S U M M A R Y Water content within the epididymis of the male reproductive system is stringently regulated to promote sperm maturation. Several members of the aquaporin (AQP) family of water channel-forming integral membrane proteins have been identified in epididymal cells, but expression profiling for this epithelium is presently incomplete, and no AQP isoform has yet been identified on basolateral plasma membranes of these cells. In this study, we explored AQP expression by RT-PCR and light microscopy immunolocalizations using peroxidase and wide-field fluorescence techniques. The results indicate that several AQPs are coexpressed in the epididymis including AQP 5, 7, 9, and 11. Immunolocalizations suggested complex patterns in the spatial distribution of these AQPs. In principal cells, AQP 9 and 11 were present mainly on microvilli, whereas AQP 7 was localized primarily to lateral and then to basal plasma membranes in a region-specific manner. AQP 5 was also expressed regionally but was associated with membranes of endosomes. Additionally, AQPs were expressed by some but not all basal (AQP 7 and 11), clear (AQP 7 and 9), and halo (AQP 7 and 11) cells. These findings indicate unique associations of AQPs with specific membrane domains in a cell type-and region-specific manner within the epididymis of adult animals.

Past studies have shown that the epithelial lining of the epididymis in adult mice deficient in protective protein cathepsin A (PPCA 2/2) becomes swollen and vacuolated as a result of an accumulation of pale lysosomes, some very large, in addition to the presence of an abundance of macrophages infiltrating the intertubular spaces. The purpose of this study was to assess the integrity of the epididymal epithelial-blood barrier in these altered mice by characterizing the distribution of claudins (Cldns) and the leakiness of tight junctions to lanthanum nitrate. A second goal was to characterize sperm motility behavior in PPCA 2/2 mice using computer-assisted sperm analyses (CASA). The results indicated that lanthanum nitrate penetrated apical junctional complexes between adjacent epithelial cells and entered the epididymal lumen in PPCA 2/2 mice but not in control PPCA +/+ mice. Immunostaining for Cldns 1, 3, 8, and 10 revealed unique patterns of expression based on cell type and region specificity in PPCA +/+ mice, which were much different in PPCA 2/2 mice. PPCA 2/2 mice showed reduced intensities of immunoreactions, complete absence of immunoreactions, and appearance of atypical cytoplasmic immunoreactions. CASA indicated that sperm counts in the PPCA 2/2 mice were 70% reduced, and among other problems, there was a fourfold higher percentage of static sperm in PPCA 2/2 mice compared with controls. These results suggest that PPCA deficiency causes structural changes to the blood-epididymal barrier as evidenced by lanthanum nitrate and Cldns expression that affects the luminal environment of the epididymis, resulting in altered sperm motility.