May 25, 1992 - and pseudopregnancy. In ovariectomized mice,. Fos was induced .... associated with c-fos expression. Several growth factors expressed.
BIOLOGY
OF
REPRODUCTION
47,
492-501
(1992)
of c-fos-like
Localization
Proteins
in the
Mouse
implantation J.
DORIS
Department
of Anatomy,
BAKER,3
Wright
State
during
the
Pen-
Period1 NAGY,
FRANK
Endometrium
and
University
GARY
School
L. NIEDER2
of Medicine,
Dayton,
45435
Ohio
ABSTRACT Fos,
which
in the
uterus
expression
during
epithelium (P4)
and
for
and On
found and
at an earlier I and
subsequent
primary
zone
cone.
These
zona
decreased days
primary and
the
was
Day
zone.
epithelium.
On
demonstrate
P4 modulate to induce
of
respond (E2)
of a heterogeneous
differentially and progesterone
for
period 1 and
plug) cells. 4 and
pop-
replacements, whereas the
Nidatory
E2 of
the
[2, 3]. Pre-sensitization occur
Differences
between
beginning is initiated
on
P4 levels stroma
1 being
beginning
and
uteri
luminal
and
of Day
4. No
luminal of Day
giant and
afternoon
of Day
in the
found
but
was
cells
that
Fos
4, while
and
in the
the
Fos
remained
absent
luminal from
the
ectoplacental
is induced it is still
in was
epitheium
Fos
was
Fos with
in non-sites. zone,
trophoblast
and for
distinguishable,
5, staining
decidual
staining
detected
epithelium
effect
epitheium
further
were
in the
added
of ovariectomized
glandular
Fos was areas
for
uterine
on
changes
and
pseudopregnancy
pregnancy
and
pseudopregnancy
[5]. The
coordinated
of Day action
4 when
during encased
imin the
the
presence
of development action of the
of a conceptus
(or an artificial blastocyst) are nec-
and
level of gene expression requires transduction of the message to the
a recognition
general
ways:
mechanism
for a subset
of genes
activated or repressed. Transcription be modulated by external stimuli (1)
membrane-permeable
may directly transcription
interact rates;
in
molecules
with nuclear receptor (2) agents that cannot
cross the cell membrane may activate second messenger systems to bring about direct changes in gene transcription;
sensiti-
and
[4].
(3)
stimuli
can
target gene expression of genes that encode
are implanta-
of E2 and
and
at the signal
such as steroids proteins to alter
implan-
and
evening
3
proliferation
pregnancy the
three
of the
impending
endometrium
molecules receptors,
nucleus,
day
Day
the
appropriate stage that mimics the
to be transcniptionally of cellular genes may
epithethe
differentiation stromal
uterus
these signal
difand
by E2 and of the
(Day
E2 enhances
sensitizes
zation
Received
in the
Fos
the
essary for transformation of the stroma into decidual tissue. Although the exact cellular and molecular mechanisms that regulate the actions of E2 and P4 as well as signals from the implanting blastocyst are unknown, the response of cells to
added
proliferation
in a proliferation
rising of the
in both
also
2 of pregnancy
and
proliferation
Accepted
progesterone
2 mg
glandular
induced
cell
endometrium the
with
E, had
type
of Fos
and
giving
secondary
mouse on
Priming
course
in luminal
P4 concomitantly
non-site
afternoon
embryonic
expression
to the steroid (P4). When
maximal
are
E2 results
Days
tation
tion
the
is composed
P4 is required
P4. Preovulatory
noted
the
in the
in the in the
in just
at the stimulus
regulated
Day
on
found
(E,).
4 of pregnancy, and
sensitize
pen-implantation
on
and
in the morning
sites
INTRODUCTION
the
epithelial
Fos Fos
proliferation and early pregnancy
cause
in sites,
detected
stromal cells [1]. The cell-type-specific ferentiation of endometrial cells during
of vaginal
of Day
induced
the
P4 before
in any
localized
determine
pellucida.
of cells which 17-estradiol
on
afternoon
in sites
E2 and
the
5, implantation
6, staining also
detected
to
was
2 mg with
Fos
by
was
appears
ovariectomized mice are given hormone causes proliferation of the epithelium
hum
to induce
Day
was
Priming
detectable
the
stroma
7, Fos
that embryro
failed
7 of pregnancy.
