HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text Full Text (PDF) All Versions of this Article: 71/1/307    most recent biolreprod.104.027748v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Baker, M. A. Articles by Aitken, R. J. Search for Related Content PubMed PubMed Citation Articles by Baker, M. A. Articles by Aitken, R. J. Agricola Articles by Baker, M. A. Articles by Aitken, R. J.

Identification of Cytochrome P450-Reductase as the Enzyme Responsible for NADPH-Dependent Lucigenin and Tetrazolium Salt Reduction in Rat Epididymal Sperm Preparations¹

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 and WST-1 reduction can be detected following addition of NADPH to many cell types, including rat epididymal sperm suspensions. Although many reports suggest that such a phenomenon is due to reactive oxygen species production, other probes—such as MCLA and luminol—that are capable of detecting reactive oxygen metabolites do not produce a chemiluminescent signal in this model system. Our aim was to purify and identify the enzyme catalyzing the NADPH-dependent lucigenin and WST-1 reduction from rat epididymal spermatozoa preparations. Here, we show the identity of this enzyme as cytochrome P450-reductase. In support of this, a homogenous preparation of this protein was capable of reducing lucigenin and WST-1 in the presence of NADPH. Moreover, COS-7 cells overexpressing cytochrome P450-reductase displayed a 3-fold increase in the aforementioned activity compared with mock-transfected cells. Immunolocalization studies and biochemical analysis suggest that the majority of the NADPH-lucigenin activity is localized to the epithelial cells present within the epididymis. These results emphasize the importance of the direct NADPH-dependent reduction of superoxide-sensitive probes by cytochrome P450-reductase even though this enzyme does not, on its own accord, produce reactive oxygen species.

¹ Funded by the Ernst-Schering Trust through the AMPPA network and also by the Australian Research Council.

² Correspondence: R. John Aitken, Discipline of Biological Sciences, The University of Newcastle, Callaghan, NSW 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

This article has been cited by other articles:

				K Sabeur and B A Ball Characterization of NADPH oxidase 5 in equine testis and spermatozoa Reproduction, August 1, 2007; 134(2): 263 - 270. [Abstract] [Full Text] [PDF]

				G. N. De Iuliis, J. K. Wingate, A. J. Koppers, E. A. McLaughlin, and R. J. Aitken Definitive Evidence for the Nonmitochondrial Production of Superoxide Anion by Human Spermatozoa J. Clin. Endocrinol. Metab., May 1, 2006; 91(5): 1968 - 1975. [Abstract] [Full Text] [PDF]

				J. Seligman, G. L. Newton, R. C. Fahey, R. Shalgi, and N. S. Kosower Nonprotein Thiols and Disulfides in Rat Epididymal Spermatozoa and Epididymal Fluid: Role of {gamma}-Glutamyl-Transpeptidase in Sperm Maturation J Androl, September 1, 2005; 26(5): 629 - 637. [Abstract] [Full Text] [PDF]

				M. A. Baker, A. Krutskikh, B. J. Curry, L. Hetherington, and R. J. Aitken Identification of Cytochrome-b5 Reductase as the Enzyme Responsible for NADH-Dependent Lucigenin Chemiluminescence in Human Spermatozoa Biol Reprod, August 1, 2005; 73(2): 334 - 342. [Abstract] [Full Text] [PDF]