Expression of receptors for ovarian steroids and prostaglandin E2 in the endometrium and myometrium of mares during estrus, diestrus and early pregnancy

Abstract

The objective of this study was to compare expression of estrogen receptor alpha (ER-α), β (ER-β), progesterone receptor (PR), as well as prostaglandin E2 type 2 (EP2) and 4 (EP4) receptors in the equine myometrium and endometrium during estrus, diestrus and early pregnancy. Tissues were collected during estrus, diestrus, and early pregnancy. Transcripts for ER-α (ESR1), ER-β (ESR2), PR (PGR), EP2 (PTGER2) and EP4 (PTGER4) were quantified by qPCR. Immunohistochemistry was used to localize ER-α, ER-β, PR, EP2 and EP4. Differences in transcript in endometrium and myometrium were compared by the ΔΔCT method. Expression for ESR1 (P < 0.05) tended to be higher during estrus than diestrus in the endometrium (P = 0.1) and myometrium (P = 0.06). In addition, ESR1 expression was greater during estrus than pregnancy (P < 0.05) in the endometrium and tended to be higher in estrus compared to pregnancy in the myometrium (P = 0.1). Expression for PGR was greater (P < 0.05) in the endometrium during estrus and diestrus than during pregnancy. In the myometrium, PGR expression was greater in estrus than pregnancy (P = 0.05) and tended to be higher during diestrus in relation to pregnancy (P = 0.07). There were no differences among reproductive stages in ESR2, PTGER2 and PTGER4 mRNA expression (P > 0.05). Immunolabeling in the endometrium appeared to be more intense for ER-α during estrus than diestrus and pregnancy. In addition, immunostaining for PR during pregnancy appeared to be more intense in the stroma and less intense in glands and epithelium compared to estrus and diestrus. EP2 immunoreactivity appeared to be more intense during early pregnancy in both endometrium and myometrium, whereas weak immunolabeling for EP4 was noted across reproductive stages. This study demonstrates differential regulation of estrogen receptor (ER) and PR in the myometrium and endometrium during the reproductive cycle and pregnancy as well as abundant protein expression of EP2 in the endometrium and myometrium during early pregnancy in mares.

Full text

Animal
Reproduction
Science
151
(2014)
169–181
Contents
lists
available
at
ScienceDirect
Animal
Reproduction
Science
jou
rn
al
hom
epage
:
w
ww.elsevier.com/locate/anir
eprosci
Expression
of
receptors
for
ovarian
steroids
and
prostaglandin
E2
in
the
endometrium
and
myometrium
of
mares
during
estrus,
diestrus
and
early
pregnancy
E.S.M.
Silvab,
K.E.
Scoggina,
I.F.
Canissoa,
M.H.T.
Troedssona,
E.L.
Squiresa,
B.A.
Balla,∗
aReproduction
Laboratory,
Maxwell
H.
Gluck
Equine
Research,
Department
of
Veterinary
Science,
University
of
Kentucky,
Lexington
KY
40546-0099,
USA
bFaculdade
de
Medicina
Veterinária
e
Zootecnia,
UNESP–Universidade
Estadual
Paulista,
Departamento
de
Reproduc¸
ão
Animal
e
Radiologia
Veterinária,
Botucatu,
São
Paulo,
CEP:
18618-970,
Brazil
a
r
t
i
c
l
e
i
n
f
o
Article
history:
Received
18
June
2014
Received
in
revised
form
31
October
2014
Accepted
3
November
2014
Available
online
10
November
2014
Keywords:
Steroid
receptor
Prostaglandin
E2
receptor
Estrous
cycle
Pregnancy
Uterus
Equine
a
b
s
t
r
a
c
t
The
objective
of
this
study
was
to
compare
expression
of
estrogen
receptor
alpha
(ER-␣),
␤
(ER-␤),
progesterone
receptor
(PR),
as
well
as
prostaglandin
E2
type
2
(EP2)
and
4
(EP4)
receptors
in
the
equine
myometrium
and
endometrium
during
estrus,
diestrus
and
early
pregnancy.
Tissues
were
collected
during
estrus,
diestrus,
and
early
pregnancy.
Transcripts
for
ER-␣
(ESR1),
ER-␤
(ESR2),
PR
(PGR),
EP2
(PTGER2)
and
EP4
(PTGER4)
were
quantified
by
qPCR.
Immunohistochemistry
was
used
to
localize
ER-␣,
ER-␤,
PR,
EP2
and
EP4.
Dif-
ferences
in
transcript
in
endometrium
and
myometrium
were
compared
by
the
CT
method.
Expression
for
ESR1
(P
<
0.05)
tended
to
be
higher
during
estrus
than
diestrus
in
the
endometrium
(P
=
0.1)
and
myometrium
(P
=
0.06).
In
addition,
ESR1
expression
was
greater
during
estrus
than
pregnancy
(P
<
0.05)
in
the
endometrium
and
tended
to
be
higher
in
estrus
compared
to
pregnancy
in
the
myometrium
(P
=
0.1).
Expression
for
PGR
was
greater
(P
<
0.05)
in
the
endometrium
during
estrus
and
diestrus
than
during
preg-
nancy.
In
the
myometrium,
PGR
expression
was
greater
in
estrus
than
pregnancy
(P
=
0.05)
and
tended
to
be
higher
during
diestrus
in
relation
to
pregnancy
(P
=
0.07).
There
were
no
differences
among
reproductive
stages
in
ESR2,
PTGER2
and
PTGER4
mRNA
expres-
sion
(P
>
0.05).
Immunolabeling
in
the
endometrium
appeared
to
be
more
intense
for
ER-␣
during
estrus
than
diestrus
and
pregnancy.
In
addition,
immunostaining
for
PR
during
preg-
nancy
appeared
to
be
more
intense
in
the
stroma
and
less
intense
in
glands
and
epithelium
compared
to
estrus
and
diestrus.
EP2
immunoreactivity
appeared
to
be
more
intense
during
early
pregnancy
in
both
endometrium
and
myometrium,
whereas
weak
immunolabeling
for
EP4
was
noted
across
reproductive
stages.
This
study
demonstrates
differential
regu-
lation
of
estrogen
receptor
(ER)
and
PR
in
the
myometrium
and
endometrium
during
the
reproductive
cycle
and
pregnancy
as
well
as
abundant
protein
expression
of
EP2
in
the
endometrium
and
myometrium
during
early
pregnancy
in
mares.
©
2014
Elsevier
B.V.
All
rights
reserved.
∗Corresponding
author.
E-mail
address:
b.a.ball@uky.edu
(B.A.
Ball).
1.
Introduction
Changes
in
the
mare’s
reproductive
tract
throughout
the
estrous
cycle
and
early
pregnancy
are
coordinated
by
http://dx.doi.org/10.1016/j.anireprosci.2014.11.001
0378-4320/©
2014
Elsevier
B.V.
All
rights
reserved.

