in the pseudopregnant endometrium. As pregnancy progressed, protein synthesis was altered in the placental/junctional zone and the non-site endometrium,.
BIOLOGY
OF
44,
REPRODUCTION
Changes
345-356
(1991)
in Endometrial
and Placental
Pregnancy
and
A. BOOMSMA,
ROBERT
A.
of Obstetrics
College
Synthesis
Pseudopregnancy
PATRICIA
Department
Protein
in the and
MAVROGIANIS,
and
of Medicine
Gynecology,
at Chicago,
and Morphology Cat1
HAROLD
University
Chicago,
during
G. VERHAGE2
of Illinois
Illinois
60680
ABSTRACT This
study
synthesis.
was
and
analyzed for light
(Biol
Reprod
1987;
munocytochemical PDP.
and
By 20 days of PDP.
deposits
began
detected
of nine was
other
progressed,
proteins
portion
implanting
cat
the
protein
protein
continued becoming
of the blastocyst
synthetic
implantation
site
of the
patterns
the
of
it continued
the
end
site
morphology
uterus
show
deep
under
in the
such
but
and
and
site the
implantation
apparently
not
differentiation
and
synthesis
The
non-site
site
and
the
non-site
but
non-site
continue
pregnancy
As
of the
in the
non-site.
endometrium,
region. to change
The
was
synthesis
endometrium
endometrium.
portion
non-
of PDP
pseudopregnant
PDP
5 weeks.
in the
of lacked
the
in
the
in
endometrium,
deep
and
deposits
days.
through
pattern
non-site
to the
large
im-
deposits
epitheium
35
and by
identified
regressed
glands
20
Boomsma
by
large
with
pseudopregnant
the
similar
surface
L-
with
by.fluorography,
was
had
glands
endometrial
that
cultured
contained
placenta The
between
in the
and
the
protein
and
then
[2j), glands
nonimplantation
found
regional
The
observed
of the
endometrium
the
and
followed previously
described
deep
site.
were
zone
unchanged
on the
cat
pattern
41:347-354)
progressed.
of implantation
the
1989;
well.developed
implantation
bodies
largely
implantation
pregnant
the
placental/junctional
it was effect
in the
electrophoresis
glands
morphology
cats
gel (PDP), and
as pregnancy
while
to
12 days,
endometrial
endometrial
pseudopregnant
Reprod
after
but
similar
in the
a significant
activity
by
but
altered
was
has
synthetic
altered site
(Biol
thicken
apoptotic
16 days,
after
significantly
and
to those
though
al.
deep
convolutions,
similar even
site
synthesis
the
and
and
polyacrylamide protein
Ct
on
blastocyst
pregnant
began
to develop extreme
implantation
Verhage
Attachment
developed,
well
of PDP
was the
protein
dometrial
and
implantation
from
different
[1])
developed
4 weeks,
in the
The
37:117-126
analysis.
cat
implanting
from
microscopy.
was
deposits
obtained
by 2.dimensional progesterone.dependent
placenta
by
maintained
of the
effect
were
synthesis
immunoblot
regions
to regress
animals
for protein
placenta
The
site
implantation
not
the
the
tissues
endometrial
and
[35Sjmethionine and also processed Verhage
to determine
undertaken
Placental
deep
en-
Thus,
the
and
alters
morphology
and
as pregnancy
pro-
gresses.
INTRODUCTION The
cat
is an
of seasonal levels
are
induced
estrous
ovulator
periods.
elevated
high
During
[3] and
the endometrium. These trophy and hyperplasia,
and and
undergoes estrus,
characteristic changes
molecular
Ovulation occurs (P) is detected
action
include
of P on
an
E2-primed
glycogen
deposition
secretion
of a P-dependent
and
recently been shown collagenase cathepsin-L
uterus
during
processing,
Accepted
September
Received
June
and (PDP)
to have [12].
significant
and later the
the
at Chicago,
840
of Obstetrics
5. Wood
[13].
The
cat has
an
that
We
effect
partments
and
10, 11]. PDP
theorize
in addition the
to the
implanting synthesis.
obvious
effects
on
cat blastocyst also This concept is sup-
of the
implanting
cat
blastocyst
on
endometrial
with
pseudopregnant
endometrium.
the protein changed
In addition,
synthetic patas pregnancy
with
Tissue
MATERIALS
AND
METHODS
supplies
were
purchased
culture
from
GIBCO
Biological Co., Grand Island, NY), and electrophoretic supplies were obtained from Bio-Rad Laboratories (Richmond, CA). Ampholines were from LKB (Uppsala, Sweden), and immunocytochemical supplies from (Grand
Island
Cappel Laboratories 1142 Ci/mmol)
and Gynecology
Street, Chicago,
days
[14].
we wanted to determine whether terns observed after implantation progressed to term.
cavity at the morula/ 6-7 days postcoitum
Department
12-13
protein synthesis and to correlate any change with morphological alterations by comparing pregnant uterine com-
25. 1990.
of Illinois
after
placenta, and thus part of the uterine wall implantation so that the fetal allantochocontact with the maternal blood vessels
the
preimplan-
homology
occurs
ported by studies in the mouse [15, 16], cow [17], and sheep [18, 19]. The major objective of this study was to determine
progesThe
[7].
synthesis
[1,2,8,
attachment
endotheliochorial is eroded during non comes into
endometrial morphology, affects endometrial protein
28, 1990.
