Departments of 1 Reproductive Biology, 2 Physiology and Biophysics, and 3 Environmental Health Sciences, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106
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ABSTRACT |
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The objective of the study was to determine the effect of retinoids on paracellular resistance across the cervical epithelium and the mechanisms involved. The experimental model was cultures of human CaSki cells on filters, which retain phenotypic characteristics of the endocervical epithelium. End points for paracellular resistance were measurements of transepithelial electrical resistance and fluxes of pyranine (a trisulfonic acid that traverses the epithelium via the intercellular space). Paracellular resistance was significantly increased in cells grown in retinoid-free medium; the effect could be blocked and reversed with all-trans-retinoic acid (tRA) and with agonists of RAR and RXR receptors but only partially with retinol. The effect of tRA was dose dependent and saturable, with a 50% effective concentration of 0.8 nM. The increases in paracellular resistance induced by vitamin A deficiency required longer incubation in retinoid-free medium than decreases in resistance induced by retinoic acid. tRA had only a minimal effect on paracellular resistance in cells maintained in regular medium. Retinoid-free medium increased and tRA decreased the relative cation mobility across CaSki cultures. Also the effects of tRA were nonadditive to those of cytochalasin D (which decreases tight junctional resistance) and additive to those of ionomycin (which decreases the resistance of the lateral intercellular space), suggesting that tRA modulates tight junctional resistance. It is concluded that vitamin A determines the degree of paracellular resistance across cervical cells by a mechanism that involves modulation of tight junctional resistance.
vitamin A; cervical mucus; intercellular lateral space; transepithelial transport; paracellular transport; permeability
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INTRODUCTION |
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THE MAIN FUNCTION of the endocervical epithelium is to regulate secretion of fluids and solutes from the blood into the cervical canal, i.e., the cervical mucus. Abnormal secretion of cervical mucus may result in infertility and may affect a woman's health (12).
Vitamin A and related ligands, the retinoids, are important regulators of female reproductive tract epithelia. Studies in vivo indicate that vitamin A deficiency leads to dryness of mucus membranes and, in particular, of the endocervix (8). In addition, the simple columnar epithelium of the endocervix undergoes squamous metaplasia in response to vitamin A deficiency, followed by keratinization (10). Treatment of human ectocervical cells (14) or ectocervical cells immortalized with human papillomavirus 16 (2) with nanomolar concentrations of natural or synthetic retinoids suppresses squamous differentiation, reduces expression of cytokeratins K5, K6, K13, K14, K16, and K17, which are markers of stratifying epithelium, and increases cytokeratins K7, K8, and K19, which are markers of the simple epithelium (6). These observations suggest that vitamin A and the retinoids direct the differentiation of cervical cells and sustain the endocervix as a simple columnar epithelium. However, the above observations do not explain the specific role of vitamin A in the regulation of mucus secretion and the mechanisms involved, because stratification does not significantly affect transepithelial transport (16).
Secretory epithelia such as the endocervix control transport via the cells (transcellular route) or via the intercellular space (paracellular route). Movement of molecules from the blood into the lumen via the transcellular route is restricted by the plasma membrane. Because the resistance of plasma membranes to passive movement of molecules is 104- to 106-fold higher than that of the paracellular pathway (25), the latter determines the overall permeability properties of the epithelium. Movement of molecules in the intercellular space is restricted by the resistance of the tight junctional complex (RTJ) and by the resistance of the lateral intercellular space (RLIS) between the tight junction and the basement lamina. The regions of the tight junction that are considered a high-resistive element are areas where plasma membranes of neighboring cells come into contact and occlude effectively the intercellular space (5). In contrast, the RLIS is considered a low-resistive element and is determined by the proximity of the plasma membranes of neighboring cells and the length of the intercellular space from the tight junctions to the basal lamina.
It was believed that transport via the intercellular space is passive and is influenced by hydrostatic or hypertonic gradients between blood and lumen that dilate the intercellular space and decrease the RLIS. More recent studies showed that the RLIS and the RTJ can be regulated (17). In view of the observations that vitamin A regulates lubrication of the female genital tract in vivo, we hypothesized that retinoids modulate paracellular resistance across the cervix by regulating the RLIS or RTJ.
