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Keywords:

  • Hydrogen peroxide;
  • lipid peroxidation;
  • disulfide bridges;
  • protamines;
  • DNA condensation;
  • DNA oxidation;
  • genomic stability

Abstract

  1. Top of page
  2. Abstract
  3. Reactive Oxygen Species and Mammalian Spermatozoa: Friends and Foes
  4. Epididymal GPx in Mammals: ROS Scavengers and Thiol Peroxidases
  5. Lessons From the Mouse GPx Knockout Models
  6. Conclusions
  7. References

ABSTRACT: The mammalian glutathione peroxidase (GPx) gene family encodes bifunctional enzymes that can work either as classical reactive oxygen species (ROS) scavengers or as thiol peroxidases, thereby introducing disulfide bridges in thiol-containing proteins. These dual effects are nowhere better demonstrated than in epididymal maturing spermatozoa, where the concomitant actions of several GPx ensure the achievement of the structural maturation of sperm cells as well as their protection against ROS-induced damage. We review here the roles played by the sperm-associated forms of GPx4 (mitochondrial GPx4 and nuclear GPx4), the secreted GPx5 protein, and the epithelial proteins GPx1, GPx3, and cellular GPx4, all functioning in the mammalian epididymis at different stages of the sperm's epididymal journey, and in different epididymis compartments.

Glutathione peroxidases (GPx; EC 1.11.1.9) belong to the classical catalytic triad of primary enzymatic antioxidant scavengers that any oxygen-consuming eukaryotic cell uses to equilibrate the generation/recycling of either physiological or toxic oxygen byproducts. Briefly, along with superoxide dismutase (SOD; EC 1.15.1.1) and catalase (CAT; EC 1.11.1.6), GPx participate in the recycling of free radicals, of activated forms of oxygen (such as hydrogen peroxide [H2O2]), and of some peroxidized compounds resulting from the rapid attacks of organic molecules by reactive oxygen species (ROS; see Figure 1). Both GPx and CAT mediate the same reaction, which is the recycling of H2O2 into water. But these 2 enzymes have specific domains and characteristics of action. Catalase is a peroxisome-located enzyme that will recycle only H2O2 and will be activated when cellular H2O2 concentrations are far above physiological levels, during a so-called oxidative burst. Such nonphysiological situations of oxidative insults are reached in several stress conditions, and in that respect CAT is an acute stress-response scavenger. GPx deals with small physiological adjustment of H2O2 concentrations in both the intracellular and extracellular compartments. GPx are also more versatile than CAT in the substrates they can metabolize, because besides H2O2 GPx can recycle organic peroxidized molecules, including those in free polyunsaturated fatty acids (PUFA) and in complex membranes such as phospholipid hydroperoxides, and therefore act both as scavengers and as repairing enzymes. Thus, although CAT is a powerful H2O2-recycling enzyme, GPx 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 in view of the range of actions devoted to H2O2 in cell physiology. To complete the picture, peroxiredoxins (PRDX; EC 1.11.1.5) also act as ROS scavengers at physiological ROS levels and are considered as regulators of H2O2 concentration and ROS-dependent signaling events (Rhee et al, 2005).

image

Figure 1. . Hydrogen peroxide (H2O2) generation and recycling by the classical superoxide dismutase (SOD)/glutathione peroxidase (GPx)/catalase (CAT)/peroxiredoxin (PRDX) system. H2O2 arises from the activity of SOD, which recycles superoxide anion (O2•) coming from oxygen catabolism. GPx, CAT, and PRDX recycle H2O2 into radical-free H2O. H2O2 concentrations are kept under precise control in and out of the cell because H2O2 has both beneficial and detrimental effects on cell physiology. On the one hand, excessive accumulation of H2O2 due to increased generation or 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 constituent starting with lipids in membranes. Excessive free radical–mediated damage 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 second messenger in signal transduction pathways, and it also modulates signal transduction cascades via its effect on cellular proteins that are sensitive to the redox state of the cell. Second, H2O2 either spontaneously, or via the action of enzymes such as disulfide isomerases, thiol peroxidases, and GPx, transforms free thiol groups carried by cysteine-containing proteins into disulfide bonds.