‘Supported by NIH Grant HD11404. 2Correspondence: Harold G. Verhage, University
in
hyper-
including the inhiand CUPED synthethat follows in the and hyperplasia,
protein
The cat embryo enters the uterine early blastocyst stage approximately
808),
(E2)
occur
glandular
1-2 days postcoitum, in the plasma a day
tation period is at first antiestrogenic, bition of Erinduced cytodifferentiation sis [6,8,9]. The progestational response glands includes additional hypertrophy
(m/c
estradiol
changes
and the synthesis and release of a weight protein (CUPED) by the uterine glands
[3-6]. terone
has the
a series
II.60612.
345
was
(Malvem, obtained
PA). L-[35S]methiOnine from New England
(sp. act. Nuclear
346
BOOMSMA
(Boston,
MA). All other
or better
were
inorganic
products
chemicals
of Sigma
of reagent
Chemical
grade
Co. (St. Louis,
MO).
Sexually in rooms
mature maintained
domestic cats at 22#{176}C with
in the Biological Resources
were individually caged a 12L: 1 2D photopeniod
Laboratory
of the University of
Illinois. Food and water were given ad libitum. Animals were anesthetized with an i.m. injection of ketamine hydrochloride (25 mg/kg body weight) supplemented with aceprom(0.6
mg/kg)
prior
to all
surgical
procedures.
Estrus
was
ture queens behavioral
determined
for estrus,
by daily
lordotic behavior. the queen was
observation On placed
and observed until coitus occurred. to remain with the male overnight. considered
to be
Day
zero
animals),
12 (3),
of the
14 (4),
ma-
the second day of with a fertile male
The queen was allowed The day of coitus was
of pregnancy.
ovulation was verified at the time presence of newly formed corpora
Coitus-induced
of laparotomy lutea. Tissue
16 (3),
18 (3),
by the taken
was
20 (3),
and
24 (2) days, and 4 (3), 5 (2), 6 (2), 7 (2), 8 (2), and 9 (2; term) weeks postcoitum. Pseudopregnancy was induced by mating estrous queens with a vasectomized 24, 28, and 35 days stage. cat
male. Tissue was taken postcoitum from a single
Pseudopregnancy
lasts
only
through
14, 16, 18, 20, animal at each 5 weeks
in the
[3].
Tissue
Collection
Tissue
was
omy in the implantation
and
Explant
collected
using
Cultures sterile
technique.
removed from then removed,
and
the
embryo
and
the placenta and and implantation
large
blood
and post-
discarded. sites were
vessels
were
The uterus separated
the intervening uterine regions (nonimplantation cats 12-24 days postcoitum, each implantation carefully opened and the embryo was removed
was from
sites). In site was after the
uterus was taken out of the animal and the implantation site and non-site was separated. In both the implantation site and nonimplantation site, the endometrium was care-
portion
from
the
underlying
tissue
separated from point between of the
the myometrium. 16 and 24 days
implantation
site
myometrium.
Endometrial and out in the presence viously then
described dialized
mM
Tris
In one postcoitum,
(placenta
and
placental explant of L-[355]methionine methods
(pH
8.2)
cultures were carried for 24 h using pre-
[1, 20]. The
(12 000-14
Polyacrylamide
000
culture
cutoff)
M
at 4#{176}C and
stored
medium
for
was
48 h against
frozen
until
2
used.
animal the
at each surface
adhering
dometrium) was separated from the underlying tissue cultured separately. In one animal at each time point 6-9 weeks of pregnancy, the surface region of the
enand from non-
Gel Electrophoresis
Lyophilized aliquots cpm of radioactivity (2-D)
SDS-PAGE
O’Farrell focusing
of culture medium containing 100 000 were subjected to two-dimensional
using
a modification
[21]. 2-D analysis was in the first dimension
amide
slab gel with a stacking
of
man
Kodak
Co.,
gel
Rochester,
in the
NY)
[22]
25 g protein 10% SDS-PAGE using
tem.
the
Proteins
Bio-Rad
Modular
then
of
dimension,
Mini
fluorograph and
for
per
flushings
con-
to one-dimensional method of Laemmli Electrophoresis
electrophoretically
nitrocellulose and immunostained Rad Immuno-Blot kit [2].
Tissue
second
media
were subjected according to the
were
method
[1, 20]. Fluorography Kodak XAR film (East-
[1]. One
tissue compartment was analyzed. Lyophilized aliquots of culture taining (1-D)
the
performed with isoelectric and a 10% SDS polyacryl-
using the Bio-Rad Protean II system was performed on dried gels using
At laparot-
pregnant animal, the uterus was exposed sites were identified. In cats 4-9 weeks
endometrium,
the
Microscopic
coitum, the implantation site was carefully opened in situ, the embryo with placenta was removed by carefully separating the placenta and junctional zone from the underlying
fully time
site was separated separately.
