1 Department of Obstetrics and Gynaecology, Third Hospital, Peking University, Peking, 100083 China
2 To whom correspondence should be addressed. Email: chenguian{at}bjmu.edu.cn
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Abstract |
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Key words: CFTR/chloride channel/human endometrium/menstrual cycle
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Introduction |
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Materials and methods |
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Human endometrial tissue
The endometrial specimens were all taken from patients, aged 2345 years old, attending the in- and out-patient clinics at our Department, between January 1999 and March 2002. Patients were undergoing hysterectomy for uterine myoma or endometriosis, or endometrial biopsy before assisted reproductive techniques, with no sign of endometrial disease. The present study was approved by the Ethics Committee at Peking University Medical Center. All patients had regular menstrual cycles (2835 days), and none were given hormonal therapy for at least 3 months before surgery. The specimens were fixed in 4% paraformaldehyde solution or immediately frozen in liquid nitrogen. Fresh samples in the proliferative phase were isolated for cell culture. The cycle dating was established by the menstrual history and histological examination according to the criteria of Noyes et al. (1975).
Culture of human endometrial epithelial cells
Human endometrial cell culture was performed according to a slightly modified method of Liu and Teng (1979). Briefly, the endometrial tissues were digested by collagenase (type II, 1 mg/ml, Gibco, New York, NY) in RPMI 1640 medium (Gibco) in a 37°C water bath for 1 h with shaking. After enzyme digestion, the suspension consisted of single stroma and fragments of epithelial glands. These two populations were separated by differential sedimentation at unit gravity. The collected epithelial cell clamps were cultured on coverglasses in Petri dishes (35 mm, Corning, NY). RPMI 1640 medium containing fetal bovine serum (FBS; 10%, Hyclone, UT), insulin (2 U/100 ml), penicillin and streptomycin (100 U/ml) was changed after 24 h. 17
-Estradiol (3.6 x 108 mol/l; Sigma, St Louis, MO) and progesterone (106 mol/l; Sigma) were added to the medium (2% FBS) according to experimental requirement. The cultured cells were subjected to immunohistochemical and in situ hybridization analysis.
Immunohistochemical detection of CFTR in human endometrium
Paraformaldehyde-fixed and paraffin-embedded human endometria were cut into 5 µm sections, then deposited on glass slides coated with 3-amino-propyltriethoxy silane (APES; Sigma). The sections were then dewaxed with xylene, rehydrated through sequentially graded ethanol and rinsed with phosphate-buffered saline (PBS). They were subsequently incubated in methanol containing 3% H2O2 for 10 min at room temperature to inhibit the endogenous peroxidase activity. Non-specific staining was blocked by a 30 min incubation with blocking serum. The specimens were then incubated at 4°C overnight with polyclonal goat anti-human CFTR antibody (1:100 dilution; Santa Cruz Biotechnology, Santa Cruz, CA) which recognizes a CFTR epitope at the N-terminus, followed by the incubation with biotinylated secondary antibody (Maixin Bio, Fuzhou, China). The CFTR antigen was visualized by using streptavidinperoxidase and 3,3'-diaminobenzidine (DAB; Sigma). The sections were counter-stained with Harris' haematoxylin. Negative controls were performed by replacing the primary antibodies with non-immune serum.
The cultured endometrial glandular cells on coverglasses were also tested for CFTR expression by using the same immunohistochemical method after fixing in cold acetone for 10 min.
