Page 1 of 40 Reproduction Advance Publication first posted on 27 January 2011 as Manuscript REP-10-0505 1 Placental development during early pregnancy in sheep: Cell proliferation, global 2 methylation and angiogenesis in the fetal placenta 3 4 5 Anna T. Grazul-Bilska, Mary Lynn Johnson, Pawel P. Borowicz, Megan Minten, Jerzy J. Bilski, 6 Robert Wroblewski, Mila Velimirovich, Lindsey R. Coupe, Dale A. Redmer and Lawrence P. 7 Reynolds. 8 9 10 Department of Animal Sciences, Center for Nutrition and Pregnancy, North Dakota State University, Fargo, ND 58108. 11 12 Short title: Fetal placental development during early pregnancy 13 14 Correspondence should be addressed to Anna T Grazul-Bilska; E-mail Anna.Grazul- 15 Bilska@ndsu.edu 1 Copyright © 2011 by the Society for Reproduction and Fertility. Page 2 of 40 16 Abstract 17 To characterize early fetal placental development, gravid uterine tissues were collected 18 from pregnant ewes every other day from day 16 to 30 after mating. Determination of: 1) 19 cell proliferation was based on Ki67 protein immunodetection; 2) global methylation was 20 based on 5-methyl-cytosine (5mC) expression and mRNA expression for DNA 21 methyltransferase (DNMT) 1, 3a and 3b; and 3) vascular development was based on smooth 22 muscle cell actin immunolocalization and on mRNA expression of several factors involved 23 in the regulation of angiogenesis in fetal membranes (FM). Throughout early pregnancy, 24 labeling index (proportion of proliferating cells) was very high (21%) and did not change. 25 Expression of 5mC and mRNA for DNMT3b decreased, but mRNA for DNMT1 and 3a 26 increased. Blood vessels were detected in FM on days 18 to 30 of pregnancy, and their 27 number per tissue area did not change. The patterns of mRNA expression for: placental 28 growth factor, vascular endothelial growth factor, and their receptors FLT1 and KDR; 29 angiopoietins 1 and 2 and their receptor TEK; endothelial nitric oxide synthase and the NO 30 receptor GUCY13B; and hypoxia inducing factor 1 alpha changed in FM during early 31 pregnancy. These data demonstrate high cellular proliferation rates, and changes in global 32 methylation and mRNA expression of factors involved in the regulation of DNA 33 methylation and angiogenesis in FM during early pregnancy. This description of cellular 34 and molecular changes in FM during early pregnancy will provide the foundation for 35 determining the basis of altered placental development in pregnancies compromised by 36 environmental, genetic or other factors. 37 38 Introduction 2 Page 3 of 40 39 The placenta is the exchange organ for all respiratory gases, nutrients, and wastes 40 between the fetal and maternal tissues (Ramsey, 1982; Faber & Thornburg, 1983). Thus, 41 placental development is critical for supplying the fetus with metabolic substrates via 42 transplacental exchange (Needham 1934, Ramsey 1982, Faber & Thornburg 1983, Morriss & 43 Boyd 1988, Reynolds et al. 2010). Many aspects of placental function play major roles in fetal 44 tissue growth including expression of specific genes, methylation patterns, vascularization, 45 hormone production and other processes (Reynolds & Redmer 2001, Reynolds et al. 2002, 2006, 46 2010, Blomberg et al 2008). Therefore, fetal development and pregnancy maintenance are 47 dependent on normal placental growth. 48 The placenta represents a type of organ which expresses a high rate of growth in order to 49 fulfill the metabolic demands of the growing fetus (Reynolds et al. 2002, 2006, 2010). 50 Although the role of hypertrophy and hyperplasia in placental growth has been recognized (Boos 51 et al. 2006, Murphy et al. 2006), very limited data are available concerning the rates and pattern 52 of cell proliferation in fetal membranes (FM) during early pregnancy. However, high 53 proliferation rates have been reported for placenta during early pregnancy in several species 54 (Blankenship & King 1994, Correia-da-Silva et al. 2004, Wei et al. 2005, Kar et al. 2007, 55 Grazul-Bilska et al. 2010). In addition, it has been demonstrated using transcriptome analysis 56 that genes which regulate trophoblast cell proliferation, cell differentiation, angiogenesis, and 57 numerous other genes which facilitate mother-fetus interactions are upregulated in fetal placenta 58 during early pregnancy in ruminants (Blomberg et al. 2008). 