Acidic pH and increasing [Ca2+] reduce the swelling of mucins in primary cultures of human cervical cells

M. Espinosa1, G. Noé1, C. Troncoso2, S.B. Ho3 and M. Villalón1,4

1 Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Santiago, 2 Unidad de Ginecología, Hospital Padre Hurtado, Santiago, Chile and 3 Department of Medicine, Veterans Affairs Medical Center and University of Minnesota, Minneapolis, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: Cervical mucus is a heterogeneous mixture of water, ions and mucins that form a hydrophilic polymer gel. Mucins, the main components of mucus, are condensed inside secretory granules and swell to become a hydrogel after exocytosis. Using human cervical secretory cell primary cultures, the effect of [Ca2+] and [H+] on the swelling velocity of mucin granules was investigated in vitro. METHODS and RESULTS: Immunocytochemistry demonstrated that estrogen and progesterone receptors were expressed in cultured secretory cells along with mucins type 1, 4, 5AC and 5B. Exocytosis of secretory cells, recorded by videomicroscopy, showed that during swelling, the radius of the secretory granule matrix followed first-order kinetics. An increase in extracellular [Ca2+] from 1 to 4 mmol/l or a reduction in pH from 7.4 to 6.5 was seen to produce a significant decrease in the velocity of swelling of the secretory granule matrix. CONCLUSIONS: The inverse relationship observed between the diffusion of the granular matrix and the extracellular [Ca2+] or [H+] suggested that changes in cation concentration might drastically affect the swelling characteristics of mucins and provide a control mechanism for the observed viscoelastic properties of mucus.

Key words: cations/cervix/mucins/primary cultures/swelling


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Human cervical mucus is a complex substance that is produced by secretory cells of the endocervix and plays an important role in the reproductive process (Wolf et al., 1980Go). During a normal menstrual cycle, mucus is scant, thick and viscous, and also forms a physical barrier that limits access of sperm to the genital tract. However, immediately before ovulation, and under estrogenic influence, the mucus thins and shows maximal penetrability by sperm (Moghissi, 1973Go). These changes in viscoelastic properties (Litt et al., 1977Go) correlate with mucus hydration, and also determine its rheological characteristics (Wolf et al., 1977aGo).

In mammals, cervical mucus—like other gels—consists of a polymer matrix and water as a solvent. In humans, around the time of ovulation when mucus is most abundant, the water content increases from 92–94% to 98% of the wet weight (Moghissi, 1973Go). Inorganic salts are also present in mucus and represent about 1% of the dry weight, the most abundant being KCl, CaCl2 and NaCl (Lippes et al., 1972Go; Moghissi, 1973Go; Casselen and Nilsson, 1984Go). Certain ionic constituents of cervical mucus show cyclic variations; that is, the ratio between NaCl and organic material content, measured in dehydrated mucus, is maximal at the time of ovulation (Schumacher, 1973Go). Furthermore, close to ovulation, CaCl2 decreases concomitantly with an increase in KCl concentration (Lippes et al., 1972Go). There is also evidence that the pH of cervical mucus changes cyclically, increasing progressively until ovulation and then decreasing during the post-ovulatory phase (Moghissi et al., 1972Go; Maas et al., 1977Go; Paulesu and Pessina, 1982Go). Although these results show that ion concentrations vary during the menstrual cycle, it is unclear how these changes could bring about the changes in viscoelastic properties that characterize each phase of the menstrual cycle.

