1 Paediatric Molecular Genetics, Institute of Molecular Medicine, Oxford University, John Radcliffe Hospital, Oxford OX3 9DS; 2 University Laboratory of Physiology, Oxford OX1 3PT; 3 Growth and Development Unit, Field Laboratory, Oxford University, Oxford OX2 8QJ; and 4 Department of Pathology, Oxford University, John Radcliffe Hospital, Oxford OX3 9DU, United Kingdom
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ABSTRACT |
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To investigate the biology of the male
genital duct epithelium, we have established cell cultures from the
ovine vas deferens and epididymis epithelium. These cells develop tight
junctions, high transepithelial electrical resistance, and a
lumen-negative transepithelial potential difference as a sign of active
transepithelial ion transport. In epididymis cultures the equivalent
short-circuit current (Isc) averaged 20.8 ± 0.7 µA/cm2 (n = 150) and was partially inhibited by
apical application of amiloride with an inhibitor concentration of 0.64 µM. In vas deferens cultures, Isc averaged 14.4 ± 1.1 µA/cm2 (n = 18) and was also inhibited by
apical application of amiloride with a half-maximal inhibitor
concentration (Ki) of 0.68 µM. The remaining
amiloride-insensitive Isc component in epididymis
and vas deferens cells was partially inhibited by apical application of
the Cl channel blocker diphenylamine-2-carboxylic
acid (1 mM). It was largely dependent on extracellular
Cl
and, to a lesser extent, on extracellular
HCO
3. It was further stimulated by
basolateral application of forskolin (10
5 M), which increased
Isc by 3.1 ± 0.3 µA/cm2 (n
=65) in epididymis and 0.9 ± 0.1 µA/cm2 (n =
11) in vas deferens. These findings suggest that cultured ovine vas
deferens and epididymis cells absorb Na+ via
amiloride-sensitive epithelial Na+ channels (ENaC) and
secrete Cl
and HCO
3
via apical cystic fibrosis transmembrane conductance regulator (CFTR)
Cl
channels. This interpretation is supported by
RT-PCR data showing that vas deferens and epididymis cells express CFTR
and ENaC mRNA.
ovine genital ducts; cystic fibrosis transmembrane conductance regulator; epithelial sodium channel
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INTRODUCTION |
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THE EPIDIDYMIS AND VAS DEFERENS form the proximal and
distal parts, respectively, of the secretory duct system of the male reproductive tract. The epithelium lining these ducts secretes and
absorbs ions, organic solutes, and water to provide an appropriate luminal environment for normal sperm maturation. Male infertility can
result from a number of factors involving the genital ducts. These may
be physical, as in the case of congenital absence of the genital ducts,
or functional, for example, inadequate fluid secretion. The
electrophysiology of genital duct function has been evaluated in a
number of model systems, including rodent and human intact ducts and
cultured cells. It has been proposed that the epididymis primarily
secretes Cl (27; see Refs. 21 and 44 for review)
following a mechanism common to many secretory epithelia. This involves
an anion accumulation step at the basolateral side of the epithelium
(possibly mediated by an
Na+-K+-2Cl
cotransporter, or
an Na+/H+ exchanger in parallel with a
Cl
/HCO
3
exchanger) with apical anion secretion through ion channels. In
addition, the genital ducts have a high luminal K+
concentration, which seems to be important for normal sperm function. In the vas deferens this may be is achieved by a characteristic maxi-K+ channel (34, 35), and the epididymis by an
ATP-activated K+ conductance (4). Different species show
important variation in the bioelectrical properties and anion secretion
along the length of the genital duct system (6).
Male infertility resulting from absence of the vas deferens or epididymal obstruction is one of the characteristic features of cystic fibrosis (CF). It is not clear whether this disruption of the genital ducts is due to malformation during development or to their obstruction by secreted material after the midtrimester of gestation (17, 18). The latter explanation seems most likely but is difficult to investigate in the human fetus in utero. Attempts to investigate the cause of genital duct obstruction in CF with use of CF mouse models have been disappointing, as these animals have intact genital ducts and are not infertile (22).
We previously showed that the CF transmembrane conductance regulator
(CFTR) is expressed in the epithelium of human genital ducts from the
midtrimester of human gestation onward (9, 16, 38). The CFTR
cAMP-activated Cl conductance is thought to make a
significant contribution to Cl
secretion by the
genital duct epithelium (27). The absence of CFTR or the presence of a
mutant CFTR protein is thought to underlie the genital duct
obstruction. Interestingly, the CF mouse genital duct expresses a
predominant Ca2+-mediated Cl
channel
that may compensate for mutation in CFTR (22). Because the causes of
male genital duct abnormalities are impossible to investigate in humans
in vivo, the aim of this study was to evaluate whether the
ovine genital duct would provide a suitable model to investigate the
role of CFTR in genital duct epithelial function.
