Molecular Human Reproduction Vol.9, No.12 pp. 807±813, 2003
DOI: 10.1093/molehr/gag099
Collagen remodelling in the guinea-pig uterine cervix at
term is associated with a decrease in progesterone receptor
expression
H.A.RodrõÂguez, L.Kass, J.Varayoud, J.G.Ramos, H.H.Ortega, M.Durando,
M.MunÄoz-de-Toro and E.H.Luque1
Laboratorio de EndocrinologõÂa y Tumores Hormonodependientes, School of Biochemistry and Biological Sciences,
Universidad Nacional del Litoral, Casilla de Correo 242, (3000) Santa Fe, Argentina
1
To whom correspondence should be addressed. E-mail: [email protected]
In human and guinea-pig parturition, progesterone withdrawal and estrogen action are not mediated by changes in their
circulating levels. Instead, these events might be promoted by changes in the responsiveness of the uterus and cervix to
progesterone and estrogen via changes in their receptors. In this study, the guinea-pig model was used to investigate whether
high levels of progesterone and estrogen at term are associated with regional changes in PR and ERa levels in uterus and cervix.
PR and ERa pro®les were established in both subepithelium and the muscular layer of the cervix and the lower uterine horns
during pregnancy, parturition and postpartum; while collagen remodelling was measured in the subepithelium. Our data
showed that collagen remodelling involved in cervical ripening is temporally and spatially associated with a decrease in PR,
whereas high expression of ERa is observed. This association was found in the subepithelium of the cervical tissue but not in the
same region of the uterus. The muscular region of the cervix and uterus also present a transiently decreased expression of PR
while ERa levels remain high. Thus, the present results indicate that, before parturition, diminished responsiveness of the
cervix to progesterone might be caused by a decrease in PR levels and that this may be the mechanism of functional
progesterone withdrawal. The guinea-pig was further validated as an animal model for human parturition studies.
Key words: cervix/estrogen receptor a/parturition uterus/progesteron receptor
Introduction
Normal parturition requires a close cooperation between uterus and
cervix. The uterus must become an active contracting organ while the
cervix must be capable of dilatation. Thus, suf®cient cervical ripening
in coordination with uterine contractions becomes a prerequisite for a
normal onset and progress of labour (Challis et al., 2000; Weiss,
2000). The failure of either cervical ripening or adequate uterine
contractions causes unsuccessful or dysfunctional parturition
(Downing and Sherwood, 1985; Sherwood, 1994; O'Brien, 1995;
Luque et al., 1998).
The functional changes in cervix and uterus related to parturition
occur mainly in two different tissue compartments: the subepithelium
and the muscular region. The rhythmic tonic contractions of labour are
produced by myometrium, while changes in the connective tissue of
cervical subepithelium appear to be the primary factors in cervical
ripening (Huszar and Walsh, 1991).
In most mammals, coordination between cervix and uterus at term
is controlled by a decrease in circulating progesterone (progesterone
withdrawal) together with an increase in estrogen levels (estrogen
activation) (Challis and Lye, 1994). However, in humans and guineapigs, the strategy for progesterone withdrawal and estrogen activation
is not associated with signi®cant changes in their serum levels
(Csapo et al., 1971; Walsh et al., 1984; Trewin and Hutz, 1999;
Mesiano et al., 2002). Instead, these events could be facilitated
through changes in the responsiveness of the uterus and cervix to
progesterone and estrogen via different mechanisms such as changes
in receptor expression pro®les or receptor accessory proteins (e.g. heat
shock proteins and/or receptor co-regulators), or a local production of
an antiprogestin or an estrogen agonist (Challis et al., 2000; Wang
et al., 2001; Mesiano et al., 2002). Moreover, it has been hypothesized
for women that sustained circulating concentrations of progesterone
are indeed required in order to reach a functional regionalization of
myometrium at term. A functional withdrawal of progesterone in the
fundal myometrium might induce activation, whereas enhanced
progesterone signalling in the lower uterine segment promotes
relaxation (Challis et al., 2000). This regional distribution would
facilitate descent of the fetus by propagation of contractions from
fundus to cervix.
Based on previous studies, it appeared expedient to investigate
whether high levels of progesterone and estrogen at term are
associated with regional changes in progesteron receptor (PR) and
estrogen receptor (ER) a levels in uterus and cervix, potentially
capable of mediating changes in the responsiveness of the cervical
stroma and myometrium. However, time-course studies of spatial
changes cannot be performed in humans. Thus, this task was
undertaken by choosing an animal model which resembles human
conditions (guinea-pig), to establish the pro®le of ERa, PR and
collagen remodelling (as a measurement of cervical functional
changes) in uterus and cervix during pregnancy, parturition and
postpartum.
