1. family and parenting
2. motherhood
3. pregnancy

REPRODUCTION
RESEARCH
Placental development during early pregnancy in sheep:
cell proliferation, global methylation, and angiogenesis
in the fetal placenta
Anna T Grazul-Bilska, Mary Lynn Johnson, Pawel P Borowicz, Megan Minten, Jerzy J Bilski,
Robert Wroblewski, Mila Velimirovich, Lindsey R Coupe, Dale A Redmer
and Lawrence P Reynolds
Department of Animal Sciences, Center for Nutrition and Pregnancy, North Dakota State University, Fargo,
North Dakota 58108, USA
Correspondence should be addressed to A T Grazul-Bilska; Email: anna.grazul-bilska@ndsu.edu
Abstract
To characterize early fetal placental development, gravid uterine tissues were collected from pregnant ewes every other day from day 16
to 30 after mating. Determination of 1) cell proliferation was based on Ki67 protein immunodetection; 2) global methylation was based
on 5-methyl-cytosine (5mC) expression and mRNA expression for DNA methyltransferases (DNMTs) 1, 3a, and 3b; and 3) vascular
development was based on smooth muscle cell actin immunolocalization and on mRNA expression of several factors involved in the
regulation of angiogenesis in fetal membranes (FMs). Throughout early pregnancy, the labeling index (proportion of proliferating cells)
was very high (21%) and did not change. Expression of 5mC and mRNA for DNMT3b decreased, but mRNA for DNMT1 and 3a increased.
Blood vessels were detected in FM on days 18–30 of pregnancy, and their number per tissue area did not change. The patterns of mRNA
expression for placental growth factor, vascular endothelial growth factor, and their receptors FLT1 and KDR; angiopoietins 1 and 2
and their receptor TEK; endothelial nitric oxide synthase and the NO receptor GUCY13B; and hypoxia inducing factor 1 a changed
in FM during early pregnancy. These data demonstrate high cellular proliferation rates, and changes in global methylation and mRNA
expression of factors involved in the regulation of DNA methylation and angiogenesis in FM during early pregnancy. This description of
cellular and molecular changes in FM during early pregnancy will provide the foundation for determining the basis of altered placental
development in pregnancies compromised by environmental, genetic, or other factors.
Reproduction (2011) 141 529–540
Introduction
The placenta is the exchange organ for all respiratory
gases, nutrients, and wastes between the fetal and
maternal tissues (Ramsey 1982, Faber & Thornburg
1983). Thus, placental development is critical for
supplying the fetus with metabolic substrates via
transplacental exchange (Needham 1934, Ramsey
1982, Faber & Thornburg 1983, Morris & Boyd 1988,
Reynolds et al. 2010). Many aspects of placental
function play major roles in fetal tissue growth including
expression of specific genes, methylation patterns,
vascularization, hormone production, and other processes (Reynolds & Redmer 2001, Reynolds et al. 2002,
2006, 2010, Blomberg et al 2008). Therefore, fetal
development and pregnancy maintenance are dependent on normal placental growth.
The placenta represents a type of organ, which
expresses a high rate of growth in order to fulfill
the metabolic demands of the growing fetus (Reynolds
q 2011 Society for Reproduction and Fertility
ISSN 1470–1626 (paper) 1741–7899 (online)
et al. 2002, 2006, 2010). Although the role of
hypertrophy and hyperplasia in placental growth has
been recognized (Boos et al. 2006, Murphy et al. 2006),
very limited data are available concerning the rates and
pattern of cell proliferation in fetal membranes (FMs)
during early pregnancy. However, high proliferation
rates have been reported for placenta during early
pregnancy in several species (Blankenship & King
1994, Correia-da-Silva et al. 2004, Wei et al. 2005,
Kar et al. 2007, Grazul-Bilska et al. 2010). In addition, it
has been demonstrated using transcriptome analysis
that genes which regulate trophoblast cell proliferation,
cell differentiation and angiogenesis and numerous other
genes that facilitate mother–fetus interactions are
upregulated in the fetal placenta during early pregnancy
in ruminants (Blomberg et al. 2008).
DNA methylation, which is catalyzed by DNA
methyltransferases (DNMTs), is generally associated
with transcriptional silencing and imprinting, principally
DOI: 10.1530/REP-10-0505
Online version via www.reproduction-online.org
530
A T Grazul-Bilska and others
occuring at cytosine residues located in dinucleotide
CpG sites and is the most extensively characterized
epigenetic mark in mammals (Hiendleder et al. 2004,
Wilson et al. 2007, Beck & Rakyan 2008). In fact,
DNMTs are required for cell differentiation during
embryonic development to regulate gene expression
through methylation mechanisms (Gopalakrishnan et al,
2008). DNMT1 is primarily considered the maintenance
methyltransferase (Bird 2002, Gopalakrishnan et al.
2008, Kim et al. 2009); however other functions of
DNMT1, such as methylation of non-CpG sites in DNA
bubbles have recently been discovered (Ross et al.
2010). Other methyltransferases, DNMT3A and
DNMT3B are responsible for establishing de novo
DNA methylation patterns (Gopalakrishnan et al. 2008).
