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Collagen and matrix metalloproteinase-2 and -9 in the ewe
cervix during the estrous cycle
M. Rodríguez-Piñón
a
,
*
, C. Tasende
a
, D. Casuriaga
a
, A. Bielli
b
, P. Genovese
b
,
E.G. Garófalo
a
a
Biochemistry Area, Department of Molecular and Cellular Biology, Universidad de la República, Montevideo, Uruguay
b
Histology and Embryology Area, Department of Morphology and Development, Veterinary Faculty, Universidad de la República,
Montevideo, Uruguay
article info
Article history:
Received 17 December 2014
Received in revised form 21 May 2015
Accepted 21 May 2015
Keywords:
Metalloproteinase
Collagen
Cervical remodeling
Estrous cycle
Ewe
abstract
The cervical collagen remodeling during the estrous cycle of the ewe was examined. The
collagen concentration determined by a hydroxyproline assay and the area occupied by
collagen fibers (%C), determined by van Gieson staining, were assessed in the cranial and
caudal cervix of Corriedale ewes on Days 1 (n ¼6), 6 (n ¼5), or 13 (n ¼6) after estrous
detection (defined as Day 0). In addition, the gelatinase activity by in situ and SDS-PAGE
gelatin zymographies and matrix metalloproteinase-2 and -9 (MMP-2 and MMP-9,
respectively) expression by immunohistochemistry were determined. The collagen con-
centration and %C were lowest on Day 1 of the estrous cycle (P <0.04), when MMP-2
activity was highest (P <0.006) and the ratio of activated to latent MMP-2 trend to be
highest (P ¼0.0819). The MMP-2 activity was detected in 73% of the homogenized cervical
samples, and its expression was mainly detected in active fibroblasts. By contrast, the
MMP-9 activity was detected in 9% of the samples, and its scarce expression was associated
with plasmocytes, macrophages, and lymphocytes. Matrix metalloproteinase-2 expression
was maximal on Day 1 in the cranial cervix and on Day 13 in the caudal cervix and was
lower in the cranial than in the caudal cervix (P <0.0001). This time-dependent increase
in MMP-2 expression that differed between the cranial and caudal cervix may reflect their
different physiological roles. The decrease in the collagen content and increase in fibroblast
MMP-2 activity in sheep cervix on Day 1 of the estrous cycle suggests that cervical dilation
at estrus is due to the occurrence of collagen fiber degradation modulated by changes in
periovulatory hormone levels.
Ó2015 Elsevier Inc. All rights reserved.
1. Introduction
The tortuous nature of the ovine cervix restricts trans-
cervical artificial insemination and embryo transfer pro-
cedures [1–3]. However, natural cervical dilatation occurs
at estrus [4], and many studies have examined the physi-
ological mechanism of cervical dilatation for transcervical
cannulation improvement [5,6]. Fibrillar collagen and
high-molecular-weight proteoglycan complexes are the
main components of the extracellular matrix (ECM) of the
cervical connective tissue [7–9]. The biochemical in-
teractions between these structural elements are critical to
the cervical remodeling process that results in cervical
dilation [10,11].
In the ewe, the proposed model for cervical dilatation at
estrus involves a central role of periestrous endocrine
changes that drive ECM remodeling processes and, conse-
quently, cervical dilatation [4–6]. These periestrous endo-
crine changes include the preovulatory increase of estradiol
and gonadotropins [4–6,12] and the activation of the
*Corresponding author. Tel.: þ598 2622 1195; fax: þ598 2628 0130.
E-mail address: marodri@adinet.com.uy (M. Rodríguez-Piñón).
Contents lists available at ScienceDirect
Theriogenology
journal homepage: www.theriojournal.com
0093-691X/$ –see front matter Ó2015 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.theriogenology.2015.05.017
Theriogenology xxx (2015) 1–9
prostaglandin E2/oxytocin (PGE2/Ox) system [13–15].
Cervical production of PGE2 stimulates smooth muscle
relaxation and hyaluronan-like glycosaminoglycan (GAG)
synthesis via an autocrine or paracrine mechanism, causing
disaggregation of collagen fibers and cervical dilatation
[16,17]. In other species, some evidence indicates that
degradation of collagen fibers may also be involved in
cervical dilatation. For example, a decrease in collagen
content was measured chemically [18] and histologically in
pregnant women at term [19]. The degradation of collagen
in the ECM depends on the activity of matrix metal-
loproteinases (MMPs), which are the only enzymes capable
of degrading denatured fibrillar collagen [20]. In particular,
the expression of MMP-2 and -9 (also called gelatinases A
and B, respectively) increases in the human cervix at the
end of pregnancy [21], indicating a likely role of MMP-2
and -9 in the cervical dilatation process.
We hypothesized the coexistence of increased collagen
fiber disaggregation and increased enzymatic collagen
degradation in the sheep cervix around the estrus. The
changes in the collagen content and distribution and in the
MMP-2 and -9 abundance and activity along the cervix of
the ewe during the estrous cycle were examined, particu-
larly at the expected time of artificial insemination and
embryo transfer (Days 1 and 6 after estrus, respectively).
