Molecular Human Reproduction, Vol. 7, No. 7, 611-616,
July 2001
© 2001 European Society of Human Reproduction and Embryology
Testis and spermatogenesis |
Expression of the transmembrane carbonic anhydrases, CA IX and CA XII, in the human male excurrent ducts
1 Departments of Anatomy and Cell Biology and 2 Clinical Chemistry, University of Oulu, Oulu, Finland, 3 Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University, School of Medicine, St Louis, MO, USA and 4 Institute of Virology, Slovak Academy of Sciences, Bratislava, Slovak Republic
Abstract
Testicular fluid is concentrated and acidified during its passage through the excurrent ducts. These processes involve bicarbonate absorption, in which carbonic anhydrases are implicated. In this study, the distribution of two transmembrane carbonic anhydrase isozymes (CA IX and CA XII) in the human excurrent ducts was investigated using isozyme-specific antibodies in conjunction with immunohistochemical and immunoblotting techniques. Specific staining for CA XII was present in the basolateral plasma membrane of the epithelial cells in the efferent ducts, predominantly in the non-ciliated cells. In the epididymal duct, CA XII was detected only in sporadic cells, which also contained CA II, thus suggesting that they are apical mitochondria-rich cells. CA IX was also localized to the basolateral plasma membrane of the epithelium in the efferent ducts, but its staining was weaker and less uniform compared to CA XII. No signal for CA IX was detected in the epididymal duct. Western blot analysis from efferent duct samples revealed specific bands for CA IX and CA XII, confirming that the immunohistochemical stainings represent these isozymes. The expression of CA XII and CA IX in the excurrent duct system and co-expression of CA XII with Aquaporin-1 in the same efferent duct epithelial cells suggest their functional involvement in ion transport and concentration processes of testicular fluid.
bicarbonate/carbonic anhydrase/efferent ducts/epididymis/immunohistochemistry
Introduction
The fluid leaving the testis is chemically modified and extensively concentrated during the passage through the testicular excurrent ducts (Robaire and Hermo, 1988
). Efferent ducts reabsorb up to 96% of the fluid released with spermatozoa from the testis (Clulow et al., 1994). Water transport in these ducts is believed to be partly mediated by Aquaporin-1 (AQP1), a water channel protein (Brown et al., 1993; Fisher et al., 1998), which is also present in the apical and basolateral plasma membranes of the renal tubules (Nielsen et al., 1993). The fluid absorption is coupled to transepithelial ion transport, as occurs in the homologous renal proximal tubule (Clulow et al., 1994). Bicarbonate ions are also effectively absorbed from the efferent duct lumen (Newcombe et al., 2000). The absorption of bicarbonate involves carbonic anhydrase (CA) enzyme, which catalyses the reversible hydration of carbon dioxide in the reaction (Sly and Hu, 1995):
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Carbonic anhydrase has been shown to be present in the efferent ducts and epididymis of various mammals (Cohen et al., 1976; Goyal et al., 1980; Kaunisto et al., 1990; Ekstedt et al., 1991; Ekstedt and Ridderstråle, 1992; Parkkila et al., 1993b; Kaunisto et al., 1995), but the isozymes have not been comprehensively identified. Previous immunohistochemical studies in our laboratory have demonstrated the presence of CA II and CA IV isozymes in the human epididymis and ductus deferens, where they have been proposed to be involved in acidification of the epididymal fluid (Kaunisto et al., 1990; Parkkila et al., 1993b). Basolateral CA activity in the efferent ducts of the boar and in the epididymis of the rabbit has also been found (Ekstedt et al., 1991; Ekstedt and Ridderstråle, 1992), but the basolateral isozyme(s) expressed in the excurrent ducts have not been identified.
To achieve a more comprehensive view of CA isozymes implicated in modification of the testicular fluid, we examined the expression of two transmembrane isozymes (CA IX and CA XII) in the human male excurrent ducts and compared their expression to AQP1. CA IX has been previously localized to the gastrointestinal epithelium (Pastorek et al., 1994; Pastoreková et al., 1997; Saarnio et al., 1998b). CA XII mRNA has been identified in a variety of tissues including kidney, colon, prostate, pancreas, ovary, testis, lung, and brain (Ivanov et al., 1998; Türeci et al., 1998), whereas CA XII protein has been demonstrated only in endometrium (Karhumaa et al., 2000), colon (Kivelä et al., 2000), and kidney (Parkkila et al., 2000). Both isozymes are expressed in the basolateral plasma membrane of the epithelial cells where CA IX has been proposed to participate in cell proliferation and/or adhesion (Pastorek et al., 1994; Závada et al., 2000), while the function of CA XII is so far unknown. Our present results show that CA XII, and, to a lesser extent, CA IX localize to the same cells as AQP1 in the excurrent tubule system, thus suggesting their functional linkage to ion transport and fluid reabsorption processes.
