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

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.

Mammalian sperm metabolism: oxygen and sugar, friend and foe

“…The initiator of lipid peroxidation is the ROS, superoxide anion, O 2 ·-, which is produced mainly as a side reaction of midpiece mitochondrial electron transport to O 2 . Apparent production of O 2 ·-via a sperm-specific NADPH oxidase (Aitken and Clarkson, 1987) has recently been shown to reflect the chemistry of the lucigenin detection system rather than actual O 2 ·-generation (Richer and Ford, 2001;Ford, 2004;Baker at al., 2004;Baker et al, 2005). The O 2 ·-anion is itself a reductant rather than an oxidant, but its conjugate acid, HOO · , is a potent oxidant and acts as initiator of peroxidative chain reactions.…”

Use of spectroscopic probes for detection of reactive oxygen species

“…Although numerous reports suggest that this phenomenon is due to reaction with ROS, other probes capable of detecting reactive oxygen metabolites, such as 2-methyl-6-( p-methoxyphenyl)-3,7-dihydroimidazo [1,2-a] pyrazine-3-one (MCLA) and luminol, do not produce a chemiluminescent signal in this model system. It is important to note that direct NAD(P)H-dependent reduction of lucigenin and WST-1 by cytochrome P450-reductase and cytochrome-b5 reductase has been demonstrated, although these enzymes do not produce ROS themselves [102,103].…”

Mammalian sperm chromatin structure and assessment of DNA fragmentation

“…Also the male germ line possesses a nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX5) that is potentially capable of generating ROS in the presence of calcium and NADPH [76,77]. Other ROS-generating systems in mammalian spermatozoa may include lipoxygenase [78], tNOX [79,80] and cytochrome P450 reductase [81].…”