Figure 4.
Effects of NAC and caspase inhibitors on the developmental status of AOH plus OTA-treated embryos. Mouse blastocysts were pre-incubated with 5 mM NAC, 300 μM Z-IETD-FMK (IETD), 300 μM Z-LEHD-FMK (LEHD) or 300 μM Z-DEVD-FMK (DEVD) for 30 min or left untreated, followed by treatment with or without AOH (0.5, 1 or 2 μM) plus 8 μM OTA or 0.5% DMSO alone (control) for a further 24 h. (A and C) ROS generation was measured by staining with 20 μM H2DCF-DA fluorescence dye. (B and C) The fluorescence intensity of intracellular ROS in each group was quantitatively analyzed using the Image J software. (D) Assessment of apoptosis via TUNEL staining. Mean numbers of apoptotic (TUNEL-positive) cells per blastocyst were calculated. Values are presented as means ± SD of five determinations. Data were obtained from 150 blastocysts per group. Different symbols indicate significant differences at P < 0.05. Scale bar = 20 μm.