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Articles |
a Renal Unit and Program in Membrane Biology, Massachusetts General Hospital, Charlestown, Massachusetts 02129
b Molecular Medicine and Renal Units, Beth Israel Deaconess Medical Center, Boston, Massachusetts 02215
c Division of Hematology, The Children's Hospital, Boston, Massachusetts 02215
d Departments of Medicine, Cell Biology,
e and Pediatrics,
f Harvard Medical School, Boston, Massachusetts 02215
g The Jackson Laboratories, Bar Harbor, Maine 04609
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Epithelial cells lining the lumen of the epididymis and vas deferens are the sites of active water, solute, and acid/base transport between the lumen and interstitial fluid [17,18]. Modifications of the tubular fluid composition that occur in the epididymis establish the appropriate environment for spermatozoa as they mature and are stored in this organ [19]. These changes include significant fluid reabsorption, sodium and chloride reabsorption, and potassium secretion [18]. Transepithelial acid/base transport by this epithelium is indicated by several lines of evidence. The luminal concentration of HCO3 becomes significantly lower than that of blood when the efferent duct fluid transits through the initial segment and intermediate zone of the epididymis, the luminal bicarbonate concentration then remaining low in the more distal parts of the epididymis and the vas deferens [18]. Luminal pH also becomes lower than blood pH [18,20, 21], indicating the occurrence of active proton transport. This acidic environment helps to maintain sperm in an immotile but viable state while they mature and are stored in the epididymis [22]. An apical Na/H-exchanger was initially proposed to be involved in acidification, on the basis of the observed requirement of sodium in this process [17]. We have more recently demonstrated that a bafilomycin-sensitive H+ATPase is responsible for a large fraction of the proton secretion measured in the vas deferens [23], and that the apical H+ATPase that is involved in this process is selectively located in specialized cells of the epithelium that lines the epididymis and the vas deferens [23, 24]. H+ATPase-rich cells in the male reproductive tract contain high levels of the cytosolic carbonic anhydrase, CAII [23,25–27], indicating concomitant bicarbonate transport during the acidification process. The role of bicarbonate in epididymal function was further suggested by studies demonstrating that the CAII inhibitor acetazolamide increases luminal pH in the cauda epididymidis [17]. We have recently shown a similar inhibitory effect of acetazolamide on proton secretion in the vas deferens [25]. Altogether, these results suggest that bicarbonate is reabsorbed by the epithelial cells.
Despite physiological evidence indicating the involvement of transepithelial bicarbonate transport in the male reproductive tract, little is known regarding the actual mechanisms responsible for this transport. A basolateral Cl/HCO3 exchange mechanism has been described in primary cultures of cauda epididymidis [28]. We have recently reported high levels of expression of an Na-HCO3 cotransporter in the basolateral membrane of epithelial cells lining the initial segment and intermediate zones of the epididymis, and low expression in the more distal parts of the epididymis [29]. In a previous study, we did not succeed in localizing AE1 in the epididymis and the vas deferens [23], but the presence of other isoforms of the AE family was not investigated. Given the absolute requirement for an SDS unmasking technique to immunolocalize AE2 in the kidney [8, 9], we reassessed the possibility that this isoform is expressed in the epididymis. Using this novel unmasking technique [30], we detected the presence of AE2 in the basolateral membrane of epithelial cells lining the lumen of the rat and mouse epididymis and vas deferens. The identity of this immunoreactive material as AE2 was further confirmed by using knockout mice null for expression of the entire AE1 gene [31].
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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An affinity-purified rabbit polyclonal antibody against the carboxyl terminal 12 amino acids 1224–1237 of mouse AE2 was used. This antibody cross-reacts with another member of the AE anion exchanger family, AE1, but does not recognize AE3 [8, 9]. For immunofluorescence microscopy, goat-anti-rabbit IgG coupled to CY3 was used (Sigma Chemical Company, St. Louis, MO), and for Western blotting, horseradish peroxidase-conjugated goat-anti-rabbit IgG was used (Jackson Immunoresearch, West Grove, PA).