59 DNA methylation, which is catalyzed by DNA methyltransferases (DNMTs), is generally 60 associated with transcriptional silencing and imprinting, principally occuring at cytosine residues 61 located in dinucleotide CpG sites and is the most extensively characterized epigenetic mark in 3 Page 4 of 40 62 mammals (Hiendleder et al. 2004, Wilson et al. 2007, Beck & Rakyan 2008). In fact, DNMTs 63 are required for cell differentiation during embryonic development to regulate gene expression 64 through methylation mechanisms (Gopalakrishnan et al, 2008). DNA methyltransferase 1 is 65 primarily considered the maintenance methyltransferase (Bird 2002, Gopalakrishnan et al. 2008, 66 Kim et al. 2009); however other functions of DNMT1, such as methylation of non-CpG sites in 67 DNA bubbles have been recently discovered (Ross et al. 2010). Other methyltransefrases, 68 DNMT3A and DNMT3B are responsible for establishing de novo DNA methylation patterns 69 (Gopalakrishnan et al. 2008). 70 Although DNA methylation is the most commonly studied mode of epigenetic regulation, 71 the process of methylation/demethylation or the expression of the enzymes that promote 72 methylation have not been investigated in detail in the placenta. It has been demonstrated that 73 around the time of gastrulation and implantation, de novo methylation reestablishes the 74 developing organism’s methylation patterns both in the embryo and in extraembryonic tissues 75 (Maccani & Marsit 2009). However, the pattern of methylation in the embryo differs from the 76 extraembryonic tissues (Monk et al. 1987; Katari et al. 2009). In human placenta collected from 77 several stages of pregnancy and at term, low expression of 5-methyl-cytosine (5mC) and relative 78 hypomethylation have been reported (Kokalj-Vokac et al. 1998, Katari et al. 2009). Many genes 79 expressed in extraembryonic tissues are imprinted (Reik et al. 2001, Myatt et al. 2006, Jansson 80 & Powell, 2007), and several of these imprinted genes are involved in regulating fetal and 81 placental growth (Reik et al. 2001, 2003, Myatt et al. 2006, Wagschal et al. 2008). Thus, DNA 82 methylation plays a significant role during embryonic and placental development in 83 physiological and pathological conditions (Kim et al. 2009). However, little is known about 84 global methylation and expression of DNA methyltransferases (DNMTs) in placental tissues 4 Page 5 of 40 85 86 during early pregnancy in any species. Vascularization of both fetal and maternal placenta is a critical factor in pregnancy 87 maintenance (Zygmunt et al. 2003, Huppertz & Peeters 2005, Arroyo & Winn 2008, Reynolds et 88 al. 2006, 2010). Fetal placental vasculogenesis, which is a result of de novo formation of blood 89 vessels, is initiated very early in pregnancy (e.g., in humans about 21 days, in rhesus monkey 90 about 19 days post conception; Kaufmann et al. 2004). Vasculogenesis is very tightly regulated 91 by angiogenic and other factors (Flamme et al. 1997, Patan 2000, Kaufmann et al. 2004, Demir 92 et al. 2007). Although expression of several factors involved in the control of angiogenesis has 93 been studied in the placenta of several species, limited information concerning expression of 94 these factors is available for FM during early pregnancy. 95 We hypothesized that the patterns of cellular proliferation, global methylation of DNA, 96 expression of several DNMTs, vascular development, and expression of factors involved in the 97 regulation of angiogenesis in FM will change as early pregnancy progresses. Therefore, the 98 objective of this experiment was to determine 1) labeling index (LI; a proportion of proliferating 99 cells), 2) global methylation based on expression of 5mC in DNA and expression of mRNA for 100 DNMT1, 3a and 3b, 3) development of blood vessels based on immunodetection of smooth 101 muscle cell actin (SMCA; a marker of pericytes and smooth muscle cells, and thus blood 102 vessels), and 4) expression of 12 factors involved in the regulation of angiogenesis and their 103 receptors including placental growth factor (PGF), vascular endothelial growth factor (VEGF) 104 and their receptors FLT1 and KDR, fibroblast growth factor (FGF) 2 and receptor 2IIIc, 105 angiopoietin (ANGPT) 1 and 2 and their receptors TEK, endothelial NO synthase (NOS3) and 106 receptor soluble guanylate cyclase (GUCY1B3), and hypoxia inducing factor 1 alpha (HIF1A) in 107 FM during early pregnancy in sheep. 