1. family and parenting
2. motherhood
3. pregnancy

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Reproduction Advance Publication first posted on 27 January 2011 as Manuscript REP-10-0505
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Placental development during early pregnancy in sheep: Cell proliferation, global
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methylation and angiogenesis in the fetal placenta
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Anna T. Grazul-Bilska, Mary Lynn Johnson, Pawel P. Borowicz, Megan Minten, Jerzy J. Bilski,
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Robert Wroblewski, Mila Velimirovich, Lindsey R. Coupe, Dale A. Redmer and Lawrence P.
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Reynolds.
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Department of Animal Sciences, Center for Nutrition and Pregnancy, North Dakota State
University, Fargo, ND 58108.
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Short title: Fetal placental development during early pregnancy
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Correspondence should be addressed to Anna T Grazul-Bilska; E-mail Anna.Grazul-
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[email protected]
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Copyright © 2011 by the Society for Reproduction and Fertility.
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Abstract
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To characterize early fetal placental development, gravid uterine tissues were collected
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from pregnant ewes every other day from day 16 to 30 after mating. Determination of: 1)
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cell proliferation was based on Ki67 protein immunodetection; 2) global methylation was
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based on 5-methyl-cytosine (5mC) expression and mRNA expression for DNA
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methyltransferase (DNMT) 1, 3a and 3b; and 3) vascular development was based on smooth
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muscle cell actin immunolocalization and on mRNA expression of several factors involved
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in the regulation of angiogenesis in fetal membranes (FM). Throughout early pregnancy,
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labeling index (proportion of proliferating cells) was very high (21%) and did not change.
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Expression of 5mC and mRNA for DNMT3b decreased, but mRNA for DNMT1 and 3a
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increased. Blood vessels were detected in FM on days 18 to 30 of pregnancy, and their
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number per tissue area did not change. The patterns of mRNA expression for: placental
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growth factor, vascular endothelial growth factor, and their receptors FLT1 and KDR;
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angiopoietins 1 and 2 and their receptor TEK; endothelial nitric oxide synthase and the NO
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receptor GUCY13B; and hypoxia inducing factor 1 alpha changed in FM during early
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pregnancy. These data demonstrate high cellular proliferation rates, and changes in global
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methylation and mRNA expression of factors involved in the regulation of DNA
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methylation and angiogenesis in FM during early pregnancy. This description of cellular
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and molecular changes in FM during early pregnancy will provide the foundation for
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determining the basis of altered placental development in pregnancies compromised by
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environmental, genetic or other factors.
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Introduction
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The placenta is the exchange organ for all respiratory gases, nutrients, and wastes
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between the fetal and maternal tissues (Ramsey, 1982; Faber & Thornburg, 1983). Thus,
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placental development is critical for supplying the fetus with metabolic substrates via
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transplacental exchange (Needham 1934, Ramsey 1982, Faber & Thornburg 1983, Morriss &
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Boyd 1988, Reynolds et al. 2010). Many aspects of placental function play major roles in fetal
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tissue growth including expression of specific genes, methylation patterns, vascularization,
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hormone production and other processes (Reynolds & Redmer 2001, Reynolds et al. 2002, 2006,
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2010, Blomberg et al 2008). Therefore, fetal development and pregnancy maintenance are
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dependent on normal placental growth.
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The placenta represents a type of organ which expresses a high rate of growth in order to
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fulfill the metabolic demands of the growing fetus (Reynolds et al. 2002, 2006, 2010).
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Although the role of hypertrophy and hyperplasia in placental growth has been recognized (Boos
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et al. 2006, Murphy et al. 2006), very limited data are available concerning the rates and pattern
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of cell proliferation in fetal membranes (FM) during early pregnancy. However, high
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proliferation rates have been reported for placenta during early pregnancy in several species
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(Blankenship & King 1994, Correia-da-Silva et al. 2004, Wei et al. 2005, Kar et al. 2007,
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Grazul-Bilska et al. 2010). In addition, it has been demonstrated using transcriptome analysis
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that genes which regulate trophoblast cell proliferation, cell differentiation, angiogenesis, and
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numerous other genes which facilitate mother-fetus interactions are upregulated in fetal placenta
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during early pregnancy in ruminants (Blomberg et al. 2008).
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DNA methylation, which is catalyzed by DNA methyltransferases (DNMTs), is generally
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associated with transcriptional silencing and imprinting, principally occuring at cytosine residues
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located in dinucleotide CpG sites and is the most extensively characterized epigenetic mark in
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mammals (Hiendleder et al. 2004, Wilson et al. 2007, Beck & Rakyan 2008). In fact, DNMTs
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are required for cell differentiation during embryonic development to regulate gene expression
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through methylation mechanisms (Gopalakrishnan et al, 2008). DNA methyltransferase 1 is
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primarily considered the maintenance methyltransferase (Bird 2002, Gopalakrishnan et al. 2008,
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Kim et al. 2009); however other functions of DNMT1, such as methylation of non-CpG sites in
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DNA bubbles have been recently discovered (Ross et al. 2010). Other methyltransefrases,
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DNMT3A and DNMT3B are responsible for establishing de novo DNA methylation patterns
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(Gopalakrishnan et al. 2008).