Until recently, most of our knowledge about regulation of transcervical transport came from studies conducted in vivo by sampling the secretions in the genital tract (20). Those studies provided information about hormonal regulation of cervical mucus production, but they failed to provide data about the cellular and molecular mechanisms involved. Part of the difficulty in studying transcervical transport phenomena has been lack of an appropriate model to study directly fluid and solute transport across the cervical epithelium.
Recently, we described an in vitro model of human cervical epithelial cells on permeable support. These cells retain phenotypic characteristics of the endocervical epithelium and can be used for flux studies and electrophysiological measurements (18). The cultured cervical epithelium is characterized by a relatively high permeability to probes that traverse the epithelium via the intercellular (paracellular) space and by a relatively low transepithelial electrical resistance (RTE) (16). Interestingly, the permeability characteristics of the cultured human cervical cells are similar to those of the endocervical epithelium in women. For example, during the proliferative phase of the cycle, glucose secretion in the cervical mucus is 2 µmol/h (13). Similarly, the subluminal-to-luminal flux of an 0.18 kDa-molecule (e.g., glucose) with cis concentration of 5 mM (the physiological extracellular concentration of glucose) across 20 cm2 [the estimated surface area of the human endocervix (21)] is 7 µmol/h (18). The similarities in the permeability characteristics between the cultured cervical epithelium and the endocervix suggest that the paracellular pathway is also the main route for transport of fluids and solutes in vivo. We used this novel experimental system to study the effects of retinoids on paracellular resistance and the mechanisms involved.
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METHODS |
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Cell culture techniques. Cell culture techniques have been described previously (15, 18). The experimental model was CaSki, a stable line of cells that expresses phenotypic markers of the endocervix (15). CaSki cells form a confluent and polarized epithelium on a permeable support, and the cells express tight junctions that occlude effectively the intercellular space (13, 15, 18, 19). The growth of CaSki cells on filters depends on the mode of plating: when plated at low density, cells will reach confluency within 1-2 wk and will form a monolayer; when plated at high density, e.g., the present study, cells will remain attached on the filter as a bi/trilayer (15). Cells were grown and subcultured in a medium enriched with fetal calf serum, as described elsewhere (15, 18). In some experiments, cells were shifted to a medium enriched with fetal calf serum that was delipidized (retinoid-free medium) to remove vitamin A. Serum delipidization was done as described with some modifications (26): fetal calf serum was added to a cooled (4°C) mixture of ethanol and acetone (1:5:5, vol/vol/vol) and mixed slowly for 4 h at 4°C. The solution was brought to room temperature and filtered twice. The protein residue was washed with ether and dried, resuspended 2:1 (vol/vol) in water, frozen, and lyophilized twice for 48 h. The residue was resuspended in water to yield a protein concentration similar to the untreated serum, and aliquots of 10× phosphate-buffered saline (pH 7.4) were added to adjust salts to isotonic conditions. In experiments using retinoid-free medium, cells were propagated on regular tissue culture plates for one or two passages (5-10 days) and then plated on collagen-coated Anocell filters (15, 18). Cells maintained in retinoid-free medium for >2 wk and propagated for three or more passages had a slow rate of proliferation, and we were unable to obtain a high enough number of cells to plate on parallel filters for experiments and controls. Levels of RTE and permeability to pyranine (Ppyr; see below) were determined 2-6 days after cells were plated on filters. Drugs were added from 1,000× stock solutions; vehicles had no effect. Cells treated with retinoids were maintained in the dark.
Changes in paracellular permeability. Changes in paracellular permeability were determined in terms of changes in Ppyr and as RTE. Before experiments, filters containing cells were washed three times and preincubated for 15 min at 37°C in a modified Ringer buffer composed of (in mM) 120 NaCl, 5 KCl, 10 NaHCO3 (before saturation with 95% O2-5% CO2), 1.2 CaCl2, 1 MgSO4, 5 glucose, and 10 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, pH 7.4, and 0.1% bovine serum albumin in volumes of 4.7-5.2 ml in the luminal and subluminal compartments (17).
Determinations of Ppyr.