170
E.S.M.
Silva
et
al.
/
Animal
Reproduction
Science
151
(2014)
169–181
ovarian
steroids
(Ginther,
1992).
Both
estrogen
and
proges-
terone
bind
to
nuclear
receptors
and
act
through
induction
of
transcription
for
a
variety
of
genes,
thus
regulating
cell
development
and
differentiation
(DeFranco,
2002).
Estrogen
mediates
its
effects
through
two
types
of
receptors
(i.e.
␣
and
␤)
encoded
by
different
genes,
ESR1
and
ESR2
(Enmark
et
al.,
1997).
Estrogen
receptor
␣
(ER-␣)
is
the
predominant
estrogen
receptor
(ER)
in
the
uterus
(Weihua
et
al.,
2000).
It
plays
a
major
role
in
the
uterotrophic
effects
of
estrogen,
as
evidenced
by
a
loss
of
cellular
proliferation
and
secretory
protein
produc-
tion
in
the
uterus
of
adult
female
ER-␣
knockout
mice
(Lubahn
et
al.,
1993).
Estrogen
receptor
␤
(ER-␤)
has
a
high
degree
of
sequence
homology
with
the
classical
ER-␣
(Hiroi
et
al.,
1999)
and
has
been
described
in
the
uterus
of
women
(Matsuzaki
et
al.,
1999),
mice
(Weihua
et
al.,
2000),
and
more
recently
the
horse
(Honnens
et
al.,
2011).
Adult
female
ER-␤
knockout
mice
were
infertile
or
had
signs
of
subfertility,
demonstrating
that
this
recep-
tor
is
important
in
female
reproduction
(Weihua
et
al.,
2000).
In
addition,
it
has
been
suggested
that
ER-␤
mod-
ulates
the
uterotrophic
effects
of
ER-␣
(Weihua
et
al.,
2000).
Progesterone
mediates
its
functions
through
two
iso-
forms
of
nuclear
receptors
(PGR
isoform
A
and
PGR
isoform
B),
which
are
encoded
by
the
same
gene
(Mote
et
al.,
2006).
The
isoform
PGR-A
plays
a
major
role
in
mediat-
ing
the
actions
of
progesterone
in
the
uterus
and
ovaries,
while
PGR-B
is
more
important
in
the
development
of
the
mammary
gland
(Mulac-Jericevic
et
al.,
2000,
2003).
Studies
with
progesterone
receptor
(PR)
in
knockout
mice
demonstrated
that
PR
is
essential
for
secondary
sexual
development
and
function
of
progesterone
targeted
organs
(Lydon
et
al.,
1995).
In
the
horse,
dynamics
of
ER
and
PR
during
the
repro-
ductive
cycle
and
early
pregnancy
have
been
primarily
described
in
the
endometrium
(Aupperle
et
al.,
2000;
Hartt
et
al.,
2005;
McDowell
et
al.,
1999;
Tomanelli
et
al.,
1991;
Watson
et
al.,
1992).
To
date,
there
are
no
stud-
ies
addressing
the
distribution
of
ER
and
PR
in
the
equine
myometrium
during
either
the
estrous
cycle
or
early
pregnancy.
Thus,
knowing
the
dynamics
of
these
steroid
receptors
during
the
estrous
cycle
and
early
pregnancy
will
allow
us
to
better
understand
normal
mare
physiology.
Prostaglandins,
in
addition
to
steroids,
are
potent
medi-
ators
in
the
female
reproductive
tract
(Sales
and
Jabbour,
2003).
Prostaglandin
E2(PGE2)
is
probably
the
most
ver-
satile
prostanoid
modulator
of
reproductive
events
(e.g.
ovulation,
fertilization,
implantation
and
parturition;
Lim
et
al.,
1997).
Effects
of
PGE2are
mediated
by
four
recep-
tor
sub-types
which
are
encoded
by
different
genes:
EP1,
EP2,
EP3
and
EP4
(Narumiya
et
al.,
1999).
Isoforms
EP2
and
EP4
are
known
as
relaxant
receptors,
which
medi-
ate
an
increase
in
cyclic
AMP
and
induce
smooth
muscle
relaxation
(Narumiya
et
al.,
1999;
Narumiya
and
Fitzgerald,
2001).
In
contrast,
EP2
is
considered
a
contractile
recep-
tor,
mediating
intracellular
Ca2+mobilization
and
induction
of
smooth
muscle
contraction.
The
isoform
EP3
is
termed
an
inhibitory
receptor,
which
mediates
decreases
in
cyclic
AMP
and
inhibits
smooth
muscle
relaxation
(Narumiya
et
al.,
1999;
Narumiya
and
Fitzgerald,
2001).
Of
all
the
PGE2receptors,
EP3
and
EP4
are
the
most
widely
distributed
throughout
the
body,
with
expression
present
in
almost
all
murine
tissues
examined
(Honda
et
al.,
1993;
Sugimoto
et
al.,
1992).
In
the
uterus,
EP2
is
the
most
abundant
sub-type
(Smock
et
al.,
1999).
Studies
in
knock-
out
mice
indicate
that
EP2,
but
not
EP1,
EP3
or
EP4,
is
associated
with
impaired
ovulation
and
reduction
in
lit-
ter
size
(Hizaki
et
al.,
1999).
Both
isoforms,
EP2
and
EP4,
are
associated
with
uterine
relaxation
during
pregnancy
in
different
mammals,
including
rats
(Papay
and
Kennedy,
2000),
mice
(Lim
and
Dey,
1997),
sheep
(Ma
et
al.,
1999),
cattle
(Arosh
et
al.,
2003),
baboon
(Smith
et
al.,
2001)
and
human
(Milne
et
al.,
2001).
Expression
and
regulation
of
the
EP2
and
EP4
receptors
in
the
uterus
vary
throughout
the
reproductive
cycle
and
pregnancy
in
multiple
species
(Arosh
et
al.,
2003;
Blesson
et
al.,
2012;
Dong
and
Yallampalli,
2000;
Milne
et
al.,
2001).
Prostanoid
receptor
EP2
is
the
major
cyclic
AMP-generating
isoform
expressed
and
regulated
in
bovine
uterine
tissues
during
the
estrous
cycle
and
early
pregnancy
(Arosh
et
al.,
2003).
No
differences
in
EP2
expression
were
observed
in
the
nonpregnant
human
uterus
across
the
menstrual
cycle;
however,
EP4
receptor
mRNA
expression
was
sig-
nificantly
higher
during
the
late
proliferative
stage
of
the
cycle
(Milne
et
al.,
2001).
In
ovariectomized
steroid-treated
mice,
uterine
PGE2receptors
are
regulated
by
estradiol
and
progesterone
(Blesson
et
al.,
2012).
In
the
mare,
EP2
trans-
cripts
were
found
to
be
increased
during
late
diestrus
and
early
pregnancy
in
the
endometrium,
while
no
variations
throughout
the
reproductive
stages
were
observed
for
EP4
(Atli
et
al.,
2010).
Nevertheless,
distribution
of
PGE2recep-
tors
in
the
endometrium,
as
well
as
EP2
and
EP4
expression
and
immunolocalization
in
the
myometrium
have
not
been
reported
throughout
the
estrous
cycle
or
pregnancy
in
mares.
Therefore,
the
objectives
of
the
present
study
were
to
compare
transcripts
and
to
describe
protein
expression
of
the
steroid
(ER-␣,
ER-␤
and
PR)
and
PGE2(EP2
and
EP4)
receptors
in
the
endometrium
and
myometrium
during
estrus,
diestrus
and
early
pregnancy
in
mares.
2.
Materials
and
methods
2.1.
Animals
and
tissue
collection
All
animal
procedures
were
completed
in
accordance
with
the
Institutional
Animal
Care
and
Use
Committee
of
the
University
of
Kentucky
(Protocol
#00843A2005).
Sixteen
clinically
healthy
mares
of
different
light
breeds,
ranging
from
4
to
18
y
of
age
were
used
in
this
study.
Uter-
ine
tissue
samples
were
obtained
postmortem
from
mares
during
estrus
(n
=
7),
diestrus
(n
=
6)
and
early
pregnancy
(n
=
3).
Estrus
was
defined
by
the
presence
of
at
least
one
preovulatory
follicle
(≥35
mm),
no
visible
corpus
luteum
and
presence
of
endometrial
edema
observed
via
transrec-
tal
ultrasonography.
Immediately
before
euthanasia,
the
mares
were
re-examined
by
transrectal
ultrasound
to
con-
firm
the
presence
of
the
preovulatory
follicle.
For
diestrus
tissues,
ovulation
was
confirmed
by
daily
transrectal
pal-
pation
and
ultrasonography,
and
mares
were
euthanized
between
9
and
13
d
post-ovulation.
Pregnant
mare
tissues

