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
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Abstract |
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Key words: cations/cervix/mucins/primary cultures/swelling
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Introduction |
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In mammals, cervical mucuslike other gelsconsists 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 9294% to 98% of the wet weight (Moghissi, 1973). 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., 1972
; Moghissi, 1973
; Casselen and Nilsson, 1984
). 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, 1973
). Furthermore, close to ovulation, CaCl2 decreases concomitantly with an increase in KCl concentration (Lippes et al., 1972
). 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., 1972
; Maas et al., 1977
; Paulesu and Pessina, 1982
). 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, 1990). Mucins are synthesized on membrane-bound ribosomes and subsequently modified in the Golgi apparatus, and then packaged into secretory granules (Forstner, 1995
). 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, 2001
). 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., 1995
; Gipson et al., 1997
). In the endocervical epithelium the most abundant mucins correspond to MUC4 and MUC5B (Audie et al., 1995
; Gipson et al., 1999
). Furthermore, MUC4 and MUC5B mRNA expression in human endocervix is inversely related to plasma levels of progesterone (Gipson et al., 1999
). Although several mucins have been partially characterized, their functional role in the cervix is unknownas 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., 1978), it is unclear which factorsother than water availabilityare 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, 1981
) 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., 1977b
), 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.
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Materials and methods |
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Primary cultures
Epithelial secretory cells of the human cervix were isolated using a modification of a previously published technique (Verdugo et al., 1990). 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 Hanks solution. The tissue was washed in Hanks 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 acidSchiff (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 receptors (ER
) and progesterone receptors (PgR), using a monoclonal antibody against ER
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
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
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., 1992) and using chicken polyclonal antibodies against the carboxyl terminal non-repetitive synthetic peptide sequence of MUCs 4, 5AC, 5B and 6 (Ho et al., 1995
). 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 510 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, 1979; Verdugo, 1984
; Verdugo et al., 1987a
). 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:
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Also in agreement with the linear theory of swelling of gels (Verdugo, 1984; Verdugo et al., 1987a
) 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:
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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, 1984; Verdugo et al., 1987a
).
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, 1979) 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 (1
) 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 (
). Finally, a value for D was obtained from Equation (2
).
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 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.
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Results |
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Discussion |
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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., 1986), the presence of granules that contained glycoprotein-like mucins (Dray-Charier et al., 1997
) and the expression of MUC1, 4, 5AC and 5B, but not MUC6 (Audie et al., 1995
; Gipson et al., 1997
; Gipson, 2001
). 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., 1985; Verdugo et al., 1987a
; Fernández et al., 1991
; Nicaise et al., 1992
). 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., 1984
; Verdugo et al., 1987b
). 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 calciums role as a cationic shielding agent (Verdugo et al., 1987a
). 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., 1987a
,b
). 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 increasesas in the present experimentsthe 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., 1984
). 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, 1924
), 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, 1981
).
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., 1977a). It is known that estradiol induces an increased water flow through the cervical epithelium by decreasing epithelial paracellular resistance (Haas et al., 1987
; Gorodeski, 2000
). 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., 1987
; Gorodeski, 2000
). During this period changes are also produced in the ionic content of the cervical canal, namely increased K+ and decreased Ca2+ (Casselen and Nilsson, 1984
), while the pH is returned to mild alkaline (near 7.4) (Lippes et al., 1972
; Maas et al., 1977
; Hunter, 1988
). 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, 1973
; Richardson et al., 1993
). In this scenario, estradiol may affect the expression of one or more mucin forms (Idris and Carraway, 2000
; Gipson et al., 2001
), 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 6
). During the luteal phase, progesterone inhibits the secretory activity of the cervical epithelium (Roelofs et al., 1983
). The water content is reduced (Gorodeski, 2000
) and the ionic constituents are altered, namely decreased K+, increased Ca2+ (Casselen and Nilsson, 1984
) and reduced pH, i.e. more acidic conditions (Hunter, 1988
). 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 6
). 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., 1999
), and suggests that an alteration in mucin expression might be responsible for the mid-cycle change in expressed mucins.
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Acknowledgements |
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Notes |
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References |
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Submitted on December 3, 2001; accepted on March 28, 2002.