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Regular Article |
2,a
a Unit of Molecular Toxicology, Institute for Medical Research and Occupational Health, 10001 Zagreb, Croatia
b Program in Membrane Biology, Massachusetts General Hospital, Charlestown, Massachusetts 02129
c Departments of Medicine and
d Pathology, Harvard Medical School, Boston, Massachusetts 02215
e Urology Clinics, Clinical Hospital "Sisters of Mercy", 10000 Zagreb, Croatia
ABSTRACT
Luminal acidification in parts of the male reproductive tract generates an appropriate pH environment in which spermatozoa mature and are stored. The cellular mechanisms of proton (H+) secretion in the epididymis and the proximal vas deferens involve the activity of an apical vacuolar H+ATPase in specialized cell types, as well as an apical Na+/H+ exchanger in some tubule segments. In this study we used Western blotting and immunocytochemistry to localize the H+ATPase in various segments of the male reproductive tract in rat and man as a first step toward a more complete understanding of luminal acidification processes in this complex system of tissues. Immunoblotting of isolated total cell membranes indicated a variable amount of H+ATPase in various segments of the rat reproductive tract. In addition to its known expression in distinct cell types in the epididymis and vas deferens, the H+ATPase was also localized at the apical pole and in the cytoplasm of epithelial cells in the efferent duct (nonciliated cells), the ampulla of the vas deferens and the ventral prostate (scattered individual cells), the dorsal and lateral prostate, the ampullary gland, the coagulating gland, and all epithelial cells of the prostatic and penile urethra. Both apical and basolateral localization of the protein were found in epithelial cells of the prostatic ducts in the lateral prostate and in periurethral tissue. Only cytoplasmic, mostly perinuclear localization of the H+ATPase was found in all epithelial cells of the seminal vesicles and in most cells of the ventral prostate and coagulating gland. No staining was detected in the seminiferous tubules, rete testis, and bulbourethral gland. In human tissue, H+ATPase-rich cells were detected in the epididymis, prostate, and prostatic urethra. We conclude that the vacuolar H+ATPase is highly expressed in epithelial cells of most segments of the male reproductive tract in rat and man, where it may be involved in H+ secretion and/or intracellular processing of the material endocytosed from the luminal fluid or destined to be secreted by exocytosis.
epididymis, male reproductive tract, penis, prostate, seminal vesicles, sperm maturation, testis, vas deferens
INTRODUCTION
After leaving the seminiferous tubules, spermatozoa transit through and are stored in the epididymis, where they progressively acquire the ability to fertilize an oocyte [1, 2]. This maturation depends on coordinated reabsorptive and secretory processes of the epithelial cells lining the male reproductive tract [1–3]. Generation of a luminal acidic pH and a low luminal bicarbonate concentration are factors that maintain spermatozoa in an immotile state during their maturation and storage [4–8]. Sperm motility is triggered following ejaculation, when the acidic fluid originating from the epididymis and vas deferens is mixed with alkaline, higher-bicarbonate secretions derived from the coagulating gland and seminal vesicles, and with the less acidic prostatic secretion [1–5].
As measured in rats in vivo, the intraluminal pH in seminiferous tubules is 7.0–7.3, and progressively acidifies to 6.5 along the epididymis [6–10]. Studies on perfused epididymis and cultured epididymal principal cells have shown the involvement of an apical Na+/H+ exchanger [11] and cytoplasmic carbonic anhydrase type II (CAII) [12–14] in this acidification process. Our discovery of a specialized population of cells in the rat epididymis [15] and proximal vas deferens [14], which contain a vacuolar-type H+ATPase (H+ pump) in their apical membrane, suggested an additional mechanism in luminal acidification. In the proximal vas deferens in vitro, this bafilomycin-sensitive H+ATPase may account for about 80% of the net H+ secretion into the lumen [14–17], whereas the Na+/H+ exchanger may be quantitatively more important in proximal regions of the epididymis. Recent immunocytochemical data have revealed the Na+/H+ exchanger isoforms, NHE1 and NHE2 [18], as well as NHE3 [19] in principal cells of some regions of the epididymis. The relative importance of the vacuolar H+ATPase in luminal acidification in some parts of the excurrent duct system is indicated by data from cadmium-treated rats, in which alkalinization of the tubular fluid was observed [9]. We have recently shown that cadmium modifies the differentiated phenotype of H+ATPase-rich cells, and directly inhibits proton secretion by these cells [20].
