The Exacerbation of Aging and Oxidative Stress in the Epididymis of Sod1 Null Mice

There is growing evidence that the quality of spermatozoa decreases with age and that children of older fathers have a higher incidence of birth defects and genetic mutations. The free radical theory of aging proposes that changes with aging are due to the accumulation of damage induced by exposure to excess reactive oxygen species. We showed previously that absence of the superoxide dismutase 1 (Sod1) antioxidant gene results in impaired mechanisms of repairing DNA damage in the testis in young Sod1−/− mice. In this study, we examined the effects of aging and the Sod−/− mutation on mice epididymal histology and the expression of markers of oxidative damage. We found that both oxidative nucleic acid damage (via 8-hydroxyguanosine) and lipid peroxidation (via 4-hydroxynonenal) increased with age and in Sod1−/− mice. These findings indicate that lack of SOD1 results in an exacerbation of the oxidative damage accumulation-related aging phenotype.


Introduction
Reactive oxygen species (ROS) generate chain reactions that can affect numerous biomolecules, including lipids, RNA, DNA, proteins [1][2][3][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][6][7][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][12][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 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][47][48][49][50][51][52][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.

Animal Model
B6.129S7-Sod1 tm1Leb /DnJ mice were purchased from the Jackson Laboratory (#003881, Bar Harbor, ME, USA). They were backcrossed with C57Bl/6NJ mice (#005304, Bar Harbor, ME, USA) and bred in-house at McGill University in the McIntyre Comparative Medicine and Animal Resources Centre. Mice were kept on a 12L:12D cycle; food and water were provided ad libitum, and the temperature was maintained at 22 • C. Young (3 to 4-month-old) and aged (18-month-old) wild-type (WT) and Sod1 null (Sod1 −/− ) mutants mice were selected for this study. The number of mice in each group was from 3 to 6. Mice were euthanized by CO 2 asphyxiation followed by cervical dislocation. Both epididymides were harvested. All animal studies were conducted in accordance with the principles and procedures outlined in the Guide to the Care and Use of Experimental Animals prepared by the Canadian Council on Animal Care (McGill Animal Care Committee protocol #4687).

Immunofluorescence Imaging and Quantification
Imaging of each full section was done with the Opera Phenix™ high-content screening system and Harmony software (Perkin Elmer, Montreal, QC, Canada) with a field overlap of 5%, at a 40× magnification. The images were further analyzed using Columbus™ system (Perkin Elmer, Montreal, QC, Canada) to quantify the signal of the immunofluorescent staining. Analyses were applied which recognized DAPI as a nucleus marker and SRC as a cytoplasmic marker to segment the cells for quantification of fluorescence intensity in each cellular compartment. The 4-HNE and 8-OHG intensities were used to determine oxidative damage in membranes and DNA, respectively. Analyses were done separately in the caput, corpus and cauda epididymides, as well as in the epithelium and in the interstitial cells (endothelial, smooth muscle, and conjunctive cells).

Statistical Analyses
All graphical data are represented as the means ± SEM. Prior to analysis, data were determined to be normally distributed. All data were then analyzed using a 3-way analysis of variance ANOVA with genotype, epididymal segment and age as variables, followed by Tukey's multiple comparisons test. All statistical analyses were done with the software Prism8 v8.0.1 (GraphPad Software, La Jolla, CA, USA). Differences among samples were considered to be significant when the p < 0.05.

Histology
Representative sections of the initial segment, caput, corpus and cauda regions of the epididymis of young and old wild-type and Sod −/− mice are shown in Figure 1. No major differences were observed between young Sod1 −/− and WT mice (3 months) with respect to either the epididymal tubule histology (Figure 1) or the interstitial tissue histology (smooth muscle cells, conjunctive tissue, vascular system) (data not shown). In old (18 months) Sod1 −/− and WT mice distinctive features included an increase in the tubule diameter all along the epididymis, a decrease in the height of epididymal cells, as well as an accumulation of spermatozoa in the initial segments, where they are usually absent. There was also an increase in luminal round cells (circles). Further, we observed the presence of large vacuoles in some epithelial cells in the corpus and cauda epididymides (arrows). The thickness of the myoid layer surrounding the epididymal tubule was increased only in the cauda epididymidis. Among these features, only two were enhanced by the absence of the SOD1 protein in this tissue. The accumulation of cellular vacuoles in the cauda epididymidis was increased in old Sod1 −/− mice compared to old WT mice. Moreover, the thickening of the myoid layer was greater in old Sod1 −/− mice than in old WT mice, but this is also visible in cauda epididymidis of young Sod1 −/− mice. In one old Sod1 −/− mouse, the accumulation of an amorphous matter in the epididymal lumen of the caput, corpus, and cauda epididymides was associated with round cells only in the cauda epididymides [53]. Thus, overall our observations indicate that aging affects the histology of the epididymis and this phenotype is enhanced by the absence of the SOD1 protein.
epididymidis. Among these features, only two were enhanced by the absence of the SOD1 protein in this tissue. The accumulation of cellular vacuoles in the cauda epididymidis was increased in old Sod1 −/− mice compared to old WT mice. Moreover, the thickening of the myoid layer was greater in old Sod1 −/− mice than in old WT mice, but this is also visible in cauda epididymidis of young Sod1 −/− mice. In one old Sod1 −/− mouse, the accumulation of an amorphous matter in the epididymal lumen of the caput, corpus, and cauda epididymides was associated with round cells only in the cauda epididymides [53]. Thus, overall our observations indicate that aging affects the histology of the epididymis and this phenotype is enhanced by the absence of the SOD1 protein.

