Sertoli cell expression of the cystic fibrosis transmembrane conductance regulator

Fredric R. Boockfor1, Rebecca A. Morris1, Dennis C. DeSimone1, D. Margaret Hunt2, and Kenneth B. Walsh3

Departments of 1 Cell Biology and Neuroscience, 2 Microbiology and Immunology, and 3 Pharmacology, University of South Carolina School of Medicine, Columbia, South Carolina 29208

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene have been associated with a number of male reproductive problems, including testis abnormalities and a reduction in germ cell quality and number. To establish at least one site of functional CFTR expression in the testis, we subjected cultured Sertoli cells to analysis of message, protein, and channel activity for CFTR. With reverse transcription-polymerase chain reaction, we obtained evidence for the presence of CFTR RNA when CFTR primers were used with RNA from cultured Sertoli cells. Western analysis performed with both anti-R and anti-C domain CFTR antibodies revealed immunoreactive material in extracts from primary Sertoli cell cultures that seemed consistent with CFTR previously identified in other cells and tissues. This led us to perform more detailed studies using the whole cell arrangement of the patch-clamp technique. Application of the membrane-soluble cAMP analog, 8-chlorophenylthio-cAMP, resulted in the activation of a Cl- current that displayed a permeability sequence of Br- > I- >=  Cl- and was blocked by diphenylamine-2-carboxylate and glibenclamide. In addition, a 13-pS conductance Cl- channel was measured in excised membrane patches exposed to the catalytic subunit of protein kinase A. When taken together, our findings of evidence of CFTR message, immunoreactive material that appeared consistent with CFTR, and Cl- channels with properties similar to those reported for CFTR provide strong evidence that Sertoli cells express a functional CFTR-like protein. The presence of CFTR in these cells may be needed to maintain the specific nutritional and fluid balance in the seminiferous tubule that is vital for normal spermatogenesis.

testis; chloride; epithelial cells; fluid balance

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

CYSTIC FIBROSIS IS AN autosomal recessive disease that is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) (29, 42). CFTR is believed to function as a cAMP-dependent protein kinase-activated Cl- channel (2, 4, 28) and is located primarily in the apical membrane of polarized epithelial cells (12, 15, 23, 33). Because epithelial cells are associated with many different tissues, the presence of mutant CFTR can result in a variety of abnormal functions, including irregular glandular or mucus secretion, intestinal blockage, and pancreatic insufficiency (39).

A growing body of evidence suggests that mutation of the CFTR gene is also associated with a severe reduction in fertility, especially in male cystic fibrosis (CF) patients. Much of this infertile state is related to developmental abnormalities that result in blockage or absence of the vas deferens or parts of the epididymis. However, the role of CFTR in the testis during spermatogenesis is suggested by observations that many germ cell types produced by men with CF are reduced in number or malformed (7, 13). Abnormalities in several different germ cell types as well as reductions in Sertoli cell numbers have been observed in CF patients (46, 50).

Mutations of the CFTR gene have also been associated with other non-CF reproductive conditions. Infertile patients with a condition called congenital bilateral absence of the vas deferens (CBAVD) were found to express mutant forms of the CFTR gene (3, 9, 35, 38). Interestingly, patients with this condition do not possess any other systemic symptoms of CF but do exhibit reproductive structural abnormalities. Although the lack of a functional vas deferens plays a major role in the infertile condition, it has been found that CBAVD patients exhibit reductions in germ cell quality and numbers as well (3, 35, 38, 45). Additional studies have revealed that mutation of the CFTR gene is also associated with congenital unilateral absence of the vas deferens, another related but not as well-studied state of infertility (8, 32). Finally, recent evidence suggests that CFTR mutation may be an important underlying cause of undefined male infertility. Van der Ven et al. (53) revealed that 17.5% of men with infertility due to sperm abnormalities have a mutation in the CFTR gene. When taken together, these observations suggest not only that CFTR-related infertility is more widespread than previously thought but also that the problem may be associated in part with a basic malfunction in the spermatogenic process.