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Although DNA methylation is the most commonly studied mode of epigenetic regulation,
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the process of methylation/demethylation or the expression of the enzymes that promote
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methylation have not been investigated in detail in the placenta. It has been demonstrated that
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around the time of gastrulation and implantation, de novo methylation reestablishes the
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developing organism’s methylation patterns both in the embryo and in extraembryonic tissues
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(Maccani & Marsit 2009). However, the pattern of methylation in the embryo differs from the
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extraembryonic tissues (Monk et al. 1987; Katari et al. 2009). In human placenta collected from
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several stages of pregnancy and at term, low expression of 5-methyl-cytosine (5mC) and relative
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hypomethylation have been reported (Kokalj-Vokac et al. 1998, Katari et al. 2009). Many genes
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expressed in extraembryonic tissues are imprinted (Reik et al. 2001, Myatt et al. 2006, Jansson
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& Powell, 2007), and several of these imprinted genes are involved in regulating fetal and
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placental growth (Reik et al. 2001, 2003, Myatt et al. 2006, Wagschal et al. 2008). Thus, DNA
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methylation plays a significant role during embryonic and placental development in
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physiological and pathological conditions (Kim et al. 2009). However, little is known about
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global methylation and expression of DNA methyltransferases (DNMTs) in placental tissues
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during early pregnancy in any species.
Vascularization of both fetal and maternal placenta is a critical factor in pregnancy
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maintenance (Zygmunt et al. 2003, Huppertz & Peeters 2005, Arroyo & Winn 2008, Reynolds et
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al. 2006, 2010). Fetal placental vasculogenesis, which is a result of de novo formation of blood
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vessels, is initiated very early in pregnancy (e.g., in humans about 21 days, in rhesus monkey
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about 19 days post conception; Kaufmann et al. 2004). Vasculogenesis is very tightly regulated
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by angiogenic and other factors (Flamme et al. 1997, Patan 2000, Kaufmann et al. 2004, Demir
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et al. 2007). Although expression of several factors involved in the control of angiogenesis has
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been studied in the placenta of several species, limited information concerning expression of
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these factors is available for FM during early pregnancy.
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We hypothesized that the patterns of cellular proliferation, global methylation of DNA,
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expression of several DNMTs, vascular development, and expression of factors involved in the
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regulation of angiogenesis in FM will change as early pregnancy progresses. Therefore, the
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objective of this experiment was to determine 1) labeling index (LI; a proportion of proliferating
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cells), 2) global methylation based on expression of 5mC in DNA and expression of mRNA for
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DNMT1, 3a and 3b, 3) development of blood vessels based on immunodetection of smooth
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muscle cell actin (SMCA; a marker of pericytes and smooth muscle cells, and thus blood
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vessels), and 4) expression of 12 factors involved in the regulation of angiogenesis and their
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receptors including placental growth factor (PGF), vascular endothelial growth factor (VEGF)
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and their receptors FLT1 and KDR, fibroblast growth factor (FGF) 2 and receptor 2IIIc,
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angiopoietin (ANGPT) 1 and 2 and their receptors TEK, endothelial NO synthase (NOS3) and
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receptor soluble guanylate cyclase (GUCY1B3), and hypoxia inducing factor 1 alpha (HIF1A) in
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FM during early pregnancy in sheep.
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Results
The length of the fetus increased (P<0.0001) ~3-fold from day 20 to day 30 of pregnancy
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(Fig. 1A). Labeling index (a proportion of proliferating cells, based on Ki67 protein detection)
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did not change significantly (P>0.2) from day 16 to day 30 of pregnancy (Fig. 1B). Overall, LI
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was 20.7±1.5% in FM, and ranged from 17 to 26%; regression analysis demonstrated a linear
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decrease (R2 = 0.110; P<0.055; Y = -0.63X + 36.3) from day 16 to day 30 of pregnancy. Ki67,
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5mC, and SMCA proteins were immunodetected in the FM throughout early pregnancy (Fig. 2A,
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B and C). Ki67 and 5mC were localized to the cell nuclei (Fig. 2A and B) but SMCA was
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localized to cytoplasm of blood vessel cells (Fig. 2C).
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Image analysis demonstrated that positive 5mC staining occupied 10.5±1.0% of cell
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nuclei in FM (range 9-13%) and significant changes were not observed throughout early
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pregnancy. However, DNA dot blot analysis demonstrated a ~2-fold decrease (P<0.003) in 5mC
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expression in FM on days 16-20 compared to days 28-30, and regression analysis demonstrated a
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cubic decrease (R2 = 0.355; P<0.0003; Y = -12.07+1.89X-0.09X2+0.001X3) throughout early
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pregnancy (Fig. 3A). Expression of DNMT1 mRNA tended (P<0.11) to increase ~2-fold from
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day 16 to day 30 (Fig. 3B), and regression analysis demonstrated a linear increase (R2 = 0.173;
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P<0.002; Y = 0.18+0.03X) throughout early pregnancy. Expression of DNMT3a mRNA
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increased (P<0.004) ~2-fold from day 16 compared with days 24-30 (Fig. 3C), but DNMT3b
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mRNA decreased (P<0.0001) ~3-fold from day 16-18 compared with days 20-22 and decreased
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by 5-fold by day 30 (Fig. 3D) of pregnancy. Regression analysis of mRNA expression for
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DNMT3a demonstrated a linear increase (R2 = 0.301; P<0.0001; y =– 0.06+0.04x) but for
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DNMT3b a cubic pattern (R2 = 0.624, P<0.0001; Y = 11.57-0.97X +0.02X2-0.0002X3) of
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decrease throughout early pregnancy.
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Blood vessels marked with SMCA were detected in FM on days 18 to 30 of pregnancy
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(Fig.3C). Overall, the number of blood vessels per FM tissue area was 1.7±0.4/10,000 µm2,
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ranged from 0-7/10,000 µm2, and did not change throughout early pregnancy.