Although DNA methylation is the most commonly
studied mode of epigenetic regulation, the process
of methylation/demethylation or the expression of the
enzymes that promote methylation has not been
investigated in detail in the placenta. It has been
demonstrated that around the time of gastrulation and
implantation, de novo methylation reestablishes the
developing organism’s methylation patterns both in the
embryo and in the extraembryonic tissues (Maccani &
Marsit 2009). However, the pattern of methylation in the
embryo differs from the extraembryonic tissues (Monk
et al. 1987, Katari et al. 2009). In human placenta
collected from several stages of pregnancy and at term,
low expression of 5-methyl-cytosine (5mC) and relative
hypomethylation have been reported (Kokalj-Vokac
et al. 1998, Katari et al. 2009). Many genes expressed
in extraembryonic tissues are imprinted (Reik et al. 2001,
Myatt 2006, Jansson & Powell 2007), and several of these
imprinted genes are involved in regulating the fetal and
placental growth (Reik et al. 2001, 2003, Myatt 2006,
Wagschal et al. 2008). Thus, DNA methylation plays a
significant role during embryonic and placental development in physiological and pathological conditions (Kim
et al. 2009). However, little is known about global
methylation and expression of DNMTs in placental
tissues during early pregnancy in any species.
Vascularization of both the fetal and maternal
placenta is a critical factor in pregnancy maintenance
(Zygmunt et al. 2003, Huppertz & Peeters 2005,
Reynolds et al. 2006, 2010, Arroyo & Winn 2008).
Fetal placental vasculogenesis, which is a result of
de novo formation of blood vessels, is initiated very early
in pregnancy (e.g. in humans about 21 days, in rhesus
monkey about 19 days post-conception; Kaufmann et al.
2004). Vasculogenesis is very tightly regulated by
angiogenic and other factors (Flamme et al. 1997,
Patan 2000, Kaufmann et al. 2004, Demir et al. 2007).
Although the expression of several factors involved in
the control of angiogenesis has been studied in the
placenta of several species, limited information
concerning the expression of these factors is available
for FM during early pregnancy.
Reproduction (2011) 141 529–540
We hypothesized that the patterns of cellular proliferation, global methylation of DNA, expression of
several DNMTs, vascular development, and expression
of factors involved in the regulation of angiogenesis
in FM will change as early pregnancy progresses.
Therefore, the objective of this study was to determine
1) labeling index (LI; a proportion of proliferating cells),
2) global methylation based on the expression of 5mC in
DNA and expression of mRNA for DNMT1, 3a, and 3b,
3) development of blood vessels based on immunodetection of smooth muscle cell actin (SMCA; a marker of
pericytes and smooth muscle cells, and thus blood
vessels), and 4) expression of 12 factors involved in
the regulation of angiogenesis and their receptors
including placental growth factor (PGF), vascular
endothelial growth factor (VEGF), and their receptors
FLT1 and KDR; fibroblast growth factor (FGF) 2 and its
receptor 2IIIc; angiopoietins (ANGPT) 1 and 2 and their
receptors TEK; endothelial NO synthase (NOS3) and its
receptor soluble guanylate cyclase (GUCY1B3); and
hypoxia inducing factor 1 a (HIF1A) in FM during early
pregnancy in sheep.
Results
The length of the fetus increased (P!0.0001) approximately threefold from day 20 to 30 of pregnancy
(Fig. 1A). Labeling index (a proportion of proliferating
cells, based on Ki67 protein detection) did not change
significantly (PO0.2) from day 16 to 30 of pregnancy
(Fig. 1B). Overall, LI was 20.7G1.5% in FM, and ranged
from 17 to 26%; regression analysis demonstrated a linear
decrease (R2Z0.110; P!0.055; YZK0.63XC36.3)
from day 16 to 30 of pregnancy. Ki67, 5mC, and
SMCA proteins were immunodetected in the FM
throughout early pregnancy (Fig. 2A, B, and C). Ki67
and 5mC were localized to the cell nuclei (Fig. 2A and B),
but SMCA was localized to cytoplasm of blood vessel
cells (Fig. 2C).
Image analysis demonstrated that positive 5mC
staining occupied 10.5G1.0% of cell nuclei in FM
(range 9–13%), and significant changes were not
observed throughout early pregnancy. However, DNA
dot blot analysis demonstrated an approximately twofold
decrease (P!0.003) in 5mC expression in FM on
days 16–20 compared with that on days 28–30,
and regression analysis demonstrated a cubic decrease
(R2Z0.355; P!0.0003; YZK12.07C1.89XK0.09X2
C0.001X3) throughout early pregnancy (Fig. 3A).
Expression of DNMT1 mRNA tended (P!0.11) to
increase approximately twofold from day 16 to 30
(Fig. 3B), and regression analysis demonstrated a linear
increase (R2Z0.173; P!0.002; YZ0.18C0.03X)
throughout early pregnancy. Expression of DNMT3a
mRNA increased (P!0.004) approximately twofold
from day 16 compared with days 24–30 (Fig. 3C), but
DNMT3b mRNA decreased (P!0.0001) approximately
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531
Early fetal placental development
P<0.0001
A
25
Length of fetus (mm)
d
20
c
c
15
b
10
b
a
5
0
20
22
24
26
28
30
Day of pregnancy
Labeling index (%)
B
40
P>0.2
30
20
10
0
16
18
20
22
24
26
28
30
Day of pregnancy
Figure 1 Crown-to-rump length of fetuses from day 20 to 30 of
pregnancy (A) and labeling index (percentage of proliferating cells;
(B) in fetal membranes on days 16–30 of pregnancy. Fetuses from days
16 and 18 were not collected and measured due to their small size
(!2 mm) and tissue transparency. a,b,c,dP!0.0001; valuesGS.E.M. with
different superscripts differ within a specific measurement.
VEGF mRNA expression increased (P!0.0001) approximately twofold on days 28 and 30 compared with days
16–20 (Fig. 4B). FLT1 mRNA expression increased
(P!0.0001) w5- to 50-fold on days 28 and 30 compared
with days 16–24 (Fig. 4C). KDR mRNA expression was 2to 11-fold greater (P!0.0001) on days 20–24 than on
days 16–18 and 26–30 (Fig. 4D).