After
removal of the reproductive tract, the anesthetized animals were given a lethal iv. dose of sodium pentobarbital.
10 (2
implantation and cultured
Uterine flushings were obtained in situ in the preimplantation (10 days postcoitum) and pseudopregnant animals as previously described [1]. The uterus was then removed, and the endometnium was carefully separated from
Animals
azine
ET AL.
sys-
transferred PDP
using
to
the
Bio-
Procedures from
the
various
compartments
was
immersion-
fixed in a HEPES-buffered solution formaldehyde/1% glutaraldehyde perature, dehydrated in graded
containing 3% for 4 h at room ethanol changes,
paratemand
embedded in Araldite [23]. For general observation, 0.5-p.m
sections
on
Sorvall MT-5000 walk, CT), placed blue.
For
ultramicrotome on glass slides,
immunocytochemistry,
were
cut
a
(Ivan Sorvall, Inc., Norand stained with toluidine serial
1-p.m
sections
were
cut, placed on acid-cleaned slides, and etched for 10 mm in saturated ethanolic sodium hydroxide. A modification of the peroxidase-antiperoxidase immunocytochemical localization procedure of Sternberger et al. [24] was used to localize PDP in the plastic-embedded sections antibody was diluted 1: 1 000 for use in this control the
sections,
preimmune
serum
was
[2, 5]. Primary procedure. For used
in place
of
primary antiserum. For electron microscopy, tissue was post-fixed in osmium tetroxide before embedding [23]. Thin sections (70 nm) were cut with a diamond knife, collected on copper grids, and stained with uranyl acetate and lead citrate.
PROTEIN
SYNTHESIS
RESULTS Preimplantation
10 days postcoitum. and
The
microscopic
structure
immunocytochemical
in the endometrium obtained mals was similar to that previously
of PDP
from 10-day postcoital aniobserved in steroid-treated summarized folded, with the surface
into the stroma. The cells of the deep endometrial were tall-columnar and contained oval nuclei with
prominent gen,
of the
localization
and pregnant animals [2, 10, 11] and is briefly here. The surface epithelium was moderately numerous convoluted glands extending from deep glands
nucleoli,
and
present
small
onstrated ithelium
a vacuolated
halo
in their
cytoplasm,
granules.
lumina.
some
A basophilic
The
presence
immunocytochemically as a strong cytoplasmic
glyco-
substance of PDP
was
was
dem-
in the deep glandular epreaction product in all of
cells observed. Table 1 lists the proteins that appeared the course of pregnancy. Table 2 outlines times thetic ure
14 days postcoitum
(Fig. 2C). PDP
observed reduced
CP2
they are present. The typical endometrial protein synpattern from cats 10 days postcoitum is shown in Fig1A and
was
similar
to that
reported
previously
[1]. Two
was
observed
was
cultured
one was
their
presence to draw any
Implantation
Site
12-14
days
postcoitum.
Implantation
sites
were
evi-
dent by 12 days postcoitum. At this time the blastocyst had expanded to fill the uterine lumen, the luminal epithelium was flattened, and the neck region of the glands was beginning to dilate. By 14 days, the luminal epithelium was still
CP3
16-24
mostly
intact
except
for
a few
Placental and endometrial major alterations in synthesis during pseudopregnancy.
TABLE
1.
Protein
M, 36000
6.0-7.0
CP1” CP2
28000 20000 29000 35000
5.0-6.0 5.0 5.0 5.6 5.0 4.8 5.6 6.3-7.3
CP4 CP5 CP6 CP7
44000
CP8
41 000 31 000
41 000 38000 47000
CP9
show and
Description
5.0-6.0
7.5
relative molecular weight. isoelectric point. CPDP = progesterone-dependent dCp = cat protein.
of penetration
proteins that cat pregnancy
jb
PD
CP3
sites
=
protein.
series of 3-5 spots series of 4 spots single spot single spot single spot single definitive spot single diffuse spot single spot series of 5 spots series of 4 spots 2 spots
by
and
spot
from
villi
parallel chyme
had
the
increased By
postcoitum.
was complete, and invading
were apparent. After more deeply into the
round maternal gan to take on of the
CP9
days
enchyme extend
conclusions.
only
12-14 days poin the preim-
for
the
deep
farthest
from other
the
CP3
CP4 CP5 CP6 CP7 CP8 CP9
*Abbreviations:
re-
were observed portion. In the CP7, was obepi-
the surface epithelium villi containing fetal 18 days, the endometrium
junctional consisted
by cytotrophoblast
and
was mes-
villi began to and to sur-
P1
NS
(Fig.