2. Materials and methods
2.1. Animals and treatments
The experiment was carried out at the experimental field
of the Veterinary Faculty of the University of Uruguay, Cane-
lones, Uruguay (35
S), during the breeding season of Corrie-
dale ewes (February through March). Animal experimentation
was performed in compliance with regulations set by the
Veterinary Faculty of the University of Uruguay. The adult
Corriedale ewes were kept under natural daylight conditions.
They grazed on native pastures and were given water ad libi-
tum. Vasectomized rams fitted with marking crayons were
keptwith the ewesfor 2 monthsbefore thestart of thestudy to
confirm the normal cyclic conditions of the ewes.
The estrus was synchronized with two doses of a PGF2
a
analogue (intramuscularly, 150 mg, Glandinex; Laboratorio
Universal, Montevideo, Uruguay) administered 6 days
apart. From Day 10 after the second PGF2
a
treatment, ewes
were checked twice daily (at 6 and 18 hours) for service
marks of two vasectomized rams carrying marking crayons
(day of estrus ¼Day 0). Seventeen ewes (bodyweight,
mean pooled standard error of the mean, 39.0 1.1 kg)
showing spontaneous estrus were slaughtered on Days 1
(n ¼6), 6 (n ¼5), or 13 (n ¼6) after the estrus detection.
The day of the estrous cycle for each animal was confirmed
by concentrations of circulating estradiol-17
b
(E2) and
progesterone [12].
2.2. Cervical samples, wet weight, and water content
The cervices were weighed and dissected at a temperature
of 0
Cto4
C into three transversal segments of equal length
labeled cranial, middle,and caudal cervical zones(2–2.5 g per
cervical zone). The cranial and caudal cervical zones were
longitudinally cut into four equal segments (500–600 mg/
segment). One longitudinal segment for each cervical zone
was used to determine the water content by drying until a
constant weight was reached at 80
C for 3 hours; this water
content was expressed as percentage humidity (%). Another
longitudinal segment was weighed, sliced, andhomogenized
in PBS buffer (1/10, wt/vol) with a Polytron Homogenizer
(Polytron Homogenizer PT-10; Kinematica AG, Littau Luzern,
Switzerland). Aliquots of homogenateswere stored at 80
C
until the spectrophotometric and SDS-PAGE zymography
assays were performed. The third longitudinal segment was
immediately fixed by immersion in buffered 4% formalde-
hyde (pH 7.4) for 24 hours and then stored in 70% ethanol for
10 days. Fixed cervices werethen dehydrated and embedded
in paraffin until histochemistry and immunohistochemistry
assays were performed. Thefourth longitudinal segment was
embedded in tissue-freezing medium without fixation and
stored at 80
C until the in situ zymographywas performed.
2.3. Collagen and total soluble protein content determined by
spectrophotometry
The collagen content was measured indirectly by a hy-
droxyproline assay adapted from Bannister and Burns [22].
Aliquots of frozen homogenates were hydrolyzed in
constantly boiling hydrochloric acid(3 N) at 90
Cfor24hours.
After a partial neutralization (pH ¼2.3) with 3-N NaOH, the
hydrolyzed samples were exposed to an oxidizing agent
(chloramine-T 7%: water: PBS buffer, 7:100:500, v:v:v) for
15 minutes at room temperature (RT). Under these conditions,
hydroxyproline was liberated by acid hydrolysis and oxidized
to a pyrrole, which then reacted with the color reagent (30 g of
4-dimethylaminobenzaldehyde, 45 mL of 60% perchloric acid,
and 250 mL of propan-2-ol) at 70
Cfor15minutes.The
absorbance of the resulting colored product was read at
550 nm. Readings were calibrated against standards prepared
from L-4-hydroxyproline (Fluka, 56250) dissolved in 0.01-N
HCl (0.5–30
m
g/mL, r¼0.9976, P <0.0001). All samples
were analyzed in a single assay, with a sensitivity of 0.5
m
g/mL
and intra-assay coefficient of variation of 7%. The collagen
concentration was calculated assuming that the hydroxypro-
line/collagen ratio is 14%[23] and was expressed relative to dry
tissue mass (mg/g of dry tissue).
The total soluble protein concentration in the aliquots of
frozen homogenates was determined by the method of
Lowry et al. [24], using BSA (Fraction V, Sigma Chemical, St.
Louis, MO, USA) as the standard (0.05–0.8 mg/mL,
r¼0.9960, P <0.0001). All samples were analyzed in a
single assay, with a sensitivity of 0.05 mg/mL and intra-
assay coefficient of variation of 4%. The total soluble pro-
tein concentrations (mg/g of dry tissue) were positively
correlated with the amount of tissue used (r¼0.7123,
n¼86, P <0.0003), showing that the total proteins
extracted were similar among cervical samples.
