Vitrification of Mouse MII Oocyte Decreases the Mitochondrial DNA Copy Number, TFAM Gene Expression and Mitochondrial Enzyme Activity

Abstract

Background: 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.

Methods: 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.

Results: 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).

Conclusion: 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.

Keywords: Cytochrome c oxidase; Reactive oxygen species; Succinate dehydrogenase; mtDNA copy number.

Figure 1.

Representative photomicrographs of cytochrome C oxidase (A and B) and succinate dehydrogenase (C and D) reaction in MII oocytes collected from two groups of study. The cytochrome C oxidase activity is shown as brown color in non-vitrified (A) and vitrified (B) MII oocytes. The succinate dehydrogenase activity is shown as purple color in non-vitrified (C) and vitrified (D) MII oocytes

Figure 2.

Distribution of mitochondria in non-vitrified (A) and vitrified (B) MII oocytes that were stained by Mitotracker green. The mitochondria are shown as green clusters within the ooplasm. The fluorescence intensity of MII oocytes in two groups of study (C). * Significant differences with non-vitrified group (p<0.05)