ANGPT1 mRNA expression was low on days 16–24 of
pregnancy and then increased (P!0.0001) w2- to
50-fold on days 26–30 of pregnancy (Fig. 4E). Expression
of ANGPT2 mRNA was not detectable on day 16 of
pregnancy but increased (P!0.0001) w3.5- to 5-fold
from day 18 to days 22–30 of pregnancy (Fig. 4F). TEK
mRNA expression increased (P!0.0001) approximately
seven- to ninefold from day 16 to days 20–24, and then
decreased on days 26–30 (Fig. 4G).
FGF2 mRNA expression increased (P!0.06) approximately four- to fivefold from day 16 to days 20–24,
and then decreased on days 26–28 (Fig. 4H), whereas
NOS3 mRNA expression increased (P!0.001) w5- to
16-fold from days 16–18 to days 22–30 of pregnancy
(Fig. 4I). GUCY1B3 mRNA expression was approximately two- to sevenfold greater (P!0.01) on day 18
than on any other day of pregnancy (Fig. 4J). HIF1A
mRNA expression was approximately one- to twofold
greater (P!0.02) on days 18, 20, and 30 than on days 16
and 24 of pregnancy (Fig. 4K).
A
B
FM
FM
threefold from day 16 to 18 compared with days 20–22
and decreased by fivefold on day 30 (Fig. 3D) of
pregnancy. Regression analysis of mRNA expression for
DNMT3a demonstrated a linear increase (R2Z0.301;
P!0.0001; yZK0.06C0.04x) but for DNMT3b a
cubic pattern (R2Z0.624, P!0.0001; YZ11.57K
0.97XC0.02X2K0.0002X3) of decrease throughout
early pregnancy.
Blood vessels marked with SMCA were detected in
FM on days 18–30 of pregnancy (Fig. 2C). Overall,
the number of blood vessels per FM tissue area was
1.7G0.4/10 000 mm2, ranging 0–7/10 000 mm2, and did
not change throughout early pregnancy.
Expression of mRNA for factors involved in the
regulation of angiogenesis including PGF, VEGF, FLT1,
KDR, ANGPT1, ANGPT2, ANGPT receptor TEK, FGF2,
NOS3, GUCY1B3, and HIF1A (Fig. 4A–H), but not for
FGFR2IIIc (data not shown) in FM changed (P!0.0001–
0.06) during early pregnancy (Fig. 4A–K). PGF mRNA
expression increased (P!0.0001) w3.5- to 34-fold from
days 16–22 to days 24–30 of pregnancy (Fig. 4A).
www.reproduction-online.org
50 um
E
C
50 um
E
D
FM
E
FM
50 um
50 um
E
Figure 2 Representative photomicrographs of immunohistochemical
staining for Ki67 (A), 5-methyl-cytosine (5mC) (B), and smooth muscle
cell actin (SMCA) (C) in uterine tissues from day 24 of early pregnancy.
Dark color represents positive staining, and pink color (nuclear fast
red staining) indicates unlabeled cell nuclei. In (A), note nuclear
staining of Ki67 (arrows) in fetal membranes (FM) and endometrium (E).
In (B), note punctate staining of 5mC in the nuclei of the majority of
cells (arrows) in FM and E, and a lack of staining in some cells
(arrowheads) in FM. In (C), note SMCA cytoplasmic staining in blood
vessels in FM (arrows) and E (arrowheads). In (D), note a lack of positive
staining in the controls in which mouse IgG was used in place of
the primary antibody.
Reproduction (2011) 141 529–540
532
A T Grazul-Bilska and others
Results of regression analysis demonstrating a pattern
of change in mRNA expression for all 12 investigated
genes involved in the regulation of angiogenesis are
presented in Table 1. Correlation coefficients for mRNA
5mC; P<0.003
A
Relative expression
2
1.6
ab
b
Discussion
b
abc
ac
1.2
ac
c
c
28
30
0.8
0.4
0
16
18
20
22
24
26
Day of pregnancy
Relative mRNA expression
B
1.5
DNMT1; P < 0.11
bc
1
a
ab
ab
16
18
20
bc
abc
c
abc
0.5
0
22
24
26
28
30
Day of pregnancy
Relative mRNA expression
C
1.5
DNMT3a ; P < 0.004
cd
bcd
abc
1
ab
ab
20
22
d
cd
a
0.5
0
16
18
24
26
28
30
Day of pregnancy
Relative mRNA expression
D
2.5
DNMT3b ; P < 0.0001
a
2
a
1.5
b
1
b
bc
bc
bc
c
24
26
28
30
0.5
0
16
18
20
22
Day of pregnancy
Figure 3 Expression of 5mC as determined by DNA dot blot assay
(A) and mRNA for DNA methyltransferases (DNMTs) 1 (B), 3a (C),
and 3b (D) in fetal membranes (FM) on days 16–30 of pregnancy.
a,b,c,d
P!0.0001–0.11; valuesGS.E.M. with different superscripts differ
within each specific gene.
Reproduction (2011) 141 529–540
expression of evaluated genes involved in the regulation
of angiogenesis are presented in Table 2. Expression of
mRNA for the majority of these genes was significantly
(P!0.0001–0.08) correlated (Table 2).
Early pregnancy is characterized by dramatic uterine
and embryonic/fetal tissue growth, differentiation, and
remodeling, and it is the critical period for establishing a
healthy pregnancy. During this critical period, maternal
recognition of pregnancy, initial attachment/implantation of FM to uterine epithelium, and initiation of
placental growth and development take place (Bowen
& Burghardt 2000, Spencer et al. 2007, 2008). In
addition, most embryonic loss occurs in early pregnancy
with rates of pregnancy losses reported as R30% in
most mammalian species and possibly O50% in
humans (Reynolds & Redmer 2001, Miri & Varmuza
2009). Thus, investigation of the fetal and maternal
placental growth during early pregnancy is needed to
establish the mechanisms that contribute to pregnancy
maintenance or loss.