Mucins are the main component of the polymer matrix and correspond to a family of very high molecular-weight polyanionic polymers which are randomly coiled and tangled to form a three-dimensional network (Verdugo, 1990Go). Mucins are synthesized on membrane-bound ribosomes and subsequently modified in the Golgi apparatus, and then packaged into secretory granules (Forstner, 1995Go). In all mucin-secreting cells, apical granules extrude their contents during exocytosis, after fusion of the granule with the plasma membranes. At the present time, 12 distinct mucin genes have been cloned and designated, in order of discovery, as MUCs 1, 2, 3, 4, 5AC, 5B, 6, 7, 8, 9, 11 and 12 (Gipson, 2001Go). Using in-situ hybridization, it has been shown that cervical epithelium expresses six of the first eight mucin genes, with the exception of MUCs 3 and 7 and the sporadic presence of MUC2 (Audie et al., 1995Go; Gipson et al., 1997Go). In the endocervical epithelium the most abundant mucins correspond to MUC4 and MUC5B (Audie et al., 1995Go; Gipson et al., 1999Go). Furthermore, MUC4 and MUC5B mRNA expression in human endocervix is inversely related to plasma levels of progesterone (Gipson et al., 1999Go). Although several mucins have been partially characterized, their functional role in the cervix is unknown—as are the viscoelastic properties of each endocervically-expressed mucin.

Although evidence exists that sperm penetrability and viscoelasticity of the cervical mucus is dependent on mucin hydration (Wolf et al., 1978Go), it is unclear which factors—other than water availability—are important in determining the high degree of mucin hydration observed during ovulation in comparison with the luteal phase. It has been observed (Tam and Verdugo, 1981Go) that cow cervical mucus hydration follows a Donnan equilibrium process. In this study, it was proposed that movement of ions (including H+), soluble proteins and water across the mucosa, along with soluble protein and ion concentration within the secreted mucus, could be the variables which determine mucus hydration and thereby its rheological properties. Although mucin hydration is the major determining factor for mucus viscoelasticity (Wolf et al., 1977bGo), it is not known how ionic variations in the milieu can affect this process.

In the present study a tissue culture technique to grow human cervical secretory cells has been developed in order to study the effect of variations in pH and in extracellular calcium concentration upon the swelling velocity of mucin granules in vitro. It was also postulated how these variations might cause the changes in mucus viscoelasticity observed during the menstrual cycle.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Specimens
Cervical tissue was obtained from patients undergoing hysterectomy for uterine leiomyoma at the Gynecology Unit of the Hospital Padre Hurtado, Santiago, Chile. Each patient provided their written consent and was informed of the purpose of the research investigation. This protocol was approved by the Ethics Committee of the Hospital Padre Hurtado. Patient selection criteria included women who required uterine surgery but who were otherwise healthy, aged between 30 and 47 years, with regular menstrual cycles and not using hormonal contraceptives. Immediately after removal of the uterus, the tissue incorporating the region from the external cervical os to the fundus was dissected under aseptic conditions. The surface of the endocervix mucosa was rinsed with Hank’s solution (Sigma Chemical Co., St Louis, MO, USA) before endocervical biopsies were obtained. Each biopsy consisted of approximately 1 cm2 of epithelium and 2–3 mm of the underlaying stroma. The biopsy was placed in Hank’s solution at room temperature and transported to the laboratory, where cultures were prepared.

Primary cultures
Epithelial secretory cells of the human cervix were isolated using a modification of a previously published technique (Verdugo et al., 1990Go). The endocervical biopsy was treated with protease type 14 (0.05%), followed by trypsin (0.01%) (both from Sigma), for 30 min at 4°C. Each enzyme was dissolved in Hank’s solution. The tissue was washed in Hank’s solution and placed in Waymouth medium (Sigma) containing penicillin G (100 units/ml), streptomycin (100 µg/ml) and amphotericin B (1 µg/ml) (all from Sigma). At this time, the mucus was aspirated with a syringe and the epithelium of the biopsy was removed mechanically using a scalpel. The epithelium was centrifuged at 300xg for 5 min, the pellet was resuspended in 1 ml Waymouth medium and then dispersed mechanically with a 1 ml syringe. Glass coverslips on which the cells were to be grown were placed into 4-well culture dishes. The cells were added and each well was filled with Waymouth medium (pH 7.4) supplemented with 20% fetal bovine serum (FBS) (Sigma), 1.2% L-glutamine, penicillin G (100 units/ml), streptomycin (100 µm/ml) and amphotericin B (1 µg/ml). Culture dishes were incubated in a controlled atmosphere of 5% CO2 and 95% O2 at 37°C. On three occasions each week, the cells were washed in serum-free medium before the addition of fresh media.

