in vitrified oocytes in comparison with non-vitrified group (p<0.05). But the intensity of succinate dehydrogenase activity was not significantly different between the two groups. The pattern of mitochondrial distribution in two groups of study was similar but the intensity of Mitotracker green in non-vitrified oocytes was significantly higher than vitrified oocytes (p<0.05).
This study showed that vitrification of mouse MII oocytes reduced the mtDNA copy number and mitochondrial cytochrome C oxidase activity by increasing ROS level, thus the subsequent embryo development may be affected.
本研究的目的是确定玻璃化冷冻后卵母细胞中活性氧（ROS）水平、线粒体DNA（mtDNA）拷贝数、酶活性以及转录因子A（TFAM）基因表达的变化。
从超排卵的成年雌性小鼠（n = 40）中收集处于中期II（MII）期的卵母细胞（n = 320）。将这些卵母细胞分为玻璃化冷冻组和非玻璃化冷冻组（每组n = 160）。对卵母细胞进行玻璃化冷冻后，评估ROS水平、mtDNA拷贝数、TFAM基因表达以及线粒体酶活性（细胞色素C氧化酶和琥珀酸脱氢酶），并与非玻璃化冷冻组进行比较。使用共聚焦显微镜下的MitoTracker绿色染色对线粒体进行可视化观察。数据采用独立样本t检验进行比较。p<0.05的值被认为具有统计学意义。
玻璃化冷冻和复温后卵母细胞的存活率为96.05%。与非玻璃化冷冻组相比，非玻璃化冷冻卵母细胞中的细胞色素C氧化酶活性强度、mtDNA拷贝数和TFAM基因表达显著降低，而玻璃化冷冻卵母细胞中的ROS水平较高（p<0.05）。但两组之间琥珀酸脱氢酶活性强度无显著差异。两组研究中线粒体分布模式相似，但非玻璃化冷冻卵母细胞中MitoTracker绿色的强度显著高于玻璃化冷冻卵母细胞（p<0.05）。
本研究表明，小鼠MII期卵母细胞的玻璃化冷冻通过增加ROS水平降低了mtDNA拷贝数和线粒体细胞色素C氧化酶活性，从而可能影响后续胚胎发育。
