The mammalian epididymis provides sperm with an environment that promotes their maturation and protects them from external stresses. For example, it harbors an array of antioxidants, including non-conventional glutathione peroxidase 5 (GPX5), to protect them from oxidative stress. To explore the role of GPX5 in the epididymis, we generated mice that lack epididymal expression of the enzyme. Histological analyses of Gpx5 -/-epididymides and sperm cells revealed no obvious defects. Furthermore, there were no apparent differences in the fertilization rate of sexually mature Gpx5 -/-male mice compared with WT male mice. However, a higher incidence of miscarriages and developmental defects were observed when WT female mice were mated with Gpx5-deficient males over 1 year old compared with WT males of the same age. Flow cytometric analysis of spermatozoa recovered from Gpx5-null and WT male mice revealed that sperm DNA compaction was substantially lower in the cauda epididymides of Gpx5-null animals and that they suffered from DNA oxidative attacks. Real-time PCR analysis of enzymatic scavengers expressed in the mouse epididymis indicated that the cauda epididymidis epithelium of Gpx5-null male mice mounted an antioxidant response to cope with an excess of ROS. These observations suggest that GPX5 is a potent antioxidant scavenger in the luminal compartment of the mouse cauda epididymidis that protects spermatozoa from oxidative injuries that could compromise their integrity and, consequently, embryo viability.

In mammals, posttesticular epididymal sperm maturation is considered an essential step in the transformation of immature testicular gametes to mature spermatozoa capable of fertilization. Reactive oxygen species (ROS) have been shown to be key actors in this maturation process, and it is now clear that ROS are central for sperm physiology in processes such as sperm maturation and capacitation. However, during epididymal maturation and storage and until the onset of fertilization, oxidative damage is a threat spermatozoa must face more than any other cells. Spermatozoa were found to be extremely sensitive to oxidative attacks correlated with lipid peroxidation, DNA damage, and impaired sperm motility, all affecting fertilization. To control the quantity of H(2)O(2) in the vicinity of male gametes, mammalian epididymis uses a panel of nonenzymatic and enzymatic scavengers, among which the glutathione peroxidase (GPx) family is largely represented. Among the various GPx proteins expressed in the mammalian epididymis, GPx4 and GPx5 occupy unique positions and functions that are reviewed in this paper. This paper underlines the importance of the GPx protein family in determining the fertilizing potential of mammalian spermatozoa. This is particularly relevant in the field of mammalian fertility and infertility as well as in the development of assisted medical procreation technologies and male gamete preservation techniques that are extensively used in human and animal reproduction programs.

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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.

Indoleamine 2,3-dioxygenase (IDO) is the first and rate-limiting enzyme of tryptophan catabolism through the kynurenine pathway. Intriguingly, IDO is constitutively and highly expressed in the mammalian epididymis in contrast to most other tissues where IDO is induced by proinflammatory cytokines, such as interferons. To gain insight into the role of IDO in the physiology of the mammalian epididymis, we studied both wild type and Ido1 ؊/؊ -deficient mice. In the caput epididymis of Ido1؊/؊ animals, the lack of IDO activity was not compensated by other tryptophan-catabolizing enzymes and led to the loss of kynurenine production. The absence of IDO generated an inflammatory state in the caput epididymis as revealed by an increased accumulation of various inflammation markers. The absence of IDO also increased the tryptophan content of the caput epididymis and generated a parallel increase in caput epididymal protein content as a consequence of deficient proteasomal activity. Surprisingly, the lack of IDO expression had no noticeable impact on overall male fertility but did induce highly significant increases in both the number and the percentage of abnormal spermatozoa. These changes coincided with a significant decrease in white blood cell count in epididymal fluid compared with wild type mice. These data provide support for IDO playing a hitherto unsuspected role in sperm quality control in the epididymis involving the ubiquitination of defective spermatozoa and their subsequent removal.Indoleamine 2,3-dioxygenase (IDO) 3 (EC 1.13.11.42) is the first and rate-limiting enzyme in Trp catabolism through the kynurenine pathway (Fig. 1). IDO is a ubiquitously expressed cytoplasmic protein typically activated by interferons (IFNs) (1-5). There is ample evidence that IDO mediates potent immunosuppression in classical immune responses as well as in fetal tolerance, tumor immune resistance, and regulation of autoimmune responses (1-3, 6 -8).Thirty years ago, Yoshida et al. (9) reported that rodent epididymal protein extracts exhibited a high IDO activity. Later, Takikawa et al. (10) demonstrated that unlike the classical cytokine-mediated expression of IDO encountered in nearly all mammalian tissues, the epididymal expression of IDO was constitutive and independent of IFN-␥. More recently, we have shown that IDO is expressed in a regionalized manner by both the principal and the apical cells of the most proximal epididymal region, the caput epididymis. To gain insights into the functions of IDO and the intermediates of the kynurenine pathway in the physiology of the mammalian epididymis, we measured the expression of IDO and related enzymes as well as the abundance of kynurenines and other Trp metabolites in both wild type (WT) and Ido1 Ϫ/Ϫ male mice. These data were correlated with light and electron microscopic analyses of epididymal epithelium, sperm count, sperm morphology, and fertility.