Materials and methods
Antibodies
The polyclonal antibody against the secretory form of human CA XII, the monoclonal antibody against human CA IX (M75) and the polyclonal rabbit antiserum to human CA II have been previously described (Parkkila et al., 1993a; Pastoreková et al., 1997; Karhumaa et al., 2000). The antibody against AQP1 (Delporte et al., 1996) was kindly provided by Dr Bruce Baum (NIH, Bethesda, MD, USA). A polyclonal antibody to human Ki-67 (Zymed Laboratories, San Francisco, CA, USA) was used to assess the proliferative activity of the epithelium (Kujat et al., 1995).
Tissue preparation
Samples of the human efferent ducts and epididymis were obtained together with routine histopathological specimens taken during surgical operations for prostatic carcinoma (n = 4). Informed consent was obtained from each patient and the research was carried out according to the provisions of the Declaration of Helsinki. The specimens were fixed in Carnoy's fluid (absolute ethanol + chloroform + glacial acetic acid 6:3:1) for 6 h at 4°C. The samples were then dehydrated and embedded in paraffin wax in a vacuum oven at 58°C. For immunohistochemistry, sections of 5 µm were cut and placed on microscope slides. For Western blotting, the same paraffin-embedded samples were deparaffinized in xylene, washed in 100% ethanol, and solubilized into sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) sample buffer (Conti et al., 1988). Fresh human excurrent duct specimens were no longer available for the study because of a change in orchiectomy technique.
Immunohistochemistry
The tissue sections were immunostained by the biotin–streptavidin complex method, employing the following steps. (i) Pretreatment of the sections with undiluted cow colostral whey for 40 min and rinsing in phosphate-buffered saline (PBS). (ii) Incubation for 1 h with the primary antibody diluted 1:100 or 1:10 (M75 hybridoma medium) in 1% bovine serum albumin (BSA)–PBS. (iii) Incubation for 1 h with biotinylated swine anti-rabbit IgG (Dakopatts, Glostrup, Denmark) or goat anti-mouse IgG (Dakopatts) diluted 1:300 in 1% BSA–PBS. (iv) Incubation for 30 min with peroxidase-conjugated streptavidin (Dakopatts) diluted 1:600 in PBS. (v) Incubation for 2 min in DAB solution containing 9 mg 3,3'-diaminobenzidine tetrahydrochloride (Fluka, Buchs, Switzerland) in 15 ml PBS + 5 µl 30% H2O2. The sections were washed three times for 5 min in PBS after incubation steps ii, iii and iv. Some sections were counterstained with haematoxylin after the immunostaining procedures. All the incubations and washings were carried out at room temperature, and the sections were finally mounted in Permount (Fisher Scientific, Fair Lawn, NJ, USA). The stained sections were examined and photographed with a Nikon Eclipse E600 microscope (Tokyo, Japan).
Western blotting
All the reagents for SDS–PAGE were from Bio-Rad Laboratories (Richmond, CA, USA). The electrophoreses were performed in a Mini-Protean electrophoresis unit (Bio-Rad Laboratories, Pierce, Rockford, IL, USA) under reducing conditions (Laemmli, 1970), using a 10% acrylamide separating gel and a 4% acrylamide stacking gel.
The proteins were transferred electrophoretically from the gels to a PVDF membrane (Millipore Corporation, Bedford, MA, USA) in a Novex Blot Module (Novex). After the transblotting, the membrane was first incubated with TBST buffer (10 mmol/l Tris–HCl, pH 7.5, 150 mmol/l NaCl, 0.05% Tween-20) containing 10% cow colostral whey (Hi-Col, Oulu, Finland) for 30 min at room temperature and then with anti-human CA XII antiserum diluted 1:200 or M75 hybridoma medium (anti-MN/CA IX antibody) diluted 1:100 in TBST buffer overnight at 4°C or for 1 h at room temperature. The membrane was washed five times for 5 min with TBST buffer and incubated for 1 h at room temperature with peroxidase-conjugated goat anti-rabbit (Bio-Rad Laboratories) diluted 1:16 000 in TBST buffer or 30 min at room temperature with alkaline phosphatase-conjugated goat anti-mouse IgG (Bio-Rad Laboratories) diluted 1:3000 in TBST buffer. After washing four times for 5 min in TBST buffer, the polypeptides were visualized by chemiluminescence substrates (Bio-Rad Laboratories).