E.S.M.
Silva
et
al.
/
Animal
Reproduction
Science
151
(2014)
169–181
171
were
collected
from
one
mare
at
54
d
and
two
mares
at
66
d
of
gestation
to
evaluate
the
steroids
and
prostaglandin
E2
expression
during
the
early
pregnancy
stage
in
which
microvillous
attachment
is
becoming
established.
Immediately
after
collection,
tissues
were
kept
on
ice
until
further
processing.
Uterine
tissues
were
dissected
into
mucosa
(endometrium)
and
muscularis
(myometrium)
and
separate
aliquots
were
preserved
in
RNAlater
(Invitrogen,
Carlsbad,
CA),
refrigerated
overnight
(4 ◦C)
and
then
frozen
and
preserved
at
−80 ◦C
until
RNA
isolation.
Uterine
sam-
ples
were
also
fixed
in
buffered
formalin
until
further
processing.
2.2.
qPCR
analysis
The
mRNA
expressions
of
ESR1,
ESR2,
PGR,
PGE2recep-
tors
subtype
EP2
(PTGER2)
and
subtype
EP4
(PTGER4)
were
quantified
by
real-time
quantitative
PCR
(qPCR).
Total
cel-
lular
RNA
was
extracted
from
endometrial
and
myometrial
samples
using
TRIzol
Reagent
(Invitrogen)
according
to
the
manufacturer’s
recommendation.
The
RNA
was
then
pre-
cipitated
with
an
equal
volume
of
isopropanol
and
1/10
volume
of
3
M
sodium
acetate
and
then
resuspended
in
deionized
bi-distilled
water.
RNA
was
quantified
via
spec-
trophotometry
(NanoDrop
ND-1000;
Agilent
Technologies,
Palo
Alto,
CA),
and
samples
with
a
260/280
ratio
of
1.95
or
greater,
and
a
260/230
ratio
of
2.0
or
greater
were
used
for
analysis.
RNA
samples
(2
␮g/reaction)
were
treated
with
rDNase
I
(Invitrogen)
for
30
min
at
37 ◦C,
followed
by
treatment
with
DNase
Inactivation
Reagent
(room
temperature
for
2
min),
RNA
was
then
reverse
transcribed
using
high
capac-
ity
cDNA
reverse
transcription
kit
and
random
hexamers
(Invitrogen).
Primers
specific
for
the
selected
transcripts
were
designed
using
Primer-BLAST
(NCBI;
Table
1).
Quantitative
PCR
was
completed
using
SYBR
Green
PCR
Master
Mix
(Invitrogen)
with
the
following
cycling
condi-
tions:
95 ◦C
for
10
min,
40
cycles
of
95 ◦C
for
15
s
and
60 ◦C
for
1
min,
and
55–95 ◦C
for
dissociation.
Each
PCR
was
per-
formed
in
duplicate.
PCR
efficiencies
were
calculated
using
LinRegPCR
(version
2012.0).
All
reactions
were
automat-
ically
pipetted
using
the
epMotion
Automated
Pipetting
Systems
(Eppendorf,
Westbury,
NY).
Specificity
of
amplifi-
cation
was
monitored
by
completing
a
dissociation
analysis
at
the
end
of
each
real-time
run
to
verify
the
amplification
of
a
single
product.
Changes
in
gene
expression
were
calculated
by
mean
threshold
cycle
(CT)
and
then
normalized
for
the
house-
keeping
gene
glyceraldehyde-3-phosphate
dehydrogenase
(GAPDH)
to
generate
delta
()
CTvalues.
GAPDH
has
been
shown
to
be
stably
expressed
across
equine
samples
obtained
from
a
wide
range
of
reproductive
tissues
(Klein
et
al.,
2011).
Changes
in
relative
abundance
of
specific
transcripts
were
examined
by
calculating
the
expression
of
the
target
transcript
relative
to
the
reference
transcript
-CTmethod
(Livak
and
Schmittgen,
2001).
2.3.
Immunohistochemistry
Protein
localization
of
ER-␣,
ER-␤,
PR,
EP2
and
EP4
in
uterine
tissue
was
investigated
by
immunohistochemistry
(IHC)
using
antibodies
previously
validated
for
the
horse
(Abd-Elnaeim
et
al.,
2009;
Alm
et
al.,
2009;
Ball
et
al.,
2013;
Parlevliet
et
al.,
2006;
Pearl
et
al.,
2011).
Endometrium
and
myometrium
were
fixed
in
10%
formalin
for
24
h.
After
the
24
h
fixation
period,
samples
were
dehydrated
and
embed-
ded
in
paraffin.
Slides
were
sectioned
at
5
␮M
for
IHC.
Immunostaining
of
tissues
was
conducted
with
␣-bovine
ER␣
(1:25,
mouse
monoclonal
SC-311,
Santa
Cruz
Biotech-
nology
Inc.,
Santa
Cruz,
CA),
human
estrogen
receptor
beta
(ER-␤)
1
isoform
(1:20,
mouse
monoclonal
MCA1974S,
AbDSerotec,
Raleigh,
NC),
␣-human
PR
(1:100,
mouse
monoclonal
PR-2C5,
Invitrogen),
␣-human
EP2
(1:50,
rab-
bit
polyclonal
101750,
Cayman
Chemical,
Minneapolis,
MN),
␣-human
EP4
(1:50,
rabbit
polyclonal
101775,
Cay-
man
Chemical).
Paraffin
sections
were
processed
with
the
Leica
BOND-MAX
system
(Leica
Microsystems,
Buffalo
Grove,
IL).
Briefly,
automated
dewaxing
and
rehydration
steps
were
followed
by
heat-induced
(100 ◦C
for
20
min)
anti-
gen
retrieval
using
the
pH
8.9
EDTA-based
ready-to-use
solution
(Leica
Biosystems)
for
the
PR,
EP2
and
EP4
anti-
bodies.
For
ER-␣
and
ER-␤,
automated
dewaxing
and
rehydration
steps
were
followed
by
heat-induced
(100 ◦C
for
30
min)
antigen
retrieval
using
the
pH
5.9
citrate-
based
ready-to-use
solution
(Leica
Biosystems).
The
slides
were
subsequently
incubated
with
3%
hydrogen
perox-
ide
(5
min),
optimally
diluted
primary
antibody
(15
min),
a
post-primary
blocking
reagent
(to
prevent
nonspecific
polymer
binding)
(8
min),
horseradish
peroxidase-labeled
polymer
(8
min),
and
diaminobenzidine
substrate
(10
min).
All
reagents
were
components
of
the
bond
polymer
refine
detection
system
(Leica
Biosystems).
Primary
anti-
bodies
were
diluted
to
optimal
concentration
using
bond
primary
antibody
diluent
(Leica
Biosystems).
Wash-
ing
steps
between
each
reagent
were
performed
using
bond
wash
solution
10×
concentrate
(Leica
Biosys-
tems)
diluted
to
a
1×
working
solution
with
distilled
water.
Negative
controls
were
prepared
with
normal
mouse
IgG
instead
of
primary
antibody
for
ER-␣,
ER-␤
and
PR
and
normal
rabbit
IgG
for
EP2
and
EP4
(data
not
shown).
Specificity
of
primary
antibody
was
determined
by
pre-
incubation
with
the
corresponding
blocking
peptide
prior
to
IHC
(EP2
receptor
blocking
peptide
and
EP4
recep-
tor
blocking
peptide,
Cayman
Chemical).
The
polyclonal
antibody
and
blocking
peptide
were
incubated
for
1
h
at
room
temperature
in
a
1:1
(v/v)
ratio.
The
mixture
was
diluted
to
the
final
working
antibody
concentra-
tion
and
processed
as
described
above
(data
not
shown).
After
staining,
slides
were
dehydrated
using
serial
ethanol
dilutions
followed
by
immersion
in
four
baths
of
100%
xylene
and
mounted
for
analysis.
Slides
were
observed
at
40×
and
100×
magnification.
Immunoblotting
was
carried
out
to
confirm
the
specificity
of
estrogen
recep-
tors
in
the
tissues.
The
specificity
of
PGE2
receptors
by
immunoblotting
was
confirmed
previously
(Ball
et
al.,
2013).
Within
tissues
(endometrium
and
myometrium),
staining
intensity
and
distribution
were
independently
described
by
three
observers
blinded
to
mare
reproductive
status
(i.e.
estrus,
diestrus
or
early
pregnancy).