In addition to the epididymis, an acidic luminal pH was measured in rat seminiferous tubules and efferent ducts [6–10]. As measured in dogs and men, the pH of prostatic fluid may depend upon the secretory state of the gland and may vary between slightly acidic (6.5–6.7) [21, 22] to slightly alkaline (7.31) [23]. The mechanisms of acidification in these and possibly other parts of the male reproductive tract in rat, and also in human, have not been examined. Thus, the purpose of our present study was to localize the vacuolar H+ATPase along the entire male reproductive tract in rat and in the accessible parts of the reproductive organ in man, as a first step in understanding the potential role of this enzyme in regulating luminal fluid composition, intracellular processes, or both, in other regions of the reproductive tract.
MATERIALS AND METHODS
Animals
Adult Wistar male rats were obtained from the breeding colony at the Institute for Medical Research and Occupational Health in Zagreb, Croatia. The care and use of rats in our experiments strictly followed recommendations in the Guide for Care and Use of Laboratory Animals, published by the U.S. National Institutes of Health. All experiments were approved by the Institutional Ethics Committee in Zagreb.
Human Tissue
Human reproductive organ specimens (epididymis, prostate, and prostatic urethra) were obtained during regular surgery from three adult men (aged 50–65 yr) with testicular or bladder neck carcinoma, or benign prostatic hyperplasia. This research was approved by the hospital ethics committee in Zagreb, Croatia, and a written voluntary agreement was obtained from patients before the surgery was performed. According to hospital protocols and information provided by the physicians, the patients had not received any medication for at least 1 mo before surgery.
Isolation of Total Cell Membranes from Rat Tissues
Rats were killed by decapitation. The reproductive tract was removed and manually dissected into distinct segments: testis, efferent ducts, epididymis (divided into caput, corpus, and cauda), vas deferens, seminal vesicles, and prostate (divided into ventral and dorsolateral lobes). The segments were carefully trimmed free of fat. In order to obtain enough membranes for immunoblotting, tissue from five to six animals was pooled and processed as one sample. The pooled tissues were finely minced, rinsed three times with ice-cold homogenizing buffer (HB; 300 mM mannitol, 12 mM Hepes/Tris, 1 mM phenylmethylsulfonyl fluoride, 10 µM benzamidine, and 0.1 µg/ml antipain pH 7.4), to remove sperm, dispersed in 15 ml HB, and homogenized for 30 sec with a High Speed Homogenization System (Kinematica GmbH, Littau-Luzern, Switzerland; setting 4 [10 000 rpm]). The homogenates were centrifuged in a refrigerated Sorvall RC5C centrifuge (DuPont Co., Wilmington, DE) at 2500 x g for 15 min. The pellets were discarded and the supernatants were centrifuged at 10 000 x g for 15 min. The resulting pellets, enriched in mitochondria and lysosomes, were discarded, and the supernatants were further centrifuged at 48 000 x g for 30 min. The final pellets (total cell membranes) were used for Western blotting.
Rat renal cortical brush-border membranes (BBM) were isolated from renal cortical homogenates by the Mg2+-aggregation method [24].
Membrane preparations were resuspended in HB to a protein concentration of 3 mg/ml, and stored in liquid nitrogen until further use. Protein was determined by the Bradford assay [25] using BSA as a standard.
