Content uploaded by Marcelo Rodríguez-Piñón
Author content
All content in this area was uploaded by Marcelo Rodríguez-Piñón on Mar 06, 2014
Content may be subject to copyright.
Content uploaded by Marcelo Rodríguez-Piñón
Author content
All content in this area was uploaded by Marcelo Rodríguez-Piñón on Mar 06, 2014
Content may be subject to copyright.
CSIRO PUBLISHING
Reproduction, Fertility and Development, 2008, 20, 350–356 www.publish.csiro.au/journals/rfd
Oestrogen and progesterone receptor binding capacity
and oestrogen receptor alpha expression (ERαmRNA)
along the cervix of cycling ewes
M. Rodríguez-PiñónA,B,C. TasendeA,P. PuimeAand E. G. GarófaloA
ABiochemistry, Department of Molecular and Cellular Biology, Faculty of Veterinary,
Montevideo, Uruguay.
BCorresponding author. Email: marodri@adinet.com.uy
Abstract. The aim of the present work was to study the oestrogen receptor (ER) and progesterone receptor (PR) binding
capacity and the oestrogen receptor alpha (ERα) mRNA concentration in cranial and caudal cervix during the ovine
oestrous cycle. Cervical samples of synchronised Corriedale ewes were obtained on Day 1 (n=7),6(n=6) or 13 (n=7)
after oestrus detection (Day 0). The ER and PR binding capacity by ligand-binding assay and the ERαmRNA concentration
by solution hybridisation in both cranial and caudal zones of the cervix were determined.The ER and PR binding capacity
were higher (P<0.005) on Day 1 than on Days 6 and 13 in both cranial and caudal zones. The ERαmRNA concentrations
were higher (P<0.0001) on Day 1 than on Days 6 and 13 only in the caudal zone. The PR binding capacity and ERα
mRNA concentration were higher (P<0.005) in the caudal than in the cranial zone on Day 1. The ER and PR expression
in the ovine cervix varied during the oestrous cycle in agreement with the known upregulation exerted by oestrogen and
downregulation exerted by progesterone. Differences in ER and PR expression along the longitudinal axis of the ovine
cervix were found, reflecting histological and functional differences between the cranial and caudal zones.
Additional keyword: ewe cervical steroid receptors.
Introduction
The ewe cervix acts as a barrier against the passage of the tran-
scervical instruments for artificial insemination (AI) (Lightfoot
and Salamon 1970; Halbert et al. 1990a, 1990b; Campbell et al.
1996) and for embryo transfer (ET) procedures (Armstrong and
Evans 1983; Kraemer 1989; Croy et al. 1999). Treatments with
oestrogen combined with relaxin (Nemec et al. 1988), oxytocin
(Khalifa et al. 1992; Flohr et al. 1999), or prostaglandin E2
(Barry et al. 1990; Mylne et al. 1992; McKelvey et al. 1997)
have been performed to increase cervical dilatation and to facil-
itate the passage through the ovine cervix into the uterine lumen.
Those studies suggest that cervical dilatation and penetrability
varies along the oestrous cycle and are under the influence of
the ovarian steroid hormones. In addition, the cervix of the ewe
supports the passage of normal motile sperm by modifications
of the physical and molecular composition of the cervical mucus
(Lee et al. 1986), associated with cyclic changes in morphology
and secretory capacity of the luminal epithelium (Moré 1984).
These events are modulated by ovarian steroid hormones.
The genomic actions of oestrogen (E) and progesterone (P)
depend on their circulating concentrations and on the target
tissue’s sensitivity in terms of their specific and high-affinity
nuclear receptor concentrations (ER and PR, respectively) (Clark
et al. 1992; Meikle et al. 2004). It is accepted that E upregulates
ER and PR expression, whilst P downregulates both recep-
tors (Ing et al. 1993; Couse et al. 2006). This regulation is
consistent with the higher uterine ER and PR protein and ERα
mRNA and PR mRNA concentrations at oestrus than in the luteal
phase found in ruminants (Miller et al. 1977; Rexroad 1981;
Vesanen et al. 1988; Ott et al. 1993; Meikle et al. 2001a;Tasende
et al. 2005b). However, data on cervical ER and PR expression
during the oestrous cycle are contradictory. Cyclic variations in
ER and PR (Stanchev et al. 1984) and in ERαmRNA and ERα
protein (Wang et al. 2000) concentrations were found in pig and
rat, respectively. On the contrary, no variations in cervical ER
concentrations were found in cow (Vesanen et al. 1991) and mare
(Re et al. 1995), species in which PR concentration varies.
In the ewe, information about the cervical ER and PR bind-
ing capacity is scarce. The presence of the cervical ER binding
protein in ovariectomised ewes (Tang and Adams 1981, 1986),
as well as the cervical ER, PR, ERαmRNA, and PR mRNA in
prepubertal lambs (Meikle et al. 2001b; Rodríguez-Piñón et al.