Molecular Human Reproduction 9(12) ã European Society of Human Reproduction and Embryology 2003; all rights reserved
807
H.A.RodrõÂguez et al.
Materials and methods
Animals
Guinea-pigs (12±15 weeks old) of the American Short Hair strain, bred at the
Department of Human Physiology (Santa Fe, Argentina) were used. Animals
were maintained in a controlled environment (22 6 2°C; lights on from 06:00
to 20:00 h), and they had free access to pellet laboratory chow and tap water.
Guinea-pigs received a supplement of green hay and ascorbic acid added at the
rate of 400 mg/l in tap water.
The animals were examined daily for vaginal opening for at least two
consecutive cycles before being assigned to the experimental groups. The day
when the vagina was fully open was designated as day 0 of the estrous cycle.
Pregnant guinea-pigs of known mating date were obtained by placing estrous
females with a buck and checking for plugs the following morning (day of plug
= day 1). In our colony delivery occurs on day 64 or 65.
All animal work was conducted in accordance with the Guide for the Care
and Use of Laboratory Animals issued by the National Academy of Sciences
(USA).
Sample collection and tissue processing
Female guinea-pigs (at least ®ve/group) were randomly assigned to the
experimental groups. Animals were killed by decapitation. Trunk blood,
uterine horns and cervical tissue were obtained at estrus, on pregnancy days 21,
42, 56 and 63, immediately after parturition (<1.5 h after they had already
delivered all pups) or in early postpartum (postpartum day 1; 24 h after
parturition). The serum was separated by centrifugation and stored at ±20°C.
Using an aseptic technique, a midline laparatomy was performed in order to
gain complete access to the genital tract. Tissue samples were ®xed by ®lling
the lumen of the genital tract with the ®xative (10% buffered formalin) for 30
min while still in situ. The organs (i.e. cervix and uterine horns) were then
immersed in the same solution for 6 h at room temperature and processed until
paraf®n embedding. Serial sections (5 mm in thickness) of whole uterus and
cervix were taken along the cervical and uterine canals. Sections were assigned
to one of the following staining procedures: haematoxylin±eosin, picrosirius±
haematoxylin or immunohistochemistry.
By gross anatomy assessment and by haematoxylin±eosin staining, we
divided cervical and uterine tissues into four regions: lower uterine horn,
uterine septum, endocervix and exocervix (Figure 1). In each region, two tissue
compartments were analysed: the subepithelium (limited by the basement
membrane and the muscular layer), and the muscular layer (Figure 1). In
previous studies with human (Montes et al., 2002) and rat (Varayoud et al.,
2001; Ramos et al., 2002) cervical tissue obtained during pregnancy and at
parturition, signi®cant changes in the cellular phenotypes in these zones were
described. Immunostaining and collagen remodelling results were expressed
according to this regionalization.
Serum progesterone and estradiol (E2) levels
Progesterone and E2 were measured in serum samples by a sequential
immunometric assay, using kits (Immulite Progesterone and Immulite
Estradiol; Diagnostic Products Corporation, USA) for in-vitro diagnostics.
Results were recorded with the Immulite Analyzer. Quality control of the
assays was performed according to the manufacturer's instructions, as well as
previously published results (Costongs et al., 1995; Rodriguez-Espinosa et al.,
1998). Cross-reactivity for both antisera (against progesterone and E2) were
evaluated by the manufacturer and found to be <1.5%. The accuracy of the
assays was tested by measuring known amounts of progesterone (10, 100 and
300 ng/ml) and E2 (25, 35 and 50 pg/ml) dissolved in guinea-pig charcoaltreated male serum. The recovery was high according to the correlation
between the amount added and the obtained results (r = 0.969 for progesterone
and r = 0.878 for E2). The intra-assay variance was calculated as the mean
coef®cient of variation of sextuplicates of a serum pool containing 10 ng/ml of
progesterone or 40 pg/ml of E2. They were 5.8 and 14.0% respectively. These
serum pooled samples were run in all assays. The inter-assay coef®cient of
variation was calculated from the results obtained from such sextuplicate
determinations in all the assays and was 7.2% for progesterone and 12.0%
for E2.
