FIGURE 2.

The naturally occurring lipid aldehydes, 4HNE and acrolein, induce mitochondrial ROS generation in human spermatozoa. A, flow cytometry of the generation of mitochondrial ROS, motility, and vitality of spermatozoa exposed to the naturally occurring electrophile, acrolein, was analyzed. The exposure conditions (2 h at 37 °C) and measurements were identical to those described in Fig. 1. Acrolein stimulated a dose-dependent increase in mitochondrial ROS (MSR), that peaked at 200 μm, coupled with a significant loss of sperm motility (filled bars) at doses that preceded a loss of cell viability (open bars). B, parallel data for 4HNE are presented. C, to confirm the physiological significance of these electrophilic effects, motility, and progressive motility were also examined with lower doses of 4HNE (6.25–50 μm) after 3 h (filled bars) and 24 h (open bars) of exposure, and highly significant inhibitory effects observed that were both time- (p < 0.001) and dose- (p < 0.001) dependent by ANOVA. D, low doses of 4HNE were also found to induce time- (p = 0.011) and dose- (p = 0.003) dependent increases in the percentage of live cells producing mitochondrial ROS, when assessed after 3 h (filled bars) and 24 h (open bars) of exposure. E, physiological significance was also emphasized by the highly significant correlations (p < 0.001) observed between the 4HNE content of human spermatozoa and their spontaneous capacity for generating ROS whether determined by the oxidation of DHE as a measure of overall cellular ROS generation (R² = 0.85, left) or MSR, focusing on the mitochondrial contribution (right, R² = 0.89). Data analyzed by ANOVA and values are presented as means ± S.E. (error bars); ***, p < 0.001; **, p < 0.01; *, p < 0.05 for differences with vehicle control by Fisher's PLSD. Analyses were based on three independent semen samples for acrolein and four independent samples for 4HNE.