On
Day
cells.
Fos
E, and
immunohistochemically
and
17-estradiol
ng
Fos was
of Day
antimesometrial
through
in stromal
was
mouse
mice,
ng
100
not
On
morning
in the
100
giving
alone
3 and
family,
expression
with
E, or
staining
of pseudopregnancy. and
the
ng
pseudopregnancy,
Day
proto-oncogene
Fos
In ovariectomized
Progesterone and
On
on
treatment
100
enhanced
by
decidual
Significantly,
endometrium
influence
following
point.
stroma.
results
plantation.
time
of non-sites
and
cell
with
epithelium
epitheium
epitheium
pseudopregnancy. of
c-los
of the
of steroids
and
2 of pregnancy
was
in luminal
in the
The
epithelium
antimesometrial
present
ulation hormones
stromal
Staining
during the
pregnancy
to the
products
effects
administration
Days
stroma.
the
prior
Fos
animals.
to protein
ascertain
an occasional
glandular
of inducing
refers
to normal
3 days
luminal
the
collectively
mouse
also
bring
ulatory proteins [6]. One known transcription gene, is an “immediate early
P4 to
May 25, 1992. May 17, 1991.
about
factor, gene”
is synthesized within minutes sponse to a variety of agents
This work was supported by Grant HI) 25236 from the National Institute of Health and Human Development. ‘Correspondence: Dr. Gary L Nieder, Department of Anatomy, Wright State University School of Medicine, Dayton, OH 45435. FAX: (513) 873-3391. address: Department of Clinical Laboratory Sciences, University of Nevada Las Vegas, Las Vegas, NV 89154.
Expression of c-fos is associated may be a change from quiescence
Child
processes gen 492
has
associated been
shown
indirect
alterations
of
by rapidly activating the expression known or putative transcriptional reg-
with
the whose
proto-oncoprotein product
after stimulation in many different with
[6,7] cell
in retypes.
a change of state. This to proliferation or with
differentiation.
to induce
c-fos
expression
Exogenous of the
estroc-fos
gene
IN THE
c-fos
in a variety the
of estrogen-responsive
human
and
breast
immature
cell
uterus
[9-11].
rat
cells rectly
by directly by activating
ulate least
c-fos
stimulating certain
[8]. The in part, due
ceptor
complex
though
the
with
parently
and
17]
and
known early
cAMP [20], following [21].
which artificial
and
state
present
study
was
it
mouse
endometrial
during
second
mes-
dramatically inof the decidual both
To establish on proteins
the
and
ment
studies,
they
received
injection
females
pseu-
a) a single
of 2 mg
injection
P4; c) a single
were
(pH this
7.4). time.
flushed with and examined of development
was flushed blastocysts dopregnancy.
was
confirmed
Sprague replace-
after
14 days
ng E2; b) a single of 100
ng E2 + 2
0800
h. A control
mice received no hormone at 1, 3, 6, 12, and 24 h after To obtain timed pregnancies females were mated with fertile The morning of a vaginal plug was
1 of pregnancy
between
h and
or pseudopregnancy. 1000
h on
Days
was
on
non-sites
based
the
Day
was oviducts
mice mice.
confirmed at were excised,
saline appropriate
and
and
macroscopic the
for
(DPBS), stage
unfertilized
oo-
of fat and
cut
were
5-mm
forty
tissue
culture
from
reaction
pieces
uterus.
separated
uterus. while
[23]
and
Sections
were
in the
fixative
then were placed incubated overnight
1-tm
sections
horizontal (pH 7.5).
from pregnancy (90-120 sections)
in a 48-well
blue
temperature, sucrose and and
of the
were
of the
were cut on a sliding in 50 mM Tris buffer
pregnancy and rially sectioned
appearance
pontamine
into
frozen
only if the blastocyst By Day 6, pregnancy
sites
appearance
and left for 1 h at room fixative containing 10%
well)
imbuffer
On Days 4 and 5 of pregone uterine horn was fixed the contralateral uterine horn
5, pregnancy
on
macroscopic
cleaned
immediately
in 0.2 M phosphate
or pseudopregnancy 1 through 3, the
by the
Beginning
sites on Days and collected dish.
