Copyright ©The Histochemical Society, Inc.

Region- and Cell-specific Differences in the Distribution of Carbonic Anhydrases II, III, XII, and XIV in the Adult Rat Epididymis

Louis Hermo, Dennis Lee Chong, Pierre Moffatt, William S. Sly, Abdul Waheed and Charles E. Smith

Department of Anatomy and Cell Biology (LH) and Faculty of Dentistry (DLC), McGill University, Montreal, Quebec; Departement de Stomatologie, Universite de Montreal, Montreal, Quebec (PM,CES); and Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, St. Louis, Missouri (WSS,AW)

Correspondence to: Dr. Louis Hermo, McGill University Department of Anatomy and Cell Biology, 3640 University St., Montreal, Quebec, Canada H3A 2B2. E-mail: louis.hermo{at}mcgill.ca


    Summary
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 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 Literature Cited
 
We employed RT-PCR followed by light microscope immunocytochemistry on St. Marie's- and Bouin's-fixed tissues to define the distribution of carbonic anhydrase (CA) isoforms in the male reproductive tract. The data revealed that CA II, III, IV, XII, and XIV were expressed in rat epididymis. Whereas CA III was found in principal cells of all epididymal regions, CA II was localized in narrow cells of the initial segment and principal cells of all regions. CA XII expression was most intense in the corpus and proximal cauda regions, where it appeared over the basolateral plasma membranes of principal cells. Narrow cells of the initial segment also revealed intense reactions, as did basal cells of the corpus and proximal cauda regions. Principal cells of the initial segment and proximal caput regions showed diffuse apical cytosolic reactions and occasional basolateral staining for CA XIV, whereas principal cells of distal regions showed more diffuse cytosolic reactions highlighting both apical and basal regions of the cell, with basal cells also being reactive. These data suggest subtle differences in cell type and subcellular- and region-specific distributions for CAs in their role of fine-tuning pH in the lumen, cell cytosol, and intervening intercellular spaces of the epididymis. (J Histochem Cytochem 53:699–713, 2005)

Key Words: RT-PCR • immunocytochemistry • principal cells • carbonic anhydrase • epididymis • pH • basal cells • region specificity • rat


    Introduction
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 Introduction
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AFTER BEING PRODUCED in the testis, sperm pass through the efferent ducts and epididymis and mature to become motile and fertile. The epithelial cells that line the epididymal duct are involved in this maturation process by activities such as secretion and endocytosis of various molecules (Hamilton 1975Go; Robaire and Hermo 1988Go; Hermo et al. 1994Go). In addition to proteins and lipids, a significant component of the luminal contents in the efferent ducts and epididymis is water (Hinton and Setchell 1993Go; Hess et al. 2002Go; Turner 2002Go; Wong et al. 2002Go). Water produced by Sertoli cells of the testis is reabsorbed mainly by the epithelial cells lining the efferent ducts, but water continues to be removed from the lumen of the epididymis, as well as secreted, to prevent dehydration of this tissue (Wong et al. 2002Go). In addition to water reabsorption, the epithelial cells of the epididymis transport ions and solutes between the lumen, cytosol, and intercellular spaces, utilizing various membrane pumps, channels, and transporter systems in conjunction with a family of enzymes known as carbonic anhydrases (CAs) (Hermo and Robaire 2002Go; Hess 2002Go; Turner 2002Go; Breton 2003Go). Collectively, these various integrated systems are responsible for maintaining proper cell volume, as well as the appropriate pH in various microenvironments (Hess 2002Go; Turner 2002Go; Wong et al. 2002Go; Breton 2003Go).

In the lumen of the epididymal duct, the concentrations of ions such as Na+ decline from the caput to the cauda epididymidis, whereas HCO3 ions show a sharp decline through the initial segment followed by a slight rise as pH changes from a starting value near pH 7.2 to a low of pH 6.6 followed by a slight rise to pH 6.8 in the cauda (Levine and Marsh 1971Go; Levine and Kelly 1978Go; Turner 1984Go; Caflisch and DuBose 1990Go; Turner 2002Go; Breton 2003Go). It has been shown in several species that this mild fluid acidification immobilizes cauda sperm, and this prevents premature activation of their acrosomal enzymes in the epididymis (Acott and Carr 1984Go; Carr et al. 1985Go; Clulow et al. 1992Go; Gatti et al. 1993Go; Hinton et al. 1995Go; Jones and Murdoch 1996Go). Although exact details of the mechanisms of this acidification are not fully understood, the enzyme CA is clearly involved (Cohen et al. 1976Go; Brown et al. 1992Go Kaunisto et al. 1995Go; Breton et al. 1999Go; Hermo et al. 2000Go; Breton 2003Go), as are apically positioned epithelial H+-ATPase proton pumps (Breton et al. 1996Go; Hermo et al. 2000Go).

