Nitric oxide-induced capacitation of cryopreserved bull spermatozoa and assessment of participating regulatory pathways

The effect of nitric oxide (NO) 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 four bulls were incubated in TALP medium in the presence of heparin (10 IU/ml) or sodium nitroprusside (SNP, 0.05-100 μM), a NO donor. The participation of NO was confirmed by the use of scavengers, i.e. methylene blue (50 100 μM) and hemoglobin (20-40 μg/ml). The role of nitric oxide synthase in heparin-induced capacitation was evaluated using enzyme inhibitors Nω-nitro-l-arginine methyl ester (l-NAME) and Nω-nitro-l-arginine (l-NA) in concentrations ranging from 1 to 500 μM. The effects of protein kinase A (PKA), protein kinase C (PKC) and protein tyrosine kinase (PTK), on NO-induced capacitation were evaluated by incubation with specific inhibitors of these enzymes (H-89, 50 μM; bisindolylmaleimide I, 0.1 μM and genistein, 3 μM). The role of hydrogen peroxide or superoxide anion in NO-induced capacitation was evaluated by incubation with catalase (20-100 μg/ml) or superoxide dismutase (SOD, 0.05-0.5 mg/ml), respectively. Capacitation percentages were determined by the fluorescence technique with chlortetracycline (CTC). SNP concentrations employed had no effect on progressive motility or sperm viability. Capacitation values of the 0.05 μM SNP treatment (31 ± 5.15%) were similar to those of heparin treated samples (33 ± 4.27%). Inhibitors of nitric oxide synthase (NOS) diminished capacitation percentages in a dose-dependent manner as did the addition of NO scavengers (P < 0.05). The presence of PKA, PKC and PTK inhibitors likewise decreased capacitation percentages (6.25 ± 0.71, 12.75 ± 1.41, 9.00 ± 1.41%, respectively). The presence of catalase or SOD in the incubation medium had no effect on capacitation percentages. These results indicate that NO may be generated by a sperm NOS during heparin-induced capacitation and that exogenous NO acts as a capacitation inducer and involves the participation of PKA, PKC and PTK as part of the intracellular mechanisms that lead to capacitation in cryopreserved bull spermatozoa.