The site of such a malfunction remains obscure. Recent reports from Buchwald's laboratory (51, 52) have shown CFTR message to be associated mainly with developing germ cells in the rat testes, raising the possibility that CFTR, if mutated, may have a direct deleterious influence on germ cell development. A second possibility is that CFTR may be important in enabling Sertoli cells to maintain the unique microenvironment in the seminiferous tubule that is necessary for proper germ cell development. As mentioned above, CFTR has been shown to be present in the membrane of several types of polarized epithelial cells (12, 15, 23, 33). The cell type in the testis that most closely fits these characteristics is the Sertoli cell. This, when coupled with the important secretory and regulatory functions of Sertoli cells during spermatogenesis (19), makes them a potential site for CFTR-related functional problems and an important candidate for investigation. In fact, recent evidence has suggested that CFTR mRNA may be present in Sertoli cells during early postnatal development (52). However, no information is available on the expression or activity of CFTR protein in the rat testes during this time period or later in reproductive life. Thus the goal of this study was first to determine whether CFTR is expressed in Sertoli cell populations from adult animals and if so to begin to assess the characteristics of this channel protein.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Preparation of Sertoli cell cultures. Sertoli cells were obtained as described previously (22). Briefly, testes from adult rats (>70 days; Harlan Sprague-Dawley, Indianapolis, IN) were decapsulated and placed into an enzyme solution. This solution consisted of collagenase-dispase (0.03%; Boehringer Mannheim Biochemicals, Indianapolis, IN) and hyaluronidase (0.05%, type 1-S, Sigma Chemical, St. Louis, MO) in minimum essential medium (MEM; GIBCO BRL, Grand Island, NY) with BSA (0.01%, fraction V; Sigma) and antibiotics [penicillin-G (100 U/ml), streptomycin (100 mg/ml), and gentamicin (50 mg/ml), all from GIBCO]. Each testis was gently shaken for 90 min during which the seminiferous tubules separated from the other testis components. With the use of a dissecting microscope, segments of these tubules were obtained and placed into a second solution containing collagenase III (0.02%; Worthington Biochemical, Freehold, NJ) and hyaluronidase (0.07% in MEM) for 30 min at 34.5°C. Tubule fragments that remained were reduced in size by repeated passage through the tip of a flame-polished Pasteur pipette. The cells were resuspended in DMEM and Ham's F-12 (DMEM-F12, 1:1 mixture) with 4.0% fetal bovine serum and antibiotics as above and plated into culture dishes for protein acquisition or coverslips for patch-clamp experiments. The cell preparations were allowed to attach for 48 h and then washed repeatedly to remove germ cells and debris. Sertoli cells used for Western analysis were cultured for at least 14 days, and the absence of myoid cell contamination was confirmed by alkaline phosphatase staining (6, 22). Rats were maintained and utilized in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Maintenance of continuous cell lines. Several cell lines were used in this study. First, T84 cells were obtained from American Type Culture Collection (Rockville, MD) and cultured as described by Cohn et al. (11). The culture medium consisted of DMEM-F12 (1:1) supplemented with fetal calf serum (5.0%; GIBCO BRL) and antibiotic-antimycotic (1.0%; Sigma). These cells were originally obtained from a human colon carcinoma; they express large amounts of CFTR protein. A second cell line, CFTR-transfected 3T3 cells, were obtained from Dr. Michael Welsh at the Howard Hughes Medical Institute, University of Iowa. These cells were transfected with the human CFTR gene; they also express large amounts of CFTR (1, 5). The medium used for culture consisted of DMEM supplemented with fetal bovine serum (10%), gentamicin (0.5%), fungizone (1.0%), and Penstrep (1.0%), all from GIBCO. A third cell line used was the ASC-17D cells. Cultures of adult Sertoli cells were immortalized with the temperature-sensitive mutant of the SV40 virus, tsA255 (40), generously supplied by Dr. K. Roberts at the University of Minnesota. The cells were cultured in DMEM containing fetal bovine serum (4%) and antimycotic-antibiotic. Unlike the other two lines that were incubated at 37°C, these cells were cultured at 33°C. Finally, a rat placental cell line, Rcho-I, was used as a control in some of the studies. These cells were supplied by Dr. Michael Soares (University of Kansas). These cells were cultured as described previously (18) at 37°C in NCTC-135 medium (Sigma) supplemented with 10% fetal bovine serum.

