Original Paper
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Acta Biochim Biophys
Sin 2008, 40: 864-872
doi:10.1111/j.1745-7270.2008.00469.x
Functional expression of cystic fibrosis transmembrane conductance
regulator in rat oviduct epithelium
Minhui Chen1, Jianyang Du1, Weijian Jiang3, Wulin Zuo1, Fang Wang1, Manhui Li1, Zhongluan Wu1, Hsiaochang Chan2*, and Wenliang Zhou1*
1 School of Life Science, Sun Yat-sen
University, Guangzhou 510275, China
2 Epithelial Cell Biology Research Center,
Department of Physiology, Faculty of Medicine, Chinese University of Hong Kong,
Shatin, NT, Hong Kong, SAR
3 Department of Pharmacology, Zhongshan
School of Medicine, Sun Yat-sen University, Guangzhou 510080, China
Received: May 06,
2008
Accepted: August
06, 2008
This work was
supported by grants from the National Natural Science Foundation of China (No.
30770817), the the National Basic Research Program of China (Nos. 2006CB504002
and 2006CB944002), and the South China National Research Center for Integrated
Biosciences in Collaboration with Zhongshan University (No. 008)
Corresponding
authors:
Wenliang Zhou:
Tel/Fax, 86-20-84110060; E-mail, [email protected]
Hsiaochang Chan:
Tel, 852-26961105; Fax, 852-26037155; Email, [email protected]
The aim of this study was to investigate
the functional expression of cystic fibrosis transmembrane conductance
regulator (CFTR) with electrophysiological and molecular technique in rat
oviduct epithelium. In whole-cell patch clamp, oviduct epithelial cells
responded to 100 ?M 8-bromoadenosine 3‘,5‘-cyclic
monophosphate (8-Br-cAMP) with a rise in inward current in Gap-free mode, which
was inhibited successively by 5 ?M CFTR(inh)-172, a CFTR specific
inhibitor, and 1 mM diphenylamine-2-carboxylate (DPC), the Cl– channel blocker. The cAMP-activated current
exhibited a linear current-voltage (I-V) relationship and time- and voltage-independent
characteristics. The reversal potentials of the cAMP-activated currents in
symmetrical Cl– solutions were close to the Cl– equilibrium, 0.5±0.2 mV (n=4). When
Cl–
concentration in the bath solution was changed from 140 mM to 70 mM and a
pipette solution containing 140 mM Cl– was used, the reversal potential shifted
to a value close to the new equilibrium for Cl–, 20±0.6 mV (n=4), as compared with
the theoretic value of 18.7 mV. In addition, mRNA expression of CFTR was
also detected in rat oviduct epithelium. Western blot analysis showed that CFTR
protein is found in the oviduct throughout the cycle with maximal expression
at estrus, and immunofluorescence and immunohistochemistry analysis revealed
that CFTR is located at the apical membrane of the epithelial cells. These
results showed that the cAMP-activated Cl– current in the oviduct epithelium was
characteristic of CFTR, which provided direct evidence for the functional
expression of CFTR in the rat oviduct epithelium. CFTR may play a role in
modulating fluid transport in the oviduct.