Histochemistry
Alcian blue stain and periodic acid–Schiff (PAS) reagent was used to verify the presence of glycoproteins in the secretory products released from cultured cells of the human cervix.

After secretory activity and swelling had been recorded, cultures were fixed in 10% formaldehyde for 30 min and stained with PAS or Alcian blue at pH 2.5. After staining, cultures were dehydrated in alcohol, mounted with Permount (Fisher Scientific, Pittsburgh, PA, USA) and examined under an Olympus microscope model BH-2 (New Jersey Scientific, Inc., USA) and photographed with a Nikon camera using 100 ASA film. Microscopic observations and colour micrographs were used to verify the presence of staining in the secreted material.

Detection of estrogen and progesterone receptors
Secretory cells were examined immunohistochemically to confirm the presence of both estrogen {alpha} receptors (ER{alpha}) and progesterone receptors (PgR), using a monoclonal antibody against ER{alpha} and a polyclonal antibody against PgR (both from Dako Corp., Carpinteria, USA). Fourteen 1-day-old primary cultures were fixed in 4% paraformaldehyde at room temperature for 30 min and then washed in phosphate-buffered saline (PBS), pH 7.4, for 15 min. These cells were incubated in acetone for 5 min at –20°C before washing with PBS. The cultures were blocked with 2% goat serum in 1% PBS/bovine serum albumin (BSA) for 45 min, followed by an overnight incubation at 4°C with primary antibody. ER{alpha} was diluted 1:70 and PgR 1:50 in blocking solution. All subsequent steps were carried out at room temperature. As controls, coverslips with cells were incubated without the primary antibody or with preimmune rabbit serum. On the following day, cells were washed in PBS for 15 min, followed by incubation with biotinylated secondary antibody diluted 1:400 for the ER{alpha} and 1:500 for the PgR, for 45 min. After a 10 min wash in PBS, coverslips were incubated for 30 min with streptavidinconjugated peroxidase diluted 1:400 in PBS for 30 min, followed by a 10 min wash in PBS. The localization of the primary antibody was visualized by incubating the cells with diaminobenzidine (Sigma) dissolved in Tris buffer (0.5 mmol/l) and hydrogen peroxide for 5 min. This treatment resulted in a brown-coloured staining. Coverslips were then dehydrated through graded ethanol solutions, cleared in xylol, and mounted with Permount. Cell cultures were observed with brightfield optics using a microscope (Olympus BH-2) and photographed with a Nikon camera and 100 ASA film.

Detection of mucins
The presence of mucins was determined by indirect immunofluorescence using a polyclonal antibody against the MUC1 cytoplasmic domain (Pemberton et al., 1992Go) and using chicken polyclonal antibodies against the carboxyl terminal non-repetitive synthetic peptide sequence of MUCs 4, 5AC, 5B and 6 (Ho et al., 1995Go). Primary cultures, on glass coverslips, were used to register exocytosis (see below) and then used for immunocytochemistry. For immunocytochemistry, the cells were washed with PBS for 5 min and fixed in paraformaldehyde 4% in PBS at room temperature for 30 min. Subsequently, the cultures were washed three times for 5 min each in PBS. One set of cultures was incubated in pure acetone at –20°C for 5 min to permeate membranes before washing three times for 5 min each in PBS. Cells were blocked in PBS/BSA 1% and 2% goat serum for 45 min at room temperature and incubated overnight at 4°C using differing dilutions of anti MUC1 (1:100), MUC4 (1:5000), MUC5AC (1:1000), MUC5B (1:1000) and MUC6 (1:4000) antibodies. Fluorescein isothiocyanate (FITC)-donkey (Research Diagnostics, Inc., New Jersey, USA) anti-chicken IgG was added at a 1:50 dilution for 45 min at room temperature for MUCs 4, 5AC, 5B and 6, and FITC-goat (Amersham Life Sciences, Arlington Heights, Illinois, USA) anti-rabbit IgG at 1:500 for 45 min for MUC1. The slides were mounted with the fluorescence stabilizer Fluoromount G (Electron Microscopy Sciences, Washington, USA). Observation and photographs were taken with an Olympus BH-2 microscope adapted for epifluorescence, and photographed with Nikon camera using 100 ASA film. Negative controls were obtained by replacing the primary antibody with preimmune chicken serum, or by omission of the primary antibody.