Reverse transcription-polymerase chain reaction. Primers for cDNA synthesis and PCR amplification were synthesized identically to those reported by Mulberg et al. (34). The primer sites are located in the middle coding region of the human CFTR gene. Specifically, the forward primer (FB5; 5'-GACTACATGGAACACATACCTTCG) was chosen in exon 14a and the reverse primer (FB7; 5'-ATAGCAAGCAAAGTGTCGGCTACTC) in exon 15. Total cellular RNA was isolated by the Qiagen RNeasy total RNA kit (Qiagen, Chatsworth, CA) and subjected to reverse transcription. First, the following reagents were combined into a final volume of 25 µl: 3.33 pmol of FB7 primer, 1× PCR buffer II (500 mM KCl and 100 mM Tris, pH 8.3), 4 mM MgCl2, 0.8 mM deoxynucleotide triphosphates (dNTPs), 8 mM dithiothreitol, 20 units RNasin (Promega, Madison, WI), and 50 units of Moloney murine leukemia virus reverse transcriptase (Stratagene, La Jolla, CA). Next, the mixture was overlaid with molecular-grade mineral oil (Sigma) and incubated at 45°C for 60 min followed by 5 min of enzyme inactivation at 95°C. The cDNA was then diluted with 125 µl of glass-distilled water (GDW), boiled for 10 min, and then placed in an ice bath before a portion of it was added to the PCR mix. For PCR, a solution was made that contained 1× PCR buffer II, 2.5 mM MgCl2, 16.7 pmol each of FB5 and FB7, 0.2 mM dNTPs, 2.5 units of Amplitaq gold (Perkin-Elmer, Foster City, CA), 5 µl of cDNA, and GDW to 100 µl. This mixture was subjected to 45 cycles at 95°C for 12 min, 96°C for 1 min, 54°C for 1 min, and 72°C for 2 min and followed by a final extension step of 72°C for 5 min. Amplification products were visualized with an Alpha-Imager 2,000 ultraviolet densitometer (Alpha Innotech, San Leandro, CA) after size fractionation through a 2% agarose gel stained with ethidium bromide. The resulting 258-bp amplification product was confirmed to be a fragment of CFTR by restriction enzyme digestion with BsaA I (which reduced the product to 225 bp as predicted) and sequence analysis (Sequenase version 2.0; Amersham, Cleveland, OH). All PCR experiments included control reactions that contained GDW rather than cDNA.

Preparation of cell lysates. Sertoli cell cultures, T84 cells, and 3T3 cells (either nontransfected or transfected with the CFTR gene), cultured as described above, were washed several times in D-PBS (Ca2+ and Mg2+ free) and then incubated with 10 mM Tris · HCl buffer (pH 8.0) containing n-octyl glucopyranoside (46 mM; Sigma) for 15-20 min. The nuclear debris was removed by centrifugation at 200 g for 5 min. The supernatant was removed, dialyzed against water, lyophilized, and then resuspended in 0.1% SDS in water.

Gel electrophoresis and Western blotting. Proteins in tissue and cell lysate or membrane preparations (~40 µg/sample) were separated by electrophoresis on SDS-7.5% polyacrylamide gels using a Bio-Rad mini PROTEAN cell (Bio-Rad, Melville, NY) by the procedure of Laemmli (30). The running buffer contained 20 mM Tris-glycine (pH 8.3) and 0.2% wt /vol SDS; runs were for 60 min at 120 V. To enhance the transfer of high-molecular-mass proteins, electroblotting of the gels was conducted using a Tris buffer containing a high concentration of glycine and no methanol (0.7 M glycine and 25 mM Tris, pH 7.7) as described by others (43). The transfers were performed in a mini Trans-Blot apparatus (Bio-Rad) (40 V for 12 h at 4°C) using polyvinylidene difluoride membranes (0.2 µm pore size; ICN, Irvine, CA). For immunodetection, membranes were first incubated in Tris-buffered saline (TBS)-Tween (0.1% Tween 20, 20 mM Tris, and 500 mM NaCl) containing 5.0% Carnation nonfat dry milk for 60 min at room temperature. Monoclonal antibodies raised to the "R" domain of the CFTR protein (Genzyme, Cambridge, MA) were diluted (1:2,000) and incubated with the blotted membranes. The R antibody was generated with a beta -galactosidase-CFTR exon 13 fusion protein and has been shown to recognize the hydrophobic R portion of the CFTR molecule and to be effective in immunoprecipitation of the CFTR molecule (24). Another antibody raised to the "C" domain of the CFTR protein was used. This antibody was generated with a glutathione S-transferase-CFTR fragment (1377-1480 amino acids) fusion protein and found to recognize the COOH-terminal amino acids in the CFTR molecule. After treatment with these antibodies, the blots were washed in TBS-Tween and incubated with a second antibody [1:1,000; horseradish peroxidase-conjugated goat anti-mouse IgG specific for the Fc region; Sigma] for 60 min, rewashed, and then visualized by the enhanced chemiluminescence method (Amersham, Arlington Heights, IL) as described in the kit. The gels were subjected to Coomassie blue stain to ensure that all proteins had transferred to the membranes.