5 Page 6 of 40 108 109 Results The length of the fetus increased (P<0.0001) ~3-fold from day 20 to day 30 of pregnancy 110 (Fig. 1A). Labeling index (a proportion of proliferating cells, based on Ki67 protein detection) 111 did not change significantly (P>0.2) from day 16 to day 30 of pregnancy (Fig. 1B). Overall, LI 112 was 20.7±1.5% in FM, and ranged from 17 to 26%; regression analysis demonstrated a linear 113 decrease (R2 = 0.110; P<0.055; Y = -0.63X + 36.3) from day 16 to day 30 of pregnancy. Ki67, 114 5mC, and SMCA proteins were immunodetected in the FM throughout early pregnancy (Fig. 2A, 115 B and C). Ki67 and 5mC were localized to the cell nuclei (Fig. 2A and B) but SMCA was 116 localized to cytoplasm of blood vessel cells (Fig. 2C). 117 Image analysis demonstrated that positive 5mC staining occupied 10.5±1.0% of cell 118 nuclei in FM (range 9-13%) and significant changes were not observed throughout early 119 pregnancy. However, DNA dot blot analysis demonstrated a ~2-fold decrease (P<0.003) in 5mC 120 expression in FM on days 16-20 compared to days 28-30, and regression analysis demonstrated a 121 cubic decrease (R2 = 0.355; P<0.0003; Y = -12.07+1.89X-0.09X2+0.001X3) throughout early 122 pregnancy (Fig. 3A). Expression of DNMT1 mRNA tended (P<0.11) to increase ~2-fold from 123 day 16 to day 30 (Fig. 3B), and regression analysis demonstrated a linear increase (R2 = 0.173; 124 P<0.002; Y = 0.18+0.03X) throughout early pregnancy. Expression of DNMT3a mRNA 125 increased (P<0.004) ~2-fold from day 16 compared with days 24-30 (Fig. 3C), but DNMT3b 126 mRNA decreased (P<0.0001) ~3-fold from day 16-18 compared with days 20-22 and decreased 127 by 5-fold by day 30 (Fig. 3D) of pregnancy. Regression analysis of mRNA expression for 128 DNMT3a demonstrated a linear increase (R2 = 0.301; P<0.0001; y =– 0.06+0.04x) but for 129 DNMT3b a cubic pattern (R2 = 0.624, P<0.0001; Y = 11.57-0.97X +0.02X2-0.0002X3) of 130 decrease throughout early pregnancy. 6 Page 7 of 40 131 Blood vessels marked with SMCA were detected in FM on days 18 to 30 of pregnancy 132 (Fig.3C). Overall, the number of blood vessels per FM tissue area was 1.7±0.4/10,000 µm2, 133 ranged from 0-7/10,000 µm2, and did not change throughout early pregnancy. 134 Expression of mRNA for factors involved in regulation of angiogenesis including PGF, 135 VEGF, FLT1, KDR, ANGPT1, ANGPT2, ANGPT receptor TEK, FGF2, NOS3, GUCY1B3 and 136 HIF1A (Fig. 4A-H), but not for FGFR2IIIc (data not shown) in FM changed (P<0.0001-0.06) 137 during early pregnancy (Fig. 4A-K). PGF mRNA expression increased (P<0.0001) ~3.5 to 34- 138 fold from days 16-22 to days 24-30 of pregnancy (Fig. 4A). VEGF mRNA expression increased 139 (P<0.0001) ~2-fold on days 28 and 30 compared with days 16-20 (Fig. 4B). FLT1 mRNA 140 expression increased (P<0.0001) ~5 to 50-fold on days 28 and 30 compared with days 16-24 141 (Fig. 4C). KDR mRNA expression was 2 to 11-fold greater (P<0.0001) on days 20-24 than on 142 days 16-18 and 26-30 (Fig. 4D). 143 ANGPT1 mRNA expression was low on days 16-24 of pregnancy and then increased 144 (P<0.001) ~2 to 50-fold on days 26-30 of pregnancy (Fig. 4E). Expression of ANGPT2 mRNA 145 was not detectable on day 16 of pregnancy, but increased (P<0.001) ~3.5 to 5-fold from day 18 146 to days 22-30 of pregnancy (Fig. 4F). TEK mRNA expression increased (P<0.001) ~7 to 9-fold 147 from day 16 to days 20-24, and then decreased on days 26-30 (Fig. 4G). 148 FGF2 mRNA expression increased (P<0.06) ~4 to 5-fold from day 16 to days 20-24, and 149 then decreased on days 26-28 (Fig. 4H), whereas NOS3 mRNA expression increased (P<0.001) 150 ~5 to 16-fold from days 16-18 to days 22-30 of pregnancy (Fig. 4I). GUCY1B3 mRNA 151 expression was ~2 to 7-fold greater (P<0.01) on day 18 than on any other day of pregnancy (Fig. 152 4J). HIF1A mRNA expression was ~1 to 2-fold greater (P<0.02) on days 18, 20 and 30 than on 153 days 16 and 24 of pregnancy (Fig. 4K). 7 Page 8 of 40 154 Results of regression analysis demonstrating a pattern of change in mRNA expression for 155 all 12 investigated genes involved in the regulation of angiogenesis are presented in Table 1. 