FIGURE 3.

Dose- and time-dependent effects of H₂O₂ on superoxide generation by human spermatozoa. A, superoxide produced by spermatozoa from two different donors with or without 100 μm H₂O₂ was measured over the indicated time course. These results are representative of the range of superoxide responses observed among a total of 94 sperm donors. B, superoxide production was measured in sperm cells stimulated for 10 min with various concentrations of H₂O₂ (0–500 μm). C, superoxide production stimulated with 100 μm H₂O₂ was determined in sperm cells preincubated with BAPTA (100 μm), DPI (10 μm), SOD (400 units/ml), or GKT136901 (0.5 μm). Superoxide production was determined as described in the legend to Fig. 2 and expressed as a percent of non-treated cells (NT). The data expressed in panels B and C are the mean ± S.E. of 6–10 independent experiments. Asterisks indicate statistical significance versus control cells without H₂O₂. *, p < 0.05; **, p < 0.01; ***, p < 0.001, and number signs indicate statistical significance versus cells stimulated with H₂O₂. #, p < 0.05; ##, p < 0.01; ###, p < 0.001. D, confocal images of spermatozoa incubated for 10 min without (NT) or with H₂O₂ were recorded using a Zeiss microscope. Each set of four photomicrograph panels corresponds to superoxide production (red fluorescence, top left), Ca²⁺ influx (green fluorescence, top right), the differential interference contrast (DIC) image (bottom left), and a merge picture of the two fluorescent images (bottom right). The scale bar indicates a size of 10 μm. DHE, dihydroethidine.