Ion channel recordings. The patch-clamp method (25) was used to record whole cell and single-channel Sertoli currents using a Warner PC-501 (Warner Instrument, Hamden, CT) or a List EPC-7 (Adams and List Associates, Darmstadt-Eberstadt, Germany) amplifier in cells cultured for 2-4 days. Measurement of whole cell Cl- currents has been described in detail previously (54). Microelectrodes were made from Gold Seal Accu-fill 90 Micropets (Fisher Scientific, Pittsburgh, PA) and had resistances of 3-4 MOmega when filled with the cesium aspartate internal solution (see below). Uncompensated series resistance in this study was typically <2 MOmega . A reference electrode made from a Ag-AgCl pellet was connected to the bath using an agar salt bridge saturated with external solution. Data were adjusted for liquid junction potentials that arose between both the pipette solution and bath solution and the reference electrode and the bath solution as described previously (54).

The standard pipette (internal) solution consisted of (in mM) 70 CsCl, 50 cesium aspartate, 2 MgCl2, 1 CaCl2, 11 EGTA, 3 ATP (K+ salt), and 10 HEPES, pH 7.3 with CsOH (total Cl- concentration = 76 mM). The ratio of EGTA to CaCl2 in the internal solution sets the free intracellular Ca2+ concentration to ~10 nM (17). The standard external solution consisted of (in mM) 132 NaCl, 4 KCl, 1 MgCl2, 1 CaCl2, 5 dextrose, and 5 HEPES, pH 7.4 with NaOH (total Cl- concentration = 140 mM). For the low Cl-, I-, and Br- external solutions, NaCl was replaced with either sodium aspartate or NaI (total Cl- concentration = 8 mM). The external and internal solutions were maintained at room temperature (22-24°C). The drugs 8-chlorophenylthio-cAMP and glibenclamide were purchased from Sigma. Diphenylamine-2-carboxylate (DPC) was obtained from Aldrich Chemical (Milwaukee, WI) and prepared from a 1 M stock solution in 1 N NaOH.

The permeability ratio of a test anion (A-) to Cl- (PA/PCl) was determined using the Goldman-Hodgkin-Katz (GHK) equation
<IT>E</IT><SUB>rev</SUB> = (<IT>RT</IT>/<IT>zF</IT>) ln {(<IT>P</IT><SUB>A</SUB>[A<SUP>−</SUP>]<SUB>i</SUB> + <IT>P</IT><SUB>Cl</SUB>[Cl<SUP>−</SUP>]<SUB>i</SUB>)/(<IT>P</IT><SUB>A</SUB>[A<SUP>−</SUP>]<SUB>o</SUB> + <IT>P</IT><SUB>Cl</SUB>[Cl<SUP>−</SUP>]<SUB>o</SUB>)}
where the intracellular Cl- concentration ([Cl-]i) is 76 mM, the extracellular Cl- concentration ([Cl-]o) is 8 mM, [A-]o is 132 mM, Erev is the reversal potential, R is the gas constant, T is the temperature (K), z is the valence, F is Faraday's constant, and PA and PCl are the permeabilities.

Steady-state single-channel records were recorded from inside-out patches of membrane exposed to the catalytic subunit of protein kinase A (Sigma) and stored on videotape using an analog data recorder (Instrutech, Great Neck, NY). Data were later digitized at 400 Hz and filtered at 200 Hz with an eight-pole Bessel filter (Frequency Devices, Haverhill, MA). Data acquisition and analyses were performed using pCLAMP (Axon Instruments, Foster City, CA) and SigmaPlot (Jandel Scientific, Corte Madera, CA) software, with channel openings determined by setting a threshold detector at the 50% level of the open-channel amplitude.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Detection of a CFTR RNA in Sertoli cells. Evidence for the presence of CFTR RNA was detected with RT-PCR. The results are presented in Fig. 1. As shown for comparison (lane A), a 258-bp product was generated when RNA from 3T3 cells that had been transfected with the CFTR gene was used as a template. This product was identical to that predicted from the CFTR primers used and was consistent with the results obtained previously in other rat tissues (20, 34). When RNA from primary Sertoli cell cultures were used, we found a similar 258-bp amplification product generated (lane B). Finally, our use of RNA from the continuous rat Sertoli cell line, ASC-17D, in the RT-PCR procedure yielded a product similar to those observed with the other cells studied (lane C). Our use of RNA from a rat placental cell line (Rcho-1; lane D) or replacement of the RNA component of RT-PCR with GDW (lane E) resulted in an absence of product formation.


