Human Reproduction Vol.17, No.8 pp.

1964–1972, 2002

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

1Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Santiago, 2Unidad de Ginecologı́a, Hospital Padre
Hurtado, Santiago, Chile and 3Department of Medicine, Veterans Affairs Medical Center and University of Minnesota, Minneapolis,
USA
4Towhom correspondence should be addressed at: Pontificia Universidad Católica de Chile, Facultad de Ciencias Biológicas,
Alameda 340, Casilla 11-D, Santiago, Chile. E-mail: mvilla@bio.puc.cl

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 (Schumacher, 1973). Furthermore, close to ovulation, CaCl2

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., 1980). 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, 1973). These
changes in viscoelastic properties (Litt et al., 1977) correlate
with mucus hydration, and also determine its rheological
characteristics (Wolf et al., 1977a).
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, 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
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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 sub-
sequently 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
© European Society of Human Reproduction and Embryology

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 unknown—
as are the viscoelastic properties of each endocervically-
expressed mucin.
Although evidence exists that sperm penetrability and visco-
elasticity of the cervical mucus is dependent on mucin hydration
(Wolf et al., 1978), 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, 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.
Materials and methods
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.,
1990). The endocervical biopsy was treated with protease type 14
Mucin swelling in primary cultures of human cervix
(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 300⫻g 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 ampho-
tericin 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 α receptors (ERα) and progesterone
receptors (PgR), using a monoclonal antibody against ERα and a
polyclonal antibody against PgR (both from Dako Corp., Carpinteria,
USA). Fourteen 1-day-old primary cultures were fixed in 4% parafor-
maldehyde 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. ERα 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 ERα and 1:500 for the PgR, for 45 min. After a 10 min wash in
PBS, coverslips were incubated for 30 min with streptavidin-
conjugated 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.
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M.Espinosa et al.
Detection of mucins
The presence of mucins was determined by indirect immunofluores-
cence 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 immunocyto-
chemistry, 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 Dia-
gnostics, 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 ⫻400, 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:
r(t) ⫽ rf – (rf – ri)⫻e–t/τ (1)
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:
D ⫽ (rf)2/τ (2)
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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 polyacryl-
amide 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
(τ). 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 τ 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. Signific-
ance was accepted when P was ⬍ 0.05.
Results
Primary cultures of human cervical secretory cells
Secretory cells were obtained after enzymatic dispersion of
the human endocervical epithelium (Figure 1A). 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 1B). 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 experi-
mentation 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 1C) and PAS
staining (Figure 1D) of the endocervical cell cultures revealed
 Mucin swelling in primary cultures of human cervix

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).

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 1E) and PgR (Figure 1F). 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.
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 B). On the other hand, in
permeabilized cells, using the same antibody, the immuno-
reactivity was detected mainly in secretory granules localized
in the cytoplasm (Figure 2D). Permeabilized cells have positive
immunoreactivity to MUCs 1, 5AC and 5B (Figure 2C, E and
F) 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 H).
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 3A. 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 3B. The increase in radius of the granule as a
function of time follows first-order kinetics with a characteristic
time (τ) 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 (1) (see Materials and
methods) gives a correlation coefficient of 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 4. Each
point corresponds to the final radius (expressed in cm2) and
characteristic time, τ (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 (2) is τ ⫽ 1/D⫻(rf)2; therefore,
the diffusion coefficient (D) is the inverse of the slope, and
this was significantly reduced (P ⬍ 0.05) from 2.25⫻10–7
cm2/s to 3.33⫻10–8 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 signific-
ant (P ⬍ 0.05) linear relationship with τ 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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M.Espinosa et al.

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.)

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 signific-
ant decrease (P ⬍ 0.05) in the diffusion coefficient, from
2.25⫻10–7 cm2/s at pH 7.4 to 1.11⫻10–8 cm2/s at pH 6.5
(Figure 5). 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 τ
⬎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.
Discussion
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
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mucus during the human menstrual cycle (Gibbons and Sell-
wood, 1973; Wolf et al., 1977a, 1977c, 1980; Haas et al.,
1987), 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., 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

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 (τ) of swelling of 1.5 s. Note that fitting of the
experimental data (o) to Equation (1) described a theoretical curve
for first-order kinetics (continuous line), and gave a statistically
significant correlation coefficient (r ⫽ 0.99; P ⬍ 0.001).
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 calcium’s
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
Mucin swelling in primary cultures of human cervix
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 2.25⫻10–7
cm2/s to 3.33⫻10–8 cm2/s. The line represents the fitting of
experimental data to the linear expression of Equation (2), where
τ ⫽ 1/D⫻(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.
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 ⫽ 1.11⫻10–8 cm2/s, while at pH 7.4
(o, n ⫽ 27), D ⫽ 2.25⫻10–7 cm2/s, with correlation coefficients of
r ⫽ 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.
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
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M.Espinosa et al.

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⫹↓], higher potassium [K⫹↑] 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⫹↑], lower potassium [K⫹↓] and acidic pH) prevents mucin swelling, thus producing a scant, viscous and low-
hydration mucus typical of the luteal phase.

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., 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 rheolo-
gical properties of mucus (Wolf et al., 1977a). It is known
that estradiol induces an increased water flow through the
1970
cervical epithelium by decreasing epithelial paracellular resist-
ance (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 modu-
lated 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 hydra-
tion of secreted mucins would be slower than in the periovula-
tory 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.
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
The authors thank Maritza Gonzalez, Nelly Farı́as 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.).
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Submitted on December 3, 2001; accepted on March 28, 2002