d12-w9
abs d12-w9 postcoitum;
d12-w9 d12-w9 d14-w9
d12-w5 var abs d12-w5 d12-w5 d18-w5
abs var d14-w9 d12-w9
abs abs var d12-w5
w5-w9
d12-w9 d12-w9 d12-d24 w4-9 (var) w4-w6
day
up
PS
d12-w5 var
d14-w9
=
lined of mesen-
Compartment
d12-d16 var
abs abs abs dlO
were
syncytiotrophoblast
IS
diOt dlO abs dlO dlO abs
d
zone
of a core
and temporal expression of pregnancy and pseudopregnancy. Tissue
PDP CP1 CP2
In
endometrial
in intensity. 16 days postcoitum,
and
TABLE 2. Compartmentalization proteins synthesized during cat
Protein
time.
superficial site (Fig.
blood vessels. By 20 days, the placenta beits mature structure (Fig. 2D). The portions
to each lined
first
studied at 14 days, the region of the implantation
separately
thelial penetration completely eroded,
However, of pregnancy
immunoreactivity,
(Fig. 1C). In this animal, CP2 and PDP in the superficial placental/endometrial endometrial portion, a new protein,
served.
also observed. over the course
as a faint
animals
forms of PDP and CP1, 3, 4, and 9 are present at this time. Two groups of high molecular weight proteins-one basic, acidic-were too variable
tall-colarge at 12-
in the deep glands, was intense after 12 days, greatly after 14 days, and absent by 16 days (data not
one of the four placental/endometrial 1D)
found
of 2A). and
plantation endometrium. By 14 days (Fig. 1B), the diffuse CP5 spot was consistently seen and was at its largest size in the complete implantation site (superficial and deep),
gion only deep
be
epithelial cells. The contained relatively to the halo granules
Alterations in protein synthesis occurred stcoitum when compared to protein synthesis
as the
can
Tissue degeneration the trophoblast and
shown).
as well
proteins
(Fig. 2A). between
among the deep glands in addition
over com-
the
of trophoblast at the interface
mitosis was evident lumnar cells of the basophilic granules
to change the tissue
where
347
CAT
the maternal tissues and was characterized by groups epithelial and stromal cells with pyknotic nuclei (Fig. The neck region of the glands was dilated (Fig. 2B),
and
the
partment
PREGNANT
small stumps was evident
Endomeirium
endometrium
IN THE
w
=
week
postcoitum;
P1
=
preimplantation endometrium; IS = implantation site (placenta and endometrium); NS = nonimplantation site endometrium; PS = pseudopregnant endometrium; var = variable synthesis; abs = absent (not synthesized). tdata in this table indicates only the presence or absence of synthesis of a particular protein and not the spot intensity.
BOOMSMA
348
ET AL.
p1
8
V
6.1
V
4.5
8
4.5
6.1
‘V
8
‘V
V
.
-.i
30
21
#{149}I.
b.
FIG. 1. Fluorographs of culture media from pregnant cats. The two forms of PDP are enclosed in brackets. Other proteins are identified by the appropriate number without the CP prefix. A: Endometrium 10 days postcoitum. B: Implantation site 14 days postcoitum. C: The deep endometrial portion of the implantation site 14 days postcoitum. D: The superficial placental/endometrial portion of the implantation site 14 days postcoitum. E: The deep endometrial portion of the implantation site 20 days postcoitum. F: The superficial placental/endometrial portion of the implantation site 20 days postcoitum. G: The deep endometrial portion of the implantation site 6 weeks postcoitum. H: The deep endometrial portion of the implantation site 7 weeks posicoitum. I: Placental/junctional zone of the implantation site 7 weeks postcoitum.
PROTEIN
SYNTHESIS
IN
THE PREGNANT
CAT
349
FIG. 2. Implantation site region from pregnant cats. A-C: 14 Days postcoitum. A: t = Trophoblast; p = Pyknotic cells. x460. B: Neck glands below the invading trophoblast. Arrow mitotic figure. x460. C: Deep glands below the invading trophoblast. Arrowheads = large basophilic granules; arrows = dense core granules. (Electron micrographs of these granules are shown in Figure 3E}. x460. D-l: 20 Days postcoitum. D: Low magnification overview of the implantation site region. I = Placental labyrinth; jz = junctional zone; n = neck glands; d = deep endometrial glands. x70. Broken lines indicate divisions between regions. E: The placental labyrinth. g = Maternal giant cells; mc = maternal capillaries; fm s = syncytiotrophoblast. x460. F: The junctional zone. g = Giant cells; t = trophoblast. x460. G: Neck glandular arrow = mitotic figure; arrowheads = dome-shaped apical tips of epithelial cells. x460. H: Deep glandular region. of PDP in the deep glandular region. Note the absence of specific immunoreactivity. x460.
=
fetal mesenchyme; c = cytotrophoblast; region. lu = Lumina of the neck glands; x460. I: Immunocytochemical localization
350
BOOMSMA
neighboring
villi
(Fig.
2E).
mellae
(Fig.
2E).
These
cytoplasm,
are believed generation
large
large
to was
maintained
as a thick
area
(Fig. 2D). epithelial
deep
present,
with
began
after
iF)
was
and
was
protein
absent
last
began
cells
(Fig.
to thin
cells
were
time
after close
were
found
(Fig.
7 weeks
in
3A).
