Nonenzymatic Epididymal Scavengers

Many molecules with intrinsic radical-scavenging activity exist in the epididymis compartment. I do not intend here to present them in an exhaustive fashion. I only focus on those that are quite classical and those that have been highlighted because they are present in unusual concentrations in this tissue.

5.3.1.1 Glutathione and Thiol-Containing Compounds

The tripeptide glutathione (GSH) is generally the most important/abundant nonenzymatic scavenger in and outside cells. Its cysteine residue provides a reactive thiol group (SH) that interacts readily with free radicals. Reduction of oxidized GSH is then ensured by GSH reductase and nicotinamide adenine dinucleotide (NADPH). It was repeatedly reported in many mammal models that GSH concentrations are rather low in the epididymis compartment, suggesting that GSH is not a major antioxidant actor in this tissue. However, it is important to note that yGT (gamma glutamyl transpeptidase), the enzyme that regenerates GSH, is quite abundantly expressed in the caput epididymidis [ 10] . In addition, data have been produced pleading in favor of a significant role of GSH in protecting epididymal sperm from oxidative insult since cauda epididymidis and stored spermatozoa have been shown to be particularly vulnerable to GSH depletion [11]. Thus, this common and powerful scavenger is most probably one important player in the global antioxidant defense strategy of the epididymis. Observations indicating that thiol content in normozoospermic semen was found to be significantly higher than in samples originating from vasectomized men [12] also support this idea and strongly suggest that the epididymis contributes significantly to the accumulation of thiol-containing antioxidants in the semen.

Thioredoxins (Trx) are other thiol-containing molecules that could indirectly act as antioxidants within the epididymis. They are 12-kDa oxidoreductase enzymes that facilitate the reduction of other proteins by cysteine thiol-disulfide exchange, found in nearly all known organisms, and have been shown to be essential for life in mammals [13]. Although Trx is an enzyme and therefore I should have evoked it in the next section of this review devoted to epididymal enzymatic scavengers, it also acts as an electron donor to peroxidases and other enzymes [14]. In that sense, Trx functions more as a disulfide intermediate rather than an enzymatic antioxidant. There is very little information regarding the level of Trx in the mammalian epididymis, so it is quite difficult to ascribe to Trx an important role in protecting epididymal spermatozoa from oxidative insult. However, it was reported that human spermatozoa do possess specific Trx called SPTRX for spermatozoa-specific Trx [15]. SPTRX expression is restricted to postmeiotic sperm cells and are considered to play roles in the extensive reorganization of sperm protein disulfide bonds that occur during either spermiogenesis or/and epididymal maturation to stabilize cytoskeletal sperm structures [15, 16].

5.3.1.2 Ascorbic Acid and Uric Acid

In addition to thiols, ascorbic acid (AA) and uric acid (UA) are the major individual antioxidants present in mammalian semen, humans included [17]. However, unlike thiol-containing molecules, semen ascorbate and urate contents of vasectomized men do not differ from those of normozoospermic men suggesting that AA and UA are unlikely to be prominent epididymal antioxidants [12]. Besides the scavenging effects AA and UA have on H2O2 and on the hydroxyl radical, respectively, they both have been proposed to be good ONOO- scavengers [18]. It is suggested that it is through this action that both molecules exert their antioxidant potential [19].

The primary role of l-carnitine is in transferring long-chain fatty acids across mito-chondrial membranes, thus facilitating oxidation within mitochondria during energy production. It is found concentrated in tissues, such as muscles, in which energy demand is high. Intriguingly, l-carnitine was shown to be present at very high concentrations in the mammalian epididymis and spermatozoa (in the mM range) far above circulating levels (in the ||M range). Epididymal intraluminal carnitine concentration increases gradually along the epididymis, reaching its maximum in the cauda compartment [20]. The prevalent view is that carnitine is not synthesized by the epididymis epithelia but rather is transported from the systemic compartment. Although, it is quite conceivable that, as the energy substrate for spermatozoa carnitine might support sperm respiration and motility, it is however difficult to understand why this should be the case in a compartment where spermatozoa are immotile. Therefore, the exact role of the high epididymal carnitine content remains an enigma. In addition to its contribution to cellular energy metabolism, l-carnitine and acetyl-l-carnitine were shown to protect cells from apoptosis in various ways [21], one being through their antioxidant properties [22]. It was suggested recently that the antioxidant properties of l-carnitine and/or acetyl-l-carnitine are not direct but secondary to their anti-inflammatory potential [23]. It is furthermore believed to be mediated at least partly by the downregulation of the proinflammatory sphingo-myelin/ceramide pathway.

