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Biology of Reproduction 64, 1509-1515 (2001)
© 2001 Society for the Study of Reproduction, Inc.

Regular Article

Expression of Cystic Fibrosis Transmembrane Conductance Regulator in Rat Efferent Duct Epithelium1

G.P.H. Leunga, X.D. Gonga, K.H. Cheunga, S.B. Cheng-Chewa, and P.Y.D. Wong2,a

a Department of Physiology, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong, China

ABSTRACT

The expression of cystic fibrosis transmembrane conductance regulator (CFTR) was studied in rat efferent ducts. Under whole-cell patch-clamp condition, efferent duct cells responded to intracellular cAMP with a rise in inward current. The cAMP-activated current exhibited a linear I–V relationship and time- and voltage-independent characteristics. The current was inhibited by the Cl- channel blocker diphenylamine 2,2'-dicarboxylic acid (DPC) in a voltage-dependent manner and reversed at 24 ± 0.5 mV, close to the equilibrium potential for Cl- (30 mV), suggesting that the current was Cl- selective. The cAMP-activated current displayed a permeability sequence of Br- > Cl- > I-. Short-circuit current measurement in cultured rat efferent duct epithelia also revealed a cAMP-activated inward current inhibitable by DPC. These electrophysiological properties of the cAMP-activated Cl- conductance in the efferent duct were consistent with those reported for CFTR. In support of the functional studies, reverse transcription polymerase chain reaction revealed the presence of CFTR message in cultured efferent duct epithelium. Immunohistochemical studies in intact rats also demonstrated CFTR protein at the apical membrane of the principal cells of efferent duct. CFTR may play a role in modulating fluid transport in the efferent duct.

epididymis, male reproductive tract

INTRODUCTION

Cystic fibrosis (CF) is the most prevalent genetic disease, affecting 1 of every 2000 newborns in the Causcasian population. It is caused by the mutation of a membrane protein called the cystic fibrosis transmembrane conductance regulator (CFTR) normally present in epithelial cells. Several hundred forms of mutation of the CFTR gene have been identified, and they are thought to be responsible for a wide spectrum of disease phenotypes. In the most severe form of gene mutation (delta 508), male patients suffer from debilitating respiratory and pancreatic insufficiency plus obstruction or agenesis of the vas deferens. However, in the least severe form of mutation, poor sperm quality appears to be the only sign in otherwise apparently healthy men [1]. This observation indicates that among the CFTR expressing organs, the male reproductive tract appears to be most vulnerable to even the mildest loss of CFTR function.

CFTR is known to be highly expressed in the epithelium of the epididymis [2], where it plays the role of a cAMP-activated chloride channel. Secretion of Cl- via CFTR provides a driving force for salt and water secretion. By effecting fluid secretion, CFTR acts counter to fluid reabsorption and serves to fine-tune the fluidity of the epididymal microenvironment [3].

The efferent duct (ductuli efferente) is a series of parallel ducts linking the rete testis with the epididymis. Its primary function is to reabsorb the fluid leaving the testis and to deliver spermatozoa at a high concentration to the epididymis, where the composition of the fluid is further modified [4]. The efferent duct is derived from the Wolffian duct of the embryonic mesonephric kidney and is homologous to the proximal tubule of the kidney [5]. As with the proximal tubule, the Na+/H+ exchanger type 3 (NHE3) is the principal transporter for Na+ reabsorption (and fluid reabsorption) in the efferent duct [6, 7]. Some Cl- channels with different characteristics have been found in the epithelial cells of the kidney tubule [8, 9]. The present study was carried out to demonstrate the presence of CFTR, a cAMP-activated Cl- channel in the epithelial cells of the efferent duct. The physiological role of CFTR in the efferent duct is also discussed.

MATERIALS AND METHODS

Chemicals

Eagle minimum essential medium (EMEM), fetal bovine serum, glutamine, and nonessential amino acids were purchased from Gibco Laboratories (New York, NY). Penicillin/streptomycin, Hanks balanced salt solution (HBSS), sodium pyruvate, 5{alpha}-dihydrotestosterone, trypsin, collagenase I, and cAMP were from Sigma (St. Louis, MO). Diphenylamine 2,2'-dicarboxylic acid (DPC) was purchased from Calbiochem (San Diego, CA). The monoclonal mouse anti-CFTR antibody (MATG 1061) was a gift from Transgene (Strasbourg, France).