CAs exist as three genetically unrelated families of proteins, of which only the {alpha} genes are present in vertebrates (Chegwidden and Carter 2000Go; Sly 2000Go). The {alpha}-CAs are monomeric zinc metalloenzymes of ~29-kDa molecular mass. So far, several different {alpha}-CA isoforms have been reported (Vince and Reithmeier 1998Go; Chegwidden and Carter 2000Go; Parkkila 2000Go; Sly 2000Go; Tripp et al. 2001Go). CAs are important for acid–base regulation, catalyzing the reversible reaction of CO2 + H2O to H+ + HCO3. Several CAs can be expressed in the same mammalian tissue, and although they may show cell type, region, and subcellular specificity, more than one CA can be expressed in the same cell type. CAs have unique functions in different cellular locations and result in a variety of diseases in their alteration or absence (Chegwidden and Carter 2000Go; Parkkila 2000Go; Sly 2000Go; Tripp et al. 2001Go). Whereas some CAs are present in the cytoplasm (I–III, VII, XIII), others are found in mitochondria (V) or salivary secretions (VI). The remaining CAs (CA IV, IX, XII, and XIV) are transmembrane proteins, with CA IV being glycosylphosphatidylinositol (GPI) anchored (Chegwidden and Carter 2000Go; Parkkila 2000Go; Sly 2000Go; Tripp et al. 2001Go).

In the adult rat efferent ducts and epididymis, only two isoforms of the CA family of proteins have been investigated to date. In the epididymis, CA IV has been well documented to be present along the apical plasma membranes of principal cells in the distal caput, the corpus, and proximal cauda regions, with maximal expression occurring in the corpus (Kaunisto et al. 1995Go,1999Go). However, results for CA II distribution have been conflicting, with localizations reported in narrow cells of the initial segment and intermediate zone (Hermo et al. 2000Go), both narrow and clear cells (Breton et al. 1996Go,1998Go) and narrow and principal cells (Kaunisto et al. 1995Go,1999Go). CA II has also been identified in the epithelial non-ciliated cells of efferent ducts (Hess 2002Go).

The epithelial cells of the different segments of the kidney nephron bear notable structural and functional similarities to those of the efferent ducts and epididymis on the basis of embryological derivations, with the proximal convoluted tubule bearing similarity to the efferent ducts and proximal epididymal regions and the collecting duct to the distal epididymal regions and the vas deferens (Hinton and Turner 1988Go; Hess 2002Go). In the kidney, many CA isoforms have been identified and are often expressed in a precise cell- and region-specific manner including CA II, IV, XII, and XIV (Mori et al. 1999Go; Sly 2000Go; Sterling et al. 2001Go; Kaunisto et al. 2002Go; Kyllonen et al. 2003Go). On this basis, it seemed reasonable to hypothesize that isoforms other than CA II and IV are probably expressed in the efferent ducts and epididymis of adult rats. This was investigated by RT-PCR using oligonucleotide primers specific for rat CA II and IV as positive controls and other isoform-specific primers for CAs known to be present in kidney and other cell types. RT-PCR results for identified isoforms were complemented with light microscope immunocytochemistry.


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Animals and Experimental Protocols
RT-PCR Experiments
Male Wistar rats (Charles River Canada; St Constant, Quebec) weighing ~100–125 g were anesthetized with halothane and decapitated. Various tissues including testis, epididymis, kidney, intestine, muscle, brain, liver, salivary glands, and bone were removed, snap frozen in liquid nitrogen, and freeze dried. Total RNA was extracted from ~100 mg of each lyophilized tissue after being homogenized with a polytron in 2.5 ml of Trizol (Invitrogen Canada Inc.; Burlington, ON, Canada). The final RNA pellet was resuspended in diethyl pyrocarbonate–treated water. RNA yields obtained by these procedures ranged from 440 µg to 1.7 mg, depending upon the tissue. The quality of the RNAs was judged to be acceptable as determined by the integrity of rRNAs when electrophoresed on formaldehyde-agarose gels and stained with ethidium bromide. RT-PCR reactions were carried out on total RNA using the One Tube Titan kit (Roche Diagnostics; Laval, QC, Canada) according to the manufacturer's recommendations and oligonucleotide primers designed specifically for CA isoforms present in rat (Table 1). Briefly, 25-µl reactions were set up on ice by mixing 500 ng total RNA, 5 µl RT 5x buffer, 0.5 µM of each primer, 0.2 mM dNTPs, 5 mM DTT, 0.5 µl Titan enzyme mix, and 6 U RNAGuard (Amersham Biosciences Inc.; Baie d'Urfé, Quebec). After a 30-min reverse transcription step at 50C, the cDNAs were subjected to the following PCR cycling conditions: 94C for 30 sec, 56C for 30 sec, and 68C for 45 sec. Eight-µl aliquots were taken after 25 and 40 cycles, loaded on 1.8% agarose gels, and visualized by ethidium bromide staining.