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Hydrogen peroxide can be considered as 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 Figure 1; Halliwell and Gutteridge, 1999). However, certain amounts of H2O2 and lipid hydroperoxides (LOOH) are necessary for normal cell physiology because these molecules also act as second messengers modulating intracellular signal transduction pathways (Seiler et al, 2008; Bartz and Piantadosi, 2010; Conrad et al, 2010; Forman et al, 2010). In addition, H2O2 and LOOH are necessary substrates for numerous enzymes that use it to mediate disulfide bridging events in thiol-containing proteins. This is the case with disulfide isomerases/thiol peroxidases. Disulfide-bridging events are one type of posttranslational modification important for protein maturation. When occurring between sulfhydryl (SH) groups on one protein, they participate in its proper folding, whereas when disulfide bonds affect different proteins, they are involved in protein-protein interactions. Both phenomena greatly contribute to the activity of the respective proteins. During the last decade it has been reported that some GPx have the ability to work as bona fide disulfide isomerases, provided they contain in their primary amino acid sequence (outside their scavenger catalytic site) a cysteine residue that will be involved in disulfide-bridging events of thiol-containing protein targets (Delaunay et al, 2002). To mediate disulfide bond formation in thiol-carrying proteins, GPx require H2O2 or LOOH as cosubstrates. Thus GPx can either neutralize H2O2 by using glutathione (GSH) as a cofactor or mediate disulfide-bridging events by using H2O2 or LOOH and thiol-containing proteins. The common factor between both reactions is the presence of H2O2 or other organic hydroperoxides in the environment.

Alvarez and Storey (1989) were 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 spermatozoa was correlated with infertility in humans (Imai et al, 2001; Foresta et al, 2002). In the last 5 years, the development of mouse GPx knockout models (Conrad et al, 2005; Chabory et al, 2009; Imai et al, 2009; Liang et al, 2009; Schneider et al, 2009) associated with infertility or subfertility have demonstrated that GPx do indeed play important roles in mammalian sperm physiology.

Reactive Oxygen Species and Mammalian Spermatozoa: Friends and Foes

  1. Top of page
  2. Abstract
  3. Reactive Oxygen Species and Mammalian Spermatozoa: Friends and Foes
  4. Epididymal GPx in Mammals: ROS Scavengers and Thiol Peroxidases
  5. Lessons From the Mouse GPx Knockout Models
  6. Conclusions
  7. References

Among the 200 or so cell types constituting a higher vertebrate, there is no cell in which H2O2 presents such a paradoxical situation as in mature spermatozoa. On the one hand, H2O2-mediated oxidative injuries of spermatozoa are a classical parameter accompanying male infertility, whether it is due to abnormal spermatogenesis, abnormal posttesticular steps of sperm maturation, aging, or pathological situations such as infection or inflammation (Aitken and Clarkson, 1987; Griveau and Le Lannou, 1997; Aitken, 1999; Agarwal et al, 2006). In vitro, oxidative insults of spermatozoa are also major parameters influencing the success of assisted reproductive technologies such as in vitro fertilization and intracytoplasmic sperm injection (ICSI) as well as artificial insemination. Spermatozoa are indeed particularly susceptible to oxidative damage for 3 major reasons. First, mature posttesticular spermatozoa are silent cells that harbor a highly compacted haploid nucleus that is due to the meiotic process and the replacement of nucleosomal histones by protamines during late stages of spermatogenesis. Both contribute to silencing the paternal chromosomes that thus will not be able to engage any transcriptional activation when challenged by extracellular or intracellular stress, including oxidative stress. Second, spermatozoa are also silent in terms of protein synthesis because upon spermiation they lose most of their cytoplasm, and consequently the subcellular organelles that support protein translation, as well as their stock of cytosolic enzymes that might dampen intracellular cell stresses, again such as oxidative stress. Third, posttesticular spermatozoa are highly reactive to oxidative injury because of the peculiar lipid composition of their plasma membrane. If there are organic components that are the targets of choice of free radicals, these are lipids, and essentially those containing PUFA, that are prone to oxidation triggered by ROS. During posttesticular (ie, epididymal) maturation of spermatozoa, both the phospholipid fraction and the proportion of PUFA in this fraction are significantly increased. This increases the fluidity of the mature sperm plasma membrane and at the same time increases its susceptibility toward oxidative attacks. On the other hand, and rather paradoxically for a cell that is in danger of oxidative injury, spermatozoa were the first cells reported to generate significant levels of ROS (Tosic and Walton, 1946). This sperm-associated ROS generation is attributable to the high mitochondrial activity of fully motile spermatozoa. Besides this point, spermatozoa also exploit ROS, especially H2O2, superoxide anion, and nitric oxide, as signaling actors in order to trigger the ultimate maturation steps in the female genital tract represented by the processes of capacitation and acrosome reaction (see for recent reviews de Lamirande et al, 1997; Baker and Aitken, 2004; O'Flaherty et al, 2006; de Lamirande and O'Flaherty, 2008). In addition, earlier on during the epididymal journey, in which spermatozoa progressively acquire their fertilizing potential, they also require the action of H2O2 that allows extensive sulfoxidation of various proteins in distinct sperm structures such as the nucleus, the acrosome, and the sperm midpiece (Cummins et al, 1986; Mate et al, 1994; Mammoto et al, 1997; Sivashanmugam and Rajalakshmi, 1997). These disulfide-bridging events occurring during epididymal sperm maturation will complete the fine shaping of this highly differentiated cell (Seligman et al, 2005).

