*3.1. 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*−/<sup>−</sup>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.

**Figure 1.** The histology of the epididymis is affected by aging in wild-type mice and even more so in *Sod1*−/−mice. Representative pictures of epididymal sections (initial segment, caput, corpus, and cauda epididymides) stained with toluidine blue from 3-month-old and 18-month-old wild-type and *Sod1*−/− mice. An increase in luminal round cells (circles) and in the number and size of clear vesicles (arrows) were observed in the epididymal epithelium of the corpus and cauda epididymides (n = 3). Scale bar: 40 μm.
