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
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ABSTRACT |
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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
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INTRODUCTION |
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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.
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MATERIALS AND METHODS |
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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 -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 M
when filled with the cesium aspartate internal solution (see below). Uncompensated series resistance in this study was typically <2 M
. 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).
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RESULTS |
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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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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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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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DISCUSSION |
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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.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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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.
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