Human Reproduction vol.11 no.l pp.212-217, 19%
Human Fallopian tubal epithelial cells in vitro:
establishment of polarity and potential role of
intracellular calcium and extracellular ATP in fluid
secretion
C J.Dickens, M.T.Comer, J.Southgate1 and
HJ.Leese 2
Department of Biology, University of York, PO Box 373,
York YO1 5YW and 'ICRF Cancer Medicine Research Unit,
St James's University Hospital, Beckett Street, Leeds LS9 7TF, UK
2
To whom correspondence should be addressed
A pure population of human Fallopian tubal epithelial cells
has been isolated by enzyme digestion, grown in primary
culture and used to explore the biochemical basis of oviduct
fluid secretion. Confluence was achieved in 3—7 days.
Immunocytochemical labelling for cytokeratins indicated
that the cells were epithelial in nature and formed extensive
desmosomal contacts, producing a polarized layer in culture. By growing the cells on collagen-impregnated filters,
a small transepithelial electrical potential difference could
be recorded, with the apical side of the cells negative with
respect to the basal side. In addition, the consumption of
glucose and the appearance of lactate were greater on the
basal than on the apical side of the cells. Because intracellular Ca 2+ ([Ca 2+ ]i) is well established as a signal transduction
agent in epithelial fluid secretion, the effect of a wide range
of agonists on [Ca 2+ ] ( in isolated tubal epithelial cells was
studied using Fura-2. The only agent which induced a
change in [Ca 2 + ]| was extracellular ATP. The transients
induced were dependent on both intracellular and extracellular calcium. ATP added to the basal side of the cells
of the polarized layer induced a transient increase in the
potential difference. The data are consistent with a potential
role for extracellular ATP in the regulation of human tubal
fluid formation.
Key words: epithelia/Fallopian tube/human tubal fluid/
intracellular calcium/purinergic receptors
Introduction
Oviduct fluid provides the milieu in which gamete transport
and maturation, fertilization and the first few days of embryo
development occur, and as such would be an appropriate
medium for in-vitro fertilization (IVF). Although the composition of oviduct fluid has been reported in a number of species
(David et al., 1969; Borland et al, 1980; Gardner and Leese,
1990; Dickens and Leese, 1994a; Dickens et al, 1995), little
is known about its mechanism of secretion. It is important to
understand the nature of oviduct fluid formation, because up
to 28% of cases of female infertility are of tubal origin (Hull
et al., 1985) and a steady flow of fluid into the lumen is likely
to be essential in maintaining mucosal integrity, as in other
212
epithelial tissues such as those which line the gut and airways.
The impairment of tuba! flow and the failure to clear cellular
debris in the lumen could compromise tubal patency.
The composition of oviduct fluid is ultimately determined
by the transport properties of the epithelial cells which line
the lumen. We have described previously a method for isolating
rabbit oviduct epithelial cells and growing them in primary
culture (Dickens et al, 1993a). When grown on coverslips
or semipermeable, collagen-impregnated filters, the cells reestablish junctions with one another and form a polarized layer
in the same orientation as that found in vivo. The advantage
of growing cells on collagen filters is that both sides of the
cell layer are readily accessible for sampling. Thus, the niters
can be mounted in a modified Ussing chamber to enable the
transport of molecules across the epithelial cells to be examined,
as well as the electrical potential difference (Dickens et al.,
1993a; Edwards and Leese, 1993). A number of filters can be
established from a single tube, making the method cost
effective and economical in the use of tissue.
We have now applied this technique to human Fallopian
tubes removed at hysterectomy. A limited immunocytochemical characterization has been carried out using antibodies
specific to epithelial cytokeratins. The electrical potential
difference across the epithelial cells has been determined, and
we have also measured the consumption of glucose and the
appearance of lactate in the basal and apical media to discover
whether there is asymmetry in metabolic terms between
the two compartments. Ca 2+ ions are well established as
intracellular mediators of fluid secretion in a wide variety of
cell types (Villereal and Byron, 1992; Berridge, 1993). Therefore we have studied the effect of a large series of agonists
on intracellular calcium concentrations ([Ca2+]j) in human
tubal epithelial cells in vitro using the fluorescent indicator
Fura-2. For this work, the cells were grown on laminin-coated
glass coverslips because the collagen-impregnated membranes
autofluoresced strongly. The only agonist which induced a
consistent effect on [Ca2+]j was extracellular ATP. Therefore
this response was pursued in terms of the nature of the
purinergic receptor involved. Preliminary accounts of some of
this work have been published previously (Dickens and Leese,
1992, 1994b; Dickens et al., 1993b).
