No abstract

We have isolated vesicular structures from mouse epididymal fluid, referred to as epididymosomes. Epididymosomes have a roughly spherical aspect and a bilayer membrane, and they are heterogeneous in size and content. They originate from the epididymal epithelium, notably from the caput region, and are emitted in the epididymal lumen by way of apocrine secretion. We characterized their membranous lipid profiles in caput and cauda epididymidal fluid samples and found that epididymosomes were particularly rich in sphingomyelin (SM) and arachidonic acid. The proportion of SM increased markedly during epididymal transit and represented half the total phospholipids in cauda epididymidal epididymosomes. The cholesterol:phospholipid ratio increased from 0.26 in the caput to 0.48 in the cauda epididymidis. Measures of epididymosomal membrane anisotropy revealed that epididymosomes became more rigid during epididymal transit, in agreement with their lipid composition. In addition, we have characterized the membrane lipid pattern of murine epididymal spermatozoa during their maturation. Here, we have shown that mouse epididymal spermatozoa were distinguished by high percentages of SM and polyunsaturated membranous fatty acids (PUFAs), principally represented by arachidonic, docosapentanoic, and docosahexanoic acids. Both SM and PUFA increased throughout the epididymal tract. In particular, we observed a threefold rise in the ratio of docosapentanoic acid. Epididymal spermatozoa had a constant cholesterol:phospholipid ratio (average, 0.30) during epididymal transit. These data suggest that in contrast with epididymosomes, spermatozoal membranes seem to become more fluid during epididymal maturation.

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.

Mitochondrial DNA (mtDNA) can undergo double-strand breaks (DSBs), caused by defective replication, or by various endogenous or exogenous sources, such as reactive oxygen species, chemotherapeutic agents or ionizing radiations. MtDNA encodes for proteins involved in ATP production, and maintenance of genome integrity following DSBs is thus of crucial importance. However, the mechanisms involved in mtDNA maintenance after DSBs remain unknown. In this study, we investigated the consequences of the production of mtDNA DSBs using a human inducible cell system expressing the restriction enzyme PstI targeted to mitochondria. Using this system, we could not find any support for DSB repair of mtDNA. Instead we observed a loss of the damaged mtDNA molecules and a severe decrease in mtDNA content. We demonstrate that none of the known mitochondrial nucleases are involved in the mtDNA degradation and that the DNA loss is not due to autophagy, mitophagy or apoptosis. Our study suggests that a still uncharacterized pathway for the targeted degradation of damaged mtDNA in a mitophagy/autophagy-independent manner is present in mitochondria, and might provide the main mechanism used by the cells to deal with DSBs.

Epididymal sperm maturation culminates in the acquisition of functional competence by testicular spermatozoa. The expression of this functional state is dependent upon a redox-regulated, cAMP-mediated signal transduction cascade that controls the tyrosine phosphorylation status of the spermatozoa during capacitation. Analysis of superoxide anion (O2(-.)) generation by rat epididymal spermatozoa has revealed a two-component process involving electron leakage from the sperm mitochondria at complexes I and II and a plasma membrane NAD(P)H oxidoreductase. Following incubation in a glucose-, lactate-, and pyruvate-free medium (-GLP), O2(-.) generation was suppressed by 86% and 96% in caput and cauda spermatozoa, respectively. The addition of lactate, malate, or succinate to spermatozoa incubated in medium -GLP stimulated O2(-.) generation. This increase could be blocked by rotenone and oligomycin (R/O) in the presence of malate or lactate but not succinate. Stimulation with all three substrates, as well as spontaneous O2(-.) production in +GLP medium, was blocked by the flavoprotein inhibitor, diphenylene iodonium. Diphenylene iodonium, but not R/O, suppressed NAD(P)H-induced lucigenin-dependent chemiluminescence. This NAD(P)H-dependent enzyme resided in the sperm plasma membrane and its activity was regulated by zinc and uncharacterized cytosolic factors. Reverse transcription-polymerase chain reaction analysis indicated that the sperm NAD(P)H oxidoreductase complex is quite distinct from the equivalent leukocyte system.

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.

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.

In mice, GPX5 is a secreted protein abundantly synthesized by the caput epididymidis. The protein is secreted as early as the initial segment of the caput and is found subsequently associated with the sperm plasma membrane in a sub-acrosomic localization. We show here that GPX5 is present in the caput and cauda epididymides lumens in three different locations: either free as a soluble protein in the caput epididymal fluid, weakly bound to caput sperm membranes, or, finally, associated to lipid-containing structures conferring to the protein a protective effect against proteolytic digestions. Within the cauda epididymidis, the amount of free GPX5 is low compared to the caput and the association with sperm membranes proved to be more solid. In both caput and cauda sperm samples, the association of GPX5 with the sperm membrane protects GPX5 from proteolytic cleavages. Protection against proteolytic digestions can be overcome by physical treatments of epididymal fluid and sperm samples such as ultrasounds or very acidic pH. These data suggest that complex phenomena and structures participate in the transfer and binding of the caput-secreted GPX5 protein to the sperm plasma membrane.