Gene expression in the placenta: Maternal stress and epigenetic responses

Abstract

Successful placental development is crucial for optimal growth, development, maturation and survival of the embryo/fetus into adulthood. Numerous epidemiologic and experimental studies have demonstrated the profound influence of intrauterine environment on life, and the diseases to which one is subject as an adult. For the most part, these invidious influences, whether maternal hypoxia, protein or caloric deficiency or excess, and others, represent types of maternal stress. In the present review, we examine certain aspects of gene expression in the placenta as a consequence of maternal stressors. To examine these issues in a controlled manner, and in a species in which the genome has been sequenced, most of these reported studies have been performed in the mouse. Although each individual maternal stress is characterized by up- or down-regulation of specific genes in the placenta, functional analysis reveals some patterns of gene expression common to the several forms of stress. Of critical importance, these genes include those involved in DNA methylation and histone modification, cell cycle regulation, and related global pathways of great relevance to epigenesis and the developmental origins of adult health and disease.

Full text

Gene Expression in the Placenta:
Maternal Stress and Epigenetic Responses
Ciprian P. Gheorghe, Ravi Goyal, Ashwani Mittal, and Lawrence D. Longo
Center for Perinatal Biology, Departments of Physiology and Obstetrics and Gynecology, Loma
Linda University School of Medicine, Loma Linda, CA 92350
Keywords
placenta; gene regulation; microarray; DNA methylation; epigenetics
Introduction
In the Western world, cardiovascular disease, along with diabetes mellitus and its
complications, are leading causes of death. A number of important risk factors have been
associated with the virtual pandemic of these killers. These include smoking, sedentary
lifestyle, high body mass index, hypertension, and so forth. Nonetheless, many individuals
who develop cardiovascular and/or metabolic disease do not have these risk factors. Thus, it
is clear that as yet unrecognized and underappreciated factors must be considered in the genesis
of these disorders. In his monumental volume Stress, Hans Selye (1907-1982) observed that
stress to the organism, in essentially any of its forms -- dietary, environmental, disease, and
others -- could result in cellular, hormonal, and related damage, with the body mounting a
response he termed the “General-Adaptation-Syndrome” (Selye, 1950). Writing years before
the nuances of biochemical and molecular mechanisms were established, Selye envisioned an
orchestrated biological defensive response to such challenges. This concept is of special
relevance to the developing fetus, as during the course of gestation a number of stresses to the
mother are known to affect placental, as well as embryonic/fetal development, many with life-
long consequences.
Along this line, a factor that has received increasing attention is the idea of “programming”
during fetal life, often as a consequence of maternal stress. Special features of antenatal
programming include: critical periods of vulnerability, failure or unsatisfactory completion of
specific developmental milestones, association with functional defects, the permanent nature
of such sequelae, and so forth (Barker, 1989a; 1989b; 1992; 1994; 1995a; 1995b; 1998;
2004; Barker et al, 1995; Nijland et al, 2008). The concept of the developmental origins of
adult health and disease, first articulated by David J.P. Barker in the mid-1980s, “... suggested
that poor nutrition in early life increases susceptibility to the effects of an affluent diet” (Barker
& Osmond, 1986a). In a related analysis, these authors noted the high correlation of
cerebrovascular accidents in the 1970s, to increased infant mortality six decades earlier during
the years 1911-1914 (Barker & Osmond, 1987). In addition, in men born from 1911 to 1930,
Barker and his group have shown an inverse correlation between the weights both at birth and
Send Correspondence to: Lawrence D. Longo, M.D., FACOG, FRCOG, Director, Center for Perinatal Biology, Loma Linda University,
School of Medicine, Loma Linda, CA 92350, E-mail: llongo@llu.edu, Tel: 909 558 4325.
Additional Authors: Ciprian P. Gheorghe: cgheorghe@llu.edu
Ravi Goyal: rgoyal@llu.edu
Ashwani Mittal: asmittal@llu.edu
NIH Public Access
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at 1 year of age to coronary artery disease as adults (Barker et al, 1989a; 1989b). A subsequent
study disclosed a similar trend with birthweight among women (Osmond et al, 1993).
Since this concept first was proposed, supporting evidence has been provided by a series of
epidemiologic studies from a number of countries and cultures. These include studies
correlating adult mortality from acute myocardial infarction with high infant mortality rates in
a given population, follow-up studies correlating adult hypertension, coronary artery disease,
and type II diabetes with low birth weight, the relation of increased mortality from coronary
artery disease to low weight at 1 yr of age, and the relation of both newborn ponderal index
[weight (g) x 102/crown-heel length (cm)2] and placental-to-fetal weight ratio to hypertension
in the adult (see Barker, 2003, and Barker et al, 1995 for review). These associations are
independent of adult life style risk factors (Barker et al, 1993). Among a number of other
maternal stress-induced sequelae are those of immune dysfunction (Götz et al, 2007; Merlot
et al, 2008), cortisol secretion later in life (Reynolds et al, 2007; Tu et al, 2007), increased
incidence of schizophrenia (Hoek et al, 1996; Hulshoff et al, 2000; Susser & Lin, 1992), and
many others (Ham & Tronick, 2006). More recently, numerous studies in experimental animals
have demonstrated a relation between intrauterine fetal stress, particularly that of maternal food
deprivation and/or emotional stress, and adult disease (Gluckman et al, 2008; Green & Hanson,
2004; Hanson & Gluckman, 2005; Jansson & Powell, 2007). Among the major known
intrauterine stresses about which the effects on subsequent adult health are largely unknown,
are maternal hypoxia and dietary imbalance.