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Expression of mRNA for factors involved in regulation of angiogenesis including PGF,
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VEGF, FLT1, KDR, ANGPT1, ANGPT2, ANGPT receptor TEK, FGF2, NOS3, GUCY1B3 and
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HIF1A (Fig. 4A-H), but not for FGFR2IIIc (data not shown) in FM changed (P<0.0001-0.06)
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during early pregnancy (Fig. 4A-K). PGF mRNA expression increased (P<0.0001) ~3.5 to 34-
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fold from days 16-22 to days 24-30 of pregnancy (Fig. 4A). VEGF mRNA expression increased
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(P<0.0001) ~2-fold on days 28 and 30 compared with days 16-20 (Fig. 4B). FLT1 mRNA
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expression increased (P<0.0001) ~5 to 50-fold on days 28 and 30 compared with days 16-24
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(Fig. 4C). KDR mRNA expression was 2 to 11-fold greater (P<0.0001) on days 20-24 than on
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days 16-18 and 26-30 (Fig. 4D).
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ANGPT1 mRNA expression was low on days 16-24 of pregnancy and then increased
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(P<0.001) ~2 to 50-fold on days 26-30 of pregnancy (Fig. 4E). Expression of ANGPT2 mRNA
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was not detectable on day 16 of pregnancy, but increased (P<0.001) ~3.5 to 5-fold from day 18
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to days 22-30 of pregnancy (Fig. 4F). TEK mRNA expression increased (P<0.001) ~7 to 9-fold
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from day 16 to days 20-24, and then decreased on days 26-30 (Fig. 4G).
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FGF2 mRNA expression increased (P<0.06) ~4 to 5-fold from day 16 to days 20-24, and
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then decreased on days 26-28 (Fig. 4H), whereas NOS3 mRNA expression increased (P<0.001)
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~5 to 16-fold from days 16-18 to days 22-30 of pregnancy (Fig. 4I). GUCY1B3 mRNA
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expression was ~2 to 7-fold greater (P<0.01) on day 18 than on any other day of pregnancy (Fig.
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4J). HIF1A mRNA expression was ~1 to 2-fold greater (P<0.02) on days 18, 20 and 30 than on
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days 16 and 24 of pregnancy (Fig. 4K).
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Results of regression analysis demonstrating a pattern of change in mRNA expression for
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all 12 investigated genes involved in the regulation of angiogenesis are presented in Table 1.
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Correlation coefficients for mRNA expression of evaluated genes involved in regulation of
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angiogenesis are presented in Table 2. Expression of mRNA for the majority of these genes was
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significantly (P<0.0001-0.08) correlated (Table 2).
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Discussion
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Early pregnancy is characterized by dramatic uterine and embryonic/fetal tissue growth,
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differentiation and remodeling, and it is the critical period for establishing a healthy pregnancy.
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During this critical period, maternal recognition of pregnancy, initial attachment/implantation of
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FM to uterine epithelium and initiation of placental growth and development take place (Bowen
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& Burghardt 2000, Spencer et al. 2007, 2008). In addition, most embryonic loss occurs in early
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pregnancy with rates of pregnancy losses reported as ≥30% in most mammalian species and
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possibly >50% in humans (Reynolds & Redmer 2001, Miri & Varmuza 2009). Thus,
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investigation of fetal and maternal placental growth during early pregnancy is needed to establish
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the mechanisms that contribute to pregnancy maintenance or loss.
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The present study demonstrated a rapid increase in fetal size, decrease in 5mC expression
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(as determined by DNA dot blot), and dramatic changes in the mRNA expression in FM of
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several factors involved in regulation of DNA methylation, angiogenesis and tissue growth
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during early pregnancy. However, the rates of cellular proliferation were maintained at a high
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level but not significantly changed. In addition, image analysis did not show any differences in
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5mC expression throughout pregnancy. The discrepancies between the results of 5mC evaluation
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by DNA dot blot and by image analyses were likely due to the lower sensitivity of
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immunohistochemistry and image analysis than dot blot analysis.
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Early embryonic development is tightly regulated and includes control of cell growth,
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proliferation and differentiation, morphogenesis, and protein synthesis and trafficking (Blomberg
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et al. 2008, Igwebuike 2009). In the present study, growth of the fetal placental was reflected by
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rapid increase of embryonic size and very high rates of cellular proliferation in FM. High rates of
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cell proliferation were also observed in the fetal placenta during early pregnancy in humans and
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monkeys (Wei et al. 2005, Korgun et al. 2006, Kar et al. 2007). Interestingly, cell proliferation
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in fetal and maternal placenta obtained after transfer of embryos created in vitro or through
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parthenogenetic activation was less than in pregnancies after natural breeding in sheep
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(Borowicz et al. 2009, Grazul-Bilska et al. unpublished). In addition, altered placental cell
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proliferation or turnover was observed in several pathological conditions including diabetes and
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trophoblastic diseases at several pregnancy stages in humans (Zhang et al. 2009; Burleigh et al.
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2004). These data suggest that altered cellular proliferation in fetal placenta is a feature of
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compromised pregnancies. However, the mechanism of regulation of cell proliferation in FM
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has not been elucidated and this subject requires additional investigation.
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Since epigenetic modifications of the genome include methylation of DNA at cytosine
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residues and histone modifications through methylation catalyzed by DNMTs, we choose to use
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5mC, and DNMT1, 3a and 3b as markers of global methylation in our study. In the present
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experiment, expression of these markers was detected in FM, and the pattern of changes differed
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during early pregnancy. Interestingly, expression of 5mC decreased during early pregnancy
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indicating demethylation was occurring in the FM. However, in our study, only one of the
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enzymes catalyzing methylation and/or demethylation, DNMT3b (Ooi & Bestror, 2008) had
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decreased mRNA expression; whereas expression of DNMT3a mRNA increased during early
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pregnancy. Therefore, we hypothesize that a specific balance exists between expression and/or
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function of DNMTs, and likely other enzymes involved in methylation and/or demethylation
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(e.g., DNA glycosylase; Zhu 2009) are present in the tissue to further regulate methylation
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processes. It is believed that genomic imprinting, regulated by methylation mechanisms, may
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play a critical role in placental biology (Maccani & Marsit 2009, Coan et al. 2005, Miri &
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Varmuza 2009). In fact, alterations in imprinting have been linked to placental pathologies
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(Tycko 2006, Wagschal & Feil 2006). Therefore, correction of the DNA methylation may offer
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new strategies for preventing pregnancy complications. However, more research is required to
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gain a better understanding of the mechanisms of imprinting and methylation in the placenta in
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order to establish a strategy for successful pregnancy outcomes.