2005) have been previously demonstrated. On the other hand,
during the postpartum period in the breeding season, the cer-
vical ER and PR concentrations were low after parturition and
increased in late postpartum, associated with the presence of
the E-active large follicles in the ovarian surface (Rodríguez-
Piñón et al. 2000). This suggests that changes in the cervical
steroid receptors are related to the levels of ovarian steroid hor-
mones. We are unaware of any reports of the cervical ER and PR
binding capacity during the oestrous cycle in ewes. However, by
immunohistochemical technique, both ER and PR were detected
© CSIRO 2008 10.1071/RD07186 1031-3613/08/030350
Cervical steroid receptors in cycling ewe Reproduction, Fertility and Development 351
in the epithelium around the time of oestrus, but not in the luteal
phase (Zhao et al. 1999). In addition, in bovine cervix, the ER
and PR immunoreactivity increased from the cranial to the cau-
dal region (Breeveld-Dwarkasing et al. 2000, 2002), suggesting
that the distribution of these steroid receptors could be different
along the longitudinal axis of the ruminant cervix.
The aim of the present work was to study the ER and PR
binding capacity and the ERαmRNA concentration of the cranial
and caudal ovine cervix in relation to levels of circulating E and
P during the oestrous cycle, including the days whenAI and ET
would be performed (Days 1 and 6 after oestrus, respectively).
Materials and methods
Experimental design
The experiment was performed at the experimental field of Vet-
erinary Faculty, Canelones, Uruguay (35◦S), during the breeding
season of Corriedale ewes (February to March). Animal experi-
mentation was performed in compliance with regulations set by
the Veterinary Faculty, University of Uruguay.
Twenty adult Corriedale ewes (bodyweight, mean ±pooled
s.e.m., 39.1 ±0.85 kg) were synchronised with two doses,
6 days apart, of a prostaglandin F2αanalogue intra-muscularly
(i.m.) (150 µg, Glandinex, Laboratorio Universal, Montevideo,
Uruguay). From Day10 of the first oestrous cycle, ewes remained
with two vasectomised rams with marking crayons and were
checked twice a day (at 600 hours and 1800 hours) for service
marks indicative of oestrus (day of oestrus=Day 0). All ani-
mals were located under natural daylength, grazed on native
pastures and given water ad libitum. The ewes were killed on
Day1(n=7), 6 (n=6) or 13 (n=7) after oestrus detection.
The whole cervices were dissected into three equal length seg-
ments named cranial, middle and caudal zones and including
all the histological layers. The cranial (next to the uterus) and
caudal (next to the vagina) zones were selected for the present
study. The tissues were frozen in liquid nitrogen and stored at
−80◦C until receptor and transcript determinations were carried
out. The number of ruptured follicles and corpus luteum (CL)
present in the ovarian surface were recorded.
For circulating P determination, blood samples were collected
daily from Day −1 of expected oestrus until the killing time in
the second oestrous cycle. For oestradiol-17β(E2) determina-
tions, the samples were collected every 8 h during the 48 h period
before the killing time. The samples were centrifuged (900gfor
15 min at 4◦C) within the first hour after collection and serum
was stored at −20◦C until hormone assays were performed.
Hormone determinations by radioimmunoassay (RIA)
Progesterone concentrations were assayed by direct solid-phase
125I radioimmunoassay (RIA) method (Count-A-Count TKPG;
Diagnostic Products Corporation, Los Angeles, CA, USA),
according to the manufacturer’s instructions and as previously
described for sheep (Garófalo and Tasende 1996). All samples
were performed in duplicate in the same assay. The sensitivity of
the assay was 0.1nm, and the intra-assay coefficient of variation
was less than 10%.
For the E2assay, the serum samples were extracted with
diethyl ether and assayed in duplicate with 125I RIA (oestra-
diol double antibody, KE2D; Diagnostic Products Corporation)
as previously described for sheep (Meikle et al. 1998; Tasende
et al. 2002). All samples were determined in the same assay. The
detection limit of the assay was 4.0pmand the intra-assay coef-
ficients of variation for three control samples were 25% (7pm),
8% (44 pm), and 6% (122 pm).
Steroid receptors by binding assay
Ligand-binding assays for ER and PR were performed in soluble
fraction of cervix as described previously (Garófalo and Tasende
1996; Rodríguez-Piñón et al. 2000). The term ‘soluble fraction’
refers to the supernatant fractions of tissue homogenates after a
high-speed centrifugation, and does not imply cellular receptor
localisation. Unless otherwise stated, the reagents were obtained
from Sigma Chemicals (St Louis, MO, USA). The frozen cer-
vical samples (300 to 500 mg) were sliced and homogenised in
Tris buffer with a Polytron homogenizer (Polytron homogenizer
PT-10, Kinematica AG, Littau Luzern, Switzerland). The solu-
ble fractions were separated by a first centrifugation at 1000g
for 15 min and then at 40 000gfor 90min. This and subsequent
procedures were carried out at 0–4◦C. The soluble fractions, in
duplicate, were incubated with five to six increasing concentra-
tions of 3H-E2(86 Ci mmol−1; 0.15–15 n m)or3H-ORG-2058
(40 Ci mmol−1; 0.5–30 nm) (Amersham International, Buck-
hinghamshire, England), for determination of the total bound
3H-labelled ligands. Identical duplicate samples were incubated
with 200-fold molar excess of either unlabelled diethylstilbestrol
or unlabelled ORG-2058, for determination of non-specifically-
bound 3H-labelled ligands. After 18h incubation free hormones
were removed and radioactivity was measured by liquid scintilla-
tion counting. Specific binding data were obtained by subtracting
non-specific binding from total binding. A linear regression test
of inverse Scatchard model analysis (Braunsberg 1984) was used
to obtain the apparent dissociation constant (Kd,nm) and the
concentration of receptor sites expressed as fmol mg−1proteins.