We already established cell cultures of human fetal genital duct cells
and showed these to express CFTR mRNA and CFTR Cl
channels in vitro (16, 17, 27). Epididymis cells have also been
cultured from adult human epididymis epithelium (10). In this report we
show that cultured ovine epididymis and vas deferens epithelial cells
express CFTR mRNA and Cl
channels and also the
epithelial Na+ channel (ENaC). These cells provide an
excellent culture system for examining the function and regulation of
the ovine CFTR gene and the CFTR protein and its role in genital duct
abnormalities associated with CF.
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MATERIALS AND METHODS |
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Cell culture. Testicles and genital ducts were removed from 10 male lambs that died perinatally for nonpathological reasons. The lambs were the progeny of Mule ewes mated with Poll Dorset or Charolais rams.
Genital ducts were dissected from the testicular capsule and divided into vas deferens and epididymis; each was minced into 1-mm3 explants and placed in a minimal volume of CMRL 1066 medium with 20% fetal bovine serum, insulin (0.2 U/ml), hydrocortisone (1 µg/ml), cholera toxin (10Electron microscopy. Cells for transmission electron microscopy were grown to confluence on permeable collagen membranes (Cellagen disks, ICN Biomedical, Aurora, OH), fixed in gluteraldehyde, postfixed in osmium tetroxide, and dehydrated. Samples were then treated with propylene oxide and embedded in epoxy resin. Thin sections were cut and stained with uranyl acetate and lead citrate and then examined with a transmission electron microscope (model 1200EX, Jeol).
Transepithelial electrical measurements.
For transepithelial studies, cells were seeded onto 12-mm-diameter
Millicell-HA culture plate inserts (Millipore, Bedford, MA). Cells were
allowed to grow to confluence, and transepithelial voltage
(Vte) and resistance (Rte) were
routinely checked using a commercially available epithelial
volt-ohm-meter (EVOM) with "chopsticks" electrodes (World
Precision Instruments, Sarasota, FL). For the resistance measurements,
the EVOM uses an alternating square-wave current of ±20 µA at 12.5 Hz. The chopsticks electrodes were immersed in the cell
culture medium bathing the apical and basolateral sides of the cultures
under sterile conditions so that cultures could be monitored repeatedly
for several days. EVOM measurements were also performed on monolayers
grown on Cellagen disks for electron microscopy, and data obtained from
Millicell inserts and Cellagen disks were pooled. Some of the Millicell inserts were subsequently transferred to modified Ussing chambers for
continuous equivalent short-circuit current (Isc)
measurements. Ussing experiments were performed using a
computer-controlled clamp device (model CVC 6, Fiebig, Berlin,
Germany), as previously described (1). In these Ussing experiments,
Rte was evaluated every 20 s by measuring the
voltage deflections induced by 200-ms symmetrical square-current pulses
of ±25 µA. Open-circuit Vte was also measured,
and the equivalent Isc was calculated according to
Ohm's law. Conventionally, a lumen-negative Vte
corresponds to a positive Isc, which may be due to
electrogenic cation absorption and/or electrogenic anion secretion.
The bath solution was identical on the apical and basolateral sides
of the epithelial monolayer and contained (in mM) 140 Na+,
4 K+, 1 Ca2+, 1 Mg2+, 124 Cl, 24 HCO
3, and 5 glucose. A reservoir connected to each half-chamber contained bath
solution, which was kept at 37°C by a temperature-controlled water
jacket. The solution was continuously recirculated from the reservoir
through the half-chamber by use of a bubble lift, which gassed the
solution with 95% O2-5% CO2, maintaining pH
at 7.4. The volume of each half-chamber was 1.8 ml, and the total
volume of circulating solution was 10 ml. Drugs were added from stock
solutions to the apical or basolateral side of the Ussing chamber.
Drugs were washed out by repeated simultaneous removal of 5 ml of
solution from both half-chamber reservoirs and readdition of the same
volume of fresh prewarmed and pregassed bath solution. This procedure
was repeated
10 times, which corresponded to a >1,000-fold dilution
of the added drug and usually appeared to be an efficient washout, as
shown by the reversibility of the amiloride effect (see
RESULTS). However, in experiments using basolateral
forskolin (10
5 M), washout appeared to
be delayed, possibly because of an "unstirred" layer phenomenon
within the filter membrane. In some experiments, cholera toxin was
omitted from the tissue culture medium 24 h before the cultures were
used in Ussing experiments to avoid downregulation of cAMP-mediated
pathways by chronic stimulation with cholera toxin.