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Very few studies have evaluated global methylation during early placental development,
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but several studies investigated methylation in the placenta at specific time points. For human
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placenta, it has been demonstrated that methylation levels measured by 5mC content increased in
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a gestational stage-dependent manner (Fuke et al. 2004), DNA methylation measured by the
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mean CpG methylation status of genes probed in a microarray analysis was decreased after in-
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vitro vs. in-vivo conception (Katari et al. 2009), and that the decrease in X chromosome-linked
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placental methylation was greater in pregnancies carrying female than male babies Cotton et al.
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(2009). Studies of embryonic and/or extraembryonic tissues during mouse development,
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demonstrated that global methylation and demethylation and expression of DNMT were stage-
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and tissue-specific (Monk et al. 1987, Trasler et al. 1996, Watanabe et al. 2002). For cows,
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global methylation in the fetal placenta on day 80 was similar for pregnancies established after
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transfer of embryos created through artificial insemination, in vitro fertilization or somatic cell
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nuclear transfer (Hiendleder et al. 2004). In our study, significant changes in global methylation,
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measured by 5mC and DNMTs mRNA expression, indicate that the pattern of methylation in FM
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is changing throughout early pregnancy. Since data concerning the methylation process in
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developing and growing placenta are extremely limited, further studies should be undertaken to
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study this process in detail.
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In the present study, blood vessels marked with SMCA were detected in FM as early as
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on day 18 of pregnancy. For human placenta, it has been demonstrated that angiogenesis,
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manifested by vascular tube formation, and presence of haemangiogenic cell cords, was evident
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21-27 days post conception, on day 32 post conception erythrocytes were observed within blood
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vessels lumen, and between days 35-42 the networks of cords were heavily connected with each
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other without any interruption (Demir et al. 1989, 2004, Torry et al. 2004, Zygmunt et al. 2003,
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Arroyo & Winn 2008, Burton et al. 2009, van Oppenraaij et al. 2009). Thus, vasculogenesis is
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initiated very early in pregnancy in order to support dramatic fetal growth.
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Fetal membrane growth and vascular development has to be tightly regulated to
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coordinate development of the fetal and maternal placenta and embryonic tissues. Therefore,
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vasculogenesis, angiogenesis and tissue growth within fetal placenta are regulated by numerous
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growth factors (Patan 2000, Zygmunt et al. 2003, Demir et al. 2007, Herr et al. 2008, Burton et
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al. 2009). In the present study, increased mRNA expression of several factors and their receptors
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involved in the regulation of angiogenesis and growth in FM was observed as pregnancy
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progressed. In fact, changes in mRNA expression of several growth/angiogenic factors and/or
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their receptors including PGF, FLT1, ANGPT1, TEK, NOS3 and HIF1A in FM paralleled
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expression of these factors in maternal placenta in sheep (Grazul-Bilska et al. 2010), indicating a
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similar role of these factors in the regulation of fetal and maternal placental growth and function.
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Although protein and/or mRNA expression of VEGF, PGF and receptors, and/or FGF2
and receptor were detected in extraembryonic tissues at specific stages of early pregnancy in
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monkeys, humans and cows (Vuorela et al. 1997, Ghosh et al. 2000, Hildebrandt et al. 2001,
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Wang et al. 2003, Demir et al. 2004, Wei et al. 2004, Pfarrer et al. 2006) the changes during
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placental development have not been evaluated for these or other species. In the present study,
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dramatic changes of expression of mRNA for members of the VEGF and ANGPT systems were
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observed. Since during early pregnancy first vasculogenesis and then angiogenesis are initiated,
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it seems that high expression of VEGF and ANGPT systems is required to regulate these
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processes. In fact, members of the VEGF family and ANGPTs are recognized as the major
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regulators of vasculogenesis and angiogenesis in the placenta (Zygmunt et al. 2003, Reynolds et
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al. 2002, 2006, Demir et al. 2007, Seval et al. 2008). In the primate placenta, protein and/or
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mRNA expression of VEGF, FLT1, KDR, ANGPT1, ANGPT2 and/or receptor TEK were
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detected during early pregnancy (Demir et al. 2004, Demir 2009, Wei et al. 2004, Seval et al.
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2008). These factors appeared to be spatio- and temporary-regulated during early pregnancy in
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primates (Demir et al. 2004, Wei et al. 2004, Kayisli et al. 2006, Seval et al. 2008). The
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increased expression of several angiogenic factors during early pregnancy indicates that these
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factors are involved in regulation of vascular development, remodeling and trophoblast function.
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However, functional studies should be undertaken to verify the specific roles of VEGF and
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ANGPT systems in placental growth and function.
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Expression of mRNA for FGF2 but not FGFR2IIIc in FM increased during early
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pregnancy in this study. Although expression of FGF2 and its receptor was detected in fetal
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placenta in sheep and other species (Wei et al. 2004, Liu et al. 2005, Kaufman et al. 2004), little
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is known about the specific role of FGF system in early placental development. However, it has
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been demonstrated that FGFs stimulate differentiation of the embryonic germ layers, and it has
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been suggested that the FGF system is involved in the regulation of growth and differentiation of
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vascular and non-vascular compartments of the placenta (Reynolds et al. 2002). In addition,
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FGF2 is a potent stimulator of cell proliferation (Reynolds & Redmer 2001); therefore, it is
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reasonable to postulate that FGF2 and its receptor are involved in the regulation of cell
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proliferation in FM.