All other
in at
through
microtome Uteri from 5-7
the
and Day
col4 of
were se(1 section!
sections
pooled in Tris buffer and randomly selected The sections were either used immediately
For processing, all sections (1/well) were well culture dish and incubated overnight
for or
were
staining. stored for
the
This sequence ran [24] and
region as defined DNA binding region
Mice 1 through
group
treatments. steroid horand pseuor vasecdesignated were
killed
7 of preg-
Dr.
is in the M-peptide encompasses the
fos peptide (amino acids
family of proteins. The antibody was Michael J. ladarola, Neurobiology NIH, been
Bethesda, well
and recognizes several family [25], hereafter
MD.
protein designated
products simply
sequence 128-152). by Curof the
a generous gift from and Anesthesiology
The
characterized
in-
placed in a 48with an affinity-
purified rabbit IgG against KVEQLSPEEEEKRRIRRERNKMAAA
has
Day
horn
lected from pregnant mice for fixation was still encased in the zona pellucida.
NIDR,
dopregnancies, tomized males.
h.
with DPBS and examined for the presence of in pregnancy and for unfertilized eggs in pseuOn the afternoon of Day 4, uteri were se-
antibody
of ovariectomized Mice were killed mone injection.
one
in pregnant
Branch,
1000
and
cytes in pseudopregnant nancy and pseudopregnancy, for immunohistochemistiy
administered
h and
5, additional
1700
Delbecco’s phosphate-buffered for fertilized eggs at the
fos
0800
excised
Pregnancy On Days
mg P4; or d) three daily injections of 2 mg P4 followed by a single injection of 100 ng E2 + 2 mg P4 on Day 4 [22]. All hormones were injected s.c. in 0.1 ml sesame oil and between
4 and
h and
in 50 mM Iris buffer (pH 7.5). Frozen sections were cubated for 20-60 mm in Tnis buffer prior to processing.
and
of 100 injection
Days
1500
up to 4 mo at - 20#{176}C in a cyroprotectant consisting of 30% sucrose, 6.2% ethylene glycol, and 1% polyvinyl pyrohidone
METHODS
ovariectomized
On
betwen
in 4% paraformaldehyde
uterus lected
period.
mice (6-10 wk of age; Harlan IN) were used. For hormone were
Uteri mersed
4#{176}C. Uteri
Animals Female ICR Swiss Dawley; Indianapolis,
killed
Immunohlstochemisby
modpro-
differential
pregnant
pseudopregnancy.
were
exog-
control of tranwere localized in
pen-implantation
AND
MATERIALS
proto-on-
that
of both the
The
milieu of pregnancy c-fos-immunoreactive
embryo
(TGFuterus
factor (EGF), (IGF-I) [14-
to the
ascertained
compartments mice
[6, 18, 19].
endometrium.
effects of steroids and the scription, c-fos-immunoreactive
sys-
with c-fos by the devel-
growth factor
is rapidly and stimulation
of at ap-
messenger
growth-factor-a [13] and by the
in response
steroids and the hormonal cell-specific induction of
dopregnant
Al-
how preg-
endocrine
factors
of c-fos
inducers
senger creased response
in the
[12].
to exogenous
is not during
levels
including epidermal insulin-like growth induced
the
steroid is, at estrogen-re-
uterus
493
target
of c-fos
changing
growth
c-fos is also
tein(s)
of the of the
and
animals
of c-fos or indithat in turn stim-
it
including transforming platelet-derived (PDGF)
known
In the
affect
rodent
The
cogene
enous ulate
may
of which have been associated Several growth factors expressed
pregnancy TGF-3,
are
c-fos.