Keywords oviduct; cystic fibrosis
transmembrane conductance regulator (CFTR); patch clamp; estrus cycle
Possessing complex physiological function, the oviduct is an
important part of the female reproductive system. It is well known that oviduct
epithelial cells secret a variety of nutrient substances and factors, which
provide an appropriate environment for a series of reproductive events,
including capacitation of sperm, maturation of oocyte and embryo development
[1,2].The mammalian oviduct is capable of active fluid secretion from the
blood into the lumen that is driven by electrogenic Cl– secretion across the oviduct epithelium [3]. Cystic fibrosis
transmembrane conductance regulator (CFTR) plays a critical role in the
regulation of ion transport in a number of exocrine epithelia, including the
lungs, intestine, pancreas and sweat gland duct [4–7]. Although CFTR
mRNA was detected in murine oviduct and cystic fibrosis (CF) mouse oviduct
exhibited defective cAMP-mediated Cl–
secretion [8–10], no direct evidence of the electrophysiological characterization
of CFTR as a cAMP-activated Cl– channel in
the oviduct has been provided with patch clamp technique. Therefore, one of the
aims of our present study was to investigate the electrophysiological
properties of CFTR in rat oviduct epithelium using the
whole-cell patch clamp technique. Additionally, we investigated the cyclic variations in the
expression of CFTR in rat oviduct, as the number of spermatozoa migrating
through the oviduct is significantly higher in estrus than in other stages
[11]; the high migration rates may result from fluid accumulation, which is
highest during estrus [12]. CFTR expression may regulate uterine fluid
production to facilitate sperm transport in different stages. We examined the
cyclic variations using Western blot analysis.
Materials and Methods
AnimalsImmature and mature female Sprague-Dawley rats were purchased from
the Animal Center of Sun Yat-sen University (Guangzhou, China). Animals were
housed and fed according to the guidelines of the Sun Yat-sen University
Animal Use Committee; all procedures were approved prior to each experiment.
Animals were kept in a room with a constant temperature (20 ?C) with a 12L:12D
photoperiod and were allowed to access food and water ad libitum.
Medium and drugs Hanks balanced salt solution, penicillin/streptomycin, Dulbeccos
modified Eagles medium/F12 (DMEM/F12), 0.25% trypsin and SuperScript One-Step
reverse transcription-polymerase chain reaction (RT-PCR) with PlatinumTaq were
purchased from Invitrogen and Gibco (Carlsbad, USA). Fetal bovine serum was
from Hyclone (Logan, USA). Collagenase, 8-bromoadenosine 3‘,5‘-cyclic
monophosphate (8-Br-cAMP), CFTR inhibitor(inh)-172 and
diphenylamine-2-dicarboxylic acid (DPC) were purchased from Sigma (St. Louis,
USA). The primers for CFTR and glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) were synthesized by the Sangong Company (Shanghai, China).
Culture of rat oviduct epitheliumImmature female Sprague-Dawley rats weighing 100–120 g were
killed by CO2 inhalation. Their lower abdomens were opened, and the oviducts were
isolated and microdissected under sterile conditions to remove fat and
connective tissues. The tissues were cut into small segments, incubated in
0.25% (W/V) trypsin for 30 min at 37 ?C and then in 1 mg/ml
collagenase I for 10 min at 37 ?C with vigorous shaking (150 strokes/min). The
cells were separated by centrifugation (800 g, 5 min). The pellets were
resuspended in DMEM/F12 containing 10% fetal bovine serum, penicillin (100
IU/ml), and streptomycin (100 mg/ml). The cell suspension was incubated at 37 ?C in 95% O2/5% CO2.
Immunofluorescence of cytokeratinParaformaldehyde-fixed primary rat oviduct epithelial cells were
washed in phosphate-buffered saline (PBS) and incubated with 1% bovine serum
albumin (BSA) for 15 min before incubation with the primary mouse anti-rat
cytokeratin antibody (Cat. No. BM0030; BOSTER, Wuhan, China) at room
temperature for 90 min. After three washes with PBS, cells were incubated for
60 min with the secondary anti-mouse IgG-fluorescein isothiocyanate (FITC)
conjugate (BOSTER) in the dark, followed by three washes with PBS. Cells were
mounted onto glass slides using a 1:1 mixture of Vectashield medium (Vector
Laboratories, Burlingame, USA) and 0.3 M Tris solution (pH 8.9). Cells were
then viewed using TCS SP2 Confocal Imaging System (Leica Microsystems,
Wetzlar, Germany).