The
number
as pregnancy
so that from
this
junctional
below the junctional zone tissue, and only a few deep
epithelial cells were present (Fig. tivity was never observed in any during this study period (data not protein
synthetic
portion
pattern
of the
observed
implantation
(Fig. days were The
1H),
the
12-24
was
endome-
tron-opaque
were
cap3A).
between
4 and
pattern
appeared
6 weeks similar
(Fig.
spot; CP5
immunoreacsite tissue deep
en-
slightly
seen
7 weeks after
20
(compare with Fig. 1E), except that CP3, 4, 5, and 9 reduced in intensity, and CP5 was frequently absent. basic synthetic pattern was generally unchanged after
CP7
mitotic figures
were
and
the
indistinct. After
epithelium
of the
14 days, the epithelial
were tall-columnar and contained in addition to halo granules (Fig. granules contained either elecmaterial
(Fig.
3E).
The
deep
en-
through 24 days was evident in 3F) and declined Giant stromal cells
observed.
protein site
synthetic
pattern
endometrium
was
of the only
Day
12-24
slightly
nonim-
different
from
after 20 days (Fig. 4D), this enlarged so that CP4 and became indistinct from each other. In addition, CP7
4-9
en-
4B),
the preimplantation endometrium at 10 days postcoitum. At 14 days postcoitum (Fig. 4C), CP5 was observed as a faint
and CP8
not
(Fig.
18 and 24 days that the junction
epithelium
or flocculent
plantation
ap-
could
became
never
Fig.
1G). After
to that
zone
of the nonimplantation (Fig. 3C). This continued
glands maintained this structure Strong PDP immunoreactivity glands through 20 days (Fig. after 24 days (data not shown).
The
(compare
of the
the luminal
dometrial postcoitum. the deep somewhat
zone only a
weeks
9
Numerous
of the deep glands basophilic granules The large basophilic
capillaries
only
days postcoitum.
in the luminal epithelium 12-16 days postcoitum
cells large 3D).
vacuoles
in the
site
pe-
Site Endometrium
were obregion
altered during the remainder of pregnancy. CP6, a protein not seen before Day 24, was observed transiently in this compartment
Nonimplantation
glands
consisted of stromal glands with cuboidal
3B). PDP implantation shown).
this
placental/junctional
in intensity. After
neck
the fetal (Fig.
Most
reduced
was and
cytotrophob-
region
zone.
was
between
below the junctional zone, 20 days, began to reduce in
by 5 weeks
the
during
media.
face
4 weeks,
distinguished
of fetal progressed
superficial
to such an extent between
junctional
perinuclear
observed
had greatly increased in intensity and was much more prominent in the placental/junctional zone than in the underlying endometrium (compare with Fig. 4A). PDP and CP6 were never observed in placental/junctional zone culture
observed site from
labyrinth became to late pregnancy. the
the
was
so that by 9 weeks
to have
The neck glandular region which was a thick layer after
dometrial
deep
cells were observed and to the syncytiotrophoblast
observed
peared to increase 3A with Fig. 2E).
dometrium connective
the
7 weeks,
21).
pattern
adjacent placental lamellae. The
few cytotrophoblastic illaries were very
after
from
After
by 20
epithelium granules
synthetic
The placental during mid
organized
giant
giant
separately
postcoitum. more
between
The
the
so that
as an intense spot. Differences superficial placental/endometrial
cultured
weeks
and
be
(Figs. was
(Fig. 1E). CP2 was only present in the superCP3 and 9 were more intense in the deep CP7 was observed in both compartments.
Numerous
size
16 days,
never
zone,
below
the glandular 2H). Although
was
by pale-
zone glands
lumina
PDP
nucleoli,
in this region 16-24 days postcoitum. PDP observed, CP5 had decreased in intensity,
trial region ficial region, region, and
at this
dilated
and (Fig.
consistent
CP2 was observed served when the
The
prominent
4A).
of the implantation site (Fig. 11) had a protein synthetic pattern similar to that found after 20 days (compare with Fig. iF), except that CP2, limited to the placental/junctional
characterized and
immunoreactivity
and
observed no longer
4-9
first
Blebbing was evident on the apicells (Fig. 2G). A general regres-
glands
PDP
A distinct
thicker
were
fingers of the junctional of the endometrial
days few glands were present, was low columnar-cuboidal
(Fig.
cells
be of decidual origin [14, 25]. Tissue deprominent among the irregular mesen-
junctional zone cal tips of these of the
cells,
nuclei,
chyme-filled trophoblastic 2D, F). The neck region
were
stromal
(Fig.
riod.
at 20 days both in the junctional zone among the villi (Fig. 2F) and between the trophoblastic Ia-
staining
sion
Giant
AL.