various
oping embryo a), TGF-13, and during TGF-a,
Estrogen
UTERUS
nancy
mature
concomitant signaling from the blastocyst sets into motion a cascade of events that
involve
tems, many expression.
the
element
characterized, in steroid
including
[8] and
transcription growth factors
of the
uterine
the uterus implantation
tissues
MCF-7
a regulatory
responses
affect
line
transcriptional effect to direct interaction
E2 and P4 have been physiological changes nancy
target
cancer
PERI-IMPIANTATION
specificity
using
western
of the c-fos as Fos. The
of the blots gene anti-
body was diluted 1:10 000 with 50 mM Tris buffer (pH 7.5) + 0.2% triton X-100 + 4% normal lamb serum + 0.1% azide (Tris-NLS-triton-azide). Sections were then rinsed twice with Iris buffer and washed with shaking at room temperature
for 30 mm
in Tris-NLS-tniton-azide.
Next,
sections
were
BAKER
494 incubated (Vectastain, h, the rinses
with 1 g/ml Vector Labs.,
of biotmnylated Burlington, CA).
biotmnylated antibody was in Iris buffer and another
triton-azide,
sections
oxidase
complex
tastain perature another
were
removed 30-mm
incubated
as per
anti-rabbit At the end
IgG of 1
and following two wash in Tris-NLS-
with
avidmn-biotmn-per-
manufacturer’s
instructions
(Vec-
Elite Kit, Vector Labs.) for 1 h at room temwith shaking. After two rinses in Tnis buffer and wash in Inis-NLS-triton-azide, sections were stained
ABC
with nium
0.025% sulfate
presence
diaminobenzidmne in 0.1 M sodium
of 0.00 18%
coverslipped,
and
H2O2.
and acetate Sections
examined.
1.5% nickel (pH 6) sulfate were
Control
placed
ammoin the
on a slide,
experiments
included
sections from tissue known to contain c-fos run in the ence or absence of the primary antibody. Sections from rat ventromedial hypothalamus were run as positive
presadult con-
trols
uter-
in preliminary
experiments
[26].
In later
ine sections from Day 1 of pregnancy positive controls. Exogenous peroxidase by pre-treating for
ET AL.
5 mm
sections before
tibody was with either
with
0.6%
processing.
assays,
were run as routine activity was blocked H2O2
in 10%
Specificity
of the
methanol primary
tested by preabsorbing 1 ml of diluted 10 p.g Fos Peptide 1 (epitope: M-peptide;
an-
anti-Fos amino
acids 128-152 human p62 Fos) or Peptide 2 (epitope: nterminal domain; amino acids 4-17) (Oncogene Science, Inc., Manhasset, NY) for 2 h at room temperature prior to incubating
with
treatment
tissue
groups,
sections.
For
a minimum
from at least 3 animals; serial amined for pregnant animals through Day 7. Reported all sections and animals
most
of 6 sections sections from the
experimental were
(90-110) afternoon
examined were exof Day 4
results were consistently seen for a given treatment group.
in
RESULTS Spec/ic
and
Nonspec(fic
With the exception munohistochemical ovariectomized ment tissue
staining
of a rare stromal cell, no specific staining was detected in the uteri
mice
that
did
(Fig. 1A). Nonspecific sections. This intense
not
receive
staining staining
hormone was was
imof
replace-
observed in the seen in the nu-
cleus and the cytoplasm of blood cells that were for the most part contained in blood vessels. Nonspecific staining (denoted by arrow in Fig. 1C and 3D) could be eliminated by pretreating This ment
ing) and interpretation munoreactive to be
the
pretreatment compromised the
positive
(Fig. 1B), were body or when 1 (amino acids tained,
however,
sections
with
H2O2
prior
to processing.