Ppyr was
determined from unidirectional (luminal-to-subluminal) fluxes, as
described previously (15, 18). Pyranine was chosen as a probe to assess
paracellular permeability, because it is a highly charged 0.51-kDa
trisulfonic acid and its concentration can be measured down to
nanomolar concentrations by fluorescence techniques. Pyranine traverses
the epithelium via the paracellular pathway, and it does not permeate
cells; cytolysis of dispersed CaSki previously incubated with 0.1 mM
pyranine does not increase pyranine fluorescence significantly above
background (not shown). We previously showed that pyranine fluxes
(Jpyr) in the
luminal-to-subluminal direction are similar to those in the
subluminal-to-luminal direction (15). Pyranine was added to the luminal
compartment, and the amount of pyranine in the subluminal compartment
was measured after 6 min. The transepithelial permeability coefficient
(Ppyr) was
calculated from the area-normalized unidirectional
Jpyr as follows:
Ppyr = Jpyr/[Pyr]cis,
where [Pyr]cis is the
concentration of pyranine in the cis
(luminal) compartment. Jpyr were
calculated as follows:
Jpyr = ([Pyr]cis · Ftrans)/(Fcis · S · t),
where Fcis and
Ftrans represent
background-corrected pyranine fluorescence in
cis and
trans compartments, respectively, S is the surface area of the filter
insert (0.6 cm2), and
t is time.
Determinations of RTE.
Changes in RTE
were determined continuously across filters mounted in a modified
Ussing chamber, as described previously (16), from successive
measurements of the short-circuit current
(Isc, normalized
to the 0.6-cm2 surface area of the
filter) and the transepithelial potential difference (PD, lumen
negative), with switching between
Isc and PD at a
rate of 20 Hz:
RTE = PD/
Isc. The
experimental design of the electrophysiological measurements, including
calibrations and controls, the significance of the potential
(V) and
Isc, and the
conditions for optimal determinations of
RTE across the
low-resistance CaSki epithelium have been described and discussed
elsewhere (13, 17). In CaSki epithelia the electrical resistance of the
filter itself as measured in the Ussing chamber is ~20
· cm
2, which is ~1.5-
to 2-fold higher than the resistance generated by the cells. To
determine the epithelium-generated resistance, the following steps were
taken: First, in every experiment we included additional controls, such
as blank filters without cells and filters containing cells grown in
regular medium with known RTE based on
previous studies. Second, each experiment began by adjusting the PD
across a blank filter without cells to zero to allow for subtraction of
the filter-generated resistance. Third, at the conclusion of some
experiments, calcium in the medium was lowered to zero by addition of 2 mM ethylene glycol-bis(
-aminoethyl ether)-N,N,N',N'-tetraacetic
acid to disrupt intercellular connections, including the tight
junctions (19), and to abolish the paracellular resistance. Under these
conditions, RTE
is similar to the resistance of the filter itself.
Determinations of the dilution potential.
Dilution potential
(Vdil) was
determined in the Ussing chamber, as described elsewhere (17).
Transepithelial
Vdil values were
determined by measuring the effect of lowering NaCl in the luminal
solution on changes in voltage generated across the epithelium. This
was done by replacing the Ringer buffer in the luminal compartment (130 mM NaCl) with low (10 mM)-NaCl solution.
Vdil is the
measured PD (voltageSL voltageL, where SL and L represent
subluminal and luminal solutions, respectively) after lowering of NaCl
in the luminal solution, corrected for the potential-electrode
asymmetry. The differences in the potentials across the tips of the
subluminal and luminal electrodes were determined by the Henderson
diffusion equation for monocations and monoanions after correction for
fluid resistivity. Changes in fluid resistivity were determined with a
conductance bridge (model 31, Yellow Springs Instrument, Yellow Springs, OH) operating at 50-60 Hz. The Henderson diffusion
equation was also used to interpret the transepithelial
Vdil in terms of ionic permeabilities, i.e., the ratio
uCl/uNa,
where uCl and
uNa are the
mobilities of Na+ and
Cl
in the intercellular
space (27).
Statistical analysis of the data. Values are means ± SD, and significance of differences among means was estimated by Student's t-test. Trends were calculated using GB-STAT version 5.3 (Dynamic Microsystems, 1995, Silver Spring, MD) and analyzed with analysis of variance. Best fit of regression equations (least-squares criterion) was achieved with SlideWrite Plus 1993 (Advanced Graphics Software, Carlsbad, CA), which uses the Levenberg-Marquardt algorithm, and analyzed using analysis of variance.