Whole-cell patch clamp recording After 2 d of culture, the rat oviduct epithelial cells were used for
patch clamping. The cell cultures were incubated in Ca2+-free
Dulbeccos PBS solution containing 1 mM ethylene glycol tetraacetic acid (EGTA)
for 10–15 min to separate the cells. The cells were then moved to a 1 ml
chamber that was fixed on a X-Y axis stage of an Olympus BX51WI immersing lens
microscopy system (Tokyo, Japan). Recording was performed at room temperature
using a Multiclamp700A amplifier and the Digidata 1322 series interface (AXON
Instrument, Foster City, USA). Signals were filtered at 10 kHz. Pclamp 9.0
software system (AXON Instrument) was used for data recording and analysis. Using a horizontal puller P-97 (Sutter Instrument, Novato, USA) and
glass pipettes, we pulled patch pipettes (1.2 mm outside diameter and 0.5 mm
inside diameter). Ionic current was recorded using the conventional whole-cell
patch clamp technique. The pipettes were filled with a solution containing 120
mM CsCl and 20 mM tetraethylammonium-Cl (pH adjusted to 7.2 with CsOH). The
bath solution contained 135 mM NaCl, 1.2 mM Na2HPO4, 2 mM MgCl2, 10 mM HEPES and 10 mM glucose
(pH 7.4). The low Cl– (70 mM) bath solution
contained 69 mM Na-glutamate, 66 mM NaCl, 1.2 mM Na2HPO4, 2 mM MgCl2, 10 mM HEPES and 10 mM glucose
(pH 7.4). The resistance of the patch pipettes was approximately 2–7 MW. Positive
pressure was applied in the patch pipette before it was immersed in the bath
solution. When the tip of the pipette attached to the cell surface, pressure
was withdrawn, and then a giga-seal between the pipette and cell membrane
formed normally. After being sucked from the pipette, the whole cell
configuration was confirmed. The oviduct cell was held at its resting potential
–30
mV in the episodic recording mode, and the voltage was clamped from –120 mV to +100
mV in 20 mV increments. In the Gap-free recording mode, the cells were held at –70 mV throughout
the period of recording. The pipette potential and liquid junction potential
were auto-compensated by the Multiclamp700A amplifier before the electrode
touched the cell.
Reverse transcription-PCRTotal RNA was isolated using TRIzol reagent (Life Technologies,
Gaithersburg, USA), and 2 mg was used to amplify CFTR using Superscript II One-Step RT-PCR with
Platinum Taq (Invitrogen). The primers for CFTR were: sense 5?-GACAACATGGAACACATACCTTCG-3? (corresponding
to nucleotides 2514–2537) and antisense 5?-TCTCGTTCGTTTCACAGTCGGTGAG-3? (corresponding
to nucleotides 2771–2747), which yielded a PCR product of 258 bp. The primers for GAPDH
were sense 5?-ACTGGCGTCTTCACCACCAT-3? and antisense 5?-TCCACCACCCTGTTGCTGTA-3?, which yielded
a PCR product of 300 bp. Reactions were carried out with the following
parameters: denaturation at 94 ?C for 1 min, annealing at 60 ?C for 45 s, and
extension at 72 ?C for 1 min, for a total of 45 cycles. PCR products were
analyzed by agarose gel electrophoresis and visualized by staining with
ethidium bromide.
Protein extraction and Western blot analysisOviduct tissue from mature female rats at different estrus stages
were cut into small pieces and disrupted with a Tenbroeck
tissue grinder in 3 ml M-PER Mammalian Protein Extraction
Reagent, and complete protease inhibitors (Roche Applied
Science, Indianapolis, USA). Homogenates were
centrifuged for 5 min at 14,000 g at 4 ?C. The supernatant was
collected and the protein concentration was determined with BCA protein assay
kit (Pierce Biotechnology, Rockford, USA). Protein extracts were aliquoted and
stored at –80 ?C. Protein was loaded onto 8% Tris-glycine
polyacrylamide gels (Cambrex, Rockland, USA). After SDS-PAGE
separation, proteins were transferred onto an Immun-Blot
polyvinylidene difluoride membrane (Bio-Rad Laboratories, Hercules, USA).