was not routinely done since the integrity of the sections
the treat(i.e., tear-
nonspecific staining did not interfere with of the results. Specific staining for c-fos-improteins was not noted when sections known for
Fos,
such
as the
Day
processed in the absence the antibody was preabsorbed 128-152; Fig. 1C). Specific when
the
antibody
was
1 pregnant
uterus
of primary antiwith Peptide staining was repreabsorbed
with
FIG. 1. Immunohistochemical localization of Fos in uterine sections. Fos immunostaining in (A) an ovariectomized mouse uterus; (B) a Day 1 pregnant mouse uterus; and (C) a Day 1 pregnant uterus after preabsorbing with c-fos peptide 1 (amino acids 128-152). (le) Luminal epithelium; (s) stromal cell; arrow denotes probable blood cells. Bar equals 100 1A.m.
c-fos Peptide
2 (amino
acids
4-17)
prior
to
IN
THE
PERI-IMPLANTATION
processing
(not
(Fig. 4D). In non-sites, thelium through Day
shown).
staining
Effects
of Steroid
Hornones
on
Uterine
Fos
In ovariectomized mice, after a single injection of 100 ng E2, no cells staining for Fos were detected at 1 h, but nuclear staining for Fos was evident at 3 h in the luminal and
glandular
epithelium
The nuclear staining 12 h (Fig. 2A), but P4 alone
(Fig.
ectomized E2, it was
of E2 on Fos with E2 (Fig. with staining ing,
failed
to when
at 6 h and persisted at 24 h. In sharp
to induce
Fos
in the
cell.
through contrast,
uteri
Since P4 is known to modify to determine if P4 influences
in the
Progesterone
E2 + 2 mg
stromal
of ovani-
the
stroma
P4 on Day
E2 and
imen nuclear was intense
was
priming 4 resulted
P4 were
increased
for
3 days
action of the effects
with
in a staining
co-administered,
staining for Fos was at 12 h (Fig. 2E), and
this
followed
regi-
by 100
pattern but
ng
similar
with
this
was
primary 6, the
reg-
detected at 1 h (Fig. 2D), still present at 24 h (Fig.
2F).
Pregnancy
and
On Days I and 2 of pregnancy Fos was detected in the luminal and
in the
creased Day
stroma
by Day 4, only
sional
(Fig. 3 (Fig.
very
staining
and
pseudopregnancy existed between
3, A-D).
Overall F), and for
stroma
cells
Fos
was
cone
(Fig.
Fos
detected
in the
deof
in occa-
pregnancy
(Fig. 3, G and H). No obvious pregnancy and pseudopregnancy
the morning of Day 4. On the embryo was still encased
was
and
differences from Day
the afternoon of Day in the zona pellucida,
endometrium,
but
the
staining
pat-
tern was not consistent throughout the uterus. Examination of serial sections showed that in some areas no staining Fos
was
hum
detected,
stained,
in other
while
areas
in other
only
areas
the
the
luminal
luminal
placement
retained
in
of embryos the
lumen
since through
the the
epithe-
nuclear stained
embryos staining
stainareas
were
not
procedure.
The cluding
fra-1,
transiently ical and
expressed pharmacological
ucts
fra-2,
of these
Fos,
act at AP-1 study by
to
binding
both
that
of early
present in the
guished. tioned
Beginning at this in order to determine
staining. luminal (Fig.
pregnant
On the epithelium 4C)
and
morning and in
just
the the
of Day
5, implantation
animals
could
sites
be
distin-
time all sites were serially secthe spatial progression of Fos of Day 5, Fos antimesometrial luminal
was
epithelium
present stroma in
in the in sites non-sites
with
stromal
cell.
duced mRNA
the
physiological
Fos
was
induced
The
was
with
rent
study
lated
antigens
results
restricted initial
from
this
against
the
has
also [25].
directed The
been
may
also
have
their
own
and
anan
127-152
(M-
acids
time
several for Fos
a conserved used in the
55 kDa,
shown proteins
glan-
Fos-related et al. used
shown to detect et al. [31] probed
to detect
rat brain,
the findings that immu-
noted at 24 h. as well as cell-
against antibody
shown
In the
from
at 3 h, maximal
reduction induction
been Papa
c-fos
with Results
luminal
noted
c-fos amino
are
the E2 stimulus, significantly re-
may be due to the particular by various antibodies. Gibbs had previously antigens [32], and
study
expression Fos staining
with found
to the
staining
the still
primarily localized also detected in an
more closely investigators
and 38 kDa proteins have been peptide antibody. The different antibody
was was
rap-
with h, and
estrogen-induced et al. [31] noted
staining at 6 h, and a significant Differences in the kinetics of Fos type specificity tigens detected
mouse
and
time course is in agreement in the whole organ [11].