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Expression of NOS3 mRNA gradually increased from day 16 to 22, and remained at a
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similar level until day 30, but the NOS3 receptor GUCY1B3 mRNA expression was enhanced
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only on day 18 of pregnancy in FM in our study. Endothelial NOS is expressed in fetal placenta
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from early pregnancy in several species (Ariel et al. 1998, Al-Hijji et al. 2003, Sladek et al.
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1997, Gagioti et al. 2000). NOS are recognized as regulators of implantation and pregnancy
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maintenance, and angiogenesis in the fetal and maternal placenta (Maul et al. 2003; Gagioti et al.
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2000), however the mechanism of NOS effects on these processes remains to be elucidated.
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Furthermore, it has been demonstrated that NOS3 expression is regulated by FGF2 and VEGF in
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ovine placental artery endothelial cells (Mata-Greenwood et al. 2008). These interactions seem
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to be reflected in our study by significant correlations between the mRNA expression of NOS3
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and expression of members of the VEGF and FGF2 systems.
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An increased HIF1A mRNA expression in FM was observed on days 18-20 of pregnancy
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in our study. This transient high expression of HIF1A mRNA may be associated with low
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oxygen levels observed during early pregnancy in several species (Rodesch et al. 1992,
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Rajakumar & Conrad 2000, Fryer & Simon 2006, Ietta et al. 2006, Pringle et al. 2007). It seems
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that HIF1A expression is decreasing after delivery of oxygen is well established through
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developing blood vessel network. A decrease of HIF1A mRNA expression from day 50 to the
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end of pregnancy in fetal placenta was observed in sheep (Borowicz et al. 2007). It has been
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clearly demonstrated using the knockout and other models that HIF activity is necessary for
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placental development, since HIF1A is involved in the regulation of placental morphogenesis,
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cell migration, angiogenesis, erythropoesis and cell metabolism, and is critical for adaptive
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responses to hypoxia (Cowden Dahl et al. 2005, Fryer & Simon 2006). In fact, HIF1A
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expression is altered in preeclampsia and IUGR placentas (Rajkumar et al. 2007; Zamudio et al.
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2007). Therefore, HIF1A may be used as a marker of compromised pregnancies.
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In summary, this study demonstrates a dramatic increase in fetal size, high cellular
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proliferation rates, a decrease in 5mC expression (as determined by DNA dot blot), a lack of
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changes in vascularization measured as the number of blood vessels per tissue area, but
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significant changes in mRNA expression of factors involved in the regulation of methylation,
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angiogenesis and tissue growth in FM during early pregnancy. Positive correlations among
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mRNA expression of several growth/angiogenic factors and/or their receptors indicate
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interactions among these factors in the regulation of development of fetal placenta. However,
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since we have evaluated expression for the factors mentioned above at the mRNA level only,
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additional studies should be undertaken to determine the pattern of protein expression and its
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relation to mRNA expression in order to better understand the process of placental growth and
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function. This description of cellular and molecular changes in FM during early pregnancy will
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provide a foundation for determining whether and how placental development is altered in
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compromised pregnancies. Furthermore, it will help to establish a baseline that can be used to
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design therapeutic treatments to restore normal fetal development in compromised pregnancies.
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Material and Methods
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Animals
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The NDSU Institutional Animal Care and Use Committee approved all animal
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procedures in this study. Gravid uteri were obtained from crossbred Western Range (primarily
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Rambouillet, Targhee, and Columbia) ewes (n=5 to 8 per day) on days 16, 18, 20, 22, 24, 26, 28,
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and 30 after mating (day of mating=day 0). At tissue collection for immunohistochemical
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staining, specimen pins were inserted completely through the uterus and FM at the level of the
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external intercornual bifurcation to maintain specimen morphology; cross sections of the entire
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gravid uterus (approximately 0.5-cm thick) were obtained using a Stadie-Riggs microtome knife
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followed by immersion in formalin or Carnoy’s solution and embedding in paraffin. For total
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cellular RNA extraction, chorioallantoic FM were dissected from the area close to the embryo,
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snap-frozen, and stored at -70 C. On days 20-30 of pregnancy, fetuses were separated from fetal
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membranes and crown-rump length of each fetus was measured. The length of fetuses on days 16
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and 18 was not determined due to the small fetal size (<2 mm) and tissue transparency.
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Immunohistochemistry
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Immunohistochemical procedures were used as described before (Grazul-Bilska et al.
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2010). Paraffin-embedded uterine tissues containing FM were sectioned at 4 µm and mounted
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onto slides. Sections were rinsed several times in PBS containing Triton-X100 (0.3%, v/v) and
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then were treated for 20 min with blocking buffer [PBS containing normal horse serum (2%,
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vol/vol)] followed by incubation with specific primary antibody for Ki67 (a marker of
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proliferating cells; 1:500; mouse monoclonal; Vector Laboratories, Burlingame, CA, USA), 5mC
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(a marker of global DNA methylation; 1:500; mouse monoclonal; Eurogentec North America,
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San Diego, CA, USA), or SMCA (a marker of pericytes and smooth muscle cells and thus blood
334
vessels; 1:150; mouse monoclonal; Oncogene Research Products; San Diego, CA, USA)
335
overnight at 4˚ C. Primary antibodies were detected by using secondary anti-mouse antibody
336
coupled to peroxidase (ImPress Kit; Vector Laboratories). The sections were then counterstained
337
with nuclear fast red (Sigma, St. Lois, MO, USA) to visualize cell nuclei. Control sections were
15
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338
incubated with normal mouse IgG (4 µg/mL) in place of primary antibody. Fetal placental cell
339
types were not identified in this study due to methodological difficulties, such as a lack of
340
specific markers for these cell types in sheep or absence of some cell types in individual tissue
341
slides; thus we used the entire fetal placenta for immunohistological and other evaluations, which
342
of course has some limitations that the reader should keep in mind.