Membranes were blocked in Tris-buffered saline (TBS)
with 5% non-fat dry milk and then incubated overnight at 4 ?C with rabbit anti-CFTR antibody (Cat. No. ACL-006; Alomone Labs,
Jerusalem, Israel) diluted (1:1000) in TBS with 2.5% non-fat dry milk. After being washed four times in TBS
with 0.1% Tween-20, membranes were incubated with donkey
anti-rabbit IgG antibody conjugated to horseradish peroxidase
(Cell Signal Technology, Beverly, USA) for 1 h at room temperature. After an
additional four washes, bindings were detected using
Western Lightning chemiluminescence reagent (Perkin Elmer Life
Sciences, Boston, USA). The gray scales on the bands of
CFTR protein were normalized to that of b-actin, which was used as
the loading control for the protein sample. The ratio meant the different
expression levels of CFTR in other three stages as compared with that of
proestrus but not simply CFTR/b-actin.
Immunofluorescence of rat oviduct frozen sectionFresh isolated oviducts were embedded in Tissue-Tek OCT compound
4583 (Sakura Finetek USA, Torrance, USA), mounted on a cutting block and frozen
at –27
?C. Sections (5 mm) were cut by a Reichert-Jung 1800 Frigocut cryostat (Leica
Microsystems, Bannockburn, USA) and stored at 4 ?C until use. Sections were
rinsed in PBS for 5 min and treated with 0.05% Triton X-100 for 1 min. Sections
were subsequently washed three times in PBS for 5 min each, incubated for 15
min in 1% (W/V) BSA in PBS with 1% sodium azide to prevent
non-specific staining and then incubated in the anti-CFTR antibody at room
temperature for 120 min. The sections were incubated with PBS alone, which used
as negative control. After three 5 min PBS washes, the FITC-conjugated
secondary antibody was applied for 1 h at room temperature, and the slides were
then rinsed again in PBS three times for 5 min. Slides were mounted in a 1:1
mixture of Vectashield medium and 0.3 M Tris solution (pH 8.9), and then viewed
under the TCS SP2 Confocal Imaging System.
Immunohistochemistry of rat oviduct paraffin sectionParaffin-embedded 5 mm sections of paraformaldehyde-fixed oviducts were dewaxed and
hydrated. Antigen was retrieved by treatment in 0.01 M citrate buffer (pH 6.0)
for 20 min in a sub-boiling water bath. After rinsing with phosphate buffered
saline Tween-20 (PBST), sections were incubated in normal blocking serum for 30
min and then with the rabbit anti-rat CFTR antibodies diluted 1:100 with
diluting buffer (PBS with 1% BSA, 0.1% gelatine and 0.05% sodium azide) at 4 ?C
overnight. Sections were washed three times with PBST and incubated with
biotinylated secondary antibody at 37 ?C for 30 min. After being washed three
times with PBST, the sections were incubated with the horseradish peroxidase
conjugated donkey anti-goat IgG antibody for 30 min and finally washed three
times with PBST. Visualization was achieved by immersing sections in a peroxidase
substrate solution 3,3?-diaminobenzidine-tetrachloride (K4011; DakoCytomation, Glostrup,
Denmark) until desired stain intensity was reached. Slides were rinsed with
pure water for 5 min, counterstained with Harris hematoxylin (Sigma),
dehydrated, and mounted for observation. Negative controls were incubated with
antigen of primary antibody.
DNA sequencingThree 258 bp CFTR PCR products (from separate RNA isolations) were
cloned into the pCR 2.1 vector (Invitrogen) using T4 DNA ligase, and they were
then used to transform “super competent” Escherichia coli
cells, which were grown for 1 d. The DNA was isolated using an isolation kit
(Promega, Madison, USA), and the plasmid was sequenced on the ABI PRISM
sequencer model 2.1.1 (Foster City, USA) and analyzed by BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi).
