BIOLOGY
OF
REPRODUCTION
Growth
and
47,
698-708
(1992)
Microvascular
Development LAWRENCE
Department
of Animal
and
Range
of the
P. REYNOLDS2
Sciences,
Uterus and
North
Dakota
during
Early
DALE
A. REDMER
State
University,
Pregnancy
Fargo,
North
in Ewes1
Dakota
58105
ABSTRACT Growth 4 ewes/day; were
increased
24 and
and
dry
weights
fresh
and
fresh
content).
Increased
creased
RNA:DNA
on Days
24 and 30 after
endometrial
of mating)
on Days
increased
and
capillary
microvascular
factors
mating,
will during
dramatic
enable early
horns
changes
by Day
us to further
the role
of the
ovine
blastocyst
(presumptive in ruminants
begins
by Days
by Day
30 [2, 3,6].
expanded uterine
blastocyst probably lumenal environment
Histological occur in
20-25
after
Until
begins
studies
in the
ruminants,
mating
including
3, 6-9]. metabolic
These events demands
sorption
but
also,
loss,
representing
with
during
vascularization
is a marked
of the
increase
15]. Quantitative cular development however, is not tors
Uterine during
June
Received
March
by the
30%
rate on
during available.
early of ewes [16,
2029 of the North 2Correspondence:
NIH
FAX:
grant
HD22559
Agric. Exp. Sts., (701) 237-8520.
fetal
of
On
the ab-
mem-
exchange
potential
early
of uterine
blood
placental
growth
pregnancy
the
It
diameter
microvessels
uterine
DNA
hypertrophy was
tissues
of the patterns
30. The (inelevated
and decreased development
and
of uterine
growth
and conceptus-derived
and
growth
to LP.R
in
and
factors
have
D.AR
off-
angiogenic however,
found
factors a more
and
vascular
flow
[13-
and
vas-
in uterine
and
placental
mammalian the role(s)
in placental complete
development
species, of these
development description
can of the
during
early
pregnancy
AND
METHODS
I
Days
12,
18, 24,
and
presence
of embryos
30 after
in uterine
presence of conceptuses sues were removed with
pregnancy
sected cation
ruminants,
Journal
been
mating,
ewes
(n
4/
=
rinses
(see
below)
was
confirmed by using a stereomicroscope (model SMZ-10; Nikon, Tokyo, Japan). For ewes on Days 18, 24, and 30 after mating, a small incision was made in each uterine horn,
secretes angiogenic facIn addition, numerous
Project
with
lumenal
of large
on Day (increased
day) were slaughtered, and their reproductive tracts were obtained. Location of the corpora lutea was recorded, and the ovaries, oviducts, cervix, and mesometrium were removed from each tract. For ewes on Day 12 after mating,
between the to the uterine
transected with the
at the uterine
discarded.
Each
was confirmed, forceps. Each
uterine body
internal body;
conceptus then was
tisbi-
horns from the external bifur(Fig. 1). Each uterine horn was
bifurcation Fig. 1), and
uterine
and uterus
horn
then
(i.e., at its confluence the uterine body was was
incised
longitudi-
nally along the mesometrial border (Fig. 1), and the lumen was rinsed gently with 50 ml of PBS (0.01 M sodium phosphate, 0.14 M NaCI, pH 7.3) to remove uterine and conceptus
by
(increased
reported
Experiment
and [1-
9, 1992.
Dakota
associated
MATERIALS
23, 1992. in part,
only
hyperplasia
of endometrial
Characterization
of previously
elevated
in ewes.