Antibodies
To localize the vacuolar H+ATPase along the male reproductive tract, two different polyclonal antipeptide antibodies against the 31-kDa ("E") subunit were used. The antibodies were raised in rabbits or chickens using an 11-amino acid peptide (CGANANRKFLD) from the C-terminal domain, coupled to KLH, as an immunogen [26]. The immunogen was synthesized by the Massachusetts General Hospital Core Facility, whereas the antibodies were raised commercially (Cocalico Biologicals, Inc., Reamstown, PA). The rabbit immune serum was used unpurified, whereas the chicken antibody was affinity-purified from chicken serum using the immunizing peptide coupled to an agarose column. Both antibodies labeled a 31-kDa protein band in rat kidney BBM, and by indirect immunofluorescence they stained various H+ATPase-positive cells along the rat nephron and epididymis, in accordance with previously published data [14, 15, 20, 27]. In both cases, the staining was completely abolished by preabsorption of the antibodies with the immunizing peptide (see later description). A monoclonal antibody against chick brain 
-tubulin was purchased from Sigma (T-9026). Secondary antibodies were purchased commercially (alkaline phosphatase-labeled goat anti-rabbit immunoglobulin G [IgG; GARAP] was from Vector Laboratories, Burlingame, CA; fluorescein isothiocyanate- or CY3-labeled goat anti-rabbit [GARF or GARCY3, respectively] or donkey anti-chicken IgG [DACF or DACCY3, respectively], and CY3-labeled goat anti-mouse IgG [GAMCY3] were from Jackson Immunoresearch, West Grove, PA).
SDS-PAGE and Western Blotting
Total cell membranes from the male reproductive tract segments and renal cortical BBMs were denatured by boiling in sample buffer (1% SDS, 12% v/v glycerol, 5%ß-mercaptoethanol, 30 mM Tris/HCl pH 6.8) for 5 min. Proteins (40 µg protein/lane) were separated through 12% SDS-PAGE Laemmli mini gels and transferred to Immobilon-P membrane (Millipore, Bedford, MA). The transfer membrane was briefly stained with Coomassie Brilliant Blue to check the efficiency of the transfer, destained, and blocked in blotting buffer (5% nonfat dry milk, 0.15 M NaCl, 1% Triton X-100, 20 mM Tris/HCl pH 7.4), followed by incubation with the anti-31-kDa subunit antibody (rabbit immune serum diluted 1:500 at 4°C overnight). The membrane was then rinsed with several changes of blotting buffer, incubated for 1 h at room temperature with the same buffer that contained GARAP, rinsed, and stained with the BCIP/NBT method (Kirkegaard and Perry Laboratories, Gaithersburg, MD). The peptide inhibition of the staining was tested using the same protocol, except that the immune serum was preincubated with the immunizing peptide (0.5 mg peptide protein/ml serum) for 2 h at room temperature prior to incubation with the transfer membrane.
Fixation and Immunocytochemistry of Rat and Human Tissues
Rats were anesthetized with Nembutal (65 mg/kg body mass, i.p.) and perfused via the left cardiac ventricle, first with aerated (95% O2/5% CO2) and temperature-equilibrated (37°C) PBS (140 mM NaCl, 4 mM KCl, 2 mM KH2PO4 pH 7.4) for 2–3 min to remove circulating blood, followed by 200 ml PLP fixative (2% paraformaldehyde, 75 mM lysine, 10 mM sodium periodate) [28] per rat. The reproductive tract was removed, manually separated into different segments, sliced, and kept in the same fixative at 4°C overnight, followed by extensive rinsing in PBS. The tissue was kept in PBS (plus 0.02% NaN3) at 4°C until further use.
Human tissue samples were briefly rinsed in ice-cold PBS to remove blood, and immersed in freshly prepared PLP fixative immediately after surgical removal. Tissues were fixed overnight at 4°C, followed by extensive rinsing and storage in PBS (plus 0.02% NaN3) at 4°C until further use.
To cut 4-µm frozen sections for indirect immunofluorescence, tissue blocks were infiltrated with 30% sucrose (in PBS) overnight, rapidly frozen in liquid nitrogen, and sectioned on a Leica Jung CM 1800 cryomicrotome. Sections were collected on gelatin-coated slides and kept frozen (at -18°C) until further use. For immunostaining, sections were rehydrated in PBS for 10 min, soaked in 1% SDS (in PBS) for 5 min to enhance antigenicity [29], extensively rinsed in PBS, blocked with 1% BSA (in PBS) for 15 min, then incubated with the primary antibody against the 31-kDa H+ATPase subunit (immune rabbit serum diluted in PBS 1:200 or affinity-purified chicken antibody diluted1:20) at 4°C overnight, rinsed in PBS, and incubated with secondary antibody (GARF/GARCY3 or DACCY3, respectively) at room temperature for 1 h. The slides were then rinsed in PBS and mounted in Vectashield (Vector Laboratories). The specificity of the primary antibodies was tested by incubating sections of each tissue with a mixture of the primary antibody (diluted as for immunocytochemistry) and the immunizing peptide (final concentration of the peptide was 0.5 mg/ml) that had been preincubated for 2 h at room temperature prior to application. In this paper we show only the specific staining (e.g., the staining that was blocked by the peptide-pretreated antibody).
