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1 July 2004 Identification of Cytochrome P450-Reductase as the Enzyme Responsible for NADPH-Dependent Lucigenin and Tetrazolium Salt Reduction in Rat Epididymal Sperm Preparations
Mark A. Baker, Anton Krutskikh, Benjamin J. Curry, Eileen A. McLaughlin, R. John Aitken
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Abstract

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.

Mark A. Baker, Anton Krutskikh, Benjamin J. Curry, Eileen A. McLaughlin, and R. John Aitken "Identification of Cytochrome P450-Reductase as the Enzyme Responsible for NADPH-Dependent Lucigenin and Tetrazolium Salt Reduction in Rat Epididymal Sperm Preparations," Biology of Reproduction 71(1), 307-318, (1 July 2004). https://doi.org/10.1095/biolreprod.104.027748
Received: 27 January 2004; Accepted: 1 May 2004; Published: 1 July 2004
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KEYWORDS
male reproductive tract
sperm
sperm capacitation
sperm maturation
stress
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