Nitric oxide is produced during heparin-induced capacitation in cryopreserved bovine spermatozoa

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

Montevideo, Uruguay

Congreso; V Meeting of the South American Group of the Society for Free Radical Biology and Medicine; 2007

Reactive oxygen species and reactive nitrogen species are involved in bovine sperm fertilizing processes. Exogenous nitric oxide (NO) induces sperm capacitation. Nitric oxide synthase (NOS) inhibitors or NO scavengers prevent capacitation induced by heparin. The aim of this work was to determine NO production during heparin-induced capacitation in cryopreserved bovine spermatozoa. NO production and capacitation were evaluated at different times in spermatozoa incubated for 45 min at 38oC: a) with heparin (10 UI/ml), b) with heparin (10 UI/ml) + L-NAME (500 mM) and c) without heparin (control). NO release was measured at 38ºC by following the oxidation of oxyhemoglobin to methemoglobin (577-591 nm) in the presence of SOD and catalase. Sperm capacitation was determined by the chlortetracycline assay (CTC). Progressive motility and sperm viability were determined by optic microscopy and eosin/nigrosin technique, respectively. The percentage of capacitation was significantly higher in spermatozoa capacitated with heparin (37.3 ± 2.1%) than the control (6.3 ± 0.6%), and the addition of L-NAME significantly diminished it (8.3 ± 0.6%). Control samples did not show NO release. Spermatozoa capacitated with heparin produced NO (1.6 ± 0.2 pmol/min/107 cells, at 30 min) reaching a plateau between 15 and 45 min of incubation. L-NAME significantly diminished NO production (0.9 ± 0.2 pmol/min/107 cells, at 30 min) and capacitation. Progressive motility and sperm viability were not affected by the different treatments.  Similar increasing patterns were observed in both NO production and capacitation, suggesting that NO plays a pivotal role in this process. Endogenous NO is produced by nitric oxide synthase during heparin-induced capacitation in cryopreserved bovine spermatozoa