Culture of Rat Efferent Duct Epithelium

All experiments on animals were conducted in accordance with the guidelines on the use of laboratory animals laid down by the Animal Research Ethics Committee of the Chinese University of Hong Kong. The procedures of primary culture of rat efferent duct epithelia were modified from the protocol published by Rozewicka et al. [10]. Male Sprague-Dawley rats weighing 200–250 g were killed by CO2 inhalation. The lower abdomens were opened, and the efferent ducts were isolated and microdissected under sterile conditions to remove fat and connective tissue. The ductules were cut into several small segments, transferred to HBSS containing 0.1% (w/v) trypsin and 0.2% (w/v) collagenase I, and incubated in a water bath at 32°C for 1 h with vigorous shaking (150 strokes/min). The tissue was separated by low-speed centrifugation (800 x g, 5 min). The supernatant was discarded, and the pellets were resuspended in HBSS containing 0.2% (w/v) collagenase I for 30 min at 32°C with vigorous shaking. After centrifugation at 800 x g for 5 min, cell plaques were resuspended in HBSS containing 0.2% (w/v) collagenase I again and subjected to repeated pipetting for 15 min. The cells were then centrifuged at 800 x g for 5 min and resuspended in EMEM containing nonessential amino acids (0.1 mM), sodium pyruvate (1 mM), glutamine (4 mM), 5{alpha}-dihydrotestosterone (1 nM), 10% fetal bovine serum, penicillin (100 IU/ml), and streptomycin (100 µg/ml). The cell suspension was incubated for 5–6 h at 32°C in 95% O2/5% CO2. During this period, fibroblasts and smooth muscle cells became attached to the bottom of the culture flask, and the epithelial cells remained suspended. The resulting epithelial cell suspension was immediately used for RNA isolation. For whole-cell patch-clamp study and short-circuit current (Isc) measurement, cells were seeded onto 35-mm dishes or wells of millipore filter assemblies with a window area of 0.2 cm2, respectively.

Whole-Cell Recording

After 5 days in culture, confluent cell monolayers were incubated in Ca2+-free Hanks solution containing 1 mM EGTA (Sigma, St. Louis, MO) for 20 min to separate the cells. Recordings were performed at room temperature using an Axopatch-1D amplifier and DigiData 1200 series Interface (Axon Instrument, Foster City, CA). Ionic current was recorded using a conventional whole-cell patch-clamp technique [11]. The cell membrane potential was held at -70 mV. Signals were filtered at 1 kHz and then digitalized with Digidata 1200. Sampling rate was set at 500 µsec. The pClamp 8 program (Axon Instrument) was used for data recording and analysis. Patch pipettes (2–5 M{Omega}) were pulled from 1.0 mm outside diameter, 0.5 mm inside diameter borosilicate glass pipettes (Sutter Instrument Co., Novato, CA) using a horizontal puller (Sutter) and were polished before use.

The pipettes were filled with a solution containing 120 mM CsCl, 20 mM tetraethylammonium-Cl, and 100 µM cAMP, pH adjusted to 7.4 with CsOH. The bath solution contained 135 mM NaCl, 1.2 mM Na2HPO4, 10 mM MgCl2, 10 mM Hepes, and 10 mM glucose (pH 7.4). In anion selectivity experiments, NaCl in the bath solution was progressively replaced by NaBr or NaI. Osmolarity of the pipette and bath solutions was adjusted to isotonic (290 mOsm) by the addition of mannitol.

Isc Measurement

Confluent efferent duct monolayers were clamped between two halves of Ussing chambers with a 0.6-cm2 window. The tissue was short-circuited with a voltage-clamp amplifier (DVC 1000; World Precision Instrument, New Haven, CT). The Isc was displayed on a pen recorder. Transepithelial resistance was obtained using Ohm's law by clamping the tissue intermittently at a voltage of 0.1 to 0.3 mV displaced from zero. The monolayers were bathed on both sides with Krebs-Henseleit solution, gassed with 95% O2/5% CO2, and warmed to 32°C. Krebs-Henseleit solution had the following composition: 117 mM NaCl, 4.7 mM KCl, 1.2 mM KH2PO4, 1.2 mM MgSO4·7H2O, 2.56 mM CaCl2·2H2O, 24.8 mM NaHCO3, and 11.1 mM glucose.

RNA Isolation and Reverse Transcription Polymerase Chain Reaction

Total RNA was isolated using TRIzol reagent (Gibco BRL), and 2 µg was used for first strand cDNA synthesis using random hexamer primers and Superscript II RNase H- Reverse Transcriptase (SuperScript Preamplification System, Gibco BRL). The resulting first strand cDNA was directly used for polymerase chain reaction (PCR) amplification.