172
E.S.M.
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al.
/
Animal
Reproduction
Science
151
(2014)
169–181
Table
1
Forward
and
reverse
primer-sequences
used
for
estrogen
receptors
␣
and
␤
(genes
ESR1
and
ESR2,
respectively),
progesterone
receptor
(PGR),
prostanoid
receptors
(PTGER2
and
PTGER4)
and
glyceraldehyde
3-phosphate
dehydrogenase
(GAPDH)
transcripts.
Gene
(Locus)
Forward
primer
sequence
(5−3)
Reverse
primer
sequence
(5−3)
ESR1
(NM
001081772)
TCCATGGAGCACCCAGGAAAGC
CGGAGCCGAGATGACGTAGCC
ESR2
(XM
001915519)
TCCTGAATGCTGTGACCGAC
GTGCCTGACGTGAGAAAGGA
PGR
(XM
001498494)
CTTCCCCGACTGCGCGTACC
TTGTGTGGCTGGAAGTCGCCG
PTGER2
(NM 001127352) CCTCCAAGCCCTTAGGTTTC
TATCCACAAGGGCCAGCTAC
PTGER4
(XM 001499068) GGTGTGCCTGGCATGGGCTT
TAGTCCCGCCCACCTCGTCC
GAPDH
(NM
001163856)
AGAAGGAGAAAGGCCCTCAG
GGAAACTGTGGAGGTCAGGA
Fig.
1.
Boxplot
showing
change
in
ESR1
mRNA
expression
during
the
estrous
cycle
and
pregnancy
in
the
endometrium
and
myometrium
of
mares.
Data
are
expressed
as
median
CTrelative
to
expression
of
the
reference
transcript
(GAPDH).
Lower
CTrepresent
greater
expression
of
ESR1
mRNA.
Within
tissues,
values
with
different
superscripts
differ
(a,b:
P
≤
0.1;
c,d:
P
<
0.05).
2.4.
Statistical
analysis
The
CTvalues
for
estrus,
diestrus
and
pregnancy
for
endometrium
and
myometrium
for
ESR1,
ESR2,
PGR,
PTGER2
and
PTGER4
were
compared
by
nonparametric
pairwise
Wilcoxon
test.
Data
for
qPCR
are
presented
as
-
CT.
A
P-value
of
0.05
or
less
was
considered
statistically
significant
and
a
P-value
of
0.1
or
less
was
considered
as
a
tendency
to
significance.
Statistical
analyses
were
carried
out
using
JMP10.0
(SAS
Institute,
Cary,
NC).
3.
Results
3.1.
Endometrial
and
myometrial
transcripts
for
steroid
and
PGE2receptors
during
estrus,
diestrus
and
early
pregnancy
Relative
mRNA
expression
of
ESR1
tended
to
be
higher
during
estrus
than
diestrus
in
both
the
endometrium
(p
=
0.1)
and
myometrium
(P
=
0.06;
Fig.
1).
In
addition,
ESR1
mRNA
expression
during
estrus
was
greater
than
dur-
ing
pregnancy
(P
<
0.05)
in
the
endometrium
and
tended
to
be
higher
in
estrus
compared
to
pregnancy
in
the
myometrium
(P
=
0.1).
Fig.
2.
Boxplot
showing
change
in
PGR
mRNA
expression
during
the
estrous
cycle
and
pregnancy
in
the
endometrium
and
myometrium
of
mares.
Data
are
expressed
as
median
CTrelative
to
expression
of
the
reference
transcript
(GAPDH).
Lower
CTrepresent
greater
expression
of
PGR
mRNA.
Within
tissues,
values
with
different
superscripts
differ
(a,b:
P
≤
0.05;
c,d:
P
<
0.1).
Expression
of
PGR
was
greater
in
the
endometrium
dur-
ing
estrus
and
diestrus
than
during
pregnancy
(P
<
0.05;
Fig.
2).
Expression
for
PGR
in
the
myometrium
was
greater
in
estrus
than
pregnancy
(P
=
0.05)
and
tended
to
be
higher
during
diestrus
than
pregnancy
(P
=
0.07).
There
were
no
differences
in
ESR2,
PTGER2
and
PTGER4
mRNA
expression
in
the
uterine
tissues
among
reproductive
stages
(P
>
0.05;
data
not
shown).
3.2.
Immunohistochemistry
Immunostaining
for
ER-␣
was
present
in
the
nucleus
and
cytoplasm
of
luminal
epithelium,
glandular
epithelium
and
stromal
nuclei
of
the
endometrium
(Fig.
3A,
C
and
E).
In
the
myometrium,
immunoreactivity
was
detected
in
the
nucleus
and
cytoplasm
of
smooth
muscle
(Fig.
3B,
D
and
F).
Subjectively,
ER-␣
immunostaining
appeared
to
be
more
intense
during
estrus
than
diestrus
and
pregnancy
in
the
endometrium,
while
no
apparent
differences
were
noted
between
the
different
stages
of
the
estrous
cycle
and
preg-
nancy
for
the
myometrium
(Fig.
3).
Immunolabeling
for
ER-␤
was
detected
in
the
nuclei
of
stroma
and
luminal
epithelium,
and
endometrial
glands
showed
nuclear
and
cytoplasmic
labeling
(Fig.
4A,
C
and
E).
In
the
myometrium,
staining
was
present
in
the
smooth

