Abstract
Nitric oxide (NO·), synthesized from L-arginine by nitric oxide synthase (NOS), is involved in sperm functionality. NOS isoforms have been detected in spermatozoa from different species, and an increment in NOS activity during capacitation has been reported. This work aims to determine the presence and localization of NOS isoforms in ram spermatozoa and analyse their possible changes during in vitro capacitation. Likewise, we investigated the effect of melatonin on the expression and localization of NOS and NO· levels in capacitated ram spermatozoa. Western blot analysis revealed protein bands associated with neuronal NOS (nNOS) and epithelial NOS (eNOS) but not with inducible NOS (iNOS). However, the three isoforms were detected by indirect immunofluorescence (IFI), and their immunotypes varied over in vitro capacitation with cAMP-elevating agents. NO· levels (evaluated by DAF-2-DA/PI staining) increased after in vitro capacitation, and the presence of L-arginine in the capacitating medium raised NO· production and enhanced the acrosome reaction. Incubation in capacitating conditions with a high-cAMP medium with melatonin modified the NOS distribution evaluated by IFI, but no differences in Western blotting were observed. Melatonin did not alter NO· levels in capacitating conditions, so we could infer that its role in ram sperm capacitation would not be mediated through NO· metabolism.
Highlights
Nitric oxide (NO·) is a free radical involved in various physiological processes, acting as a signalling molecule
Western blot analysis revealed protein bands associated with neuronal nitric oxide synthase (NOS) and epithelial NOS but not with inducible NOS
Constitutive isoforms are responsible for physiological NO· levels in somatic cells, whereas inducible NOS is involved in the production of higher levels under intracellular or extracellular inflammatory stimuli [14,15,16]
Summary
Nitric oxide (NO·) is a free radical involved in various physiological processes, acting as a signalling molecule. Nitric oxide synthase (NOS) is the enzyme responsible for NO· production through the conversion of L-arginine to L-citrulline. Three isoforms of this enzyme have been identified to date: type I or neuronal (nNOS), type II or inducible (iNOS) and type III or endothelial (eNOS). Constitutive isoforms are responsible for physiological NO· levels (nM levels) in somatic cells, whereas inducible NOS is involved in the production of higher levels (μM levels) under intracellular or extracellular inflammatory stimuli [14,15,16]
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