Protein concentrations were determined by the method of Lowry
et al. (1951). The protein concentration was positively correlated
with the amount of tissue used in the receptor assay, showing
that the protein extraction procedure was similar in the different
cervical samples.
ERαmRNA by solution hybridisation assay
The method used was previously validated for cervical ovine
tissue (Meikle et al. 2001b). The hybridisation probe used was
derived from plasmids containing 360 bp cDNAs from the ovine
ERα, generously supplied by Dr N. Ing, Texas A&M Univer-
sity, TX, USA (Ing et al. 1996). Total nucleic acids (TNA)
were obtained by digesting homogenised (Polytron homogenizer
PT-10, KinematicaAG) cervical tissues (200–250mg) with pro-
teinase K in a buffer containing sodium dodecyl sulfate (SDS)
followed by subsequent extraction with phenol-chloroform. The
TNA content in the samples was determined spectrophotomet-
rically at 260 nm and expressed as absorbance relative units
(ARU). The 35S-UTP-labelled cRNA was hybridised overnight
at 70◦C to TNA samples. The hybridisations were performed in
352 Reproduction, Fertility and Development M. Rodríguez-Piñón et al.
duplicates at two different concentrations in 40µL of hybridi-
sation formamide buffer under two drops of paraffin oil. After
hybridisation, samples were treated with 1mL of RNase buffer
containing 40 µg RNase A, 118 U RNase T1 (Boeringer-
Mannheim, Mannheim, Germany), and 100 µg calf thymus
DNA for 45min at 37◦C to digest unhybridised RNA. Labelled
hybrids protected from RNase digestion were precipitated with
trichloroacetic acid and collected on filters (Whatman GF/C,
Whatman Nederland B.V., Hertogenbosch, The Netherlands).
Radioactivity was determined in a liquid scintillation counter. All
samples from the experiment were determined in the same assay
and the intra-assay CV was 16%.The ERαmRNA concentrations
were expressed as amolARU−1.
Statistical analysis
The ERαmRNA, ER and PR concentrations and Kdvalues were
analysed by analysis of variance using a mixed model (Statistical
Analysis Systems; SAS Institute, Cary, NC, USA) that included
the fixed effects of day of oestrous cycle (Day 1, 6 or 13), cervical
zone (cranial or caudal), and their interaction. Progesterone and
E2serum concentrations were analysed by repeated-measures
(Mixed Proc), and the statistical model included the effects of
time of sampling, day of oestrous cycle, and their interaction.
The correlation procedure available in SAS was used to study
the relationship between ER, PR, ERαmRNA, and hormone
concentrations. For the correlations between receptor and hor-
mone concentrations, the mean of the E2or P concentrations
pooled samples of 24, 16, 8, and 0 h (for E2), and 24 and 0h (for
P) before the killing time, were considered. The results were
expressed as the least square mean ±pooled s.e.m. The level of
significance was P<0.05, except where otherwise specified.
Results
Structures on the ovarian surface and hormone
concentrations
All ewes killed on Day 1 had one ruptured follicle and those
killed on Days 6 and 13 had one corpus luteum on the ovarian
surface. There was a significant effect of time of sampling on
P serum concentrations (P<0.0001). Progesterone concentra-
tions were low from Day −1 to Day 1 after oestrus, and then
increased significantly starting from Day 3 to Day 13 (Fig. 1).
There was a significant effect of time of sampling on E2serum
concentrations (P<0.001). Maximum E2concentrations were
found between 24 and 8h before oestrus detection. After this
time E2concentrations decreased and remained at basal levels
(Fig. 1).
Steroid receptor binding capacity
A single, saturable and high-affinity binding site for E and P
were found in all samples. The Kdvalues were not affected by
day of cycle or cervical zone. The Kdmeans (±pooled s.e.m.,
n=40, nm) for ER and PR were 0.56 ±0.06 and 1.04 ±0.07,
respectively.
There was a significant effect of day of oestrous cycle on ER
concentrations (P<0.005), but there was no effect of cervical
zone. The ER concentrations were higher on Day 1 than on Days
0
2
4
6
8
10
12
14
16
21012345678910 14131211
Day of oestrous cycle
Progesterone (nmol L1)
0
5
10
15
20
25
30
Oestradiol-17 (pmol L1)
Progesterone
Oestradiol-17
*
*
*
Fig. 1. Serum concentrations (mean ±pooled s.e.m.) of P (nm) and E2
(pm) of ewes killed (arrows) on Days 1 (n=7), 6 (n=6) or 13 (n=7)
after oestrus (Day 0). Values marked with an asterisk differ significantly
(P<0.001).
6 and 13 in both cranial and caudal zones (Fig. 2a). There were
no differences in ER concentrations between cranial and cau-
dal zones on Days 1 and 6, while on Day 13 ER concentration
tended to be lower (P=0.054) in the cranial than in the caudal
zone (Fig. 2a). The ER concentrations were positively correlated
with E2concentrations only in the caudal zone (r=0.48, n=20,
P<0.05), and were negatively correlated with P concentrations
in both cranial (r=−0.43, n=20, P<0.05) and caudal zones
(r=−0.69, n=20, P<0.001).