The two primers used for amplifying CFTR were sense 5'-GCCAGGGCTAGCAGTCTTCATTTTACTGAG-3' (corresponding to nucleotides 3045–3075) and anti-sense 5'-AGCCTCGAGCACTAGAGCCGGGTCTCTTGC-3' (corresponding to nucleotides 4419–4448) [12], which yielded a PCR product of 1.4 kilobases (kb). Reactions were carried out with the following parameters: denaturation at 94°C for 1 min, annealing at 60°C for 1 min, and extension at 72°C for 1.5 min, for a total of 30 cycles. PCR products were analyzed by agarose gel electrophoresis and visualized by staining with ethidium bromide. The PCR products of the expected sizes were confirmed by sequencing.

Immunohistochemistry

Paraffin-embedded sections of Bouin-fixed efferent ducts (3 µm thick) were dewaxed and hydrated. Antigen was retrieved by treatment in 0.01 M citrate buffer (pH 6.0) for 5 min in a microwave oven. Sections were then rinsed twice with pure water and incubated in methanol containing 3% H2O2 for 15 min. After rinsing with pure water and PBS, sections were incubated in normal blocking serum (Vectastain Elite ABC kit, Vector PK-6101; Vector Laboratories, Burlingame, CA) for 30 min and then with the monoclonal mouse anti-CFTR antibodies (MATG 1061; Transgene SA, France), diluted 1:100 with diluting buffer (PBS with 0.01% Triton X-100, 0.01% Tween 20, and 0.1% BSA) at 4°C overnight. Sections were washed three times with PBS and incubated with biotinylated secondary antibody (ABC kit) for 30 min. After washing three times with PBS, the sections were incubated with the ABC reagent for 30 min and finally washed again three times with PBS. Visualization was achieved by immersing sections in a peroxidase substrate solution (VIP substrate kit; Vector) until desired stain intensity developed. Slides were rinsed with pure water for 5 min, counterstained with Lillie-Mayer hematoxylin (Merck, Darmstadt, Germany), dehydrated, and mounted for observation. Negative controls were obtained by omission of primary antibodies.

Statistical Analysis

Results are expressed as means ± SEM. Comparisons between groups of data were made by the Student t-test. A P value of less than 0.05 was considered significant.

RESULTS

Activation of Cl- Current by cAMP in the Rat Efferent Duct Epithelial Cell

Figure 1A shows the time course of whole-cell current activation in cultured efferent duct cells by cAMP. No current was detected when the pipette solution contained only 120 mM CsCl and 20 mM TAE-Cl. Addition of 100 µM cAMP into the internal solution resulted in an inward current at -70 mV. The cAMP-activated whole-cell current profile elicited by a series of voltages exhibited time- and voltage-independent characteristics with a linear I–V relationship (Fig. 1, B and C). The chloride channel blocker DPC blocked the cAMP-activated Cl- current when the membrane potential was held at negative potentials (Fig. 1, D and E). This voltage-dependent blockade by DPC was in line with the characteristic of CFTR previously reported in other epithelial cells [13, 14].



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  		FIG. 1. Characteristics of cAMP-stimulated whole cell current. A) Time course of whole cell current stimulated by 100 µM cAMP in efferent duct epithelial cells. At the time indicated, 500 µM DPC was added to the bath. Membrane potential was held at -70 mV. Dashed line represents zero current level. Whole-cell current recording prior to (B) and after (C) cAMP stimulation or after cAMP stimulation followed by DPC (D). The voltage protocol used consisted of a stepwise depolarization, in steps of 20 mV, from -100 mV to +100 mV, with a holding potential of -70 mV. E) Average I–V relationship measured 180 msec following the onset of the pulse, obtained from the cell at rest (triangles), cAMP stimulation alone (squares), or cAMP stimulation followed by DPC addition (circles). Each value represents the mean ± SEM (n = 6)

Anion Selectivity Sequence of the cAMP-Dependent Cl- Conductance

In the presence of a Cl- gradient, 140 mM in pipette solutions and 40 mM in bath solutions, cAMP (100 µM) activated a whole-cell current that had a reversal potential (Erev) of 24 ± 0.5 mV (Fig. 2), close to the calculated Cl- equilibrium potential of 30 mV. The anion selectivity sequence for the cAMP-activated conductance was studied by examining the permeability of a number of monovalent anions relative to that of Cl- (PX/PCl). The shift in reversal potential was measured upon replacement of Cl- with the test anions in the bath solution (n = 6, Fig. 3), and PX/PCl was calculated from the Goldman-Hodgin-Katz equation. Substitution of Cl- by Br- and I- in the bath solution caused the Erev to shift from 0.2 ± 1.43 to -15.7 ± 3.55 and 19.9 ± 1.29, respectively. The relative permeability sequence of the cAMP-dependent Cl- conductance thus obtained was Br- (1.3) > Cl- (1.0) > I- (0.72), consistent with that reported for CFTR in a number of epithelial cells [15–18].