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Fig. 1.   Generation of a cystic fibrosis transmembrane conductance regulator (CFTR) amplification product (258 bp) by RT-PCR from Sertoli cell cultures. Samples of RNA from 3T3 cells transfected with the CFTR gene (lane A), primary adult Sertoli cell (SC) cultures (lane B), cultures of ASC-17D cells (lane C), cultures of Rcho-1 cells (lane D), and a glass-distilled water (GDW) control (lane E) were subjected to RT-PCR using CFTR-specific primer pairs. Mobility of the molecular mass markers is indicated to right.

Identification of a CFTR-like protein in Sertoli cell preparations. Western analysis was used to determine whether a CFTR-like protein was present in Sertoli cells. To test our ability to detect CFTR-like protein, we first attempted to identify this substance in lysates of T84 cells, a cell line well recognized to produce large amounts of CFTR protein (11). As shown in Fig. 2 (top), we were able to identify an immunoreactive band (~180 kDa) associated with these cells when an antibody that was raised to the R domain of the CFTR molecule was used for detection. Previous studies have shown that the major glycosylated form of human CFTR protein ranges from ~165 to 200 kDa (11, 14, 43). The band identified in these preparations falls well within this size range. Sertoli cell lysates (top, lane B) were then subjected to the same procedure and found to contain an immunoreactive protein that was similar in mobility characteristics to that found with T84 cells (11). Finally, 3T3 cells that had been transfected with the CFTR gene were subjected to this procedure and found to contain this high-molecular-mass protein as well (top, lane C). As expected, nontransfected 3T3 cells did not exhibit immunoreactivity in this Western analysis (top, lane D). To confirm these findings (Fig. 2, bottom), lysates from cultures of primary Sertoli cells (lane A) and CFTR-transfected 3T3 cells (lane B) were subjected to Western analysis using an anti-C antibody for detection. As shown, the results from this procedure were consistent with those obtained using the anti-R antibody. Although small differences can be visualized in results obtained with the two antibodies, it appears that in each case both Sertoli and CFTR-transfected 3T3 cells contained immunoreactive material in the 180-kDa range, which seemed to be consistent with signals detected in cells known to contain CFTR.


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Fig. 2.   Identification of CFTR-like protein in whole cell preparations. Top: protein from cultures of T84 cells (A), primary Sertoli cells (B), 3T3 cells transfected with the CFTR gene (C), and nontransfected 3T3 cells (D) were subjected to Western analysis using a monoclonal antibody raised against the R portion of the CFTR molecule. Bottom: proteins from primary Sertoli cell cultures (A) or cultures of CFTR-transfected 3T3 cells (B) were also subjected to Western analysis, but a monoclonal antibody raised against the COOH-terminal domain of the CFTR molecule was used. Protein extracts were separated on a SDS-7.5% polyacrylamide gel, transferred to polyvinylidene difluoride, and probed with the anti-R or the anti-C antibody, and the immunoreactive proteins were detected with chemiluminescence. Mobility of molecular mass markers (kDa) is indicated with each blot.

Measurement of cAMP-sensitive Cl- current in Sertoli cells. Figure 3A shows background currents measured from an adult Sertoli cell using the whole cell arrangement of the patch-clamp technique. Currents were measured during voltage steps applied to various potentials from a holding potential of -30 mV. Addition of the membrane-soluble cAMP analog 8-chlorophenylthio-cAMP, to stimulate protein kinase A, resulted in the activation of time-independent inward and outward currents (cAMP). As shown in Fig. 3B, the current vs. voltage (I-V) relationship for the Sertoli cell cAMP-sensitive current displayed outward rectification when measured with 76 mM Cl- in the pipette and 140 mM Cl- in the bath. In addition, the Erev for the cAMP-sensitive current was close to the equilibrium potential for Cl- (ECl) (Erev = -11 ± 1 mV, n = 10 cells, ECl = -15 mV), suggesting that this current may result from the activity of Cl--selective channels. External application of the arylaminobenzoate compound DPC, which blocks the CFTR Cl- channel, caused a complete inhibition of the cAMP-sensitive current at a concentration of 1 mM (Fig. 3) (n = 5 cells). A smaller, voltage-dependent block of the Sertoli cell current was observed with a 10-fold lower concentration of DPC (Fig. 4A). In three experiments, 100 µM DPC blocked the cAMP-sensitive current by 25 ± 3% at -90 mV and 5 ± 1% at +60 mV. Because DPC may have nonspecific inhibitory actions at these concentrations, the effect of the sulfonylurea compound glibenclamide was also examined on the current (Fig. 4B). Glibenclamide (100 µM) produced a strong block of the Sertoli cell current (70-80%, n = 3 cells), which did not show significant voltage dependence (Fig. 4).