Measurement of collagen remodelling
Collagen remodelling was evaluated by measuring the intensity of
birefringence with the picrosirius±polarization method, as previously described
(Luque et al., 1996; Montes, 1996; Kass et al., 2001). Brie¯y, images were
recorded using a Dplan 3100 objective (numeral aperture = 1.25) of systematic
randomly selected ®elds of the stroma (n > 40 ®elds) with an Olympus BH2
microscope and a Sony Exwave HAM colour video camera (Sony Electronics,
Inc., USA). Image analysis was performed using the Image Pro-Plus 4.1.0.1
system (Media Cybernetics, USA). Using Auto-Pro macro language, an
automated standard sequence operation was created to measure intensity of
birefringence. In this method, the intensity of collagen birefringence evaluated
by polarization microscopy is an indication of collagen remodelling, since only
orientated collagen molecules present a bright birefringence. Normally,
collagen ®bres form thick bundles of densely packed and regularly arranged
®bres, and they appear as brightly birefringent structures throughout the whole
microscopic ®eld. When collagen ®bres are not dense and/or not regularly
arranged, they are weakly birefringent. A decrease in the intensity of
birefringence shows a transformation from hard and unyielding to a soft,
swollen and ¯exible structure. Thus, the intensity of birefringence is inversely
related to collagen remodelling.
Immunohistochemical detection of ERa and PR
Figure 1. Gross anatomy and histology of guinea-pig cervical and uterine
tissues stained with picrosirius±haematoxylin showing the regionalization
used in the present study. Immunostaining and collagen remodelling
evaluations were performed in two tissue compartments [subepithelium, S
(from the basement membrane to the muscular layer) and muscular layer, M]
of each selected region. Ep = epithelium; EGC = endometrial gland cells; US
= uterine septum. Genital tract, scale bar = 5 mm; histological sections, scale
bar = 100 mm.
808
Immunostaining was performed as previously described (MunÄoz-de-Toro et al.,
1998). After deparaf®nation, microwave pretreatment (antigen retrieval) was
performed; primary antibodies were incubated overnight at 4°C. For ERa, a
mouse monoclonal antibody (1:80 dilution) raised to recombinant ER fusion
protein (clone CC4-5; Novocastra Laboratories Ltd, UK) was used. PR was
immunolocalized by applying a monoclonal antibody diluted 1:100 (clone PRAT 4.14; Af®nity Bioreagents, USA). Reaction was developed with biotin±
streptavidin±peroxidase complex (ABC kit, Novocastra).
The quanti®cation was performed in samples obtained from animals (at least
®ve/experimental group) from mid-pregnancy to early postpartum. Two
sections per animal that included the four selected regions (endocervix,
exocervix, septum and lower uterine horns, as shown in Figure 1) were
evaluated. The quanti®cation was carried out in each one of these regions.
Inside each region, results were expressed considering two different tissue
compartments: ®broblast of the subepithelium and smooth muscle cells of the
muscular layer. At least 1000 cells/tissue compartment were counted. All
immunostained nuclei, regardless of intensity, were scored as positive. Results
were expressed as the percentage ratio of the total number of the corresponding
cell type.
Collagen remodelling, PR and ERa expression in the cervix and uterus
Western blot analysis
To our knowledge, this is the ®rst time that these antibodies have been used on
guinea-pig samples. Thus, the speci®city has been tested by us using Western
blot analysis of protein extracts obtained from intact uterine horns of mouse
(used as positive control) and guinea-pig. Uterine horns were dissected and
immediately frozen in liquid nitrogen. Ice-cold lysis buffer [1% Nonidet P40,
0.5% sodium deoxycholate, 0.1% sodium dodecyl sulphate (SDS), 2 mmol/l
EDTA, 50 mmol/l sodium ¯uoride, 1 mmol/l sodium vanadate, 0.200 mg/ml
aprotinin (all from Sigma, USA) in phosphate-buffered saline, pH 7] was
added. The tissue was dounce-homogenized; 100 mg/ml phenylmethylsulphonyl ¯uoride was added, and the solution was incubated on ice for 30
min. The homogenate was centrifuged at 4°C for 20 min at 14 000 g and the
supernatant was collected. Fifty mg of protein per lane was resolved by 7%
SDS±polyacrylamide gel electrophoresis, transferred to nitrocellulose
membranes (Protoblot; Schleicher and Schuell, USA), and reacted with antiERa 1:50 (clone CC4-5; Novocastra) or anti-PR 1:100 (clone PR-AT 4.14;
Af®nity Bioreagents). The peroxidase-conjugated secondary antibody
(Amersham Pharmacia Biotech, Argentina) was used at a 1:500 dilution, and
the reaction was visualized with the ECL detection kit (Amersham Pharmacia
Biotech).
Negative controls were performed with blots not exposed to the primary
antibodies.
bands were absent when primary antibody was omitted. Isoforms B,
A1 and C have already been described in the human uterus (Wei et al.,
1990) and B, A1, A2 and C in rats (Ogle et al., 1997).