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  		FIG. 2. Effect of extracellular Cl- concentration on cAMP-activated whole-cell current in efferent duct epithelial cell. Whole-cell current recorded in the presence of extracellular 140 mM (squares) or 40 mM (circles) Cl-. Each value represents the mean ± SEM (n = 5)



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  		FIG. 3. Effect of extracellular Cl- substitution on cAMP-activated whole-cell current in efferent duct epithelial cell. Whole-cell current recorded in the presence of Br- (squares), Cl- (circles), or I- (triangles). Each value represents the mean ± SEM (n = 5)

Isc Study in Efferent Duct Epithelial Cells

Rat efferent duct monolayers clamped in an Ussing chamber developed a transepithelial potential difference of 0.4 ± 0.1 mV (n = 6), apical side negative, and a basal Isc of 2.5 ± 0.2 µAcm-2 (n = 6). The transepithelial resistance was about 100 {Omega}cm2. Addition of cAMP (100 µM) to the basolateral side of the epithelium caused a significant increase of Isc by 4 ± 0.2 µAcm-2 (n = 6). Consistent with the result of patch-clamp study, addition of DPC to the apical side inhibited the Isc response to cAMP (Fig. 4).



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  		FIG. 4. Effect of cAMP on Isc in efferent duct epithelium. Cyclic AMP (100 µM) was added to the basolateral side, followed by 500 µM DPC added apically. Horizontal lines indicate zero Isc. Each record is representative of six different experiments

Identification of CFTR in Epithelial Cells of the Efferent Duct by Reverse Transcription PCR

Reverse transcription PCR (RT-PCR) was used to study the expression of CFTR in the efferent duct (Fig. 5). The rat cauda epididymidis was used as a positive control because CFTR was expressed in this tissue [17]. The PCR products of CFTR (1.4 kb) were amplified by RT-PCR from RNA isolated from epithelial cells of the efferent duct and cauda epididymal epithelia. These products were confirmed by sequencing (data not shown).



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  		FIG. 5. RT-PCR analysis of CFTR mRNA in the efferent duct. PCR products are seen in reactions using oligonucleoside primer pairs for CFTR. Positive control with rat cauda epididymidis cDNA indicates the expected size of the amplified fragment (1.4 kb). Negative control was performed with no RT added. DNA size markers are indicated on the left

Immunolocalization of CFTR in Efferent Duct

The apical surface of the principal cells of efferent duct was highly stained with CFTR antibody, whereas that of the ciliated cells were not stained (Fig. 6, B and C). Control experiments in which the primary antibody was omitted resulted in no positive labeling (Fig. 6A).



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  		FIG. 6. Immunolocalization of CFTR in rat efferent duct. A) Control efferent duct section in which primary antibodies were omitted. B) Efferent duct section labeled with anti-CFTR antibody. Immunoreactivity of CFTR was detected at the apical membrane of the principal cells. C) Arrows indicate two ciliated cells with negative CFTR staining. L, Tubular lumen

DISCUSSION

Previous work in our laboratory has shown that the rat efferent duct is capable of secreting anions electrogenically when stimulated by humoral agents that increase intracellular cAMP [19, 20]. In agreement with these studies, the present work showed that exogenous cAMP stimulated transepithelial Cl- transport across the efferent duct epithelium (Fig. 4), presumably by activating an apically placed chloride channel, also found in the rat epididymis [2, 3]. Although CFTR plays the role of a cAMP-activated Cl- channel, some of the cAMP-activated Cl- channels found in the epithelia of the airway, choroid plexus, and small intestine exhibited characteristics different from those of CFTR [21–23]. In view of these discrepancies, different approaches were used in this study to see whether the cAMP-activated Cl- conductance in the efferent duct was indeed mediated by CFTR.

In the whole-cell patch-clamp study, several lines of evidence suggested that the cAMP-activated Cl- current in the efferent duct epithelium was characteristic of CFTR [15–18]. First, The cAMP-activated Cl- current was time and voltage independent and exhibited a linear I–V relationship (Fig. 1B). Second, the halide selectivity sequence for the cAMP-activated Cl- current was Br- > Cl- > I- (Fig. 3), in agreement with the sequence for CFTR. Third, as with CFTR, the cAMP-activated Cl- current was inhibited by DPC in a voltage-dependent manner (Fig. 1D) [13, 14]. In support of these functional data, RT-PCR detected CFTR gene expression in the efferent duct (Fig. 5), and immunohistochemistry demonstrated CFTR protein at the apical side of the principal cells of the efferent duct epithelium (Fig. 6). However, the apical membrane of the ciliated cells did not appear to be stained (Fig. 6).

