Protective effect of antioxidants on the pre-maturation aging of mouse oocytes

Abstract

Pre-maturation aging of immature oocytes may adversely affect the fate of an oocyte. Oxidative stress is one of the most detrimental factors affecting oocyte developmental competence and maturation during aging. In this study, experiments were designed to examine whether supplementation of antioxidants in a culture medium could protect immature mouse oocytes from damages caused by oxidative stress. Mouse oocytes at germinal vesicle stage were prevented from meiosis resumption and cultured in a medium with or without antioxidants for 12-36 h to allow oocytes to undergo aging. After aging, oocytes were cultured for maturation. Nuclear maturation, mitochondria activity, spindle morphology and DNA integrity were examined after maturation. It was found that antioxidants had protective effects on the oocytes in terms of nuclear maturation, functional mitochondria, spindle morphology and DNA integrity. As aging time was prolonged from 12 to 36 h, the protective effect of antioxidants became more obvious. However, as compared with oocytes without aging, it was found that aging significantly inhibited nuclear maturation, impaired mitochondria function, and damaged the spindle and DNA. These results indicate that pre-maturation aging is detrimental to oocytes' competence to undergo maturation and other cellular activities, and antioxidants can protect oocytes from damages caused by aging.

Figure 1

Effects of antioxidants on oocyte maturation after pre-maturation aging and IVM. Oocytes at GV stage were directly cultured (Fresh) or pre-maturation aging for 12 (a) 24 (b) and 36 h (c) in a medium supplemented with (AO) or without (control) antioxidants before IVM. *P < 0.05; **P < 0.01; ***P < 0.001. No. of oocytes at each group was shown in the bracket. AO: antioxidants.

Figure 2

Effects of antioxidants on mitochondrial distribution in oocytes at MII stage after pre-maturation aging and IVM. Mitochondrial distribution patterns in oocytes were detected by fluorescence microscopy using MitoTracker Red. (a) Shows polarized distribution. (b) Shows homogeneous distribution. Both a and b were considered as normal mitochondria distribution patterns. (c) Shows large heterogeneous clump distribution, which was considered as abnormal mitochondria distribution pattern. Bar = 10 µm. (d) Shows proportions of oocytes with abnormal mitochondria distribution after 12 h pre-maturation aging and IVM. (e) Shows proportions of oocytes with abnormal mitochondria distribution after 24 h pre-maturation aging and IVM. (f) Shows proportions of oocytes with abnormal mitochondria distribution after 36 h pre-maturation aging and IVM. *P < 0.05; **P < 0.01; ***P < 0.001. No. of oocytes at each group was shown in the bracket. AO: antioxidants.

Figure 3

Effects of antioxidants on spindle morphology and chromosome alignment in oocytes at MII stage after pre-maturation aging and IVM. (a) Shows an oocyte with a normal spindle and chromosome alignment. (b) Shows an oocyte with an abnormal spindle and chromosome alignment. Green: a-Tubulin; Red: chromosomes. (Bar = 10 µm). (c) Shows percentages of oocytes with abnormal spindle and/or misaligned chromosomes in oocytes after 12 h pre-maturation aging and IVM. (d) Shows percentages of oocytes with abnormal spindle and/or misaligned chromosomes in oocytes after 24 h pre-maturation aging and IVM. (e) Shows percentages of oocytes with abnormal spindle and/or misaligned chromosomes in oocytes after 36 h pre-maturation aging and IVM. *P < 0.05; **P < 0.01; ***P < 0.001. No. of oocytes at each group was shown in the bracket. AO: antioxidants.

Figure 4

Effects of antioxidants on DNA integrity in the oocytes at GV stage after pre-maturation aging and IVM. Fluorescence images show DNA and γ-H2AX in oocytes at GV stage after pre-maturation aging and IVM. (a) Shows an oocyte with slight DNA damage. (b) Shows an oocyte with moderate DNA damage. (c) Shows an oocyte with severe DNA damage. Red: γ-H2AX; Blue: DNA. (Bar = 10 mm). (d) Shows levels of fluorescence intensity of γ-H2AX in oocytes after 12 h pre-maturation aging and IVM. (e) Shows levels of fluorescence intensity of γ-H2AX in oocytes after 24 h pre-maturation aging and IVM. (f) Shows levels of fluorescence intensity of γ-H2AX in oocytes after 36 h pre-maturation aging and IVM. *P < 0.05; **P < 0.01; ***P < 0.001. No. of oocytes at each group was shown in the bracket. AO: antioxidants.