The placenta, a fetomaternal organ joining mother and offspring during pregnancy in mammals,
serves as an endocrine organ in the “maternal-placental-fetal” complex, in addition to its role
in the exchange of respiratory gases, a multitude of nutrients, an immunologic barrier, and
other functions. As has been recognized for many years, compromised placental function can
have both short- and long-term consequences for the developing conceptus. In the present
review, we examine the current state of knowledge of placental gene expression responses to
maternal stress such as hypoxia, protein deficiency, and caloric excess. For the most part these
studies are in rodents, however, when applicable, we also review those studies of placental
gene expression in the human. (We will not review thoroughly the field of antenatal origins of
adult health and disease, nor the role of environmental toxins in developmental disorders, as
these topics have been reviewed in extenso elsewhere). Importantly, beyond mere description,
we attempt to place these gene expression changes into a framework of the biochemical
pathways and molecular mechanisms, by which stresses to the maternal organism can result
in alterations of great biologic and epigenetic importance to the developing embryo and fetus.
Finally, we consider important issues for future investigation, i.e., questions that probe the
limits of our understanding.
Why Study Gene Expression in the Placenta?
Successful placental development is crucial for optimal growth, maturation, and survival of
the embryo/fetus. The placenta not only nurtures the fetus, but protects it from harmful waste
products by acting as an excretory route, and also presents an immunologic barrier between
the maternal and fetal circulatory beds. Although the nucleus of every cell in the body carries
a complete set of DNA, these cells differ in function with placental and embryological
development consisting of an elegantly orchestrated switching of genes on and off in the
transition from single fertilized cell to fully formed placenta and fetus. Deviation in the normal
gene expression pattern may lead to altered placental phenotype, as well as a modified
phenotype of the conceptus. This is evidenced by the numerous lethal embryonic null mutants
secondary to placental failure. The mouse has been employed as a useful model of placental
development. While the mouse placenta is not identical to its human counterpart, many studies
have shown that similar cell lineages are largely conserved, and similar genes direct placental
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development in both species. Placental cell lineages derive from trophoectoderm precursors.
The mural trophoectoderm differentiates into primary trophoblast giant cells, while the polar
trophoectoderm gives rise to the extraembryonic ectoderm and the ectoplacental cone. In many
mammals, including the mouse, the extraembryonic ectoderm forms the chorion that fuses with
the allantois, an outgrowth of extraembryonic mesoderm, at around embryonic day 8 (E8) to
form the placental labyrinthine layer. The spongiotrophoblast layer of the murine placenta
derives from ectoplacental precursor cells and forms the middle layer of the placenta, also
known as the junctional zone. The outermost placental cells are the trophoblast giant cell layer.
In addition to the primary trophoblast cells derived from the mural trophoectoderm, secondary
trophoblast giant cells are derived from the spongiotrophoblast. Later in placental development,
around E12.5, glycogen-filled trophoblast cells appear in the spongiotrophoblast layer.
Although their function is unclear, these cells express several important gene products, and
migrate into the decidua later in pregnancy. Several reviews have detailed placental cell
lineages, and some of the genes involved in their differentiation (Cross, 2005; Simmons &
Cross, 2005).
Recent studies reveal some of the fundamental mechanisms underlying placental development
(Cross et al, 2003; Daoud et al, 2005; Gheorghe et al, 2006; Hemberger, 2007; Sood et al,
2006; Tanaka et al, 2000). Numerous genes are required for proper development of the
placenta, and their number has increased greatly, in part, due to the discovery of numerous
lethal embryonic null mutants secondary to placental failure (Adams et al, 2000; Schorpp-
Kistner et al, 1999; Schreiber et al, 2000; Yamamoto et al, 1998). For example, the disruption
of many genes, including growth factors, transcription factors, extracellular matrix proteins,
and proteins involved in cell signaling, leads to embryonic lethality secondary to placental
failure (Rossant & Cross, 2001). In human trophoblast in vitro, several gene classes are strongly
up- and down-regulated in the course of differentiation (Aronow et al, 2001). Another study
compared differentially expressed genes between the murine placenta and the embryo itself at
E12.5 (Tanaka et al, 2000). Microarray analysis has provided insights into aspects of the genetic
mechanisms of development, cell growth both normal and abnormal, responses to stress, and
numerous other processes (Chu et al, 1998; Gasa et al, 2004; Iyer et al, 1999).
To What Extent is Placental Gene Expression Altered During Gestation?