9 weeks
2E). Fetal blood vessels were present within the mesenchyme, and maternal blood vessels were found between observed invading
El
were
observed
weeks
postcoitum.
epithelium
in the
as faint spots after The non-site
irregular continued
20 days. nature of the to increase.
surBe-
tween 7 and 9 weeks postcoitum, the surface appeared extremely villous-like in nature, with epithelial-lined structures containing a core of stroma projecting into the uterine lumen (Fig. 3G). The columnar merous long, apical microvilli
epithelium at this time
A regression of the glandular weeks so that by 6-7 weeks
epithelium the number
jecting
deep
into
the
endometrium
contained (Fig. 3G).
began after 4 of glands pro-
declined,
and
thelial cells were cuboidal with few cytoplasmic (Fig. 3H). Basophilic secretion was absent in the lumina of the few remaining glands after 9 weeks. never detected by immunocytochemistry dular cells during this period (data not cells were not observed.
nu-
the
epi-
granules glandular PDP was
in the deep glanshown). Giant stromal
A different protein synthetic pattern was observed 5-7 weeks postcoitum (Fig. 4E). The diffuse CP5 spot that began to enlarge CP4 and
after 20 days CP5 were seen
continued to do so. as one large diffuse
By 4 weeks, spot. After 5
PROTEIN
FIG.
3.
A:
Placental labyrinth from
a cat 9 weeks
SYNTHESIS
postcoitum.
c
=
IN THE
Cytotrophoblast;
PREGNANT
fc
=
351
CAT
fetal
capillaries;
s
=
syncytiotrophoblast;
mc
=
maternal
capillaries; g = giant cells. x480. B: Deep endometrial glands below the placenta from a cat 9 weeks postcoitum. x530 C: Surface epithelium from the nonimplantation site endometrium 14 days postcoitum. Arrow = mitotic figure. x480. D: Deep endometrial gland from the nonimplantation site endometrium 14 days postcoitum. Arrowheads = large basophilic granules; arrows = dense core granules. (Electron micrographs of these granules are shown in E.) x480. E: Electron micrograph of the deep endometrial glands from the nonimplantation site endometrium 14 days postcoitum. Arrowheads = large basophilic granules seen in the light micrographs; arrow = dense core granule in the same cell. x13000. F: Immunocytochemical localization of PDP in the deep glands from the non-site 20 days postcoitum. Note the presence of specific immunostaining in the cytoplasm of the epithelial cells. x530. G: Uterine luminal surface from the nonimplantation site endometrium 7 weeks postcoitum. x480. H: Deep glandular region from the nonimplantation site endometrium 7 weeks postcoitum. x480. I: Deep endometrial glands from a pseudopregnant cat 24 days postcoitum. Arrows = apoptotic bodies. x480.
352
BOOMSMA
ET AL.
p1 6.1
8
4.5
4.5
6.1
8
4.5
6.1
V
VV
V
V
V
V
V
8
V MW
a
x103
-
68
..
43
-
68
..
43
.
30
7s...
(‘
___
5
9
-e
I
-c
7
(. 0
=
4p
.
t
4
7) 3 9
21
FIG. 4. Fluorographs of culture media of tissue taken from pregnant and pseudopregnant cats. The two forms of PDP are enclosed in brackets. Other proteins are identified by the appropriate number without the CP prefix. A: Deep endometrial portion of the implantation site 9 weeks postcoitum. (Compare with Fig. 1H.) B: Placental/junctional zone of the implantation site 9 weeks postcoitum. C: Nonimplantation site endometrium 14 days postcoitum. D: Nonimplantation site endometrium 20 days postcoitum. E: Nonimplantation site endometrium 7 weeks postcoitum. F: Superficial portion of the nonimplantation site endometrium 9 weeks postcoitum. G: Superficial portion of the nonimplantation site endometrium 7 weeks postcoitum. (Compare with E.) H: Deep portion of the nonimplantation site endometrium 7 weeks postcoitum. (Compare with Fig. 1H.) I: Endometrium from a pseudopregnant cat 5 weeks postcoitum.
weeks,
CP8
and CP2 PDP was altered
was
consistently
seen
PROTEIN
SYNTHESIS
as a prominent
region,
IN
THE
PREGNANT
MWx1O3
was seen for the first time in this compartment. absent after 5-6 weeks. This pattern was slightly after
8 and
9 weeks
(Fig. 4F) in that a few spots
slightly more basic than CP3 were large CP4/5 region and the intense
seen. Note the extremely CP8 spots, and how this
differs from
in the deep
the pattern observed
implantation
site endometrium (Fig. 4A) and superficial placental/junctional zone (Fig. 4B) cultures from the same animal. After 6-9 weeks, the non-site from one of two animals
-“43
gion
(compare Figs. 4E and 4G) CP4/5 region, and a prominent appeared
to be very
site endometrium CP2, the enlarged
The
had CP2, the CP8. The deep
to the
deep
enre-
implantation
(compare Figs. 1H and 4H) and CP4/5 region, and the prominent
Pseudopregnant
lacked CP8.
was
well
developed
numerous convoluted The epithelial cells
14 days
glands radiating of the deep glands
postcoiaway from were tall-
columnar, containing a vacuolated cytoplasm and cytoplasmic granules. After 20-24 days, the glands appeared to be similar;
however,
evidence
for
served (Fig. 3!). Apoptosis 5 weeks postcoitum. The numerous
after
5 weeks,
lumnar epithelial munoreactivity but not
appeared shown).
apoptotic
cell
and
to diminish The surface
the
cytoplasm
in intensity epithelium
as cells
death
was
continued to be evident glands did not appear
14
did
it
were
low
co-
granules. PDP pseudopregnancy,
im-
after 5 weeks did not develop
in the nonimplantation never observed. The
pattern in this tissue in the nonimplantation (Fig. 41).