DNA Oxidation
DNA oxidation was assessed by quantitative immunostaining of the oxidized guanine in the nucleus (8-OHdG). Immunofluorescent staining was detected in both epithelial and interstitial cells of the different regions of the epididymis (initial segment, caput, corpus, and cauda) (Figure 2). In 18month-old mice, staining was more intense in the cauda epididymidis than in the rest of the tissue; further, this increase was more pronounced in the distal cauda epididymidis than in the proximal cauda epididymidis. Quantification of nuclear 8-OHdG immunofluorescent staining (Figure 2 bar graph) indicated that the major increase in DNA oxidation was observed in the cauda epididymidis of 18-month-old Sod1 −/− mice. Statistical analysis by 3-way ANOVA (age, genotype, region, Table 2) revealed that the main source of variation in the DNA oxidation levels was the Sod1 −/− genotype; in

DNA Oxidation
DNA oxidation was assessed by quantitative immunostaining of the oxidized guanine in the nucleus (8-OHdG). Immunofluorescent staining was detected in both epithelial and interstitial cells of the different regions of the epididymis (initial segment, caput, corpus, and cauda) (Figure 2). In 18-month-old mice, staining was more intense in the cauda epididymidis than in the rest of the tissue; further, this increase was more pronounced in the distal cauda epididymidis than in the proximal cauda epididymidis. Quantification of nuclear 8-OHdG immunofluorescent staining (Figure 2 bar graph) indicated that the major increase in DNA oxidation was observed in the cauda epididymidis of 18-month-old Sod1 −/− mice. Statistical analysis by 3-way ANOVA (age, genotype, region, Table 2) revealed that the main source of variation in the DNA oxidation levels was the Sod1 −/− genotype; in addition, there were interactions between epididymal regions and age and between age and genotype. addition, there were interactions between epididymal regions and age and between age and genotype.

Lipid Peroxidation
Lipid peroxidation was evaluated by immunostaining of 4-hydroxynonenal (4-HNE) protein adducts, a by-product of lipid peroxidation. Longitudinal sections of the epididymides of 18-month-old WT and Sod1 −/− mice revealed major differences in 4-HNE staining intensity and its regional distribution (Figure 3). Staining was absent in the initial segment and the caput epididymidis and was found almost exclusively in the corpus and the cauda epididymides. Further, in 18-month-old Sod1 −/− mice, it appeared to be higher in the distal than in the proximal cauda epididymidis. The 4-HNE staining was detected mainly on cytoplasmic membranes, particularly on the apical membranes; in highly stained cells, it was also visible on intracellular and nuclear membranes. Quantitative analyses were done for staining on all cell surfaces. In the caput epididymidis, staining was indistinguishable to that seen in the control sections without primary antibody. The 3-way ANOVA analysis on quantitative data clearly indicated that there was a significant difference between the WT and Sod1 −/− genotypes and that this effect was amplified by the age of the mice ( Table 2).

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 effects 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][47][48][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 −/− , 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 segment 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 affected 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 segment 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 affect 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 suggest that Sod1 −/− mice constitute a valuable model for better understanding aging in the epididymis.

Conclusions
Aging is a progressive biological process that is characterized by an accumulation of various physiological and molecular changes, including an increase in oxidative damage to different biomolecules, leading to cell and tissue dysfunctions. In this study, we evaluated the impact on the epididymis of removing SOD1 using Sod1 −/− mice. We demonstrated that, using this rodent model, the aging phenotype is exacerbated by increased oxidative damage.