Expression of CFTR and ENaC mRNA. Cells were harvested with a cell scraper, and total RNA was prepared with an RNeasy kit (Quiagen). RNA was extracted from intact genital ducts by guanidinium isothiocyanate extraction (8). RT-PCR was performed as described previously (except RNA was transcribed using Superscript RT), with amplification of the ovine C fragment (bases 1806-2597) of the CFTR cDNA (37). RNA semiquantitation was provided by simultaneous RT-PCR of the housekeeping gene, subunit c of sheep mitochondrial ovine ATP synthase (25).
For the detection of ENaC transcripts, RNA was transcribed using Superscript RT (Life Technologies, Paisley, UK) and random hexamer primers (Pharmacia, Freiburg, Germany). Sequence information for the sheep ENaC is not available. Therefore, primers were chosen that correspond to regions highly conserved between ![]() |
RESULTS |
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Cell cultures.
Cultures of vas deferens and epididymis epithelium were established
from 10 pairs of lamb genital ducts. Epithelial cells migrated
from explants 48-72 h after establishment of cultures on Primaria
flasks (Becton Dickinson). Figure
1 shows the morphology of
epididymis (A and B) and vas deferens (C and
D) cells in culture. The morphology is similar to that of human
genital duct epithelial cells (17), with at least two predominant cell
types in culture, one showing a more cuboidal appearance. The cells
were routinely passaged with trypsin-EDTA and maintained their
differentiated morphology for at least four passages at split ratios of
1:2-3. This yielded 6-8 × 75 cm2 flasks
from each genital duct. It was essential to monitor cultures closely
for fibroblast contamination at the initial stages. Fibroblasts were
removed by a wash in 0.25% trypsin-1 mM EDTA or by physical disruption
with a cell scraper. Cells were successfully cryopreserved in 95%
fetal bovine serum and 5% DMSO.
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Electrophysiological characterization of the epididymis and vas
deferens epithelial cells.
For these experiments, epididymis cells were derived from different
primary cultures and used in passages 2-6. Rte
and Vte of cells grown on permeable supports were
repeatedly monitored using chopstick electrodes connected to an EVOM.
At 4-5 days after seeding, the epididymis cells had developed an
average Rte of 1,325 ± 34 · cm2 and a lumen-negative
Vte of
21.1 ± 0.8 mV (mean ± SE,
n = 179). In vas deferens monolayers derived from three
different primary cultures and seeded onto permeable supports at
passage 3, similar Rte and
Vte measurements averaged 889 ± 104
· cm2 and
10.6 ± 1.2 mV
(n = 22), respectively, 4 days after seeding. Thus, when grown on a permeable support, epididymis and vas
deferens cells formed epithelial monolayers with a high
Rte consistent with the presence of tight junctions
as detected by electron microscopy (see above). Moreover, the
lumen-negative Vte indicated the presence of active
transepithelial ion transport, which could be electrogenic absorption
of cations and/or electrogenic secretion of anions.
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Expression of CFTR and ENaC.
To investigate whether cultured ovine epididymis and vas deferens cells
express the CFTR gene and subunits of ENaC, we performed RT-PCR analysis. Figure 9 shows the
amplification of the C fragment (bases 1806-2597) of the ovine
CFTR cDNA in RNA extracted from three sets of genital ducts established
in culture and from intact genital duct tissue. Although the RT-PCR is
not quantitative, comparison of the CFTR-derived product with the ovine
ATP synthase product amplified in the same reaction gives a measure of
the variation in the CFTR levels in different genital duct cell
cultures. Figure 10 shows a 370-bp RT-PCR
product generated by ENaC-specific primers in RNA from epididymis
(lane 4) and vas deferens (lane 5).
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DISCUSSION |
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Male infertility due to absence of the vas deferens or epididymal
abnormalities is characteristic of CF. This has generated a research
interest in the biology of the male genital duct epithelium in this
disease. It is known that CFTR mRNA is expressed from the midtrimester
of gestation and that at this gestational age CF genital ducts are
generally morphologically normal (13, 16). It is not clear whether the
ducts fail to continue normal development during later gestation in CF
or whether they become obstructed by secretory deposits, as happens in
the pancreatic ducts (16, 29). Epithelial cell cultures that we
established from human fetal male genital ducts (17) express the
CFTR gene and the CFTR cAMP-activated Cl
channel in addition to other Cl
conductances and a
significant maxi-K+ channel (27, 35, 44).