This study demonstrated a rapid increase in fetal size,
decrease in 5mC expression (as determined by DNA dot
blot), and dramatic changes in the mRNA expression
in FM of several factors involved in the regulation of
DNA methylation, angiogenesis, and tissue growth
during early pregnancy. However, the rates of cellular
proliferation were maintained at a high level but not
significantly changed. In addition, image analysis did not
show any differences in 5mC expression throughout
pregnancy. The discrepancies between the results of
5mC evaluation by DNA dot blot and image analyses
were likely due to the lower sensitivity of immunohistochemistry and image analysis than dot blot analysis.
Early embryonic development is tightly regulated and
includes control of cell growth, proliferation and
differentiation, morphogenesis, and protein synthesis
and trafficking (Blomberg et al. 2008, Igwebuike 2009).
In this study, growth of the fetal placenta was reflected by
rapid increase in embryonic size and very high rates of
cellular proliferation in FM. High rates of cell proliferation were also observed in the fetal placenta during
early pregnancy in humans and monkeys (Wei et al.
2005, Korgun et al. 2006, Kar et al. 2007). Interestingly,
cell proliferation in the fetal and maternal placenta
obtained after transfer of embryos created in vitro or
through parthenogenetic activation was less than in
pregnancies after natural breeding in sheep (Borowicz
et al. 2009; AT Grazul-Bilska, PP Borowicz, DA Redmer
& LP Reynolds 2010, unpublished observations).
In addition, altered placental cell proliferation or
turnover was observed in several pathological conditions
including diabetes and trophoblastic diseases at several
pregnancy stages in humans (Burleigh et al. 2004, Zhang
et al. 2009). These data suggest that altered cellular
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533
Early fetal placental development
16 18 20 22 24 26 28 30
a
16 18 20 22 24 26 28 30
0
Relative mRNA expression
Relative mRNA expression
c
c
b
a
0.1
a
a
a
a
16 18 20 22 24 26 28 30
0.08
Relative mRNA expression
bc
bc
b
ab
0.04
a
0
16 18 20 22 24 26 28 30
a
c
bc
1.2
bc
ab
a
0
16 18 20 22 24 26 28 30
0.5
b
b
0.8
d
0.4
0.2
a
0
a
a
0
16 18 20 22 24 26 28 30
0.1
c
bc
bc
bc
0.2
abc abc
ab
0.1
a
0
16 18 20 22 24 26 28 30
Day of pregnancy
HIF1A ; P<0.02
K
a
a
a
0
16 18 20 22 24 26 28 30
Day of pregnancy
FGF2 ; P<0.06
0.3
Day of pregnancy
a
a
a
16 18 20 22 24 26 28 30
0.3
a
bc
ab
ab
b
a
cd
bc
0.4
a
a
0.2
H
cd
0.6
b
Day of pregnancy
TEK ; P<0.0001
0.4
0.2
ab ab
16 18 20 22 24 26 28 30
GUCY1B3 ; P<0.01
J
c
c
a
c
c
NOS3 ; P<0.001
0.4
a
ab
0
Day of pregnancy
1.6
0.8
bc
0.4
G
0.12
Day of pregnancy
I
c
0.6
Day of pregnancy
ANGPT2 ; P<0.0001
F
0.4
0.2
a
0.8
Day of pregnancy
ANGPT1 ; P<0.0001
0.3
bc
0
Day of pregnancy
E
ab ab
Relative mRNA expression
a
0
0.1
ab
Relative mRNA expression
a
a
0.2
Relative mRNA expression
a
cd
KDR ; P<0.0001
D
d
1.2
Relative mRNA expression
b
FLT1 ; P<0.0001
C
d
0.3
Relative mRNA expression
2
Relative mRNA expression
Relative mRNA expression
c
c
1
VEGF ; P<0.0001
B
c
3
Relative mRNA expression
PGF ; P<0.0001
A
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
Day of pregnancy
b
a
bc
b
ac
ac ac
a
16 18 20 22 24 26 28 30
Day of pregnancy
Figure 4 Expression of mRNA for placental growth factor (PGF; A), vascular endothelial growth factor receptor (VEGF; B), VEGF receptor FLT1 (C),
VEGF receptor KDR (D), angiopoietin (ANGPT) 1 (E), ANGPT2 (F), ANGPT receptor TEK (G), fibroblast growth factor 2 (FGF2; H), endothelial nitric
oxide synthase (NOS3; I), NOS3 receptor GUCY1B3 (J), and hypoxia inducible factor (HIF) 1A (K) in fetal membranes (FM) on days 16–30 of
pregnancy. a,b,c,dP!0.0001–0.06; valuesGS.E.M. with different superscripts differ within each specific gene.
proliferation in the fetal placenta is a feature of
compromised pregnancies. However, the mechanism
of regulation of cell proliferation in FM has not been
elucidated, and this subject requires additional
investigation.
Since epigenetic modifications of the genome include
methylation of DNA at cytosine residues and histone
modifications through methylation catalyzed by
DNMTs, we choose to use 5mC and DNMT1, 3a, and
3b as markers of global methylation in our study. In this
study, expression of these markers was detected in FM,
and the pattern of changes differed during early
pregnancy. Interestingly, expression of 5mC decreased
during early pregnancy indicating that demethylation
was occurring in the FM. However, in our study, only one
of the enzymes catalyzing methylation and/or demethylation, DNMT3b (Ooi & Bestor 2008), had decreased
mRNA expression; whereas expression of DNMT3a
mRNA increased during early pregnancy. Therefore,
we hypothesize that a specific balance exists between
Table 1 Regression analysis of angiogenic genes in fetal membranes (FM) from early pregnancy.