E.S.M.
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/
Animal
Reproduction
Science
151
(2014)
169–181
173
Fig.
3.
Representative
photomicrographs
of
the
equine
endometrium
(A,
C
and
E)
and
myometrium
(B,
D
and
F)
immunostained
for
estrogen
receptor
␣
(ER-
␣)
during
estrus
(A,
B),
diestrus
(C,
D)
and
early
pregnancy
(E,
F).
Insets
show
higher
magnification
of
each
tissue
(scale
bar
=
40
␮m).
In
the
endometrium,
immunolabeling
of
ER␣
was
present
in
the
nucleus
and
cytoplasm
of
luminal,
glandular
epithelium
and
in
the
nucleus
of
the
stroma
during
estrus
(A),
diestrus
(C)
and
(E)
early
pregnancy
(54–66
d).
In
the
myometrium,
positive
ER-␣
staining
was
observed
in
the
nucleus
and
cytoplasm
of
the
smooth
muscle,
during
estrus
(B),
diestrus
(D)
and
early
pregnancy
(F).
muscle
and
stroma
(Fig.
4B,
D
and
F).
Subjectively,
the
intensity
of
immunostaining
for
ER-␤
did
not
differ
appre-
ciably
between
cycle
stages
within
tissues
(Fig.
4).
During
estrus
and
late
diestrus,
immunostaining
for
PR
was
detected
mostly
in
the
nuclei
of
luminal
and
glandular
epithelium,
as
well
as
in
the
stromal
nuclei
within
the
endometrium
(Fig.
5A,
C
and
E).
Immunopos-
itive
PR
was
also
present
in
smooth
muscle
(nucleus
and
cytoplasm)
in
the
myometrium
(Fig.
5B,
D
and
F).
Labeling
in
early
pregnancy
appeared
to
be
more
intense
in
stromal

174
E.S.M.
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/
Animal
Reproduction
Science
151
(2014)
169–181
Fig.
4.
Representative
photomicrographs
of
the
endometrium
(A,
C
and
E)
and
myometrium
(B,
D
and
F)
immunostained
for
estrogen
receptor
␤
(ER-␤)
during
estrus
(A,
B),
diestrus
(C,
D)
and
early
pregnancy
(E,
F).
Insets
show
higher
magnification
of
each
tissue
(scale
bar
=
40
␮m).
In
the
endometrium,
immunostaining
for
ER-␤
was
present
in
the
nucleus
of
luminal
epithelium
and
stroma,
as
well
as
in
the
nucleus
and
cytoplasm
of
the
glands
during
estrus
(A),
diestrus
(C)
and
early
pregnancy
(E).
In
the
myometrium,
positive
ER-␤
staining
was
observed
in
the
stroma
and
in
the
smooth
muscle
nuclei
and
cytoplasm
in
all
reproductive
phases
(B,
D
and
F).
nuclei
and
less
intense
in
glands
and
epithelium
compared
to
estrus
and
diestrus
(Fig.
5E).
In
the
myometrium,
label-
ing
appeared
to
be
stronger
during
estrus
and
pregnancy
(Fig.
5B
and
F).
Immunoexpression
for
EP2
was
localized
primarily
in
the
cytoplasm
of
luminal
epithelium,
glandular
epithe-
lium
and
in
the
stroma
within
endometrium
(Fig.
6C).
In
the
myometrium,
positive
staining
was
observed
in
the

E.S.M.
Silva
et
al.
/
Animal
Reproduction
Science
151
(2014)
169–181
175
Fig.
5.
Representative
photomicrographs
of
the
endometrium
(A,
C
and
E)
and
myometrium
(B,
D
and
F)
immunostained
for
progesterone
receptor
(PR)
during
estrus
(A,
B),
diestrus
(C,
D)
and
early
pregnancy
(E,
F).
Insets
show
higher
magnification
of
each
tissue
(scale
bar
=
40
␮m).
In
the
endometrium,
immunolabeling
of
PR
was
present
mostly
in
the
nucleus
of
luminal,
glandular
epithelium
and
stroma
during
estrus
(A)
and
diestrus
(C).
Labeling
in
early
pregnancy
(54–66
d)
appeared
to
be
more
intense
in
stromal
nuclei
and
less
intense
in
glands
and
epithelium
during
early
pregnancy
(E).
In
the
myometrium,
positive
PR
staining
was
observed
in
the
nucleus
and
cytoplasm
of
the
smooth
muscle
during
estrus
(B),
diestrus
(D)
and
early
pregnancy.
cytoplasm
and
nuclei
of
smooth
muscle
(Fig.
6D)
and
in
vascular
endothelium
in
the
myometrium
(Fig.
6B
and
F).
Subjectively,
EP2
immunolabeling
was
more
intense
dur-
ing
pregnancy
and
estrus
than
diestrus
in
endometrium
and
myometrium
(Fig.
6).
Immunostaining
for
EP4
was
present
mostly
in
the
cyto-
plasm
of
luminal
and
glandular
epithelium
(Fig.
7A)
and
in
the
cytoplasm
of
the
smooth
muscle
(Fig.
7B)
during
estrus,
diestrus
(Fig.
7C
and
D)
and
early
pregnancy
(Fig.
7E
and
F).
Subjectively,
the
intensity
of
immunostaining