Immunofluorescence Microscopy
Sexually mature male Sprague-Dawley rats, CD1 mice, and SV129/C57BL6J hybrid mice of AE1 -/- and +/+ genotypes were studied. Anesthetized animals were perfused via the left ventricle with PBS (0.9% NaCl in 10 mM sodium phosphate buffer, pH 7.4) followed by paraformaldehyde-lysine-periodate fixative [32]. The epididymis and proximal vas deferens were dissected, further fixed overnight at 4°C, and washed in PBS, as previously described [23, 24]. Tissues were cryoprotected in 30% sucrose for at least 1 h, and 4-µm cryostat sections were made using a Reichert FC4 Ultracryomicrotome (Reichart Jung, Nossloch, Germany). Sections were picked up on Fisher Superfrost Plus charged glass slides (Fisher Scientific Co., Pittsburgh, PA) and stored at 4°C.
For indirect immunofluorescence labeling, sections were hydrated for 5 min in PBS and treated for 4 min (rat) and 10 min (mouse) with SDS (1% in distilled water), an antigen retrieval technique that we have recently adapted for immunocytochemistry [30], and which is essential to reveal AE2 antigenicity in the kidney [8] and epididymis/vas deferens (present study). Sections were washed twice for 5 min each time in PBS and then blocked in a solution of 1% BSA/PBS/sodium-azide for 10–15 min. Primary antibodies at concentrations of 3.0 µg/ml (rat) or 0.76 µg/ml (mouse) were applied in a moist chamber, for either 1.5–2.0 h at room temperature, or overnight at 4°C, and then rinsed twice for 5 min each time in a high-salt PBS (2.7% NaCl in 10 mM sodium phosphate buffer) and washed once for 5 min in normal PBS. In competition experiments, an irrelevant peptide, an AE1 peptide containing amino acids (aa) 917–929, or an AE2 aa 1224–1237 peptide was mixed with primary antibody at 12 µg/ml and allowed to incubate for at least 1 h at room temperature before use (rat). For mouse sections, primary antibody and peptide (24 µg/ml) were applied simultaneously. The secondary antibody was applied (2.5 µg/ml) for 1 h at room temperature and washed as above.
Rat sections were mounted in a 2:1 mixture of Vectashield (Vector Laboratories, Burlingame, CA) mounting medium/1.5 M TRIS solution (pH 8.9) and photographed using a Nikon FXA epifluorescence microscope (Nikon Instruments, Garden City, NY) on Kodak TMAX 400 film (Eastman Kodak, Rochester, NY) push-processed to 1600 ASA. Mouse sections were mounted in PBS with 0.2% n-propylgallate in 50% glycerol and photographed using an Olympus BH2 epifluorescence microscope (Olympus America, Melville, NY). When the results of peptide antigen competition experiments were to be compared, photos were taken at fixed exposure times and printed under identical conditions.
Immunoblotting
Mice were perfused through the left ventricle with PBS (pH 7.4) maintained at 37°C. Tissue samples were removed onto an iced surface, cut into smaller pieces with a razor blade, transferred to 2 ml of ice-cold lysis buffer containing protease inhibitors (Mini-Complete; Boehringer-Mannheim, Indianapolis, IN), and homogenized using a small Potter and a 27-gauge needle/syringe. Lysis buffer consisted of 15 mM NaCl, 10 mM TRIS (pH 7.4), 1% Nonidet P-40, 200 µM PMSF, and 1.5 tablets of Mini-Complete Protease Inhibitor Cocktail per 10 ml. After determination of their protein content (BCA Assay; Pierce, Rockford, IL), homogenates were diluted 4:1 in 5-strength Laemmli (reducing) sample buffer (Boston BioProducts, Ashland, MA), immediately vortexed, and set on ice for 20–30 min. The homogenates were then clarified by centrifugation (2 min at 14 000 rpm), and the supernatant was loaded on the gel.
Samples were loaded at 100 µg protein per lane onto SDS polyacrylamide 4–16% gradient gels, and separated using the Laemmli method [33]. Proteins were transferred in a semi-dry transfer cell (Trans-Blot SD; Bio-Rad, Hercules, CA) onto supported nitrocellulose transfer membrane (Schleicher & Schuell, Keene, NH). The quality of transfer was checked by protein staining of the membrane with Ponceau Red, and of the residual protein in the gel with Coomassie brilliant blue. After destaining, membranes were blocked for 1 h at room temperature in Blotto (5% nonfat dry milk/0.05% Tween-20/tris-buffered saline, pH 7.5).