Materials and methods
Cell culture
Human Fallopian tubal epithelial cells were isolated by the method
of Dickens et al. (1993a), which is similar to that devised by Kimber
et al. (1993) for mouse uterine epithelial cells and is, in turn, a
modification of that used by Glasser et aL (1988). Tubes at a variety
© European Society for Human Reproduction and Embryology
Human Fallopian tubal epithelial cells in culture
of hormonal stages, as determined from the date of the last menstrual
period, were removed from pre-menopausal patients attending for
hysterectomy who had given informed consent Briefly, the tubes
were washed in Hank's balanced salt solution without calcium
and magnesium (Ca-Mg-free HBSS; Gibco, Life Technologies Ltd,
Paisley, UK), slit along their length and the muscular tissue cut away.
The mucosa was chopped into 1 cm segments and incubated in CaMg-free HBSS containing 0.5% type I trypsin (Sigma Chemical Co.,
St Louis, MO, USA), 2.7% pancreatin (Gibco) for 1 h at 4°C,
followed by 1 h at room temperature. The enzyme medium was
removed, Ca-Mg-free HBSS added and the epithelial cells vortexed
into suspension. After centrifugation at 250 g for 5 min, the cells
were washed in Ca-Mg-free HBSS. This process was repeated twice
and the cells finally resuspended in pre-warmed gassed culture
medium. Cell integrity was tested by the ability to exclude the dye
Trypan Blue (0.4% w/v). More than 95% of the cells in suspension
were found to be viable on this basis.
The culture medium was a 1:1 mixture of Dulbecco's modified
Eagle's medium (Sigma) and Nutrient mixture Ham's F-12 (Sigma)
with the following additions: 0.1% bovine serum albumin (ICN
Pharmaceuticals Ltd, Oxfordshire, UK), 270 IU/ml penicillin (Gibco),
270 fig/ml streptomycin (Gibco), 2.5% heat-inactivated fetal calf
serum (Gibco), 2.5% Nu serum (Becton-Dickinson, Bedford, MA,
USA), 2.5 mM glutamine (Sigma) and 20 Jig/ml fungizone (Gibco).
The cells were plated onto laminin-coated (Sigma) 10 mm-diameter
glass coverslips or, for transcellular studies, onto semipermeable
collagen-impregnated filters (ICN Pharmaceuticals Ltd). The latter
were placed in multiwell plates. In all, 250 p.1 of cell suspension
containing at least 1.0X106 cells/ml were plated on the top of the
filters, and 450 (il of fresh medium added underneath. The cells were
placed in a humidified incubator at 37°C containing 5% CO2 in air.
The medium from above and below the filters was replaced every
24 h for the first 3 days in culture, and every 48 h thereafter. The
cells had usually become confluent 3—7 days after being placed in
culture. The resistance across the cells on the filter was measured
using an epithelial voltmeter, 'EVOM', connected to art Endohm
chamber (World Precision Instruments Inc., Sarasota, FL, USA). The
mean resistance across the cells was 151 ± 25 il (n = 8).
Immunocytochemical studies
Immunolabelling was carried out as described previously (Soumgate
et aL, 1987; Dickens et aL, 1993a). Briefly, isolated cells from the
Fallopian tube (as above) were plated onto 12-well 'Multispot' glass
slides (ICN Pharmaceuticals Ltd) containing identical medium and
conditions to the cultures on collagen filters. After 96 h, the slides
were rinsed in phosphate-buffered saline (PBS; 137 mM NaCl,
2.7 mM KC1, 3.2 mM Na2HPO4 and 1.47 mM KH2PO4, pH 7.2),
fixed in two changes of a freshly prepared mixture of methanol and
acetone (v/v), and air dried.
Primary antibodies LDS103 (anti-cytokeratins 7 and 8; Southgate
et aL, 1987) and DP2.15 (anti-desmoplakins 1 and 2; ICN Pharmaceuticals Ltd) were applied to individual wells of the slides and
incubation continued at room temperature for 1 h. Slides were rinsed
in PBS, re-fixed in methanol:acetone and air dried. Goat anti-mouse
immunoglobulin G conjugated to fluorescein isothiocyanate (FTTC;
Southern Biotechnology Associates, obtained via Eurogenetics,
Teddington, UK) was applied to each well and incubated for 30 min
at room temperature. Slides were washed in several changes of PBS
containing 0.25% Tween 20, rinsed in distilled water and air dried.