For the rat efferent duct, double staining was performed to identify ciliated and nonciliated cell types in this epithelium. Sections were first incubated overnight with rabbit or chicken antibody against the 31-kDa H+ATPase subunit at 4°C, as described in detail above, rinsed in PBS, then incubated with the monoclonal anti--tubulin antibody (diluted 1:50 in PBS) at room temperature for 2 h. After rinsing in PBS, sections were incubated with GAMCY3 for 1 h, rinsed in PBS, incubated for 1 h with GARF or DACF, washed again in PBS, and mounted in Vectashield.
All sections were examined with a Zeiss-Opton fluorescence microscope and photographed using Kodak TMAX 400 film.
Unless specified differently, all the chemicals used in this work were purchased from Sigma (St. Louis, MO). Protein standards were from GIBCO BRL (Rockville, MD).
Presentation of the Data
The immunoblots are representative of similar findings in three different membrane preparations, whereas the immunocytochemical figures represent data obtained on tissue sections from three to four rats.
RESULTS
Immunoblotting of the 31-kDa H+ATPase Subunit in Total Cell Membranes
Immunoblotting of the total cell membrane preparations with a rabbit polyclonal antibody to the 31-kDa H+ATPase subunit is shown in Figure 1. As shown in panel A, the immune serum strongly labeled a 31-kDa band in renal cortical BBM, which was used as a positive control. It also recognized a 31-kDa protein in membranes isolated from various reproductive tract segments. The intensity of the 31-kDa band was greatest in membranes from the cauda epididymidis and dorsolateral prostate, intermediate in the corpus epididymidis and ventral prostate, and weak in other segments. This band was barely detectable in testicular and seminal vesicle membranes. The weak bands of <31 kDa in membranes from the kidney cortex and dorsolateral prostate probably represent degradation products of the 31-kDa protein. In some membranes, but not in the renal BBM, the serum also labeled some proteins between 55 and 65 kDa. The 
60-kDa band was the strongest in membranes from the seminal vesicles and dorsolateral prostate.
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In order to check if the labeled bands are specifically related to the H+ATPase 31-kDa subunit, selected membrane preparations were blotted with rabbit immune serum that had been preabsorbed with the immunizing peptide. As shown in Figure 1B, the 31-kDa protein band, labeled with native immune serum in renal BBM and total cell membranes of the epididymis cauda and dorsolateral prostate (-PEPTIDE, lanes 1, 2, and 3, respectively), was strongly reduced and abolished with the peptide-pretreated immune serum (+PEPTIDE, lanes 5, 6, and 7, respectively). In the preparation of seminal vesicle membranes, a 31-kDa band was not detectable; however, the 60-kDa band in the membranes of dorsolateral prostate and seminal vesicles remained unaffected in blots incubated with the peptide-pretreated serum. We conclude that the 60-kDa protein band represents nonspecific staining, and is not related to the vacuolar H+ATPase.
Immunocytochemical Localization of the Vacuolar H+ATPase in the Male Rat Reproductive Tract
Previous immunocytochemical data, generated with a polyclonal antibody to the 56-kDa H+ATPase subunit [15] and a monoclonal antibody to the 31-kDa H+ATPase subunit [14], revealed the vacuolar H+ pump in specialized cells along the rat epididymis and proximal vas deferens. By using native and peptide-pretreated rabbit immune serum to the 31-kDa subunit protein, we first checked the specificity of immunostaining in sections of the selected tissues; as shown in Figure 2, A, C, and E, the native immune serum stained cells in the epididymal cauda, dorsal prostate, and seminal vesicle, respectively; whereas the staining was absent in tissue sections incubated with the peptide-pretreated immune serum (Fig. 2, B, D, and F, respectively). Similar immunocytochemical data were obtained by using the affinity-purified chicken antibody (data not shown). This experiment indicated that 1) the immunostaining with both rabbit immune serum and affinity-purified chicken antibody was specific for the 31-kDa H+ATPase subunit, and 2) the 60-kDa protein, observed with the rabbit immune serum by immunoblotting did not appear as a false positive staining in immunocytochemical experiments. All other tissues in the following experiments were similarly tested to verify if the staining was specific.