Thus, ROS exert dual actions on spermatozoa, being both beneficial and detrimental. This ambiguous situation is very well illustrated in the epididymis, where ROS-mediated beneficial actions (sulfoxidation) occur while spermatozoa are incapable of protecting themselves from the inherent damaging effect of ROS. To avoid oxidative insults of the maturing male gametes, the epididymal environment is involved in a balancing act, because on the one hand, it provides sufficient H2O2 to allow optimal disulfide-bridging events, whereas on the other hand, it also provides ROS scavengers to protect spermatozoa. A very fine control of the ROS generation or recycling balance is therefore expected to operate in the luminal compartment of the mammalian epididymis. This is corroborated by the observations that we and others have made, that the mammalian epididymis expresses a complex array of primary antioxidant scavengers, including several GPx that seem to occupy a central position in maintaining this balance.

Epididymal GPx in Mammals: ROS Scavengers and Thiol Peroxidases

  1. Top of page
  2. Abstract
  3. Reactive Oxygen Species and Mammalian Spermatozoa: Friends and Foes
  4. Epididymal GPx in Mammals: ROS Scavengers and Thiol Peroxidases
  5. Lessons From the Mouse GPx Knockout Models
  6. Conclusions
  7. References

We, and others, have shown that the mammalian epididymis expresses several GPx and, to date, it is the organ in which one can find expressed, although at different levels and in different subterritories, most of the known GPx, from GPx1 to GPx8 (Drevet, 2000, 2006). Within the GPx multigenic family, 4 members (GPx1, GPx3, GPx4, and GPx5) are particularly well represented and characterized and are located in the epididymal epithelium, in the luminal compartment, and in spermatozoa. Figure 2 presents a scheme of the localization of these GPx in the mouse epididymis, the picture being approximately the same in all the mammals that have been tested (reviewed in Vernet et al, 2004). Briefly, GPx1, GPx3, and cGPx4 are cytosolic enzymes expressed by the epididymal epithelial cells (essentially principal cells), whereas GPx5 is a secreted protein. GPx5 and GPx3 are quantitatively the most abundant GPx in the whole epididymis, representing altogether more than 95% of the epididymal GPx at both the mRNA and protein levels. Their differences essentially reside in the fact that GPx3 is a cytosolic enzyme increasingly expressed from the caput to the cauda epithelia, whereas GPx5 is a secreted enzyme whose expression and secretion is restricted to the caput epithelium. Another difference between GPx3 and GPx5 is the fact that GPx3 is a classical selenium-dependent GPx, whereas GPx5 belongs to the noncanonical selenium-independent GPx (together with GPx6 and the predicted GPx7 and GPx8). Although it has long been suspected to be an inefficient GPx, we have demonstrated that GPx5 and other selenium-independent GPx can act as true H2O2 scavengers, as expected of the selenium-dependent members (Vernet et al, 1996, 1999; Herbette et al, 2007; Chabory et al, 2009). GPx1 and cGPx4 are expressed all along the epididymis epithelium at low levels compared with the other epididymal GPx. Both are cytosolic enzymes. Thus, the epididymal epithelium is protected mainly by GPx3, whereas the luminal compartment of the epididymis is protected by GPx5. In many mammals, GPx5 is a major secretion of the proximal epididymis duct (Belleannée et al, 2011). It moves along the epididymal duct with maturing spermatozoa and accumulates with them in the caudal storage compartment. The strong GPx5 caudal luminal content together with the high cauda epithelial cytosolic expression of GPx3 suggests that this territory is involved in protecting sperm cells and the epididymal tissue from peroxidative injuries.