2.4. Collagen distribution determined by van Gieson staining
Van Gieson’s picrofuchsin stain was used to observe the
connective tissue fibers in deparaffinized and rehydrated
cervical sections (5
m
m). Sections were stained with iron
hematoxylin for 10 minutes, washed in running water for
M. Rodríguez-Piñón et al. / Theriogenology xxx (2015) 1–92
15 minutes, and rinsed with distilled water. After differ-
entiation with 0.5% acid alcohol for 2 minutes, the slides
were stained with van Gieson stain (alcoholic picric acid
and acid fuchsine) for 5 minutes, dehydrated, and mounted
in Entellan (Merck, Darmstadt, Germany). All cervical
samples were analyzed in a single assay. After a general
inspection of each slide, the stroma was divided into four
histologic compartments (SHC) depending on cell density
and localization according to Rodríguez-Piñón et al. [15]:
superficial fold stroma (SFS), deep fold stroma (DFS), su-
perficial wall stroma (SWS), and deep wall stroma (DWS).
To estimate the percentage of collagen fibers (red stain), a
quantitative image analysis was performed on selected
digitized images captured by an Olympus microscope and
infinity camera connected to a computer. Ten digitized
images (400) of systematic randomly selected fields of
each SHC for two slides per cervical zone per ewe were
analyzed separately by removing the other SHCs (Photo-
shop 6.0). Color-discrimination software (Image-Pro Ex-
press 6.0) was used to apply a threshold for red staining by
a color detection system; it creates a binary image from
which the percentage of the total area that contains
collagen fibers (%C) is automatically estimated.
2.5. Gelatinase and collagenase activity detected by in situ
zymography
Frozen tissue sections were examined for gelatinase and
collagenase activity using in situ zymography. Fluorescein
conjugates DQ gelatin from pig skin and DQ collagen type 1
from bovine skin (Molecular Probes, Inc., Eugene, OR, USA)
were used as substrates. Seven-micron sections were cut
from each frozen cervical segment, placed on poly-L-
lysine–coated slides, fixed in 10% buffered formalin for
5 minutes at 48
C, and washed three times with cold Tris-
buffered saline (TBS). Nuclear counterstaining was per-
formed with propidium iodide (Molecular Probes) diluted
1:50 (wt/vol) in TBS for 8 minutes at RT. After washing, the
slides were maintained in a darkened TBS bath at 48
C
until use. The desired substrate (DQ gelatin or DQ collagen)
was dissolved to a final concentration of 25 mg/mL in a
mixture of 2% gelatin, 2% sucrose, and 0.02% sodium azide
in TBS. The substrate solution was layered over the tissue
section, covered with a coverslip, and incubated in a
darkened humidity chamber at 37
C for 16 hours. For
control sections, the broad-spectrum MMP inhibitor 1,10-
phenanthroline (Sigma) was added at a final concentra-
tion of 10 mM and incubated at 37
C for 1 hour before
counterstaining. Each section was viewed using an
Olympus Corp. (Birkerød, Denmark) fluorescent micro-
scope with a fluorescein isothiocyanate filter.
2.6. MMP-2 and -9 activity detected by gel electrophoresis
(SDS-PAGE zymography)
Aliquots of frozen homogenates were diluted 1:1 in a 50-
mM Tris/HCl sample buffer containing 10% glycerol, 2% SDS,
and 0.0025% bromophenol blue at pH 7.6 and incubated at
37
C for 1 hour before electrophoresis. The samples were
loaded (10
m
L) onto 1-mm-thick polyacrylamide gels (10%)
copolymerized with gelatin (2.5 mg/mL, G9391; Sigma) and
electrophoresed at 100 V for 2 hours. After washing, the gels
were incubated in 50-mM Tris/HCl buffer (5 mM of CaCl
2
,
200 mM of NaCl, and 0.005% Brij 35, Sigma, pH 7.8) for
24 hours at 37
C. The gels were then stained with 1% Coo-
massie blue R-250 (Sigma) and destained. Gelatin degrada-
tion was manifest as clear zones on a blue-stained gel. The
gels were scanned, and the images were analyzed using the
menu option Analyze->Measure in ImageJ 1.46r to measure
the area and number of pixels for each clear band, then
converted to standard units using the Set Scale function. The
concentration of both latent (L) and activated (A) forms of
MMP-2 and -9 in the samples were calculated from cali-
bration curves prepared from human recombinant stan-
dards (M9445, Sigma; 5–0.08 ng/10
m
L, r¼0.9654 and
r¼0.9794 for L and A MMP-2 forms, respectively,
and M8945, Sigma; 0.5–0.008 ng/10
m
L, r¼0.9629 and
r¼0.9756 for L and A MMP-9 forms, respectively,
P<0.0001). Low, medium, and high MMP-2 and -9 standard
concentrations were loaded in each gel, with interassay
coefficients of variation of 11%, 8%, and 26%, and 6%, 11%, and
23%, respectively. Two samples were treated with 20-mM
EDTA (Sigma) as negative controls, and two were treated
with 1-mM p-aminophenylmercuric acetate (Sigma), for a
metalloproteinase-specific activation test. The activities of
both L and A forms of MMP-2 and -9 in the homogenates
were expressed in ng/mg protein using the total soluble
protein concentration (see Section 2.4), and the A/L ratios
were calculated for both MMP-2 and -9 isoenzymes.
