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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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