image

Figure 2. . Glutathione peroxidase (GPx) localizations in the mammalian epididymis. Schematic representation of the GPx expression by the epididymal epithelium. GPx5 is abundantly expressed by the caput epithelium and the protein is secreted into the epididymal duct. The luminal GPx5 protein accompanies spermatozoa in transit and is stored with them in the cauda lumen. GPx3 is a cytosolic GPx increasingly expressed by the epididymal epithelium from the caput to the cauda. Besides these 2 major GPx, the epididymal epithelium (proximal to distal) expresses at lower levels the cytosolic GPx (GPx1 and cGPx4). In addition, epididymal spermatozoa carry 2 sperm-specific isoforms of GPx4, the mitochondria-associated mGPx4 (in the sperm midpiece) and the nucleus-associated nGPx4.

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To complete the picture of the epididymal localization of mammalian GPx, one should note that spermatozoa themselves carry GPx proteins. These sperm-bound GPx have been added to the spermatozoa during testicular spermatogenesis. This is the case for the sperm nucleus–associated isoform of GPx4 (snGPx4 or nuclear GPx4, nGPx4) and the mitochondria-associated isoform of GPx4 (mGPx4). Along with the cytosolic or cellular GPx4 variant (cGPx4), mGPx4 and snGPx4 arise from differential expression of the single-copy GPx4 gene (Pushpa-Rekha et al, 1995; Godeas et al, 1997; Pfeifer et al, 2001; Maiorino et al, 2003; Moreno et al, 2003). Both sperm-associated mGPx4 and nGPx4 are precisely localized to the midpiece compartment and to the nucleus (Figure 2), respectively, during the final cyto-differentiation step of spermatogenesis.

Lessons From the Mouse GPx Knockout Models

  1. Top of page
  2. Abstract
  3. Reactive Oxygen Species and Mammalian Spermatozoa: Friends and Foes
  4. Epididymal GPx in Mammals: ROS Scavengers and Thiol Peroxidases
  5. Lessons From the Mouse GPx Knockout Models
  6. Conclusions
  7. References