Fos
epithelium
[30].
in the
steroids
the current study correlate of Gibbs et al. [26]. These for
transcription
of the rat uterus 2 h after of cells expressing Fos
at 6 h. This concentrations
noreactivity
complexes
pregnancy.
somewhat different from the of Fos in the rat uterus. Papa in the epithelium with the number
usually
of the jun
proteins
is modulated
but decreased at 24 h. Fos endometrial epithelium, but
occasional
and
mice given E2 replacement at 3 h, maximal at 6-12
antigens with an antibody gion in the amino terminus.
morning
found decidua
of physiologprotein prod-
heterodimeric
to modulate Fos
exogenous
staining
the
was
is rapidly
to a variety [27-29]. The
form
sites
showed
pattern
that
participate
family
that
By the
7, staining mesometnial
also detected in the embryonic cells and in the ectoplacental
fos-B,
directed
in
detected in the secondfrom the primary zone
On Day and the
in response stimuli
genes,
proto-oncogene that
and
peptide) Fos-related
noted.
Day site
c-fos, the cellular homolog of the is a member of a gene family, in-
proto-oncogene a-fos oncogene,
viral
antibody
non-sites
in the on
5E).
Staining for Fos was not detected on the afternoon of Day 4 of pseudopregnancy (Fig. 4B). The uteri of pseudopregnant animals were examined through Day 6 with no further and
and
F). Beginning the implantation
DISCUSSION
dular
epithelium
as well as the antimesometrial stoma showed ing (Fig. 4A). It was not possible to correlate with
for
epithelium
4, E and within
Day 7 Fos was including giant
idly in ovariectomized protein(s) detectable
morning
noted
in both
1 through 4, while was
staining by the
luminal
Staining for Fos was zone, but was absent
On
hormone
and pseudopregnancy, and glandular epithelium
3, E and
faint
epithelial
Pseudopregnancy
in the
and the luminal epithelium. in the deep secondary zone
uterus
Fos during
found
(Fig. 5, A-C). ary decidual
This
Uterine
Fos remained in the luminal epi7. In sites, on the afternoon of Day 5
decidual zone (Fig. embryo was detectable
(Fig. SD). trophoblast,
induction. When P4 was given in conjunction 2C), cells positive for Fos were noted at 3 h still detectable at 24 h. The intensity of stain-
especially
men.
in an occasional
was intense was decreased
2B)
mice. important
and
495
UTERUS
several
Fos-re-
44 kDa,
41 kDa,
to bind boun’d course
recur-
to the Mby a given
of induction.
496
BAKER
FIG.
2.
Immunohistochemical
istration; (B) 12 days P4 priming.
localization
h after P2 administration; (le) Luminal epithelium;
of
Fos
in
ovariectomized
(C) 12 h after co-administration (ge) glandular epithelium; and
ET AL.
following the administration of steroids hormones: (A) 12 h after E2 adminof E2 + P4; (D) 1 h, (E) 12 h, and (F) 24 h after E2 administration following 3 (s) stromal cell; (st) stroma. Bar equals 100 m.
mice
FIG.
3.
Immunohistochemical
pseudopregnancy; Day
(st)
4 of pregnancy;
stroma.
A-F and
(C) Day
localization
of
2 of pregnancy;
(H) morning
H: bar equals
of Day
100
Fos
(Dl Day
on
Days
4 of pseudopregnancy.
p.m;
G-1.