156 Correlation coefficients for mRNA expression of evaluated genes involved in regulation of 157 angiogenesis are presented in Table 2. Expression of mRNA for the majority of these genes was 158 significantly (P<0.0001-0.08) correlated (Table 2). 159 Discussion 160 Early pregnancy is characterized by dramatic uterine and embryonic/fetal tissue growth, 161 differentiation and remodeling, and it is the critical period for establishing a healthy pregnancy. 162 During this critical period, maternal recognition of pregnancy, initial attachment/implantation of 163 FM to uterine epithelium and initiation of placental growth and development take place (Bowen 164 & Burghardt 2000, Spencer et al. 2007, 2008). In addition, most embryonic loss occurs in early 165 pregnancy with rates of pregnancy losses reported as ≥30% in most mammalian species and 166 possibly >50% in humans (Reynolds & Redmer 2001, Miri & Varmuza 2009). Thus, 167 investigation of fetal and maternal placental growth during early pregnancy is needed to establish 168 the mechanisms that contribute to pregnancy maintenance or loss. 169 The present study demonstrated a rapid increase in fetal size, decrease in 5mC expression 170 (as determined by DNA dot blot), and dramatic changes in the mRNA expression in FM of 171 several factors involved in regulation of DNA methylation, angiogenesis and tissue growth 172 during early pregnancy. However, the rates of cellular proliferation were maintained at a high 173 level but not significantly changed. In addition, image analysis did not show any differences in 174 5mC expression throughout pregnancy. The discrepancies between the results of 5mC evaluation 175 by DNA dot blot and by image analyses were likely due to the lower sensitivity of 176 immunohistochemistry and image analysis than dot blot analysis. 8 Page 9 of 40 177 Early embryonic development is tightly regulated and includes control of cell growth, 178 proliferation and differentiation, morphogenesis, and protein synthesis and trafficking (Blomberg 179 et al. 2008, Igwebuike 2009). In the present study, growth of the fetal placental was reflected by 180 rapid increase of embryonic size and very high rates of cellular proliferation in FM. High rates of 181 cell proliferation were also observed in the fetal placenta during early pregnancy in humans and 182 monkeys (Wei et al. 2005, Korgun et al. 2006, Kar et al. 2007). Interestingly, cell proliferation 183 in fetal and maternal placenta obtained after transfer of embryos created in vitro or through 184 parthenogenetic activation was less than in pregnancies after natural breeding in sheep 185 (Borowicz et al. 2009, Grazul-Bilska et al. unpublished). In addition, altered placental cell 186 proliferation or turnover was observed in several pathological conditions including diabetes and 187 trophoblastic diseases at several pregnancy stages in humans (Zhang et al. 2009; Burleigh et al. 188 2004). These data suggest that altered cellular proliferation in fetal placenta is a feature of 189 compromised pregnancies. However, the mechanism of regulation of cell proliferation in FM 190 has not been elucidated and this subject requires additional investigation. 191 Since epigenetic modifications of the genome include methylation of DNA at cytosine 192 residues and histone modifications through methylation catalyzed by DNMTs, we choose to use 193 5mC, and DNMT1, 3a and 3b as markers of global methylation in our study. In the present 194 experiment, expression of these markers was detected in FM, and the pattern of changes differed 195 during early pregnancy. Interestingly, expression of 5mC decreased during early pregnancy 196 indicating demethylation was occurring in the FM. However, in our study, only one of the 197 enzymes catalyzing methylation and/or demethylation, DNMT3b (Ooi & Bestror, 2008) had 198 decreased mRNA expression; whereas expression of DNMT3a mRNA increased during early 199 pregnancy. Therefore, we hypothesize that a specific balance exists between expression and/or 9 Page 10 of 40 200 function of DNMTs, and likely other enzymes involved in methylation and/or demethylation 201 (e.g., DNA glycosylase; Zhu 2009) are present in the tissue to further regulate methylation 202 processes. It is believed that genomic imprinting, regulated by methylation mechanisms, may 203 play a critical role in placental biology (Maccani & Marsit 2009, Coan et al. 2005, Miri & 204 Varmuza 2009). In fact, alterations in imprinting have been linked to placental pathologies 205 (Tycko 2006, Wagschal & Feil 2006). Therefore, correction of the DNA methylation may offer 206 new strategies for preventing pregnancy complications. However, more research is required to 207 gain a better understanding of the mechanisms of imprinting and methylation in the placenta in 208 order to establish a strategy for successful pregnancy outcomes. 