[11, 12]. Associated during
17).
were
abundance
growth
devel-
capillary beds [1,6, 10). the majority of embryonic
placenta
in the
architecture
that
glands, tissues
transplacental
pregnancy
information
endometrium early pregnancy
Accepted Supported,
no.
early
uterine
however,
implantation
for meeting conceptus via
secretions
approximately
occurs
processes and
only
the
upon the needs [2].
organogenesis,
for
were
with
2, vascularity
(increased mating,
horns
normal patterns of uterine and placental growth must be obtained. The purpose of this study, therefore, was to provide a qualitative and quantitative description of uterine
established
growth of uterine and fetal placental
fetal that
30,
Day
growth and be evaluated,
therefore,
the
attachment
stages,
between the maternal and is not surprising, therefore, spring,
is well
described
glandular at later
the
with maternal to implantation,
mating,
are critical not of the developing
of uterine
branes
and
embryonic
opment of chorionic areolae, vascularization of maternal
13-15
membranes to Interdigitation
is highly dependent for its metabolic
between
associated
and 30 after mating (3of gravid uterine horns
tissues and uterine flushings of several including those of ewes [17-20]. Before
[1-5].
30 after
have
interval
by Days
cotyledon) is analogous
Day
uterine
were
growth
after mating, and attachment of fetal uterine mucosa begins by Days 15-18 of fetal membranes caruncles, which
on
on Days 12, 18, 24, weight and dry weight
pregnancy.
INTRODUCTION Expansion
24
in uterine
24 after
define
ewes
In experiment
development
observed
for
1, fresh
of nongravid
Day
tissues.
microvascular
were
on
uterine
of uterine
In addition,
those
horns
of gravid
ratios)
plexus)
were evaluated In experiment
whereas
uterine
weights
dry
uterus
experiments.
of gravid
in uterine
development
angiogenic
30 after
mating, and
of the
in two
protein:DNA
thickness)
of a subepithelial and
development
and microvascular Day 0 = day
article
fluids.
Fresh
weight
of each
uterine
horn
determined. A cross section was obtained from the portion of each uterine horn (-1 cm thick, obtained
1782.
698
then
was
middle at the
GROWFH
UTERINE
AND
oviductal of uterine
VASCUL8RI1Y
end horn
lated (see
mesometrial external
IN
as the below).
E4eriment
border
bifurcation
EARLY
On
FIG. 1.
Schematic
representation
cation from which cross sections were the right uterine horn. For a schematic section, see Figure 2.
of ovine
uterus, dorsal view. The loobtained is indicated (=‘===) for representation of a uterine cross
level
of the
external
bifurcation;
of endometrium
and
Fig.
1), and
myometrium
dissected. Although myometrial samples trium, both endometrial and myometrial predominantly
of endometrial
and
samples were
myometrial
(----1
En-
obtained stored
determine of each
its dry uterine
weight:fresh horn
was
dry weight:fresh weight weight. The DNA content
weight
ratio.
calculated
as the
The
dry
product
ratio and the uterine of each uterine horn
horn was
each
tract,
PBS
(v/v)
1%
+
both
middle
mm
thick,
ewes
uterine
lidocaine
followed
obtained
Park, After
IL) according fixed tissues
(Day
12) or conceptus
J?NA, and
myometrium
was to
at the
tissues
Protein
(Fig.
weight of the
Tissue
fresh calcu-
DNA
arteries
by
20-30
level
of the
Schematic which
(Days
18-30)
was
cedures,
respectively
1 was
(PBS + 3 mM (Brinkmann, analyzed
for
[22, 23].
caruncular
sections
Standards
of a cross obtained.
section
from
the
middle
portion
of a uterine
were
endo-
J metrium
horn.
See
Figure
homogenized
and
uterine
were
confirmed
NaN3 + Westbury,
gland
representation
bifur-
1 mM NY).
concentrations
to diphenylamine
intercaruncular
cross
of Car-
external
lumen from
ml
tissue to fix for 15 mm, of each uterine horn
experiment
were
RNA according
J circular
2.
were
Concentrations
2) from
homogenates and
blood vessel
FIG.