176
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/
Animal
Reproduction
Science
151
(2014)
169–181
Fig.
6.
Representative
photomicrographs
of
the
endometrium
(A,
C
and
E)
and
myometrium
(B,
D
and
F)
immunostained
for
prostaglandin
E2
receptor
isoform
2
(EP2)
during
estrus
(A,
B),
diestrus
(C,
D)
and
early
pregnancy
(E,
F).
Insets
show
higher
magnification
of
each
tissue
(scale
bar
=
40
␮m).
In
the
endometrium,
immunolabeling
of
EP2
was
present
in
the
cytoplasm
of
luminal
and
glandular
epithelium
and
in
the
stroma
during
estrus
(A),
diestrus
(C)
and
early
pregnancy
(54–66
d)
(E).
In
the
myometrium,
positive
EP2
staining
was
observed
mostly
in
the
cytoplasm
of
the
smooth
muscle
(D)
and
in
vascular
endothelium
(arrows).
for
EP4
did
not
differ
within
tissues
among
cycle
stages
(Fig.
7).
Immunoblots
for
ER-␣
and
ER-␤
against
proteins
extracted
from
the
endometrium
revealed
proteins
of
approximately
62
and
55
kD,
respectively
(Fig.
8).
4.
Discussion
Here
we
report
the
protein
and
mRNA
expression
of
ER-
␣,
ER-␤,
PR,
EP2
and
EP4
in
the
equine
endometrium
and
myometrium
during
the
estrous
cycle
and
early
pregnancy.

E.S.M.
Silva
et
al.
/
Animal
Reproduction
Science
151
(2014)
169–181
177
Fig.
7.
Representative
photomicrographs
of
the
endometrium
(A,
C
and
E)
and
myometrium
(B,
D
and
F)
immunostained
for
prostaglandin
E2
receptor
isoform
4
(EP4)
during
estrus
(A,
B),
diestrus
(C,
D)
and
early
pregnancy
(E,
F).
Insets
show
higher
magnification
of
each
tissue
(scale
bar
=
40
␮m).
In
the
endometrium,
immunolabeling
of
EP4
was
present
in
the
cytoplasm
of
luminal
and
glandular
epithelium
during
estrus
(A),
diestrus
(C)
and
early
pregnancy
(54–66
d)
(E).
In
the
myometrium,
positive
EP4
staining
was
observed
in
the
cytoplasm
of
smooth
muscle
(B)
during
all
reproductive
stages
(B,
D
and
F).
This
study
is
apparently
the
first
to
compare
transcripts
and
describe
protein
expression
of
the
steroid
receptors
ER-␣,
ER-␤,
PR
and
PGE2receptors
(isoforms
EP2
and
EP4)
in
the
myometrium
of
the
mare,
during
estrus,
diestrus
and
early
pregnancy.
Increased
endometrial
transcripts
for
ESR1
during
estrus
and
decreased
during
late
diestrus
have
been
reported
in
ruminants
(Kimmins
and
MacLaren,
2001;
Martin
et
al.,
2008;
Spencer
and
Bazer,
2002)
and
in
the
horse
(Gebhardt
et
al.,
2012;
Hartt
et
al.,
2005).
In

178
E.S.M.
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/
Animal
Reproduction
Science
151
(2014)
169–181
Fig.
8.
Immunoblot
analysis
of
ER-␣
(A)
and
ER-␤
(B)
receptor
gene
expression
in
estrous
endometrium
protein
lysates
(lane
1,
10
␮g).
Molec-
ular
weight
markers
are
indicated.
ovariectomized
mares,
estradiol
treatment
resulted
in
an
increase
of
endometrial
ESR1
(McDowell
et
al.,
1999).
It
has
been
suggested
that
expression
of
uterine
sex
hormone
receptors
are
stimulated
by
estradiol
and
down-
regulated
by
progesterone
(Aupperle
et
al.,
2000;
Hartt
et
al.,
2005;
Kimmins
and
MacLaren,
2001;
Spencer
and
Bazer,
2002;
Watson
et
al.,
1992).
In
the
present
study,
endometrial
transcripts
for
ESR1
tended
to
be
higher
during
preovulatory
period
than
late
diestrus.
As
demon-
strated
by
the
aforementioned
studies,
our
results
indicate
that
estrogen
secreted
during
the
preovulatory
phase
appeared
to
increase
the
endometrial
transcripts
for
ESR1
and
continuous
exposure
to
progesterone
during
diestrus
down-regulated
its
expression.
Similar
to
ESR1
transcripts,
progesterone
receptor
mRNA
expression
in
the
endometrium
has
been
reported
to
increase
during
estrus
and
decrease
during
diestrus
(Hartt
et
al.,
2005;
Gebhardt
et
al.,
2012;
McDowell
et
al.,
1999);
however,
there
were
no
differences
for
PGR
endometrial
transcripts
between
estrus
and
late
diestrus
in
the
present
study.
Endometrial
transcripts
for
ESR1
and
PGR
during
early
pregnancy
(10–20
days)
have
been
described
and
com-
pared
to
corresponding
days
of
the
estrous
cycle
in
mares
(Hartt
et
al.,
2005;
McDowell
et
al.,
1999).
In
these
stud-
ies,
the
authors
demonstrate
a
differential
regulation
of
ER-␣
and
PR
mRNA
starting
from
day
15
of
pregnancy,
showing
lower
expression
compared
to
the
corresponding
days
of
the
estrous
cycle.
To
our
knowledge,
the
ESR1
and
PGR
transcripts
during
the
early
gestational
period
evalu-
ated
here
(54
and
66
days)
have
not
been
examined,
which
represent
the
period
of
time
in
which
microvillous
placen-
tation
is
becoming
established,
thus
an
important
period
for
fetal
development.
In
the
present
study,
ESR1
and
PGR
endometrial
transcripts
were
lower
during
54
and
66
days
of
pregnancy
as
compared
to
preovulatory
phase.
It
is
not
known
whether
the
down-regulation
during
these
stages
of
pregnancy
is
due
to
the
presence
of
the
conceptus
(Spencer
and
Bazer,
2002)
or
to
maintenance
of
high
circulating
pro-
gesterone
levels
(McDowell
et
al.,
1999).
Interestingly,
there
were
no
differences
in
expression
of
endometrial
ER-␤
mRNA
in
any
of
the
reproduc-
tive
stages
evaluated.
Previously,
a
positive
correlation
between
endometrial
transcripts
of
ESR1
and
ESR2
were
observed
throughout
the
equine
estrous
cycle
(Honnens
et
al.,
2011).
In
addition,
there
were
effects
of
estrous
cycle
day
on
the
expression
of
ESR1
and
ESR2
(Honnens
et
al.,
2011).
Since
we
compared
ESR2
during
the
preovulatory
period,
late
diestrus
and
early
pregnancy
in
postmortem
specimens,
and
did
not
evaluate
its
expression
at
other
time
points
of
the
estrous
cycle,
we
could
therefore
have
missed
differences
in
ESR2
mRNA
expression
across
different
days
of
the
reproductive
cycle.
Differences
in
the
methodology
applied
in
the
previous
study
(Honnens
et
al.,
2011)
and
herein
could
also
account
for
discrepancies
in
the
results.
In
the
myometrium,
ESR1
expression
tended
to
be
greater
during
estrus
than
diestrus,
although
no
appar-
ent
immunolabeling
change
for
ER-␣
was
observed
in
the
reproductive
stages
evaluated.
Moreover,
there
were
no
differences
in
the
myometrium
expression
of
PGR
mRNA
detected
between
estrus
and
diestrus
groups,
although
PR
immunoreactivity
in
the
tissue
appeared
to
be
increased
in
estrus
compared
to
diestrus.
It
is
well
known
that
transcripts
only
partially
explain
the
protein
concentra-
tions
present
in
the
tissue
(De
Sousa
Abreu
et
al.,
2009)
and,
together
with
the
knowledge
that
different
processes
can
regulate
mRNA
and
protein
production
and
degra-
dation
(Vogel
and
Marcotte,
2012),
post-transcriptional
regulatory
factors
could
explain
the
differences
between
transcripts
and
proteins
in
a
given
estrous
cycle
stage
and
reproductive
tissue
found
in
this
study.
Furthermore,
this
is
apparently
the
first
study
to
address
the
transcripts
and
protein
expression
of
ER-␤
in
the
myometrium
during
estrus,
diestrus
and
early
pregnancy.
However,
there
was
no
evidence
for
differential
regula-
tion
of
ER-␤
within
the
tissue
among
the
reproductive
stages.
Unlike
ER-␣,
it
appears
that
ER-␤
is
constitutively
expressed
in
the
myometrium.
It
has
been
reported
that
periovulatory
mares
exhibit
an
increased
level
of
ER-␣
in
luminal
epithelium,
glan-
dular
epithelium
and
stroma
in
relation
to
mares
in
diestrus
(Hartt
et
al.,
2005).
An
apparent
increased
immu-
nostaining
of
ER-␣
during
estrus
was
also
observed
in
the
endometrium
in
the
current
study.
In
female
reproduc-
tive
tract,
ER
has
been
associated
with
cell
proliferation
(Buchanan
et
al.,
1998;
Lai
et
al.,
2000),
and
in
the
bovine
uterus,
ER
expression
may
be
associated
with
increased
glandular
growth
in
preparation
for
secretion
of
histotroph
to
support
pregnancy
(Kimmins
and
MacLaren,
2001).
It
is
likely
that
the
abundant
expression
of
ER
during
estrus
is
meant
to
prepare
the
uterus
for
the
subsequent
luteal
phase
and
a
potential
pregnancy.
Unlike
ruminants,
where
ER
is
absent
in
the
luminal
and
glandular
epithelium
during
pregnancy
(Spencer
and
Bazer,
2002),
we
localized
abundant
ER-␣
and
ER-␤
in
both
cell
types
at
54
and
66
days
of
gestation.
This
is
in
agree-
ment
with
the
findings
of
Wilsher
and
Allen
(2011)
in
early
pregnant
mares
(20–68
days
of
pregnancy).
It
is
known
that
the
mare’s
pregnant
uterus
is
exposed
to
endogenous
estrogen
from
embryonic
origin
as
early
as
day
10
after
ovulation
(Choi
et
al.,
1997;
Zavy
et
al.,
1979).
In
addition,