Estrogen and progesterone serum levels from midpregnancy to early postpartum
Patterns of progesterone and E2 serum levels from mid-pregnancy to
early postpartum are shown in Figure 3. Levels of both steroids at
estrus are also shown and, as expected, low progesterone and high E2
values were found. High progesterone levels were recorded during
Statistics
Statistical analyses were performed using the Kruskal±Wallis one-way analysis
of variance. Signi®cance between groups was determined by Dunn post-test,
and correlations were performed using Pearson analysis (Siegel, 1956).
Results
Speci®city of antisera used to detect ERa and PR
Western blot analysis of protein extracts showed that antibodies used
in immunohistochemistry speci®cally detected ERa and PR in guineapig tissue. Figure 2a shows that ERa protein was detected in mouse
and guinea-pig uterus, and it did not appear when primary antibody
was omitted (negative control). Figure 2b shows that PR-A and PR-B
isoforms were present in the mouse uterine extracts whereas in the
immunoblot of guinea-pig uterus four PR isoforms were revealed. The
mol. wt pro®le of the PR variants observed in guinea-pig was: B (~110
kDa), A1 (~90 kDa), A2 (~76±82 kDa) and C (~60±64 kDa). These
Figure 2. Immunoblot analysis of estrogen receptor (ER) a (a) and
progesterone receptor (PR) (b) in mouse and guinea-pig uterine tissues.
Immunoreactive forms are indicated. Protein extract from mouse uterus was
used as a control. In all cases (+) and (±) correspond to incubation with or
without the antibodies respectively. The position and size of the molecular
weight standards (MW) are indicated on the right (in kDa).
Figure 3. Serum levels of progesterone (P4) and estradiol (E2) in guinea-pig at estrus (Es), along pregnancy [days (D) 42, 56 and 63], immediately after
parturition (iAP) and at postpartum day 1 (PPD1). Sequential immunometric assay was performed according to Materials and methods. Values are the mean 6
SEM from at least ®ve guinea-pigs. Vertical dashed line denotes time of delivery. Means with different letters are signi®cantly different (P < 0.05).
809
H.A.RodrõÂguez et al.
pregnancy and immediately after parturition. A sharp fall was
observed on postpartum day 1 when progesterone reached estrous
values. E2 showed a slight increase in serum levels from day 42 to day
63. No signi®cant changes in E2 were observed during parturition,
whereas on postpartum day 1 it decreased signi®cantly compared with
day 63.
Collagen remodelling in the cervix and uterus from
mid-pregnancy to early postpartum
The intensity of birefringence, as an indication of collagen remodelling, was measured in the subepithelium of the cervix and uterus
(Figure 4a and b). Two clearly differentiated stages, regarding
collagen remodelling, were de®ned in the cervix. From day 21 to day
56, a high intensity of birefringence was scanned as an indication of
the presence of a dense and highly organized cervical collagen
framework. In contrast, a dramatic decrease in the intensity of
birefringence was observed during the perinatal period (day 63,
immediately after parturition and postpartum day 1), re¯ecting a
widespread reduction in density and orientation of collagen ®bre
bundles together with an intense collagen remodelling. In the uterus,
even though intensity of birefringence was lower than in cervical
tissue, an early signi®cant decrease from day 21 to day 42 was
detected; after that, the intensity of birefringence remained low and no
changes were recorded during the period studied.
PR and ERa expressions in the cervix and uterus from
mid-pregnancy to early postpartum
PR and ERa expressions were evaluated in the subepithelium and
muscular cells of the endocervix, exocervix, septum and lower uterine
horns from pregnant females on day 42 throughout postpartum day 1.
No different results were found either between endocervix and
exocervix, nor between septum and lower uterine horns. Therefore,
the regions are hereafter simpli®ed as cervix and uterus respectively.
Figure 4. Collagen remodelling in the subepithelium of the cervix and uterus
during gestation [days (D) 21, 42, 56, 63] immediately after parturition (iAP)
and at postpartum day 1 (PPD1). Picrosirius±polarization method and image
analysis were used to quantify the intensity of birefringence (expressed in
arbitrary units) as a measurement of collagen remodelling. Note that the
lowest intensity of birefringence indicates the highest collagen remodelling.
Bars represent mean (+ SEM) intensity of birefringence from at least ®ve
animals/group. Means with different letters differ signi®cantly (P < 0.05).