Nitrocellulose was incubated with primary antibody used at 0.076 µg/ml in Blotto, in the presence of 24 µg/ml irrelevant peptide or peptide antigen, for 1 h at room temperature. Secondary antibody was diluted 1:10 000 in Blotto, and incubated for 1 h at room temperature. Washes between and after incubations were done in 1% powdered nonfat milk and repeated 4 times for 10 min each time. Detection of antibody binding was performed with ECL+Plus reagents (Amersham Life Sciences, Little Chalfont, Bucks, UK), using Kodak X-Omat Blue XB-1 film.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Figure 1 shows the presence of AE2 polypeptide in the mouse epididymis. The major immunoreactive band displays a molecular mass of ~180 kDa, slightly higher than noted in stomach [11] and choroid plexus [10]. This molecular size value might be consistent with the presence in the band of the AE2a and/or AE2b polypeptides, but not of the ~20-kDa shorter form AE2c [9, 34]. The immunoreactivity was abolished in the presence of peptide antigen (lane 2), but not in the presence of irrelevant peptide (lane 1). As evident from the immunocytochemical studies presented below, the ~100-kDa band represents erythroid AE1 from erythrocytes contaminating the tissue used for the blot.
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AE2 Immunostaining in Rat Epididymis
Indirect immunofluorescence staining of the epididymis for AE2 was dependent upon pretreatment of the tissue sections with SDS, an antigen-retrieval technique [30], before application of the primary antibody. The anti-AE2 antibody produced strong basolateral staining in epithelial cells in the initial segment (Fig. 2A) and in the intermediate zone (Fig. 2B). A similar but weaker staining was also observed in the caput epididymidis (not shown). The staining became progressively weaker in the corpus epididymidis (not shown) and re-intensified in the cauda epididymidis (Fig. 3A) and vas deferens (Fig. 3B). In some cases, intracellular structures compatible with Golgi apparatus were visible (not shown). This Golgi staining was stronger on initial segment and intermediate zone sections that were not pretreated with SDS. Residual erythrocytes were also labeled because of cross-reactivity of the antibody with AE1 (Fig. 2).
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The basolateral staining described above was obtained using an antibody raised against the AE2 COOH-terminal peptide. Since this antibody also cross-reacts with the COOH-terminal sequence of AE1, we performed control experiments to determine the nature of the AE isoform detected. Competition experiments were done on cryostat sections of the initial segment and ductuli efferentes using an irrelevant peptide (Fig. 4A), the AE1 COOH-terminal peptide aa 917–929 (sharing amino acid identity in 8 of 13 aa with the mouse AE2 COOH-terminal sequence; Fig. 4B), and the AE2 COOH-terminal peptide aa 1224–1237 (Fig. 4C). Figure 4A shows strong basolateral membrane staining of epithelial cells in the initial segments and of ciliated cells of the ductuli efferentes. This basolateral staining is not affected by the presence of an irrelevant peptide. Figure 4B shows that immunostaining in the presence of AE1 peptide is only slightly attenuated. This minimal competition of immunostaining by AE1 is consistent with the previously reported weak competition of AE2 immunostaining by AE1 C-terminal peptides in stomach [11] and kidney cells [8]. In contrast, the presence of AE2 peptide completely abolished basolateral staining in epithelial cells in the initial segment and ciliated cells in ductuli efferentes (Fig. 4C). Persistence of punctate intracellular staining in the ductuli efferentes indicated its nonspecificity. Thus, the basolateral staining that is revealed using the SDS unmasking treatment in rat epididymis is fully consistent with expression of AE2 polypeptide. However, the slight reduction of staining that was observed in the presence of AE1 C-terminal peptide might still indicate a low level of expression of AE1 polypeptide in epididymis. We further tested this possibility by comparing the localization of AE2 anion exchanger in normal mouse epididymis with the epididymis of the AE1 knockout mouse.
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AE2 Immunostaining in Normal and AE1 Knockout Mouse Epididymis
Figure 5 shows immunolocalization of AE2 in normal mouse epididymis. Strong basolateral membrane staining was observed in epithelial cells of the initial segment (Fig. 5A) and of the caput epididymidis (Fig. 5C). No detectable staining was observed in the corpus epididymidis (not shown), and a weaker staining was seen in the cauda epididymidis (Fig. 5E). Residual erythrocytes were also stained. As shown in the right panels of Figure 5, a complete inhibition of immunoreactivity was produced by preincubation of the antibody with the AE2 C-terminal peptide antigen, indicating expression of this isoform in mouse epididymis.