E.S.M.
Silva
et
al.
/
Animal
Reproduction
Science
151
(2014)
169–181
179
there
is
an
increase
in
circulating
estrogen
concentrations
of
luteal
origin
from
day
38
of
pregnancy
onwards
(Daels
et
al.,
1991).
In
pigs,
it
has
been
reported
that
embryonic
estrogens
stimulate
an
increase
in
histotroph
secretion
and
that
they
also
act
in
a
paracrine
fashion
on
the
luminal
and
glandular
epithelia
to
increase
growth
factors,
which,
in
turn,
stimulate
development
of
the
trophoblast
(Ka
et
al.,
2001).
Perhaps
the
presence
of
ER
in
the
luminal
and
glan-
dular
epithelium
in
the
mare
during
early
pregnancy
also
plays
an
important
role
in
the
uterine
secretory
function,
although
these
mechanisms
have
not
yet
been
elucidated
in
horses.
During
early
pregnancy
(54
and
66
days),
we
observed
reduced
PR
mRNA
in
the
endometrium
as
compared
to
estrus
and
diestrus,
as
well
as
reduced
immunolabeling
in
luminal
and
glandular
epithelium;
however,
stronger
labeling
appeared
to
be
present
in
the
stroma,
as
previously
reported
for
pregnant
ewes
(Spencer
and
Bazer,
2002)
and
mares
(Wilsher
et
al.,
2011).
Spencer
and
Bazer
(2002)
sug-
gested
that
the
actions
of
progesterone
on
endometrial
epithelia
during
most
of
gestation
appear
to
be
medi-
ated
by
the
endometrial
stroma,
which
remains
PR-positive
throughout
pregnancy.
In
addition,
it
has
been
suggested
that
the
remodeling
of
the
endometrial
glandular
epithe-
lium
that
occurs
during
pregnancy
may
require
the
absence
of
PR
expression
(Spencer
and
Bazer,
1995).
In
pregnant
ewes,
PR
become
absent
in
uterine
epithelia
when
circu-
lating
concentrations
of
progesterone
are
high
(Spencer
and
Bazer,
2002).
In
the
mouse
uterine
epithelium,
proges-
terone
inhibits
cell
proliferation
(Tong
and
Pollard,
1999).
Therefore,
it
is
likely
that
progesterone
activated
PR
inhibits
epithelial
morphogenesis
through
a
negative
effect
on
pro-
gression
of
the
cell
cycle
(Spencer
and
Bazer,
2002).
In
ruminants,
a
decrease
of
PR
in
the
glandular
epithelium
may
be
essential
to
the
pregnancy
dependent
hyperplasia
and
hypertrophy
of
the
endometrial
glands
(Spencer
and
Bazer,
2002).
Considering
that
these
glands
remain
active
and
nurturing
the
developing
fetus
in
a
histotrophic
man-
ner
until
the
end
of
gestation
in
mares
(Allen,
2001),
it
is
likely
that
PR
decrease
in
glandular
epithelium
also
have
to
take
place
to
enable
growth
of
the
endometrial
glands.
In
addition
to
endometrial
stroma,
PR
remains
abun-
dant
also
in
the
myometrium
between
20
and
140
days
of
gestation
in
ewes
(Spencer
and
Bazer,
2002).
Similar
find-
ings
were
observed
in
the
present
study
at
54
and
66
days
of
pregnancy
in
mares,
despite
the
reduced
mRNA
expres-
sion
in
comparison
to
estrus
and
diestrus.
It
has
been
stated
that
progesterone
and
progestagens
are
responsible
for
uterine
quiescence
during
pregnancy
(Ousey
et
al.,
2003).
These
hormones
reduce
uterine
contractility
by
hyperpo-
larizing
the
myometrium
and
by
reducing
the
numbers
of
gap
junctions
and
receptors
for
contractile
agents
in
the
myometrium
(Thorburn
et
al.,
1988;
Thorburn,
1993).
Thus,
abundant
PR
in
equine
myometrium
during
early
preg-
nancy
may
be
necessary
for
myometrial
quiescence
and
normal
fetal
development.
Expression
of
prostaglandin
E2
synthase
during
late
diestrus
(10
and
15
days
after
ovulation)
and
early
pregnancy
(15
days)
has
been
described
in
equine
endometrium,
and
no
significant
variations
in
PGES
mRNA
levels
were
found
(Boerboom
et
al.,
2004).
On
the
other
hand,
Atli
et
al.
(2010)
detected
increased
PTGER2
and
prostaglandin
E2
synthase
transcripts
during
late
diestrus
(13.5–14
days
after
ovulation),
early
pregnancy
(14–22
days)
and
decreased
transcripts
during
estrus,
while
PTGER4
expression
did
not
change
throughout
the
eval-
uated
stages.
Gebhardt
et
al.
(2012)
reported
increased
PTGER2
on
days
8
and
12
in
mare’s
endometrium
and,
in
contrast
to
the
results
found
by
Atli
et
al.
(2010),
highest
PTGER4
on
days
16
and
0
(day
of
ovulation)
of
the
cycle.
In
cows,
PGE2receptor
transcripts
PTGER2
and
PTGER4
do
not
appear
to
change
throughout
the
estrous
cycle
in
the
endometrium
and
myometrium
(PTGER2)
or
are
undetected
in
both
uterine
tissues
(PTGER4;
Arosh
et
al.,
2003).
In
the
present
study,
PTGER2
and
PTGER4
transcripts
did
not
vary
significantly
between
preovulatory
estrus,
late
diestrus
and
pregnancy
(54
and
66
days)
in
either
endometrium
or
myometrium.
Additional
studies
on
PGE2
receptor
expression
during
the
estrous
cycle
and
preg-
nancy
are
necessary
to
clarify
these
conflicting
results
Despite
the
lack
of
increased
PTGER2
transcripts,
we
observed
intense
EP2
immunolabeling
in
all
cell
types
of
the
endometrium
and
myometrium
during
54
and
66
days
of
pregnancy,
in
contrast
to
apparently
reduced
staining
in
estrus
and
diestrus.
In
cows,
elevated
EP2
protein
in
both
tissue
was
also
described
during
early
pregnancy
(18
days
post
ovulation;
Arosh
et
al.,
2003).
In
mares,
Stout
and
Allen
(2002)
have
demonstrated
that
PGE2is
the
predom-
inant
prostaglandin
secreted
by
the
equine
conceptuses
by
30
days
post-ovulation.
These
authors
suggested
that
PGE2produced
by
equine
conceptus
are
involved
in
uterine
functions
important
to
the
development
and
nutrition
of
the
conceptus,
since
it
stimulates
increase
in
uterine
blood
flow
and
vascular
permeability
that
increase
nutrient
sup-
ply
(Lewis,
1989).
Moreover,
it
is
possible
that
elevated
EP2
in
uterine
smooth
muscle
during
pregnancy
is
due
to
the
action
of
PGE2in
uterine
quiescence
(Arosh
et
al.,
2003),
as
EP2
has
been
considered
a
relaxant
receptor
in
the
myometrium
of
different
species
(Ma
et
al.,
1999;
Smith
et
al.,
2001).
In
this
way,
the
observation
of
a
greater
EP2
immunolabeling
at
days
54
and
66
of
pregnancy
in
our
study
suggest
that,
in
this
specific
gestational
age,
EP2
also
plays
an
important
role
for
pregnancy,
either
for
concep-
tus
nutrition
or
myometrial
quiescence.
However,
the
exact
function
of
EP2
during
pregnancy
requires
further
informa-
tion.
Prostaglandin
E2
receptor
EP4
showed
reduced
tis-
sue
immunostaining
and
no
apparent
changes
during
estrous
cycle
and
pregnancy
in
both
endometrium
and
myometrium
in
this
study.
Likewise,
low
levels
of
EP4
were
reported
in
uterine
tissues
during
estrous
cycle
and
early
pregnancy
in
cows
(Arosh
et
al.,
2003).
Although
PTGER4
expression
has
been
described
during
the
estrous
cycle
and
early
pregnancy
in
mares
(Atli
et
al.,
2010;
Gebhardt
et
al.,
2012),
there
are
no
EP4
protein
changes
reported
during
reproductive
stages
in
the
species.
Further
studies
may
be
necessary
to
clarify
the
role
of
EP4
transcripts
and
protein
in
uterine
tissues.
In
conclusion,
our
results
indicate
that
ER-␣
and
PR
mRNA
expression
in
the
myometrium
show
differen-
tial
regulation
between
estrus,
late
diestrus
and
early
pregnancy
(54
and
66
days),
as
in
the
endometrium.