There was a significant effect of day of oestrous cycle
(P<0.0001) and cervical zone (P<0.0004) on PR concen-
trations, but there was no interaction between them. The PR
concentrations were higher on Day 1 than on Days 6 and 13
in both cranial and caudal zones (Fig. 2b). The PR concentra-
tions were lower in the cranial than in the caudal zone on Day 1
(Fig. 2b). In both cervical zones, PR concentrations were posi-
tively correlated with E2concentrations (cranial zone, r=0.58,
n=20, P<0.01; caudal zone, r=0.44, n=20, P<0.05),
and negatively correlated with P concentrations (cranial zone,
r=−0.76, n=20, P<0.0001; caudal zone, r=−0.70, n=20,
P<0.0005).
Independently of the day of oestrous cycle, there was a pos-
itive correlation between ER and PR concentrations in both
cranial (r=0.52, n=20, P<0.01) and caudal zones (r=0.80,
n=20, P<0.00001), indicating a positive relationship between
the expression of these two receptors.
ERαmRNA concentrations
There was a significant effect of day of oestrous cycle
(P<0.0001), cervical zone (P<0.005), and interaction
between them (P<0.05) on ERαmRNA concentrations. In
the caudal zone, ERαmRNA concentrations were higher on
Day 1 than on Days 6 and 13 (Fig. 3). In contrast, no differ-
ences between days were found in the cranial zone (Fig. 3).
The ERαmRNA concentrations on Day 1 were lower in the
Cervical steroid receptors in cycling ewe Reproduction, Fertility and Development 353
0
50
100
150
200
250
(a)
(b)
1613
Day of oestrous cycle
ER (fmol mg1 proteins)PR (fmol mg1 proteins)
Cranial
Caudal
Cranial
Caudal
0
250
500
750
1000
1250
1500
1613
Day of oestrous cycle
a
b*
b
a
b*
b
a
d
cd
b
cd
c
Fig. 2. Concentrations (fmol mg−1proteins, mean ±pooled s.e.m.) of
oestrogen receptor (ER, panel a) and progesterone receptor (PR, panel b)
binding proteins in cranial and caudal cervical zones of ewes killed on Days
1(n=7),6(n=6) or 13 (n=7) after oestrus (Day 0). Bars marked with
different letters differ significantly within days and zones (P<0.005) and
bars marked with asterisks tended to be different (P=0.054).
0
20
40
60
80
100
1613
Day of oestrous cycle
ER mRNA (amol ARU1)
a
a
a
b
a
a
Cranial
Caudal
Fig. 3. Oestrogen receptor αmessenger RNA (ERαmRNA) concentra-
tions (amolARU−1, mean ±pooled s.e.m.) in cranial and caudal cervical
zones of ewes killed on Days 1 (n=7),6(n=6) or 13 (n=7) after oestrus
(Day 0). Bars with different letters are significantly different within days and
zones (P<0.05).
cranial than in the caudal zone (Fig. 3). The ERαmRNA con-
centrations in the caudal zone were correlated positively with E2
(r=0.59, n=20, P<0.01) and negativelywith P concentrations
(r=−0.74, n=20, P<0.0005). There were no correlations
between ERαmRNA and E2and P concentrations in the cra-
nial zone. There was a positive correlation between ERαmRNA
and ER concentrations only in the caudal zone (r=0.52, n=20,
P<0.05).
Discussion
In the present work we demonstrated that the expression of ER
and PR in the ovine cervix varied during the oestrous cycle as
well as along the longitudinal cervical axis.
High-affinity receptor binding proteins for oestrogen and
progesterone were found in all cervical samples studied.
The Kdvalues for ER and PR in all days of the oestrous cycle
and all cervical zones studied were similar, suggesting that vari-
ations in the sensitivity of the ovine cervix to E and P may
not depend on changes of affinity of the receptors, but rather
on their binding capacity (ER and PR cervical concentrations).
The cervical ER and PR Kdvalues were similar to those found
in adult (Rodríguez-Piñón et al. 2000) and prepubertal ewes
(Meikle et al. 2001b; Rodríguez-Piñón et al. 2005), as well as in
other E and P ovine target tissues (Garófalo and Tasende 1996;
Meikle et al. 2000; Tasende et al. 2005a, 2005b).
The cervical ER and PR concentrations were higher on Day 1
than on Days 6 and 13 of the oestrous cycle.This steroid receptor
pattern is consistent with the stimulatory effect of the high E2
levels found previous to oestrus and the inhibitory effect of the
high P levels found during the luteal phase.The present findings
in ovine cervix are in agreement with the E upregulation and
P downregulation of the ER and PR expression found in ovine
uterus during the oestrous cycle (Miller et al. 1977; Rexroad
1981; Ott et al. 1993; Tasende et al. 2005b). The existence of
this ovarian steroid hormone regulation on the expression of ER
and PR in ovine cervix during the oestrous cycle was confirmed
by the positive correlation found between E2levels and ER (at
least in caudal zone) and PR concentrations, and the negative
correlation between P levels and ER and PR concentrations.