2.7. MMP-2 and -9 histology distribution assessed by
immunohistochemistry
Sections (5
m
m) of cervical samples were deparaffinized
and rehydrated before antigen retrieval treatment, con-
sisting of steam heating in 10-mM sodium citrate buffer, pH
6.0 for 30 minutes at 100
C. After rinsing, endogenous
peroxidase activity was blocked by 30% hydrogen peroxide
in methanol for 10 minutes at RT in a humidified chamber.
To prevent nonspecific reactions, samples were incubated
with normal horse serum (Vectastain Elite ABC Kit; Vector
Laboratories, Burlingame, CA, USA) for 30 minutes at RTand
then treated with a primary antibody. Goat polyclonal
MMP-2 and -9 primary antibodies against the C-terminus
amino acidic sequence of human MMPs (hMMPs) were
used (K-20, sc-8835 and C-20, sc-6840, respectively, Santa
Cruz Biotechnology, Inc., Santa Cruz, CA, USA); they
recognize both L and A forms. Primary antibodies (dilution
1/100) were incubated at RT for 1 hour. Replacement of the
primary antibody with an equivalent concentration of
normal horse serum (sc-2025; Santa Cruz Biotechnology)
was used as a negative control. After primary antibody
binding, sections were rinsed and incubated with a bio-
tinylated horse antimouse IgG secondary antibody (Vec-
tastain Elite ABC Kit, Mouse IgG, Cat # PK-6102) at a
dilution of 1:200 in normal horse serum for 60 minutes.
After rinsing, the sections were incubated in a horseradish
avidin–biotin peroxidase complex (Vectastain Elite ABC Kit)
for 60 minutes and then in 3,3
0
-diaminobenzidine (DAB Kit,
sk-4100; Vector Laboratories) for 60 seconds. All sections
were counterstained with Mayer’s hematoxylin and
mounted with Entellan. Due to scarce and sporadic positive
M. Rodríguez-Piñón et al. / Theriogenology xxx (2015) 1–93
immunostaining obtained with the MMP-9 antibody, a
quantitative image analysis was only performed on the
selected digitized images for MMP-2 immunostaining. The
number of positive (brown stained) and negative (blue
stained) cells of each SHC (defined in Section 2.4)was
counted for a sample of 1000 cells at a magnification
of 400. Brown-stained cells were considered to be posi-
tive, irrespective of the intensity of the color. The propor-
tion of positive cells relative to the total numberof cells was
calculated (%MMP-2), and the MMP-2–positive cell density
was estimated (MMP-2 cells/mm
2
) using the area mea-
surement tool of the software (Image-Pro Express 6.0).
2.8. Statistical analysis
Data were analyzed by ANOVA using the MIXED pro-
cedure implemented in Statistical Analysis Systems (SAS
Institute, Cary, NC, USA). The model included the fixed ef-
fects of day of estrous cycle (Days 1, 6, or 13), cervical zone
(cranial or caudal), and their interactions. For immunohis-
tochemistry and histochemistry determinations, the
ANOVA model also included the fixed effect of SHC (SFS,
DFS, SWS, or DWS) and their interactions. For cervical
weight, only the effect of day of estrous cycle was consid-
ered. The %MMP-2 and the MMP-2–positive cell density
showed skewed and nonnormal distributions (Kolmo-
gorov–Smirnov Test, Statgraphics Centurion XV, 2011).
Before the analysis, these variables were log transformed
and the variance homogeneity between groups was
confirmed (Statgraphics Centurion XV, 2011). The results
are expressed as the least-square mean pooled standard
deviation, and P <0.05 was considered statistically signif-
icant, unless otherwise specified.
3. Results
3.1. Cervical weight and water content
The cervical wet weight (g) was greater on Day 1 than
on Days 6 and 13 (10.4 0.4, 7.4 0.5, and 7.9 0.9,
respectively, P <0.005). No effect of day of estrous cycle or
cervical zone on water content was found, which ranged
from 66% to 74%.
3.2. Collagen and total soluble protein content
A significant effect of day of estrous cycle on collagen
concentration (P <0.04) was found. The collagen concen-
tration was lower on Day 1 than on Days 6 and 13 (Table 1).
There was an effect of day of estrous cycle on total soluble
protein concentration (P <0.0002). The total soluble pro-
tein concentration was lower on Days 1 and 6 than on Day
13 (Table 1).
3.3. Collagen distribution
There was an effect of day of estrous cycle (P <0.002),
cervical zone (P <0.0001), and SHC (P <0.0001) on %C, as
well as an interactive effect among stroma types (P <0.005).
The %C was lower on Days 1 and 6 thanon Day 13 (37.8 1. 7% ,
37.9 1.5%, and 43.0 1.8%, respectively) and was greater in
the cranial than in the caudal cervix (47.1 1.4% and
32.7 2.7%, respectively). The %C differed between all SHCs
evaluated (25.0 2.1%, 34.4 3.7%, 44.5 2.8%, and
53.0 1.8 % fo r SF S, D FS , SWS, a nd DWS, r es pe ct ively).
