Figure 1. Apoptosis and sTNF-α release during culture of CCs. CCs recovered from newly ovulated oocytes were cultured in CZB medium alone or with 200-µM H2O2 (+). At different times of the culture, rates of apoptotic cells were observed after Hoechst or annexin-V staining, and concentrations of sTNF-α in CM were measured by ELISA. Both the Hoechst and annexin-stained smears were observed under a fluorescence microscope (Original magnification ×400). Micrographs (A), (B) and (C) show Hoechst-stained smears of CCs cultured for 0 h or 24 h in CZB alone or CZB with H2O2, showing approximately 6%, 54%, and 85% apoptotic cells, respectively. Micrographs (F) and (G) show Hoechst and annexin-V double-stained smears of CCs cultured for 0 and 24 h, respectively, in CZB with H2O2, showing approximately 13% and 94% apoptotic cells, respectively. The photos are merged images with Hoechst and annexin-V colored blue and green, respectively. Graphs (D) and (H) show percentages of apoptotic CCs revealed by Hoechst and annexin-V staining, respectively. Each treatment was repeated 3-4 times with each replicate containing CCs from about 60-90 freshly recovered (0 h) COCs or from one well of cultured cells. Graph E shows levels of sTNF-α in CM, and each treatment was repeated 3 times with each replicate including 100 µl of CM from one culture well. a–e: Values with a different letter above bars differ significantly (P < 0.05).