Figure 5. Immunohistochemistry for progesterone receptor (PR) and estrogen receptor (ER) a in the subepithelium and muscular regions of the cervix and
uterus during gestation [days (D) 42, 56, 63] immediately after parturition (iAP) and at postpartum day 1 (PPD1). Evaluated regions are de®ned in Materials
and methods and receptors were expressed as percentage of positive cells. Bars represent mean (+ SEM) percentage of positive cells from at least ®ve animals/
group. Means with different letters differ signi®cantly (P < 0.05, Kruskal±Wallis and Dunn post-test).
810
Collagen remodelling, PR and ERa expression in the cervix and uterus
Figure 6. Relationships between intensity of birefringence and progesterone
receptor (PR) (a) or estrogen receptor (ER) a (b) expression in
subepithelium of the cervix during gestation [days (D) 42, 56 and 63] and
parturition. PR and ERa exhibited signi®cantly positive and negative
correlations respectively with the intensity of birefringence in the cervix,
although not in the uterus (data no shown). r = correlation coef®cient.
In cervical subepithelium, PR expression decreased before parturition (day 63) and it remained low until postpartum day 1 (Figure 5),
whereas in the uterus subepithelium, high expression of PR was
observed throughout gestation and parturition, decreasing sharply on
postpartum day 1 (Figure 5). In contrast, in the cervix and uterus, the
subepithelial expression of ERa was high before and during
parturition, decreasing at postpartum day 1 (Figure 5).
In the muscular region of cervix and uterus, a signi®cant decrease in
PR expression was found before parturition (day 63), whereas at
parturition this expression reached high values again, similar to days
42±56. High levels of ERa expression were displayed from late
pregnancy (day 56) with no changes around parturition in both organs.
Correlation between collagen remodelling and steroid
receptor expression
During pregnancy and parturition, in the cervical subepithelium, we
have found that PR expression is positively correlated to the intensity
of birefringence (Figure 6a, r = 0.8168, P < 0.001). On the other hand,
the subepithelial cervical expression of ERa showed a negative
correlation to the intensity of birefringence (Figure 6b, r = ±0.7353, P
< 0.01). These associations between PR and ERa expressions and the
intensity of birefringence were not observed in the uterus (r = 0.1796,
P > 0.01 and r = ±0.04820, P > 0.01 respectively). Taking into account
that the intensity of birefringence is inversely related to collagen
remodelling (see Materials and methods), these correlations indicate
that the characteristic process of collagen remodelling in the cervix is
simultaneously associated with a decrease in PR and an increase in
ERa expressions. Interestingly, we also found a negative correlation
between ERa and PR expressions in the subepithelium of the cervix
Figure 7. Relationships between estrogen receptor (ER) a and progesterone
receptor (PR) expression in the subepithelium of the cervix (a) and uterus
(b) during gestation [days (D) 42, 56 and 63] and parturition. r = correlation
coef®cient.
(Figure 7a), while no correlation was observed in the uterus
(Figure 7b).
Discussion
In the present study, using the guinea-pig as an animal model, we
demonstrate that at parturition, collagen remodelling involved in
cervical ripening is temporally and spatially associated with a
decrease in PR while a high expression of ERa is observed. This
association was found in the subepithelium of the cervical tissue but
not in the same region of lower uterine horns. Before parturition, the
muscular region of the cervix and uterus also present a decreased
expression of PR whereas ERa levels remain high.
Previously, we have demonstrated that in the rat, collagen
remodelling in the uterine cervix is evidence of the cervical softening
that allows its dilatation and a normal delivery (Luque et al., 1996,
1998). Studying the connective tissue of the uterus and cervix in nonpregnant versus women in labour, GranstroÈm et al. (1989) found that
collagen remodelling occurs in both organs at term. In guinea-pig,
collagen remodelling also took place in the subepithelium of the
cervix and uterus at parturition. However, we have observed that this
process occurs earlier in the uterus than in the cervix. Temporal
differences in the onset of collagen remodelling in the uterus versus
cervix could be explained by the fact that during pregnancy the uterus
must relax to accommodate the growing fetus, whereas the cervix
must keep ®rm and closed to hold the uterine contents until term.