In SFS, the %C was greater on Days 1 and 6 than on Day
13 in the cranial cervix, whereasit was lower on Day 1 than
on Day 13 in the caudal cervix (Table 2). In DFS, the %C was
greater on Day 1 than on Days 6 and 13 in the cranial cervix
and was greater on Day 1 than on Day 13 in the caudal
cervix. In SWS and DWS, no significant difference between
days in the %C was found in the cranial cervix. However, it
increased from Day 1 to Day 13 in SWS and from Days 1 and
6 to Day 13 in DWS in the caudal cervix (Fig. 1).
3.4. Gelatinase and collagenase activity estimated by in situ
zymography
Using in situ analysis, we found that gelatinase and
collagenase activity were localized primarily in the extra-
cellular space, although pericellular localization was also
visible (not shown). Negative controls performed with 1,10-
phenanthroline (a zinc ion chelator) completely inhibited
the fluorescence, showing that both gelatinase and colla-
genase activities were due to MMP activity. The gelatinase
activity was localized in the epithelium and in both fold
and wall stromata (Fig. 2A), whereas the collagenase ac-
tivity was restricted to the epithelium (Fig. 2B).
3.5. MMP-2 and -9 activity detected by SDS-PAGE
zymography
Using SDS-PAGE, two intense bands that migrated at
72 and 62 kDa and two weaker ones that migrated at 92
and 86 kDa were found, corresponding to the clear bands
obtained with the L and A forms of hMMP-2 and -9 stan-
dards, respectively (Fig. 3A). A clear band of w130 t o
140 kDa of unknown origin was found in four cervical
Table 1
Collagen and total soluble protein concentration (mg/g of dry tissue), gelatinase activity (ng/mg of protein) of latent (L) and activated (A) forms of matrix
metalloproteinase-2 (MMP-2), and A/L ratio in cervices of ewes on Days 1, 6, and 13 after estrus (Day ¼0).
Days after the estrous
detection (Day 0)
Collagen concentration
(mg/g dry tissue)
Total soluble proteins
concentration (mg/g dry tissue)
L MMP-2
(ng/mg of protein)
A MMP-2
(ng/mg of protein)
A/L ratio
Day 1 (n ¼6) 151 15
a
270 18
a
8.0 2.8
a
22.3 6.3
a
3.4 1.1
Day 6 (n ¼5) 200 18
b
294 25
a
2.4 1.0
b
2.4 1.1
b
1.0 0.4
Day 13 (n ¼6) 201 12
b
411 20
b
ddd
a,b
Values (mean standard error of the mean) within each column that are marked with different letters differ significantly (P <0.04).
On day 13, the L and A forms of MMP-2 were detected only in one (1.0 ng/mg of protein) and two samples (1.1 and 0.2 ng/mg of protein), respectively.
M. Rodríguez-Piñón et al. / Theriogenology xxx (2015) 1–94
samples (Fig. 3A, line 5). A clear band of gelatinase activity
at w200 kDa in the hMMP-9 standard and in two of the 34
cervical samples was found (Fig. 3B, line 1); these bands
were artifacts of MMP-9 dimerization. Treatment of sam-
ples with EDTA removed both MMP-2 and -9 gelatinase
activity bands, whereas p-aminophenylmercuric acetate
treatment reduced the bands corresponding to the L-form
and increased the bands corresponding to the A-form
(Fig. 3B, line 1), indicating that gelatinase activity is spe-
cifically exerted by MMPs.
The gelatinase activity corresponding to the L and A
forms of MMP-2 was detected in 25 of the 34 cervical
samples, whereas gelatinase activity corresponding to the L
and A forms of MMP-9 was only detected in three of the 34
cervical samples (Fig. 3A). Therefore, only bands corre-
sponding to the L and A MMP-2 forms were quantified
(Table 1).
There was an effect of day of estrous cycle (P <0.006) on
the activity of both L and A MMP-2 forms and a tendency on
the effect of day of estrous cycle on A/L ratio (P ¼0.0819).
The L and A MMP-2 activities were higher on Day 1 than on
Day 6, and they were only detected in one and two samples
on Day 13, respectively. The ratio of A/L MMP-2 trends to be
higher on Day 1 than on Day 6 (P ¼0.0819).
3.6. Characterization of MMP-2 and -9 by
immunohistochemistry
The MMP-2 immunostaining was localized in the
extracellular space for all SHCs and was primarily detected
in active fibroblasts but also in inactive fibroblasts and
occasionally leukocytes (Fig. 4A, B). Weak and sporadic
MMP-9 immunostaining was detected, which was limited
to the cytosol of some stromal cells. Positive cells were
mainly plasmocytes, some macrophages, a few lympho-
cytes, and very few fibroblasts (Fig. 4A, C). Therefore, only
the immunohistochemical signal corresponding to MMP-2
was quantified.
There was an effect of cervical zone (P <0.0001), SHC
(P <0.007), and an interaction between day of estrous cycle
and cervical zone (P <0.0001) on %MMP-2. The %MMP-2
(%) was lower in the cranial (0.32 0.01%) than in the
caudal (1.96 0.06%) cervix. It was lower in DWS than in
DFS and SWS and trends to be lower in SFS than in SWS
(0.49 0.03%, 1.14 0.06%, 1.34 0.06%, and 0.24 0.02%
in SFS, DFS, SWS, and DWS, respectively). The %MMP-2
decreased throughout the estrous cycle in the cranial cervix
but was higher on Day 13 than on Days 1 and 6 in the caudal
cervix (Table 3).