Labelled cultures were viewed using a Zeiss Axioplan microscope
equipped with epifluorescent illumination and a specific FTTC filter.
Specificity controls included the omission of primary antibody and
Figure 1. Indirect immunofluorescence labelling of human tubal
primary cell cultures. Cultures were grown on glass slides and fixed
after 6 days in culture. (A) LDS103 staining of cytokeratins 7 and
8. (B) DP2.15 staining of desmoplakins showing localization to
desmosomes at the cell borders. Bars = 12.5 |im.
the use of primary antibody HB16, a hamster-specific anti-keratin as
negative control.
Transepithelial electrical potential difference
All experiments were carried out with cells which were confluent
across the whole of the filter surface, as judged by their appearance
under low power light microscopy. If the cells were not confluent,
the transepithelial potential difference was zero and the resistance
was low (<20 Q).
A filter containing the polarized cells was clamped between the
two halves of a modified Perspex Ussing chamber. The system was
placed in a water jacket at 37°C. The balanced salt solution was
added to each half of the chamber and gassed with a stream of 5%
CO2 in O2 to ensure adequate mixing and maintain the solutions at
pH 7.4. Silicon bungs were inserted into the tops of the chambers to
prevent evaporation. The bungs contained small inlet ports which
213:
C J.Dickens et aL
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APICAL GLUCOSE CONSUMPTION
BASAL GLUCOSE CONSUMPTION
APICAL LACTATE APPEARANCE
BASAL LACTATE APPEARANCE
Figure 2. Glucose consumption and lactate production by human
tubal epithelial cells grown on collagen supports. Apical and basal
measurements were made every 24 h over days 4—10 in cultures
from a single patient. Bars show the mean ± SEM of readings
from four filters.
allowed samples to be added during the experiments. Agar bridges
were inserted into the chambers and linked via 3 M KG to matched
calomel electrodes. These were connected to a Vibron electrometer
and the output displayed on a chart recorder, enabling the potential
difference across the cell layer to be monitored continuously.
Media sampling experiments
The apical and basal media from the cells grown on collagen filters
were removed after each 24 h culture period for up to 10 days and
stored frozen at -20°C until analysed. Glucose and lactate were
measured on a COBAS Mira Autoanalyser (Roche Instruments,
Welwyn Garden City, UK), as described by Edwards and Leese (1993).
Measurement of intracellular calcium
The cells grown on coverslips were loaded with 10 (iM Fura-2AM
(Calbiochem, Nottingham, UK) in cell culture medium at 37°C for
30-60 min. The coverslips were then mounted in a Perspex chamber
(volume -200 |il) on the stage of a Nikon Diaphot microscope. Mean
measurements of intracellular calcium were obtained by monitoring
the fluorescence obtained from a 2.02 mm2 area of confluent cells
viewed under the microscope. A balanced salt solution (117.94 mM
NaCl, 4.78 mM KC1, 1.19 mM KH2PO4, 1.19 mM MgSO4-7H2O,
5.56 mM glucose, 1.71 mM CaCl2-2H2O, 4.15 mM NaHCC^ and
20.85 mM HEPES at pH 7.4) at 37CC was superfused across the cells
at a rate of 5-8 rruVmin. A nominally calcium-free solution was made
by omitting calcium from the salt solution and adding 10 mM EGTA.
The Nikon Diaphot microscope was fitted with a xenon light source
and a computer-driven excitation filter wheel (Newcastle Photometric
Systems, Newcastle upon Tyne, UK), such that the cells were excited
alternately with light of wavelengths 340 and 380 run. Light emitted
from the cells was measured at wavelengths >520 nm using a long
band pass filter. The emission at the two excitation wavelengths was
ratioed to give a value related to the free intracellular calcium, which
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DAY
214
300
Time
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o
1.51.00.5
100
200
300
400
Time (s)
500
600
Figure 3. (A) Effect of 100 uM ATP on the calcium ratio in
confluent human tubal epithelial cells. (B) Effect of 100 (im ATP in
the absence and presence of extracellular calcium in confluent
human tubal epithelial cells. Bars represent the periods during
which the cells were exposed to calcium-free medium and ATP.
was displayed on a computer screen. Background fluorescence was
automatically subtracted from the experimental data using a reading
from a cell-free area of laminin-coated glass coverslip.
Salt solutions with accurately known calcium concentrations
(Molecular Probes Inc., Eugene, OR, USA) containing 10 |iM of the
pentapotassium salt of Fura-2 (Calbiochem) were used at 22°C with
no cells present to obtain an approximate calibration of the system.