209 Very few studies have evaluated global methylation during early placental development, 210 but several studies investigated methylation in the placenta at specific time points. For human 211 placenta, it has been demonstrated that methylation levels measured by 5mC content increased in 212 a gestational stage-dependent manner (Fuke et al. 2004), DNA methylation measured by the 213 mean CpG methylation status of genes probed in a microarray analysis was decreased after in- 214 vitro vs. in-vivo conception (Katari et al. 2009), and that the decrease in X chromosome-linked 215 placental methylation was greater in pregnancies carrying female than male babies Cotton et al. 216 (2009). Studies of embryonic and/or extraembryonic tissues during mouse development, 217 demonstrated that global methylation and demethylation and expression of DNMT were stage- 218 and tissue-specific (Monk et al. 1987, Trasler et al. 1996, Watanabe et al. 2002). For cows, 219 global methylation in the fetal placenta on day 80 was similar for pregnancies established after 220 transfer of embryos created through artificial insemination, in vitro fertilization or somatic cell 221 nuclear transfer (Hiendleder et al. 2004). In our study, significant changes in global methylation, 222 measured by 5mC and DNMTs mRNA expression, indicate that the pattern of methylation in FM 10 Page 11 of 40 223 is changing throughout early pregnancy. Since data concerning the methylation process in 224 developing and growing placenta are extremely limited, further studies should be undertaken to 225 study this process in detail. 226 In the present study, blood vessels marked with SMCA were detected in FM as early as 227 on day 18 of pregnancy. For human placenta, it has been demonstrated that angiogenesis, 228 manifested by vascular tube formation, and presence of haemangiogenic cell cords, was evident 229 21-27 days post conception, on day 32 post conception erythrocytes were observed within blood 230 vessels lumen, and between days 35-42 the networks of cords were heavily connected with each 231 other without any interruption (Demir et al. 1989, 2004, Torry et al. 2004, Zygmunt et al. 2003, 232 Arroyo & Winn 2008, Burton et al. 2009, van Oppenraaij et al. 2009). Thus, vasculogenesis is 233 initiated very early in pregnancy in order to support dramatic fetal growth. 234 Fetal membrane growth and vascular development has to be tightly regulated to 235 coordinate development of the fetal and maternal placenta and embryonic tissues. Therefore, 236 vasculogenesis, angiogenesis and tissue growth within fetal placenta are regulated by numerous 237 growth factors (Patan 2000, Zygmunt et al. 2003, Demir et al. 2007, Herr et al. 2008, Burton et 238 al. 2009). In the present study, increased mRNA expression of several factors and their receptors 239 involved in the regulation of angiogenesis and growth in FM was observed as pregnancy 240 progressed. In fact, changes in mRNA expression of several growth/angiogenic factors and/or 241 their receptors including PGF, FLT1, ANGPT1, TEK, NOS3 and HIF1A in FM paralleled 242 expression of these factors in maternal placenta in sheep (Grazul-Bilska et al. 2010), indicating a 243 similar role of these factors in the regulation of fetal and maternal placental growth and function. 