Results
Immunohistochemistry
The epithelium of the human efferent ducts stained positively for both CA IX and CA XII (Table I). CA XII was present in almost all epithelial cells in the efferent ducts (Figure 1A) and it was mainly localized to the basolateral plasma membrane (Figure 1B, E). The less intense intracellular signal probably represents the newly synthesized enzyme in the endoplasmic reticulum, as has previously been shown in CHO-K1 cells expressing CA XII (Karhumaa et al., 2000).
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CA XII has recently been localized in the human kidney, where its segmental, cellular and subcellular distribution as well as its colocalization with AQP2 suggested a role in the regulation of water homeostasis (Parkkila et al., 2000
). Since water transport in the excurrent ducts is partly mediated by AQP1, we immunolocalized CA XII and AQP1 in parallel tissue sections to elucidate whether these two proteins are coexpressed. The staining for AQP1 was observed in the efferent ducts (Figure 1C
) and it was confined to the same tubules as CA XII (compare Figure 1A,C). AQP1 staining was predominantly located in the apical plasma membrane of the epithelial cells, while the basolateral staining was faint but still existed (Figure 1D). In addition, the plasma membranes of the smooth muscle cells surrounding the ducts and the endothelial cells of blood capillaries were positively stained as reported earlier in the rat (Brown et al., 1993). Moreover, CA XII was predominantly expressed in the non-ciliated cells of the efferent ducts (Figure 1E), where AQP1 is also known to be expressed (Brown et al., 1993). The staining of CA XII was faint or absent in the ciliated cells (Figure 1E). In the epididymal ducts only sporadic epithelial cells were positive for CA XII. The same cells were also found to express CA II, suggesting that they represent apical mitochondria-rich cells (Table I, Figure 1F, G). Figure 1H provides an example of controls for the specificity of the antibody for CA XII. The positive staining seen in the efferent ducts was blocked in sections exposed to antibody that was pretreated with recombinant CA XII protein (100 µg/microscope slide).
Staining of excurrent ducts for CA IX revealed that this isozyme was also expressed in the basolateral plasma membrane of the efferent duct epithelium (Figure 1I), although the staining was less uniform compared to CA XII. In some tubule sections, CA IX was present in almost all ciliated and non-ciliated epithelial cells (Figure 1I, J), whereas only sporadic cells were positively stained in some other sections (Figure 1K). Epididymal duct epithelium was completely negative for CA IX (Table I).
To determine whether the expression of CA IX correlated to cellular proliferation activity in the efferent ducts, the tissue sections were also immunostained for Ki-67. Only sporadic nuclei in the efferent duct epithelium stained for Ki-67 (Figure 1L), suggesting that this epithelium had relatively low proliferative activity. CA IX was expressed in almost all epithelial cells in some parts of the efferent ducts, and thus its expression pattern did not correlate with Ki-67 (compare panels I, J, and L showing the maximum frequency of CA IX- and Ki-67-positive cells). Control stainings using normal rabbit serum or omitting the primary antibody (data not shown) were negative.
Western blotting
Anti-human CA IX antibody recognized a specific, diffuse 54 to 58 kDa polypeptide band of CA IX (Pastorek et al., 1994) in the efferent duct samples (Figure 2). Anti-human CA XII antibody identified a single, diffuse 40–45 kDa polypeptide band of CA XII (Figure 2) (Karhumaa et al., 2000). The diffuse bands were probably due to deglycosylation and proteolytic degradation of the polypeptides in the paraffin-embedded samples.
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Discussion
The fluid leaving the testis is chemically modified and concentrated during its passage along the testicular excurrent ducts. Composition of the fluid is critical for the maturation and storage of spermatozoa. Efferent ducts are responsible for absorbing up to 96% of the luminal fluid produced by the seminiferous tubules (Clulow et al., 1994). The water reabsorption in these ducts is coupled to active transport of ions, mainly sodium and chloride (Ilio and Hess, 1994). The present finding that CA IX and CA XII are expressed in the basolateral plasma membranes of the epithelial cells in efferent ducts, suggests that these isozymes may participate in transepithelial ion transport, and thereby in water absorption from the ducts. Na+ and Cl– ions absorbed from the lumen could be transported across the basolateral membrane via a Cl–/HCO3– exchanger or Na+/HCO3– co-transporter. It has recently been shown that Cl–/HCO3– exchanger isoform, AE2, is expressed in the basolateral plasma membrane of the epithelium of rat efferent ducts and epididymis (Jensen et al., 1999b). In efferent ducts, the protein was expressed in the ciliated cells (Jensen et al., 1999b). In addition, they found Na+/HCO3– co-transporter protein, NBC, in the basolateral surface of epididymal epithelium (Jensen et al., 1999a). CA could prevent the interstitial accumulation of HCO3– transported by Cl–/HCO3– exchange or Na+/HCO3– co-transport. Alternatively, NBC might need basolateral CA to eliminate CO32– gradients across the membrane, as suggested for the renal proximal tubule (Müller-Berger et al., 1997). However, expression and localization of these ion transporters in human excurrent ducts is not known at present. Interestingly, a recent report by Tsuruoka and colleagues indicated an important role for basolateral CA in bicarbonate and fluid absorption in rabbit proximal tubule. They showed that inhibition of basolateral CA by impermeable inhibitor resulted in a 62% decrease in HCO3– and a 31% decrease in fluid absorption (Tsuruoka et al., 2001).