Mucin swelling
Secretory cells that were growing in isolation were selected to study swelling during the process of exocytosis. Before fusion with the plasma membrane, the content of each granule swelled rapidly for a period of 5–10 s to form small microspheres on the periphery of the secretory cell. This process was monitored under a phase-contrast light microscope (Nikon, Japan) at a magnification of x400, and recorded at a rate of 30 frames per second using a Sony Hi8 video camera (model T-5000). Images of the expanding exocytosed granules were digitized from the monitor at rates of 10, 15, 20, 25 and 30 frames per second using the Imagepro program. The optical resolution of the recording system determined the minimum detectable radius for each single granule, and was defined as time zero during swelling analysis.

Previous observations have indicated that the swelling of mucus is governed by the same physical principles that govern the swelling of synthetic polymer gels (Tanaka and Fillmore, 1979Go; Verdugo, 1984Go; Verdugo et al., 1987aGo). For example, the deformation of the expanding mucin microspheres, observed during degranulation in secretory cells, follows typical first-order kinetics, where the time course of the radial expansion r(t) is related to the initial radius (ri) and final radius (rf) by:


Also in agreement with the linear theory of swelling of gels (Verdugo, 1984Go; Verdugo et al., 1987aGo) is the proportional relationship between the characteristic time and the square of the final radius (rf)2, where the coefficient of diffusion (D), i.e. the ability to expand, of the mucin network is:


The coefficient of diffusion is an extremely sensitive parameter by which the effect of environmental conditions on the swelling of polymer gels including mucus, may be elucidated (Verdugo, 1984Go; Verdugo et al., 1987aGo).

In the present study, the time course of swelling was evaluated by measuring the diameter of the circular images formed by the secreted material, following a modification of a previously published method (Tanaka and Fillmore, 1979Go) for monitoring the swelling of polyacrylamide hydrogels. The increase in the radius in function of time (experimental values) was recorded during the process of exocytosis of each granule. A theoretical fitting of the experimental values of the radius was then inserted into Equation (1Go) using the statistical analysis program SYSTAT 5.0 (Systat, Inc., 1989). The program calculated the values for initial radius (ri), final radius (rf) and time ({tau}). Finally, a value for D was obtained from Equation (2Go).

Ionic effect
Fourteen 1-day-old primary cultures were used to evaluate the effect of Ca2+ and pH on the swelling kinetics of mucins. At the time of experimentation, the culture medium was replaced by a control buffer containing 144 mmol/l NaCl, 1 mmol/l HEPES, 1 mmol/ MOPS, 1 mmol/l CaCl2, at 37°C, pH 7.4 and 288 mOsm. Both the Ca2+ effect and the pH effect were evaluated by increasing the CaCl2 concentration from 1 to 4 mmol/l, or by decreasing the pH from 7.4 to 6.5 in the same buffer. Video recordings of individual cells were recorded.

Statistical analysis
To evaluate the statistical differences between slopes of the resultant diffusion curves (1/m = D), it was determined whether the values of (rf)2 and {tau} of granules of mucin followed a normal distribution (using the SYSTAT 5.0 statistics program). In this analysis, both parameters were normally distributed. Using the statistical program S-Plus 2000, the analysis of covariance (ANCOVA) was used to test the significance of the regression coefficient for each experimental condition. Significance was accepted when P was < 0.05.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Primary cultures of human cervical secretory cells
Secretory cells were obtained after enzymatic dispersion of the human endocervical epithelium (Figure 1AGo). Morphological analysis of the primary cultures determined the presence of two cellular forms: first, a more abundant cell type that corresponded to mesenchymal cells that formed clusters; and second, cells that corresponded to a pleomorphic form, which were characterized as secretory cells observed in the active process of secretion (Figure 1BGo). The endocervical secretory cells in vitro were characterized by a slow growth rate and a well-differentiated phenotype after approximately 2 weeks in culture. The highest percentage of secretory cells was observed after 12–14 days in culture, during which period the experimentation was performed. During secretion, the mucin granule content swelled rapidly for about 5–10 s, forming small microspheres on the periphery of the secretory cell. Spheres of swollen granular content detached slowly from the surface of the cell and annealed to form large aggregates of mucus, which became totally dispersed after 30 min and covered the surface of the cell. Alcian blue (pH 2.5) (Figure 1CGo) and PAS staining (Figure 1DGo) of the endocervical cell cultures revealed that both the secretory product and the mucus on the cell surface were positive for acidic glycoprotein-type mucins. Primary cultures of the human cervix also expressed ER (Figure 1EGo) and PgR (Figure 1FGo). Immunoreactivity to both receptors was localized to the nucleus of the secretory cells and in the abundant mesenchymal cells present in the culture. These results suggested that secretory cells grown in vitro maintain the ability to express ER and PgR as they do in vivo.