Western blot analysis
To substantiate further a cyclic dependence of CFTR protein expression in human endometria, western blot analysis was performed. The frozen human endometrial samples were lysed in a ice-cold buffer containing 50 mmol/l TrisHCl, 150 mmol/l NaCl, 100 µg/ml phenylmethylsulphonyl fluoride (PMSF), 1% Triton X-100, 0.02% sodium azide (pH 7.4). Tissue lysates were centrifuged for 10 min at 1000 g to remove non-lysed cells; the supernatants were spun again at 12 000 g for 20 min at 4°C. Aliquots of the total proteins (100 µg) were electrophoresed in an 8% SDSpolyacrylamide gel for separation, then electroblotted to a polyvinylamide difluoride (PVDF) membrane. Following transfer, the membrane was saturated with 5% skimmed milk in Tris-buffered saline (TBS) containing 0.5% Tween-20 for 2 h, and then incubated with a 1:100 dilution of the goat anti-human CFTR antibody (Santa Cruz Biotechnology) at 4°C overnight, followed by an alkaline phosphatase-conjugated antibody (1:500 dilution) for further incubation for 1 h at 37°C. The CFTR protein was finally visualized using 4-nitroblue tetrazolium chloride (NBT) plus 5-bromo-4-chloro-3-indolyl-phosphate (BCIP) solution.
In situ hybridization
The presence of CFTR mRNA in human cyclic endometria and cultured epithelial cells was detected by the in situ hybridization method described previously (Engelhardt et al., 1992). The 862 bp antisense and 978 bp sense CFTR probes were prepared from human CFTR cDNA which was subcloned into pBluescript plasmids, linearized with XbaI or HindIII, then transcribed from either the T7 or T3 promoters to generate single-stranded cRNA probes. The cRNA probes were labelled with digoxigenin (DIG) RNA labelling mix (Roche Molecular Biochemicals, Mannheim, Germany). In brief, the labelling process was carried out at 37°C for 2 h. Then, DNase I was added to remove template DNA. The riboprobes were precipitated with LiCl and ethanol, and stored in diethylpyrocarbonate (DEPC)-treated water at 80°C. To promote riboprobe entry, the tissue sections and cultured cells fixed on coverglasses were rinsed with 1 mol/l HCl for 10 min and then digested with proteinase K (Sigma) at 37°C for 30 min, and post-fixed in 4% paraformaldehyde. The hybridization was performed at 42°C overnight by using 500 ng/ml of DIG-labelled hCFTR RNA probe. After serially washed in 2x SSC containing 50% formamide, 2x SSC, 0.1x SSC at 37°C for 30 min, the sections were incubated with horse serum (1:100 dilution) for 1 h, and then with the anti-DIG antibody (1:500 dilution) conjugated with alkaline phosphatase for 1 h at room temperature. The CFTR mRNA staining was visualized by the precipitate of dark purple colour developed via the presence of NBT/BCIP solution under a light microscope.
Whole-cell patchclamp measurement
Cells were plated on coverglasses at low density. Current recordings were obtained using a patchclamp amplifier (Inbio PCII-C patch clamp, Inbio Instruments, Wuhan, China). Patch pipettes were pulled from glass on a microelectrode puller (Narishige, Japan) to a resistance of 46 M. In the whole-cell recordings, the pipette solution contained 140 mmol/l CsCl, 2 mmol/l MgCl2, 0.5 mmol/l EGTA, 10 mmol/l glucose, 2 mmol/l Mg-ATP and 5 mmol/l HEPES (pH 7.35, 298 mOsm/kg). The bath solution contained 170 mmol/l TrisHCl, 1 mmol/l MgCl2, 2.5 mmol/l CaCl2, 5 mmol/l HEPES and 10 mmol/l glucose (pH 7.4, 325 mOsm/kg). The membrane was voltage-clamped to a holding potential of 70 mV and stepped to levels between 80 and + 80 mV at 20 mV intervals. The whole-cell currents were filtered at 1 kHz, and digitized. All the patchclamp recordings were made at room temperature.
Statistical analysis
Immunohistochemical and in situ hybridization stainings in human endometrial tissues were graded visually by using an arbitrary scale (0 = absent, 1 = light, 2 = moderate, 3 = intense) by two independent observers who were blinded to clinical information. While the computerized densitometric analyses were performed by the CAMIS system (Micheal-Audi Technology, Beijing, China) to measure the presence of CFTR mRNA and protein in cultured human endometrial glandular cells. The integrated optical density (IOD) was measured according to grey scale and represents the comparative concentration of CFTR protein or mRNA in the pictures. At least five images per slide were evaluated.
