Lessons from the Mouse GPx Knockout Models

]n 2009, the group of Marcus Conrad (Germany) generated a transgenic mouse model in which the mGPx4 was disrupted [52] by introducing an in-frame transla-tional stop into the mitochondrial leader sequence of mGPx4. Analysis of this model revealed that mGPx4-~ mice are viable, in contrast to early embryonic lethality recorded in the GPx4~'~ mice in which the somatic isoform (cGPx4) as well as the two sperm-specific variants, mGPx4 and snGPx4, were absent [72, 73]. 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 sperm axoneme [52]. In addition and confirming the disulfide-bridging function of the protein mGPx4, deficient spermatozoa exhibit a higher protein thiol content and their phe-notype resembles what occurs in severe selenodeficiency situations [74, 75] . Not surprisingly also, sperm motility was significantly reduced in mGPx4-~ males. The authors showed that male infertility could be bypassed by intra-cytoplasmic sperm injection (ICSI), suggesting that the male gametes were inefficient because they 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 by Liang et al. [51] and Imai et al. [50]. Using a different strategy, Liang and collaborators generated trans-genic mouse strains that carried mutations inhibiting either the expression of cyto-solic 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, male infertility results, essentially because of structural malformations of the sperm midpiece. The strategy used by Imai's group was to establish a spermatocyte-specific GPx4 knockout mouse with 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 [52] .

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 [72, 73] . When spermatozoa from these nGPx4-~ animals were investigated more closely, they did not show any obvious phenotype. When spermatozoa from nGPx4-~ animals were compared with those of wild-type (WT) animals, they were found to show a delay in completion of post-testicular sperm nucleus compaction. In the caput epididymis of nGPx4-~ animals, sperm nuclei were less compacted than in spermatozoa from the caput epididymis of WT animals. This delayed compaction was resumed later on since there was no difference in the state of sperm nuclei compaction for spermatozoa collected from the cauda compartment of nGPx4-~ and WT animals [49]. 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 disulfide isomerases probably compensate for the lack of nGPx4 expression when the sperm cells travel along the epididymal tubule. Another possibility is that the cytosolic isoform (cGPx4), which is still expressed in testis of the nGPx4_/" mice, may partly back up nGPx4 deficiency since it is small enough to enter the nuclear pore [49, 76] . 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 sufficient H2O2 in the epididymal lumen.

The epididymal 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 seleno-cysteine (SeCys) residue in its catalytic site [6, 8] 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 this particularity, the scavenger activity of GPx5 was questioned. In cannonical GPx, if the SeCys residue was replaced by a cysteine amino acid, there was a dramatic drop in the enzyme activity [77]. However, it was 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 [53], It was also demonstrated that mice subjected to a selenium-free diet, depleting their Se-dependent GPx activities, show an overall increase in peroxidative injury in all tissues except in the epididymis, where GPx5 mRNA and protein levels were increased, backing up the failing Se-dependent activities [54], 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 [9]. Lack of GPx5 expression in the epididymial 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 sul-foxide 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 upregulated the three cytosolic GPx normally expressed there (GPx1, GPx3, and cGPx4). Upregulation of these epididymal GPx was sufficient to maintain the total GPx activity of the tissue at a normal value. Transcription of epithelial catalase was also increased in the cauda epididymidis of GPx5-~ animals, reinforcing the idea that the tissue was facing an increase in H2O2 [9] since catalase metabolizes only this substrate. These observations suggest that luminal ROS and especially H2O2 accumulate in the cauda compartment when GPx5 is 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, indicated by the increase in 8-oxo-deoxyguanosine residues (8-oxodG) associated with increased fragmentation and a slight nuclear decompaction state when compared to WT cauda-stored spermatozoa [9]. Although PRDX were not investigated in that study, it is possible that they also contributed to protect the cauda epididymidal epithelium and spermatozoa against the pro-oxidant situation generated in the GPx5-deficient context since several PRDX were very recently shown to be present on spermatozoa [78, 79],

Interestingly, in the caput epididymidis, sperm nuclei of the GPx5-~ animals were significantly more condensed than those of WT animals, suggesting that absence of H2 O2 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 in sperm nucleus protamines. If this is the correct hypothesis, then GPx5 that is secreted early 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 H2 O2 on spermatozoa that leads to DNA oxidation, increasing fragmentation, nucleus decompaction, and lipid peroxidation [ 9] . Spermatozoa themselves may contribute to this situation since 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 [80]. Thus, it is possible that cauda-stored spermatozoa, although not in optimal conditions of oxygen tension, pH, and energy substrate to sustain full mitochondrial activity, may contribute to the generation of free radicals via a leakage of the electron transport chain.