Chemicals and supplies. Pyranine was obtained from Molecular Probes (Eugene, OR). Anocell (Anocell-10) filters were obtained from Anotec (Oxon, UK). 9-cis-Retinoic acid and SRI-11217 were synthesized in the Department of Chemistry, Allergen (Irvine, CA). All other chemicals were obtained from Sigma Chemical (St. Louis, MO). Agonists were prepared as 1,000× stock solutions in saline or in dimethyl sulfoxide and kept in the dark.
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RESULTS |
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Effects of retinoid-free medium on paracellular resistance.
Baseline levels of
RTE were 10 ± 1 · cm
2,
and baseline levels of
Ppyr were 16 ± 2 s · cm
1 · 10
6,
which are similar to values reported previously (13, 15-18). To
study the effects of vitamin A deficiency on paracellular resistance, cells were shifted to retinoid-free medium for 1-14 days.
Incubation in retinoid-free medium increased
RTE in a
time-related manner, and after 14 days
RTE increased
from 10 ± 1 to 21 ± 3
· cm
2
(Fig.
1A;
P < 0.01). Similar effects were
obtained relative to changes in
Jpyr; because
permeability relates reciprocally to resistance, we expressed the
changes in Jpyr
in terms of the inverse of
Ppyr, i.e.,
1/Ppyr. As is
shown in Fig. 1B, after 14 days of incubation in retinoid-free medium,
1/Ppyr increased
from 7 ± 1 to 9 ± 1 s · cm
1 · 10
4 (Fig.
1B; P < 0.02). Retinoid-free medium did not affect cell viability (not
shown).
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Effects of all-trans-retinoic acid on paracellular resistance.
Addition of 10 nM all-trans-retinoic
acid (tRA) to cells grown in retinoid-free medium prevented the
increases in RTE
and in 1/Ppyr
(Fig. 1); retinoid treatment did not affect cell viability (not shown).
To clarify the effect of retinoids on paracellular resistance, cells
previously incubated for 10 days in retinoid-free medium were treated
with 10 nM tRA, and
RTE and
1/Ppyr were
determined after 2 or 6 additional days. tRA did not have an acute
effect on paracellular resistance (not shown). In contrast, longer
incubations with the retinoid decreased
RTE from 22 ± 3 to 9 ± 2 · cm
2
(Fig. 2A;
P < 0.01), and most of the decrease
in RTE occurred after 2 days of incubation with tRA. tRA also decreased
1/Ppyr from 11 ± 1 to 6 ± 2 s · cm
1 · 10
4
within 2 days of addition of the retinoid (Fig.
2B; P < 0.01).
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Sensitivity and specificity of the effects of retinoids on
paracellular resistance.
The effects of tRA on
RTE and on
1/Ppyr were dose
related. Decreases in
RTE and
1/Ppyr were
observed at 1010 M tRA and
reached saturation at
~10
8 M (Fig.
4A). The
dose-response curves of the effects of tRA on RTE and
1/Ppyr were
sigmoidal, with a 50% effective concentration (EC50) of 0.7 ± 0.2 nM for
decreases in RTE
and 0.9 ± 0.3 nM for decreases in
1/Ppyr (Fig.
4A). The dose-response curves for
RTE and
1/Ppyr could be
fitted to a modified Hill equation, with Hill coefficients
(n) of 0.9 ± 0.2 and 1.0 ± 0.2, respectively (P < 0.01). These
results indicate that low concentrations of tRA are sufficient to lower
paracellular resistance across cervical cells.
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Retinoids decrease the RTJ. The above results indicate that retinoids decrease paracellular resistance across human cervical cultures. To determine whether retinoids decrease RLIS or RTJ, we studied the effects of tRA on Vdil and on uCl/uNa. The rationale was that cation selectivity is a property of the tight junctions and changes in uCl/uNa reflect changes in RTJ (25). Transepithelial Vdil was established by replacing the luminal solution with a buffer containing low NaCl (10 mM), and levels of uCl/uNa were determined as described in METHODS. Absolute levels of Vdil and uCl/uNa across cultures incubated in retinoid-free medium were significantly lower than those across cultures incubated in regular medium (Table 1), indicating that removal of vitamin A from the culture medium increased transcervical cation selectivity. In contrast, treatment with tRA increased absolute levels of Vdil and uCl/uNa, indicating that tRA decreases cation selectivity.
