Role of nitric oxide on quality of freshly ejaculated bull spermatozoa during heparin-induced in vitro capacitation.
The objective of this study was to assess the effects of nitric oxide (NO) on heparin-induced capacitation in vitro of fresh bull sperm, through the addition of Nomega-nitro-l-arginine methyl ester (L-NAME, a NO-synthesis inhibitor) and l-arginine (L-Arg, a NO-synthesis precursor) to the capacitation medium. In Experiment 1, different concentrations of L-NAME (0.1, 1, 10mM) and of L-Arg (10mM) were added to the capacitation medium. Sperm motility and vigor were subjectively appraised using direct light microscopy; sperm membrane integrity was examined using a 2% Trypan blue solution while the concentration of nitrate/nitrite (NO(3)(-)/NO(2)(-)) was determined by using the Griess method over a 5h capacitation period. The addition of 10mM L-NAME has inhibited NO synthesis, sperm progressive motility, sperm vigor and sperm membrane integrity (P<0.05) as compared to control. The addition of 10mM L-Arg to the capacitation medium increased all variables evaluated in comparison to the control (P<0.05). In Experiment 2, mitochondrial activity was assessed through the MTT test (3-(4,5-dimetylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), and sperm capacitation was assessed through the test of penetration in homologous oocytes after addition of the 10mM L-NAME, and of the 10mM L-Arg. The addition of 10mM L-NAME caused mitochondrial activity (40%) and the percentage of oocytes penetrated (77%) to decrease in relation to the control (P<0.05). After addition of 0.6mM L-Arg+10mM L-NAME, partial reversal of mitochondrial activity did occur (only 20%). The addition of 10mM L-Arg increased the percentage of oocytes penetrated as compared to control (21%) (P<0.05). These results indicate that: (1) NO is involved in control of progressive sperm motility, vigor, membrane integrity, and mitochondrial activity along the period of heparin-induced capacitation of fresh bovine sperm via NOS/NO; (2) adequate L-Arg/NO concentrations into the capacitation medium can potentiate heparin action or act independently for increasing the number or the quality of capacitated sperm.
Leal
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Centro de Ciências e Tecnologias Agropecuárias, Universidade Estadual do Norte Fluminense Darcy Ribeiro, Parque Califórnia, Campos dos Goytacazes, RJ, Brazil.
Caldas-Bussiere
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MC
Carvalho
C S Paes de
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Viana
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Quirino
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CR
D000818:Animals; D001120:Arginine / Q000494:pharmacology; D002417:Cattle; D002462:Cell Membrane / Q000187:drug effects / Q000648:ultrastructure; D004542:Ejaculation; D005260:Female; D006493:Heparin / Q000494:pharmacology*; D008297:Male; D008928:Mitochondria / Q000187:drug effects / Q000502:physiology; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D009569:Nitric Oxide / Q000494:pharmacology*; D009865:Oocytes / Q000502:physiology*; D012661:Semen / Q000187:drug effects / Q000502:physiology; D013075:Sperm Capacitation / Q000187:drug effects*; D013084:Sperm-Ovum Interactions / Q000187:drug effects / Q000502:physiology; D013094:Spermatozoa / Q000187:drug effects*
D009569:Nitric Oxide; D006493:Heparin; D001120:Arginine; D019331:NG-Nitroarginine Methyl Ester