GPx4 Knockout Models—

Both the sperm-associated and sperm-restricted GPx4 variants (mGPx4 and nGPx4) have been shown to be associated with intracellular proteins and to function as disulfide isomerases rather than as classical ROS-scavenging GPx. Concerning the sperm midpiece–located mGPx4, it has been estimated that this isoform constitutes up to 50% of the sperm midpiece protein content that embeds the helix of mitochondria (Ursini et al, 1999). For that reason it was proposed that mGPx4 is the selenoprotein of the sperm midpiece, a role given earlier to a protein called sperm mitochondria-associated cysteine-rich protein (Kleene, 1994). In the sperm midpiece, the mGPx4 protein is suggested to be more a structural protein than an active enzyme because it has been shown to have completely lost its solubility and its scavenging enzymatic properties (Ursini et al, 1999). It is, however, probable that the sperm midpiece–located mGPx4 is involved in local structural reorganization based on protein disulfide-bridging events. It has been shown that disulfide bonds in the late stages of spermatogenesis and during epididymal transit are important for several sperm structures (besides the nucleus), such as the plasma membrane, the midpiece, and the acrosome (Cummins et al, 1986; Mate et al, 1994; Francavilla et al, 1996; Mammoto et al, 1997; Sivashanmugam and Rajalakshmi, 1997). In the sperm midpiece, during spermiogenesis, it has been shown that mitochondria attach to outer dense fiber proteins of the axoneme and that disulfide bonds in several proteins are involved in this process. As a result, the spermatid cytoplasm is reduced and the sperm plasma membrane is connected to the sperm midpiece. In addition, it has been shown that the acrosome contains the greatest relative amount of disulfides, before the head and the tail in guinea pig spermatozoa (Huang et al, 1984), suggesting that there are regionalized disulfide-bridging events during sperm maturation. The group of M. Conrad (Schneider et al, 2009) generated a transgenic mouse model in which the mGPx4 was disrupted via the introduction of an in-frame translational stop into the mitochondrial leader sequence of mGPx4. The analysis of this mouse model reveals that mGPx4−/− mice are viable, contrary to the GPx4−/− mice (in which the somatic isoform[cGPx4] as well as the 2 sperm-specific variants [mGPx4 and snGPx4] are absent) that die during early embryogenesis (Imai et al, 2003; Yant et al, 2003). Interestingly, the mouse mGPx4−/− model showed male infertility associated with impaired sperm integrity. Essentially and quite logically, mGPx4−/− spermatozoa showed important structural abnormalities in the midpiece region, leading to an increase in bent flagella, sperm heads detached from the flagellum, abnormal distribution of mitochondria along the midpiece, and abnormal organization of the axoneme (Schneider et al, 2009). In addition, and confirming the disulfide-bridging function of the protein mGPx4, deficient spermatozoa exhibit a higher protein thiol content, and their phenotype resembles what occurs in severe selenodeficiency situations (Flohé, 2007; Shalini and Bansal, 2008). Also not surprisingly, sperm motility was significantly reduced in mGPx4−/− males. The authors showed that male infertility could be bypassed by ICSI, suggesting that the male gametes were unable to move properly as a consequence of sperm midpiece structural abnormalities and not because of their incapacity to initiate fertilization. Confirmation of these findings with regard to mGPx4 function was reported in Liang et al (2009) and Imai et al (2009). Using a different strategy, Liang et al (2009) generated transgenic mouse strains that carried mutations inhibiting the expression of either cytosolic or mitochondrial GPx4 and, consequently, overexpressing the other isoform. Their data confirmed that the mitochondrial GPx4 variant is testis- and male germ cell–specific. They also confirmed that when mGPx4 is not expressed, it leads to male infertility, essentially because of structural malformations of the sperm midpiece. The strategy used by Imai et al (2009) was to establish a spermatocyte-specific GPx4 knockout mouse via the Cre-loxP system. Again, this new transgenic mouse model showed oligoasthenozoospermia resulting in male infertility, confirming that a decrease in GPx4 activity in spermatozoa results in male infertility in mice.