5.3.1.4 Taurine/Hypotaurine

Taurine (Tau) is one of the most abundant free amino acids in several tissues and it is known to have beneficial physiological effects, including an antioxidant potential [24], The presence of taurine and hypotaurine in significant quantities in the male genital tract and their actions on spermatozoa have long been reported [25, 26]. More precisely, taurine was reported to be particularly abundant in the cauda epididymidis with a concentration largely exceeding that found in the caput [27] . Although Tau is incapable of directly scavenging the classical ROS such as superoxide anion, hydroxyl radical, and H2O2 [28], there are numerous studies suggesting that it is an effective inhibitor of ROS generation (reviewed in [29]). One of the mechanisms put forward is that taurine may upregulate/restore antioxidant enzymes, including superoxide dismutase (SOD) and glutathione peroxidase (GPx) [30]. Another potential indirect antioxidant action of taurine could be the prevention of mitochondria-associated ROS generation as a result of calcium accumulation. Taurine interferes with calcium overload [31] by its osmolyte effect on the stimulation of Na2+/Ca2+ exchanger [29]. Recently, several papers have underlined the mitochondrial action of taurine and its effect in attenuating mitochondrial ROS production [29, 32]. Briefly, taurine was shown to form conjugates with mitochondrial tRNAs, thus facilitating the translation of mitochondrial proteins and in fine energy production. If not present in adequate quantity, this could lead to impaired electron transport flux, lower rate of ATP generation, superoxide anion generation, and mitochondria damage. Therefore, in the cauda epididymis, taurine could be essential for maintaining an adequate energy balance and limiting the rate of superoxide generation [29].

5.3.1.5 Clusterin

Clusterin (Clu) is one of the major secretions of the mammalian epididymis epithelium accounting, for example, for 30% of the human epididymis secretome [33], Clusterin is also named apolipoprotein J (Apo-J). It is a 75-80 kDa disulfide-linked heterodimeric glycoprotein quite conserved between many species and quite ubiquitously expressed in most mammalian tissues and body fluids, including semen. It contains a cysteine-rich domain stabilized by five disulfide bridges. Because clus-terin can partner with a broad range of molecules, it is difficult to assign it a particular function. It is now considered that clusterin functions as an extracellular chaperone protecting cells from various stresses, including oxidation. For some authors, clus-terin is best viewed as a marker of cell response to pro-oxidant situations. This would be in agreement with the idea that the epididymis luminal environment is in a pro-oxidant context, where clusterin is present to exert its chaperone function.

5.3.1.6 Albumin/Lactoferrin

As was the case with clusterin, albumins are major components of the mammalian epididymis fluid. Albumin possesses two types of antioxidant properties [34]. First, it is involved in free radical trapping and, second, it specifically binds metals ions, such as copper and iron, thus preventing the production of free radicals by these transition metals [35]. Lactoferrin, which is also present in the mammalian epididymal fluid, also provides an indirect antioxidant protection by blocking iron, thus avoiding its immediate reaction with H2O2 along the Fenton/Haber-Weiss biochemical pathway. Albumin-free radical-trapping property is mediated by a sulfhydryl group (Cys34) that can form disulfide with several compounds. Through this reduced Cys34, albumin may scavenge hydroxyl radicals as well as reactive nitrogen

BENEFICIAL

Intracellular/extracellular modulators of signal transduction Disulfide-bridging effector pathways (spontaneous and enzyme-mediatec)

GPx, PRDX

Fenton & Haber-Weiss reactions

DETRIMENTAL

Lipids Proteins Glucides Nucleic acids

Oxidation

IMPAIRED CELL FUNCTIONS CELL DEATH

Fig. 5.2 Hydrogen peroxide (H2O2) generation and recycling by the classical SOD/GPx/catalase enzymatic triad. H2O2 arises from the activity of superoxide dismutase (SOD) that recycles superoxide anion (O^-) coming from oxygen catabolism. Glutathione peroxidases catalase and perox-iredoxins recycle H2O2 into something neutral, H2O. H2O2 concentrations are precisely kept under control in and out the cell because H2O2 has both beneficial H2O2 and detrimental effects on cell physiology. On the one hand, excessive accumulation of H2O2 due to increased generation or/and defective recycling will lead (in the presence of iron (Fe) and oxygen via the classical Fenton and Haber-Weiss biochemical reactions) to the production of very aggressive free radicals. Eukaryotic cells have no enzymatic equipment to deal with these free radicals that will damage every cell constituents starting with lipids in membranes. Excessive free radical-mediated damages will first impair cell functions and could, if not properly counteracted, lead to cell death. On the other hand, H2O2 is necessary for some physiological processes. First, it acts as a secondary messenger in signal transduction pathways and it also modulates signal transduction cascades via its effect on cellular proteins that are sensible to the redox state of the cell. Second, H2O2 transforms free thiol groups carried by cysteine-containing proteins into disulfide bonds, either spontaneously or via the action of enzymes, such as disulfide isomerases, thiol peroxidases, and glutathione peroxidases species, such as peroxynitrite (ONOO-), that are powerful oxidants [34]. In addition, albumins bind with high affinity to long-chain fatty acids and PUFAs, perhaps protecting them from lipid peroxidation [ 36] . With all these antioxidant effects, albumin in the epididymal fluid should contribute to the protection of spermatozoa against oxidative insults.

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