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Figure 1. Morphological analysis of primary cultures of human cervical epithelium. (A) Two cell types are found in the culture: a more elongated form identified as mesenchymal cells (arrow with asterisk); and a round cell defined as a secretory cell (arrow). (B) Higher magnification of a secretory cell indicates that after exocytosis, swollen secretory granules (arrows) remained attached to the cell surface. (C, D) Stained secretory granules, localized on the cell surface (arrows), shows a positive reaction to Alcian blue (C) and periodic acid–Schiff staining (D). Expression of estrogen (E) and progesterone receptors (F), after 14 days in culture, indicates that immunoreactivity to both receptors is localized in the nucleus, of mesenchymal cells (arrow with asterisk) and secretory cells (arrow).

 
Primary cultures of endocervical epithelium express different types of mucins
In epithelial cell culture, MUC4 immunoreactivity to non-permeabilized cells was localized mainly at the surface of the secretory cell (Figure 2A and BGo). On the other hand, in permeabilized cells, using the same antibody, the immunoreactivity was detected mainly in secretory granules localized in the cytoplasm (Figure 2DGo). Permeabilized cells have positive immunoreactivity to MUCs 1, 5AC and 5B (Figure 2C, E and FGo) with a granular staining pattern similar to MUC4. No immunoreactivity to MUC6 was observed (data not shown). No positive staining was observed in negative control cultures (Figure 2G and HGo).



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Figure 2. (A–H) Immunohistochemical localization of mucins in primary cultures of endocervical epithelium. A phase-contrast image of the cell culture is shown in (A). Immunoreactivity to MUC4 in non-permeabilized secretory cells is localized mainly on the cell surface (B). Immunohistochemical localization of MUC1 (C), MUC4 (D), MUC5AC (E) and MUC5B (F) in permeabilized cells. Immunoreactivity was detected mainly in secretory granules localized in the cytoplasm. (G, H) No reaction was observed in negative control cultures, where the primary antibody was replaced with preimmune chicken serum. (A and G are phase-contrast images of the treatment B and H respectively.)

 
Swelling of mucin-containing secretory granules in vitro
A composition of digital images from a single mucin secretory granule during its expansion to the extracellular space, as recorded by videomicroscopy, is shown in Figure 3AGo. A total of 10.9 s was required for the granule matrix to reach its final radius of 2.0 µm. A typical time course of the swelling of a single human cervical secretory cell granular matrix is shown in Figure 3BGo. The increase in radius of the granule as a function of time follows first-order kinetics with a characteristic time ({tau}) of swelling of 1.5 s (the time in which the granule reaches two-thirds of its final radius). The fitting of the experimental data to the theoretical curve for a first-order of radial expansion described by Equation (1Go) (see Materials and methods) gives a correlation coefficient of r = 0.99 (P < 0.001).



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Figure 3. (A) Digital image composition of the expansion of a single mucin granule during exocytosis, recorded by videomicroscopy. The volume of the granule increased as a function of time, and reached a final radius of 2 µm in ~11 s. The time interval between each picture is 454 ms. (B) Radius increase of the granule during swelling. The radius of the granule increased ( ’) as a function of time, and followed first-order kinetics, with a characteristic time ({tau}) of swelling of 1.5 s. Note that fitting of the experimental data (o) to Equation (1Go) described a theoretical curve for first-order kinetics (continuous line), and gave a statistically significant correlation coefficient (r = 0.99; P < 0.001).

