*3.2. 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.



\* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001, \*\*\*\* *p* < 0.0001.

**Figure 2.** Aging induces an accumulation the nucleic acid damage in the epididymis (initial segment, caput, corpus, and cauda epididymides) which is enhanced in *Sod1*−/−mice. Representative pictures of sections of the epididymis from 3-month-old and 18-month-old wild-type and *Sod1*−/−mice after immunostaining of oxidized nucleic acids (8-OHG, red); the nuclei are counterstained with DAPI (blue). Immunostaining negative controls (no primary antibody) are displayed for the caput and cauda epididymides. Scale bar: 40 μm. 8-OHG staining was quantified separately in the epididymal epithelium (clear histograms) and in the interstitial tissue (dashed histograms). \*\* *p* < 0.01, \*\*\* *p* < 0.001 (3-way ANOVA, n = 4–5).