The molecular basis of placental development remains incompletely understood. Recent
studies have begun to shed some light on this process, and numerous genes have been
demonstrated to be essential for the proper differentiation of placental cell lineages and fetal
survival. Unfortunately, the details of the interactions and effects of these genes are unclear.
In an effort to understand this process at a more fundamental level, we examined gene
expression patterns in the developing murine placenta at days E10.5 E12.5, E15.5, and E17.5,
testing the hypothesis that from E10.5 until E17.5, numerous placental genes are up- or down-
regulated to a significant degree, and that specific functional groups of genes are regulated at
the different developmental ages (Gheorghe et al, 2006). To examine gene clustering and
functional analysis of pathways, we focused on those genes most highly regulated by
development. At E10.5, several functional categories were over-represented, including genes
involved in angiogenesis and blood vessel development, morphogenesis, and organogenesis,
and genes involved in lipid metabolism and transport. At E12.5, over-represented gene
categories were involved in cell cycle control and RNA binding proteins. At E15.5, notably
over-represented were genes involved in cellular transport and cell growth and maintenance.
At E17.5, we noted the up-regulation of an over-abundance of genes involved in the regulation
of transcription and numerous proteins that localize to the nucleus.
This study identified several subsets of genes highly regulated during placental development.
Clustering according to their expression patterns, suggests that at crucial times during placental
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ontogeny particular subsets of diverse genes are induced or repressed in concert (Fig. 1). Genes
up-regulated early in placental development clearly underlie the rapid tissue growth, cell
proliferation, and vascular development occurring during this period. At E10.5, genes involved
in several key processes were strongly up-regulated. These include angiogenesis, lipid
metabolism, and cell cycle regulation. Genes such as ELK3, c-fos induced growth factor,
plasminogen (the precursor to angiostatin), serine (or cysteine) proteinase inhibitor clade F
member 1, all are involved in blood vessel morphogenesis, and were up-regulated at E10.5. At
both E10.5 and E12.5 cyclin, D1, cyclin E2, cyclin C, MAD 2, pleiotrophin, BRCA 2, which
are involved in cell cycle control also are up-regulated strongly. At E12.5, ribosomal genes
were notably up-regulated, as were several genes involved in lipid transport and metabolism
including: apolipoprotein B, apolipoprotein C-II, lysophospholipase, microsomal triglyceride
transfer protein and adiponectin receptor 1. As must be evident, lipid transport and metabolism
are important for the proper fetal development (Shekhawat et al, 2003) and disruption of lipid
transporters leads to embryonic lethality (Farese et al, 1996;Gimeno et al, 2003). Previous null
mutant experiments have identified several of these genes as embryonic lethal, further
confirming their importance in development. For instance, Cops2 mutants died soon after
implantation (Lykke-Anderson et al, 2003), Pten mutants died at E9.5 secondary to placental
failure (Yamamoto et al, 1998), and connexin 43 mutants died shortly after birth, due to cardiac
and vascular abnormalities (Reaume et al, 1995).
Genes such as growth hormone releasing hormone prolactin-like protein I, secretin, and
chorionic somatomammotropin hormone 2 also were upregulated during the course of placental
development. Recent studies in the sheep suggest that growth hormone releasing hormone
regulates the expression of both placental growth hormone and lactogen (Lacroix et al,
2002). Insulin-like growth factor II (IGF-II) and Insulin-like growth factor binding protein 2,
genes have been shown to be expressed in the placenta (Zollers et al, 2001), were up-regulated
from E10.5 to E12.5. Previous studies have shown that after E12.5 IGF-II is mainly produced
by trophoblast glycogen cells (Redline et al, 1993); however, in our study it was up-regulated
at E12.5 and later (Gheorghe et al, 2006). IGF-II also appears to have key functions in placental
transport and permeability (Sibley et al, 2004). A number of prolactin-like proteins have been
shown to be regulated with development, such as: prolactin-like protein C 1, prolactin-like
protein F, prolactin-like protein I, prolactin-like protein K. The prolactin gene family in the
mouse has at least 26 identified members (Wiemers et al, 2003), and several studies have shown
that in the placenta this gene family performs key reproductive and regulatory functions (Ain
et al, 2003; 2004). In the near-term human placenta, mRNA for a number of factors associated
with angiogenesis (vascular endothelial growth factor and annexin V) and homeostasis
(plasminogen activator factor, thrombomodulin, and others) are widely distributed (Chinni et
al, 2008). Circulating fetal fibrocytes, and perhaps other cells, also play a role in the
development of the placenta and the umbilical arteries and vein (Kim et al, 2008).
To What Extent is Placental Gene Expression Altered by Maternal Hypoxia?