343
Image analysis
344
For each tissue section, images were taken at 400x (Ki67 staining), 600x (5mC staining) or
345
200x (SMCA staining) magnification, using an Eclipse E600 Nikon microscope and digital
346
camera for 5-40 randomly chosen fields (0.025 mm2 per field) from areas containing FM. To
347
determine LI, the percentage of 5mC positive staining in cell nucleus or the number of blood
348
vessels per FM tissue area, an image analysis system (Image-Pro Plus, Media Cybernetics, Inc.,
349
Bethesda , MD, USA) was used as described previously (Grazul-Bilska et al. 2010). The LI was
350
calculated as the percentage (%) of proliferating Ki67-positive cells out of the total number of
351
cells within an FM tissue area.
352
DNA dot blot assay
353
DNA dot-blot analysis of 5mC was based on modifications of previously described
354
methods (Tao et al. 2004, Park et al. 2005). DNA was isolated from FM tissues homogenized in
355
Tri-Reagent (Molecular Research Center, Cincinnati, OH, USA). Purified DNA (0.5 µg) was
356
denatured by adding NaOH and EDTA to final concentrations of 0.4 N NaOH and 10 mM
357
EDTA, heated to 100oC for 10 min, followed by cooling to 4oC, and then neutralized with an
358
equal volume of cold (4oC) 2 M ammonium acetate. Denatured DNA was spotted onto Ambion
359
BrightStar-Plus nylon membrane (Ambion/Applied Biosystems, Austin, TX, USA) using the
360
BRL HYBRI-DOT Manifold (Bethesda Research Laboratories, Gaithersburg, MD, USA). The
16
Page 17 of 40
361
DNA was cross-linked to the membrane for 2 min with the CL-1000 Ultraviolet Crosslinker
362
(UVP, Upland, CA, USA) and then dried. After wetting in dH2O, the membrane was blocked
363
with 5% skim milk in phosphate-buffered saline + 0.1% Tween 20 (PBST) by rocking for 3 h at
364
room temperature. The membrane was probed with a 1:2000 dilution in 2% milk-PBST of
365
monoclonal mouse antibody against 5mC (Eurogentec North America) by rocking at 4oC
366
overnight. The membrane was washed three times for 10 min each in PBST before incubation
367
with a 1:5000 dilution of HRP-conjugated anti-mouse secondary antibody in 2% milk-PBST
368
with rocking for 1 h at room temperature. After three washes in PBST, the membrane was
369
incubated with ECL Plus Western blotting reagent (GE Healthcare; Piscataway, NJ, USA) and
370
the chemiluminescense of 5mC was detected and quantified using the AlphaEaseFC imager
371
(Alpha Innotech, San Leandro, CA, USA). After detection of 5mC, the membrane was stained
372
with 0.02% methylene blue for DNA quantification and the relative dot intensity was measured
373
with the AlphaEaseFC imager. Each sample was normalized to its DNA concentration by
374
dividing the 5mC signal intensity of the sample by the dot intensity of methylene blue.
375
Quantitative Real-Time RT-PCR
376
All procedures for determining the expression of mRNA of several genes in ovine tissues
377
by RT-PCR have been reported previously (Redmer et al. 2005, Johnson et al. 2006, Grazul-
378
Bilska et al. 2010). Briefly, snap-frozen FM tissues were homogenized in Tri-Reagent
379
(Molecular Research Center) according to the manufacturer’s specifications. The quality and
380
quantity of total RNA were determined via capillary electrophoresis using the Agilent 2100
381
Bioanalyzer (Agilent Technologies, Wilmington, DE, USA). Real-time RT-PCR reagents,
382
probes, and primers were purchased from and used as recommended by Applied Biosystems
383
(Foster City, CA, USA). For each sample, 30 ng total RNA was reverse transcribed in triplicate
17
Page 18 of 40
384
20-µl reactions using random hexamers. Sequence-specific Taqman probes and primers were
385
designed using the Primer Express Software from Applied Biosystems, and sequences for 12
386
factors involved in the regulation of angiogenesis have been published before (Redmer et al.
387
2005, Johnson et al. 2006, Grazul-Bilska et al. 2010). The sequences of probes and primers for
388
DNMT1, 3a and 3b are presented in Table 3. The ABI PRISM 7000 was used for detection of
389
sequences amplified at 60oC typically for 40 or 45 cycles (Applied Biosystems). Quantification
390
was determined from a relative standard curve of dilutions of the cDNA generated from tcRNA
391
pooled from placentomes collected on day 130 of pregnancy. Expression of each gene was
392
normalized to expression of 18S ribosomal RNA (rRNA) in a multiplex reaction using the
393
human 18S pre-developed assay reagent (PDAR) from Applied Biosystems. The PDAR
394
solution, which is primer limited and contains a VIC- labeled probe, was further adjusted by
395
using one-fourth the normal amount, so that it would not interfere with amplification of the
396
FAM-labeled gene of interest. Standard curves were also generated with the multiplex solution,
397
and the quantity of 18S rRNA and the gene of interest were determined using each specific
398
standard curve. The concentrations of mRNA were then normalized to 18S rRNA by dividing
399
each of the mRNA values by their corresponding 18S rRNA value (Grazul-Bilska et al., 2010).
400
Statistical Analysis
401
Data were analyzed using the general linear models (GLM) procedure of SAS and
402
presented as means ± SEM with the main effect of day of pregnancy (SAS Institute 2010).
403
When the F-test was significant (P<0.05), differences between specific means were evaluated by
404
using the least significant differences test (Kirk 1982). The SAS procedure PROC REG was
405
used for regression analysis and PROC CORR was used to calculate simple linear correlations
406
between specific variables.
