Immunolocalization of CA II and H+V-ATPase in epithelial cells of the mouse and rat epididymis

Acidification of the epididymal lumen has been suggested to play an importa nt role in sperm functions; however, the cell types. pumps, and mechanisms involved have not been fully addressed. In this study, carbonic anhydrase I I (CA II) and a 67-kd subunit of Neurospora crassa vacuolar proton adenosin etriphosphatase (H+V-ATPase) pump were immunolocalized using light microsco py and electron microscopy (EM) in the epididymis of rats and mice. In both animals, narrow cells, identified in the initial segment and intermediate zone of the epididymis, contained numerous small vesicles in their apical r egion, often cup-shaped in appearance. In the mouse but not rat, these cell s also possessed numerous cisternae of smooth endoplasmic reticulum, sugges ting steroid synthesis; and cytoplasmic blebs of their apical cell surface, which appeared to detach, suggesting apocrine secretion. Anti-CA II antibo dy was immunocytochemically localized in the light microscope within narrow cells but not over any other cell types of the entire epididymis. Anti-H+V -ATPase antibody was also localized in narrow cells of the initial segment and intermediate zone; as well as clear cells of the caput, corpus, and cau da regions. Using EM, gold particles for anti-CA II and H+V-ATPase antibodi es were noted in the apical region of narrow cells in relation to the numer ous, small, cup-shaped vesicles. Although CA II was mainly located in the c ytosol near these vesicles, H+V-ATPase appeared on their delimiting membran e and on the apical plasma membrane of these cells. A similar distribution was noted for H+V-ATPase in clear cells. The nature of the small vesicles o f the apical region of narrow cells was examined with electron-dense fluid phase tracers that were introduced into the epididymal lumen. The tracers a ppeared within these vesicles and a few endosomes 1 hour after injection, s uggesting that they contact the apical plasma membrane. Since these vesicle s are also related to CA II and H+V-ATPase, the data suggests that, as the site of proton production, the vesicles recycle to and from the apical cell surface, and in this way, deliver protons to the epididymal lumen for acid ification. Clear cells and their expression of H+V-ATPase may also serve in this function. In summary, both narrow and clear cells appear to be involv ed in luminal acidification, an activity that may be essential for sperm as they traverse and are stored in the epididymis.