As noted above, a number of stressors can lead to altered placental and fetal growth and
development. Of great importance in this regard, is the less than optimal supply of oxygen
(O2), e.g., hypoxia. Hypoxia has been identified as a major stressor in development, and is
believed to be a contributing cause to placental pathology such as that associated with
preeclampsia (Austgulen et al, 2004; Challier & Uzan, 2003). Hypoxia can lead to low birth
weight and intrauterine growth restriction (IUGR) and disease of the newborn such as persistent
pulmonary hypertension (Zamudio, 2003). Little is known, however, about the adaptive
mechanisms involved in the placental responses to suboptimal oxygen availability. Several
studies have attempted to harness the power of microarray and proteomic analysis to elucidate
responses to hypoxia in cultured human cytotrophoblasts (Hoang et al, 2001), and in rat
embryos and placentas (Huang et al, 2004). As with other cell types, oxygen is a critical
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regulator of the normal trophoblast cell development, which undergo differentiation and/or
proliferation in response to varying O2 concentrations. Acting through aryl receptor nuclear
transporter and hypoxia-inducible factor 1α (HIF-1α), oxygen regulates placental cell
phenotypes and gene expression (Adelman et al, 2000). Hypoxic stress can lead to placental
cell death and dysregulation of vasculogenesis, which negatively affects the development of
the placental vascular bed (Kingdom et al, 2000). In response to exposure to high altitude,
long-term hypoxia, the ovine placentomes undergo significant structural changes (Penninga &
Longo, 1998), and the vasculature displays significant increases in capillary density, vessel
tortuosity, and a decrease in diffusion distance from maternal to fetal blood (Krebs et al,
1997). The human placenta also shows significant morphologic and morphometric changes in
response to high altitude hypoxia (Zhang et al, 2002). Nonetheless, essentially nothing is
known about the molecular basis of these changes.
To understand in greater detail the role of hypoxia in placental gene expression, we tested the
hypothesis that hypoxia-induced altered placental morphology is accompanied by significant
changes in expression profiles. We compared gene expression levels between the normal
murine placenta at E17.5, and that from dams exposed to 0.5 atmosphere hypoxia (10.5%
O2) for 48 hours from E15.5 to E17.5. In response to this stress, some of the most highly up-
regulated genes were those related to metabolism (alpha-keto reductase family-1 member 7,
mitochondrial solute carrying protein, acetyl-coenzyme A synthetase 2, and NADPH oxidase
4), oxygen transport (erythroid associated factor, hemoglobin Y beta-like embryonic chain,
erythrocyte protein band 4.2), proteolysis (cathepsin G, kallikrein 4, dipeptidase 1, serine
protease 32), cell death (Bcl-like 2, perforin 1, glutathione peroxidase 1), and metabolism of
reactive oxygen species (Gheorghe et al, 2007). Of particular note, several genes related to
DNA methylation and epigenetic control were up-regulated (DNA methyltransferase 3B,
methyl-CpG binding domain protein 1, RNA binding motif protein 3). Of the chromosomal
distribution of genes up-regulated by hypoxia, we noted an over-abundance of genes from
chromosome 14 (9.5% of the regulated genes as compared to 3.5% of genes on the array).
Many of these correspond to the granzyme family of proteases.
Several of these genes have been noted previously to be regulated by hypoxia and/or involved
in angiogenesis and metabolic responses. For instance, NAPDH oxidase 4 has been identified
as a potential O2 sensor and regulator of HIF-1α (Zhu et al, 2002). Ferrocheloelastase is
involved in heme metabolism, and has been shown to be up-regulated by hypoxia (Liu et al,
2004). Aminolevulinic acid synthase 2, glutathione peroxidase 1, and peroxiredoxin 2 are
involved in the metabolism of reactive oxygen species; their activity, and expression levels
have been identified as regulated by hypoxia (Abu-Farha et al, 2005; Mysore, 2005).
Glycophorin A is involved in erythroid differentiation, and is regulated by erythropoietin
(Gubin et al, 1999). Lactotransferrin is an antioxidant involved in iron metabolism and in
scavenging of free radicals under hypoxic conditions (Morris et al, 1995). As noted above,
hypoxia upregulated several members of the granzyme gene family. These are serine proteases
expressed by lymphocytes, and thought to be involved in T-cell-mediated cytotoxicity.
Granzymes are released along with perforin by natural killer (NK) cells and cytotoxic T
lymphocytes, and trigger apoptosis in target cells through several mechanisms. A number of
studies have demonstrated placental expression of granzymes, and they may play a broader
role in placental development (Allen et al, 1998; Hirst et al, 2001). We also observed an up-
regulation of perforin, which suggests that in response to hypoxia a greater number of NK cells,
which have been shown to mediate a number of important functions, invade the placenta
(Parham, 2004). Thus, the data suggest that granzymes not only play a role in normal placental
development, but also are involved in hypoxia-mediated responses.