Changes in ERa and PR levels in cervical tissue might be
responsible for the histofunctional adaptations of this organ associated
811
H.A.RodrõÂguez et al.
with delivery. Existing data indicate that species without a fall in
progesterone prior to parturition (guinea-pigs, non-human primates
and humans) respond to antiprogestins with an increase in myometrial
responsiveness and cervical ripening, but not always by the induction
of pre-term birth (Elger, 1987; Hegele-Hartung et al., 1989; Chwalisz,
1991, 1994). Overall, these studies indicate that, previous to
parturition, the myometrium and cervix of guinea-pigs undergo a
conditioning step (i.e. increased responsiveness) which can be
mediated through PR. Despite high progesterone concentrations in
maternal blood, spontaneous increase in myometrial responsiveness
and cervical ripening physiologically occur in humans and guineapigs before parturition (Chwalisz, 1994). Our results support these
®ndings by showing that, before parturition, diminished responsiveness of the cervix to progesterone might be caused by a decrease in PR
levels and this may be the mechanism of functional progesterone
withdrawal. Since it is well established that progesterone suppresses
ERa expression in the reproductive tract (Haluska et al., 1990), this
might allow the increased ERa expression that we observed around
parturition in cervical subepithelium. Such a phenomenon suggests a
decrease in progesterone action. On the other hand, a high expression
of ERa in cervical subepithelium could raise its responsiveness to
circulating estrogen, leading to an active process of collagen
remodelling that could contribute to cervical effacement. Dilatation
of the guinea-pig cervix is accompanied by an active degradation of
subepithelial collagen type I by a stromal collagenase, and this process
is stimulated by E2 and blocked by progesterone (Rajabi et al.,
1991a,b,c, 1995). Even though our data are in agreement with these
studies, the hypothesis presented here needs to be tested in future
experiments.
On the other hand, our study showed a transient decrease in PR
levels in the muscular region of the cervix and uterus at day 63 of
gestation, after which PR levels return to high values. This means that
parturition occurs with high levels of PR in the myometrium,
indicating that the functional progesterone withdrawal in this tissue
compartment would not be the result of a decreased expression of PR
only and/or that PR-reduced expression on day 63 is enough to
promote later changes in organ responsiveness. It has been recently
reported that, in human myometrium at term, responsiveness to
progesterone is controlled by the expression of PR-A relative to PR-B
and that a signi®cant increase in this ratio underlies functional
progesterone withdrawal (Mesiano et al., 2002). In this study, we have
found that the guinea-pig uterus has similar PR isoforms as described
for women (Wei et al., 1990; Challis et al., 2000). Thus, the possibility
that myometrial progesterone responsiveness is related to changes in
expression of PR-A relative to PR-B might also be considered as a
probable mechanism of myometrial functional progesterone withdrawal in guinea-pigs.
Classically, the guinea-pig has been considered an adequate animal
model to study human gestation and parturition. This concept has been
supported, in part, by the similarities of both species in the serum
pro®les of sex steroids during pregnancy and parturition (Trewin and
Hutz, 1999). The results presented here support the usefulness of the
guinea-pig model by con®rming previous ®ndings and describing
more similarities with humans. We found that guinea-pig uterus has
PR isoforms similar to those described for women (Wei et al., 1990;
Challis et al., 2000). As reported for women (Stjernholm et al., 1996;
Wang et al., 2001), we found that guinea-pig cervical subepithelium
has a signi®cant down-regulation of PR immediately before parturition and a decrease of ERa in the cervical muscular region at
postpartum.
In summary, we provide evidence that in guinea-pig parturition
high circulating levels of progesterone and estrogen at term are
associated with changes in the expression of PR in the uterine cervix.
812
The decreased sensitivity of the cervix to progesterone may be
mediated through a reduced PR expression in the subepithelium. The
results found in the muscular region do not convincingly demonstrate
the functional progesterone withdrawal hypothesis. In fact, it suggests
that another mechanism between steroid hormones and their nuclear
receptors could be involved at the end of gestation and parturition (i.e.
changes in isoform ratio). Our laboratory is currently performing
experiments to clarify this issue. In addition, we demonstrated some
events that extend the classical concept by which the guinea-pig's
reproductive system resembles that of women. Thus, we believe that
the guinea-pig model holds great promise for studies of the
pathophysiology of parturition in women and for therapeutic strategy
design.
Acknowledgements
We are grateful to Mr Juan Grant and Mr Juan C.Villarreal for animal care, to
Professor Alfredo Castro-Vazquez (School of Medicine, Mendoza, Argentina),
to Drs Miguel Nanni and Silvia Sandoz (ParanaÂ, Argentina) for steroid assay
assistance, and to Patricia E.RodrõÂguez (ParanaÂ, Argentina) for grammatical
revision of the manuscript. This study was supported by grants from the
Argentine National Council for Science and Technology (CONICET) (PIP 528/
98), Universidad Nacional del Litoral (CAI+D 027-195) and the Argentine
National Agency for the Promotion of Science and Technology (ANPCyT)
(PICT-99 N° 5-7001).
References
Challis, J.R.G. and Lye, S.J. (1994) Parturition. In Knobil, E. and Neill, J.D.