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Table 1

Oligonucleotide primer sequences designed for RT-PCR detection of the various rat carbonic anhydrase isoforms

 
Light Microscope Experiments
Adult male Sprague Dawley rats (Charles River Canada) weighing ~300–350 g were maintained on 12-hr light and dark cycles and given lab chow and water ad libitum. The rats were anesthetized with phenobarbitol (65 mg/kg) and the testes, efferent ducts, and epididymides of four animals per group were fixed by vascular perfusion with either Bouin's or St. Marie's fixative (95% ethanol, 1% acetic acid) via the abdominal aorta for 10 min (Cyr et al. 1999Go). Following perfusions, various tissues were removed, and the epididymides were sliced open so that subsequent preparations would include all major regions—the initial segment, intermediate zone, caput, corpus, and cauda (Hermo et al. 1991Go)—in a single section. The tissues were immersed in fresh fixative for 72 hr and then were dehydrated and embedded in paraffin. Tissue sections at 5-µm thickness were cut and mounted on glass slides. The Animal Care Committee of McGill University approved all protocols described in these experiments.

Dako Envision+ System Kit
Primary antibodies used in this study were mouse monoclonal CA III (1:100) (courtesy of Spectral Diagnostics; Toronto, ON, Canada), rabbit anti-mouse CA XII (1:100) (Tureci et al. 1998Go), rabbit anti-mouse CA XIV (1:100) (Mori et al. 1999Go), and rabbit anti-bovine CA II antibody (Biodesign; Saco, ME).

Sections of Bouin's and St. Marie's fixed tissues were deparaffinized with Histoclear and rehydrated in a series of 100%, 100%, 95%, 80%, 70%, and 50% ethanol solutions and distilled water, respectively. During hydration, residual picric acid was neutralized by using a 70% ethanol solution containing 1% lithium carbonate. After hydration, endogenous peroxidase activity was blocked for 5 min with Dako peroxidase blocking reagent (DakoCytomation; Mississauga, ON, Canada) followed by washing. Localizations of CA II, III, XII, and XIV were performed using the Dako Envision+ Peroxidase diaminobenzidine (DAB) kit. Washings between each step were done for 10 min using a buffer solution containing 0.05 M Tris, 0.3 M NaCl, and 0.1% Tween 20, pH 7.2–7.6. Substrate–chromogen solutions were prepared by adding one drop of liquid DAB + chromogen with an additional 4 µl to 1 ml of the buffered substrate. The sections were counterstained for 10 sec in a 1:5 diluted solution of 0.1% methylene blue and 0.1% thionin, washed, and quickly back dehydrated to Histoclear. Coverslips were then mounted with Permount.

Immunofluorescence
Primary antibodies used were rabbit anti-mouse CA XII (1:100) (Tureci et al. 1998Go) and rabbit anti-mouse CA XIV (1:100) (Mori et al. 1999Go); the secondary antibody was an AlexaFluor 594-labeled goat anti-rabbit IgG (1:250) (Molecular Probes; Eugene, OR).

Sections of St. Marie's fixed tissues were deparaffinized as per the DAKO protocol. Blocking of nonspecific binding sites was done on some sections using the Dako protein block serum-free solution for 20 min (DakoCytomation), followed by incubations with primary antibody for 1.5 hr at room temperature. The sections were washed and incubated for 30 min with AlexaFluor 594-labeled goat anti-rabbit IgG. Following this step, nuclei were stained by incubating the sections for 5 min in a 300-nM 4',6-diamidino-2-phenylindole dihydrochloride (DAPI) solution at room temperature. Coverslips were mounted using Vectashield Hard Set aqueous mounting medium (Vector Laboratories; Burlington, ON, Canada). The sections were examined and photographed on a Zeiss Axioskop 2 motorized light microscope equipped with variable intensity FluorArc epifluoresence mercury lighting and AxioCam HR color digital camera (Carl Zeiss Canada; Montreal, QC, Canada).