1,
2,
and
3 of
2 of pseudopregnancy;
2: bar equals
(le) Luminal
50 m.
pregnancy
(El Day
and
pseudopregnancy:
3 of pregnancy;
epithelium,
(gel glandular
(F) Day
(A)
Day
1 of
pregnancy;
3 of pseudopregnancy;
epithelium;
(myo)
myometrium;
(G-1,
(B) Day 1 of 2) morning of (s) stromal
cell;
FIG. 4. lmmunohistochemical localization of Fos on Days 4 and 5 of pregnancy and pseudopregnancy. (A) Afternoon of Day 4 of pregnancy; afternoon of Day 4 of pseudopregnancy; (C) pregnancy site on morning of Day 5; (D) non-site on the morning of Day 5 of pregnancy; (E) afternoon Day 5 pregnancy site; (F) enlargement of area outlined in (E). (am st) Antimesometrial stroma; (le) luminal epithelium. (pdz) primary decidual zone; stromal cell; (st) stroma. Bar equals 100 m.
(B)
of (s)
IN
c-fos
FIG. 5. lmmunohistochemical localization of area outlined in (A); (C) site approximately embryonic trophoblast on Day 7. (em) Embryo; p.m; B: bar equals 50 p.m.
It is somewhat was
detected
staining
has
not
uterus [26,31]. fect on stromal epithelial,
surprising after
cells
that
been
reported
respond
PERI-IMPLANTATION
499
UTERUS
of Fos on Days 6 and 7 of pregnancy. (A) Site adjacent to embryo on the morning of Day 6; (B) enlargement 200 p.m from embryo on Day 6; (D) site on Day 7; and (E) enlargement of area outlined in (D) showing (mdec) mesometrial decidua; (sdz) secondary decidual zone (tb) trophoblast. A end C-E: bar equals 100
stromal
E2 administration
staining, since
in this
Estrogen is known, cells. Stromal and
THE
cell
however myometrial,
to E2 in the
albeit
rare,
E2-induced
immature
type
Fos
in the
rat
to have an efas well as rat
uterus
[33].
The
response
by hypertrophy
is marked and
hyperplasia
by initial [34].
hyperemia The
nuclear
followed estrogen-
binding sites are still present in both the epithelium and stroma in the mature rat uterus [35] and E2 alone has been shown to cause alterations in stromal cells. For example, slight increases in the granular component of nucleoli are
BAKER
500 noted
in
stromal
cells
when
ovariectomized
ceive E2 replacements [36]. proto-oncogenes, including liferative
changes.
triol
16a-estradiol
and
significantly sible that proliferation
c-fos,
example, can
was
activities no
ministered
for mice.
tent with studies showing a lack the rat uterus [9, 26]. Progesterone E2
intensified
and
prolonged
for Fos, additional
h)
E2 administration.
tion
on
uterine
es-
estradiol
to be
via Fos
Fos
when
This
finding
since
P4 was
ad-
response concurrently
to P4 in with
immunohistochemical
P4 is known
endometrial
shown to modulate the proto-oncogene, c-myc,
cells
to modulate
[1], and
E2-responsive in the mouse
E2 1
E2 ac-
recently
stance
[39], an
P4 was
areas.
from Days when
Fos
was
detected
in
myometrium, and to the hormonal
the
luminal
and
the stroma. This milieu resulting
E2 administration following 3 days of P4 priming. On 3 and early Day 4 of pregnancy and pseudopregnancy, the uterus is primarily under the influence of P4,
staining for Fos was not detected. This pattern reflects the failure of P4 alone to induce Fos in ovariectomized animals. Fos was also not detected on subsequent days of pseudopregnancy (Days 5 and 6) when the uterus remains under P4 dominance Beginning noted time. nant
[I]. on the
between Staining uterus.