Gene
Regression type
P value
PGF
VEGF
FLT1
KDR
ANGPT1
ANGPT2
TEK
FGF2
FGFR2
NO3S
GUCY1B3
HIF
Exponential sigmoidal
Quadratic
Exponential
Cubic
Exponential sigmoidal
Exponential sigmoidal
Exponential sigmoidal
Exponential sigmoidal
–
Cubic
–
Cubic
!0.0001
!0.0001
!0.0001
!0.0001
!0.0001
!0.0001
!0.0001
0.0002
NS
0.0002
NS
0.007
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R2
0.7904
0.5432
0.7788
0.4568
0.7860
0.7741
0.4610
0.3033
–
0.3393
–
0.2250
Equation
YZ5.284!107 exp (0.996XK0.016X 2)
YZ0.249K0.021XC0.001X 2
YZ0.0007 exp (0.231X)
YZK13.535C1.724XK0.069X 2C0.0009X 3
YZ4.453!10K12 exp (1.621XK0.027X 2)
YZ4.083!10K19 exp (3.138XK0.061X 2)
YZ2.823!10K8 exp (1.367XK0.028X 2)
YZ1.267!10K11 exp (1.855XK0.036X 2)
–
YZ1.542K0.537XC0.039X 2K0.0007X 3
–
YZK11.594C1.666XK0.075X 2C0.001X 3
Reproduction (2011) 141 529–540
534
A T Grazul-Bilska and others
Table 2 Correlation coefficients for mRNA expression of angiogenic factors in fetal membranes.
VEGF
PGF
VEGF
FLT1
KDR
0.735
0.750
P!0.0001 P!0.0001 NS*
0.779
–
P!0.0001 NS
FLT1
–
NS
KDR
–
ANGPT1
ANGPT2
0.783
P!0.0001
0.775
P!0.0001
0.802
P!0.0001
K0.249
P!0.08
0.544
P!0.0001 NS
0.458
P!0.0007 NS
0.485
P!0.0003 NS
0.851
NS
P!0.0001
0.343
P!0.01
NS
0.424
–
P!0.002
–
ANGPT1
–
ANGPT2
TEK
TEK
FGF2
FGFR2IIIc NOS3
NS
0.260
P!0.05
NS
0.274
P!0.0001
0.237
P!0.09
0.416
P!0.002
NS
0.422
P!0.002
NS
0.341
P!0.01
0.499
P!0.0002
NS
NS
0.438
P!0.001
–
NS
FGF2
GUCY1B3
0.453
P!0.0009 NS
0.510
P!0.0001 NS
0.248
P!0.08
NS
NS
0.358
P!0.01
0.472
P!0.0005
0.488
P!0.0003
0.335
P!0.02
–
NS
–
NS
NS
NS
0.367
P!0.008
NS
NS
NS
NS
K0.327
P!0.02
NS
NS
NS
NS
NS
NS
0.459
P!0.0007
NS
0.265
P!0.06
FGFR2IIIc
NOS3
GUCY1B3
HIF1A
–
*NS, not statistically significant. PR0.1.
expression and/or function of DNMTs, and likely other
enzymes involved in methylation and/or demethylation
(e.g. DNA glycosylase; Zhu 2009) are present in the
tissue to further regulate methylation processes. It is
believed that genomic imprinting, regulated by methylation mechanisms, may play a critical role in placental
biology (Coan et al. 2005, Maccani & Marsit 2009, Miri
& Varmuza 2009). In fact, alterations in imprinting have
been linked to placental pathologies (Tycko 2006,
Wagschal & Feil 2006). Therefore, correction of the
DNA methylation may offer new strategies for preventing
pregnancy complications. However, more research is
required to gain a better understanding of the
mechanisms of imprinting and methylation in the
placenta in order to establish a strategy for successful
pregnancy outcomes.
Very few studies have evaluated global methylation
during early placental development, but several studies
investigated methylation in the placenta at specific time
points. For human placenta, it has been demonstrated
that methylation levels measured by 5mC content
increased in a gestational stage-dependent manner
(Fuke et al. 2004), that DNA methylation measured by
the mean CpG methylation status of genes probed in a
microarray analysis was decreased after in vitro versus
in vivo conception (Katari et al. 2009), and that the
decrease in X chromosome-linked placental methylation
was greater in pregnancies carrying female than male
babies Cotton et al. (2009). Studies of embryonic and/or
extraembryonic tissues during mouse development
demonstrated that global methylation and demethylation
and expression of DNMT were stage and tissue specific
(Monk et al. 1987, Trasler et al. 1996, Watanabe et al.
2002). For cows, global methylation in the fetal placenta
on day 80 was similar for pregnancies established
Reproduction (2011) 141 529–540
after transfer of embryos created through artificial
insemination, in vitro fertilization, or somatic cell
nuclear transfer (Hiendleder et al. 2004). In our study,
significant changes in global methylation, measured by
5mC and DNMTs mRNA expression, indicate that the
pattern of methylation in FM is changing throughout
early pregnancy. Since data concerning the methylation
process in developing and growing placenta are
extremely limited, further studies should be undertaken
to study this process in detail.
In this study, blood vessels marked with SMCA were
detected in FM as early as on day 18 of pregnancy. For
human placenta, it has been demonstrated that angiogenesis, manifested by vascular tube formation, and
presence of hemangiogenic cell cords, was evident
21–27 days post-conception; on day 32 post-conception,
erythrocytes were observed within blood vessels lumen,
and between days 35 and 42, the networks of cords
were heavily connected with each other without any
interruption (Demir et al. 1989, 2004, Zygmunt et al.
2003, Torry et al. 2004, Arroyo & Winn 2008, Burton et al.