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By using rabbit immune serum to the 31-kDa subunit protein, we confirmed the presence of H+ATPase-positive cells along the rat epididymis and vas deferens (Fig. 3); the antibody strongly stained the tall columnar narrow or apical cells in the initial segment (Fig. 3A), low columnar clear cells in the caput (not shown) and corpus epididymidis (Fig. 3B), cuboidal clear cells in the cauda epididymidis (Fig. 3C), and columnar clear cells in the proximal vas deferens (Fig. 3D). The cells in the initial segment exhibited a strong apical staining. A narrow neck of stained cytoplasm between the cell apex and the base gave these cells a typical wineglass shape. In the corpus and cauda epididymidis, positive cells were more numerous and, along strong apical staining, they also had a substantial intracellular staining. Most principal cells, particularly in the corpus and cauda, exhibited a weak, fine granular intracellular staining, concentrated subapically (Fig. 3, B and C, respectively). In the vas deferens, the specific cells had strong apical and limited intracellular staining, whereas other cells exhibited only a weak, fine granular intracellular staining. A similar pattern of staining was observed with the affinity-purified chicken anti-31-kDa H+ATPase subunit antibody (data not shown). These data are similar to those we have previously reported [14–17, 20] and confirm the utility and specificity of the antibodies used in the present study.
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No specific staining with either rabbit or chicken antibodies to the H+ATPase 31-kDa subunit was detectable in testicular seminiferous tubules and rete testis (data not shown). However, in the efferent ducts, which connect the rete testis to the initial segment of the epididymis and where the epithelium consists of columnar nonciliated (predominant cell type) and ciliated cells [30–33], the H+ATPase was localized to the apical and cytoplasmic domains on nonciliated cells (Fig. 4A). The ciliated cells, which are recognizable by abundant tubulin-rich apical cilia (Fig. 4C), were devoid of the apically located H+ATPase staining (Fig. 4B). The ciliated cells, however, showed a weak intracellular staining beneath the nucleus.
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As shown in our previous [14] and present experiments (Fig. 3D), the H+ATPase-rich cells in the proximal vas deferens vary in shape from cuboidal to low columnar. The middle and distal portion of the vas deferens contained no H+ATPase-positive cells (not shown). However, in the ampulla of the vas deferens, high columnar principal cells exhibited very weak, diffuse intracellular staining, and a strong apical staining was found in rare cells that had a long and slender appearance (Fig. 5A).
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The rat seminal vesicle is lined by a simple columnar epithelium that exhibited a granular staining for H+ATPase, concentrated in the supranuclear (subapical) region (Fig. 5B). The staining intensity varied among animals and even in different parts of the same organ, and was completely absent following preabsorption of the antibody with the peptide (Fig. 2F).
The rat coagulating gland, which lies in the curvature of the seminal vesicles, consists of simple columnar epithelial cells characterized by many short microvilli on their luminal surfaces [1]. In sections of this gland, two different staining patterns were found. In most animals, the majority of glandular cells had a diffuse H+ATPase in the subapical cytoplasm, whereas occasional cells had a more uniformly stained cytoplasm (Fig. 5C). In some animals, however, limited regions of the gland showed a sharp staining of the apical membrane of many cells in addition to cells having the diffuse subapical or intracellular staining pattern (Fig. 5D).
The ampullary gland is located adjacent to the dorsal wall of the urethra, where the ampulla of the vas deferens enters the prostate [34]. Its glandular epithelium contains two cell types: most cells are low cuboidal and showed a weak intracellular and variable apical staining for H+ATPase, whereas other cells are low columnar and were intensely stained in their apical region, and less intensely in the cytoplasm (Fig. 6A). These H+ATPase-rich cells were similar in appearance to the clear cells in the corpus and cauda epididymidis.