Immunochemical
Detection culture
nant cats were munochemically
followed the site endometrium
(data the site, protein
changes obthrough
(Fig.
media analyzed staining
of PDP from
on Western
pregnant
and
Blots
deep
from
for the presence of PDP by imi-D Western blots with the PDP
5). PDP immunoreactivity
both
endometrial
the
superficial
regions
after
tivity was present in nonimplantation ture media samples through 4-5 observed in the in all endometrial (through
PL/FL
DISCUSSION This study clearly demonstrates that in the cat the implanting blastocyst has a differential effect on endometrial protein tion
synthesis when
was
present
in cul-
5 weeks).
placental/endometrial
and
16 days. PDP immunoreacsite weeks,
endometrial culbut was no longer
non-site after 6 weeks. PDP was cultures from pseudopregnant
and
morphology
compared
at the site of implanta-
to nonimplantation
on Day 12 [26]. Penetration of the uterine luminal had begun by 14 days postcoitum, was complete 16,
and
the
placenta
attained
and
detected animals
pseudo-
occurred epithelium Day
dissolution
and
endometrium.
by
Zona
site
pregnant
attachment
its mature
structure
by Day 20. Changes in protein synthesis were correlated with these events. Among the most prominent changes were the transient increase in the synthesis of CP5 12-16 days the appearance of PDP
of CP2
after 14 days, and
by 16 days. These
changes
observed in the non-site or pseudopregnant This study also shows that the morphology
pseudopreg-
ture media as 28 000 and 36000 M forms. PDP immunoreactivity in pregnant animals was present in the implantation site after 12 days, declined after 14 days, and was absent
NS
Uterine flushings from pseudopregnant cats were also analyzed for PDP immunoreactivity. PDP was observed in all flushings and was present in all three molecular weight forms: 28 000, 36 000, and 41 000 (Fig. 5).
disappearance
antibody
NS/DI
FIG. 5. Western blot immunostained with PDP antibody. Aliquots of complete implantation site (IS), nonimplantation site endometrium INS), deep implantation site endometrium (Dl), or superficial placental/endometrial (PL) culture media from pregnant cats 12, 14, and 16 days postcoitum and a uterine flushing (FL) from a pseudopregnant cat 20 days postcoitum were used.
postcoitum,
Explant
16
NS/IS
ob-
through to be as
of the
cells contained fewer was present throughout
extreme convolutions and giant stromal synthetic served 5 weeks
12
IS
Endometrium
endometrium
mm, with the lumen.
similar
and
-
-.
from each time point was separated into a superficial and a deep region on the basis of differences in tissue density, and these two regions were cultured separately. The superficial region appeared to be similar to the unseparated non-site larged
353
CAT
were
the not
endometrium. and protein
synthetic activity of the postimplantation cat uterus continues to change as pregnancy progresses towards term. The protein synthetic activity of the placental/junctional zone was altered such that by term, CP2 synthesis was reduced while CP7 activity was greatly enhanced; these changes in synthetic activity were The
deep
additional ing the the
endometrium
below
the
with
structural changes.
placenta
morphological and protein synthetic remainder of pregnancy. The superficial
non-site
alterations pearance and CP8.
correlated
developed in
villous-like
structures
showed
little
change durregion of and
protein synthetic activity including of CP2 and the enhanced production The deep region of the non-site developed
showed the apof CP4/5 a glan-
354 dular
structure
and
protein
synthetic
pattern
similar
found in the deep implantation site endometrium. The synthesis of some proteins appeared to be primarily
to a particular
regions
are
uterine
composed
compartment;
of multiple
cell
BOOMSMA
ET AL.
to that
fragmentation,
limited
however, types
and
these thus
specific cellular localization of the proteins is unknown. was associated primarily with the placental/junctional of the implantation synthesized later plantation site sively embryonic to the tional
site by the
as well. origin
development zone, since
diately
precedes
of decidual its appearance the
giant
cells
uterus
coworkers
and interimplantation Nieder and Macon
help
and
secretion
in the cat. Preliminary that a protein in these
but
not
The
use
of CP2
cultures nologically
with
the related
as a marker
may
same molecular weight as CP2 is immuto the low molecular weight form of in-
sulin-like growth-factor-binding ciated with the decidual CP5 was associated with
protein, a molecule assocells of the primate uterus [27]. the early phases of implantation
and later with the development It may be that CP5 is related that is in temporal primarily associated
of the nonimplantation site. to the epithelial hyperplasia
conjunction with the
with its synthesis. placental/junctional
CP7 zone
was and
CP8 with the upper region of the non-site. CP1 has electrophoretic similarities to retinol-binding protein found in pig conceptus [28] and uterine [29] secretions. Future studies using antibodies generated against these proteins clarify the cellular localization issue, similar to our studies
where
we
demonstrated
sence of PDP is linked the deep endometrial
that
to the glands.
