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 microM), a NO* donor. The participation of NO* was confirmed by the use of scavengers, i.e. methylene blue (50,100 microM) and hemoglobin (20-40 microg/ml). The role of nitric oxide synthase in heparin-induced capacitation was evaluated using enzyme inhibitors Nomega-nitro-L-arginine methyl ester (L-NAME) and Nomega-nitro-L-arginine (L-NA) in concentrations ranging from 1 to 500 microM. 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 microM; bisindolylmaleimide I, 0.1 microM and genistein, 3 microM). The role of hydrogen peroxide or superoxide anion in NO*-induced capacitation was evaluated by incubation with catalase (20-100 microg/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 microM 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.
Rodriguez
P C
PC
Biological Chemistry, School of Veterinary Sciences, University of Buenos Aires, Chorroarín 280, Buenos Aires C1427 CWO, Argentina.
O'Flaherty
C M
CM
Beconi
M T
MT
Beorlegui
N B
NB
D000818:Animals; D002374:Catalase / Q000378:metabolism; D002417:Cattle; D015925:Cryopreservation / Q000662:veterinary*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019833:Genistein / Q000378:metabolism; D006493:Heparin / Q000494:pharmacology; D006706:Homeostasis; D006861:Hydrogen Peroxide / Q000378:metabolism; D008297:Male; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D009569:Nitric Oxide / Q000494:pharmacology*; D020030:Nitric Oxide Donors; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors; D019335:Nitroarginine / Q000494:pharmacology; D009599:Nitroprusside / Q000494:pharmacology; D011493:Protein Kinase C / Q000378:metabolism; D012662:Semen Preservation / Q000662:veterinary*; D013075:Sperm Capacitation / Q000187:drug effects*; D013094:Spermatozoa / Q000502:physiology*
D004791:Enzyme Inhibitors; D020030:Nitric Oxide Donors; D009599:Nitroprusside; D019335:Nitroarginine; D009569:Nitric Oxide; D006493:Heparin; D006861:Hydrogen Peroxide; D019833:Genistein; D002374:Catalase; D019001:Nitric Oxide Synthase; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D019331:NG-Nitroarginine Methyl Ester