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To further confirm that the epithelial cell labeling observed with the anti-AE2 antibody was attributable entirely to AE2 expression and was not the result of cross-reactivity with AE1, we examined the epididymis of AE1-/- mice [31]. As shown in Figure 6A, epithelial cells from the initial segment of a normal mouse epididymis presented strong basolateral staining. Residual erythrocytes were also stained in this section. Epididymis in the AE1-/- mouse showed a normal gross morphology. A similar region of epididymis from the AE1-/- mouse still showed strong basolateral staining (Fig. 6B). This result further demonstrated the presence of AE2 in the basolateral membrane of mouse epididymis.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The epididymis is involved in net transepithelial bicarbonate reabsorption that results in the establishment of a luminal bicarbonate concentration < 15% of that in blood [36]. Luminal acidification in the distal portions of the epididymis is inhibited by acetazolamide [17, 25], and carbonic anhydrase type II (CAII) is concentrated in H+ATPase-rich cells of the epididymis and vas deferens [23, 26, 27, 39], indicating a role for carbonic anhydrase in bicarbonate reabsorption.
By analogy with other proton-secreting and bicarbonate-reabsorbing epithelia, basolateral HCO3- extrusion in epithelial cells can be mediated by a Cl-/HCO3- AE. Such a function is mediated by the kidney isoform of AE1 in proton-secreting intercalated cells in the kidney [5], and is probably mediated by AE2 in the thick ascending limb of Henle [8, 40]. HCO3- reabsorption can also be achieved by the activity of a basolateral Na+/HCO3- co-transporter, functioning in the efflux mode, similar to that predominating in renal proximal tubules [41–43]. We have recently shown that an Na+/HCO3- co-transporter is located in the basolateral plasma membrane of epithelial cells of the epididymis and the vas deferens [29]. The expression of this protein, as well as its respective mRNA, was considerably greater in the caput epididymidis than in the cauda and vas deferens, indicating that other bicarbonate transporters might be involved in bicarbonate transport in the more distal regions of the male reproductive tract.
Our previous attempts to localize one member of the AE family, AE1, proved unsuccessful, but the presence of other AE proteins was not investigated [23]. The purpose of the present study was to determine whether the Cl/HCO3 exchanger, AE2, was present in the epididymis and vas deferens, in view of our recent reports demonstrating the necessity of performing unmasking SDS treatment to reveal its antigenicity in other tissues [8, 9]. Our results show that AE2 is abundantly expressed by epithelial cells of the rat and mouse initial segment and intermediate zone, where it is located on the basolateral membrane. The caput and cauda epididymidis and the vas deferens also contain basolateral AE2, but the corpus epididymidis does not show detectable levels of this protein.
The antibody used in the present study cross-reacts with AE1 [8], as also evident in Figure 1. It was, therefore, necessary to demonstrate that the immunoreactivity observed in the epididymis derived from AE2 and not AE1. The fact that the epithelial immunostaining was completely abolished when the antibody was preincubated with the AE2 polypeptide, while it was only moderately affected after preincubation with the AE1 polypeptide, represents strong evidence that the reaction can be attributed to detection of AE2. The unaltered epithelial immunostaining detected in the epididymis of AE1 knockout mice further confirmed the presence of AE2 in the basolateral membrane of epithelial cells of the male reproductive tract.
AE2 has been implicated in numerous functions including net transepithelial acid/base and NaCl transport, cell volume regulation, and intracellular pH regulation [1, 8,14]. Cl/HCO3 exchange by AE2 is regulated by intracellular pH [15] and is activated under hypertonic conditions [12] and by NH4+ concentrations physiologically present in kidney, gut, and other tissues [13]. The presence of AE2 in the basolateral membrane of epithelial cells of the proximal parts of the epididymis correlates with the low luminal concentration of bicarbonate that is reached in these segments. This indicates that basolateral AE2 might contribute, in parallel with the recently described sodium bicarbonate co-transporter (NBC) protein [29], to net bicarbonate reabsorption. The presence of AE2 in both principal cells and H+ATPase-rich cells is intriguing. H+ATPase-rich cells are the apical/narrow cells in the caput epididymidis and the clear cells in the cauda epididymidis [24, 39]. These cells are also enriched in CAII, and we have shown that apical/narrow cells contain basolateral NBC. The presence of an additional bicarbonate transporter in their basolateral membrane provides these cells a complete machinery for net proton secretion coupled to bicarbonate reabsorption. AE2 might therefore be involved in net proton secretion by H+ATPase-rich cells.
