Abstract
Physiological and pathological processes in spermatozoa involve the production of reactive oxygen species (ROS), but the identity of the ROS-producing enzyme system(s) remains a matter of speculation. We provide the first evidence that NOX5 NADPH oxidase is expressed and functions in human spermatozoa. Immunofluorescence microscopy detected NOX5 protein in both the flagella/neck region and the acrosome. Functionally, spermatozoa exposed to calcium ionophore, phorbol ester, or H(2)O(2) exhibited superoxide anion production, which was blocked by addition of superoxide dismutase, a Ca(2+) chelator, or inhibitors of either flavoprotein oxidases (diphenylene iododonium) or NOX enzymes (GKT136901). Consistent with our previous overexpression studies, we found that H(2)O(2)-induced superoxide production by primary sperm cells was mediated by the non-receptor tyrosine kinase c-Abl. Moreover, the H(V)1 proton channel, which was recently implicated in spermatozoa motility, was required for optimal superoxide production by spermatozoa. Immunoprecipitation experiments suggested an interaction among NOX5, c-Abl, and H(V)1. H(2)O(2) treatment increased the proportion of motile sperm in a NOX5-dependent manner. Statistical analyses showed a pH-dependent correlation between superoxide production and enhanced sperm motility. Collectively, our findings show that NOX5 is a major source of ROS in human spermatozoa and indicate a role for NOX5-dependent ROS generation in human spermatozoa motility.
Highlights
The identity of the reactive oxygen species (ROS)-producing enzyme(s) in human spermatozoa remains uncertain
Immunoblot analysis of sperm lysates using NOX1 or NOX4 antibody resulted in no detectable signals, whereas appropriate immunoreactive bands were detected in lysates of CaCo2 and kidney cells (Fig. 1A), which are known to express NOX1 and NOX4, respectively
Because we have shown that superoxide production by phagocyte NOX2 is regulated by H2O2, we ensured that leukocytes did not account for the superoxide signal stimulated by H2O2 in sperm samples by discarding all semen samples in which leukocytes were evident by microscopic observation
Summary
The identity of the reactive oxygen species (ROS)-producing enzyme(s) in human spermatozoa remains uncertain. Generation of ROS is required for sperm capacitation, the final maturation steps associated with hyperactive motility and a physiological acrosome reaction [7, 8]. Despite evidence for both physiologic and pathologic effects of ROS in spermatozoa, the identity of the ROS-producing enzyme(s) remains uncertain. Numerous studies showed similarity between the ROS-producing enzyme in spermatozoa and the enzymatic system expressed in phagocytes In these cells, the major ROS precursor is the superoxide anion, which is generated by the singleelectron reduction of molecular oxygen by the NOX2 NADPH oxidase complex. We present data documenting both the expression and function of NOX5 in human spermatozoa
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