= 3/ were
to standard histological procedures [21]. were obtained, the presence of embryos
in 10 vol PBS-EDTA buffer EDTA) by use of a polytron
metriumi
location
(n tracts
tubing (0.76 mm i.d x L22 mm o.d.; NJ) and perfused, by use of a 10manual pressure, with 20-30 ml of
mesometrium
myo-
concentration
To evaluate cellular growth of uterine tissues, DNA, RNA, and protein concentrations were determined as described previously [22, 23]. Briefly, -1 g each of endometrium and
-
of each uterine horn subsequently lyophilized
For
with PE-60 Parsippany, and gentle
DNA,
from each uterine horn, frozen in liqat 70#{176}C, and later analyzed for DNA
concentration. The remainder weighed, frozen at -70#{176}C,and
DNA
in all ewes.
dometrial and myometrial samples were frozen in liquid nitrogen and stored at -70#{176}Cuntil analyzed for tissue DNA, RNA, and protein concentrations. In addition, a sample of whole uterus (endometrium, myometrium, and perimetrium) was uid nitrogen,
and
cation; Fig. 1) was immersed in Carnoy’s fixative for 4-6 h and then transferred to 70% ethanol. These uterine tissues were embedded in paraffin (Paraplast Plus; Baxter, McGaw
perimeconsisted tissues.
weight
noy’s fixative [21]. After allowing the a cross section of the middle portion
carefully
included samples
of fresh
cannulated Clay Adams, ml syringe
(-5
g each)
product
2
removed.
bifurcation
699
EWES
Days 12, 18, 24, and 30 after mating, were slaughtered, and their reproductive
day)
internal
PREGNANT
1 for
orcinol DNA
of proType
I
700
REYNOLDS
TABLE
1.
Fresh and
weights
dry
and
DNA
content
of uterine
horns
from
AND ewes
REDMER during
early
pregnancy. Day
Item
Fresh wt
Dry
12
Side
wta
DNA
Ig)
(g)
27.5 27.9
±
Gravid Nongravid
4.5 4.5
±
91.9 100.6
± ±
Gravid
(mg)
content’
Gravid Nongravid
Nongravid “Means ± SEM (n = 4 ewes/day). Fresh wt and dry wt of gravid uterine horns of all other uterine horns. bDNA content of the gravid uterine horn was
from from sue
on Days
24 and
greatest
(p
calf thymus and RNA Type IV from Sigma, St. Louis, MO). Concentration homogenates
was
et al. [24] with
determined
BSA (Fraction
by the
V, Sigma)
tent of DNA was used as an index ratios of RNA:DNA and protein:DNA of tissue
hypertrophy
OF
REPRODUCTION
Growth
and
47,
698-708
(1992)
Microvascular
Development LAWRENCE
Department
of Animal
and
Range
of the
P. REYNOLDS2
Sciences,
Uterus and
North
Dakota
during
Early
DALE
A. REDMER
State
University,
Pregnancy
Fargo,
North
in Ewes1
Dakota
58105
ABSTRACT Growth 4 ewes/day; were
increased
24 and
and
dry
weights
fresh
and
fresh
content).
Increased
creased
RNA:DNA
on Days
24 and 30 after
endometrial
of mating)
on Days
increased
and
capillary
microvascular
factors
mating,
will during
dramatic
enable early
horns
changes
by Day
us to further
the role
of the
ovine
blastocyst
(presumptive in ruminants
begins
by Days
by Day
30 [2, 3,6].
expanded uterine
blastocyst probably lumenal environment
Histological occur in
20-25
after
Until
begins
studies
in the
ruminants,
mating
including
3, 6-9]. metabolic
These events demands
sorption
but
also,
loss,
representing
with
during
vascularization
is a marked
of the
increase
15]. Quantitative cular development however, is not tors
Uterine during
June
Received
March
by the
30%
rate on
during available.
early of ewes [16,
2029 of the North 2Correspondence:
NIH
FAX:
grant
HD22559
Agric. Exp. Sts., (701) 237-8520.