2009, van Oppenraaij et al. 2009). Thus, vasculogenesis
is initiated very early in pregnancy in order to support
dramatic fetal growth.
FM growth and vascular development have to be
tightly regulated to coordinate the development of the
fetal and maternal placenta and embryonic tissues.
Therefore, vasculogenesis, angiogenesis, and tissue
growth within the fetal placenta are regulated by
numerous growth factors (Patan 2000, Zygmunt et al.
2003, Demir et al. 2007, Herr et al. 2009, Burton et al.
2009). In this study, increased mRNA expression of
several factors and their receptors involved in the
regulation of angiogenesis and growth in FM was
observed as pregnancy progressed. In fact, changes in
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Early fetal placental development
mRNA expression of several growth/angiogenic factors
and/or their receptors including PGF, FLT1, ANGPT1,
TEK, NOS3, and HIF1A in FM paralleled the expression
of these factors in maternal placenta in sheep (GrazulBilska et al. 2010), indicating a similar role of these
factors in the regulation of fetal and maternal placental
growth and function.
Although protein and/or mRNA expression of VEGF,
PGF and receptors, and/or FGF2 and receptor was
detected in extraembryonic tissues at specific stages of
early pregnancy in monkeys, humans, and cows
(Vuorela et al. 1997, Ghosh et al. 2000, Hildebrandt
et al. 2001, Wang et al. 2003, Demir et al. 2004, Wei
et al. 2004, Pfarrer et al. 2006), the changes during
placental development have not been evaluated for
these or other species. In this study, dramatic changes in
the expression of mRNA for members of the VEGF and
ANGPT systems were observed. Since during early
pregnancy first vasculogenesis and then angiogenesis
are initiated, it seems that high expression of VEGF and
ANGPT systems is required to regulate these processes.
In fact, members of the VEGF family and ANGPTs are
recognized as the major regulators of vasculogenesis and
angiogenesis in the placenta (Reynolds et al. 2002,
2006, Zygmunt et al. 2003, Demir et al. 2007, Seval
et al. 2008). In the primate placenta, protein and/or
mRNA expression of VEGF, FLT1, KDR, ANGPT1,
ANGPT2, and/or receptor TEK was detected during
early pregnancy (Demir et al. 2004, Wei et al. 2004,
Seval et al. 2008, Demir 2009). These factors appeared
to be spatio- and temporal-regulated during early
pregnancy in primates (Demir et al. 2004, Wei et al.
2004, Kayisli et al. 2006, Seval et al. 2008). The
increased expression of several angiogenic factors
during early pregnancy indicates that these factors are
involved in the regulation of vascular development,
remodeling, and trophoblast function. However,
functional studies should be undertaken to verify the
specific roles of VEGF and ANGPT systems in placental
growth and function.
Expression of mRNA for FGF2 but not for FGFR2IIIc in
FM increased during early pregnancy in this study.
Although the expression of FGF2 and its receptor was
detected in the fetal placenta in sheep and other species
(Kaufmann et al. 2004, Wei et al. 2004, Liu et al. 2005),
little is known about the specific role of FGF system in
early placental development. However, it has been
demonstrated that FGFs stimulate differentiation of the
embryonic germ layers, and it has been suggested that
the FGF system is involved in the regulation of growth
and differentiation of vascular and non-vascular compartments of the placenta (Reynolds et al. 2002). In
addition, FGF2 is a potent stimulator of cell proliferation
(Reynolds & Redmer 2001); therefore, it is reasonable to
postulate that FGF2 and its receptor are involved in the
regulation of cell proliferation in FM.
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535
Expression of NOS3 mRNA gradually increased from
day 16 to 22 and remained at a similar level until day 30,
but the NOS3 receptor GUCY1B3 mRNA expression
was enhanced only on day 18 of pregnancy in FM in our
study. Endothelial NOS is expressed in the fetal placenta
from early pregnancy in several species (Sladek et al.
1997, Ariel et al. 1998, Gagioti et al. 2000, Al-Hijji et al.
2003). NOS are recognized as regulators of implantation
and pregnancy maintenance, and angiogenesis in the
fetal and maternal placenta (Gagioti et al. 2000, Maul
et al. 2003); however, the mechanism of NOS effects on
these processes remains to be elucidated. Furthermore, it
has been demonstrated that NOS3 expression is
regulated by FGF2 and VEGF in ovine placental artery
endothelial cells (Mata-Greenwood et al. 2008). These
interactions seem to be reflected in our study by
significant correlations between the mRNA expression
of NOS3 and expression of members of the VEGF and
FGF2 systems.
An increased HIF1A mRNA expression in FM was
observed on days 18–20 of pregnancy in our study. This
transient high expression of HIF1A mRNA may be
associated with low oxygen levels observed during
early pregnancy in several species (Rodesch et al.
1992, Rajakumar & Conrad 2000, Fryer & Simon 2006,
Ietta et al. 2006, Pringle et al. 2007). It seems that HIF1A
expression is decreasing after delivery of oxygen is well
established through developing blood vessel network.
A decrease in HIF1A mRNA expression from day 50 to
the end of pregnancy in the fetal placenta was observed
in sheep (Borowicz et al. 2007). It has clearly been
demonstrated using the knockout and other models that
HIF activity is necessary for placental development,
since HIF1A is involved in the regulation of placental
morphogenesis, cell migration, angiogenesis, erythropoiesis, and cell metabolism, and is critical for adaptive
responses to hypoxia (Cowden Dahl et al. 2005, Fryer
& Simon 2006). In fact, HIF1A expression is altered
in preeclampsia and IUGR placentas (Rajakumar et al.
2007, Zamudio et al. 2007). Therefore, HIF1A may be
used as a marker of compromised pregnancies.
