Male Reproductive Tract

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

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 catalysing 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.

Key words: Male Reproductive Tract • Sperm • Sperm capacitation • Sperm maturation • Stress

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