The sperm nucleus–specific isoform of GPx4 (nGPx4) was shown to result from differential expression of its gene owing to the use of an alternative promoter located in the first intron (Moreno et al, 2003). This results in the expression of a GPx4 isoform having an N-terminus sequence rich in arginine residues, allowing its nuclear localization and binding to chromatin (Pfeifer et al, 2001). In sperm nuclei, the GPx4 variant has been proposed to act as a protamine thiol peroxidase responsible for stabilizing the condensed chromatin by cross-linking protamine disulfides (Pfeifer et al, 2001). Condensation of sperm chromatin is an essential process in sperm differentiation, which starts during postmeiotic spermatogenesis with the replacement of somatic histones by transition proteins and finally by protamines. It appears that the sperm DNA-packaging process is not totally completed when spermatozoa leave the testis and that it goes on in the early stages of epididymal maturation. During epididymal transit, oxidation of protamine thiols plays an important role in compacting sperm DNA further and also locking it in that highly condensed state. The cross-linking of protamine disulfides induced by ROS is comparable to GSH oxidation and peroxide reduction catalyzed by GPx. Therefore, it has been proposed that the spermnucleus GPx4 variant uses protamine cysteine residues as reducing partners and acts as a protamine thiol peroxidase (Pfeifer et al, 2001). For its activity, the nGPx4 isoform would not depend on GSH availability, which decreases significantly in late spermatogenesis and early maturation during epididymal transit. In agreement with that hypothesis is the observation that in selenium-deficient animals, in which the concentrations of selenium-dependent GPx such as nGPx4 are greatly reduced, nearly all sperm cells recovered from the vas deferens possess incompletely compacted nuclei. In addition, in vitro experiments have shown that dithiothreitol provokes rat sperm DNA decondensation, an effect that is restored by adding H2O2 (Pfeifer et al, 2001). Finally, it has been shown that the use of an nGPx4 inhibitor blocks the condensation of sperm DNA. Together, these data strongly support the idea that the sperm nucleus–located GPx4 variant is responsible for protamine disulfide bridging within the sperm nucleus. In 2005, Conrad et al generated a transgenic mouse model in which they specifically abolished the expression of the sperm nucleus GPx4 isoform. In contrast to the full GPx4 knockout, nGPx4−/− animals are viable and fully fertile, suggesting that the nGPx4 isoform is not responsible for the developmental defects observed when all the GPx4 isoforms are deleted (Imai et al, 2003; Yant et al, 2003). When spermatozoa from these nGPx4−/− animals were investigated more closely, they did not show any obvious phenotype. When spermatozoa from nGPx4−/− animals were compared to those of wild-type (WT) animals, it appeared that there was a delay in the completion of posttesticular sperm nucleus compaction. In the caput epididymis of nGPx4−/− animals, sperm nuclei were less compacted than spermatozoa from the caput epididymis of WT animals. This delayed compaction was resumed later on, because there was no difference in the state of sperm nuclei compaction for spermatozoa collected from the cauda compartment of nGPx4−/− and WT animals (Conrad et al, 2005). These data support the idea that nGPx4 acts as a thiol peroxidase on thiol-containing sperm nuclear protamines in the caput compartment of the epididymis. The fact that normal sperm-DNA compaction is recovered in spermatozoa stored in the cauda compartment of the nGPx4−/− animals suggests that one or more other thiol peroxidases most likely compensate for the lack of nGPx4 expression as the sperm cells travel along the epididymal tubule. Another possibility is that the cytosolic isoform (cGPx4), which is still expressed in testis of nGPx4−/− mice, may partly back up nGPx4 deficiency because it is small enough to enter the nuclear pore (Weis, 1998; Conrad et al, 2005). Finally, although H2O2 is commonly believed to be rather inefficient in mediating -S-S-bridging directly, one cannot exclude the idea that spontaneous disulfide bridging occurs during epididymal migration of sperm cells providing there is enough H2O2 in the epididymal lumen to sustain it.