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Fig. 3.   Measurement of a cAMP-sensitive Cl- current in adult Sertoli cells. A: whole cell background currents recorded during 40-ms voltage steps applied from a holding potential of -30 to -90 mV through +60 mV in 10-mV increments. Currents were measured under basal conditions and after the addition of 1 mM 8-chlorophenylthio-cAMP (cAMP) in the presence or absence of 1 mM diphenylamine-2-carboxylate (DPC). B: current-voltage (I-V) relationship for cAMP-sensitive Cl- current obtained in the presence and absence of 1 mM DPC. Each point represents mean ± SE for 5 experiments. Values for DPC were obtained between 120 and 180 s after addition of the drug to the bath.


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Fig. 4.   Pharmacology of the Sertoli cell cAMP-sensitive Cl- current. A: I-V relationship for cAMP-sensitive Cl- current obtained in the presence and absence of 100 µM DPC. Block of the current was voltage dependent, with a 24% decrease at -90 mV and a 2% decrease at +60 mV. B: I-V relationship for cAMP-sensitive Cl- current obtained in the presence and absence of 100 µM glibenclamide (GLB). Block of the current was independent of the voltage, with a 71% decrease at -90 mV and a 80% decrease at +60 mV.

The CFTR Cl- current displays a linear I-V relationship when measured with nearly symmetrical concentrations of Cl- across the plasma membrane (2) and is reduced during substitution of Cl- with I- (1, 48). When the Cl- concentration in the pipette was increased to 126 mM, the I-V relationship for the Sertoli cell cAMP-sensitive current became linear in shape (Fig. 5A). In addition, as would be predicted for a Cl--selective current, reducing external Cl- from 140 to 8 mM caused the Erev for the current to shift from -1 mV (ECl = -3 mV) to +50 mV (ECl = +69 mV). Partial replacement of external Cl- with I- caused a strong but reversible decrease in the Sertoli cell cAMP-sensitive current (Fig. 5B). Overall, in five cells examined, the average slope conductance decreased by 46% in the presence of external I-. However, I- caused no significant change in the Erev of the current when compared with external Cl- (shift in Erev = -4 + 3 mV, n = 5 cells, P > 0.05). In contrast, substitution of external Cl- with Br- caused a significant shift in Erev (-7 + 1 mV, n = 4 cells, P < 0.05). Using the GHK equation, the PBr /PCl and PI/PCl were determined to be 1.22 and 1.03, respectively. This suggests that the permeability sequence of this channel is Br- > I- >=  Cl-.


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Fig. 5.   Properties of a cAMP-sensitive Cl- current in adult Sertoli cells. A: I-V relationship for cAMP-sensitive Cl- current measured with 126 mM Cl- in the pipette and either 140 mM Cl- external solution (ECl = -3 mV) or 8 mM Cl- external solution (ECl = +69 mV) in the bath. B: I-V relationship for the cAMP-sensitive current measured with 76 mM Cl- in the pipette and either Cl- external solution or I- external solution in the bath. Each point represents mean ± SE for 5 experiments. Slope conductance was measured over the linear part (-20 to +20 mV) of the I-V relationship.

In an attempt to identify the single-channel currents that might underlie the whole cell cAMP-sensitive current shown in Figs. 3-5, inside-out patches of membrane were obtained from the Sertoli cells and the cytosolic surface was exposed to catalytic subunit of protein kinase A (175 nM) with 3 mM MgATP. Internal and external solutions bathing the patch contained 146 mM Cl-. One successful experiment is displayed in Fig. 6, which shows single-channel currents recorded at various membrane potentials. The open probability of the channel, calculated by dividing the sum of the open times by the total acquisition period, was 0.22 at -60 mV. The slope conductance for the protein kinase-activated channel was 13 ± 1 pS, and the interpolated Erev was 1 ± 1 mV (ECl = 0 mV) (n = 3 patches). Channel activity was seen in the presence but not the absence of this catalytic subunit. This small-conductance Cl- channel was observed in 3 of 20 patches exposed to this catalytic subunit.


