Characterization of reactive oxygen species induced effects on human spermatozoa movement and energy metabolism

Reactive oxygen species (ROS) inhibit sperm movement and have been implicat ed in male infertility. In this study, we determined the effects of specifi c ROS produced by activated leukocytes on human spermatozoa and investigate d their metabolic site of action. We used chemiluminescence and electron pa ramagnetic resonance (EPR) to characterize the ROS generated by both blood and seminal leukocytes. We also determined the effects of these ROS on sper m energy metabolism using biochemical analyses and flow cytometry. Both blo od and seminal leukocytes produced the same characteristic ROS which were d etermined to be hydrogen peroxide (H2O2) and superoxide radicals (O-2(.-)). EPR using the spin trapping technique indicated that superoxide radical-de pendent hydroxyl radicals (HO.) were also generated. ROS generated by PMA-s timulated blood leukocytes (2-5 X 10(6)/ mi) caused inhibition of sperm mov ement in 2 h (p < .01). Using the hypoxanthine/xanthine oxidase (0.5 U/ml) system to generate ROS, we determined that spermatozoa ATP levels, after RO S treatment, were reduced approximately eight-fold in 30 min (0.10 x 10(-10 ) moles/10(6) sperm cells) compared to control (0.84 x 10(-10) moles/10(6) sperm cells) (p < .01). Sperm ATP reduction paralleled the inhibition of sp erm forward progression. Neither superoxide dismutase (100 U/ml) nor dimeth yl sulfoxide (100 mM) reversed these effects; however, protection was obser ved with catalase (4 X 10(3) U/ml). Flow cytometric analyses of sperm treat ed with various doses of H2O2 (0.3 mM-20.0 mM) showed a dose-dependent decr ease in sperm mitochondrial membrane potential (MMP), however, at low conce ntrations of H2O2, sperm MMP was not significantly inhibited. Also, sperm M MP uncoupling with CCCIP had no effect on either sperm ATP levels or forwar d progression. These results indicate that H2O2 is the toxic ROS produced b y activated leukocytes causing the inhibition of both sperm movement and AT P production. O-2(.-) and HO. do not play a significant role in these proce sses. Low concentrations of H2O2 causing complete inhibition of sperm movem ent and ATP levels inhibit sperm energy metabolism at a site independent of mitochondrial oxidative phosphorylation. (C) 1999 Elsevier Science Inc.