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The prostate gland of a sexually mature rat can be anatomically separated into paired ventral, dorsal, and lateral lobes [34]. All lobes consist of a simple columnar epithelium, with cells characterized by a large Golgi complex, but fine structural distinctions exist among them [34]. Distinct patterns of H+ATPase staining were detected in the different prostatic lobes. In some regions of the dorsal prostate, all epithelial cells in the alveoli showed a strong and sharp fluorescence of the luminal membrane and a variable, punctate intracellular staining (Fig. 6B). In other regions, however, the staining was much weaker or even absent (not shown). The abundance of the strongly stained, weakly stained, and unstained regions varied among different animals, indicating its possible dependency on the functional state of the organ. In the lateral prostate, the staining pattern also depended on the precise region examined. Epithelial cells in alveoli positioned more laterally (Fig. 6C) had a brightly stained luminal membrane and a weakly stained cytoplasm. In addition, occasional cells with a large nucleus showed a strong apical staining with less intracellular fluorescence. In the lateral prostate adjacent to the prostatic urethra, sharp staining was present at the apical and basolateral plasma membrane in almost all cells of the prostatic ducts (Fig. 6D). Finally, in the ventral prostate epithelium, two types of H+ATPase-positive cells were detected (Fig. 6E); most cells lining the alveoli had a relatively strong supranuclear staining, whereas the rarer, wineglass-shaped cells with a large nucleus had a bright apical and a weaker cytoplasmic staining.
In the submuscular stroma that surrounds the prostatic urethra, the prostatic ducts were stained both luminally and basolaterally, the staining of the luminal membrane being much stronger (Fig. 7A). Furthermore, H+ATPase was also detectable in the epithelium lining the urethra, as shown in Figure 7B; the apical surface of the transitional stratified epithelium in the prostatic urethra was brightly stained, and the underlying cells were stained intracellularly. Also, the penile urethra, lined with stratified epithelium throughout most of its length, showed a bright specific staining of the luminal surface and a somewhat weaker staining of the lateral cell membranes (Fig. 7C). Similar to the findings in other male reproductive tract segments, shown in this paper, the epithelial staining of the prostatic and penile urethra was absent with the peptide-blocked antibody (data not shown).
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Finally, in the paired bulbourethral (Cowper's) gland we observed no specific staining with the anti-31-kDa H+ATPase subunit antibodies (data not shown).
Immunolocalization of the Vacuolar H+ATPase in Human Male Reproductive Tract
The distribution of the vacuolar H+ATPase was studied in human epididymis, prostate, and urethra. In the small number of samples available for this study, the majority of cells in the columnar epithelium of the corpus epididymidis were not stained with the anti-H+ATPase antibodies. However, scattered clusters of 5–6 H+ATPase-positive cells and some single cells were present through the entire epithelium (Fig. 8, A and B). The cells in clusters had a strong apical staining and weaker intracellular staining, whereas bright apical and diffuse cytoplasmic staining was observed in some individual cells.
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The human prostate consists of tubuloalveolar glands that surround the proximal urethra. In the majority of prostatic alveoli, H+ATPase-related specific staining was not detectable. In some alveoli, however, a specific bright staining was present at the apical domain of most epithelial cells (Fig. 8C), whereas in others, the individual cells had a strong intracellular staining (Fig. 8D).
The luminal surface of the human prostatic urethra was brightly stained with the anti-31-kDa subunit antibody, whereas the cells in the stratified epithelium showed a much weaker staining of the entire cell membrane (Fig. 8E).
DISCUSSION
Our recent identification of H+-secreting cells in the rat epididymis and vas deferens [14, 15] indicates that the acidic pH of the epididymal lumen in vivo [8–10] may be partially generated by the vacuolar type H+ATPase. Some other segments of the male reproductive tract also have an acidic lumen [6–10], but the mechanism of H+ secretion in these regions has not been examined. Our present data demonstrate that various cell types in the efferent duct, ampulla of the vas deferens, seminal vesicles, coagulating gland, ampullary gland, prostate, and urethra also express high levels of the vacuolar H+ATPase either at the cell surface or in the cytoplasm. Seminiferous tubules and rete testis were unstained, which agrees with the absence of a significant H+ATPase band by immunoblotting of the testicular total cell membranes. The seminiferous tubular fluid has a reported pH of between 7.0 and 7.3 in rats [6–9], and the vacuolar H+ATPase is probably not the mechanism responsible for the modest degree of acidification relative to plasma that occurs in this segment.