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Fig. 6.   Measurement of a small-conductance Cl- channel in adult Sertoli cells. A: single Cl- channels measured from an excised, inside-out patch of Sertoli cell membrane and recorded in the presence of 175 nM of the catalytic subunit of protein kinase A and 3 mM ATP. Channel activity was not evident in the absence of protein kinase A. Single-channel currents are shown in the presence of symmetrical 146 mM Cl- at the indicated potentials. Downward deflections represent inward current, and upward deflections indicate outward current. B: I-V relationship for the protein kinase-activated Cl- channel shown in A. Conductance and extrapolated reversal potentials in this experiment were 14 pS and +1 mV, respectively.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Our results demonstrate clearly that CFTR is expressed in rat Sertoli cells, and this is supported by several lines of evidence. First, using primers designed to exon 14a and 15 of the CFTR coding region, we identified a RT-PCR product of predicted size consistent with the CFTR sequence when RNA from Sertoli cell cultures were used as a template. These primers are well characterized and have been used by others to detect CFTR mRNA in rat tissue (34). The product identified is consistent with that found in a 3T3 cell line transfected with the CFTR gene. These CFTR-transfected 3T3 cells have been shown to express large amounts of CFTR (2, 5). Finally, our demonstration of this product in ASC-17D cultures provides additional evidence for CFTR RNA expression by Sertoli cells. This ASC-17D clonal line was established by treatment of Sertoli cell cultures derived from sexually mature rats with a temperature-sensitive mutant of the SV40 virus. These cells have been well characterized and shown to express several markers of Sertoli cell function with no expression of indicators of other testis cell types (40). The Sertoli cell origin of these clonal cells further strengthens the likelihood that CFTR message associated with these and our primary cultures is due to Sertoli cells. Thus, on the basis of these results, it seems clear that rat Sertoli cells express CFTR message.

To determine whether CFTR message was translated into a functional protein, we first used Western analysis. With this approach, we were able to detect immunoreactive bands in Sertoli cell preparations using antibodies to two domains of CFTR that seemed to be consistent with bands detected in human tissues (11, 12, 14, 43) and in rat uteri (41) and liver (21) found to contain CFTR. Recent cloning and analysis of the rat CFTR gene have revealed a very high sequence homology between the rat and the human proteins in the primary structure (20). In addition, we observed that T84 cells, a human colon cancer cell line found to express CFTR protein, contained a comparable high-molecular-mass immunoreactive protein. The presence of immunoblot signals in CFTR-transfected but not untransfected 3T3 cells in our studies also suggested that a CFTR-like protein was detected. These immunoblot studies were not conclusive, but we believe they provided enough evidence to justify performing more detailed patch-clamp studies on these cells. Further work will be necessary to determine whether these immunoreactive bands represent authentic CFTR and whether such processes as extensive glycosylation and alternate splicing contribute to the synthesis of this CFTR-like molecule. Information of this type will be important in the elucidation of the precise similarities or differences between this and human CFTR.

Our use of a patch-clamp approach revealed that Sertoli cells express a Cl- channel consistent with the characteristics of CFTR. We found that stimulation of cAMP-dependent protein kinase in the Sertoli cells resulted in the activation of a time-independent Cl- current. The Sertoli cell Cl- current displayed an outward-rectifying I-V relationship when measured with asymmetric concentrations of Cl- across the plasma membrane but had a linear I-V relationship with nearly symmetrical concentrations of Cl-. This Cl- current was inhibited by the drugs DPC and glibenclamide and was reduced during external substitution of Cl- with I-. The channel displayed an anion permeability of Br- > Cl-. Most importantly, application of the catalytic subunit of protein kinase A to excised patches of membrane resulted in the activation of a small-conductance Cl- channel that displayed long openings. These properties are similar to those reported for the CFTR Cl- current measured in T84 cells (10) as well as other heterologous cells (1, 2, 5, 39, 47) expressing the CFTR Cl- channel.

In contrast to the present results, which show an equal permeability of the Sertoli cell channel for Cl- and I-, the human CFTR channel is believed to have a permeability sequence of Cl- > I- (1). In addition, the Sertoli cell single Cl- channel displayed a higher conductance (13 pS) than that reported for the human epithelial CFTR channel (10 pS) (5, 47). However, the CFTR Cl- channels found in guinea pig ventricular myocytes (37, 54) and rat epididymal (27) and pancreatic cells (23) have a permeability for I- that is close to that of Cl-. The cardiac CFTR channel also displays a relatively high single-channel conductance at room temperature (13-15 pS) (16). This suggests that differences in I- permeability, as well as conductance, may be related to tissue or species variability in the ion pore of the CFTR protein. Part of the variability from tissue to tissue may result from differences in location or ambient conditions. Such factors may be particularly important for tissues in the testis, an organ that functions at several degrees below normal body temperature. Interestingly, Denning et al. (14) reported that processing of CFTR in cells transfected with a mutant gene is altered when the culture temperature is changed, raising the possibility that CFTR processing is temperature sensitive.