 
Swelling velocity of mucin granules decreases as extracellular [Ca2+] increases
The effect on the swelling velocity of mucin granules from secretory cells in vitro by increasing the extracellular Ca2+ concentration from 1 to 4 mmol/l is shown in Figure 4Go. Each point corresponds to the final radius (expressed in cm2) and characteristic time, {tau} (expressed in seconds) for single secretory granules after exocytosis in each experimental condition. The swelling velocity of the exocytosed secretory material at 4 mmol/l Ca2+ is much less than that at 1 mmol/l Ca2+. The linear expression of Equation (2Go) is {tau} = 1/Dx(rf)2; therefore, the diffusion coefficient (D) is the inverse of the slope, and this was significantly reduced (P < 0.05) from 2.25x10–7 cm2/s to 3.33x10-8 cm2/s for 1 and 4 mmol/l Ca2+ respectively. Although an heterogeneous population of swollen granules based on their final radius was observed, a statistically significant (P < 0.05) linear relationship with {tau} was found for both experimental conditions, with a correlation coefficient of r = 0.90 for 1 mmol/l Ca2+ and r = 0.53 for 4 mol/l Ca2+. This suggests that if divalent charges of calcium ions were present in the medium, they could neutralize the negative charges of the mucins and thus reduce the presence of repelling forces that would favour expansion of the granular matrix.



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Figure 4. Effect of extracellular calcium concentration on the diffusion coefficient of mucin granular matrix in vitro. An increase in Ca2+ concentration from 1 mmol/l (o, n = 27) to 4 mmol/l(, n = 17) produced a significant decrease (P < 0.05) in the diffusion coefficient of the polymeric network, from 2.25x10-7 cm2/s to 3.33x10-8 cm2/s. The line represents the fitting of experimental data to the linear expression of Equation (2Go), where {tau} = 1/Dx(rf)2, with correlation coefficients for each experimental condition of r = 0.904 and r = 0.533 respectively. Note that increasing the calcium concentration in the extracellular medium reduced the mucin swelling velocity of the granular matrix.

 
Velocity of mucin swelling decreases with increased acidity of the extracellular medium
In comparison with primary cultures at pH 7.4, cultures exposed to an acidic extracellular medium (pH 6.5) showed slower-expanding mucin granules. This represented a significant decrease (P < 0.05) in the diffusion coefficient, from 2.25x10-7 cm2/s at pH 7.4 to 1.11x10-8 cm2/s at pH 6.5 (Figure 5Go). It was noted that lowering the pH led to a dramatic reduction in the swelling capacity of the granules, and this resulted in one group of very slow-swelling granules with {tau} >20 s, and another group of granules with very little or almost no swelling. These observations suggested that increasing [H+] in the extracellular medium is an important factor in determining the degree of hydration and velocity of mucin swelling. Furthermore, this result was consistent with previous observations which demonstrated that an acidic environment can mimic the native granular conditions which favour a condensed state of the mucin matrix.



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Figure 5. Effect of extracellular pH on the diffusion coefficient of mucin granular matrix in vitro. An increase in the proton concentration of the extracellular medium produced a significant decrease (P < 0.05) in the diffusion coefficient (D) of the network. At pH 6.5 (>>, n = 12), D = 1.11x10-8 cm2/s, while at pH 7.4(o, n = 27), D = 2.25x10-7 cm2/s, with correlation coefficients ofr = 0.803 and r = 0.90 respectively. This result was consistent with the observation that an acidic environment can mimic the native granular condition, which favours a condensed state of the granular matrix.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The present study provided evidence that primary cultures of human cervical secretory cells express different types of mucins. Furthermore, during exocytosis, swelling velocity of the mucins granules depends on the pH and the extracellular calcium concentration. Although it is known that mucin hydration determines the rheological characteristics of cervical mucus during the human menstrual cycle (Gibbons and Sellwood, 1973; Wolf et al., 1977aGo, 1977cGo, 1980Go; Haas et al., 1987Go), the mechanisms that control mucin hydration have not been determined. The present results provide evidence that changes in pH and calcium affect mucin swelling, and also suggest that regulation of the ionic milieu in the cervical canal is part of the mechanism that controls mucin hydration and the viscoelastic properties of mucus.

