Abstract
Reactive oxygen species (ROS) play an essential role in mammalian sperm capacitation. NADPH oxidase 5 (NOX5) has been described as the main source of ROS production in some mammalian spermatozoa, such as human and equine. On the other hand, melatonin can decrease cellular ROS levels and regulates NOX activity in somatic cells. Therefore, the objectives of this work were (1) to identify NOX5 in ram spermatozoa and analyze its possible changes during in vitro capacitation and (2) to investigate the effect of melatonin on NOX5 expression and localization and on superoxide levels in capacitated ram spermatozoa. Protein bands associated with NOX5 were detected by Western blot analysis. Likewise, indirect immunofluorescence (IIF) revealed six different immunotypes for NOX5, which varied throughout in vitro capacitation. Superoxide (O2⋅–), evaluated by DHE/Yo-Pro-1, rose after in vitro capacitation and in the presence of the calcium ionophore A23187 but decreased in the presence of the NOX inhibitor GKT136901. GKT also reduced the percentage of capacitated and acrosome-reacted spermatozoa that had increased during incubation in capacitating conditions. The presence of melatonin at micromolar concentrations avoided the increment in O2⋅– and the changes in NOX5 immunotypes provoked by capacitation. In conclusion, NOX5 is present in ram spermatozoa and the changes in its distribution, associated with sperm capacitation, can be prevented by melatonin. To this extent, it could imply that melatonin exerts its antioxidant role, at least in part, by modulating NOX5 activity during ram sperm capacitation.
Highlights
Reactive oxygen species (ROS), such as superoxide anion (O2·−), are involved in sperm functionality under physiological conditions
Immunofluorescence analysis revealed the presence of the NADPH oxidase 5 (NOX5) enzyme in ram spermatozoa, and six different immunotypes were distinguished (Figure 2): immunotype 1, with labeling only at the midpiece; immunotype 2, with labeled acrosomal region; immunotype 3, labeling the apical edge; immunotype 4, labeling the apical edge and postacrosomal region; immunotype 5, labeling acrosomal and postacrosomal regions; and immunotype 6, with postacrosomal labeling
It is commonly assumed that the generation of ROS in spermatozoa would be mediated by autonomous NADPH oxidase activity and that certain levels of ROS would be necessary for some physiological events
Summary
Reactive oxygen species (ROS), such as superoxide anion (O2·−), are involved in sperm functionality under physiological conditions. The enzymes responsible for this superoxide production are the NADPH oxidases (NOX) (Bedard and Krause, 2007; Maghzal et al, 2012) and, the NOX5 in mammal spermatozoa (Ghanbari et al, 2018). This isoform has some characteristics that differentiate it from other NOX family members (reviewed by Touyz et al, 2019). There is no NOX5 gene or protein in murine spermatozoa, which indicates that, at least in this species, another source must be involved in the superoxide generation (Vernet et al, 2001), such as mitochondria (Koppers et al, 2008) or inflammatory leukocytes (Whittington et al, 1999)
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