All three sequences obtained were 100% homologous to region 2514–2771 of the rat CFTR
gene.
Statistical analysis Results were expressed as mean±SEM, and n indicated the number
of experiments. Data were analyzed using Students t-test. A P
value less than 0.05 was considered to be statistically significant.
Results
cAMP elicited whole-cell currents in cultured oviduct epithelial
cellsPath clamp recordings were applied on rat oviduct epithelial cells
after two days of primary culture, which were confirmed by immunofluorescence
staining of cytokeratin (Fig. 1), to see whether oviduct epithelial
cells functionally exhibit CFTR conductance. The current recordings were obtained
using pipette and bath solutions containing symmetrical Cl– concentrations (containing intracellular 140 mM Cl– and extracellular 140 mM Cl–, respectively). Under a continuous recording with a –70 mV holding
potential, bath application of 8-Br-cAMP (100 mM) elicited inward
whole-cell currents [Fig. 2(A)]. Series steps of voltage-clamp
stimulation from –120 mV to +100 mV at 20 mV increments were applied to the cell
before application of 8-Br-cAMP [Fig. 2(B)]. The cAMP-activated
whole-cell currents demonstrated that they were independent of the stimulation
time [Fig. 2(C)].
Effect of CFTR(inh)-172 and DPC on the cAMP-stimulated Cl– conductanceThe currents were maximally activated by cAMP and were then
suppressed by subsequent application of 5 mM CFTR(inh)-172, a specific
CFTR channel blocker, and 1mM DPC, the Cl– channel blocker [Fig. 2(A)]. The whole-cell membrane current
after cAMP stimulation followed by 5 mM CFTR(inh)-172, 5 mM CFTR(inh)-172 and 1 mM
DPC, and 1 mM DPC alone, respectively, in response to square voltage pulses
from a holding potential at –30 mV to a potential between –120 mV and +100 mV in 20 mV
increments [Fig. 2(DF)]. The mean percent inhibitions of CFTR(inh)-172
and DPC were 71.5% (n=4) and 75.2% (n=4), respectively. The current-voltage
(I-V) relationship obtained from the cell at rest, cAMP stimulation alone, cAMP
stimulation followed by CFTR(inh)-172 or cAMP stimulation followed by both
CFTR(inh)-172 and DPC is shown in Fig. 3.
Demonstration of the involvement of Cl– in cAMP-induced currentsThe I-V relationship demonstrated that the cAMP-activated
whole-cell currents had a linear relationship with gradually increased
clamping voltages [Fig. 4(A)]. The reversal potentials of the
cAMP-induced currents in symmetrical Cl– solutions were close to the Cl– equilibrium, 0.5±0.2 mV (n=4). In order to further identify
whether the cAMP-activated whole-cell currents were mediated by Cl–, and not through any non-selective conductance, we performed
experiments in which the Cl– concentration
in the bath was reduced from 140 mM to 70 mM, while a pipette containing 140 mM
Cl– was used. The reversal potential shifted to a
value close to the new equilibrium for Cl–, 20±0.6 mV (n=4), as compared with the theoretic value of
18.7 mV calculated according to the Nernst function and based on the present
experimental conditions [Fig. 4(B)]. The results suggested that Cl– mediates currents activated by extracellular cAMP.
Identification of CFTR in epithelial cells of the oviduct by RT-PCRRT-PCR was used to study the mRNA expression of CFTR in the
oviduct (Fig. 5). The PCR products of CFTR (258 bp) were
amplified by RT-PCR from RNA isolated from the oviducts epithelium, and the
epididymis was used as positive control. No product was detected from choroid
plexus, which was used as a negative control. GAPDH standard product was
expressed in all the tissues. These products were confirmed by sequencing (data
not shown).