Similar cyclic variations in cervical steroid receptor expres-
sion during the oestrous cycle were also reported for ER and PR
in the pig (Stanchev et al. 1984) and for ERαin the rat (Wang
et al. 2000). On the contrary, cyclicvariations in cervical ER con-
centration were not found in cows (Vesanen et al. 1991) or mares
(Re et al. 1995), although the cervical PR concentrations in the
same animals were higher in the non-luteal than in the luteal
phase. In ovine cervix, the epithelial ER and PR immunoreactiv-
ity were detected in the first three days of the oestrous cycle, but
were not detected during the luteal phase (Zhao et al. 1999). In a
preliminary study we immunolocalised the ERαin the cervical
epithelium of cycling ewes, and the percentage of ERαposi-
tive cells was higher around the oestrus than in the luteal phase
(M. Rodríguez-Piñón, P. Genovese, R. González, P. Puime and
A. Bielli, unpubl. data).
The pattern of variation of the cervical ER and PR bind-
ing capacity during the oestrous cycle was similar to that found
for the oxytocin receptor (OxR) binding capacity, with a maxi-
mum seen around the oestrus (Matthews and Ayad 1994). These
results suggest that one of the E and P target genes in the
cervix could be the OxR, since the sex ovarian hormones are
important regulators of the OxR gene (Gimpl and Fahrenholz
354 Reproduction, Fertility and Development M. Rodríguez-Piñón et al.
2001). The ovarian steroid hormones could modulate several
oxytocin effects on the cervix of cycling ewes by up or down-
regulation of the OxR. Since oxytocin stimulates the release
of prostaglandin E2 from the cervical mucosa of perioestrous
cows (Fuchs et al. 2002), some of the oxytocin effects on the
ruminant cervix could be mediated via paracrine release of
prostaglandin. Additionally, E could also directly modulate the
activity of prostaglandin E2, altering the ratio of content between
their different receptor subtypes, as was demonstrated in cervix
of ovariectomised ewes (Schmitz et al. 2006). The fact that the
post-mortem depth of cervical penetration was greater in non-
luteal than in luteal phase ewes (Kershaw et al. 2005) could
suggest that a degree of natural cervical relaxation at oestrus may
be a consequence of the previous high circulating E2levels act-
ing via its nuclear receptor.The return of the cervical ER and PR
binding capacity to the minimal values on Day 6 of the oestrous
cycle, as well as the decrease in the cervical oxytocin receptor
binding capacity early in the luteal phase (Matthews and Ayad
1994), suggests that the increment in cervical penetrability using
these hormones could be less successful at the ET than at the
AI time.
The caudal zone showed higher ERαmRNA concentration on
Day 1 of the oestrous cycle than during the luteal phase, while no
differences in the ERαmRNA concentration during the oestrous
cycle was found in the cranial zone. The ERαmRNA concen-
trations in the caudal zone were positively correlated with the
E2and negatively correlated with the P circulating levels, sug-
gesting that the E2upregulation and P downregulation of the
ERαare predominately at transcriptional level. The temporal
association between cervical ERαmRNA and ER protein con-
centrations are in agreement with the results found for the ovine
uterus (Ott et al. 1993), and support the hypothesis of a transcrip-
tional mechanism to control the ERαexpression in the caudal
zone. The lack of relationship found between the ERαmRNA
and ER protein concentrations in the cranial zone suggest that
the expression of ERαcould be regulated in a different manner,
depending on the cervical zone. Recently, no differences in the
ERαmRNA concentrations in the follicular and the luteal phases
were found, but the cervical zone studied was not indicated (van
Lier et al. 2006).
Interestingly, on Day 1 of the oestrous cycle, both ERαmRNA
and PR concentrations were higher in the caudal than in the
cranial zone, suggesting a differential level of ER and PR expres-
sion along the longitudinal axis of the cervix. This could reflect
histological (Moré 1984) and functional (Hawk et al. 1978) dif-
ferences between cranial and caudal regions of the ovine cervix.
For example, the cranial cervix of the ewe appears to be more
critical than the caudal cervix for sperm transport (Hawk et al.
1978). Similarly, the percentage of ER and PR immunoposi-
tive cells increases from the uterine to the vaginal ends of the
bovine cervix (Breeveld-Dwarkasing et al. 2002). This gradi-
ent of positive ER and PR cells along the cervix was associated
with regional differences in cell density (Breeveld-Dwarkasing
et al. 2000) and collagen content (Breeveld-Dwarkasing et al.
2003). These findings are relevant for the interpretation of results
obtained from in vivo sampling of the cervix, when only one
region of the cervix can be reached or when different sampling
regions are compared. The differential sensitivity to the ovarian
steroid hormones in cranial and caudal zones of the ovine cervix
could be relevant to the development of localised hormonal
treatments for the increase of cervical penetrability.
In conclusion, the expression of ER and PR in the ovine cervix
is higher at oestrus, decreases during the luteal phase, and is
higher in the caudal than in the cranial cervix, reflecting their
histological and functional differences.
Acknowledgements
We would like to thank P. Rubianes and I. Sar tore for technical assistance and
animal experimentation, F. Perdigón and L. Sosa for providing the animals,
and Dr N. Ing for providing the ovine ER cDNA. The present study received
financial support from the University of Uruguay-CSIC, PEDECIBA, Vet-
erinary Faculty-CIDEC, and Ministry of Education and Culture–DINACYT,
Uruguay.
