PEROXIREDOXINS ARE ESSENTIAL IN MAINTAINING HUMAN SPERM VIABILITY AND DNA INTEGRITY

MARÍA C. FERNÁNDEZ; ADEL R. MOAWAD; CRISTIAN O´FLAHERTY

Encuentro; 27TH ANNUAL McGILL UROLOGY RESEARCH DAY; 2017

Introduction and aim: Our lab has been pioneer to show that a family of antioxidant called peroxiredoxins (PRDXs) has a major role in the protection against oxidative stress and regulation of capacitation in spermatozoa. In fact, we reported that 60-90% of infertile men have sperm with inactive PRDXs. However, how spermatozoa maintain PRDXs active remains unknown. Here, we aimed to determine the impact of different reactive oxygen species (ROS) levels produced by inhibiting specific players of the PRDX system on sperm viability and DNA oxidation. Experimental Approach: Highly motile spermatozoa from healthy donors were incubated with thiostrepton (TSP) or conoidin A (Con-A), both inhibitors of 2-Cys PRDXs, and MJ33 (inhibitor of PRDX6 calcium independent phospholipase A2 (PRDX6-PLA2) activity) for 2h at 37°C. We further inhibited players of the PRDX re-activation system using auranofin, ezatiostat and hexylglutathione, inhibitors of thioredoxin (TRX) reductase, of glutathione-S-transferase (GST) pi and mtGST1, respectively and with ethacrynic acid (GSH adduct). Also, NADPH donors of the TRX system were inhibited with dehydroepiandrosterone or sodium oxalomalate (inhibitors of glucose-6-P-dehydrogenase and NADP-isocitrate dehydrogenase, respectively). Sperm viability was assessed using Sytox blue-calcein labeling, ROS levels (using fluorescent probes) and 8-deoxyguanosine (8-OHdG) labeling (using an anti-8-OHdG antibody) by flow cytometry.Results: We observed a significant decrease in viable cells when incubated with the highest concentration of TSP and a dose dependent decrease for Con-A and MJ33, Auranofin, ezatiostat and ethacrynic acid compared to controls (p