It has been shown that the mammalian epididymis expresses several GPx and, to date, it is the organ in which, although at different levels and in different subterritories, most of the known GPx are expressed, from GPxl to GPx8 [6, 8]. Within the GPx multigenic family, four members (GPxl, GPx3, GPx4, and GPx5) are particularly well represented and characterized and are located in the epididymal epithelium, luminal compartment, and spermatozoa. Figure 5.3 presents a diagram of the localization of these GPx in the mouse epididymis, the picture being approximately the same in all the mammals that have been tested so far (reviewed in ). Briefly, GPxl, GPx3, and cGPx4 are cytosolic enzymes expressed by the epididymal epithelial cells (essentially principal cells) while 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 both at 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 are restricted to the caput epithelium. Another difference between GPx3 and GPx5 is the fact that GPx3 is a classical selenium-dependent enzyme, whereas GPx5 belongs to the noncanonical selenium-independent group (together with GPx6, and the predicted GPx7 and GPx8). Although long suspected to be an inefficient GPx, it has been demonstrated that GPx5 and other selenium-independent forms can act as true H2O2 scavengers [9, 53-55]. 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 epididymis epithelium is mainly protected 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 . 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 epididy-mal tissue from peroxidative injuries. The importance of GPx5 as a luminal scavenger in human has been questioned because here the GPx5 gene is subjected to differential expression giving birth to two distinct transcripts. The most abundant transcript was shown to be devoid of residues important for its GPx-catalytic function , and thus the GPx5 variant from this transcript should therefore not code for an active ROS scavenger. However, this GPx5 isoform does contain appropriate cysteine residues that could allow it to work as a disulfide intermediate/exchanger like the other GPx. The second human GPx5 transcript does encode the full-length and presumably active enzyme. In addition, GPx6, a gene closely related to GPx5, probably coming from a quite recent duplication event, is also significantly expressed in the human epididymis, where it could back up GPx5 ,
To complete the picture of the epididymal localization of mammalian GPx, it should be noted that spermatozoa themselves carry GPx proteins which are 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 [59-63]. Both the sperm-associated mGPx4 and nGPx4 are precisely localized in the sperm midpiece compartment and in the nucleus (Fig. 5.3), respectively, during the final cytodifferentiation step of sper-matogenesis. 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 of GPx4 constitutes up to 50% of the sperm midpiece protein content embedding the helix of mitochondria  , For this reason, it was proposed that mGPx4 was the selenoprotein of the sperm midpiece, a role given earlier to a protein called SMCP for Sperm Mitochondria-associated Cysteine-rich Protein  . In the sperm midpiece, the mGPx4 protein is suggested to be more a structural protein than an active enzyme since it has been shown to have completely lost its solubility and scavenging enzymatic properties  . 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, mid-piece, and acrosome [66-70]. In the sperm midpiece, during late spermatogenesis, 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 (SS), compared with the head and the tail in guinea-pig spermatozoa  . suggesting that there are regionalized disulfide-bridging events during sperm maturation. Regarding the sperm nucleus-specific isoform of GPx4 (nGPx4), it was shown to result from differential expression of its gene owing to the use of an alternative promoter located in the first intron . 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 . In sperm nuclei, the GPx4 variant has been proposed to act as a protamine disulfide isomerase responsible for stabilizing the condensed chromatin by cross-linking protamine disulfides . Condensation of sperm chromatin is an essential process in sperm differentiation, which starts during postmeiotic sper-matogenesis 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 continues 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 a 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 sperm-nucleus GPx4 variant could use protamine cysteine residues as reducing partners and could act as a protamine disulfide isomerase . For its activity, the nGPx4 isoform would thus not depend on GSH availability, which decreases significantly in late spermatogenesis and early maturation within the epididymis transit. In agreement with this 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 . Finally, it has been shown that the use of an nGPx4 inhibitor blocks the condensation of sperm DNA. Altogether, these data strongly support the idea that the sperm nucleus-located GPx4 variant is responsible for protamine disulfide bridging within the sperm nucleus.
Thus, neither mGPx4 nor nGPx4 appears to have direct scavenging functions in epididymal spermatozoa, whereas GPx5 is thought to work as a true epididymal luminal scavenger.
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