It is appreciated that this is not an absolute method of calibration in
that the dye may behave in a different manner inside the cells, and
that elements such as viscosity and protein binding are unaccounted
for (Williams and Fay, 1990); however, it does provide some
quantification of the ratio data. Therefore results are expressed in
terms of the ratio value, with the approximate conversion to nanomoles
included for comparison. There was some variation in the calcium
ratio data with different batches and ages of cells. Therefore all
comparative studies were carried out on cells derived from the same
patient and on the same day of culture.
Expression of results
Results arc expressed as the mean ± SEM, with the number of
determinations in parentheses. The significance of differences between
mean values was assessed using Student's /-test, unless otherwise
stated.
Results
Immunocytochemistry
The two antibodies (LDS103 specific for cytokeratin isotypes
7 and 8, and DP2.15 specific for desmoplakins) reacted
Human Fallopian tuba) epithelial cells in culture
ATP
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2
E
5
•a
6.
1OOyM
ATP
1mM
1.51.00.5-
Conoantratlon
0.0
0
2
4
Time
6
8
(mln)
Figure 5. The effect of adding 100 urn ATP to the basal side of a
polarized layer of human tubal epithelial cells mounted in an
Ussing chamber, p.d. = potential difference.
Figure 4. (A) Dose-response chart for the effect of five
concentrations of ATP on the calcium ratio in human tuba]
epithelial cells. (B) Comparison of the effect of 100 (iM ADP, UTP
and ATP on the calcium ratio in human tubal epithelial cells.
Results are expressed as the mean ± SEM. The number of readings
is shown in parentheses at the base of each column.
intensely
showing
reformed
polarized
with the cells grown on the glass slides (Figure 1),
that the cells were epithelial in nature and had
extensive junctions when in culture to give a
layer.
Media sampling experiments
Samples of apical and basal media taken every 24 h from cells
grown on collagen filters were analysed for glucose and lactate
over the period 4-10 days in culture. Data for four filters from
a single culture from the same patient are shown in Figure 2.
For each successive 24 h period, glucose consumption and
lactate appearance were -2-fold greater at the basal than the
apical pole of the cells. Preliminary data suggest that no
differences were apparent for cells taken at different stages of
the menstrual cycle.
Transepithelial potential difference
Confluent human tubal epithelial cells derived from a number
of patients gave a potential difference of 0.12 ± 0.12 mV
(n = 32) with the apical side of the cells grounded.
Intracellular calcium
All experiments were carried out on confluent isolated epithelial
cells that had been in primary culture for between 2 and 9
days. The average ratio value for [Ca2+]j, derived from eight
different cultures, was 1.11 ± 0.02 (n = 100), corresponding
to 121 nM Ca 2+ . Superfusing the cells with a calcium-free
solution induced a nominal change in ratio from a mean resting
value of 1.19 ± 0.04 to 1.13 ± 0.06 (not significant, n = 6).
The following agents were added to the cells but had no
reproducible effect on [Ca2+],: 1 mM dibutyryl cAMP, 1100 |iM forskolin, 100 )iM isoproterenol, 100 p.M propranolol,
100 |iM phentolamine, 1 mM acetylcholine, 1-100 |lM
carbachol, 1 (iM oestradiol and 1 nM progesterone. However,
the addition of ATP to the solution superfusing the cells
induced a rapid transient increase in intracellular calcium (n =
32; see Figure 3A). A series of 18 dose-response experiments
with concentrations of ATP ranging from 100 nM to 1 mM
showed that the maximal change in [Ca2+]j was obtained using
100 |iM ATP (see Figure 4A).
In a group of seven experiments, 100 pM ATP was applied
in the absence of extracellular calcium, inducing a change in
the calcium ratio of 1.29 ± 0.42; if extracellular calcium was
returned whilst still in the presence of ATP, a second calcium
transient ensued with a change in calcium ratio of 1.67 ±
0.48, significantly greater than the first peak (P = 0.019; see
Figure 3B). In other words, this response was dependent on
the presence of both intracellular and extracellular calcium.
To investigate the nature of the purinergic receptor, comparative studies were carried out using 100 uJvl ATP, 100 |iM
ADP and 100 \LM UTP. The change in ratio was 2.20 ± 0.20
for ATP, 1.80 ± 0.27 for UTP and 1.53 ± 0.13 for ADP
(Figure 4B).
Effect of ATP on transepithelial potential difference
ATP to a final concentration of 100 |iM was added to the
basal side of the cells grown on collagen filters and mounted
in the Ussing chamber. A transient rise in the potential
difference to a peak of 1.52 ± 0.20 mV (n = 6) was observed,
which rapidly returned to resting levels (Figure 5).
