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BOR - Papers in Press, published online ahead of print April 27, 2005.
Biol Reprod 2005, 10.1095/biolreprod.104.037960
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BIOLOGY OF REPRODUCTION 73, 334–342 (2005)
DOI: 10.1095/biolreprod.104.037960
© 2005 by the Society for the Study of Reproduction, Inc.

Identification of Cytochrome-b5 Reductase as the Enzyme Responsible for NADH-Dependent Lucigenin Chemiluminescence in Human Spermatozoa1

Mark A. Baker , Anton Krutskikh 3 , Benjamin J. Curry , Louise Hetherington , and R. John Aitken 2 

The ARC Centre of Excellence in Biotechnology and Development, Reproductive Science Group, School of Environmental and Life Sciences, and Hunter Medical Research Institute, University of Newcastle, Callaghan, New South Wales 2308, Australia

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.

cytochrome-b5 reductase, female reproductive tract, lucigenin, reactive oxygen species, sperm, sperm capacitation, sperm maturation, spermatozoa, WST-1

1 Supported by the ARC Centre of Excellence in Biotechnology and Development, Ernst-Schering Trust through the AMPPA network, and the Australian Research Council.

2 Correspondence: R. John Aitken, Discipline of Biological Sciences, University of Newcastle, Callaghan, New South Wales 2308, Australia. FAX: 61 2 4921 6308; jaitken{at}mail.newcastle.edu.au

3 Current address: Institute of Reproductive and Developmental Biology, Imperial College London, Du Cane Road, London W12 0NN, United Kingdom






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Copyright © 2005 by the Society for the Study of Reproduction.