Data were analysed using the WilcoxonMannWhitney U-test, analysis of variance (ANOVA) and Student's t-test where appropriate. A P-value of <0.05 was considered statistically significant.
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Results |
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Discussion |
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In this paper, the immunohistochemical analysis of CFTR antigen used a specific antibody directed against the N-terminus of CFTR. The results showed that CFTR immunostaining was detected in a few cases (one in five) in the early proliferative endometrium, but was seen in epithelial and glandular cells at the mid proliferative phase. The strong staining was found in the late proliferative and all secretory phases. Interestingly, we not only observed a CFTR signal localized on the apical surface of epithelial cells, but it was also detected in the basolateral membrane. This is the same as reported by Cohn (1991), but the possible role of CFTR in the basolateral membrane needs further study. Our data provide evidence that in human endometrial epithelium, CFTR protein exists in the uterine inner mucosal lining. Furthermore, the expression may depend on estradiol levels. Rochwerger and Buchwald (1993)
reported that estrogen stimulated CFTR mRNA and protein in rat uterine endometrial cells. Our results also found low CFTR protein expression at the late proliferative phase when estradiol rises. The western blot analysis also showed a similar pattern of phase dependency in CFTR protein expression with the expected molecular size from cyclic human endometrial tissue. To confirm this finding, we cultured human endometrial epithelial glandular cells in vitro, and found that estradiol significantly increased the contents of CFTR protein in the primary cultured cells, while progesterone alone, or together with estradiol, has no effect on CFTR production in those cultured cells. Sweezey et al. (1996)
observed progesterone inhibition of CFTR expression using the cultured pancreatic epithelial cell model. In our work, the effect of progesterone on CFTR protein expression is different between in vivo and in vitro studies. We observed that the CFTR protein synthesis induced by estradiol could be abolished by adding progesterone to the culture medium simultaneously in vitro, whereas immunohistochemistry revealed a high level of CFTR protein in the whole secretory phase endometrium, coincident with the progesterone-dominated condition. This phenomenon may account for the serial action of estradiol and progesterone during the normal menstrual cycle. Once the CFTR protein is induced by estradiol stimulation during the proliferative phase, it will be resident for a long time in the cell plasma membrane. Lukacs et al. (1993)
reported that the functional half-life of CFTR is >24 h; even after blocking protein synthesis for 72 h, the magnitude of the cAMP-activated depolarization remains at >40% of control conditions.
CFTR mRNA expression was also detected in cyclic endometrial tissues and cultured epithelial glandular cells using in situ hybridization. Significant levels of CFTR mRNA were seen in most of the late proliferative and a few of the early secretory endometrial tissues just around the ovulating period dominated by the estradiol peak, while CFTR mRNA was absent in late secretory phases under the effect of progesterone. This appearance conformed with what was seen in conditioned cell culture. The high level of CFTR mRNA was only detected in cultured epithelial glandular cells supplemented with estradiol, but not with progesterone, regardless of whether estradiol was present or not. The estradiol-upregulated and progesterone-downregulated CFTR mRNA in human endometrial epithelial cells is the same as reported in animal experiments (Rochwerger and Buchwald, 1993; Trezise et al., 1993
; Rochwerger et al., 1994
; Mularoni et al., 1995
, 1996
; Lee et al., 2001
; Chan et al., 2002
).
A disagreement is evident between the CFTR mRNA and protein expression in late secretory endometrium. The mRNA signal was negative in all four late secretory endometrial samples while continuous expression of CFTR proteins has been detected in epithelial cells. The discrepancy could be attributed to the comparatively long half-lives of CFTR protein. On the other hand, it is also possible that a lower sensitivity of our in situ hybridization methods limits our detection. Mularoni et al. (1996) detected the continuous CFTR mRNA expression in human endometrium using competitive RTPCR and indicated that there was a lower level of CFTR mRNA during the secretory phase, consistent with our result.