In the efferent duct, the ciliated cells may assist in sperm movement from the rete testis to the epididymis, whereas the major function of nonciliated cells may be to reabsorb the large amount of fluid secreted by the testes, and assist in sperm concentration [6, 30, 32, 33]. Like the renal proximal tubule cells, they express the aquaporin (AQP)-1 water channel in both apical and basolateral membranes [32]. A bright apical and punctate intracellular staining for the vacuolar H+ATPase in nonciliated cells is also a feature of the cells in proximal tubules [27]. Efferent ducts are a site of intense H+ secretion [6], and the apically located H+ATPase in nonciliated cells could contribute to this process. The extensive punctate intracellular staining in nonciliated cells probably reflects H+ATPase located on endosomes and other intracellular organelles [1].
The presence and function of the H+ATPase in specialized cells along the epididymis and proximal vas deferens have been previously described by our laboratories [14–17]. In addition, we now show that a few tall and slender H+ATPase-positive cells, similar to narrow cells in the initial segment of the epididymis, can be also found in the extreme distal portion of the vas deferens—the ampulla. The regional specialization of vas deferens was highlighted by our recent demonstration that the AQP-2 water channel is located in principal cells of the distal part but not the proximal part of this duct [35].
The seminal vesicles secrete a viscous, protein-rich fluid of a slightly alkaline pH (7.6–8.0) that accounts for about 70% of the total ejaculate volume [3, 36, 37]. The 31-kDa H+ATPase subunit was located exclusively intracellularly in these cells, which is probably associated with organelles of the secretory pathway, and which is well developed in these protein-secreting cells [1, 3]. However, the 31-kDa protein band was barely detectable in immunoblots of total cell membranes from this tissue. This indicates that preparations of total cell membranes from the seminal vesicles and other tissues, as performed in our studies, are not significantly contaminated with intracellular membranes. Thus, abundance of the 31-kDa band in total cell membranes from other tissues probably reflects its localization in plasma membranes that are enriched in our "total cell membrane" preparations.
Whereas in most parts of the coagulating gland the H+ATPase was also localized inside the cells, in some sections we found regions of the epithelium in which cells exhibited a marked apical staining. By analogy with cells in the epididymis and vas deferens, such an apical localization points to a possible role of these cells in luminal H+ secretion. Coagulating gland epithelial cells may, therefore, exist in different functional states even in the same glandular regions.
Our immunocytochemical studies suggest that the ampullary gland epithelium may also be active in H+ secretion; all epithelial cells in this gland were positive for H+ATPase. The low cuboidal cells, which are the majority cell type, showed heterogeneous localization of the staining, suggesting different functional states with respect to H+ secretion. The minority cell population with a rough luminal surface, which probably results from extensive microvillar projections of the apical cell membrane, had an extremely bright apical H+ATPase staining. This appearance is similar to clear cells in the corpus and cauda epididymidis [15, 17], and to A-type (proton secreting) intercalated cells in the renal collecting duct [27, 38], suggesting that H+ secretion may be the major function of these cells.
The prostate secretes a colorless fluid of variable pH that is rich in proteolytic enzymes [21–23]. Membrane fractions from the anatomically and histologically distinct [34] ventral and dorsolateral lobes of this tubuloalveolar gland contained either very little, or abundant H+ATPase, respectively. Immunocytochemistry revealed considerable heterogeneity among the different lobes of the rat prostate, which may reflect functional differences in H+ secretion. The strong apical H+ATPase staining in most alveoli of the dorsal and lateral prostate indicates that practically all epithelial cells could contribute to H+ secretion in these lobes. However, some alveoli in both lobes were stained weakly or not at all, indicating that various portions of the respective epithelia may exist in different functional states. Their staining patterns may reflect a variable content of H+ATPase molecules in the apical membrane that can be regulated by trafficking (exocytosis and endocytosis) of the H+ATPase-carrying intracellular vesicles, as described previously for intercalated cells in the kidney [38] and for H+ATPase-rich cells in the epididymis [16, 17]. In addition, some acini in the prostatic lateral lobe and the periurethral tissue exhibit both apical and basolateral H+ATPase staining. Based on their localization and appearance, these ducts probably drain the prostatic lobes into the urethra. Contrary to epithelial cells in the dorsal and lateral lobes, most cells in the prostatic ventral lobe had a strong and exclusively intracellular staining for the H+ATPase. The supranuclear staining probably represents H+ATPase associated with the prominent Golgi complex in these cells [3]. The occasional wineglass-shaped cells, with a strong apical staining, are similar to "narrow" cells in the epididymis and some parts of the vas deferens, and may be directly involved in H+ secretion.