Detection of oviduct CFTR protein expression by Western blot
analysisThe protein expression of CFTR in the oviduct at different estrus
stages was also examined by Western blot analysis. The CFTR antibody raised
against a peptide (C)KEETEEEVQDTRL, corresponding to amino acid residues 1468–1480 of
cytoplasm, the C-terminal part of human CFTR. The major immunoreactive band of
CFTR and b-actin displayed a molecular mass of about 170 kDa in rat oviduct
and 42 kDa in epididymis as positive control [Fig. 6(A,B)]. In addition,
b-actin
was detected in choroid plexus as a negative control, but CFTR was not
detected. CFTR protein was detected in the oviduct throughout the estrus cycle
and in the expression of estrus and metestrus. However, it was not detected in
diestrus, and it was significantly lower in the expression of proestrus [Fig.
6(C)].
Immunolocalization of CFTR in oviductTo investigate the location of CFTR in oviduct, we stained sections
from rat oviduct using immunofluorescence and immunohistochemistry technique.
The apical surface and cytoplasm of the epithelial cells of rat oviduct were
highly stained with CFTR antibody [Fig. 7(A), Fig. 8(A)], and
there was no immunoreactivity in negative control [Fig. 7(D), Fig.
8(B)]. Bright field and converged images of CFTR immunofluorescent stained
rat oviduct sections were shown in [Fig. 7(B)] and [Fig. 7(C)],
respectively.
Discussion
The oviduct provides an electrolyte environment for ovum pickup,
sperm transport, ovum capacitation, sperm capacitation, ovum fertilization,
zygote development and zygote transport [13–15], but the regulation and
mechanism of fluid movement across the epithelium remain poorly understood.
The present study demonstrated that cAMP stimulated an inward current in
cultured rat oviduct epithelia in whole-cell patch clamp. Several lines of evidence
suggested that the cAMP-activated Cl–
current in the oviduct epithelial cell was characteristic of CFTR [6,16–20]. First,
elevating cellular cAMP stimulates whole-cell Cl–-selective conductance and exhibited time- and voltage-independent
characteristics. Second, this conductance has a linear I-V relationship. Third,
the cAMP-activated Cl– current is suppressed
in a voltage-independent manner by 5 mM CFTR(inh)-172, a specific CFTR channel
blocker [21,22]. In support of these functional data, the detection of CFTR mRNA and protein immunolocalization in rat oviduct confirm earlier
reports [8,23], which suggested the presence of this channel in the
oviduct epithelium. As a cAMP-activated Cl– channel,
CFTR plays a critical role in electrolyte and fluid secretion. When it was
stimulated by intracellular cAMP, the channel opened and allowed Cl– efflux. In addition, the estrus cycle-dependent protein expression
of CFTR in the present study was consistent with observed cyclic changes in
CFTR mRNA expression [8]. Moreover, estrogen may function as a physiological
regulator of CFTR in the oviduct [23]. Therefore, enhanced expression of CFTR
at estrus may result in a higher rate of chloride secretion and thus greater
fluid accumulation.The presence of functional CFTR in rat oviduct epithelial cells
revealed in our study should help us better understand its role in oviduct
secretion and in the pathophysiology of cystic fibrosis. Previous reports
indicated that females with CF have reduced fertility probably due to thick,
dense cervical mucus that presents a barrier for sperm penetration [24].
However, CFTR mutant in endometrium and oviduct, which results in abnormal
electrolyte secretion [25,26], may be an important cause of infertility in CF
female patient.In summary, this study showed that the cAMP-activated Cl– current in the oviduct epithelium was characteristic of CFTR, which
provided direct physiological evidence for the expression of CFTR in the rat
oviduct epithelium. The present results also suggested the capability of the
oviduct to secrete chloride ion at different stages of the estrus cycle. Cyclic
variations in the expression of CFTR likely alter the composition of oviductal
fluid to permit successful reproductive events at different times. The
functional expression of CFTR indicates that CFTR may play a role in
modulating fluid transport in the oviduct.
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