The efferent duct is homologous to the proximal tubule of the kidney. During development of the metanephric kidney, the Wolffian duct undergoes transformation into the efferent duct and proximal kidney tubule [5]. Functionally, the efferent duct and epididymis resemble the nephron in many respects [3, 4], and Cl- secretion via CFTR occurs in primary cultures of inner medullary collecting duct cells [24, 25] and distal convoluted tubule cells [9, 26–28]. In contrast, the presence of CFTR in the proximal tubule is controversial. Although CFTR mRNA was detectable in the proximal tubule and CFTR protein was located at the apical membrane [29, 30], the CFTR-mediated Cl- conductance has not been identified in the proximal tubule [9]. Unlike in the proximal tubule, CFTR chloride current was detected in the efferent duct.

Under normal conditions, large fluxes of electrolytes move across the efferent duct in both absorptive and secretory directions. The absorptive flux is driven by the NHE3 located on the apical membrane of the epithelium [6, 7], whereas the secretory flux is driven by Cl- secretion through apical Cl- channels. In the epididymis, and presumably in the efferent duct, various transporters in the basolateral membrane take up Cl- actively from the interstitial fluid into the cell cytosol so that the intracellular chloride activity is held above its electrochemical equilibrium. When the cells are stimulated, the rise in intracellular cAMP activates CFTR, which allows Cl- efflux into the lumen [20, 31]. Generally, fluid reabsorption in the efferent duct is supposed to be held at a relatively constant tonic rate under the influence of sex hormones [32, 33], whereas secretion is subject to rapid control by neurotransmitters and hormones [19]. Under basal conditions, the absorptive flux in the epididymis predominates over the secretory flux so that there is a net reabsorption of Na+ and water. However, under stimulated conditions, when the secretory flux is increased to a level that exceeds the absorptive flux, a net water secretion ensues. In the efferent duct, although it is unlikely that the secretory flux could ever exceed the absorptive flux to generate a net ductal secretion, chloride secretion by the cells may still act as a counterbalance to absorption and prevent excessive dehydration of the ductal lumen. In this way, the apical chloride channels (CFTR) safeguard a smooth and appropriate rate of flow of testicular sperm into the epididymis.

In addition to regulating NaCl and fluid transport in renal tubule [34], Cl- secretion via CFTR contributes to cyst formation in autosomal polycystic kidney diseases [35, 36]. There is a growing body of evidence that similar cyst formation in the efferent duct can lead to atrophy and obstruction of the male reproductive tract [37–39]. This observation supports the hypothesis that regulation of Cl- secretion via CFTR in the efferent duct has an impact on male fertility.

In addition to functioning as a cAMP-regulated Cl- channel, CFTR may also act as a regulator of other channels or transporters in the efferent duct epithelium. Electroneutral absorption of NaCl across intestinal epithelia occurs via parallel Na+/H+ and Cl-/HCO3- exchangers. These processes were inhibited by cAMP in normal mice but not in CFTR knockout mice [40], implying that CFTR is necessary, either directly or indirectly, for the expression of cAMP-dependent inhibition of NaCl absorption. This idea was supported by the finding that additional cellular factors are required for cAMP to inhibit the Na+/H+ exchanger [41]. Moreover, the Na+/H+ exchanger regulatory factor (NHERF) is necessary for the cAMP-dependent inhibition of NHE3 [42, 43]. The C-terminal end of CFTR contains a sequence that specifically binds to the PDZ1 domain of NHERF [44], suggesting the probable interaction between CFTR and the Na+/H+ exchanger. Another membrane transport protein with which CFTR interacts is the water channel (aquaporin). CFTR-dependent activation of aquaporin-3 and water permeability has been shown in normal but not in CF airway epithelial cells [45]. Aquaporin-1 was found to be localized at both the apical and basolateral side of the efferent duct epithelium [46, 47]. Therefore, the interaction of CFTR with aquaporin may open up new areas of investigation into fluid transport in the male reproductive system.

This study provided direct evidence for the expression of CFTR in the rat efferent duct epithelium. Through its role as a cAMP-activated chloride channel and/or regulator of other membrane transport proteins, CFTR appears to be important in efferent duct epithelial function.

FOOTNOTES

First decision: 11 December 2000.

1 This work was supported by the Research Grant Council of Hong Kong. Back

2 Correspondence. FAX: 852 2603 5022; patrickwong{at}cuhk.edu.hk Back

Accepted: January 4, 2001.

Received: October 23, 2000.

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