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Title
Identification of cytochrome-b5 reductase as the enzyme responsible for NADH-dependent lucigenin chemiluminescence in human spermatozoa
Author/Creator
Baker, Mark A.;  Krutskikh, Anton;  Curry, Benjamin J.;  Hetherington, Louise;  Aitken, Robert John
Description
Lucigenin-dependent chemiluminescence together with 2-[4-iodophenyl]-3-[4-nitrophenyl]-5-[2,4-disulfophenyl]-2H tetrazolium monosodium salt (WST-1) reduction can be detected following addition of NADH to many cell types, including human sperm suspensions. Although many reports suggest that such a phenomenon is due to reactive oxygen species production, other oxygen detecting metabolite probes, such as MCLA and luminol, do not produce a chemiluminescent signal in this model system. The enzyme responsible for NADH-dependent lucigenin chemiluminescence was purified and identified as cytochrome-b5 reductase. In support of this concept, COS-7 cells overexpressing cytochrome-b5 reductase displayed at least a 3-fold increase in the previously mentioned activity compared with mock-transfected cells. Fractions containing cytochrome-b5 reductase were capable of inducing both lucigenin-dependent chemiluminescence and WST-1 reduction. Oxygen radicals clearly did not mediate the cytochrome b5-mediated activation of these probes in vitro since neither luminol nor MCLA gave a chemiluminescence response in the presence of the enzyme and the cofactor NADH. These results emphasize the importance of the direct NADH-dependent reduction of these putative superoxide-sensitive probes by cytochrome-b5 reductase even though this enzyme does not, on its own accord, produce reactive oxygen species.
Relation
Biology of Reproduction Vol. 73, no. 2, p. 334-342
Date
2005
Publisher
Society for the Study of Reproduction
Keyword(s)
cytochrome-b5 reductase;  female reproductive tract;  lucigenin;  reactive;  oxygen species;  sperm;  sperm capacitation;  sperm maturation;  spermatozoa;  WST-1;  oxygen species generation;  superoxide anion production;  human sperm;  capacitation;  bis-n-methylacridinium;  necrosis-factor-alpha;  radical;  production;  tyrosine phosphorylation;  tetrazolium salt;  oxidase;  activity;  redox activity
Resource Type
journal article
Identifier
http://hdl.handle.net/1959.13/24450
Identifier
ISSN:1529-7268
Language
eng
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Subject
"Biology of Reproduction"
 
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