Enzymatic Scavengers

Primary antioxidant enzymes work in a tightly associated manner constituting the commonly called antioxidant catalytic triad (Fig. 5.2). SOD (EC 1.15.1.1) recycles the spontaneous formation of superoxide anion and doing so generates H2O2.

To adapt H2O2 concentrations precisely, cells rely on three enzymes, GPx (EC 1.11.1.9), catalase (CAT; EC 1.11.1.6), and peroxiredoxins (PRDX: EC 1.11.1.5). GPx and PRDX activities cover both the intracellular and the extracellular compartments while CAT located in peroxisomes confers an additional protection to the cytoplasmic compartment of most cells. These H2O2-recycling enzymes do not work in the same way. Catalase is switched on in situations of acute stress during the so-called oxidative burst when enormous amounts of H2 O2 are produced. For more physiological adjustments of H2O2 concentrations, cells rather rely on GPx and PRDX. In addition, while catalase only recycles H2O2, GPx are more versatile in their substrate preferences. This extends the protecting role of GPx since, unlike CAT, in addition to H2 O2 GPx can also recycle organic peroxidized molecules, such as phospholipid hydroperoxides. GPx therefore act both as scavengers and repairing enzymes. Thus, although catalase is a powerful H2O2-recycling enzyme, GPx and PRDX are viewed as the key regulators of H2O2 concentration and consequently of H2O2-mediated attacks in and around most cells. This is a particularly important role considering the range of actions devoted to H2 O2 in cell physiology. Roughly, H2O2 is a "Dr. Jekyll and Mr. Hyde" molecule. When present in above-physiological concentrations, it gives rise to very aggressive free radicals (OH', OH-) via the classical Fenton and Haber-Weiss biochemical reactions against which eukaryotic cells are devoid of efficient protection. Excessive generation of such free radicals will affect all organic cellular components ranging from lipids in membranes to nuclear DNA material, ultimately leading to cell death (see Fig. 5.2). However, certain amounts of H2O2 and lipid peroxides (LOOH) are necessary for normal cell physiology since these molecules also act as secondary messengers modulating intracellular signal transduction pathways [37-39]. In addition, H2O2 and LOOH are necessary substrates for numerous enzymes that use them to mediate disulfide-bridging events in thiol-containing proteins. This is the case of disulfide isomerases/thiol peroxidases. Disulfide-bridging events are one type of posttranslational modification important for protein maturation. When occurring between sulfydryl (SH-) groups on one protein, they participate in proper folding while when disulfide bonds affect different proteins they are involved in protein-protein interactions. Both phenomena greatly contribute to the activity of the two proteins. During the last decade, it has been reported that some GPx can work as bona fide disulfide isomerases, provided they contain, in their primary amino acid sequence (outside of their scavenger catalytic site), a cysteine residue that will be involved in disulfide bridging of thiol-containing protein targets [40]. To mediate disulfide bonds in thiol-carrying proteins, GPx require H2O2 or LOOH as cosubstrate. Thus, GPx can either catabolize/neutralize H2 O2 using GSH as a cofactor or/and mediate disulfide-bridging events using H2O2 or LOOH and thiol-containing proteins. The common point between these reactions is the presence of H2 O2 or other organic hydroperoxides in the environment. Because GPx are bifunctional enzymes that can either work as antioxidant scavengers or as thiol peroxidases, they are with H2O2 at the center of the "oxygen paradox" in the mammalian epididymis. This is detailed below in the context of the epididymis and spermatozoa.

5.3.2.1 Superoxide Dismutase

The mammalian epididymis expresses both the copper/zinc form of SOD (Cu-Zn SOD1) as well as a secreted form of the SOD enzyme (eSOD or SOD3). Both catalyze the dismutation of superoxide anion to generate H2 O2. Although commonly seen as an antioxidant enzyme, to my view SOD is more a pro-oxidant enzyme, since it transforms a short-lived and low-permeable free radical into an activated form of oxygen, H2 O2, that can readily pass any cell membrane. SOD1 is highly expressed throughout the epididymis epithelium from the caput down to the cauda [41]. That is not very surprising since this epithelium has an active metabolism requiring sustained mitochondrial activity, the major cytosolic source of superoxide anion. Thus, SOD1 may be responsible for epithelial production of H2 O2 part of which may reach the luminal compartment, where it could potentially harm spermatozoa. However, to counteract this, epithelial epididymal cells express, at quite high levels, various GPx (see below) that deal with the production and the circulation of H2O2 [6-8]. In addition to SOD1, the cauda epididymis epithelium also expresses SOD3, a secreted SOD that is closely associated with spermatozoa [42]. The anti-oxidant relevance of this sperm-associated SOD is difficult to apprehend. It has been suggested that this SOD could perhaps participate in H2 O2 signaling during redox-regulated tyrosine phosphorylation events associated with the induction of motility and the initiation of capacitation [43-45].

5.3.2.2 Glutathione Peroxidases

As said above, the mammalian GPx gene family encodes bifunctional enzymes that can work either as classical ROS scavengers or as disulfide isomerases, thus introducing disulfide bridges in thiol-containing proteins. These dual effects are nowhere else better illustrated than in epididymal maturing spermatozoa, where the concomitant actions of several GPx ensure the achievement of structural maturation of sperm cells as well as their protection against ROS-induced damage. I review here the roles played by the sperm-associated forms of GPx4 (mitochon-drial GPx4 and nuclear GPx4), the secreted GPx5 protein as well as the epithelial proteins, GPx1, GPx3, and cGPx4 (cellular GPx4), all functioning in the mammalian epididymis at different stages of spermatozoa epididymal journey and in different compartments. Alvarez and Storey [46] were among the first to point out the role played by GPx in protecting mammalian spermatozoa from loss of motility caused by spontaneous lipid peroxidation. Many years later, it was reported that failure of the expression of a GPx in the spermatozoa was correlated with infertility in human [47, 48]. These last 5 years, the development of mouse GPx knockout models [9, 49-52] associated with infertility or subfertility has demonstrated that GPx do play important roles in epididymal sperm protection and more largely in mammalian sperm physiology.

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Fig. 5.3 Glutathione peroxidase localizations in the mammalian epididymis. Diagrammatic representation of GPx expressed by the epididymis epithelium. GPx5 is abundantly expressed by the caput epithelium and the protein is secreted in the epididymal duct. The luminal GPx5 protein accompanies transiting spermatozoa and is stored with them in the cauda luminal compartment. GPx3 is cytosolic and increasingly expressed by the epididymis epithelium from the caput to the cauda. Besides these two major GPx, the epididymis epithelium (proximal to distal) expresses the cytosolic GPx (GPxl and cGPx4) at lower levels. In addition, epididymal transiting spermatozoa carry two sperm-specific isoforms of GPx4, the mitochondria-associated mGPx4 (in the sperm midpiece) and the nucleus-associated nGPx4

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