References
Armstrong, D. T., and Evans, G. (1983). Factors influencing success
of embryo transfer in sheep and goats. Theriogenology 19, 31–42.
doi:10.1016/0093-691X(83)90121-8
Barry, D. M., van Niekerk, C. H., Rust, J., and van der Walt, T.
(1990). Cervical embryo collection in sheep after ripening of the
cervix with prostaglandin E2 and estradiol. Theriogenology 33, 190.
doi:10.1016/0093-691X(90)90614-Y
Braunsberg, H. (1984). Mathematical analysis of data from receptor assay. In
‘Clinical Interest of Steroid Hormone Receptors in Breast Cancer. Recent
Results in Cancer Research’. (Eds G. Leclerq, S. Toma, R. Paridaens and
J. C. Heuson.) pp. 18–31. (Springer-Verlag: Berlin, Germany.)
Breeveld-Dwarkasing, V. N. A., van der Weijden, G. C., Taverne, M. A. M.,
and van Dissel-Emiliani, F. M. F. (2000). The bovine cervix during the
oestrous cycle: regional differences in morphology and density of steroid
hormone receptors. Reprod. Domest. Anim. 35, 120–124.
Breeveld-Dwarkasing, V. N. A., de Boer-Brouwer, M., Möstl, E.,
Soede, N. M., van der Weijden, G. C., Taverne, M. A. M., and
van Dissel-Emiliani, F. M. F. (2002). Immunohistochemical distribu-
tion of oestrogen and progesterone receptors and tissue concentrations
of oestrogens in the cervix of non-pregnant cows. Reprod. Fertil. Dev.
14, 487–494. doi:10.1071/RD02005
Breeveld-Dwarkasing, V. N. A., de Boer-Brouwer, M., te Koppele, J. M.,
Bank, R. A., van der Weijden, G. C., Taverne, M. A. M., and
van Dissel-Emiliani, F. M. F. (2003). Regional differences in water
content, collagen content, and collagen degradation in the cervix
of non-pregnant cows. Biol. Reprod. 69, 1600–1607. doi:10.1095/
BIOLREPROD.102.012443
Campbell, J. W., Harvey, T. G., McDonald, M. F., and Sparksman, R. I.
(1996). Transcervical insemination in sheep: an anatomical and histo-
logical evaluation. Theriogenology 45, 1535–1544. doi:10.1016/0093-
691X(96)00121-5
Clark, J. H., Schrader, W. T., and O’Malley, B. W. (1992). Mechanisms
of steroid hormones action. In ‘Textbook of Endocrinology’. (Eds
J. D. Wilson and D. W. Foster.) pp. 35–90. (WB Saunders: Philadelphia,
USA.)
Couse, J. F., Hewitt, S. C., and Korach, K. S. (2006). Steroid receptors in the
ovary and uterus. In ‘Knobil and Neill’s Physiology of Reproduction’.
(Ed. J.D. Neill.) pp. 593–678. (ElsevierAcademic Press: St. Louis, USA.)
Croy, B. A., Prudencio, J., Minhas, K., Ashkar, A. A., Galligan, C.,
Foster, R. A., Buckrell, B., and Coomber, B. L. (1999). A prelimi-
nary study on the usefulness of huil-8 in cervical relaxation of the ewe
for artificial insemination and for embryo transfer. Theriogenology 52,
271–287. doi:10.1016/S0093-691X(99)00128-4
Cervical steroid receptors in cycling ewe Reproduction, Fertility and Development 355
Flohr, S. F., Wulster-Radcliffe, M. C., and Lewis, G. S. (1999). Techni-
cal note: development of a transcervical oocyte recovery procedure for
sheep. J. Anim. Sci. 77, 2583–2586.
Fuchs, A. R., Graddy, L. G., Kowalski, A. A., and Fields, M. J. (2002).
Oxytocin induces PGE2release from bovine cervical mucosa in vivo.
Prostaglandins Other Lipid Mediat. 70, 119–129. doi:10.1016/S0090-
6980(02)00017-5
Garófalo, E. G., and Tasende, C. (1996). Uterine estrogen and progesterone
receptors in prepubertal ewe: distribution in myometrium, endometrium
and car uncles. Vet. Res. 27, 177–183.
Gimpl, G., and Fahrenholz, F. (2001). The oxytocin receptor system:
structure, function and regulation. Physiol. Rev. 81, 629–683.
Halbert, G. W., Dobson, H., Walton, J. S., and Buckrell, B. C. (1990a). The
structure of the cervical canal of the ewe. Theriogenology 33, 977–992.
doi:10.1016/0093-691X(90)90060-7
Halbert, G. W., Dobson, H., Walton, J. S., and Buckrell, B. C. (1990b).
A technique for transcervical intrauterine insemination of the ewes.
Theriogenology 33, 993–1010. doi:10.1016/0093-691X(90)90061-W
Hawk, H.W.,Conley, H. H., and Cooper,B. S. (1978). Number of sperm in the
oviducts, uterus, and cervix of the mated ewe as affected by exogenous
oestradiol. J. Anim. Sci. 46, 1300–1308.
Ing, N. H., Tsai, S. Y., and Tsai, M. J. (1993). Progesterone and estrogen.