215
C J.Dickens et aL
Discussion
We have shown that epithelial cells derived from the human
Fallopian tube may be isolated by enzymatic digestion and
grown in primary culture. The cells assume confluence after
3-7 days. Immunocytochemical analyses demonstrated that a
pure population of epithelial cells had been isolated and that
the cells formed extensive desmosome junctions in culture. A
small but reproducible transepithelial potential difference was
maintained, as well as an asymmetric distribution of glucose
disappearance and lactate appearance, confirming earlier data
in the rabbit (Edwards and Leese, 1993) and human (Leese
et aL, 1994); the latter data were obtained for culture over
successive 48 h periods. Together, these findings provide good
evidence that the cells form a polarized layer in culture.
Although me potential difference measured across the cell
layer was low, suggesting that the cells form a leaky epithelium
in culture, as in vivo (Leese, 1988), it was of the same order
as that reported by Leung et aL (1995; -1.5 mV) and Cox and
Leese (1995; -1.0 mV) for polarized mouse and bovine oviduct
epithelial cells respectively.
The method for isolating oviduct epithelium yielded a
population of cells in which the mean resting [Ca 2+ ]; varied
significantly with respect to the day of culture; therefore
comparative studies were carried out on cells from the same
day of the same culture. The removal of extracellular calcium
had a negligible effect on [Ca2+]i, indicating that the cells
were not dependent on the leakage of Ca 2+ across the plasma
membrane to maintain a stable resting level.
Out of a wide range of agonists tested, only the addition of
extracellular ATP had an effect; it induced a large transient
increase in [Ca2+]i that rapidly returned to resting concentrations. Several reports have been published recently showing
diat extracellular ATP induces intracellular calcium transients
in human (Dickens and Leese, 1994a; Hill et al., 1994), bovine
(Cox and Leese, 1993, 1994, 1995), mouse (Leung et aL,
1995) and primate (Villalon et aL, 1995) oviduct epithelial
cells. A similar response to ATP has been reported in a wide
variety of cell types (for reviews see Gordon, 1986; Dubyak
and El-moatassim, 1993): human airway epithelium (Mason
et aL, 1991), rabbit airway epithelium (Hansen et al., 1993)
and bovine aortic endothelial cells (Lynch et aL, 1992). The
use of various purinergic agonists revealed the following order
of potency: ATP > UTP > ADP, a pattern which most closely
resembles a P ^ receptor (Dubyak and El-moatassim, 1993).
The response to ATP was derived from both intracellular and
extracellular sources, suggesting that the ATP has a dual effect:
on channels in the plasma membrane as well as on intracellular
calcium stores. This differs from that generally accepted
for P ^ receptors, which usually stimulate calcium release
exclusively from
intracellular stores (Dubyak and
El-moatassim, 1993).
The addition of extracellular ATP induced a transient
increase in the potential difference that rapidly returned to
resting concentrations over a similar time course to the calcium
response. This suggests that the change in potential difference
could be related to [Ca 2+ ]; directly, i.e. the movement of
calcium across the cell membrane caused the change in
216
potential difference across the cells, or perhaps indirectly
through calcium-gated potassium channels, whereby a change
in [Ca 2+ ], can alter the gating characteristics of a K + channel
and so cause a change in the potential difference.
The physiological role of this response in the oviduct is
unclear. Within the Fallopian tube, in vivo, extracellular ATP
could come from a number of sources, such as release on the
breakage of intact cells, exocytotic release from secretory
granules or the release of cytosolic ATP through plasma
membrane pores or channels. Extracellular ATP increases the
secretion of mucin from cultured goblet cells from the airways,
and the ciliary activity of epithelial cells derived from frog
palate (Dubyak and El-moatassim, 1993). These observations
provide clues as to the possible action in the oviduct. Cumulus
cells, spermatozoa or leukocytes present in the oviduct lumen
could release ATP themselves, or stimulate the release of ATP
from the tubal epithelium. This in turn, via changes in [Ca 2+ ]j,
could lead to an increased exocytosis of secretory granules
into the lumen of the tube and/or an increase in ciliary activity.
These actions could benefit the processes of sperm capacitation,
oocyte maturation, fertilization or early embryo development
by the provision of appropriate secretory products.
Acknowledgements
We thank S.D.Maguiness and A.Palmer for obtaining patient consent
and supplying Fallopian tubes. This work was supported by the UK
Medical Research Council.
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Received on August 1, 1995; accepted on September 28, 1995
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