Negative controls were performed for each experiment and consisted of exposing sections to solutions containing all components except primary antibody (polyclonal and monoclonal) or normal rabbit serum (polyclonal antibodies). Positive controls were also done using sections from tissues known to express specific CA isoforms (e.g., CA III, skeletal muscle; CA IV, kidney; CA XIV, liver).


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 Materials and Methods
 Results
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 Literature Cited
 
CA Expression in Epididymis by RT-PCR
Rat oligonucleotide primers specific for 10 of the 15 known isoforms of CA were designed from sequences available at GenBank (Table 1). All primer pairs except for CA XII were derived from different exons of the genes to prevent possible nonspecific amplification due to contaminating genomic DNA. The specificity and efficacy of the oligonucleotide primers were first tested by running RT-PCR reactions using RNA isolated from tissues reported to contain specific isoforms (e.g., bone marrow, CA I; muscle, CA III; kidney, CA IV, etc.). Agarose gel electrophoresis revealed specific amplification products for each primer, as illustrated in the top panel of Figure 1 for CA I, III–VII, IX, and XIV. A single amplification band observed for every isoform except CA III, which yielded a slightly more intense nonspecific lower-size band, attested to the specificity of the primers. RT-PCR analyses performed with all primer pairs using RNA isolated from the epididymis indicated the presence of CA II, III, IV, XII, and XIV in this tissue (Figure 1, bottom panel). Although not quantitative, the results suggested that expression levels of CA XII and CA XIV were especially abundant in the epididymis, compared with CA II, III, and IV (Figure 1, bottom panel). The specificity of the signal detected for CA XII was further assessed in identical reactions run without the RT step. The latter samples gave no detectable amplification products (not shown).



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Figure 1

Ethidium bromide–stained agarose gels illustrating amplification fragments detected following 25 and 40 RT-PCR cycles with total RNA isolated from various control tissues (top panel) and the epididymis (bottom panel) and oligonucleotide probes listed in Table 1 for each carbonic anhydrase isoform examined. Molecular weight ladders (M) are indicated on the left side of the top and bottom panels (bp, base pairs).

 
Light Microscope Immunocytochemical Localizations of CAs II, III, XII, and XIV in the Adult Rat Efferent Ducts and Epididymis
Control sections of efferent ducts and epididymis (all regions) incubated in solutions lacking a primary antibody or incubated with normal rabbit serum instead of a primary polyclonal antibody consistently showed no staining of the epithelium or luminal and intertubular contents (Figures 2A and 2B).



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Figures 2 and 3

Figure 2 Control sections of the distal initial segment (A) and cauda (B) epididymidis of adult rats incubated in solutions containing all components except primary antibody. No peroxidase reaction product is present over the entire epithelium, including principal (P), narrow/apical (long arrows), basal (small arrowheads), and clear (large arrowheads) cells, sperm in the lumen (Lu), and contents of the intertubular space (IT). Magnification, x260.

Figure 3 Immunoperoxidase staining in different regions of the epididymis in sections incubated with anti-carbonic anhydrase (CA) III antibody. In the initial segment (A), a moderate reaction is evident over principal cells (P), whereas in the caput (B), corpus (B inset) and proximal cauda (C) regions, they display an intense reaction; in the distal cauda region (D), both reactive (P) and unreactive (open arrows) principal cells are present. Reactions with the anti-CA III antibody are evident over the cytosol of principal cells (A–D). No reaction is evident over narrow cells (long arrow). Clear cells (large arrowheads) of the caput (B), corpus (B inset) and cauda regions are unreactive as are basal cells (small arrowheads). In the epididymal lumen (Lu), the cytoplasmic droplets of sperm are unreactive in the various epididymal regions (A,B), whereas the smooth muscle cells (Sm) surrounding the tubules of the distal cauda epididymidis show intense reactivity (D). IT, intertubular space. Magnification (A,B), x325; (C,D), x200.

 
Non-ciliated and ciliated cells of efferent ducts and principal cells, but not narrow cells, in the initial segment of the epididymis were weakly reactive with the anti-CA III antibody (Figure 3A). This was in contrast to principal cells in the caput (Figure 3B), corpus (Figure 3B inset), and proximal cauda (Figure 3C), which appeared progressively more intensely reactive with the anti-CA III antibody. In the distal cauda region (Figure 3D), principal cells showed a variable staining pattern ranging from intense to completely unreactive. Immunoreactions with the anti-CA III antibody were uniform throughout the cytoplasm of principal cells, and there was no obvious staining along their microvillar borders (Figures 3A–3D). Both clear cells and basal cells in the caput, corpus, and cauda regions were unreactive (Figures 3B–3D). In the epididymal lumen, the cytoplasmic droplets of sperm were unreactive in the various epididymal regions (Figures 3A–3D), whereas smooth muscle cells surrounding the tubules of the cauda epididymidis were intensely reactive with the anti-CA III antibody (Figure 3D).