Again, of particular note in our study was the significant up-regulation of genes involved in
DNA methylation and epigenetic control: DNA methyltransferase 3b and methyl CpG binding
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domain protein 1 (Gheorghe et al, 2007). As noted below, epigenetic modification is an
important mechanism of gene expression regulation that does not involve modification of the
DNA sequence, but rather DNA methylation and histone modification. These changes in
expression patterns may be of importance in development, genomic imprinting, and the
development of cancer (Turek-Plewa & Jagodzinski, 2005). The generation of reactive oxygen
species (ROS) and the manipulation of glutathione metabolism have been shown to regulate a
number of cellular processes, including DNA methylation (Fratelli et al, 2005). As depicted
in Fig. 2, this suggests complex genetic regulation that commences with hypoxia and leads to
alternations that result in long-term programming. In turn, this activates the DNA methylation
machinery, and ultimately leads to long-term changes in the organism unrelated to modification
of the nucleotide sequence. A key to unraveling this mechanism will be the identification of
the targets for altered methylation and subsequent long-term down-regulation of transcription.
Among the down-regulated genes, most notable were several transcription factors
(transformation-related protein 63, doublesex- and mab-3-related transcription factor 1, glial
cells missing homolog 1, zinc finger, imprinted 1), cell cycle (cyclin M2), cell structure (keratin
complex 2 basic, gene 8, procollagen C-proteinase enhancer protein, fibromodulin). We have
presented a list of the genes up- or down-regulated and their known function, and verified the
regulation of several of these genes using real time PCR (Gheorghe et al, 2007). In response
to hypoxia, placental morphology also was altered significantly e.g. having a greater vascular
density and containing many more red blood cells.
Several other studies have examined gene expression changes in response to hypoxia at the
global level. One study catalogued the hypoxic-induced responses in the rat embryo to hypoxic
exposure for both 24 hours and 11 days. Glycolysis-related genes, calcium homeostasis-related
genes, and inflammatory genes (particularly as related to oxidative stress) were up-regulated,
while cell growth-related genes were down-regulated (Huang et al2004). Other studies also
have examined human trophoblast responses in-vitro to low oxygen tension. Observed were
up-regulation of antioxidants (superoxide dismutase) and glycolysis-related genes, and the
upregulation of glutathione-S-transferase (Nelson et al, 2003; Roh et al, 2005). In addition,
gene expression changes in placentas from pregnancies complicated by pre-eclampsia and
IUGR have been catalogued. In particular, H4 histone was down-regulated in women with
severe pre-eclampsia (Chen et al, 2006; Soleymanloo et al, 2005), and up-regulation of
glutathione-S-transferase was observed in human placentas from women at high altitude
(3,100m) (Chen et al, 2006; Roh et al, 2005). These studies have highlighted the diverse manner
in which placental cells respond to hypoxic stress.
In the placenta of patients that experienced chronic hypoxic ischemia, mRNA levels of both
leptin and insulin-like growth factor (IGF)-1 were upregulated significantly (Trollmann et al,
2007). In near-term placental explants cultured in 1% O2, expression of the tumor suppressor
protein p53, that promotes cell cycle arrest or apoptosis, was significantly elevated (Heazell
et al, 2008). Exposure of human placental villous explants to 3% O2 for 48 h, resulted in
significant increase in endoglin, a co-receptor for transforming growth factor (TGF-β3)
pathway (Yinon et al, 2008). In the placentae of patients with severe preeclampsia at 31±2
weeks gestation, mRNA of the antioxidant protein glutathione reductase was reduced
significantly, while that for thioredoxin peroxidase was increased (Vanderlelie et al, 2008).
This suggests that oxidative stress may play a key role in the pathophysiology of the placentae
in cases of preeclampsia.
In summary, the murine placenta appears to respond to hypoxia through several adaptive
mechanisms. These include up-regulation of genes associated with erythropoiesis, increases
in heme and iron metabolism, and in genes involved in proteolysis and peptidolysis. These
varied responses suggest that the placenta responds by increasing its oxygen carrying capacity,
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increasing metabolic and antioxidant responses, and initiating tissue growth, turnover, and
remodeling. Studies in both the human (Roh et al, 2005) and sheep (Krebs et al, 1997; Penninga
& Longo, 1998) have demonstrated that the placenta undergoes multiple morphological and
genetic changes in response to prolonged hypoxia. Hypoxic insults secondary to preeclampsia,
maternal smoking, or exposure to high altitude can contribute to placental insufficiency and
may lead to intrauterine growth restriction (Jones & Fox, 1980; Levi & Nelson, 2000;
Reshetnikova et al, 1994; Spira et al, 1997). The exact mechanisms of these changes are not
understood. Extracellular matrix remodeling, the modulation of apoptosis, altered cellular
metabolism, and epigenetic changes all appear to be crucial steps in the physiological
adaptations of the placenta to hypoxia. It is hoped that these, and other studies, will provide
insights at the molecular level into these mechanisms and important clinical problems.
To What Extent is Placental Gene Expression Altered by Maternal Protein Restriction?