There was an effect of cervical zone (P <0.0001) and
SHC (P <0.0004) on MMP-2–positive cell density. The
MMP-2 cells density was lower in the cranial (569 106)
than in the caudal (2917 401) cervix. It was higher in SFS,
DFS, and SWS than in DWS (1644 265, 1180 213,
2070 367, and 409 89, respectively).
4. Discussion
We reported for the first time in the ovine cervix that
the collagen content is lower and MMP-2 activity is higher
in estrus than in the luteal phase of the natural estrous
cycle.
Table 2
Area occupied by collagen fibers expressed as a percentage (%C) in different stromal histologic compartments of the cervices of ewes on Days 1, 6, and 13 after
estrus (Day ¼0).
Days after the estrous
detection (Day 0)
SFS DFS SWS DWS
Cranial Caudal Cranial Caudal Cranial Caudal Cranial Caudal
Day 1 (n ¼6) 36.0 1.1
a
13.6 4.3
a
58.5 4.1
a
24.1 2.6
a
51.2 0.8 18.4 2.7
a
48.8 1.5 52.3 1.9
a
Day 6 (n ¼5) 37.4 1.1
a
17.5 3.7
ab
48.4 3.3
b
13.4 2.2
b
51.0 1.2 40.1 2.3
b
52.1 4.3 45.7 1.4
a
Day 13 (n ¼6) 26.5 2.2
b
22.4 5.0
b
47.2 3.2
b
18.4 2.7
ab
55.3 2.3 51.9 5.6
c
51.4 1.2 69.5 2.3
b
a,b,c
Percentages (mean standard error of the mean) within a column that are marked with different letters differ significantly (P <0.005).
SFS, DFS, SWS, and DWS are histologic compartments.
Abbreviations: DFS, deep fold stroma; DWS, deep wall stroma; SFS, superficial fold stroma; SWS, superficial wall stroma.
Fig. 1. Images of collagen fibers with van Gieson staining from superficial wall stroma of the caudal cervix from the same ewe on Days 1 (A) and 13 (B) after estrus
detection (Day 0). Note the lower density of fibers on Day 1 than on Day 13.
M. Rodríguez-Piñón et al. / Theriogenology xxx (2015) 1–95
Both the cervical collagen concentration and distribu-
tion were lower 1 day after estrus detection compared with
during the luteal phase, suggesting that either the pre-
ovulatory estrogen levels increase collagen degradation or
the progesterone luteal levels inhibit it. Consistent with
these interpretations, the cervical collagen concentration
and distribution remain unchanged throughout most of
ovine pregnancy but decrease in the final month [25], when
circulating progesterone begins to decrease and estrogen
increases [26]. Because the collagen concentration was
expressed with respect to the dry tissue and the water
content did not change during the estrous cycle, the
decrease in collagen 1 day after estrus detection is due to a
genuine collagen degradation and not a dilution effect. In
the cervix of cycling cows, both water and collagen content
(based on dry tissue) were not associated with the pro-
gesterone status [27], suggesting interspecific differences
in the magnitude of collagen degradation during cervical
softening at estrus.
The wet weight of the cervix was higher 1 day after
estrus detection than in the luteal phase, despite similar
water contents; this is in agreement with the increase in
cervical wet weight without changes in water content re-
ported by Regassa and Noakes [25] during ewe pregnancy.
Interestingly, there was maximal cervical weight around
the estrus, when the collagen content, total soluble pro-
teins, cell proliferation [28], and nuclear density [15] were
minimal. These data suggest that the increase in the cer-
vical wet weight around estrus is due to an increase in the
nonprotein component of the cervical ECM. Hyaluronan-
like GAGs are 80% to 90% of all GAGs in the sheep cervix
and increase before the LH preovulatory surge in the cervix
of estrus-synchronized ewes [16,17]. Overall data suggest
that the ovine cervical dilatation around the estrus is a
consequence of a tissue remodeling process involving both
an increase in collagen degradation and an increase in
hyaluronan-like GAG synthesis, which have been suggested
in the human cervix during pregnancy [7,29].
In the present work, the observed gelatinase activity in
the cervix of cycling ewes was found by in situ and SDS-PGE
gelatin zymographies. Using in situ zymography, we
observed both collagenase and gelatinase activities in the
luminal epithelium but only gelatinase activity in
the stroma. The stromal gelatinase activity may be due to
the presence of MMP-2 and -9 (gelatinases) because the
immunostaining of both MMP-2 and -9 was restricted to
stroma. The gelatinase activity bands corresponding to the L
and A forms of MMP-2 were detected in all cervical samples,
but slight bands of the L and A MMP-9 forms were detected
in very few samples. Immunohistochemical findings
confirmed these observations because MMP-2–positive
cells were detected in all samples, but MMP-9–positive cells
were scarce detected or undetected. These results suggest
that the gelatinase activity in the cervix of cycling ewes is
predominantly caused by the MMP-2 isoenzyme. In addi-
tion, MMP-2 immunostaining was mainly associated with
active fibroblasts, whereas MMP-9 immunostaining was
associated with cells related to immune and inflammatory
processes. These data are in agreement with those of Stygar
et al. [21], who reported that stromal fibroblasts of the
human cervix are the main source of MMP-2, whereas
MMP-9 is restricted to leukocytes. The scarce MMP-9 ac-
tivity levels and its association with cells related to immune
and inflammatory processes suggest that this type of pro-
cesses are not predominant in the cervical ECM modifica-
tions during the estrous cycle. No reports describing the
occurrence of inflammatory and immune-mediated–like
processes were found in ruminant cervices during the
estrous cycle. In the ovine cervix, although an increase in
cervical interleukin 8 (a proinflammatory cytokine) was
detected at estrus [30], administration of interleukin 8 had
no effect on cervical penetrability [31].