Many aspects of the pathology of CF are difficult to investigate in humans and so necessitate the evaluation of alternative animal models. CF knockout mice have been useful in investigating certain aspects of CF pathology. However, they are inadequate for the study of CF disease in the airway, pancreas, and genital ducts, as they do not exhibit pathology in these organs. The limitations of CF mouse models have led to a search for alternative animal models of CF. Ovine and human development and respiratory physiology have been shown to be similar (11, 26, 39, 40). Furthermore, we have isolated the ovine CFTR cDNA and evaluated its expression through development into adult life (37). The ovine and human CFTR genes show close parallels at the sequence level and in their expression patterns. These similarities, together with recent advances in cloning technology (2, 41), have resulted in the sheep being promoted as a suitable large animal model of CF (15). To investigate the physiology of ovine genital ducts and their similarity to the human vas deferens and epididymis, we have established primary cultures of ovine genital duct epithelial cells.
The cultured cells were morphologically similar to those derived from
human genital duct epithelium (10, 17). When cultured on permeable
supports, vas deferens and epididymis cells formed monolayers with a
high Rte, indicating the presence of tight
junctions, as confirmed by electron microscopy. Moreover, our
transepithelial measurements provided evidence for the presence of
active transepithelial ion transport. We observed an
Isc component sensitive to amiloride and another
component sensitive to the Cl channel blockers DPC
and NPPB. These findings indicate that the observed basal
Isc can be attributed to electrogenic
Na+ absorption and Cl
secretion in sheep
male genital duct epithelial monolayers.
The epididymis epithelium can function as a Cl and
HCO
3 secretory epithelium (5, 21, 27),
and this secretion may be stimulated by a variety of agents known to
increase intracellular cAMP (5, 27, 44). The stimulatory effect of
forskolin on Isc in ovine epididymis cultures
described here is consistent with these observations. The underlying
secretory channels are believed to be CFTR Cl
channels, since they are known to be activated by cAMP, and CFTR transcripts have been shown to be expressed in the genital duct epithelium. CFTR Cl
channels are known to be
permeable to various polyatomic anions, including
HCO
3 (14, 31), with an anion
permeability sequence NO
3 > Cl
> HCO
3 > formate > acetate (23). Thus it is conceivable that the
HCO
3-dependent secretory Isc component observed in the present study may
also be mediated via CFTR Cl
channels. Indeed, it
has been estimated that in rat epididymis CFTR may account for 70% of
HCO
3 secretion and the
Na+-HCO
3 cotransporter may
account for 30% (5). Furthermore, single-channel patch-clamp
recordings on cultured human fetal epididymis cells have demonstrated
the presence of small-conductance forskolin-activated
Cl
channels with biophysical properties similar to
CFTR Cl
channels (27).
The inhibitory effect of millimolar DPC that we have described is
consistent with the inhibition of CFTR Cl channels
and is in agreement with previous findings in rat epididymis (19). The
Cl
channel blocker NPPB (100 µM) also partially
inhibited Isc in ovine epididymis cells, a finding
that is similar to observations on cultured rat epididymis epithelium,
where apical NPPB was shown to inhibit Isc with a
Ki of ~50 µM (43). NPPB is a potent blocker of
large-conductance outwardly rectifying Cl
channels
that have been described in excised patches of numerous epithelial
preparations, including cultured human fetal epididymis cells (27).
Ca2+-activated Cl
channels have been
demonstrated in whole cell patch-clamp recordings in rat epididymis
cells (7) and may also be inhibited by NPPB. Hence, it is possible that
CFTR Cl
channels are not the only channels
contributing to Cl
secretion in genital duct
epithelia (21). However, the molecular nature of these other
Cl
channels is not clear. In this study, the
functional evidence for expression of CFTR-associated
Cl
channels is supported by the detection of CFTR
mRNA in ovine epididymis and vas deferens cells by RT-PCR.