Several lines of evidence demonstrate that nutritional deprivation of the pregnant mother may
have deleterious consequences for the progeny. For instance, a shocking “experiment” in
humans was that during World War II of the “Hunger Winter” in Amsterdam and Western
Holland from November 1944 until the Allied victory in May 1945. This tragedy provides
useful lessons on the effects of caloric restriction/malnutrition on fetal development and disease
prevalence in adulthood. During this seven month period, the caloric ration fell from ∼2400
to 400-800 calories per day, less than 25% of the recommended intake for adults. Although
children, and to some extent pregnant and lactating women, received extra rations during the
early part of this disastrous famine, they too suffered severe dietary deficiency (Roseboom et
al, 2001a). In essence, upon reaching adulthood, the infants that were small at birth had
significantly greater prevalence of cardiovascular disease, type II diabetes (Kyle & Prichard,
2006; Painter et al, 2005a; Roseboom et al, 2001a; 2001b; Stein & Susser, 1975; Stein et al,
2004), and mood and personality disorders (Godfrey, 1998). Those fetuses exposed to maternal
caloric restriction in mid-gestation had a much greater incidence of pulmonary disease,
including bronchitis (Lopuhaa et al, 2000), and renal disease as evidenced by microalbuminuria
(Painter et al, 2005b). Females who were conceived during the famine also had a much higher
prevalence of obesity as adults (Ravelli et al, 1999), and both males and females showed
atherogenic lipid profiles (Roseboom et al, 2000). Concommently during WWII, the people
of St. Petersburg and surrounding area of Russia were subjected to severe dietary restrictions
due to interdiction of food supplies by the German army. The children born under these
conditions were not only small for gestational age, but also developed health problems later in
life (Neugebauer et al, 1999). However, records of the long-term sequelae of these individuals
are not as clear as those in Holland (Lind, 1984; Ravelli et al, 1976). Importantly, the
mechanisms of these in utero “programming” effects are unknown.
Epidemiologic data on the role of maternal nutrition in determining the long-term health of
offspring derives largely from the studies of Barker and colleagues (Barker, 1995b; 2003;
Barker & Clark, 1997; Barker & Osmond, 1986a; 1986b). Studies in several countries have
correlated maternal dietary deficiencies that result in the newborn infant being small for
gestational age, or growth restricted, with the prevalence of cardiovascular disease (Barker &
Osmond, 1986b; Barker et al, 1989a), type 2 diabetes (Hales et al, 1991; Ravelli et al, 1998),
and numerous other conditions in the adult. Maternal nutritional deprivation may influence
placental growth and morphology, alter the hormonal milieu of the developing fetus, and cause
subsequent cardiovascular, hormonal and behavioral consequences in the adult (Barker,
1992; 1994; 2003; Barker & Osmond, 1986a; 1986b; Barker et al, 1989; Gluckman et al,
2008).
The epidemiologic observations made in human subjects have been confirmed in animal
models, and have led to speculation regarding the cellular mechanisms of changes in the
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placenta, and their effects on the developing fetus (Armitage et al, 2004; Hoet & Hanson,
1999). An important question is the extent to which these observed effects result from an overall
caloric restriction, as opposed to a qualitative component in the diet that triggers the responses.
Evidence from animal studies points to protein deprivation as a major factor in these defects.
For example in the rat, the growth reducing effects of a low calorie diet can be reversed only
by a dietary increase in protein levels, while vitamin supplements and caloric increases through
carbohydrates did not reverse the effects observed (Hsueh et al, 1967). Other studies have
revealed that dietary amino acid balance is a key mediator of some of the cardiovascular and
metabolic effects observed in response to protein deprivation (Boujendar, 2003). Overall, the
several studies indicate that nutritional deprivation, and protein restriction in particular, can
have immediate deleterious effects on the placenta and the fetus, and may result in long-term
sequelae that extend into adulthood.
In a recent study in the mouse, we tested the hypothesis that moderate maternal protein
deprivation would alter gene expression patterns in the placenta. We compared gene expression
levels between normal placentas at E17.5, and those from pregnancies in which the mothers
were exposed to seven days of protein deprivation (i.e., 10% protein by weight versus the 20%
of normal chow) from E10.5 to E17.5. Of particular note, a number of genes involved in the
p53 oncogene pathway were up-regulated. In addition to p53 itself, its positive regulators Zmis,
Jmy, and Hipk2, as well as genes activated by p53 (Inpp5d, Cebpa), were induced (Figure 3A).
These p53 pathway proteins are important regulators of cell growth and proliferation. This
pathway serves as a G1 checkpoint, and arrests growth and/or induces apoptosis in response
to cellular damage. Mutations in the p53 gene have been implicated in a number of cancers
and other pathological processes (Ryan et al, 2001). Hipk2, an upstream regulator of p53,
activates its transcriptional activity and pro-apoptotic activities through phosphorylation at Ser
46 (Hoffman et al, 2002). Cebpa, is a transcription factor induced by p53, and mediates some
of the downstream effects of p53 activation (Yoon & Smart, 2004). Among the gene ontology
classes most over-represented in the up-regulated group, we noted the mitogen-activated
protein kinase pathway, regulators of apoptosis (Bcl2-like 2, p53, endophilin, Fas-activated
serine/threonine kinase), negative regulators of cell growth (farnesyltransferase CAAX box
beta, cadherin 5, CCAAT/enhancer binding protein (C/EBP) alpha, inositol polyphosphate-5-
phosphatase D, p53), and negative regulators of cellular metabolism (nuclear receptor co-
repressor 2, histone deacetylase 7A, SPEN homolog, transcriptional regulator). Acting in
concert, activation of these genes could result in growth restriction during pregnancy (Fig. 3B).