the
maintenance
presence
may PDP
or
ab-
or regression
of
The protein synthetic pattern and presence of immunoreactive PDP in the deep glands observed in the nonimplantation site endometrium was similar to that seen in the
pseudopregnant
the implantation between the suggest
the
animal site non-site
absence
but
different
from
region. These observed and pseudopregnant of an embryonic
effect
that
seen
in
similarities endometrium on the
non-site
at least through 4-5 weeks. However, the luminal surface of the non-site became increasingly convoluted with an increase in mitotic activity, a phenomenon not observed in the pseudopregnant animal. This may indicate some form of interaction between the implantation site or the nonvilbus portions of the trophoblast and the non-site endometrium. A close structural and functional relationship between
these
[30-32]. This was characterized
regions
in the
the
time
first by
cat
that
heterochromatin
has
been
apoptosis,
observed a form margination,
previously of cell
death
nuclear
[35-371,
have on
found
with
the
in the
postcoitum
[11.
an effect
endometrial
protein
suggested
that
in with-
as happens
20 days demonstrated
[38]
[33,34],
been
of the
synthesis.
changes
in the
endometrial protein synthetic pattern observed at implantation in the pig are due to estradiol from the conceptus. In the mouse, the rate of synthesis and secretion of uterine proteins has been shown to vary between the implantation
that
the issue of decidualization in our laboratory suggest
of studies
cells
It has
in association
hormones
blastocyst
protein
compartment.
uterus.
cat approximately
Geisert
and
by neighboring cat
species
implanting
believed to be decidual cells of stromal origin [14, 25]. The synthesis of CP2 in the non-site may indicate the presence of predecidual cells since giant cells were not observed in clarify studies
in the
of stimulatory
A number
are
phagocytosis
of other
CP2 zone
that
and observed
pseudopregnant
rules out an excluCP2 may be related
of the
the drawal
cells in the placental/juncin these cultures imme-
appearance
been
the
during early pregnancy, but was superficial region of the nonimThis apparently for this protein.
has
synthetic
areas of the [15] demonstrated activity
of the
same
pregnant
and
nant mouse uterus, and, in another uterine protein synthesis were correlated of certain
all, of the
mouse
uterine horn [16]. a difference in the
blastocyst
blastocyst-induced
pseudopreg-
study, alterations in with the synthesis
[391. Many,
proteins changes
in the
mouse
can be induced by use of artificialdeciduogenic stimuli [16,40]. Conceptus culture media have been shown to selectively induce the synthesis of two bovine endometrial proteins in culture [17] and to enhance the synthesis of some proteins in sheep The asynchronous
endometrial epithelial cell cultures [41]. transfer of Day 6 sheep embryos to Day
recipients results in the acceleration of the endometrial secretory response to one similar to the synchronous trans4
fer control may be due
The observed effects to the action of interferon
in the sheep and since the ovine
[42].
bovine conceptus ally related to the
proteins, oTP-1 alpha interferons
alpha protein
have been shown [18, 19,44,451. The
interferons synthesis
action between
and bTP-1, are structur[43], and oTP-1 and the
the cat conceptus
to alter nature
and
dometrium remains to be elucidated. Alterations in the presence of enzymes trophoblastic origin demonstrated (for ity was demonstrated
endometrial of the inter-
the underlying
en-
of maternal
and
during the implantation period has been review, see Denker [46]). Enzyme activin the cat by use of a variety of his-
tochemical tests, and the proteinase relation to both the trophoblast and
activity observed the endometrial
dular
a cathepsin-B-like
crypts
cathepsin-L-like onstrated metrial flushings
was
cow and
determined enzyme
to [26,46].
be
Similarly,
we
have
in glanor dem-
that PDP, a protein localized in the deep endoglands and in maximal concentration in uterine at the time of implantation [1, 2], has significant
homology with cathepsin-L teolytic activity (unpublished declines in the implantation plantation but is maintained
[12] and has cathepsin-L prodata). Our observation that PDP site soon after the onset of imin the nonimplantation
site is
consistent with variations in the activity of other enzymes in the rabbit, rat, hamster, and guinea pig as determined by enzyme histochemistry [47]. The function of these enzymes in the implantation process is not clear. It has been suggested that a complex system of proteinases and inhib-
PROTEIN
[48]. It is interesting
itors plays a role in implantation note that the cathepsin-like Denker and coworkers men
and
the
dilated
the deep glandular It may be that the stored vated
SYNTHESIS to
activity observed in the cat by [26] was present in the uterine lu-
neck
region
of the
glands
regions or the glandular cathepsin-like protein PDP
but
not
epithelial is inactive
in
cells. while
within the deep glandular epithelium and is actionly once it is secreted. In support of this hypothesis,
PDP has been found in uterine flushings in three molecular weight forms [2], similar to cathepsin-L detected in other systems, not all of which are active [49]. Future work with PDP may help clarify the role of enzymes during implantation
in the
IN THE
CAT
PREGNANT
blastocyst
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355
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