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

Abstract

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.

On methods for the detection of reactive oxygen species generation by human spermatozoa: analysis of the cellular responses to catechol oestrogen, lipid aldehyde, menadione and arachidonic acid

“…The problem with this probe is that the chemistry of its chemiluminescence is very complex. Activation of the probe requires a one-electron reduction, rather than the oneelectron oxidation associated with luminol-dependent chemiluminescence (Aitken et al, 2012a (Baker et al, , 2005.…”

“…Thus, any enzyme capable of effecting NBT reduction using NADH or NADPH as an electron donor, will generate a response that masquerades as a ROS signal (Baker et al, , 2005. So, while O 2…”

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.…”

The spermatozoa structure peculiarities of the subgenus Sumeriomys (Rodentia, Arvicolinae, Microtus)

“…The most functionally important zone is a medium part of the tail, which provides energy for spermatozoon movement. Apparently, deficiency of ATP in mitochondrion (Baker et al, 2005;Zhabin et al, 2010) can be the cause of decreasing in mobility of gametes, which generally are settling down in an intermediate zone of a tail, a midpiece (Bierła, 2006). It is shown also that ability of spermatozoa in laboratory mice to way-ahead movements and fertilization is connected with energy release in glycolysis (Miki et al, 2004).…”