Nitric oxide, superoxide anion and hydrogen peroxide production during heparin-induced capacitation cryopreserved bovine spermatozoa

RODRIGUEZ PC; VALDEZ LB,; ZAOBORNYJ T; BOVERIS A; BECONI MT

Santiago, Chile

Congreso; Free Radicals and Antioxidants in Chile 2009: VI Meeting of the Society for Free Radical Biology and Medicine, South American Group; 2009

Society for Free Radical Biology and Medicine, South American Group

The aim of this work was to quantify the endogenous NO, O2- and ONOO- production during heparin-induced capacitation of cryopreserved bovine spermatozoa. Oxygen uptake, NO and O2- production, and capacitation were evaluated at different times in spermatozoa incubated for 45 min at 38¨¬C: a) with heparin (10 UI/ml), b) with heparin (10 UI/ml) + L-NAME (500mM) or DPI (2 ¥ìM) and c) without heparin (control). A hyperbolic increase as a function of time was observed for heparin-dependent capacitation, O2 uptake, and NO production. Conversely, O2- production was increased during the first 15 min of incubation, showing a decrease from this time until 45 min. At 15 min of heparin incubation, a three-fold increase in O2 consumption (5.9 ¡¾ 0.6 nmol/min.107 cells), an enhance in NO release (1.7 ¡¾ 0.4 nmol/min.107 cells), and a five-fold increase in O2- production (1.3 ¡¾ 0.07 nmol/min.107 cells), respect to the non capacitated spermatozoa, were observed. The addition of L-NAME decreased NO production (50%), while the addition of DPI decreased O2- production (80%). Peroxynitrite production at 15 min of sperm capacitation was calculated taking into account NO and O2- generation and the second-order rate constant of the reaction between these species (k1= 1.9 x 1010 M-1 s-1). This value results about 1.7 nmol/min.107 cells, indicating that NO concentration is limiting for ONOO- formation in the incubation medium. To conclude, heparin-induced capacitation of cryopreserved bovine spermatozoa enhances NO and O2- production, generating an increase in ONOO- production.