In ‘Genes in Mammalian Reproduction’. (Ed. R. B. L. Gwatkin.) pp.
271–291. (Wiley-Liss: NewYork, USA.)
Ing, N. H., Spencer, T. E., and Bazer, F. W. (1996). Estrogen enhances
endometrial oestrogen receptor gene expression by a posttranscriptional
mechanism in the ovariectomized ewe. Biol. Reprod. 54, 591–599.
doi:10.1095/BIOLREPROD54.3.591
Kershaw, C. M., Khalid, M., McGowan, M. R., Ingram, K., Leethongdee, S.,
Wax, G., and Scaramuzzi, R. J. (2005). The anatomy of the sheep
cervix and its influence on the transcervical passage of an insemi-
nating pipette into the uterine lumen. Theriogenology 64, 1225–1235.
doi:10.1016/J.THERIOGENOLOGY.2005.02.017
Khalifa, R. M., Sayre, B. L., and Lewis, G. S. (1992). Exogenous oxytocin
dilates the cervix in ewes. J. Anim. Sci. 70, 38–42.
Kraemer, D. C. (1989). Embryo collection and transfer in small ruminants.
Theriogenology 31, 141–148. doi:10.1016/0093-691X(89)90571-2
Lee, C. N., Clayton, M. K., Bushmeyer, S. M., First, N. L., and Ax, R. L.
(1986). Glycosaminoglycans in ewe reproductive tracts and their influ-
ence on acrosome reactions in bovine spermatozoa in vitro.J. Anim. Sci.
63, 861–867.
Lightfoot, R. J., and Salamon, S. (1970). Fertility of ram spermatozoa frozen
by the pellet method. II. The effects of method of insemination on
fertilization and embryonic mortality. J. Reprod. Fertil. 22, 399–408.
Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951).
Protein measurement with the folin phenol reagent. J. Biol. Chem. 193,
265–275.
Matthews, E. L., and Ayad, V. J. (1994). Characterization and localiza-
tion of a putative oxytocin receptor in the cervix of the oestrous ewe.
J. Endocrinol. 142, 397–405.
McKelvey, W. A. C., McEvoy, T. G., Dingwall, W. S., Robinson, J. J.,
Lindsay, E., King, M. E., FitzSimons, J., and Mylne, M. J. A. (1997).The
use of exogenous hormones to facilitate transcervical embryo recovery
in sheep and their effect on embryo development. Theriogenology 47,
369. doi:10.1016/S0093-691X(97)82496-X
Meikle, A., Tasende, C., Garófalo, E. G., and Forsberg, M. (1998). Prim-
ing effect of exogenous oestradiol on luteinizing hormone secretion in
prepubertal lambs. Anim. Reprod. Sci. 54, 75–85. doi:10.1016/S0378-
4320(98)00147-X
Meikle, A., Forsberg, M., Sahlin, L., Masironi, B., Tasende, C., Rodríguez-
Piñón, M., and Garófalo, E. G. (2000). A biphasic action of oestradiol on
oestrogen and progesterone receptor content in the lamb uterus. Reprod.
Nutr. Dev. 40, 283–293. doi:10.1051/RND:2000132
Meikle, A., Sahlin, L., Ferraris, A., Masironi, B., Blanc, J. E., Rodríguez-
Irazoqui, M., Rodríguez-Piñón, M., Kindahl, H., and Forsberg, M.
(2001a). Endometrial mRNA expression of oestrogen and proges-
terone receptors and insulin-like growth factor-I (IGF-I) throughout the
bovine oestrous cycle.Anim. Reprod. Sci. 68, 45–56. doi:10.1016/S0378-
4320(01)00143-9
Meikle, A., Garófalo, E. G., Rodríguez-Piñón, M.,Tasende, C., and Sahlin, L.
(2001b). Regulation by gonadal steroids of oestrogen and progesterone
receptors along the reproductive tract in female lambs. Acta Vet. Scand.
42, 161–169. doi:10.1186/1751-0147-42-161
Meikle, A., Tasende, C., Sosa, C., and Garófalo, E. G. (2004). The role of
sex steroid receptors in sheep female reproductive physiology. Reprod.
Fertil. Dev. 16, 385–394. doi:10.1071/RD04036
Miller, B. G., Murphy, L., and Stone, G. M. (1977). Hormone receptor levels
and hormone, RNA and protein metabolism in the genital tract during
the oestrous cycle of the ewe. J. Endocrinol. 73, 91–98.
Moré, J. (1984). Anatomy and histology of the cervix uteri of the ewe: new
insights. Acta Anat. (Basel) 120, 156–159.
Mylne, M. J. A., McKelvey, W.A. C., Fernie, K., and Matthews, K. (1992).
Use of a transcervical technique for embryo recovery in sheep. Vet. Rec.
130, 450–451.
Nemec, L. A., Loskutoff, N. M., Lasley, B. L., Fields, M. J., Bowen, M. J.,
and Kraemer, D. C. (1988). The influence of relaxin, with steroid pre-
treatment, on cervical dilatation in ovariectomized ewes. Theriogenology
29, 282. doi:10.1016/0093-691X(88)90110-0
Ott, T. L., Zhou, Y., Mirando, M. A., Stevens, C., Harney, J. P., Ogle, T. F.,
and Bazer, F. W. (1993). Changes in progesterone and oestrogen recep-
tor mRNA and protein during maternal recognition of pregnancy and
luteolysis in ewes. J. Mol. Endocrinol. 10, 171–183.
