**1. Introduction**

Reactive oxygen species (ROS) generate chain reactions that can affect numerous biomolecules, including lipids, RNA, DNA, proteins [1–4]. The consequences of these biochemical reactions are part of the normal cellular function but, in excess, they can be deleterious and severely damage major cellular processes. We will focus on the impact of oxidative stress during aging in the male reproductive system and, specifically, in the epididymis. Most studies on the effects of aging on the epididymis were done using the Brown Norway rat model, a reliable model of aging [5–8]. The major characteristics of aging in the epididymis are a thickening of the basement membrane along the epididymis, a decreased integrity and functionality of the blood-epididymis barrier [9], a segment-dependent variation of each cell type distribution; in particular a decrease in the proportion of principal, clear, and basal cells and an increase in the number of halo cells in the entire epididymis was found [10]. Halo cells are immune cells and their increased number in the epididymal epithelium may reflect a lack of balance of the immune steady-state of the epididymis, in accordance with the proposed general hallmarks of aging [11].

Aging is a dynamic and continuous process with time-dependent physiological and molecular changes. Some of the major hallmarks of aging include changes in telomere length, genetic instability and an accumulation of epigenetic modifications, mitochondrial dysfunction, a decrease in the stem cell reserve, and chronic inflammation [11–13]. In 1956, a "Free Radical Theory of Aging" was proposed that states that aging is the consequence of an accumulation of oxidative damage on biomolecules [14,15].

In most species, aging is associated with an accumulation of oxidized biomolecules (proteins, nucleic acids, and lipids), resulting in the dysregulation of metabolism, with an increase in the leak of superoxide anions by the mitochondria and a decrease in the ability of cells to scavenge ROS and to repair oxidative damage. ROS play a major role in the aging process via their action on various signaling pathways, including insulin/insulin like growth factor 1 (IGF1), mammalian target of rapamycin kinase (mTOR) and AMP kinase (AMPK) [16–21]. The null mutation of *Igf1* in mice induces a shrinking of the epididymis from the corpus epididymidis to the vas deferens due to a major decrease in the sperm cell content of the tubule [22]. *Igf1* gene expression is induced in the epididymis of orchidectomized adult rats and is under an androgenic control [23].

ROS play essential roles in male reproduction. One of these is the production of disulfide bridges between cysteine thiol groups; this biochemical reaction is mandatory for the post-translational maturation of numerous proteins and various enzymatic reactions. During spermiogenesis, sperm DNA is compacted first through replacement of histones by transition proteins and then by cysteine-rich protamines [24,25]. Therefore, numerous disulfide bonds are generated when condensing sperm nucleus undergo 3D re-organization during spermatid elongation and spermatozoal maturation in the epididymis. Mice knocked out for the mitochondrial isoform of *Gpv4* (mGPX4) are infertile, due to non-motile spermatozoa with abnormal mid-piece and broken flagella [26,27]. Another form of this enzyme that is considered to play a major role in this process is the sperm nuclear isoform of GPX4 (snGPX4) [28–31]. The *snGpx4*−/− mice are viable and fertile, but their sperm chromatin shows a delay in chromatin condensation during maturation in the epididymis that is linked to a decrease in disulfide bridging in the caput epididymal sperm. Finally, the acrosome matrix is structured and held by numerous disulfide bridges and the inhibition of the disulfide isomerase enzymatic activity by sperm pre-treatment decreases the fertilization level during sperm-egg fusion in vitro [32].

ROS are also an essential component of the redox signaling pathways during sperm capacitation in the female tract. This process involves a complex group of sperm modifications to prepare the spermatozoa to undergo flagellar hyperactivity and the acrosome reaction, both necessary to the journey through the female tract and for sperm-egg recognition and fusion. Numerous reviews have provided excellent description of this process [33,34] and the role of ROS in this phenomenon [35–37].

However, despite the obvious need of ROS for sperm production, maturation, and function, spermatozoa are highly sensitive to oxidative damage. ROS can modify purine and pyrimidine bases, break nucleic acid backbone, and create covalent bonds intra-/inter-strands, and between nucleic acids and proteins [38]. The main target of ROS is guanine due to its lower redox potential, with the major by-product of the DNA oxidation being 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-OHdG); this oxidative DNA damage can be lethal for cells. While spermatogonia and spermatocytes have extensive DNA repair machinery, condensing spermatids and spermatozoa lose their DNA repair capacity [39–41].

Lipids are also targets of ROS, especially sterols and polyunsaturated fatty acids; these lipids affect the fluidity of the sperm cytoplasmic membrane. Lipid peroxidation can result in the generation of numerous toxic side products (alkanes, aldehydes, and acids), including 4-hydroxy-2-nonenal (4-HNE) which is highly cytotoxic, a molecule that is commonly used as a marker of lipid peroxidation. Lipid peroxidation decreases the fluidity of cell membranes, a major concern for sperm motility and ability to undergo capacitation and sperm-egg fusion [42,43].

Redox balance must be tightly maintained to limit the negative effects of ROS on reproductive tissues and spermatozoa, particularly during epididymal maturation when sperm are devoid of the ability to react to their environment. Consequently, the epididymal epithelium must play a protective role. A wide array of antioxidant enzymes is present in the epididymis, both in the epithelium and the luminal fluid; these include superoxide dismutases (SODs), catalase, glutathione transferases, glutathione peroxidases, and peroxiredoxins [44].

SODs are a family of metalloproteins composed of three different members, located in different cellular compartments and associated with different metal ions. SOD1, or Cu/ZnSOD is located in the intermembrane space of mitochondria, in the nucleus and in the cytoplasm [45]. Mutations of the *Sod1* gene have been associated with human pathologies such as amyotrophic lateral sclerosis [46]. Male mice lacking the ability to synthesize SOD1 are fertile but in vitro studies demonstrate that several characteristics of spermatozoa from *Sod1*−/− mice are affected, including higher lipid peroxidation, decreased motility, impaired capacitation ability, and a low fertilizing ability in in vitro fertilization [47].

*Sod1*−/− mice with a C57Bl/6 genetic background provide an interesting rodent model to test the free radical theory of aging and the mechanisms involved in the aging process. The phenotype of C57Bl/6 mice during aging is well characterized, as this animal model is often used for aging studies [48,49]. Despite a relatively normal phenotype and health during development and young adulthood, *Sod1*−/− mice have a reduced lifespan of 20.8 ± 0.7 (mean ± S.D.) months compared to the lifespan of wild-type mice of 29.8 ± 2.1 months [50]. The absence of SOD1 leads to extensive oxidative damage in various tissues including the intestine, liver, ovary, and testis [46–53] as early as 3 months of age and even in fetal fibroblasts [54]. In previous studies of the effects of the lack of SOD1 on the male reproductive system, we reported an impact on the male germline with a decreased ability to scavenge ROS, an increased number of oxidized spermatozoa, and a reduced DNA repair machinery [53]. In the present study, we examine the effects of aging on the epididymis of wild-type and *Sod1*−/− mice.