Among down-regulated genes, particularly striking were those related to nucleotide
metabolism. For selected genes, we confirmed these results using qRT-PCR.
Another potentially important finding, is that protein deprivation altered the expression of
several genes involved in DNA methylation, histone acetylation, and epigenetic regulation of
gene expression. The expression levels of histone deacetylase 7A and methionine
adenosyltransferase II, alpha were elevated several fold. Histone acetylation triggers changes
in chromatin structure, and regulates transcriptional availability of genes. In turn, histone
deacetylation increases histone affinity for DNA, thereby repressing transcription (Bulger,
2005). Methionine adenosyltransferase II alpha synthesizes AdoMet the direct precursor used
for DNA methylation by methyltransferases (Mao et al, 1998). Histone 2 (h3c2) is down-
regulated, along with Mcm6 and telomeric repeat binding factor 1. These proteins contribute
to DNA replication, stability, and structure (O’Connor et al, 2004; Yu et al, 2004). In the
placenta of patients with preeclampsia, phosphorylation of extracellular signal-regulated
kinase1/2 was significantly less frequent in the invasive trophoblasts, as compared to control
(Moon et al, 2008). In another study, in placentas of preeclamptic patients, contrary to
expectations, polymorphisms of several enzymes associated with oxidative stress (copper/zinc
superoxide dismutase, manganese superoxide dismutase, glutathione-S-transferase, and
others) did not differ from controls (Zhang et al, 2008). In contrast, in the placentas of patients
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with HELLP (Hemolysis, Elevated Liver Enzymes, Low Platelets) syndrome, genes encoding
vascular endothelial growth factor receptor, leptin, and several other proteins, were up-
regulated, as compared with the placenta of both normal control patients and those with
preeclampsia (Buimer et al, 2008).
Because the various tissues and organ systems undergo critical, often brief, periods of growth
and development during fetal life (Winick & Noble, 1966), “programming” as a consequence
of maternal stress should not be unexpected, with insults to the developing organism having
consequences later in life’s course. Studies in ruminants also have demonstrated that under-
nutrition can have profound consequences for the fetus. In sheep, restricted maternal nutrition
in early to mid-gestation was associated with an increase in placental weight, an increase in
crown-rump length, and lower fetal to placental weight ratios (Heasman et al, 1998). Maternal
under-nutrition also altered cardiovascular homeostatic regulation by the renin-angiotensin
system, and exposed the lambs to higher levels of glucocorticoids (Edwards et al, 1999), and
development of hypertension (Dodic et al, 2001). Protein restriction in bovines also resulted
in an increase in placental weight and altered placental morphology (Perry et al, 1999).
Studies in rodents have shown similar effects. In rats, maternal protein restriction triggers
hypertension in the pups in adulthood (Langley & Jackson, 1994), probably by augmentation
of the pups’ renin-angiotensin system. In the spontaneously hypertensive rat placenta, several
proteins, angiotensin receptor type I and inducible nitric oxide synthase (NOS), were up-
regulated, while angiotensin converting enzyme and peroxisome proliferator-activated
receptors alpha and gamma were downregulated (Raso et al, 2008). An alteration of placental
glucocorticoid (GC) metabolism also was observed in placentae of rats fed a protein restricted
diet, namely the activity of 11β-hydoxysteroid dehydrogenase that metabolizes
glucocorticoids. This placental enzyme, which normally protects the pups from maternal
glucocorticoid excess, was reduced in protein restricted rats (Langley-Evans et al, 1996), thus
exposing the fetus to abnormally high GC concentrations. Elevated circulating cortisol
concentrations, with modified responsiveness of the hypothalamic-pituitary-adrenal axis, and
elevated mean arterial blood pressure with increased left ventricular wall thickness and mass,
also were observed in guinea pigs in which the dam received only 70% of normal chow during
either the first or second half of gestation. Some of these changes persisted in the F2 generation
(Bertram et al, 2008). Another hormonal alteration in nutritionally deprived rat pups, was an
increase in somatostatin expression in the periventricular nucleus. This led to much lower levels
of growth hormone, and had deleterious effects on the growth of the pups post-partum
(Huizinga et al, 2000). Fetal undernourishment also led to neuronal sequelae. The facial motor
nucleus in pups was under-developed, resulting in decrease in the ability of pups to suckle and
chew (Perez-Torrero et al, 2001). These observations also may relate to the epidemiologic
findings, noted above, that abnormal antenatal nutrition may be associated with the
development of schizophrenia and other mental illness.