Interestingly, the levels of the activated form of cervical
MMP-2 were approximately 10 to 20 times higher around
the estrus, when cervical penetrability is maximal [4], than
during the luteal phase. Concomitant with this increase in
MMP-2 activity 1 day after estrus detection, a decrease in
the cervical collagen concentration and the percentage of
collagen fibers was found. Raynes et al. [32] failed to detect
changes in cervical collagenase activity in ewes during
gestation using a semisynthetic collagen-like substrate.
The higher levels of cervical MMP-2 activity found after
estrous detection could result from the induction of
MMP-2 expression by estrogen during the preovulatory
peak and/or the lack of luteal progesterone. The MMP
hormonal regulation could occur at the transcriptional and
posttranscriptional levels via changes in the rate of
messenger RNA synthesis and/or stability (half-life) [33].
Fig. 2. Gelatinase (A) and collagenase (B) activities by in situ zymography (400) in the cervixof cyclingewes. Note the presence of gelatinase activity in both the
epithelium and stroma, whereas the collagenase activity was restricted to the epithelium.
M. Rodríguez-Piñón et al. / Theriogenology xxx (2015) 1–96
Anuradha and Thampan [34] reported that E2-mediated
enhancement of collagenase activity in the rat uterus is
inhibited by actinomycin D and cycloheximide, indicating a
transcriptional inductive effect of estrogens on collagenase
expression. In agreement with this, an increase in uterine
MMP-2 expression at 48 hours after E2 treatment has been
observed in rats [35]. The relatively prolonged latency be-
tween the E2 stimulus and the increase in uterine MMP-2
expression could be explained, at least in part, by the 12-
to 36-hour half-life of MMP transcripts [36]. Data suggest
that the increase in cervical MMP-2 activity 1 day after
estrus detection could reflect a previous stimulatory effect
of preovulatory estrogens on MMP-2 expression, occurring
before the onset of estrus, under maximal concentrations of
circulating E2 [37]. This early preovulatory estrogen-
stimulatory effect on MMP-2 expression could be main-
tained during the estrus via high levels of cervical estrogen
receptors [12,15]. In addition, the MMP-2 A/L ratio trends to
be higher on Day 1 after estrus detection than during the
luteal phase, indicating that the estrogen-induced increase
in MMP-2 activity may be due to an increase in both protein
expression and enzyme activation.
The increased activity of MMP-2 1 day after the onset of
estrus may be due to an inductive effect of preovulatory es-
trogens, but other stimulatory factors cannot be ruled out.
Cervical PGE2 production increases in response to LH [38]
and Ox [39,40] in the bovine cervix, although both PGE2
receptortypes 4 and 2 have been reported in the cervix of the
ewe [16,41,42] which can mediate MMP secretion [43,44].In
an elegant in vitro experiment in the human cervix, PGE2
treatment decreased the incorporation of [
3
H] glycine but
increased [
3
H] glucosamine, precursors of collagen, and
GAGs in samples obtained during the follicular phase, and
the oppositewas found in samples obtained during the luteal
phase [45]. Overall, the data report an association between
various hormones at the control of cervical collagen
remodeling. Moreover, low-molecular-weight hyaluronic
acid (HA) can induce collagenase and gelatinase activity in
the rabbit cervix [46].
The rearrangement and dissociation of collagen fibers
and bundles is thought to contribute to cervical relaxation
in the ewe at estrus [47,48]. Kershaw et al. [48] found a
higher proportion of collagen relative to smooth muscle
before the LH surge than during the medium luteal phase
(on Day 9), and this result was attributed to estrogen-
induced separation of collagen bundles and fibers via HA
accumulation [16,17]. In the present work, a pre-LH stage
was not assessed; however, 1 day after estrous detection
(probably after the LH preovulatory surge), the area occu-
pied by collagen fibers and the collagen concentration were
lower, and the cervical MMP-2 activity and A/L ratio were
higher than they were in the late luteal phase (on Day 13).
