**4. Discussion**

Various theories have been proposed to explain the complex physiological and molecular modifications that occur during aging [13]. Among the potential causes and/or consequences of aging are an increase in oxidative stress and the accumulation of oxidative damage on many cellular components. Thus, to study the e ffects and mechanisms of aging in the male reproductive system, we have investigated various animal models, particularly rats and mice, with or without a genetically induced oxidative stress [53,56]. In this study, we analyzed the consequences of aging and oxidative stress in the epididymis, the site where spermatozoa become mature, using *Sod*−/− mice. *Sod1*−/− mice have a decreased ability to protect against ROS attack, leading to an accumulation of oxidative damage even in young animals, and have a reduced lifespan [46–49].

We analyzed the histology of the epididymis of 3- and 18-month-old wild-type and *Sod1*−/−mice. We identified some features that are indicative of a decline in epididymis structure and function with aging. The most obvious phenotypic change observed in old WT and *Sod1*−/−mice is the appearance of vacuoles in the epididymal epithelial cells, selectively in the corpus and the cauda epididymides. This feature was described previously in the corpus and proximal cauda epididymides of old Brown Norway rats [10]. The vacuoles found in principal cells were identified as giant lysosomes and lipid droplets, suggesting that the digestion/recycling system and/or the intracellular trafficking are dysregulated during aging. Further analyses in these old Brown Norway rats demonstrated a variation of the expression of glutathione S-transferases, enzymes that play an important role in detoxification of electrophiles. Thus, it would be interesting to study the effects of aging on the expression of genes/proteins in the epididymis of the aging *Sod1*−/−mice to determine if the same process occurs in mice.

Interestingly, we observed an increase in the number of round cells in the epididymal lumen of old mice compared to young mice; the nature of these cells is not clear. These cells were identified as round spermatids in some animal models of spermatogenic arrest but previous analyses of the testis in *Sod1*−/− mice did not reveal this phenotype [49]. Chronic and systemic inflammation is a hallmark of aging [11,12]. The increase in the number of halo cells (resident immune cells of the epididymis) along the epithelium during aging [57], in conjunction with the demonstration of dendritic cells [58], indicates a close interaction between the immune system and the epididymis. Therefore, it is interesting to speculate that these intraluminal round cells could be infiltrating immune cells, passed through the less impermeable blood-epididymis barrier of old animals [9]. An analysis of the blood-epididymis barrier and of the potential immune markers on the intraluminal round cells would be necessary to test this hypothesis,

The increase in the tubule diameter along the epididymis in old (18 month) *Sod1*−/<sup>−</sup>, accompanied by a decrease in epithelial cell height, is consistent with previous observations in C57Bl/6NJ mice [53]. Surprisingly, opposite histological features of aging have been observed in old Brown Norway rats [6]. In the hamster, the diameter of the lumen also decreases whereas epithelial height does not change between young and old animals [59].

Spermatozoa were present consistently in the lumen of the initial segmen<sup>t</sup> of the epididymis of young and old *Sod1*−/− mice compared to wild-type animals. This may be due to a decrease in the ability of the epididymis to transport spermatozoa. This transport is dependent on peristaltic contractions of the smooth muscle layer surrounding the tubule, pressure from the testicular fluid and new spermatozoa, and the movement of stereocilia at the apical pole of epithelial cells [60]. Another indication that the ability of the epididymis to transport spermatozoa is a ffected by aging is the increase in the smooth muscle layer in the distal cauda epididymidis where spermatozoa are stored between two ejaculations.

A progressive increase in the nucleic acid oxidation in the lumen of the tubule, from the caput epididymidis to the distal cauda epididymidis, was observed in 18-month-old mice, but not in 3-month-old wild-type mice. This phenomenon could be due to the increased quantity of spermatozoa contained in the lumen as long as they travel down the epididymis and to the increased generation of ROS by spermatozoa during aging [61].

Finally, we assessed 4-HNE immunofluorescent staining as a marker of lipid peroxidation. Lipid peroxidation appeared to be absent in the initial segmen<sup>t</sup> and the caput epididymidis and was only visible in the corpus and the cauda epididymidis. The staining was low in 3-month-old mice and increased with aging. As for the DNA oxidation, it increased along the tubule. The apical membranes of epithelial cells appeared to be more sensitive to lipid peroxidation. The reasons for the apical susceptibility to oxidative damage is unknown. It could be due to the direct contact with the luminal fluid and spermatozoa, a potential source of ROS, or to the high activity of this membrane (exocytosis, endocytosis, sensing of the luminal contents) which generates numerous reactions, another possible source of ROS.

The absence of SOD1 in the mouse epididymis did not a ffect overall oxidation as assessed by markers of DNA and lipid oxidation in 3-month-old mice. Other members of the SOD family may compensate for this loss and the repair machinery of the various cell components seems able to deal with the low level of oxidized biomolecules. However, oxidized nucleic acids and peroxidized lipids increased strikingly in the epididymal tissue of 18-month-old *Sod1*−/− mice, even compared to the old wild-type mice. Histological analyses revealed an increase in the quantity and size of vacuoles in the epididymal epithelial cells of the corpus and the cauda epididymides and an increase in the thickening of the myoid cell layer in the distal cauda epididymidis in old *Sod1*−/− mice compared to old wild-type mice. Thus, there is clearly a worsening of the aging phenotype in the mouse epididymis in the absence of SOD1. These findings sugges<sup>t</sup> that *Sod1*−/− mice constitute a valuable model for better understanding aging in the epididymis.