244 245 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 11 Page 12 of 40 246 monkeys, humans and cows (Vuorela et al. 1997, Ghosh et al. 2000, Hildebrandt et al. 2001, 247 Wang et al. 2003, Demir et al. 2004, Wei et al. 2004, Pfarrer et al. 2006) the changes during 248 placental development have not been evaluated for these or other species. In the present study, 249 dramatic changes of expression of mRNA for members of the VEGF and ANGPT systems were 250 observed. Since during early pregnancy first vasculogenesis and then angiogenesis are initiated, 251 it seems that high expression of VEGF and ANGPT systems is required to regulate these 252 processes. In fact, members of the VEGF family and ANGPTs are recognized as the major 253 regulators of vasculogenesis and angiogenesis in the placenta (Zygmunt et al. 2003, Reynolds et 254 al. 2002, 2006, Demir et al. 2007, Seval et al. 2008). In the primate placenta, protein and/or 255 mRNA expression of VEGF, FLT1, KDR, ANGPT1, ANGPT2 and/or receptor TEK were 256 detected during early pregnancy (Demir et al. 2004, Demir 2009, Wei et al. 2004, Seval et al. 257 2008). These factors appeared to be spatio- and temporary-regulated during early pregnancy in 258 primates (Demir et al. 2004, Wei et al. 2004, Kayisli et al. 2006, Seval et al. 2008). The 259 increased expression of several angiogenic factors during early pregnancy indicates that these 260 factors are involved in regulation of vascular development, remodeling and trophoblast function. 261 However, functional studies should be undertaken to verify the specific roles of VEGF and 262 ANGPT systems in placental growth and function. 263 Expression of mRNA for FGF2 but not FGFR2IIIc in FM increased during early 264 pregnancy in this study. Although expression of FGF2 and its receptor was detected in fetal 265 placenta in sheep and other species (Wei et al. 2004, Liu et al. 2005, Kaufman et al. 2004), little 266 is known about the specific role of FGF system in early placental development. However, it has 267 been demonstrated that FGFs stimulate differentiation of the embryonic germ layers, and it has 268 been suggested that the FGF system is involved in the regulation of growth and differentiation of 12 Page 13 of 40 269 vascular and non-vascular compartments of the placenta (Reynolds et al. 2002). In addition, 270 FGF2 is a potent stimulator of cell proliferation (Reynolds & Redmer 2001); therefore, it is 271 reasonable to postulate that FGF2 and its receptor are involved in the regulation of cell 272 proliferation in FM. 273 Expression of NOS3 mRNA gradually increased from day 16 to 22, and remained at a 274 similar level until day 30, but the NOS3 receptor GUCY1B3 mRNA expression was enhanced 275 only on day 18 of pregnancy in FM in our study. Endothelial NOS is expressed in fetal placenta 276 from early pregnancy in several species (Ariel et al. 1998, Al-Hijji et al. 2003, Sladek et al. 277 1997, Gagioti et al. 2000). NOS are recognized as regulators of implantation and pregnancy 278 maintenance, and angiogenesis in the fetal and maternal placenta (Maul et al. 2003; Gagioti et al. 279 2000), however the mechanism of NOS effects on these processes remains to be elucidated. 280 Furthermore, it has been demonstrated that NOS3 expression is regulated by FGF2 and VEGF in 281 ovine placental artery endothelial cells (Mata-Greenwood et al. 2008). These interactions seem 282 to be reflected in our study by significant correlations between the mRNA expression of NOS3 283 and expression of members of the VEGF and FGF2 systems. 284 An increased HIF1A mRNA expression in FM was observed on days 18-20 of pregnancy 285 in our study. This transient high expression of HIF1A mRNA may be associated with low 286 oxygen levels observed during early pregnancy in several species (Rodesch et al. 1992, 287 Rajakumar & Conrad 2000, Fryer & Simon 2006, Ietta et al. 2006, Pringle et al. 2007). It seems 288 that HIF1A expression is decreasing after delivery of oxygen is well established through 289 developing blood vessel network. A decrease of HIF1A mRNA expression from day 50 to the 290 end of pregnancy in fetal placenta was observed in sheep (Borowicz et al. 2007). It has been 291 clearly demonstrated using the knockout and other models that HIF activity is necessary for 13 Page 14 of 40 292 placental development, since HIF1A is involved in the regulation of placental morphogenesis, 293 cell migration, angiogenesis, erythropoesis and cell metabolism, and is critical for adaptive 294 responses to hypoxia (Cowden Dahl et al. 2005, Fryer & Simon 2006). In fact, HIF1A 295 expression is altered in preeclampsia and IUGR placentas (Rajkumar et al. 2007; Zamudio et al. 296 2007). Therefore, HIF1A may be used as a marker of compromised pregnancies. 