In summary, this study demonstrates a dramatic
increase in fetal size, high cellular proliferation rates,
decrease in 5mC expression (as determined by DNA dot
blot), and lack of changes in vascularization measured
as the number of blood vessels per tissue area, but
significant changes in mRNA expression of factors
involved in the regulation of methylation, angiogenesis,
and tissue growth in FM during early pregnancy. Positive
correlations among mRNA expression of several
growth/angiogenic factors and/or their receptors indicate
interactions among these factors in the regulation of
development of fetal placenta. However, since we have
evaluated the expression for the factors mentioned above
at the mRNA level only, additional studies should be
undertaken to determine the pattern of protein
expression and its relation to mRNA expression in
Reproduction (2011) 141 529–540
536
A T Grazul-Bilska and others
order to better understand the process of placental
growth and function. This description of cellular and
molecular changes in FM during early pregnancy will
provide a foundation for determining whether and how
placental development is altered in compromised
pregnancies. Furthermore, it will help to establish a
baseline that can be used to design therapeutic
treatments to restore normal fetal development in
compromised pregnancies.
Material and Methods
Animals
The NDSU Institutional Animal Care and Use Committee
approved all animal procedures in this study. Gravid uteri were
obtained from crossbred Western Range (primarily Rambouillet, Targhee, and Columbia) ewes (nZ5 to 8 per day) on days
16, 18, 20, 22, 24, 26, 28, and 30 after mating (day of mating
Zday 0). At tissue collection for immunohistochemical
staining, specimen pins were inserted completely through the
uterus and FM at the level of the external intercornual
bifurcation to maintain specimen morphology; cross sections
of the entire gravid uterus (w0.5 cm thick) were obtained using
a Stadie–Riggs microtome knife followed by immersion in
formalin or Carnoy’s solution and embedding in paraffin. For
total cellular RNA extraction, chorioallantoic FM were
dissected from the area close to the embryo, snap-frozen, and
stored at K70 8C. On days 20–30 of pregnancy, fetuses were
separated from FMs, and crown–rump length of each fetus was
measured. The length of fetuses on days 16 and 18 was not
determined due to the small fetal size (!2 mm) and tissue
transparency.
CA, USA), 5mC (a marker of global DNA methylation; 1:500;
mouse monoclonal; Eurogentec North America, San Diego,
CA, USA), or SMCA (a marker of pericytes and smooth muscle
cells and thus blood vessels; 1:150; mouse monoclonal;
Oncogene Research Products; San Diego, CA, USA) overnight
at 4 8C. Primary antibodies were detected by using secondary
anti-mouse antibody coupled to peroxidase (ImPress Kit;
Vector Laboratories). The sections were then counterstained
with nuclear fast red (Sigma) to visualize cell nuclei. Control
sections were incubated with normal mouse IgG (4 mg/ml) in
place of primary antibody. Fetal placental cell types were not
identified in this study due to methodological difficulties, such
as a lack of specific markers for these cell types in sheep or
absence of some cell types in individual tissue slides; thus we
used the entire fetal placenta for immunohistological and other
evaluations, which of course has some limitations that the
reader should keep in mind.
Image analysis
For each tissue section, images were taken at 400! (Ki67
staining), 600! (5mC staining), or 200! (SMCA staining)
magnification, using an Eclipse E600 Nikon microscope and
digital camera for 5–40 randomly chosen fields (0.025 mm2 per
field) from areas containing FM. To determine LI, the
percentage of 5mC positive staining in cell nucleus or the
number of blood vessels per FM tissue area, an image analysis
system (Image-Pro Plus, Media Cybernetics, Inc., Bethesda,
MD, USA) was used as described previously (Grazul-Bilska
et al. 2010). The LI was calculated as the percentage (%) of
proliferating Ki67-positive cells out of the total number of cells
within an FM tissue area.
DNA dot blot assay
Immunohistochemistry
Immunohistochemical procedures were used as described
before (Grazul-Bilska et al. 2010). Paraffin-embedded uterine
tissues containing FM were sectioned at 4 mm and mounted
onto slides. Sections were rinsed several times in PBS
containing Triton X-100 (0.3%, v/v) and then were treated for
20 min with blocking buffer (PBS containing normal horse
serum (2%, vol/vol)) followed by incubation with specific
primary antibody for Ki67 (a marker of proliferating cells;
1:500; mouse monoclonal; Vector Laboratories, Burlingame,
DNA dot blot analysis of 5mC was based on modifications of
previously described methods (Tao et al. 2004, Park et al.
2005). DNA was isolated from FM tissues homogenized in TRI
Reagent (Molecular Research Center, Cincinnati, OH, USA).
Purified DNA (0.5 mg) was denatured by adding NaOH and
EDTA to final concentrations of 0.4 M NaOH and 10 mM
EDTA, heated to 100 8C for 10 min, followed by cooling to
4 8C, and then neutralized with an equal volume of cold (4 8C)
2 M ammonium acetate. Denatured DNA was spotted onto
Ambion BrightStar-Plus Nylon Membrane (Ambion/Applied
Table 3 Sequence of TaqMan primers and probes for DNMT1, DNMT3a, and DNMT3b.