Re, G., Badino, P., Novelli,A., Di Renzo, G. F., Severino, L., De Liguoro, M.,
and Ferone, M. R. (1995). Distribution of cytosolic oestrogen and pro-
gesterone receptors in the genital tract of the mare. Res. Vet. Sci. 59,
214–218. doi:10.1016/0034-5288(95)90004-7
Rexroad, C. E., Jr (1981). Estrogen and progestagen binding in the
myometrium of the ewe. I. During the oestrous cycle. J. Anim. Sci. 53,
1057–1069.
Rodríguez-Piñón, M., Tasende, C., Meikle, A., and Garófalo, E. G. (2000).
Estrogen and progesterone receptors in the ovine cervix during the
postpartum period. Theriogenology 53, 743–750. doi:10.1016/S0093-
691X(99)00271-X
Rodríguez-Piñón, M., Meikle, A.,Tasende, C., Sahlin, L., and Garófalo, E. G.
(2005). Differential oestradiol effects on estrogen and progesterone
receptors expression in the oviductand cer vix of immature ewes. Domest.
Anim. Endocrinol. 28, 442–450. doi:10.1016/J.DOMANIEND.2005.
02.011
Schmitz, T., Levine, B. A., and Nathanielsz, P. W. (2006). Localization and
steroid regulation of prostaglandin E2 receptor protein expression in
ovine cervix. Reproduction 131, 743–750. doi:10.1530/REP.1.00767
Stanchev, P., Kunavongkrit, A., Edqvist, L. E., and Eriksson, H. (1984).
Receptors for estrogens and progesterone in the porcine cervix. Therio-
genology 21, 757–766. doi:10.1016/0093-691X(84)90021-9
Tang, B. Y., and Adams, N. R. (1981). Oestrogen receptors and metabolic
activity in the genital tract after ovariectomy of ewes with permanent
infertility caused by exposure to phyto-oestrogens. J. Endocrinol. 89,
365–370.
Tang, B. Y., and Adams, N. R. (1986). Apparently-normal oestrogen receptor
system in ovariectomized ewes with impaired response to oestrogen after
prolonged grazing on oestrogenic pasture. J. Endocrinol. 109, 251–255.
Tasende, C., Rodríguez-Piñón, M., Meikle, A., Forsberg, M., and
Garófalo, E. G. (2002). Estrogen and progesterone receptor content in
the pituitary gland and uterus of progesterone-primed and gonadotropin-
releasing hormone-treated anoestrus ewes. Theriogenology 57, 1719–
1731. doi:10.1016/S0093-691X(02)00665-9
356 Reproduction, Fertility and Development M. Rodríguez-Piñón et al.
Tasende, C., Forsberg, M., Rodríguez-Piñón, M., Acuña, S., and
Garófalo, E. G. (2005a). Experimentally-induced subnormal or nor-
mal luteal phases in sheep: reproductive hormone profiles and uterine
sex steroid receptor expression. Reprod. Fertil. Dev. 17, 565–571.
doi:10.1071/RD05001
Tasende, C., Rodríguez-Piñón, M., Acuña, S., Garófalo, E. G., and
Forsberg, M. (2005b). Corpus luteum life span and pituitary oestro-
gen and progesterone receptors in cyclic and gonadotrophin-releasing
hormone-treated anoestrus ewes. Reprod. Fertil. Dev. 17, 721–726.
doi:10.1071/RD05058
van Lier, E., Meikle, A., Eriksson, H., and Sahlin, L. (2006). Insulin-like
growth factor-I (IGF-I) and thioredoxin are differentiallyexpressed along
the reproductive tract of the ewe during the oestrous cycle and after
ovariectomy. Acta Vet. Scand. 48, 5. doi:10.1186/1751-0147-1-5
Vesanen, M., Isomaa, V., Alanko, M., and Vihko, R. (1988). Cytosol estro-
gen and progesterone receptors in bovine endometrium after uterine
http://www.publish.csiro.au/journals/rfd
involution postpartum and in the estrous cycle. Anim. Reprod. Sci. 17,
9–20. doi:10.1016/0378-4320(88)90041-3
Vesanen, M., Isomaa, V., Alanko, M., and Vihko, R. (1991). Bovine uterine,
cervical and ovarian estrogen and progesterone receptor concentrations.
Anim. Reprod. Sci. 26, 61–71. doi:10.1016/0378-4320(91)90066-9
Wang, H., Eriksson, H., and Sahlin, L. (2000). Estrogen receptors αand βin
the female reproductive tract of the rat during the oestrous cycle. Biol.
Reprod. 63, 1331–1340. doi:10.1095/BIOLREPROD63.5.1331
Zhao, Y., Williams, L. M., Hannah, L. T., Ross, A. W., Mc Kelvey, W. A. C.,
and Robinson, J. J. (1999). Oestrogen and progesterone receptor
immunoreactivity and c-fos expression in the ovine cervix. J. Reprod.
Fertil. 115, 287–292.
Manuscript received 8 October 2007, accepted 13 December 2007