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DISCUSSION |
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The present results indicate that retinoids regulate the paracellular resistance of CaSki cells: incubation in retinoid-free medium increased the resistance, and retinoids, at nanomolar concentrations, prevented the increase in resistance and restored it to baseline levels. We were able to incubate the cells in retinoid-free conditions only for ~2 wk; the paracellular resistance increased twofold, but the increase in resistance did not reach a plateau (Fig. 1). It is possible that longer in vivo exposure to vitamin A deficiency could increase paracellular resistance even more.
Retinoids altered ion mobilities across the cervical cultures; because cation selectivity is a property conferred by the tight junctions (25), the present results suggest that retinoids modulate RTJ. This conclusion is also supported by the experiments with cytochalasin D and ionomycin. We used cytochalasin D to disrupt actin organization and the cytoskeleton. Changes in the cytoskeleton can affect cell structure, cell volume, and the volume of the intercellular space, i.e., RLIS. Changes in the cytoskeleton can also disrupt transmembrane intercellular mechanisms (30), and possibly also the tight junctions, and affect RTJ. Cytochalasin D decreased uCl/uNa, suggesting a decrease in RTJ. The tRA-induced decrease in RTE was nonadditive to the decrease in resistance induced by cytochalasin D, suggesting that retinoids and cytochalasin D activate a common paracellular mechanism, possibly the RTJ. In CaSki cells, ionomycin also decreases RTE, but in contrast to cytochalasin D, the effect is mediated by decreases in RLIS (13). Because tRA and ionomycin had additive effects on RTE, it is suggested that ionomycin and tRA do not operate via a common paracellular mechanism, namely, that the decrease in resistance induced by retinoids is not mediated by decreases in RLIS.
Little is known about how retinoids affect the tight junctional complex. Lack of an acute effect on RTJ rules out retinoid-induced conformational changes of existing tight junction proteins (17). Whether retinoids modulate synthesis and/or assembly of tight junction proteins remains to be seen.
The cellular mechanisms of retinoid action in cervical cells are only partly understood. The effects of retinoids cannot be explained by enhanced apoptosis (23) or by toxic effects on cells, because 1) the retinoid concentrations used in the present study did not cause cell death, 2) the increase in RTE produced by retinoid-free medium was reversible, and 3) retinoids had minimal effects on paracellular resistance in cells grown in medium containing normal serum. The effects of retinoids cannot also be explained by changes in cell density or stratification, because retinoids have no significant effect on the number of CaSki cells grown on filters (not shown); more importantly, cell density contributes <5% to the RTE in cervical cultures, and the paracellular resistance depends mainly on the tight junctions (16).
Retinoids may bind to intracellular retinoid-binding proteins (9), the function of which is to sequester retinoids and to regulate their metabolism. Although information about their expression in the human cervix is not available, cellular retinol- and retinoid acid-binding proteins (CRBP and CRABP) are expressed in the cervix of rats in the columnar epithelium (28). The prolonged effects of retinoids in our system that persist long after the retinoids are removed from the medium may result from retinoid association with these intracellular binding proteins.
The effects of retinoids on gene expression are not directly mediated
via the binding proteins. These effects are mediated via interaction
with the nuclear retinoid receptors. The nuclear retinoid receptor
family consists of six members, RAR-, RAR-
, RAR-
, RXR-
,
RXR-
, and RXR-
(11), that function as ligand-activated trans-acting factors. These receptors
are similar in structure but are expressed in a tissue-specific manner,
suggesting a specific role for each family member. The RARs bind to tRA
and related ligands, whereas the RXRs bind to
9-cis-retinoic acid and various synthetic RXR-selective ligands (2). Previous studies show the
expression of RAR and RXR receptors and mRNA in mammalian cervical
cells, but the types, levels, and regulation of the receptors vary
(1-3, 7, 22, 24).
