Relocalization of the V-ATPase B2 subunit to the apical membrane of epididymal clear cells of mice deficient in the B1 subunit

Abstract

Ϫ/Ϫ mice do not develop metabolic acidosis and are fertile. While B1 is located in the apical membrane of narrow and clear cells, the B2 subunit localizes to subapical vesicles in wild-type mouse, rat and human epididymis. However, a marked increase (84%) in the mean pixel intensity of B2 staining was observed in the apical pole of clear cells by conventional immunofluorescence, and relocalization into their apical membrane was detected by confocal microscopy in B1 Ϫ/Ϫ mice compared with B1 ϩ/ϩ . Immunogold electron microscopy showed abundant B2 in the apical microvilli of clear cells in B1 Ϫ/Ϫ mice. B2 mRNA expression, determined by real time RT-PCR using laser-microdissected epithelial cells, was identical in both groups. Semiquantitative Western blots from whole epididymis and cauda epididymidis showed no variation of B2 expression. Finally, the luminal pH of the cauda epididymidis was the same in B1 Ϫ/Ϫ mice as in B1 ϩ/ϩ (pH 6.7). These data indicate that whereas overall expression of B2 is not affected in B1 Ϫ/Ϫ mice, significant redistribution of B2-containing complexes occurs from intracellular compartments into the apical membrane of clear cells in B1 Ϫ/Ϫ mice. This relocation compensates for the absence of functional B1 and maintains the luminal pH in an acidic range that is compatible with fertility. male reproductive tract; male fertility; luminal acidification; proton pump; vacuolar H ϩ ATPase SPERMATOZOA DEVELOP their ability to become motile and to fertilize an egg as they pass through the male excurrent ducts, which are composed of efferent ducts, epididymis, and vas deferens. Considerable changes in the composition of luminal fluid occur in the epididymis and include net water, Na ϩ , Cl Ϫ , and HCO 3 Ϫ reabsorption, K ϩ secretion, in addition to secretion and resorption of numerous proteins (2,17,18,30,36,37,58,59). In addition, the lumen of the epididymis and vas deferens is maintained acidic compared with blood, and has a low bicarbonate concentration (36, 37). Low pH and bicarbonate are required for sperm maturation, for the prevention of premature activation of acrosomal enzymes, and for maintaining sperm in a quiescent, immotile state during their maturation and storage in the epididymis and vas deferens (1,2,16,26,32). Thus a defect in the acidification capacity of epididymis and vas deferens might have important consequences on the maturation and storage of spermatozoa, and, consequently, on male fertility (45).In mammals, the epididymis is composed of one to three highly coiled tubules divided into several morphologically distinct regions, including the initial segments, caput, corpus, and cauda epididymidis. These regions are further segmented into intraregional segments, which all contribute to the establishment of a succession of microenvironments in which spermatozoa mature and are concentrated, transported, and stored (19,31,49). The composition of the luminal microenvironment is controlled by distinct epithelial cell types: principal cells, narrow cells and clear cells line the ent...

Regulation of V-ATPase recycling via a RhoA- and ROCKII-dependent pathway in epididymal clear cells

“…Immunogold electron microscopy was performed to localize the V-ATPase, as we have previously described (2,15,57). Small pieces of PLP-fixed epididymis were embedded at Ϫ45°C using HM20 resin (Electron Microscopy Sciences) in a Leica EM AFS, and then ultrathin sections were cut using a Leica Ultracut EM UC7 ultramicrotome (Leica Microsystems, Bannockburn, IL) and collected on Formvar-coated, gilded nickel grids.…”

Regulation of luminal acidification in the male reproductive tract via cell–cell crosstalk

“…Differential expression of a particular set of isoforms in different cell types controls the sub-cellular localization of the V-ATPase holo-enzyme (Hurtado-Lorenzo et al, 2006;Kawasaki-Nishi et al, 2001a;Kawasaki-Nishi et al, 2001b;Pietrement et al, 2006;Sun-Wada et al, 2003;Sun-Wada et al, 2004;Toyomura et al, 2003). In the epididymis, subunits A, B1, B2, C1, C2, G1, G3, E, a1, a4, d1 and d2 are all enriched in the apical domain of narrow and clear cells (Da Silva et al, 2007a;Paunescu et al, 2004;Pietrement et al, 2006). In addition, subunits A and a2 were detected in intracellular structures closely associated with the trans-Golgi network of all epithelial cells (Pietrement et al, 2006).…”

“…Such isoform replacement occurs in the absence of B1 in B1-knockout (KO) mice, where the compensatory insertion of B2 with the plasma membrane-bound V-ATPase allows VATPase-dependent proton transport to occur across the membrane of epididymal clear cells and renal intercalated cells (Da Silva et al, 2007a;Paunescu et al, 2007). Accordingly, male mice lacking the B1 subunit do not develop dRTA and are not infertile (Da Silva et al, 2007a). By contrast, in humans harboring B1 mutations, B2 replacement does not appear to take place, which results in the development of dRTA.…”

Channels for water efflux and influx involved in volume regulation of murine spermatozoa

“…It is unlikely that these effects are due to abnormal metabolism through phloretin's inhibition of glucose transport via the glucose transporter, since the BWW medium contains the respiratory substrates lactate and pyuvuate. Although phloretin can inhibit AQP9 (Castle 2005), where antibodies have been used on epididymal sections, there are no reports of this AQP in luminal spermatozoa (Ruz et al 2006, Da Silva et al 2007. Experiments designed to determine the possible alternative RVD mechanism in the presence of quinine and phloretin, leading to the observed water efflux, showed that both TEA and furosemide were able, in the same animals, to delay the onset of RVD.…”