(eds), The Physiology of Reproduction. Raven Press, New York, pp. 985±
1031.
Challis, J.R.G., Matthews, S.G., Gibb, W. and Lye, S.J. (2000) Endocrine and
paracrine control of birth at term and preterm. Endocrine Rev., 21, 514±550.
Chwalisz, K., Fahrenholz, F., Hackenberg, M., Gar®eld, R. and Elger, W.
(1991) The progesterone antagonist onapristone increases the effectiveness
of oxytocin to produce delivery without changing the myometrial oxytocin
receptor concentrations. Am. J. Obstet. Gynecol., 165, 1760±1770.
Chwalisz, K. (1994) The use of progesterone antagonist for cervical ripening
and as an adjunct to labour and delivery. Hum. Reprod., 9 (Suppl. 1), 131±
161.
Costongs, G.M., van Oers, R.J., Leerkes, B. and Janson, P.C. (1995)
Evaluation of the DPC IMMULITE random access immunoassay
analyzer. Eur. J. Clin. Chem. Clin. Biochem., 33, 887±892.
Csapo, A.I., Knobil, E., van der Molen, H. and Wiest, W.G. (1971) Peripheral
plasma progesterone levels during human pregnancy and labor. Am. J.
Obstet. Gynecol., 110, 630±632.
Downing, S.J. and Sherwood, O.D. (1985) The physiological role of relaxin in
the pregnant rat I: the in¯uence of relaxin on parturition. Endocrinology,
116, 1200±1205.
Elger, W., FaÈhnrich, M., Beier, S., Qing, S.S. and Chwalisz, K. (1987)
Endometrial and myometrial effects of progesterone antagonists in pregnant
guinea pigs. Am. J. Obstet. Gynecol., 157, 1065±1074.
GranstroÈm, L., Ekman, G., Ulmsten, U. and MalmstroÈm, A. (1989) Changes in
the connective tissue of corpus and cervix uteri during ripening and labour
in term pregnancy. Br. J. Obstet. Gynecol., 96, 1198±1202.
Haluska, G.J., West, N.B., Novy, M.J. and Brenner, R.M. (1990) Uterine
estrogen receptors are increased by RU486 in late pregnant rhesus macaques
but not after spontaneous labor. J. Clin. Endocrinol. Metab., 70, 181±186.
Hegele-Hartung, C., Chwalisz, K., Beier, H.M. and Elger, W. (1989) Ripening
of the uterine cervix of the guinea-pig after treatment with the progesterone
antagonist onapristone (ZK 98,299): an electron microscopy study. Hum.
Reprod., 4, 369±377.
Huszar, G.B. and Walsh, M.P. (1991) Relationship between myometrial and
cervical functions in pregnancy and labor. Semin. Perinatol., 2, 97±117.
Kass, L., Ramos, J.G., Ortega, H.H., Montes, G.S., Bussmann, L.E., Luque,
E.H. and MunÄoz-de-Toro, M. (2001) Relaxin has a minor role in rat
mammary gland growth and differentiation during pregnancy. Endocrine,
15, 263±269.
Luque, E.H., Ramos, J.G., Rodriguez, H.A. and MunÄoz-de-Toro, M. (1996)
Dissociation in the control of cervical eosinophilic in®ltration and
Collagen remodelling, PR and ERa expression in the cervix and uterus
collagenolysis at the end of pregnancy or after pseudopregnancy in
ovariectomized steroid-treated rats. Biol. Reprod., 55, 1206±1212.
Luque, E.H., MunÄoz-de-Toro, M., Ramos, J.G., RodrõÂguez, H.A. and
Sherwood, O.D. (1998) Role of relaxin and estrogen in the control of
eosinophilic invasion and collagen remodeling in rat cervical tissue at term.
Biol. Reprod., 59, 795±800.
Mesiano, S., Chan, E.C., Fitter, J.T., Kwek, K., Yeo, G. and Smith, R. (2002)
Progesterone withdrawal and estrogen activation in human parturition are
coordinated by progesterone receptor A expression in the myometrium. J.
Clin. Endocrinol. Metab., 87, 2924±2930.
Montes, G.S. (1996) Structural biology of the ®bres of the collagenous and
elastic systems. Cell Biol. Int., 20, 15±27.
Montes, G.S., Zugaib, M., Joazeiro, P.P., Varayoud, J., Ramos, J.G., MunÄozde-Toro, M. and Luque, E.H. (2002) Phenotypic modulation of ®broblastic
cells in the mucous layer of the human uterine cervix at term. Reproduction,
124, 783±790.
