Does Melatonin Exert Its Effect on Ram Sperm Capacitation Through Nitric Oxide Synthase Regulation?

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...

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Bibliographic Details
Published in:International journal of molecular sciences 2020-03, Vol.21 (6), p.2093
Main Authors: Miguel-Jiménez, Sara, Carvajal-Serna, Melissa, Calvo, Silvia, Casao, Adriana, Cebrián-Pérez, José Álvaro, Muiño-Blanco, Teresa, Pérez-Pe, Rosaura
Format: Article
Language:English
Subjects:
Acrosome reaction
Arginine
Capacitation
Cyclic AMP
Enzymes
Immunofluorescence
Isoforms
Labeling
Localization
Melatonin
Metabolism
Motility
Nitric oxide
Nitric-oxide synthase
Physiology
ram spermatozoa
Sperm
Spermatozoa
Western blotting
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Summary: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.
ISSN:1422-0067
1661-6596
1422-0067
DOI:10.3390/ijms21062093