Fig. 3. Gelatin zymography of cervical sample homogenates of ewes during the estrous cycle. (A) Lines 1, 2, and 3: 10
m
L of mixed human recombinant standards
of latent and activated matrix metalloproteinase (MMP)-2 (72 and 62 kDa, respectively; Sigma–Aldrich, M9445; 5.0, 0.62, and 0.08 ng in each line, respectively)
and latent and activated MMP-9 (92 and 86 kDa, respectively; Sigma–Aldrich, M8945; 0.5, 0.062, and 0.008 ng in each line, respectively). Lines 4, 5, and 6: 10
m
Lof
cervical sample homogenates of different ewes on Day 1 after estrus (Day 0). (B) Line 1: 10
m
L of cervical sample homogenates. Line 2: 10
m
L of cervical sample
homogenates preincubated with 1-mM p-aminophenylmercuric acetate (APMA). Note the clear bands of approximately 130 to 140 kDa of unknown origin in
panel A, line 5 and 200 kDa at the top of panel B, line 1.
Fig. 4. Immunohistochemical detection of matrix metalloproteinase-2 and -9 (MMP-2 and MMP-9, respectively). Negative controls omitted primary antibodies
(bar ¼50
m
m, A), MMP-2 (bar ¼20
m
m, B), and MMP-9 (bar ¼10
m
m, C). Note that the MMP-2–positive immunostaining localized pericellularly to active
fibroblasts (arrows, B), whereas the MMP-9–positive immunostaining localized in the cytoplasm of plasmocytes (arrows, C).
M. Rodríguez-Piñón et al. / Theriogenology xxx (2015) 1–97
Both Kershaw et al. [48] and the present report indicate
that collagen fiber disaggregation occurs from the late
luteal phase until ovulation, followed by enzymatic
collagen degradation. These results strongly suggest that
the dissociated and partially denatured collagen fibers that
result from HA accumulation during the early stage of the
follicular phase are degraded by MMP-2 during the late
follicular phase. Thus, around the estrus, both disaggrega-
tion and degradation of cervical collagen fibers coexist and
cooperate for the remodeling of the ECM. The temporal
relationship between the cervical collagen degradation and
the periovulatory hormonal environment should be taken
into account in the design of treatments for induction of
cervical dilation in artificial insemination and embryo
transfer techniques.
Interestingly, although the percentage of MMP-2–
positive cells did not change during the estrous cycle, its
pattern of variation differed between cranial and caudal
cervical regions, suggesting that there is a differential time-
dependent stimulation of MMP2 protein synthesis along
the longitudinal axis of the cervix. The increase in MMP-2
expression before ovulation (on Day 13 after estrus detec-
tion) in the caudal cervix could play a permissive role for
upward progress of sperm at copulation [49] by inducing
cervical relaxation. The increase of MMP-2 expression
around the time of ovulation (on Day 1 after estrus detec-
tion) in the cranial cervix could be related to its role as a
spermatic reservoir [49] by softening the cervical folds. This
differential MMP-2 protein expression between the cranial
and caudal cervix could be regulated directly or indirectly
through local tissue- or cell-dependent factors that interact
with regulation via ovarian steroid hormones [20]. Differ-
ential expression of the receptors of estrogen, progesterone,
LH, FSH, Ox, and PGE2, as well as of COX-2 and hyaluronan
synthase enzymes between the cranial and caudal cervix
has also been reported in the ewe [12,15,16,48,50,51].
Interestingly, the percentage and density of MMP-2
protein-positive cells were greater in the caudal than in
the cranial cervix, and the percentage of collagen fibers was
lower in the caudal than in the cranial cervix, suggesting a
high potential rate of collagen degradation in the caudal
cervix. The MMP-2–positive cell density showed a similar
pattern of variation to the total nuclear density previously
reported [15]. Both the MMP-2–positive cells and the total
nuclear density were lower in the cranial than in the caudal
cervix, and maximum in SWS and minimum in DWS, sug-
gesting that the MMP-2–positive cell density is strongly
influenced by the total cell density. Overall, these data
indicate molecular and functional differences between the
cranial and caudal ovine cervix; these differences are
important considerations in the design of local treatment
protocols to induce cervical dilatation.
In this study, a decrease in the cervical collagen levels
around the ovine estrus was reported, which occurs
concomitant with an increase in the activity of MMP-2,
which is produced by stromal fibroblasts and activated by
changes in periovulatory hormone levels. Additionally, a
time-dependent differential increase in MMP-2 expression
along the longitudinal axis of the cervix was detected,
occurring before ovulation in the caudal cervix and around
the time of ovulation in the cranial cervix, reflecting their
different physiological roles.
Acknowledgments
The authors would like to thank P. Rubianes for technical
assistance, M. Marco and G. Lin for technical assistance in
gelatin zymography,and C. Maeda Takiya, V. Samoto, and A.
Dantas Medeiros for the generous and selfless technical
assistance in in situ gelatin zymography. The authors
received financial support from Comisión Sectorial de
Investigación Científica (CSIC) and Programa de Desarrollo
de Ciencias Básicas (PEDECIBA), Universidad de la Repúb-
lica; Comisión de Investigación y Desarrollo Científico
(CIDEC) and Programa de Posgrados, Facultad de Veter-
inaria; Fondo Clemente Estable, Agencia Nacional de
Investigación e Innovación (ANII); and Dirección Nacional
de Ciencia y Tecnología (DINACYT), Ministerio de Educación
y Cultura, Uruguay.
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