Oligonucleotide
Nucleotide sequence
Accession numbera
Sheep DNMT1 FP
Sheep DNMT1 RP
Sheep DNMT1 probe
Sheep DNMT3a FP
Sheep DNMT3a RP
Sheep DNMT3a probe
Sheep DNMT3b FP
Sheep DNMT3b RP
Sheep DNMT3b probe
5 0 - CCT GGG TCC ACG GTG TTC -3 0
5 0 - CCA CCC ATG ACC AGC TTC A -3 0
5 0 -(6FAM) AGA GTA CTG CAA CGT CCT -(MGBNFQ)-3 0
5 0 - TGT ACG AGG TAC GGC AGA AGT G -3 0
5 0 - GGC TCC CAC AAG AGA TGC A -3 0
5 0 -(6FAM) ATG TCC TCG ATG TTC CG -(MGBNFQ)-3 0
5 0 - AGC GGC AGG CGA TGT CT -3 0
5 0 - GAG AAC TTG CCA TCA CCA AAC C -3 0
5 0 -(6FAM) CTG GAC CCA CCG CAT -(MGBNFQ)-3 0
NM_001009473
HQ202740
HQ202741
FP, forward primer; RP, reverse primer.
a
Nucleotide sequences for ovine-specific genes were obtained from the National Center for Biotechnology Information (NCBI, 2010) database.
Reproduction (2011) 141 529–540
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Early fetal placental development
Biosystems, Austin, TX, USA) using the BRL HYBRI-DOT
Manifold (Bethesda Research Laboratories, Gaithersburg, MD,
USA). The DNA was cross-linked to the membrane for 2 min
with the CL-1000 Ultraviolet Crosslinker (UVP, Upland, CA,
USA) and then dried. After wetting in dH2O, the membrane
was blocked with 5% skim milk in PBSC0.1% Tween 20
(PBST) by rocking for 3 h at room temperature. The membrane
was probed with a 1:2000 dilution in 2% milk–PBST of
monoclonal mouse antibody against 5mC (Eurogentec North
America) by rocking at 4 8C overnight. The membrane was
washed three times for 10 min each in PBST before incubation
with a 1:5000 dilution of HRP-conjugated anti-mouse
secondary antibody in 2% milk–PBST with rocking for 1 h at
room temperature. After three washes in PBST, the membrane
was incubated with ECL Plus Western blotting reagent (GE
Healthcare; Piscataway, NJ, USA), and the chemiluminescence
of 5mC was detected and quantified using the AlphaEaseFC
imager (Alpha Innotech, San Leandro, CA, USA). After
detection of 5mC, the membrane was stained with 0.02%
methylene blue for DNA quantification, and the relative dot
intensity was measured with the AlphaEaseFC imager. Each
sample was normalized to its DNA concentration by dividing
the 5mC signal intensity of the sample by the dot intensity of
methylene blue.
Quantitative real-time RT-PCR
All procedures for determining the expression of mRNA of
several genes in ovine tissues by RT-PCR have previously been
reported (Redmer et al. 2005, Johnson et al. 2006, GrazulBilska et al. 2010). Briefly, snap-frozen FM tissues were
homogenized in TRI Reagent (Molecular Research Center)
according to the manufacturer’s specifications. The quality and
quantity of total RNA were determined via capillary electrophoresis using the Agilent 2100 Bioanalyzer (Agilent Technologies, Wilmington, DE, USA). Real-time RT-PCR reagents,
probes, and primers were purchased from and used as
recommended by Applied Biosystems. For each sample,
30 ng total RNA were reverse transcribed in triplicate 20 ml
reaction volume using random hexamers. Sequence-specific
TaqMan probes and primers were designed using the Primer
Express Software from Applied Biosystems, and sequences for
12 factors involved in the regulation of angiogenesis have been
published before (Redmer et al. 2005, Johnson et al. 2006,
Grazul-Bilska et al. 2010). The sequences of probes and
primers for DNMT1, 3a, and 3b are presented in Table 3. The
ABI PRISM 7000 was used for detection of sequences amplified
at 60 8C typically for 40 or 45 cycles (Applied Biosystems).
Quantification was determined from a relative standard curve
of dilutions of the cDNA generated from tcRNA pooled from
placentomes collected on day 130 of pregnancy. Expression of
each gene was normalized to the expression of 18S rRNA in a
multiplex reaction using the human 18S pre-developed assay
reagent (PDAR) from Applied Biosystems. The PDAR solution,
which is primer limited and contains a VIC-labeled probe, was
further adjusted by using one-fourth the normal amount, so that
it would not interfere with amplification of the FAM-labeled
gene of interest. Standard curves were also generated with the
multiplex solution, and the quantity of 18S rRNA and the gene
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537
of interest were determined using each specific standard curve.
The concentrations of mRNA were then normalized to 18S
rRNA by dividing each of the mRNA values by their
corresponding 18S rRNA value (Grazul-Bilska et al. 2010).
Statistical analysis
Data were analyzed using the general linear models procedure
of SAS and presented as meansGS.E.M. with the main effect of
day of pregnancy (SAS Institute 2010). When the F-test was
significant (P!0.05), differences between specific means were
evaluated by using the least significant differences test (Kirk
1982). The SAS procedure PROC REG was used for regression
analysis, and PROC CORR was used to calculate simple linear
correlations between specific variables.
Declaration of interest
The authors declare that there is no conflict of interest that
could be perceived as prejudicing the impartiality of the
research reported.
Funding
This project was supported by the USDA grant (2007-01215)
to L P Reynolds and A T Grazul-Bilska, the NIH grant
(HL64141) to L P Reynolds and D A Redmer, the ND EPSCoR
AURA grant to Anna T Grazul-Bilska and Megan Minten, the
ND Space Grant Fellowship Program award to Megan Minten,
and the NIH grant (P20 RR016741) from the INBRE program of
the NCRR, NIH, to A T Grazul-Bilska and L P Reynolds.
Acknowledgements
The authors would like to thank Dr Eric Berg, Dr Kimberly
Vonnahme, Ms Tammi Neville, Mr James D Kirsch, Mr Kim C
Kraft, Mr Robert Weigl, Mr Tim Johnson (deceased), Mr Terry
Skunberg, and other members of our laboratories and
department for their assistance.
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Received 7 December 2010
First decision 23 December 2010
Accepted 27 January 2011
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