In the present study, newly developed culture techniques permitted functional secretory cells of human cervix to be grown. The cultured cervical epithelial cells displayed a well-differentiated phenotype in culture, which implied a maintained capacity of secretion, the presence of nuclear receptors for estrogen and progesterone (Press et al., 1986Go), the presence of granules that contained glycoprotein-like mucins (Dray-Charier et al., 1997Go) and the expression of MUC1, 4, 5AC and 5B, but not MUC6 (Audie et al., 1995Go; Gipson et al., 1997Go; Gipson, 2001Go). The presence of these characteristics provided the confidence that these cells were a good model with which to study the expansion of mucin secretory granules in vitro.

Calcium and hydrogen ions have been measured in high concentrations in a wide variety of secretory granules, including mucin secretory granules (Izutsu et al., 1985Go; Verdugo et al., 1987aGo; Fernández et al., 1991Go; Nicaise et al., 1992Go). In the present study, it was shown that an extracellular Ca2+ concentration of 4 mmol/l caused a decrease in the swelling velocity of mucin granules of about one order of magnitude compared with a Ca2+ concentration of 1 mmol/l (control condition). This was similar to the effect observed for mucin swelling velocity in the rabbit and dog trachea (Steiner et al., 1984Go; Verdugo et al., 1987bGo). A larger impact was shown upon acidification of the extracellular medium, where a 20-fold reduction on mucin matrix expansion velocity was observed in comparison with that in controls. The decrease in swelling rate that was induced by Ca2+ may be explained by calcium’s role as a cationic shielding agent (Verdugo et al., 1987aGo). Mucins are highly condensed inside secretory granules, and their polyanionic nature should prevent condensation unless their negative charges are appropriately shielded by calcium and acidic pH. By contrast, if the cationic shield is lost, then the polyanionic charges will produce a rapid expansion of the mucin polymer matrix (Verdugo et al., 1987aGo,bGo). The swelling of mucins during product release, as was observed in the present study, is an ‘explosive’ event. As Ca2+ is released from the granule, the negative charges of mucins become unshielded, and these repel each other, thereby producing the expansion and release of the secretory product. The release of the shielding ions from the granule must be determined by a concentration gradient between the intragranular compartment and the extracellular space. Thus, if the concentration of Ca2+ in the extracellular space increases—as in the present experiments—the release of Ca2+ from charged sites in the mucin chains should decrease, and this explains the slowing down in the swelling of the mucin matrix. In previous investigations it was observed that the monovalent ions increased the fluidity of gels in a concentration-dependent manner (Crowther et al., 1984Go). In this respect, further studies are required to determine the effect of monovalent ions as well as the effect of variations in ionic proportions on the expansion of the mucin matrix. Furthermore, it is also important to correlate the specific mucin types expressed on a single granule and their swelling velocity in different ionic conditions, in order to understand the functional contribution of each mucin to the rheological properties of mucus. Based on the evidence reported herein, it is proposed that expansion of the cervical mucin is governed by a Donnan equilibrium process (Donnan, 1924Go), where the velocity of expansion of mucins is dependent on the pH and ionic concentration of the extracellular medium to which the cell is exposed (Tam and Verdugo, 1981Go).