Human cervical cells, including CaSki, express RAR and RXR receptors (1, 2, 22). The present results suggest that the effects of retinoids on RTJ are mediated via the retinoid receptor mechanism: 1) The tRA-induced changes in resistance required 3-24 h of treatment, a length of time compatible with gene regulation. Lack of an acute, immediate effect rules out activation of surface receptors or a nonspecific chemical reaction. 2) The effects on RTE were sensitive to low levels of retinoids that are in the physiological range for vitamin A (8) and correlate with their affinity for the RARs and/or the RXRs (8, 10, 28). 3) The dose-response effects of tRA on RTE and 1/Ppyr were saturable and could be fitted by a modified Hill equation with n = 1 for both end points. A possible interpretation of this result is that the effect of retinoids is mediated by receptors with a single binding site for the ligand. 4) The change in RTE correlated with the affinity of the tested retinoid for the nuclear retinoid receptors; thus retinol, a weak retinoid (10), was a weak regulator. In view of the rapid decrease in resistance in response to retinoids that was observed in vitamin A-deficient cells, our results also suggest that in CaSki cells the retinoid receptor(s) that mediates the response is expressed constitutively and abundantly.
Previous studies indicate that RAR-selective ligands are important regulators of gene expression and differentiation in cervical cells (1, 2, 22). In contrast, RXR ligands have been shown to be relatively inactive (2, 22). This does not appear to be the case for the effects on paracellular resistance, inasmuch as SRI-11217, an RXR-selective ligand, is an effective regulator of RTE in CaSki cells. The present results suggest a role for RXR-specific receptors in regulation of cervical cell paracellular resistance.
Plasma levels of vitamin A in the human are tightly regulated (8), and major changes in cervical RTJ in vivo are unlikely to be determined by vitamin A per se, but rather by the status of tissue retinoid receptors or the rate of vitamin A conversion to more active metabolites (i.e., tRA). Previous studies in rodents suggest that the concentration of the receptors in the cervix changes during the estrous cycle (4, 28, 29). Because sex hormones modulate cervical mucus production (12), the present results raise the possibility that steroid hormones regulate cervical mucus secretion indirectly, by modulating tissue levels of retinoid receptors or retinoid metabolism, and that retinoids, in turn, regulate tight junction occlusion.
The present results are important for understanding regulation of fluid and solute transport across the cervix. Relatively small changes in paracellular resistance may affect significantly the quantity of fluid that accumulates in the cervical canal (the cervical mucus). An increase in paracellular resistance is likely to diminish the amount of cervical mucus, because changes in paracellular resistance are inversely related to changes in permeability (27). The present results indicate doubling of the paracellular resistance after 2 wk of incubation in retinoid-free conditions, i.e., a decrease in permeability by one-half. As was mentioned previously, it is possible that longer exposure to vitamin A in vivo will further increase the paracellular resistance and effectively block production of the cervical mucus.
Changes in paracellular resistance may also affect the composition of cervical mucus, because decreases in permeability will decrease initially the transport of larger molecules and vice versa (16). This implies that when the permeability is increased, more fluid and a relatively larger amount of higher-molecular-weight molecules will be secreted from the blood into the lumen. This prediction is supported in part by the characteristics of the cervical mucus in women. Compared with the early estrogenic phase, during the late estrogenic (preovulatory) phase of the cycle the total amount of cervical mucus increases 10- to 15-fold and the amounts of Ca2+ and Mg2+ that are secreted in the mucus increase by 1.2- to 1.5-fold (12). In contrast, the amounts of glucose and fructose increase 10- to 25-fold (12). A possible mechanism for these differences is that estrogens, like retinoids, also increase the permeability of the endocervical epithelium. This subject is being studied in our laboratory.
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ACKNOWLEDGEMENTS |
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This study was supported by National Institute of Child Health and Human Development Grants HD-00977 and HD-29924 (to G. I. Gorodeski) and a grant from the American Institute for Cancer Research (to R. L. Eckert).
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FOOTNOTES |
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Address for reprint requests: G. I. Gorodeski, University MacDonald Women's Hospital, University Hospitals of Cleveland, 11100 Euclid Ave., Cleveland, OH 44106.
Received 7 February 1997; accepted in final form 8 July 1997.
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