fetal
of
On
the ab-
mem-
exchange
potential
early
of uterine
blood
placental
growth
pregnancy
the
It
diameter
microvessels
uterine
DNA
hypertrophy was
tissues
of the patterns
30. The (inelevated
and decreased development
and
of uterine
growth
and conceptus-derived
and
growth
to LP.R
in
and
factors
have
D.AR
off-
angiogenic however,
found
factors a more
and
vascular
flow
[13-
and
vas-
in uterine
and
placental
mammalian the role(s)
in placental complete
development
species, of these
development description
can of the
during
early
pregnancy
AND
METHODS
I
Days
12,
18, 24,
and
presence
of embryos
30 after
in uterine
presence of conceptuses sues were removed with
pregnancy
sected cation
ruminants,
Journal
been
mating,
ewes
(n
4/
=
rinses
(see
below)
was
confirmed by using a stereomicroscope (model SMZ-10; Nikon, Tokyo, Japan). For ewes on Days 18, 24, and 30 after mating, a small incision was made in each uterine horn,
secretes angiogenic facIn addition, numerous
Project
with
lumenal
of large
on Day (increased
day) were slaughtered, and their reproductive tracts were obtained. Location of the corpora lutea was recorded, and the ovaries, oviducts, cervix, and mesometrium were removed from each tract. For ewes on Day 12 after mating,
between the to the uterine
transected with the
at the uterine
discarded.
Each
was confirmed, forceps. Each
uterine body
internal body;
conceptus then was
tisbi-
horns from the external bifur(Fig. 1). Each uterine horn was
bifurcation Fig. 1), and
uterine
and uterus
horn
then
(i.e., at its confluence the uterine body was was
incised
longitudi-
nally along the mesometrial border (Fig. 1), and the lumen was rinsed gently with 50 ml of PBS (0.01 M sodium phosphate, 0.14 M NaCI, pH 7.3) to remove uterine and conceptus
by
(increased
reported
Experiment
and [1-
9, 1992.
Dakota
associated
MATERIALS
23, 1992. in part,
only
hyperplasia
of endometrial
Characterization
of previously
elevated
in ewes.
[11, 12]. Associated during
17).
were
abundance
growth
devel-
capillary beds [1,6, 10). the majority of embryonic
placenta
in the
architecture
that
glands, tissues
transplacental
pregnancy
information
endometrium early pregnancy
Accepted Supported,
no.
early
uterine
however,
implantation
for meeting conceptus via
secretions
approximately
occurs
processes and
only
the
upon the needs [2].
organogenesis,
for
were
with
2, vascularity
(increased mating,
horns
normal patterns of uterine and placental growth must be obtained. The purpose of this study, therefore, was to provide a qualitative and quantitative description of uterine
established
growth of uterine and fetal placental
fetal that
30,
Day
growth and be evaluated,
therefore,
the
attachment
stages,
between the maternal and is not surprising, therefore, spring,
is well
described
glandular at later
the
with maternal to implantation,
mating,
are critical not of the developing
of uterine
branes
and
embryonic
opment of chorionic areolae, vascularization of maternal
13-15
membranes to Interdigitation
is highly dependent for its metabolic
between
associated
and 30 after mating (3of gravid uterine horns
tissues and uterine flushings of several including those of ewes [17-20]. Before
[1-5].
30 after
have
interval
by Days
cotyledon) is analogous
Day
uterine
were
growth
after mating, and attachment of fetal uterine mucosa begins by Days 15-18 of fetal membranes caruncles, which
on
on Days 12, 18, 24, weight and dry weight
pregnancy.
INTRODUCTION Expansion
24
in uterine
24 after
define
ewes
In experiment
development
observed
for
1, fresh
of nongravid
Day
tissues.
microvascular
were
on
uterine
of uterine
In addition,
those
horns
of gravid
ratios)
plexus)
were evaluated In experiment
whereas
uterine
weights
dry
uterus
experiments.
of gravid
in uterine
development
angiogenic
30 after
mating, and
of the
in two
protein:DNA
thickness)
of a subepithelial and
development
and microvascular Day 0 = day
article
fluids.
Fresh
weight
of each
uterine
horn
determined. A cross section was obtained from the portion of each uterine horn (-1 cm thick, obtained
1782.
698
then
was
middle at the
GROWFH
UTERINE
AND
oviductal of uterine
VASCUL8RI1Y
end horn
lated (see
mesometrial external
IN
as the below).
E4eriment
border
bifurcation
EARLY
On
FIG. 1.
Schematic
representation
cation from which cross sections were the right uterine horn. For a schematic section, see Figure 2.
of ovine
uterus, dorsal view. The loobtained is indicated (=‘===) for representation of a uterine cross
level
of the
external
bifurcation;
of endometrium
and
Fig.