The tubuloalveolar bulbourethral gland, the size and function of which are strongly androgen-dependent [3], secretes a clear, mucus-like fluid of unknown pH. The absence of labeling with the anti-31-kDa H+ATPase antibodies indicates that the proton secretion in this gland, if present at all, does not occur via this pump.
The strong H+ATPase staining of cells in the stratified epithelium of the prostatic and penile urethra indicates that these are not simply inert conduits for semen, but may be actively involved in ion transport processes. The contribution of urethral acidification to the final pH of the ejaculate, however, is probably limited. Rather, the acidic surface along the urethra may have a protective effect. The acidic pH of urethral secretions may be hormonal- and age-dependent, and may influence the viability and infectivity of various microbes. Alternatively, by analogy with some other epithelia [39], proton secretion may be coupled to other ion transport events in the urethra without any change in pH.
H+ATPase-rich cells were also present in the human male reproductive tract. Single positive cells were scattered among H+ATPase-negative cells in the epididymis and some prostatic acini, and distinct clusters of positive cells were also found in the epididymis. In some prostatic acini and in the urethra, the majority of epithelial cells expressed abundant H+ATPase. While the lack of suitable material precluded a more extensive examination of H+ATPase distribution in human tissues, these findings are consistent with a physiological role for the vacuolar H+ATPase in acidifying the lumen of segments of the human reproductive tract. However, although these studies indicate that the H+ATPase-positive cells are clearly present in the human male reproductive tract, we cannot exclude the possibility that the basic disease (carcinoma of the testis or bladder neck, or benign prostatic hyperplasia) directly or indirectly influenced the pattern of distribution of the antigen in the studied epithelium.
In conclusion, our data show that cells positive for the vacuolar H+ATPase are present in most segments of the male reproductive tract in rat, as well as in the human epididymis, prostate, and urethra. In some epithelial cells, the protein is concentrated at the apical pole (efferent duct, epididymis, vas deferens, ampulla of the vas deferens, coagulating gland, ampullary gland, and dorsal and lateral prostatic lobes), implying a potential role in acidification of the luminal fluid. Proton secretion in more distal segments and accessory glands may be important for adjusting the pH of the final ejaculate to its slightly acidic level [21]. Other cells, whose main function may be the synthesis and secretion of various proteins and/or processing of the material endocytosed from the luminal fluid (most cells in the epididymis, seminal vesicles, and ventral prostate), exhibited only an intracellular localization of the antigen. While the role of vesicle acidification in various intracellular processes is well established, the precise function of the high level of H+ATPase expression in epithelial cells of different reproductive tissues remains to be examined in future studies.
ACKNOWLEDGMENTS
The authors acknowledge the technical assistance of Mrs. Eva Hersak.
FOOTNOTES
First decision: 8 December 2000.
1 This work was supported by grants 0022111 (to C.M.H-K.) and 00220101 (to I.S.) from the Croatian Ministry of Science and Technology; by a Fogarty International Research Collaborative Award, 1-R03-TW01057-01 (to I.S. and D.B.); and by the National Institutes of Health, grant DK38452 (to D.B. and S.B.). 
2 Correspondence: Ivan Saboli, Unit of Molecular Toxicology, Institute for Medical Research and Occupational Health, Ksaverska cesta 2, P.O. Box 291, HR-10001 Zagreb, Croatia. FAX: 385 1 467 3303; sabolic{at}imi.hr
Accepted: January 24, 2001.
Received: November 3, 2000.
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