afternoon
pregnancy
of Day
and
4, differences
pseudopregnancy
for Fos differed in various areas Certain areas showed Fos staining
were
for the
Although sue sections reflects the
it was
not
possible
to localize
at this time, it may initial establishment
and non-sites. The expression nancy, but not pseudopregnancy,
of Fos
on late Day is significant
tis-
pattern sites
Day
sensitivity when the endometrium is receptive to a signal promoting decidualization from the blastocyst [2, 3, 23]. It is noteworthy that Fos is first expressed in the luminal epithelium during this period of receptivity since the signal for decidualization is believed to be transduced to the stromal cells via the epithelium [38]. This study supports the idea that the embryo exerts its influence via a diffusible sub-
into
showed
in the
the
[40], and
RNA
and
Day
embryos Beginning site areas.
timesometrial
luminal
since
these
sites. about
by
the
The
die
noted epithelium
to stain proximity
for Fos. This response to the implanting
embryonic
in
on
Day
ectoderm
recently
shown
a!.
Fos
mRNA
the
luminal
epithe-
by the
blasto-
degenerative
5 [42], embryo
but mesoalso failed
to be associated
have
from
previously
re-
nuclei and
7.5-day
and
also
have
embryos.
although collected temporal
pen-implantation
with
for Fos in the Fos was localin the ectopla-
trophoblastic
did not detect Fos in the Day 6 embryo, reports that 3.5-day blastocysts freshly uterus did stain positively for Fos. The Fos expression more study.
no staining in the anthe
8 of pregnancy
cfos
for
in
signal
with
[41,43]
Fos
Fos
somehow
of Day of the
the
of for
surrounding
appears blastocyst.
et
for
has
may
apoptosis
the evening in the vicinity
Adamson
deep
Fos
pattern
In this study, the first definitive staining embryo was noted on Day 7 of pregnancy; ized in giant cells of the trophoblast and staining
for
staining
making
by
changes metrial
cone.
in the
on Day 6, there was This is not surprising epithehium
cells by
areas.
7 of pregnancy
between
was
staining
decidua; Adamson [41] prein the maternal decidua from
non-site
through
in site
epithelium
the primary decidto the secondary
Fos
that
8) implantation to speculate
hum refractory. for Fos in the cyst
for
earliest
staining
luminal
Fos as was had moved
maternal proteins
c-fos
Fos
5, the
7, staining is the
4 of pregsince this
expression of Fos is apparently due to the influence of the embryo that is still encased in the zona pellucida. The timing of this expression coincides with the period of uterine
and
[23].
of Day
This
in site
non-sites
ported
in the
on
zone.
been reported viously reported
cental
embryos
cells
afternoon
zone;
secondary
first
be that this staining of future implantation
trophoblast
incorporation
stromal
By the
decidual
of the pregonly in scat-
tered luminal epithelia cells, while in other areas Fos was present in both the luminal epithelium and the antimesometrial stroma; still other regions showed no Fos activity.
the
permeability
was still immunoreactive for ual zone. On Day 6, staining
spacing
cycle,
in uridine
in vascular
7.5-day (our Day It is interesting
induction of another uterus [22].
between
Staining for Fos correlates both temporally and spatially with decidual changes on Days 5-7 of pregnancy. On the morning of Day 5, the entire luminal epithelium and many
the
epithelium, the state is similar
contact
an increase
staining
previous
actual
increase
On Days 1 and 2 of pregnancy and pseudopregnancy, when the uterus is responding to ovulatory E2 and P4 from glandular endocrine
since
uterine epithelium does not take place until the embryo hatches from the zona pehlucida a few hours later [23]. Other investigators have reported uterine responses to the zonaencased embryo including nucleolar changes in the stroma
antimesometrial
is consis-
while 3 days of priming with P4 before effect of inducing Fos earlier (within
staining had the after
both
only
[37]. Thus, it is posfunction aside from mature mouse uterus.
of Fos given the
uterus
but
appear
immunoreactivity
to ovariectomized
rat
c-fos,
not
re-
shown to induce subsequent pro-
in the
induce
do
rodents
been without
stimulates DNA synthesis some E2-dependent stromal is induced via Fos in the
P4-mediated there
For
E2 has
ET AL.
We
Adamson from the aspect of
embryo
requires
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