Previously published studies have suggested that the main determinant of viscosity is hydration, and water content has indeed been shown to be a critical determinant of the rheological properties of mucus (Wolf et al., 1977aGo). It is known that estradiol induces an increased water flow through the cervical epithelium by decreasing epithelial paracellular resistance (Haas et al., 1987Go; Gorodeski, 2000Go). On the basis of this evidence, and on the observations of the present study, it is suggested that a mechanism of swelling of cervical mucus which integrates the swelling of mucins with processes modulated by ovarian hormones could be based on the principle of the Donnan equilibrium. Here, the rheology of human cervical mucus would be regulated principally by an ionic and water exchange through the cervical epithelium by the type of secreted mucins and by the control that ovarian hormones carry out on both processes. In a Donnan system, each of these control mechanisms would have a specific role in the regulation of mucus hydration and rheology. These three factors would be conjugated during the phases of the menstrual cycle in the following way: by the effect of estradiol increasing the flow of water towards the cervical lumen during the periovulatory phase (Haas et al., 1987Go; Gorodeski, 2000Go). During this period changes are also produced in the ionic content of the cervical canal, namely increased K+ and decreased Ca2+ (Casselen and Nilsson, 1984Go), while the pH is returned to mild alkaline (near 7.4) (Lippes et al., 1972Go; Maas et al., 1977Go; Hunter, 1988Go). The extracellular medium with these characteristics would favour a more rapid hydration of the mucin chains and, as a consequence, a decrease in mucus viscosity. On the other hand, estrogens increase cervical secretion (Moghissi, 1973Go; Richardson et al., 1993Go). In this scenario, estradiol may affect the expression of one or more mucin forms (Idris and Carraway, 2000Go; Gipson et al., 2001Go), or may alter a combination of both the mucin forms and their hydration capacity, thus producing an abundance of mucus with increased alkaline and hydrated properties which are characteristic of the periovulatory phase (Figure 6Go). During the luteal phase, progesterone inhibits the secretory activity of the cervical epithelium (Roelofs et al., 1983Go). The water content is reduced (Gorodeski, 2000Go) and the ionic constituents are altered, namely decreased K+, increased Ca2+ (Casselen and Nilsson, 1984Go) and reduced pH, i.e. more acidic conditions (Hunter, 1988Go). Under these conditions, the velocity of hydration of secreted mucins would be slower than in the periovulatory phase, and thus viscous mucus would be produced (Figure 6Go). It has been found recently that the expression of MUC4, which is a characteristic of secretory cells, is stimulated by estradiol treatment and inhibited by progesterone in vitro (M.Villalón, unpublished data). This regulation by ovarian hormones on MUC4 expression is in accordance with the studies shown by others (Gipson et al., 1999Go), and suggests that an alteration in mucin expression might be responsible for the mid-cycle change in expressed mucins.



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Figure 6. Schematic representation of a proposed mechanism for mucus hydration during the human menstrual cycle. (A) At about the time of ovulation, and under estrogenic influence (E2), the flow of water increases towards the cervical lumen; synthesis and secretion of specific mucins are augmented and a specific ionic environment is present (i.e. lower calcium [Ca2+{downarrow}], higher potassium [K+{uparrow}] and alkaline pH), that favours mucin swelling. These conditions produce a hydrated and abundant mucus during the periovulatory phase. (B) By contrast, under the influence of progesterone (P4), water content, synthesis and secretion of specific mucins are diminished, and the ionic environment (i.e. higher calcium [Ca2+{uparrow}], lower potassium [K+{downarrow}] and acidic pH) prevents mucin swelling, thus producing a scant, viscous and low-hydration mucus typical of the luteal phase.

 
In summary, on the basis of results obtained with the mechanistic model presented herein, further studies are required to increase our understanding of the cellular and physicochemical mechanisms responsible for changes in the rheological properties of mucus. These data might also help in developing new fertility strategies based on controlling the viscoelastic properties of mucus.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The authors thank Maritza Gonzalez, Nelly Farías and Lucy Messen for their technical assistance, and Dr. Gareth Owen for comments on the manuscript. These studies were supported by FONDECYT grants 8980008 and the Mellon Foundation CONRAD Program (to M.V.) and a Merit Review Award, Research Service of the Department of Veterans Affairs (to S.H.).


    Notes
 
4 To whom correspondence should be addressed at: Pontificia Universidad Católica de Chile, Facultad de Ciencias Biológicas, Alameda 340, Casilla 11-D, Santiago, Chile. E-mail: mvilla{at}bio.puc.cl Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
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Submitted on December 3, 2001; accepted on March 28, 2002.