FIGURE 5.

Time course analysis of the cascade of events set in motion when human spermatozoa are exposed to lipid aldehydes. A, exposure to physiological amounts of 4HNE (50 and 100 μm) for up to 24 h at 37 °C resulted in a rapid stimulation of mitochondrial ROS (MSR), that was maximal after 4 h but then declined over the ensuing 20 h. Significant changes in mitochondrial membrane potential (MMP) were also evident within 3 h of 4HNE exposure and declined to minimal levels by 24 h. The inhibition of sperm motility with 4HNE paralleled mitochondrial membrane potential disruption, being significant at both 50 and 100 μm within 4 h of 4HNE exposure and reaching undetectable levels within 24 h. The remaining aspects of apoptosis assessed in this study including caspase activation, annexin V binding to the surface of viable cells, and lipid peroxidation, were minimally affected during the first 4 h of 4HNE exposure but then became maximal at 24 h. Open bars, control incubations; filled bars, 4HNE-treated. B, cytochrome c release from the mitochondria was elevated in a dose-dependent manner after a 3-h incubation with 4HNE (25–200 μm). Upper panel shows Western blots whereas lower panel presents densitometric scans from three independent experiments. C, under the conditions of this experiment, DNA damage, whether measured in terms of 8OHdG formation or TUNEL positivity, did not start to increase dramatically until the cells had been exposed to 4HNE for 48–72 h and thus represents an extremely late event in the apoptotic pathway. Open bars, control incubations; filled bars, 4HNE-treated. 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.