180
E.S.M.
Silva
et
al.
/
Animal
Reproduction
Science
151
(2014)
169–181
Endometrial
expression
of
ESR2
and
protein
immunolabel-
ing
of
ER-␤
did
not
vary
among
any
of
the
reproductive
stages
evaluated
here.
In
addition,
it
is
likely
that
ER-
␤
is
constitutively
expressed
in
the
myometrium
during
periovulatory
estrus,
late
diestrus
and
early
pregnancy.
Moreover,
it
is
possible
that
the
apparent
reduced
immu-
nostaining
observed
for
PR
in
luminal
and
glandular
epithelium
during
pregnancy
may
have
to
occur
to
enable
pregnancy
in
the
mare,
as
described
for
other
species.
This
study
also
demonstrates
reduced
EP4
immunostaining
and
lack
of
apparent
variation
between
the
reproductive
stages,
whereas
intense
EP2
immunolabeling
in
the
endometrium
and
myometrium
during
early
pregnancy.
We
suggest
that
EP2
plays
an
important
role
during
gestation,
either
for
myometrium
quiescence
and
conceptuses
nutrition
and
development,
which
still
needs
further
clarification.
Conflict
of
interest
The
authors
have
no
conflicts
of
interest
to
declare.
Acknowledgments
This
study
was
supported
by
the
Albert
Clay
endowment
in
Equine
Reproduction
and
by
Department
of
Veterinary
Science
of
the
University
of
Kentucky.
The
FAPESP
founda-
tion
(Fundac¸
ão
de
Apoio
a
Pesquisa
e
Ensino
do
Estado
de
São
Paulo,
Brasil)
is
also
acknowledged
for
supporting
the
first
author
during
the
execution
of
this
study
through
the
training
abroad
program
of
graduate
students.
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