Sections incubated with the anti-CA II antibody also showed reactions over the non-ciliated and ciliated cells of the efferent ducts (not shown). Narrow cells of the initial segment were intensely reactive, as were principal cells across the entire epididymis, with the reaction being cytoplasmic (Figures 4A and 4B). Clear and basal cells of the entire epididymis were consistently unreactive (Figures 4A and 4B). However, the cytoplasmic droplets of sperm in the epididymal lumens showed an intense reaction (Figure 4B), as did the cytoplasm of elongating spermatids of the seminiferous epithelium in the testis (not shown). Smooth muscle cells of the cauda epididymidis were also intensely reactive with the anti-CA II antibody (not shown).



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Figure 4

Immunoperoxidase staining with anti-CA II antibody reveals a strong reaction over narrow cells (long arrows) of the initial segment (A). In addition, an intense cytosolic reaction is noted over principal cells (P) of the entire epididymis (A,B). No visible staining is found in clear cells (large arrowheads) or basal cells (small arrowheads) throughout the epididymis (A,B). The cytoplasmic droplets (small arrows) of sperm in the lumen (Lu) are reactive (B). Magnification, x325 for each.

 
Reactions with the anti-CA XII antibody in efferent ducts were spotty and restricted to the basolateral plasma membranes of only an occasional non-ciliated cell (Figure 5A, right side). In the initial segment, only narrow cells showed prominent reactions localized basolaterally (Figure 5A, left side). Few principal cells showed a basolateral reaction in the proximal initial segment (Figure 5A), with more being evident moving from the proximal caput to the distal caput epididymidis (Figure 5B). However, the most conspicuous reaction was seen in the corpus and proximal cauda epididymidis (Figures 5C and 5D). In these regions, heavy deposits of reaction product accumulated along the lateral plasma membranes of adjacent principal cells, the principal/clear-cell interface, and along the base of the epithelium (Figures 5C and 5D). The basal reaction represented staining of the basal plasma membrane of principal and basal cells (Figures 5C and 5D). In the distal cauda epididymidis, a reaction appeared only between some adjacent principal cells and in sporadic areas of the base of the epithelium (Figure 5E). Halo cells were reactive, and this was especially evident in the initial segment where the overall staining pattern of the epithelium did not mask their presence (Figure 5B). Clear cells of the entire epididymis were consistently unreactive (Figure 5E). Smooth muscle cells of the cauda epididymidis were unreactive for CA XII. In the lumen, the principal piece of the tails of sperm showed a reaction (Figure 5A), but the reaction was most intense over the tails of the elongating step 19 spermatids in testicular sections (Figure 5A inset). Immunofluorescent staining with the anti-CA XII antibody confirmed the above results and revealed that the strongest reactions were observed in the corpus and proximal cauda epididymidis, where it delineated the basolateral plasma membranes of adjacent principal cells and the principal/clear-cell interface and was distributed along the base of the epithelium, which included staining of basal cells. No staining of clear cells was noted along the entire epididymis (Figures 6A–6D).



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Figure 5

Immunoperoxidase staining with anti-CA XII antibody is seen over the basolateral plasma membranes of only a few non-ciliated cells (thick arrows) of the efferent ducts, whereas ciliated cells appear unreactive (double arrows) (A, right side). In the initial segment (A, left side), CA XII immunoreactivity is detected exclusively over the basolateral plasma membranes of narrow cells (long arrows); no visible reaction is apparent over principal cells (P) or basal cells (small arrowhead) in this region. In the distal initial segment, a basolateral reaction is apparent between some adjacent principal cells (B). However, the strongest reaction is seen in the corpus (C) and proximal (D) cauda epididymidis, where heavy deposits of reaction product (thick arrows) accumulate along the lateral plasma membranes in the majority of adjacent principal cells (C,D). An intense reaction also appears along the base of the epithelium, suggestive of staining along the basal plasma membrane of principal cells as well as the thin attenuated processes of basal cells (small arrowheads) (C,D). An intense reaction is also present around the central body of basal cells, occupying their nucleus (C,D). In the distal cauda epididymidis (E), the reaction diminishes such that it is visible only between some adjacent principal cells (thick arrows) and in relation to sporadic areas of the base of the epithelium. Clear cells (large arrowheads) and their basal plasma membranes throughout the epididymis are consistently unreactive (E). Halo cells of the initial segment of the epididymis (B) reveal an intense reaction along their plasma membranes (curved arrows). An intense reaction is also seen over the principal piece of the tails of elongating spermatids of the testis (S) (A inset). IT, intertubular space. Magnification, x325 for each.