MunÄoz-de-Toro, M., Maf®ni, M., Kass, L. and Luque, E.H. (1998)
Proliferative activity and steroid hormone receptor status in male breast
carcinoma. J. Steroid Biochem. Mol. Biol., 67, 333±339.
O'Brien, W.F. (1995) Cervical ripening and labor induction: progress and
challenges. Clin. Obstet. Gynecol., 38, 221±223.
Ogle, T.F., Dai, D., George, P. and Mahesh, V.B. (1997) Stromal cell
progesterone and estrogen receptors during proliferation and regression of
the deciduas basalis in the pregnant rat. Biol. Reprod., 57, 495±506.
Rajabi, M.R. and Singh, A. (1995) Cell origin and paracrine control of
interstitial collagenase in the guinea pig uterine cervixÐevidence for a low
molecular weight epithelial cell-derived collagenase stimulator. Biol.
Reprod., 52, 516±523.
Rajabi, M.R., Dodge, G.R., Solomon, S. and Poole, A.R. (1991a)
Immunochemical and immunohistochemical evidence of estrogenmediated collagenolysis as a mechanism of cervical dilatation in the
guinea pig at parturition. Endocrinology, 128, 371±378.
Rajabi, M.R., Solomon, S. and Poole, A.R. (1991b) Hormonal regulation of
interstitial collagenase in the uterine cervix of the pregnant guinea pig.
Endocrinology, 128, 863±871.
Rajabi, M.R., Solomon, S. and Poole, A.R. (1991c) Biochemical evidence of
collagenase-mediated collagenolysis as a mechanism of cervical dilatation
at parturition in the guinea pig. Biol. Reprod., 45, 764±772.
Ramos, J.G., Varayoud, J., Bosquiazzo, V.L., Luque, E.H. and MunÄoz-deToro, M. (2002) Cellular turnover in the rat uterine cervix and its
relationship to estrogen and progesterone receptor dynamics. Biol. Reprod.,
67, 735±742.
Rodriguez-Espinosa, J., Otal-Entraigas, C., Gascon-Roche, N., Mora-Bungues, J.,
Urgell-Rull, E., Bordas-Serrat, J.R. and Viscasillas-Molins, P. (1998)
Analytical and clinical performance of an automated immunoassay system
(Immulite) for estradiol in serum. Clin. Chem. Lab. Med., 36, 969±974.
Sherwood, O.D. (1994) Relaxin. In Knobil, E. and Neill, J.D. (eds), The
Physiology of Reproduction. Raven Press, New York, pp. 861±1009.
Siegel, S. (1956) Nonparametric Statistics for the Behavioral Sciences.
McGraw-Hill, New York, pp. 215±224.
Stjernholm, Y., Sahlin, L., Akerberg, S., Elinder, A., Eriksson, H.A.,
MalmstroÈm, A. and Ekman, G. (1996) Cervical ripening in humans:
potential roles of estrogen, progesterone, and insuline-like growth factor-I.
Am. J. Obstet. Gynecol., 174, 1065±1071.
Trewin, A.L. and Hutz, R.J. (1999) Guinea pig female. In Knobil, E. and Neill,
J.D. (eds), Encyclopedia of Reproduction. Academic Press, New York, pp.
583±588.
Varayoud, J., Ramos, J.G., Joazeiro, P.P., Montes, G.S., MunÄoz-de-Toro, M.
and Luque, E.H. (2001) Characterization of ®broblastic cell plasticity in the
lamina propria of the rat uterine cervix at term. Biol. Reprod., 65, 375±383.
Walsh, S.W., Stanczyk, F.Z. and Novy, M.J. (1984) Daily hormonal changes
in the maternal, fetal, and amniotic ¯uid compartments before parturition in
a primate species. J. Clin. Endocrinol. Metab., 58, 629±639.
Wang, H., Stjernholm, Y., Ekman, G., Eriksson, H. and Sahlin, L. (2001)
Different regulation of oestrogen receptors a and b in the human cervix at
term pregnancy. Mol. Hum. Reprod., 7, 293±300.
Wei, L.L., Gonzalez-Aller, C., Word, W.M., Miller, L.A. and Horwitz, K.B.
(1990) 5¢-Heterogeneity in human progesterone receptor transcripts predicts
a new amino-terminal truncated ``C''-receptor and unique A-receptor
messages. Mol. Endocrinol., 4, 1833±1840.
Weiss, G. (2000) Endocrinology of parturition. J. Clin. Endocrinol. Metab.,
85, 4421±4425.
Submitted on April 28, 2003; resubmitted on July 11, 2003;
accepted on July 28, 2003
813