The GPx5 Knockout Model—

The epididymally secreted GPx5 knockout model has brought some clear evidence that the epididymal lumen contains significant amounts of H2O2 that could be available for spontaneous or enzyme-mediated sulfoxidation. It has also been shown that epididymal GPx5 is a true ROS scavenger protecting epididymis-transiting sperm cells from ROS-mediated loss of integrity. The epididymis-specific GPx (GPx5) occupies a special position in the GPx family and was initially suspected not to behave as a true GPx. The peculiarity of GPx5 is the absence of the selenocysteine (SeCys) residue in its catalytic site (Ghyselinck et al, 1993), contrary to the other well-studied members of the mammalian GPx family (GPx1 to GPx4). In GPx5, the SeCys residue is replaced by a cysteine residue. Because of that, the scavenger activity of GPx5 was questioned, because in canonical GPx, if the SeCys residue of canonical GPx was replaced by a cysteine, there was a dramatic drop in the enzyme activity (Maiorino et al, 1995). However, we have shown in vitro that GPx5-transfected mammalian cells survive much better in oxidative conditions (increasing H2O2 concentrations in the cell medium) than control cells, suggesting that GPx5 at least in vitro is efficient in recycling H2O2 (Vernet et al, 1996). We have also demonstrated that mice subjected to a selenium-free diet, depleting their Se-dependent GPx activities, show an overall increase in peroxidative injury in every tissue except the epididymis, where GPx5 mRNA and protein levels are increased, backing up the failing Se-dependent activities (Vernet et al, 1999). This strongly suggests that in vivo as well, the Se-independent GPx5 protein acts as a true scavenger. Final clues proving the real scavenging role of GPx5 in the epididymal environment came from the generation and analysis of a mouse strain that does not express GPx5 (Chabory et al, 2009). Lack of GPx5 expression in the epididymal lumen of the GPx5−/− animals established an oxidative stress in the cauda epididymidis. GPx5 deficiency was not followed by any change in the ratio of free thiols to sulfoxide in spermatozoa, suggesting that GPx5 has nothing to do with disulfide-bridging events and therefore behaves as a conventional ROS-scavenging GPx. To cope with the pro-oxidative situation in the cauda compartment, the cauda epididymidal epithelium of the GPx5−/− animals transcriptionally up-regulated the 3 cytosolic GPx normally expressed there (GPx1, GPx3, and cGPx4). Up-regulation of these epididymal GPx was sufficient to maintain the total GPx activity of the tissue to a normal value. Transcription of the epithelial cytosolic CAT was also increased in the cauda epididymidis of the GPx5−/− animals, reinforcing the idea that the tissue was facing an increase in H2O2 (Chabory et al, 2009) because CAT only metabolizes this substrate. These observations suggest that luminal ROS and especially H2O2 accumulate in the cauda compartment when GPx5 was no longer present. Despite the antioxidant response of the tissue, we have shown that the cauda epithelium of the GPx5−/− animals suffers oxidative injuries. This was also the case for the cauda-stored spermatozoa. In particular, cauda-stored spermatozoa in GPx5−/− animals showed a higher level of DNA oxidation, shown by the increase in 8-oxo-deoxyguanosine residues associated with increased fragmentation and a slight nuclear decompaction state compared with WT cauda-stored spermatozoa (Chabory et al, 2009). Although PRDX were not investigated in that study, it is possible that they also contributed to protect the cauda epididymis epithelium and spermatozoa against the pro-oxidant situation generated in the GPx5-deficient context, because several PRDX were very recently shown to be present on spermatozoa (Manandhar et al, 2009; O'Flaherty and de Souza, 2011).

Interestingly, in the caput epididymidis, sperm nuclei of the GPx5−/− animals were significantly more condensed than those of WT animals, suggesting that absence of H2O2 recycling via GPx5 in the caput luminal compartment left more H2O2 available for the disulfide-bridging activity of the nGPx4 protein or favored spontaneous disulfide-bridging events of sperm nucleus protamines. If this is the correct hypothesis, then GPx5 that is secreted in the caput lumen indirectly participates in sperm DNA compaction by regulating the luminal epididymal concentration in H2O2. In the cauda compartment of the GPx5−/− animals, we hypothesize that what we see are the results of prolonged exposure to the damaging effect of H2O2 on spermatozoa that leads to DNA oxidation, increasing fragmentation, nucleus decompaction, and lipid peroxidation (Chabory et al, 2009). Spermatozoa themselves may contribute to this situation, because it has been reported that sperm mitochondria are inactive and devoid of membrane potential in the caput, whereas they are completely mature and functional, showing a membrane potential, in the cauda epididymidis (Koppers et al, 2008). Thus, it is possible that cauda-stored spermatozoa, although they are not in optimal conditions of oxygen tension, pH, and energy substrate to sustain full mitochondrial activity, might contribute to the generation of free radicals via a leakage of the electron transport chain.

Oxidative damage of cauda-stored spermatozoa have been shown to increase in aging GPx5−/− animals (Chabory et al, 2009). The oxidative insults on the sperm DNA recorded in over-12-month-old GPx5−/− males provoked a phenotype of subfertility when these males were mated with WT female mice of proven fertility. We have observed a significant decline in male fertility that was not due to impaired fertilization but to a clear rise in developmental defects, miscarriages, and perinatal mortality (Chabory et al, 2009). In the absence of an effect on fertilization rate, and because the female mice were perfectly normal, the types of defects in embryos generated from aging GPx5−/− males indicate that loss of sperm DNA integrity is responsible. It has thus been assumed that oxidation of sperm DNA explains the effects recorded in the offspring of aging GPx5−/− males, as has been suggested elsewhere (Aitken, 2009; Aitken and De Iuliis, 2009; De Iuliis et al, 2009). Such developmental defects due to alterations of the paternal chromosomal material have already been reported in humans (Tesarik et al, 2004, 2006).