297 In summary, this study demonstrates a dramatic increase in fetal size, high cellular 298 proliferation rates, a decrease in 5mC expression (as determined by DNA dot blot), a lack of 299 changes in vascularization measured as the number of blood vessels per tissue area, but 300 significant changes in mRNA expression of factors involved in the regulation of methylation, 301 angiogenesis and tissue growth in FM during early pregnancy. Positive correlations among 302 mRNA expression of several growth/angiogenic factors and/or their receptors indicate 303 interactions among these factors in the regulation of development of fetal placenta. However, 304 since we have evaluated expression for the factors mentioned above at the mRNA level only, 305 additional studies should be undertaken to determine the pattern of protein expression and its 306 relation to mRNA expression in order to better understand the process of placental growth and 307 function. This description of cellular and molecular changes in FM during early pregnancy will 308 provide a foundation for determining whether and how placental development is altered in 309 compromised pregnancies. Furthermore, it will help to establish a baseline that can be used to 310 design therapeutic treatments to restore normal fetal development in compromised pregnancies. 311 Material and Methods 312 Animals 313 The NDSU Institutional Animal Care and Use Committee approved all animal 314 procedures in this study. Gravid uteri were obtained from crossbred Western Range (primarily 14 Page 15 of 40 315 Rambouillet, Targhee, and Columbia) ewes (n=5 to 8 per day) on days 16, 18, 20, 22, 24, 26, 28, 316 and 30 after mating (day of mating=day 0). At tissue collection for immunohistochemical 317 staining, specimen pins were inserted completely through the uterus and FM at the level of the 318 external intercornual bifurcation to maintain specimen morphology; cross sections of the entire 319 gravid uterus (approximately 0.5-cm thick) were obtained using a Stadie-Riggs microtome knife 320 followed by immersion in formalin or Carnoy’s solution and embedding in paraffin. For total 321 cellular RNA extraction, chorioallantoic FM were dissected from the area close to the embryo, 322 snap-frozen, and stored at -70 C. On days 20-30 of pregnancy, fetuses were separated from fetal 323 membranes and crown-rump length of each fetus was measured. The length of fetuses on days 16 324 and 18 was not determined due to the small fetal size (<2 mm) and tissue transparency. 325 Immunohistochemistry 326 Immunohistochemical procedures were used as described before (Grazul-Bilska et al. 327 2010). Paraffin-embedded uterine tissues containing FM were sectioned at 4 µm and mounted 328 onto slides. Sections were rinsed several times in PBS containing Triton-X100 (0.3%, v/v) and 329 then were treated for 20 min with blocking buffer [PBS containing normal horse serum (2%, 330 vol/vol)] followed by incubation with specific primary antibody for Ki67 (a marker of 331 proliferating cells; 1:500; mouse monoclonal; Vector Laboratories, Burlingame, CA, USA), 5mC 332 (a marker of global DNA methylation; 1:500; mouse monoclonal; Eurogentec North America, 333 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 Page 16 of 40 338 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 Page 19 of 40 407 408 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 References 424 Al-Hijji J, Andolf E, Laurini R & Batra S 2003 Nitric oxide synthase activity in human 425 trophoblast, term placenta and pregnant myometrium. Reproductive Biology and 426 Endocrinology 1 51. 427 Ariel I, Hochberg A & Shochina M 1998 Endothelial nitric oxide synthase immunoreactivity in 428 early gestation and in trophoblastic disease. Journal of Clinical Pathology 51 427-431. 19 Page 20 of 40 429 Arroyo JA & Winn VD 2008 Vasculogenesis and angiogenesis in the IUGR placenta. Seminars in Perinatology 32 172-177. 430 431 432 433 434 435 436 Beck S & Rakyan VK 2008 The methylome: approaches for global DNA methylation profiling. Trends in Genetics 24 231-237. Bird A 2002 DNA methylation patterns and epigenetic memory. Genes and Development 16 621. 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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.
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