Inhibition of Sirt1/Sod2 pathway abolishes the melatonin-mediated improvement of aged oocyte quality. (A) Representative images of spindle morphologies and chromosome alignment in aged, melatonin-administered and melatonin-administered + EX527-treated oocytes. Metaphase I oocytes were immunostained with α-tubulin-FITC antibody to display the spindles and counterstained with Hoechst to display the chromosomes. Scale bar, 3 μm. (B) The rate of aberrant spindle with misaligned chromosome was recorded in aged (n = 32), melatonin-administered (n = 39) and melatonin-administered + EX527-treated (n = 34) oocytes. (C) Representative images of euploid and aneuploid MII eggs. Chromosome spreading was performed to count the number of chromosomes in aged, melatonin-administered and melatonin-administered + EX527-treated oocytes. Scale bar, 5 μm. (D) The rate of aneuploid eggs was recorded in aged (n = 30), melatonin-administered (n = 31) and melatonin-administered + EX527-treated (n = 26) oocytes. Aging: oocytes from aged mice administered with PBS; Mel: oocytes from aged mice administered with melatonin; Mel + EX527: oocytes from aged mice administered with melatonin were treated by EX527. Data were presented as mean percentage (mean ± SEM) of at least three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.