Taken together, these data clearly demonstrate that GPx5 is an important luminal scavenger that protects cauda sperm cells from the damaging effects of H2O2. The physiological importance of GPx5 during aging has been highlighted, in agreement with the well-known free radical theory of aging, which maintains that a decline in ROS-scavenging activities with age allows free radicals to affect cell constituents and cell physiology in many ways. GPx5 therefore appears as quite an important enzyme that ultimately contributes to the maintenance of sperm DNA integrity and consequently to embryo viability. When it is absent, sperm DNA oxidation is too extensive for the reparative capacities of the oocyte, leading to abnormal developments. Without this protective protein, male mice run a high risk of siring offspring with developmental defects, including some severe enough to lead to miscarriage. This could be particularly relevant clinically for the fertility of the aging male and could also have an important effect on assisted reproductive technologies (Francavilla et al, 1996; Baker and Aitken, 2005; Aitken et al, 2008; Aitken, 2009; Aitken and De Iuliis, 2009; Chabory et al, 2009; Thomson et al, 2009), in which cryopreservation of the male gametes and micromanipulation in different media can be the sources of oxidative insults on the paternal chromosomal set.

Conclusions

  1. Top of page
  2. Abstract
  3. Reactive Oxygen Species and Mammalian Spermatozoa: Friends and Foes
  4. Epididymal GPx in Mammals: ROS Scavengers and Thiol Peroxidases
  5. Lessons From the Mouse GPx Knockout Models
  6. Conclusions
  7. References

The data presented in this review clearly illustrate the ambiguous situation existing in maturing epididymal spermatozoa. On the one hand, spermatozoa use ROS to mediate disulfide-bridging events that are necessary for the completion of their structural modifications. Sperm DNA compaction is one of these structural changes that are not completed when spermatozoa enter the epididymal tubule. The increased sperm DNA compaction ensured by protamine sulfoxidation in the epididymis is a crucial phenomenon that serves both to protect paternal DNA from mutational effects and to reduce the volume of the sperm head, allowing an optimum velocity of mature spermatozoa, both being critical for the success of fertilization. On the other hand, these ROS-mediated sulfoxidation events have to be particularly well balanced, because spermatozoa are particularly susceptible to oxidative insults that may have dramatic effects on their integrity and consequently on their fertilizing potential. GPx proteins appear to be the masters in controlling this fine equilibrium, acting either as disulfide isomerases or true GPx H2O2 scavengers. Whereas nGPx4 uses H2O2 or other organic hydroperoxides to perform sulfoxidation of protamines, which further compacts the sperm nucleus and locks it in this condensed state, the luminal GPx5 protein controls the amount of luminal H2O2 available for optimal sulfoxidation and also protects maturing spermatozoa against H2O2-mediated damage. This fine interplay between GPx proteins and H2O2 during the last steps of the generation of fully competent spermatozoa in the male genital tract may explain why oxidative stress is such a frequent parameter associated with male infertility, whether it comes from infections and infiltrating leucocytes, environmental toxicants, metabolic syndromes, aging, situations that are known to lead to excessive generation of ROS, or alteration of ROS scavengers.

References

  1. Top of page
  2. Abstract
  3. Reactive Oxygen Species and Mammalian Spermatozoa: Friends and Foes
  4. Epididymal GPx in Mammals: ROS Scavengers and Thiol Peroxidases
  5. Lessons From the Mouse GPx Knockout Models
  6. Conclusions
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Footnotes
  1. Supported by a grant-in-aid from the French Ministry of Higher Education, CNRS, INSERM, Ernst Schering Research Foundation, and CONRAD (CONtraceptive Research and Development).