 


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Figure 6

Immunofluorescent staining in the corpus (A,B) and proximal cauda (C,D) epididymidis at low (A,C) and high (B,D) magnifications in sections incubated with anti-CA XII antibody. Intense staining (arrows) is evident over the lateral plasma membranes of adjacent principal cells, the principal/clear interface, and along the base of the epithelium in association with basal cells and the base of principal cells. Clear cells are unreactive (arrowheads). Lu, lumen; IT, intertubular space. Magnification (A,C), x200; (B,D), x500.

 
With the anti-CA XIV antibody, basolateral plasma membranes of non-ciliated cells in the efferent ducts were outlined by immunofluorescent staining (Figures 7A and 7B). In the epididymis, apical, and/or basal plasma membrane domains of principal cells and adjacent areas of their cytosol were reactive in a region-specific manner, with sporadic region-specific lateral plasma membrane reactions. In the initial segment and proximal caput regions, the reaction was predominantly over the apical region of principal cells, with basolateral staining being evident between only some principal cells (Figures 7C and 7D). In the distal caput, corpus (Figures 7E and 7F), and cauda (Figures 7G and 7H) regions, both apical and basal regions were reactive, masking any possible staining of the basolateral plasma membranes of principal cells. Basal staining often included the basal cells. Clear cells were consistently unreactive throughout the entire epididymis (Figures 7G and 7H). The smooth muscle cells enveloping the epididymal tubules of the cauda epididymidis were intensely reactive with the anti-CA XIV antibody (Figures 7G and 7H).



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Figure 7

Immunofluorescent staining of efferent ducts (A,B) and initial segment (C,D) at low (A,C) and high (B,D) magnifications in sections incubated with anti-CA XIV antibody. In (A,B), intense basolateral staining (arrows) is evident between adjacent epithelial cells. In (C,D), an apical reaction is seen over principal cells (curved arrows), and basolateral staining is associated with several principal cells (arrows). Lu, lumen; IT, intertubular space. Magnifications: (A,C) x200; (B,D) x500. Immunofluorescent staining of the corpus (E,F) and cauda (G,H) epididymidis at low (E,G) and high (F,H) magnifications in sections incubated with anti-CA XIV antibody. In both regions, the reaction in principal cells is both apical (curved arrows) and basal (arrows), masking basolateral staining. There is no reaction over clear cells (arrowheads). Smooth muscle cells enveloping the epididymal tubules are intensely reactive (dark arrows). Lu, lumen; IT, intertubular space. Magnification (E,G), x200; (F,H), x500.

 

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 Materials and Methods
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 Literature Cited
 
In the present study, the distribution of the CA family of proteins along the efferent ducts and epididymis, as summarized in Figure 8, reveals complex patterns of expression, dependent on cell types, regions, and membrane-specific domains. In the efferent ducts, the cytosolic isoforms, CA II and CA III, are expressed in epithelial non-ciliated cells, confirming CA II expression in these cells (Hess 2002Go). In addition, CA XIV is highly expressed along the basolateral plasma membranes of these cells (Figure 8). The non-ciliated cells remove up to 90% of the water emanating from the testis (Crabo 1965Go; Clulow et al. 1998Go; Hess 2002Go). To this end, these cells express a variety of water protein channels in the aquaporin (AQP) family, along both apical and basolateral plasma membranes (Fisher et al. 1998Go; Elkjaer et al. 2000Go; Pastor-Soler et al. 2001Go; Zhou et al. 2001Go; Badran and Hermo 2002Go; Hermo et al. 2004Go). This activity concentrates sperm in the initial segment, an activity that appears to be crucial for sperm maturation (Hess et al. 1997Go; Zhou et al. 2001Go). The presence of AQPs in non-ciliated cells, along with CA II, III, and XIV, the apical sodium hydrogen exchanger NHE3, and the CFTR chloride channel (Zhou et al. 2001Go; Kaunisto and Rajaniemi 2002Go; Wong et al. 2002Go), suggest a collaborative effort in moving water and ions through the epithelium, in addition to fine-tuning the pH of the lumen and intercellular spaces.