1), and
myometrium
dissected. Although myometrial samples trium, both endometrial and myometrial predominantly
of endometrial
and
samples were
myometrial
(----1
En-
obtained stored
determine of each
its dry uterine
weight:fresh horn
was
dry weight:fresh weight weight. The DNA content
weight
ratio.
calculated
as the
The
dry
product
ratio and the uterine of each uterine horn
horn was
each
tract,
PBS
(v/v)
1%
+
both
middle
mm
thick,
ewes
uterine
lidocaine
followed
obtained
Park, After
IL) according fixed tissues
(Day
12) or conceptus
J?NA, and
myometrium
was to
at the
tissues
Protein
(Fig.
weight of the
Tissue
fresh calcu-
DNA
arteries
by
20-30
level
of the
Schematic which
(Days
18-30)
was
cedures,
respectively
1 was
(PBS + 3 mM (Brinkmann, analyzed
for
[22, 23].
caruncular
sections
Standards
of a cross obtained.
section
from
the
middle
portion
of a uterine
were
endo-
J metrium
horn.
See
Figure
homogenized
and
uterine
were
confirmed
NaN3 + Westbury,
gland
representation
bifur-
1 mM NY).
concentrations
to diphenylamine
intercaruncular
cross
of Car-
external
lumen from
ml
tissue to fix for 15 mm, of each uterine horn
experiment
were
RNA according
J circular
2.
were
Concentrations
2) from
homogenates and
blood vessel
FIG.
= 3/ were
to standard histological procedures [21]. were obtained, the presence of embryos
in 10 vol PBS-EDTA buffer EDTA) by use of a polytron
metriumi
location
(n tracts
tubing (0.76 mm i.d x L22 mm o.d.; NJ) and perfused, by use of a 10manual pressure, with 20-30 ml of
mesometrium
myo-
concentration
To evaluate cellular growth of uterine tissues, DNA, RNA, and protein concentrations were determined as described previously [22, 23]. Briefly, -1 g each of endometrium and
-
of each uterine horn subsequently lyophilized
For
with PE-60 Parsippany, and gentle
DNA,
from each uterine horn, frozen in liqat 70#{176}C, and later analyzed for DNA
concentration. The remainder weighed, frozen at -70#{176}C,and
DNA
in all ewes.
dometrial and myometrial samples were frozen in liquid nitrogen and stored at -70#{176}Cuntil analyzed for tissue DNA, RNA, and protein concentrations. In addition, a sample of whole uterus (endometrium, myometrium, and perimetrium) was uid nitrogen,
and
cation; Fig. 1) was immersed in Carnoy’s fixative for 4-6 h and then transferred to 70% ethanol. These uterine tissues were embedded in paraffin (Paraplast Plus; Baxter, McGaw
perimeconsisted tissues.
weight
noy’s fixative [21]. After allowing the a cross section of the middle portion
carefully
included samples
of fresh
cannulated Clay Adams, ml syringe
(-5
g each)
product
2
removed.
bifurcation
699
EWES
Days 12, 18, 24, and 30 after mating, were slaughtered, and their reproductive
day)
internal
PREGNANT
1 for
orcinol DNA
of proType
I
700
REYNOLDS
TABLE
1.
Fresh and
weights
dry
and
DNA
content
of uterine
horns
from
AND ewes
REDMER during
early
pregnancy. Day
Item
Fresh wt
Dry
12
Side
wta
DNA
Ig)
(g)
27.5 27.9
±
Gravid Nongravid
4.5 4.5
±
91.9 100.6
± ±
Gravid
(mg)
content’
Gravid Nongravid
Nongravid “Means ± SEM (n = 4 ewes/day). Fresh wt and dry wt of gravid uterine horns of all other uterine horns. bDNA content of the gravid uterine horn was
from from sue
on Days
24 and
greatest
(p
calf thymus and RNA Type IV from Sigma, St. Louis, MO). Concentration homogenates
was
et al. [24] with
determined
BSA (Fraction
by the
V, Sigma)
tent of DNA was used as an index ratios of RNA:DNA and protein:DNA of tissue
hypertrophy