18
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407
408
Declaration of interest
409
The authors declare that there is no conflict of interest that could be perceived as prejudicing the
410
impartiality of the research reported.
411
412
Funding
413
This project was supported by USDA grant (2007-01215) to LPR and ATGB, NIH grant
414
(HL64141) to LPR and DAR, ND EPSCoR AURA grant to ATGB and MAM, ND Space Grant
415
Fellowship Program award to MAM, and by NIH grant (P20 RR016741) from the INBRE
416
program of the NCRR, NIH to ATGB and LPR.
417
418
Acknowledgements:
419
The authors would like to thank Dr. Eric Berg, Dr. Kimberly Vonnahme, Ms. Tammi Neville,
420
Mr. James D. Kirsch, Mr. Kim C. Kraft, Mr. Robert Weigl, Mr. Tim Johnson (deceased), Mr.
421
Terry Skunberg and other members of our laboratories and department for their assistance.
422
423
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List of Figures
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Fig. 1. Crump to rump length of fetuses from day 20 to 30 of pregnancy (A) and labeling index
671
(% of proliferating cells; B) in fetal membranes on days 16-30 of pregnancy. Fetuses from day
672
16 and 18 were not collected and measured due to their small size (<2 mm) and tissue
673
transparency. a,b,c,dP<0.0001-0.05; values ± SEM with different superscripts differ within a
674
specific measurement.
675
676
Fig. 2. Representative photomicrographs of immunohistochemical staining for Ki67 (A), 5-
677
methyl cytosine (5mC; B) and smooth muscle cell actin (SMCA; C) in uterine tissues from day
678
24 of early pregnancy. Dark color represents positive staining and pink color (nuclear fast red
679
staining) indicates unlabeled ell nuclei. In (A), note nuclear staining of Ki-67 (arrows) in fetal
680
membranes (FM) and endometrium (E). In (B), note punctate staining of 5mC in nuclei of the
681
majority of cells (arrows) in FM and E, and a lack of staining in some cells (arrowheads) in FM.
682
In (C), note SMCA cytoplasmic staining in blood vessels in FM (arrows), and E (arrowheads). In
683
(D), note a lack of positive staining in the controls in which mouse IgG was used in place of the
684
primary antibody.
685
686
Fig. 3. Expression of 5mC as determined by DNA dot blot (A), and mRNA for DNA
687
methyltransferase (DNMT) 1 (B), 3a (C) and 3b (D) in fetal membranes (FM) on days 16-30 of
688
pregnancy. a,b,c,dP<0.0001-0.06; values ± SEM with different superscripts differ within each
689
specific gene.
690
31
Page 32 of 40
691
Fig. 4. Expression of mRNA for placental growth factor (PGF; A), vascular endothelial growth
692
factor receptor (VEGF; B), VEGF receptor FLT1 (C), VEGF receptor KDR (D), angiopoietin
693
(ANGPT) 1 (E), ANGPT2 (F), ANGPT receptor TEK (G), fibroblast growth factor-2 (FGF2; H),
694
endothelial nitric oxide synthase (NOS3; I), NOS3 receptor GUCY1B3 (J) and hypoxia inducible
695
factor (HIF) 1A (H) in fetal membranes (FM) on days 16-30 of pregnancy.
696
a,b,c,d
P<0.0001-0.06; values ± SEM with different superscripts differ within each specific gene.
32
Page 33 of 40
Fig. 1
25
A; P<0.0001
40
Labeling index (%)
Length of fetus (mm)
d
20
c
15
10
c
b
b
a
5
B; P>0.2
30
20
10
0
0
20
22
24
26
28
Day of pregnancy
16
30
18
20 22 24 26 28
Day of pregnancy
30
Page 34 of 40
A
B
FM
FM
E
C
E
D
FM
FM
E
FM
EE
Page 35 of 40
A; 5mC; P<0.003
Relative expression
2
1.6
ab
b
b
abc
1.2
ac
ac
c
c
0.8
0.4
0
16
18
20
22
24
26
28
30
Relative mRNA expression
Fig. 3.
1.5
B; Dnmt1; P=0.12
bc
1
ab
ab
18
20
a
abc
1
ab
d
cd
ab
a
0.5
0
16
18
20 22 24 26 28
Day of pregnancy
30
Relative mRNA expression
Relative mRNA expression
cd
abc
0
16
22
24
26
28
30
Day of pregnancy
C; Dnmt3a; P<0.004
bcd
abc
c
0.5
Day of pregnancy
1.5
bc
2.5
D; Dnmt3b; P<0.0001
a
2
a
1.5
b
1
b
bc bc
0.5
bc
c
0
16
18
20 22 24 26 28
Day of pregnancy
30
Page 36 of 40
A; PGF: P<0.0001
3
c
Relative mRNA expression
Relative mRNA expression
Fig. 4.