The characteristics of CFTR function on cultured endometrial glandular cells in estradiol-supplemented medium were studied by using the whole-cell patchclamp techinique monitoring ion channel activities. Our results first revealed the elevated Cl current in human endometrium induced by FSK, an adenylate cyclase activator, and exhibited a linear IV relationship in a time- and voltage-independent manner. We further confirmed that the cAMP-activated Cl current in cultured endometrial epithelial cells is sensitive to DPC, a chloride channel blocker. These data agreed well with CFTR features reported in other epithelial cells (Schwiebert et al., 1994; Boockfor et al., 1998
).
CFTR, as a cAMP-activated Cl channel, plays a critical role in electrolyte and fluid secretion. When it was stimulated by intracellular cAMP, the channel opened and allowed Cl efflux. The expression of functional CFTR in human endometrial epithelium in a cycle-dependent manner was thought to be associated with the composition of human uterine fluid. Twenty years ago, Casslén and Nilsson (1984) first compared the concentrations of inorganic ions between human uterine fluid and serum, and found that the concentration of potassium and calcium, but not chloride, varied cyclically, both having lower values at mid cycle than that in the proliferative and luteal phases. The permeability for cations and anions is considered an active process. The present study provided significant evidence showing the abundant CFTR mRNA and protein expressed in endometrial epithelial cells around the ovulatory period. It may facilitate sperm migration because active Cl secretion drives Na+ and fluid from plasma into the uterine lumen to produce the optimal electrolyte composition and sufficient fluid volume. This work is also the first to reveal that the CFTR protein, but not mRNA, exists in endometrial epithelium throughout the menstrual cycle except the early proliferative phase. The uterine electrolyte environment may vary with the cycle, but the regulation and mechanism of fluid movement across the epithelium remain poorly understood. Previous reports indicated that prostaglandin E2 may induce Cl secretion involved by CFTR to contribute to the higher Cl concentration during blastocyst implantation in the rat (Fong and Chan, 1998
; Deachapunya and O'Grady, 1998
). Another study of human tubal electrophysiology showed that platelet-activating factor (PAF) released by gametes or embryos could stimulate chloride ion movement across the tubal epithelium, thereby increasing the rate of production of human oviductal fluid (Downing et al., 2002
). Though little is known about the electrolyte composition of human uterine fluid during the implantation window because of the difficulty in collecting samples accurately, thick and dense uterine fluid is considered to benefit implantation (Chien et al., 2002
). However, as some cytokines, including interleukin-1
(IL-1
), can stimulate CFTR mRNA expression (Nakamura et al., 1992
; Besancon et al., 1994
; Cafferata et al., 2000
), high levels of IL-1
have been observed in women with recurrent failed embryo transfer (Inagaki et al., 2003
) presumed to produce excessive uterine fluid.
In addition, CFTR may act as a regulator of other channels and transporters including the amiloride-sensitive Na+ channel and Cl/HCO3 exchangers (Lee et al., 1999). In mouse endometrium, activation of CFTR can inhibit Na+ absorption (Chan et al., 2001
). A study by Wang et al. (2003)
demonstrated that CFTR in mouse endometrium can mediate bicarbonate secretion directly, defects in which may result in impaired sperm capacitation and decreased fertilizing capacity. Variable CFTR expression was detected in human endometrium; the absence of CFTR-mediated bicarbonate secretion, as well as the thick cervical mucus, might also account for the lower female fertility in CF. The effect of CFTR on implantation, however, is less clear.
In conclusion, in the present study, we found that CFTR mRNA and protein were localized in human endometrial epithelial cells and the amounts varied in a cyclic manner, CFTR expression in cultured glandular cells was up- and downregulated by estradiol and progesterone, respectively, and that CFTR in human endometrium functions as a cAMP-activated Cl channel. Further research into CFTR regulation may improve our knowledge about human reproductive physiology and pathophysiology, which may provide better treatments in some cases of infertility, such as defective uterine bicarbonate secretion.
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Acknowledgements |
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Submitted on March 19, 2004; accepted on August 11, 2004.
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