Oxidative damage of cauda-stored spermatozoa has been shown to increase in aging GPx5-~ animals [9], The oxidative insults on 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 [ 9] . In the absence of impact on fertilization rate and because female mice were perfectly normal, the type of defects observed 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 [ 81-83]. Such developmental defects due to alterations of paternal chromosomal material have already been reported in humans [84, 85],

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 development. Without this protective protein, male mice run a higher 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 also have an important effect on assisted reproductive technologies [9, 68, 82, 86-88] in which cryopreservation of the male gametes and micromanipulation in different media can be the sources of oxidative insults on the paternal chromosomal set.

5.3.4.1 Catalase

Among the primary antioxidant enzymes that have been studied in the epididymis, catalase is the least represented both at the mRNA and protein levels [89-91]. Since catalase is a cytosolic and not a secreted antioxidant, its contribution in protecting epididymal spermatozoa is only indirect. It is interesting to note that the cauda epididymis of GPx5-deficient animals responds to increased oxidative stress by transcriptional upregulation of cauda epithelial cell catalase. This demonstrates that, in a situation of oxidative burst, catalase is called on to protect the epididymis epithelium from H2O2-mediated damage. It, thus, limits the detrimental impacts of permeable H2O2 on the epididymis cell wall and has only a secondary role in protecting luminal spermatozoa from oxidative insults.

5.3.4.2 Indoleamine Dioxygenase

Indoleamine 2,3-dioxygenase (IDO) was once proposed to be a putative antioxi-dant in the mammalian epididymis because it uses as a cofactor the superoxide anion to catalyze the oxidative degradation of tryptophan into kynurenines and because, uniquely, this tissue shows constitutive expression of this enzyme. However, IDO is a heme oxygenase which, in the presence of oxygen, will readily regenerate as many superoxide anions as it consumes so that the intrinsic antioxi-dant impact of IDO is negligible. Some of the by-products of IDO activity are known to have antioxidant and pro-oxidant properties. We have recently shown that IDO activity in the epididymis leads essentially to the formation of kynurenic acid and 3-OH kynurenine [5]. Several reports have shown that 3-OH kynurenine induces ROS generation, mainly H,O2 and hydroxyl radicals, in vitro in primary striatal cultures as well as in various neuronal cell lines [92-96] . However, other reports demonstrated antioxidant properties of 3-OH kynurenine in the vascular system, the eye lens, and the brain [97-100]. Whether the epididymal kynurenines have antioxidant properties will have to wait further investigations.

5.3.4.3 Peroxiredoxins

PRDX are enzymes with a dual role as scavengers of ROS and modulators of ROS signaling. In mammals, six PRDX genes have been characterized, four of which, PRDX1 and PRDX4 to 6, have been found on spermatozoa at different subcellular localizations, such as head, acrosome, mitochondrial sheat, and flagellum [79] . They essentially originate from the expression of the corresponding genes in the testis during spermatogenesis. Although they may act as antioxidants in these discrete sperm locations, it is likely that they act essentially as modulators of ROS-signaling events leading to the ultimate maturation of these cells upon capacitation and acrosome reaction [ 45] . Spermatozoa PRDX are themselves susceptible to oxidative stress since it was shown that they are modified when spermatozoa are challenged with H2O2 impairing their signaling function [79].

5.3.4.4 Glutathione S-transferase

Glutathione ^-transferases (GSTs) constitute a family of enzymes that catalyze the conjugation of GSH to various compounds and doing so protect cellular constituents from oxidative attacks. They are not considered as primary antioxidants although in some situations GST harbors a GSH-dependent peroxidase activity and therefore may assist primary scavengers in recycling H2O2 [101]. The mammalian epididymis has been shown to express some GST isoforms, such as Yo and Yb1 of the mu subfamily [102, 103]. Spermatozoa have also been shown to carry GST of the mu and pi subclasses [104] at a significant level. Whether they are active players in protecting the epididymis epithelium and spermatozoa from peroxidative injuries is difficult to say with our present knowledge.

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100 Pregnancy Tips

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