Peroxynitrite participates in mechanisms involved in capacitation of cryopreserved cattle

Abstract

The effect of peroxynitrite (ONOO −) on 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 μM), 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-1 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 μM H-89, 0.1 μM bisindolylmaleimide I, and 3 μM genistein, respectively). Capacitation percentages were determined by the fluorescence technique with chlortetracycline (CTC) and true acrosome reaction was determined by trypan blue and Differential-Interferential Contrast (DIC). SIN-1 concentrations employed had no effect on progressive motility or sperm viability. Capacitation values of 10 μM 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 fluid induced acrosome reaction in spermatozoa previously capacitated with 10 μM 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 the 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 heparin- or SNP-induced capacitation. Exogenous ONOO − acts as a capacitation inducer and involves the participation of PTK, as part of the intracellular mechanisms that lead to capacitation in cryopreserved bovine spermatozoa.