c
c
2
b
1
a
a
a
16
18
a
0
20 22 24 26 28
Day of pregnancy
0.3
B; VEGF; P<0.0001
d
cd
0.2
0.1
a
ab
ab
ab
bc
a
0
30
16
18
20 22 24 26 28
Day of pregnancy
30
1.2
d
0.8
c
bc
0.4
a
a
16
18
ab
ab
ab
0
20 22 24 26 28
Day of pregnancy
Relative mRNA expression
Relative mRNA expression
C; FLT1; P<0.0001
0.6
D; KDR; P<0.0001
b
b
b
0.4
a
a
a
0.2
a
a
0
16
30
18
20 22 24 26 28
Day of pregnancy
30
0.4
c
c
0.3
0.2
b
a
0.1
0
a
a
16
18
a
a
20 22 24 26 28
Day of pregnancy
Relative mRNA expression
Relative mRNA expression
E; ANGPT1; P<0.0001
30
0.12
F; ANGPT2; P<0.0001
0.08
bc
bc
b
ab
0.04
a
0
16
c
c
18
20 22 24 26 28
Day of pregnancy
30
G; TEK; P<0.0001
d
Relative mRNA expression
bc
bc
0.4
ab
ab
0.2
a
0
16
1.6
18
20 22 24 26 28
Day of pregnancy
I; NOS3; P<0.001
c
c
ab
0.4
a
a
0
0.7
0.6
0.5
18
20 22 24 26 28
Day of pregnancy
K; HIF1A; P<0.02
b
b
ac
ac
a
0.4
0.3
0.2
0.1
0.0
16
18
20 22 24 26 28
Day of pregnancy
bc
30
bc
c
bc
0.2
ab
0.1
abc
abc
a
0
0.5
18
20 22 24 26 28
Day of pregnancy
b
0.4
0.3
0.2
30
J; GUCY1B3; P<0.01
a
a
a
a
a
0.1
a
a
0
16
30
bc
ac
a
H; FGF2; P<0.06
16
bc
0.8
0.3
30
c
bc
1.2
16
Relative mRNA expression
cd
cd
0.6
Relative mRNA expression
0.8
Relative mRNA expression
Relative mRNA expression
Page 37 of 40
18
20 22 24 26 28
Day of pregnancy
30
Page 38 of 40
Table 1. Regression analysis of angiogenic genes in FM from early pregnancy.
Gene
PGF
Regression type
P value
R2
Equation
Exponential sigmoidal P<0.0001 0.7904 Y = 5.284 x 10 7 e0.996 X - 0.016 X2
VEGF
Quadratic
FLT1
Exponential
KDR
Cubic
P<0.0001 0.5432 Y = 0.249 - 0.021 X + 0.001 X2
P<0.0001 0.7788 Y = 0.0007 e0.231 X
P<0.0001 0.4568 Y = -13.535 + 1.724 X – 0.069 X2 + 0.0009 X3
ANGPT1
Exponential sigmoidal P<0.0001 0.7860 Y = 4.453e-12 e1.621 X - 0.027 X2
ANGPT2
Exponential sigmoidal P<0.0001 0.7741 Y = 4.083e-19e3.138 X - 0.061 X2
TEK
Exponential sigmoidal P<0.0001 0.4610 Y = 2.823e-8 e1.367 X - 0.028 X2
FGF2
Exponential sigmoidal P=0.0002 0.3033 Y = 1.267e-11 e1.855 X - 0.036 X2
FGFR2
NO3S
GUCY1B3
HIF
-Cubic
NS
--
--
P=0.0002 0.3393 Y = 1.542 - 0.537 X + 0.039 X2 - 0.0007 X3
--
NS
Cubic
P=0.007
--
--
0.2250 Y = -11.594 + 1.666 X - 0.075 X2 + 0.001 X3
Page 39 of 40
Table 2. Correlation coefficients for mRNA expression of angiogenic factors in fetal membranes.
PGF
VEGF
FLT1
KDR
VEGF
0.735
P<0.0001
-
FLT1
0.750
P<0.0001
0.779;
P<0.0001
KDR
-
NS
NS*
NS
-
ANGPT1
ANGPT2
TEK
FGF2
FGFR2
IIIc
NOS3
GUCY1B
3
*NS, not statistically significant, P>0.1
ANGPT1
0.783
P<0.0001
0.775
P<0.0001
0.802
P<0.0001
-0.249
P<0.08
-
ANGPT2
0.544
P<0.0001
0.458
P<0.0007
0.485
P<0.0003
NS
0.343
P<0.01
-
FGFR2
IIIc
TEK
FGF2
NS
NS
NS
0.260
P<0.06
NS
0.851
P<0.0001
NS
0.422
P<0.002
NS
0.424
P<0.002
NS
0.341
P<0.01
0.499
P<0.0002
NS
-
NS
-
NS
0.274
P<0.05
0.237
P<0.09
0.416
P<0.002
NS
0.438
P<0.001
-
NOS3
0.453
P<0.0009
0.510
P<0.0001
0.248
P<0.08
GUCY1B
3
NS
NS
0.367
P<0.008
NS
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
-0.327
P<0.02
NS
NS
NS
NS
NS
NS
NS
0.459
P<0.0007
-
NS
HIF1A
NS
-
NS
NS
0.265
P<0.06
Page 40 of 40
Table 3. Sequence of TaqMan primers and probes for Dnmt1, Dnmt3a, and Dnmt3b.
Oligonucleotidea
Nucleotide Sequence
Sheep Dnmt1 FP
5’- CCT GGG TCC ACG GTG TTC -3’
Sheep Dnmt1 RP
5’- CCA CCC ATG ACC AGC TTC A -3’
Sheep Dnmt1 Probe
5’-(6FAM) AGA GTA CTG CAA CGT CCT -(MGBNFQ)-3’
Sheep Dnmt3a FP
5’- TGT ACG AGG TAC GGC AGA AGT G -3’
Sheep Dnmt3a RP
5’- GGC TCC CAC AAG AGA TGC A -3’
Accession numberb
NM_001009473
HQ202740
Sheep Dnmt3a Probe 5’-(6FAM) ATG TCC TCG ATG TTC CG -(MGBNFQ)-3’
Sheep Dnmt3b FP
5’- AGC GGC AGG CGA TGT CT -3’
Sheep Dnmt3b RP
5’- GAG AAC TTG CCA TCA CCA AAC C -3’
HQ202741
Sheep Dnmt3b Probe 5’-(6FAM) CTG GAC CCA CCG CAT -(MGBNFQ)-3’
a
FP, Forward primer and RP, Reverse primer.
b
Nucleotide sequences for ovine-specific genes were obtained from the National Center for Biotechnology Information
(NCBI, 2010) database.