Peroxynitrite participates in mechanisms involved in capacitation of cryopreserved cattle

The effect of peroxynitrite (ONOO-) oil the capacitation rates of cryopreserved bull spermatozoa and the participation of protein kinases in the capacitation process were evaluated. A pool of spermatozoa from five bulls Was incubated in Tyrode's albumin lactate pyruvate (TALP) medium in the presence of heparin 10 IU/ml), sodium nitroprusside (SNP, 50 nM), a nitric oxide donor or 3-morpholinosydnonimine (SIN-1, 1-20 mu M), a ONOO- donor. The participation of ONOO- was evaluated at 15, 30 and 45 min and confirmed by using a specific scavenger, uric acid (2-20 mM). Spermatozoa capacitated with SIN-I were incubated with ovarian follicular fluid of cattle to evaluate their ability to undergo acrosome reaction. The role of ONOO- during capacitation induced by heparin or nitric oxide was evaluated by the addition of uric acid. The participation of protein kinase A (PKA), protein kinase C (PKC) and protein tyrosine kinase (PTK) in capacitation induced by ONOO- was evaluated by incubation with specific inhibitors (50 mu M H-89, 0.1 mu M bisindolylmaleimide 1, and 3 mu W genistein respectively). Capacitation percentages were determined by the fluorescence technique with chlortetracycline (CTC) and true acrosome reaction was determined by trypan blue and Contrast (DIC). SIN-1 concentrations employed had no effect oil progressive motility or sperm viability. Capacitation Values of 10 mu M SIN-1 treatment (23 +/- 2%) were significantly greater with respect to the control (4.6 +/- 1.62%). At 15 min of incubation the greatest capacitation was observed (P < 0.05). reaching a plateau between 15 and 45 min. Follicular induced acrosome reaction in spermatozoa previously capacitated with 10 mu M SIN-1 (P < 0.05). Uric acid prevented SIN-1-induced capacitation and significantly diminished capacitation induced by heparin or SNP. The addition of PKA and PKC inhibitors failed to modify file capacitation induced by SIN-1 (27.4 +/- 3.85 and 24.8 +/- 4.75. respectively,). Genistein. a PTK inhibitor. produced a significant capacitation decrease (8.6 +/- 5.5%). These results indicate that endogenous ONOO- may be generated during heparinor SNP-induced capacitation. Exogenous ONOO- acts as a capacitation inducer and involves file participation of PTK, as part of the intracellular mechanisms that lead to capacitation in cryopreserved bovine spermatozoa. (C) 2007 Elsevier B.V. All rights reserved.