To strengthen the hypothesis on the role of basolateral CA in water absorption, we compared the localization of CA XII and CA IX to a water channel protein, AQP1, which is known to be expressed in the non-ciliated cells of efferent duct epithelium of rat and marmoset monkey (Brown et al., 1993; Fisher et al., 1998), where it is thought to be involved in water absorption. Distinct co-localization of AQP1 with CA XII observed in the efferent ducts is compatible with the role of basolateral CA in water absorption. Recent studies in our laboratory have shown that CA XII is also expressed in the renal proximal tubule (Parkkila et al., 2000), where the presence of AQP1 is well-documented (Nielsen et al., 1993). In addition, the segmental, cellular and subcellular distribution of CA XII, and its co-localization with AQP2 in the human kidney, further strengthens the role of CA XII in the regulation of water homeostasis (Parkkila et al., 2000). Moreover, CA XII is widely expressed in other water absorbing epithelia, including gallbladder (S.Parkkila, personal communication) and colon (Kivelä et al., 2000).
The expression of CA IX in both ciliated and non-ciliated epithelial cells in some tubule sections of efferent ducts and its absence in other sections may reflect a more complex function compared to CA XII. CA IX together with CA XII may be involved in ion transport events and fluid concentration, but due to its different distribution pattern CA IX may also have additional functions in the male excurrent ducts. Whereas CA XII is widely distributed in normal tissues (Ivanov et al., 1998; Türeci et al., 1998; Karhumaa et al., 2000; Kivelä et al., 2000; Parkkila et al., 2000), CA IX is more restricted to neoplastic cells (Liao et al., 1994; McKiernan et al., 1997; Turner et al., 1997; Saarnio et al., 1998a; Vermylen et al., 1999) linking it to cell proliferation and transformation processes. The function of CA IX has also been linked to non-malignant cell proliferation (Saarnio et al., 1998b). However, we found here a poor correlation between the localization of CA IX and the proliferation marker Ki-67, suggesting that CA IX is not implicated in growth regulation in the epithelium of the efferent ducts. Other functions for CA IX have also been proposed, such as intercellular communication (Pastoreková et al., 1997) and cell adhesion (Závada et al., 2000). Although the role of CA IX in cell–cell and cell–matrix contacts cannot be excluded in excurrent ducts, its segmental pattern of expression hints, however, at other functions.
In the epididymal duct, CA XII was expressed in a subpopulation of epithelial cells which also contained CA II, suggesting that these were apical mitochondria-rich cells (Kaunisto et al., 1990). These cells correspond to rat narrow and clear cells which are involved in acidification of the epididymal fluid (Brown et al., 1992; Martínez-García et al., 1995). Luminal proton secretion involves basolateral bicarbonate exit, which could be mediated via AE2 or NBC, since these proteins are present also in the basolateral membranes of narrow and clear cells in the rat epididymis (Jensen et al., 1999a,b). Basolateral CA in these cells might facilitate basolateral CO32– and/or HCO3– transport as suggested above.
The present results show that the human efferent ducts express CA IX and CA XII in the basolateral membranes where they may be involved in multiple functions including transepithelial acid-base transport and water absorption. The presence of CA XII in apical mitochondria-rich cells in the epididymal duct suggests its implication also in luminal acidification.
Acknowledgements
We are grateful to Dr Bruce Baum for providing anti-AQP1 antibody. We also thank Ms Lissu Hukkanen and Ms Seija Leskelä for their skilful technical assistance. The work was supported by the grants from Sigrid Juselius Foundation to S.P., from Bayer Corporation to J.P., from the Slovak Scientific Grant Agency to S.P., and from National Institutes of Health (DK 40163) to W.S.S.
Notes
5 To whom correspondence should be addressed at: Department of Anatomy and Cell Biology, Box 5000, FIN-90014 University of Oulu, Finland. E-mail: karhumaa{at}usa.net 
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Submitted on February 15, 2001; accepted on April 27, 2001.
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