AE2 might also contribute to net bicarbonate absorption by principal cells, provided that an apical entry route for bicarbonate is present. These cells are enriched in the apically bound CAIV [27] and have a limited amount of CAII [27, 39]. No apical acid extrusion mechanism has yet been described in principal cells in situ. Previous studies demonstrated that acetazolamide impairs acidification in the cauda epididymidis [17] and vas deferens [25]. However, no effect of in vivo acetazolamide administration was shown on luminal pH in the caput and corpus epididymidis [21]. It was proposed that bicarbonate reabsorption, and not proton secretion, is the mechanism of acidification in the proximal parts of the epididymis [21].
We have recently shown that, in the adult rat, the number of H+ATPase-rich cells is considerably higher in the cauda epididymidis than in the caput epididymidis [39], and that these cells are responsible for the majority of proton secretion in isolated vas deferens in vitro [23]. It therefore appears that proton secretion or bicarbonate reabsorption, or both, are produced by distinct mechanisms in the different regions of the epididymis. Whereas acidification seems to be independent of the activity of carbonic anhydrase in the proximal segments, in more distal regions CAII might play a greater role in generating H+ and HCO3- that are transported apically and basolaterally, respectively, by polarized transporters. In the proximal regions of the epididymis, basolateral AE2 might, therefore, contribute to significant bicarbonate reabsorption by principal cells, and to transepithelial proton and bicarbonate transport in apical/narrow cells. Regarding the more distal regions, it is interesting to note that we have recently reported that net proton secretion by the vas deferens, which was inhibited by SITS (4-acetamido-4'-isothiocyanato-stilbene-2, 2'-disulfonic acid), was nevertheless independent of the presence of chloride [25]. This result is not consistent with a role for basolateral AE2 in net proton secretion by clear (or H+ATPase-rich) cells in the distal portions of the male reproductive tract.
AE2, in contrast to AE1, can confer the potential for regulatory cell volume increase when expressed in Xenopus oocytes [14]. It is expressed in kidney thick ascending limb cells [8, 40], which are subject to a hypertonic environment [2]. The lumen of the epididymis is maintained as slightly hypertonic [18], and it is therefore possible that AE2 plays a volume regulatory role in this organ. The concomitant action of an Na/H exchanger with the AE2 Cl/HCO3 exchanger is required for regulatory volume increase to occur, because of net entry of NaCl followed by osmotically driven water [2]. Although the presence of Na/H exchanger(s) in epithelial cells of the epididymis has not yet been reported, it is reasonable to hypothesize their expression.
AE2 is activated when intracellular pH rises [12, 15]. This characteristic is appropriate for a role in HCO3 reabsorption, if accompanied by maintenance of intracellular [HCO3] above its electrochemical equilibrium concentration. Cl/HCO3 exchange is also known to contribute to the housekeeping regulation of intracellular pH in a variety of cell types [1]. Determination of the role of AE2 in intracellular pH regulation in the epithelial cells of the epididymis will require further studies.
Luminal bicarbonate plays a key role in triggering capacitation, a process that allows spermatozoa to interact with and fertilize the egg [44, 45]. During capacitation, a series of destabilizing events occur that eventually lead to sperm death [45]. Initiation of premature capacitation in vivo might, therefore, represent a threat for sperm survival during storage in the epididymis. The establishment of a low HCO3- concentration in the lumen of the epididymis is likely to contribute to maintaining an optimum environment for proper sperm storage and viability.
In summary, our present data show that the AE2 Cl/HCO3 exchanger is present in the basolateral plasma membrane of epithelial cells of rat and mouse epididymis and vas deferens. This protein may be involved in transepithelial acid-base transport, and in addition it may have a role in regulation of intracellular pH or cell volume.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Sylvie Breton, Renal Unit and Program in Membrane Biology, Massachusetts General Hospital East, 149 13th Street, Charlestown, MA 02129. FAX: 617 726 5669; sbreton{at}receptor.mgh.harvard.edu
3 L.J. Jensen and A.K. Stuart-Tilley share the first author position.
4 Current address: Zoophysiological Laboratory, August Krogh Institute, 13 Universitetsparken, DK-2100, Kbh. O, Denmark.
Accepted: May 13, 1999.
Received: March 11, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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