This work shows that an overload of dietary cholesterol causes complete infertility in dyslipidemic male mice (the Liver X Receptor-deficient mouse model). Infertility resulted from post-testicular defects affecting the fertilizing potential of spermatozoa. Spermatozoa of cholesterol-fed lxr−/− animals were found to be dramatically less viable and motile, and highly susceptible to undergo a premature acrosome reaction. We also provide evidence, that this lipid-induced infertility is associated with the accelerated appearance of a highly regionalized epididymal phenotype in segments 1 and 2 of the caput epididymidis that was otherwise only observed in aged LXR-deficient males. The epididymal epithelial phenotype is characterized by peritubular accumulation of cholesteryl ester lipid droplets in smooth muscle cells lining the epididymal duct, leading to their transdifferentiation into foam cells that eventually migrate through the duct wall, a situation that resembles the inflammatory atherosclerotic process. These findings establish the high level of susceptibility of epididymal sperm maturation to dietary cholesterol overload and could partly explain reproductive failures encountered by young dyslipidemic men as well as ageing males wishing to reproduce.

This article is available online at http://www.jlr.org tozoa to fully mature cells ( 1 , 2 ). Principal aspects of epididymal transformation include functional maturation, sperm concentration, storage of the spermatozoa in a quiescent state, and removal of degenerating cells. Functional maturation of these spermatozoa requires interaction of sperm with luminal fl uid, whose composition is regulated by absorption and secretion activities of the epididymal epithelium ( 3 ).The sperm plasma membrane is the site of dramatic changes throughout the development of these cells, and, in the epididymis, the remodeling process includes important modifi cations in lipid composition as well as repositioning of lipid and protein components to different membrane domains ( 4 ). The lipid composition of mammalian spermatozoa is specifi c with a large amount of plasmalogen phospholipids (PLs), a high content of long-chain PUFAs, mostly arachidonic acid (20:4, n-6), docosapentaenoic acid (22:5, n-3), and docosahexaenoic acid (22:6, n-3), and relatively low cholesterol:PL ratios ranging from 0.24 for caput epididymidis sperm to 0.29 for cauda epididymidis sperm in mice ( 5 ). This ratio is an indicator of membrane fl uidity, and although it is quite stable during epididymal transit, membrane fl uidity was shown to increase during this step ( 6 ). Earlier work showed a significant decrease of the two major PLs, phosphatidylcholine and phosphatidylethanolamine, in mouse spermatozoa from cauda epididymidis compared with those from caput epididymidis ( 5 ). During epididymal maturation, stearic acid (C18:0) decreased also, whereas palmitic acid (C16:0) increased. Among the PUFAs, docosapentaenoic and docosahexaenoic acids rose signifi cantly in the cauda epididymidis. Cholesterol content also declined by roughly 65% during epididymal maturation ( 5 ). These modifi cations have been supposed to prepare sperm cells for capacitation and acrosome reaction that take place in the Abstract Mammalian spermatozoa undergo important plasma membrane maturation steps during epididymal transit. Among these, changes in lipids and cholesterol are of particular interest as they are necessary for fertilization. However, molecular mechanisms regulating these transformations inside the epididymis are still poorly understood. Liver X receptors (LXRs), the nuclear receptors for oxysterols, are of major importance in intracellular cholesterol homeostasis, and LXR ؊ / ؊ -defi cient male mice have already been shown to have reduced fertility at an age of 5 months and complete sterility for 9-month-old animals. This sterility phenotype is associated with testes and caput epididymides epithelial defects. The research presented here was aimed at investigating how LXRs act in the male caput epididymidis by analyzing key actors in cholesterol homeostasis. We show that accumulation of cholesteryl esters in LXR The epididymis is an essential organ for reproductive physiology as sperm cells start the process of posttesticular maturation during their transit in this or...

The epididymis maintains a state of immune tolerance towards spermatozoa while also protecting them and itself against infection and acute inflammation. The immunosuppressive enzyme indoleamine 2,3-dioxygenase 1 (Ido1) participates in this delicate local equilibrium. Using the mouse Ido1−/− model, we show here that the absence of IDO1 expression leads in the epididymis but not in serum to (1) an increase in the inflammatory state as evidenced by changes in the content of cytokines and chemokines, (2) the engagement of a Th1-driven inflammatory response as evidenced by changes in the Th17/Treg as well as Th1/Th2 equilibria, as well as (3) differences in the content of lipid intermediates classically involved in inflammation. Despite this more pronounced inflammatory state, Ido1−/− animals succeed in preserving the local epididymal immune situation due to the activation of compensatory mechanisms that are discussed.