Our findings provide evidence of functional CFTR expression in Sertoli cells, strengthening the possibility that this cell type may be an important focus of CFTR-related problems in spermatogenesis. The expression of CFTR by Sertoli cells has been suggested by reports from others. Using cRNA probes for CFTR, Tresize et al. (52) observed a pattern of hybridization in 10-day-old rat testes that was consistent with Sertoli cell expression. This signal was diffuse and located throughout the seminiferous epithelium. Because of the extensive array of Sertoli cell cytoplasmic extensions surrounding developing germ cells, this type of signal was thought to reflect the diffuse nature of Sertoli cell cytoplasm. In another study using in situ hybridization, Tizzano et al. (49) observed the presence of CFTR hybridization in human infant and adult testis tissue. As with the studies above, the hybridization in these studies was modest (3-4 times over background) and located throughout the seminiferous epithelium, likely reflecting Sertoli cell expression. It is well recognized that Sertoli cells secrete many of the nutritional and regulatory components that are responsible for modulation and maintenance of germ cells (19) during spermatogenesis. In fact, these components form much of the specialized microenvironment within the seminiferous tubule that is necessary for proper germ cell development. One of the most important Sertoli cell functions in creating and maintaining this microenvironment is the control of fluid balance. In other tissues, such as the lung, pancreas, and intestine, the CFTR Cl- channel plays an important role in fluid and electrolyte transport (39). It would be quite reasonable to suggest that Sertoli cell alterations of this microenvironment, which may occur with CFTR mutation, could result in abnormal germ cell production. Our findings of functional CFTR expression in Sertoli cells extend the earlier evidence obtained by in situ hybridization and further suggest that Sertoli cell expression of CFTR may be important in testis function. Of course, as mentioned earlier, deleterious influences on germ cell development may occur by direct expression of mutant CFTR by germ cells. The expression of CFTR message in developing germ cell types has been observed in both rats and mice (52). When taken together, it appears that whether one or both sites of CFTR expression in the testis is involved, the observation of abnormal germ cell development in several conditions associated with CFTR mutation suggests that normal expression of CFTR may be required for successful spermatogenesis.

Other sites of CFTR expression in the male genital tract have also been identified and associated with infertility. The most well studied of these are the epididymis and the vas deferens (for reviews, see Refs. 35, 36, and 56). The vas deferens is frequently observed to be blocked or absent in patients with mutations in the CFTR gene. In fact, an absence of a patent vas deferens has been identified as one of the most prominent clinical indicators of CFTR-related reproductive problems (26). The epididymis in CF patients has also been observed to be obstructed by dehydrated secretions (26) and may well be due to malfunction of the CFTR Cl- channels. In studies using mice in which the CFTR gene is disrupted, it was determined that cAMP-regulated Cl- conductance that was normally present in the epididymides was eliminated (31). Such a change would be quite likely to alter mucous composition and may well result in eventual blockage of the structure. For many years, it was generally accepted that the inability to transport spermatozoa from the testis because of a blockage of the epididymis or vas deferens was the reason for CFTR-related infertility. Recently, a group of investigators found a strong correlation between infertility and the presence of mutant CFTR expression even when these tissues appeared structurally normal. Van der Ven et al. (53), when investigating underlying causes of male idiopathic infertility, found that 17.5% of men with infertility due to sperm abnormalities had a mutation in the CFTR gene. Thus it appears that several facets of mutant CFTR expression must be considered to more fully understand its impact on male reproduction. Future efforts directed toward each part of the male reproductive system, including the testis, should provide valuable insight into the wide array of processes that appear to be influenced by mutant CFTR expression and contribute to infertility in the male.

    ACKNOWLEDGEMENTS

We thank Dr. K. Roberts at the University of Minnesota for generously providing the ASC-17D cells, Dr. M. Welsh at the Howard Hughes Medical Institute, University of Iowa, for supplying the 3T3 cells transfected with CFTR, and Dr. M. Soares at the University of Kansas for kindly providing the Rcho-1 cells.

    FOOTNOTES

This work was supported by a grant from the Cystic Fibrosis Society (to F. R. Boockfor) and National Heart, Lung, and Blood Institute Grant HL-45789 (to K. B. Walsh).

Preliminary findings were presented in abstract form at the 27th Annual Meeting of the Society for the Study of Reproduction, Ann Arbor, MI, 24-27 July 1994.

Address for reprint requests: F. R. Boockfor, Dept. of Cell Biology and Neuroscience, School of Medicine, Univ. of South Carolina, Columbia, SC 29208.

Received 19 June 1997; accepted in final form 5 January 1998.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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