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Figure 8

Schematic drawing of the efferent ducts and different epididymal regions illustrating expression of CA II, III, XII, and XIV in the various epithelial cell types (only areas of maximal expression are illustrated). In the efferent ducts, both non-ciliated (NC) and ciliated (Ci) cells express cytosolic CA II and III, whereas the NC cells show basolateral CA XIV staining. In the initial segment, narrow cells (N) express CA II cytosolically, and CA XII is expressed basolaterally. In the initial segment and caput epididymidis, principal cells (P) express CA II and III in their cytosol, whereas CA XIV is present apically and basolaterally. In the corpus and cauda regions, principal cells express cytosolic CA II and III, CA XII basolaterally, and CA XIV in both apical and basal regions. Basal cells (B) express CA XII and XIV, whereas clear cells (C) are unreactive for all CA isoforms.

 
The epithelial ciliated cells of the efferent ducts also express CA II, III, and XIV, (Figure 8) along with AQP-1 and -10 and the chloride/bicarbonate anion exchanger AE2 (Bagnis et al. 2001Go; Badran and Hermo 2002Go; Hermo et al. 2004Go). Thus, together with neighboring non-ciliated cells, ciliated cells must assist in maintaining the pH of the efferent duct lumen and intercellular spaces using a slightly different group of solute transporters.

The distribution of CA II in the rat epididymis is a matter of dispute among investigators. Aside from universal agreement regarding its localization to narrow cells of the initial segment, it has been suggested that CA II is expressed in clear cells and principal cells (Kaunisto et al. 1995Go,1999Go; Breton et al. 1996Go; Hermo et al. 2000Go). In the present study, CA II was localized to narrow cells of the initial segment and to principal cells of all regions, except the distal cauda, where its expression was patchy, but on no occasion was CA II expression found in clear cells (Figure 8).

In the present study, CA III was localized to principal cells of the epididymis but not to narrow or clear cells (Figure 8). CA III, like CA II, is cytosolic, suggestive of redundancy of functions in principal cells. However, CA II is an enzyme of much higher efficiency than CA III, performing functions in respiration and acid–base balance, and is more widely expressed in mammalian cells than is CA III. Interestingly, the CA II knockout mouse model does not have a phenotype (males are fertile; Zhou et al. 2001Go), and this may be attributed to the fact that CA III is distributed throughout the epididymis in the same cells that express CA II.

In addition to carrying out reversible hydration reactions, CA III has two free thiols that scavenge oxygen radicals during oxidative stress (Cabiscol and Levine 1995Go). In the epididymis, the luminal environment is oxygen rich, and unsaturated fatty acids present in sperm membranes are susceptible to oxidative damage (Hinton et al. 1995Go). Sperm generate reactive oxygen species, such as superoxide anion (O2) and hydrogen peroxide (H2O2), especially in the cauda region, where the sperm are stored (Alvarez and Storey 1982Go,1984Go; Aitken 2002Go). To this end, principal cells throughout the epididymis protect sperm from harmful free radical injury by expressing numerous isoforms of glutathione S-transferase (GST), gamma glutamyl transferases, and other free radical scavengers (Palladino and Hinton 1994Go; Papp et al. 1995Go; Aitken 2002Go). Thus, expression of CA III in the epididymis, as well as in the efferent ducts, may also serve in a protective role for sperm. Interestingly, although basal cells express various GSTs, these cells did not express CA III. On the other hand, clear cells do not express GSTs (Papp et al. 1995Go), and these cells also showed no staining for CA III or any other CA isoform in the present study (Figure 8).

CA XII appears to be maximally expressed in the corpus and proximal cauda epididymidis, where it is localized to the basolateral plasma membranes of adjacent principal cells (Figure 8), corresponding to similar localizations in analogous regions of the kidney (Parkkila 2000Go). Weak staining for CA XII in proximal convoluted tubules (Parkkila 2000Go) correlates with similar expression in the efferent ducts, inasmuch as both of these ducts derive from similar embryological origins. CA XII has been localized in varying degrees to the efferent ducts and epididymis of humans (Karhumaa et al. 2001Go).

In the epididymis, the vacuolar H+-ATPase pump has been localized apically to narrow cells of the initial segment (Breton et al. 1996Go,1998Go; Hermo et al. 2000Go). Together with CA II expression in these cells, it has been suggested that these two players could be involved in acidification of the epididymal lumen of this region to help maintain sperm in a quiescent state as they acquire the capacity for motility during their transit down the duct (Carr et al. 1985Go; Hinton et al. 1995Go; Jones and Murdoch 1996Go). In addition, narrow cells express CA XII basolaterally (Figure 8), which together with basolateral expression of AE2, NBC1, and NBC3 (Jensen et al. 1999aGo,bGo; Pushkin et al. 2000Go), presumably assists in fine-tuning the pH of these cells and their extracellular environment. Interestingly, clear cells express vacuolar H+-ATPase (Breton et al. 1996Go, 1998Go; Hermo et al. 2000Go), yet no CA isoform has been identified within these cells (Figure 8).

