Resveratrol attenuates doxorubicin-induced meiotic failure through inhibiting oxidative stress and apoptosis in mouse oocytes

Figures

• 

  Figure 1. Effect of different concentrations of DXR and RES on oocyte meiotic maturation. (A) Microscopy images of oocytes morphologies in control, DXR treatment and RES-supplemented group, oocytes exhibited bigger first polar body (black arrowhead) after DXR treatment. Bar = 200 μm. (B) The oocyte maturation rate was recorded in control and DXR treatment groups. (C) Effect of different concentrations of RES on oocyte maturation in DXR-treated oocytes. The number of oocytes used was shown above the according column. (D) The rate of abnormal meiosis was recorded in control, DXR and RES-supplemented oocytes. All experiments were repeated at least 3 times with more than 30 oocytes examined for each experimental condition. Results were represented as means ± SEMs. * means P < 0.05, **means P < 0.01, *** means P < 0.001.

• 

  Figure 2. Effect of RES treatment on spindle morphology and chromosome alignment in DXR-treated oocytes. Oocytes were immunolabeled with anti-α-tubulin antibody to visualize spindle and counterstained with Hoechst to observe the chromosomes. (A) Representative images of spindle morphologies and chromosome alignment in control, DXR-treated and RES-supplemented oocytes. Green, α-tubulin; blue, DNA. Bar = 20 μm. (B) Representative images of chromosomes alignment in control, DXR-treated and RES-supplemented oocytes. Blue, DNA. Bar = 5 μm. (C) Rate of aberrant spindle was recorded control, DXR-treated and RES-supplemented oocytes. (D) Rate of misaligned chromosomes was recorded in control, DXR-treated and RES-supplemented oocytes. All experiments were repeated at least 3 times with more than 30 oocytes examined in each experimental condition. Results were represented as means ± SEMs. *means P < 0.05, **means P < 0.01.

• 

  Figure 3. Effect of RES treatment on ROS levels in DXR-treated oocytes. (A) Representative images showed ROS levels in control, DXR-treated and RES-supplemented oocytes, Bar = 100 μm. (B) The fluorescence intensity of ROS was measured in control, DXR-treated and RES-supplemented oocytes. *means P < 0.05, **means P < 0.01. (C) Relative expressions of antioxidant genes Cat, Sod1, Sod2 and Gpx3 mRNA levels in oocytes. β-actin was used as a housekeeping gene. *means P < 0.05, **means P < 0.01. All experiments were performed in triplicates and the data were represented as the means ± SEM.

• 

  Figure 4. RES rescued DXR-induced apoptosis through DNA-damage-P63-Caspase3 pathway in mouse oocytes. (A) Representative immunofluorescence images showing the expression of γ-H2AX in mouse oocytes. Green, γ-H2AX, Blue, DNA, Bar = 20 μm. (B) The relative immunofluorescence intensity of γ-H2AX was measured in control, DXR-treated and RES-supplemented oocytes. Experiments were repeated at least 3 times with more than 30 oocytes examined for each group. Data were presented as means ± S.E.M of three independent experiments. **means P < 0.01, *** means P < 0.001. (C) Protein levels of γ-H2AX, P63 and Active-Caspase3 were examined by Western blotting in control, DXR-treated and RES-supplemented oocytes. GAPDH was used as a loading control. The clean backgrounds for the active-Caspase-3, γ-H2AX and GAPDH is due to the exposure.