Amiloride has been reported to have no effect on the
Isc of cultured rat epididymis epithelial cells
(19). In contrast, we detected a large amiloride-sensitive
Isc component in cultured ovine epididymis and vas
deferens epithelia. Our findings appear to be consistent with the in
vivo situation, as studies in isolated and microperfused rat cauda
epididymis have demonstrated the presence of amiloride-sensitive
Na+ and fluid reabsorption in this tissue with a
Ki for amiloride of 1.6 µM (45, 46). Moreover,
our data are consistent with recent findings in mouse epididymis
cultures, in which apical application of
104 M amiloride also inhibited a
component of the equivalent Isc. Interestingly, the
mouse epididymis cells also showed a cAMP-stimulated Cl
secretory response in addition to the
amiloride-sensitive Isc component (22). In sheep
genital duct epithelia we have demonstrated a high sensitivity of the
Isc to amiloride with a Ki of
0.6-0.7 µM. This suggests that the rate-limiting Na+
entry step occurs via highly amiloride-sensitive apical Na+
channels that most likely belong to the ENaC family (3). This conclusion is further supported by our RT-PCR evidence, which demonstrates the presence of ENaC transcripts in sheep epididymis and
vas deferens epithelial cells. Thus our data suggest that ENaC is the
channel responsible for Na+ absorption in genital duct
epithelia. To our knowledge, similar data are not available for other
species, and it will be interesting to see whether ENaC is also
expressed in human genital duct epithelia.
Epithelial Na+ channels may be inhibited during activation
of Cl secretion in cells expressing both CFTR and
ENaC, which suggests that there is a regulatory relationship between
these two conductances (12, 20, 30, 36). However, in the genital duct
epithelium, it is not clear whether CFTR and ENaC are separately
expressed in different cell types or coexpressed in the same cells
where they may interact. This question warrants further evaluation.
In conclusion, we have established cultured ovine epididymis and vas deferens cells, which differentiate into transporting epithelia in vitro and will have multiple applications.
First, they will provide an excellent tool for examination of the
transepithelial transport properties of genital duct epithelia, which
appear to absorb Na+ via an amiloride-sensitive ENaC and
secrete Cl via apical CFTR Cl
channels. The coexpression of these two channels in genital duct epithelia may be interesting in the light of recent observations that
CFTR and ENaC may have a regulatory relationship and that this
interaction may be central to CF airway disease. Indeed, it has been
suggested that, in normal airway epithelium, CFTR constitutively suppresses Na+ channel activity, whereas
in CF airway epithelial cells, i.e., in the absence of normal CFTR,
Na+ channels are hyperactive (24, 36). Such a regulatory
mechanism may also be important for the pathophysiology of male
infertility in CF, where lack of Cl
secretion may
cause hyperabsorption of Na+ by the genital duct epithelia.
Second, these cells will provide an additional useful tool in studies to elucidate the mechanisms of regulation of expression of the CFTR gene. One approach to finding the elements conferring tight tissue-specific and temporal regulation of CFTR expression has been to identify DNase I hypersensitive sites associated with the locus (32, 33). This type of approach is rendered much more useful if chromatin is available from primary cell populations that express the CFTR gene endogenously, rather than relying on transformed cell lines. Similarities in the patterns of expression of CFTR in sheep and humans suggest that these two species may share common regulatory elements (37; unpublished data). Evaluation of the ovine CFTR gene control elements in the cultured primary genital duct cells will enable the future in vivo analysis of developmental expression of the CFTR gene.
Third, the similarities between the ovine and human cultured male genital duct cells suggest that once an ovine model of CF becomes available (15), it should be suitable for evaluating the pathology of the genital duct associated with CF and its progression through gestation.
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ACKNOWLEDGEMENTS |
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We are grateful to Cliff Hanson and Dr. John Cuffe for assistance and Dr. Mike Gray for helpful discussions.
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FOOTNOTES |
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* M. Bertog and D. J. Smith contributed equally to this work.
This work was supported by the Cystic Fibrosis Research Trust, the Edward Penley Abraham (EPA) Research Fund, the Association Française de Lutte Contre La Mucoviscidose and the Wellcome Trust (D. J. Smith and A. Harris), the Wellcome Trust (A. Bielfeld-Ackermann, C. Korbmacher, and D. J. P. Ferguson), and the Deutscher Akademischer Austauschdienst and the EPA Cephalosporin Fund (M. Bertog).
Part of this work was presented at the North American Cystic Fibrosis Conference, Montreal, in October 1998 and The Physiological Society, Southampton, in September 1998, and published in abstract form [Paediatr Pulmonol Suppl 17: 216-217, 1998, and J Physiol (Lond) 513: 60P, 1998].
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: A. Harris, Paediatric Molecular Genetics, Institute of Molecular Medicine, Oxford University, John Radcliffe Hospital, Oxford OX32 9DS, UK (E-mail:aharris{at}molbiol.ox.ac.uk).
Received 20 October 1998; accepted in final form 17 December 1999.
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