Peroxynitrite participates in mechanisms involved in capacitation of cryopreserved cattle.
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 microM), 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 microM H-89, 0.1 microM bisindolylmaleimide I, and 3 microM 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 microM 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 microM 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.
Rodriguez
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Biological Chemistry, School of Veterinary Science, University of Buenos Aires, Chorroarín 280, C1427 CWO Buenos Aires, Argentina. prodriguez@fvet.uba.ar
Beconi
M T
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D020101:Acrosome Reaction / Q000187:drug effects; D000818:Animals; D002417:Cattle / Q000502:physiology*; D015925:Cryopreservation / Q000379:methods / Q000662:veterinary*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D005260:Female; D015571:Follicular Fluid / Q000378:metabolism; D019833:Genistein / Q000494:pharmacology; D006493:Heparin / Q000494:pharmacology*; D007211:Indoles / Q000494:pharmacology; D008297:Male; D008301:Maleimides / Q000494:pharmacology; D008981:Molsidomine / Q000031:analogs & derivatives / Q000494:pharmacology; D030421:Peroxynitrous Acid / Q000378:metabolism*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D012662:Semen Preservation / Q000379:methods / Q000662:veterinary*; D013075:Sperm Capacitation / Q000187:drug effects*; D013094:Spermatozoa / Q000187:drug effects* / Q000201:enzymology
D007211:Indoles; D008301:Maleimides; D047428:Protein Kinase Inhibitors; D030421:Peroxynitrous Acid; C002385:linsidomine; D006493:Heparin; D008981:Molsidomine; D019833:Genistein; D011505:Protein-Tyrosine Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; C088060:bisindolylmaleimide