Basal cells of the corpus and proximal cauda regions show maximal expression of CA XII (Figure 8), and AQP-3 is also known to be present in the membranes of these cells (Hermo et al. 2004Go). Halo cells of the initial segment expressed CA XII, but no data exist on associated ion transporters. Considered as monocytes and/or lymphocytes (Robaire and Hermo 1988Go; Flickinger et al. 1997Go; Serre and Robaire 1999Go), halo cells, like basal cells, presumably fine-tune the pH of their respective environments by means of CA XII.

CA XIV, a transmembrane protein, highlighted apical and/or basal plasma membrane domains of principal cells and adjacent areas of their cytosol in a region-specific manner, with sporadic region-specific lateral plasma membrane reactions (Figure 8). The apical and basal cytosolic reactions may indicate the presence of CA XIV within vesicles destined for apical or basal plasma membrane domains, although electron microscopy was not performed. CA IV, a GPI-anchored protein, has already been localized to the apical plasma membranes of principal cells of the distal caput, corpus, and proximal cauda regions of the rat epididymis, with maximal staining in the corpus region, and this correlates with similar distribution in the human epididymis (Parkkila et al. 1993Go; Kaunisto et al. 1995Go,1999Go). The present finding of CA XIV mainly in apical areas of principal cells of proximal epididymal regions suggests that CA XIV functions in these regions, rather than CA IV, which resides more distally. In distal regions, CA XIV resides both apically and basally in association with principal and basal cells (Figure 8). The intensity of CA XIV expression along the base of the epithelium at distal epididymal sites correlates with the presence of CA XII in distal regions, in both principal cells and basal cells, suggesting redundancy of CA functions or the fact that each may perform slightly different functions.

The staining intensity for CA III in principal cells of the distal cauda epididymidis is variable and not apparent in every cell. Similarly, basolateral CA XII expression in principal cells of the distal initial segment and caput epididymidis is patchy, as is that for basolateral expression of CA XIV in principal cells of proximal regions. This type of staining pattern is not uncommon for the epididymis. Indeed, expression of many proteins, including secretory, lysosomal, and protective proteins, occurs in a patchy manner, described as a checkerboard staining pattern whereby a given cell type in a given cross-sectional profile of tubule shows varying degrees of reactivity or complete unreactivity for a given protein (Hermo et al. 1991Go,1992Go,1994Go,1998Go; Rankin et al. 1992Go; Veri et al. 1993Go; Papp et al. 1995Go). It has been suggested that cells showing this pattern of reactivity may not be expressing a particular protein of interest, or that they may be out of synchrony with one another in expressing it (Hermo et al. 1991Go). The significance of such a phenomenon has yet to be resolved, but it clearly extends to the CAs themselves.

Establishing an acidic pH and a low bicarbonate concentration in the epididymal lumen keeps sperm in a quiescent state (Babcock et al. 1983Go; Acott and Carr 1984Go; Okamura et al. 1985Go; Tajima et al. 1987Go; Holm and Wishart 1998Go; Gatti et al. 1993Go). Changes in bicarbonate composition in the lumen trigger sperm motility and activation of their acrosomal enzymes, leading to sperm death (Lee and Storey 1986Go). Altering the ionic concentration of the luminal fluid has significant effects on epithelial secretion of proteins and other compounds, which could affect sperm maturation (Au and Wong 1980Go). Thus, monitoring the proper ionic composition of the epididymal lumen in a sequential manner down the duct has relevance to sperm maturation and viability. The coordinated activities of the various epithelial cells of the epididymis, by expressing or coexpressing different CA family members of proteins in conjunction with various channels and transporters in a cell type, subcellular, and region-specific manner, must ultimately lead to the desired pH of the epididymal lumen essential for proper sperm function.


    Acknowledgments
 
This work was supported by a Canadian Institutes of Health Research grant to LH and a National Institute of Dental and Craniofacial Research (National Institutes of Health) Grant (DE-013237) to CES.

We thank Victoria Smith and Neelum Jamal for technical assistance during the course of this study. We also thank Haitham Badran for preparing the schematic drawing shown in Figure 8.


    Footnotes
 
Received for publication November 11, 2004; accepted January 11, 2005


    Literature Cited
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 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
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