In several animal models, in addition to the potential deleterious effects referenced above, a
positive aspect of nutritional deprivation in the adult is that of prolonged lifespan and reduced
cancer rates. A proposed mechanism for these benefits is that nutritional restriction without
severe malnutrition inhibits cellular proliferation and induces apoptosis. This effect has been
shown in mice lacking p53, in which -/- and +/- mutants have lowered spontaneous cancer
rates when fed a calorically reduced, but otherwise complete, diet (Hursting et al, 2004). In
the adult and aging animal, nutritional restriction has been shown to have beneficial effects
that increased life span (Nikolich-Zugich & Messaoudi, 2005). A different picture has emerged
in the fetus, however. As discussed above, caloric and protein deprivation have been shown to
trigger fetal programming of adult disease, and lead to an increased prevalence of metabolic
disorders in adulthood (Barker, 1995b; 1998; Barker & Clark, 1997). In the developing fetus,
numerous animal studies have shown negative long-term effects of caloric and protein
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deprivation on the cardiovascular, renal, nervous system and metabolism (for review see
McMillan & Robinson, 2005). A different form of nutritional compromise, that of placental
restriction in sheep by removal of the endometrial caruncles in the nonpregnant ewe prior to
mating, alters the expression of a number of genes associated with adipogenesis in adipose
tissue of the fetus (Duffield et al, 2008). These findings emphasize the interrelation of placental
development and its gene expression, to development of the fetus and its repertoire of gene
expression.
To What Extent is Placental Gene Expression Altered by Maternal Caloric Excess?
Because maternal obesity poses an increased risk to the fetus during pregnancy, and has long-
term consequences for the progeny, we tested the hypothesis that maternal caloric excess effects
growth-related gene expression changes in the placenta. We fed female C57BL/65 mice a
hypercaloric diet (20% fat, 38% sugar) or standard chow for six weeks prior to mating and
throughout pregnancy. Near-term (E18), the dams were euthanized. We measured gene
expression changes in the placenta, and performed pathway analysis on regulated genes.
Maternal overfeeding was associated with a two-fold increase in body fat mass, with several
genes related to obesity, diabetes, DNA methylation, and the transforming growth factor-beta
(TGF-β) pathway being differentially expressed (Poston et al, in preparation). The TGF-β
superfamily comprises ∼30 growth and differential factors, including several TGF-βs, activins,
inhibins, and other growth and cell cycle control factors (Goumans & Mummery, 2000; Kitisin
et al, 2007; Massague et al, 2000; Roberts & Mishra, 2005; Roberts & Wakefield, 2003). Thus,
our findings may have important implications for placental growth and epigenetic regulation.
In other studies in mice, the chow was supplemented with methyl supplements (Wolff et al,
1998) or folic acid, vitamin B-12, choline, and betaine to enhance metabolism of cellular methyl
donors (S-adenosylmethionine) (Waterland & Jirtle, 2003). These interventions resulted in
altered coat color phenotype with concomitant increase in DNA methylation at the Avy locus.
Conversely, in mice fed a methyl-donor-deficient diet that lacked folic acid, vitamin B-12, and
choline the imprinted Igf2 gene was down-regulated with altered DNA methylation (Waterland
et al, 2006a). Human studies also have demonstrated effects in the placenta on maternal dietary
supplementation (Rush et al, 1984).
What is the Role Epigenetics in Placental Gene Expression?
During the course of life and reproduction, cells store information that has been handed down
from their ancestors, and that will be transmitted to their descendents. For the most part, this
“memory” is encoded in the sequence of nucleic acids that comprise the DNA of the genome,
the genotype or entire compliment of genes that provides the stability and accurate heritability
from generation to generation. Much traditional research has explored the combined effects of
genetics and the environment in germline mutations of the coding and promoter regions of
genes. In addition, cells can inherit and transmit information that is not part of the genomic
sequence. This “epigenetic” [from Greek, upon, over, or beyond conventional genetic], cellular
memory involves the heritable transmission of gene expression patterns that persist through
cell division, but do not involve an alteration in DNA sequence. Epigenetic processes act in a
cell specific, temporally-regulated manner to direct development, differentiation,
organogensis, and related processes. Some have compared epigenetic mechanisms to the
software to orchestrate and/or modulate the DNA hardware. One major class of epigenetic
mechanisms termed “cytoplasmic”, is determined by cis-acting factors associated with DNA
methylation and/or histone modification by acetylation/methylation/phosphorylation. DNA
with accompanying histones are packaged in nucleosomes, the core of which contains an
octamere of histone proteins. Four basic forms of histones (H2A, H2B, H3, and H4, as well as
minor variants), are encircled by 146 base pairs of DNA (Finch et al, 1977); a fifth histone,
H1, serves as a linker protein (Bernstein et al, 2007). The histone modifications noted above,
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and DNA methylation, confer a great increase in the regulatory capacity of each nucleosome,
allowing specific functions such as DNA repair and gene activation to be modulated in the
appropriate manner (Sarma & Reinberg, 2005). Enzymes critically associated with these
nucleosomal modifications include: DNA methyltransferases (DMT), histone
acetyltransferases (HAT), histone methyltransferase (HMT), histone deacetylases (HDAC),
histone demethylases (HDM), and others (Dodd et al, 2007; Klose et al, 2006). It is by these
nucleosomal modifications, with their influence on proximate genes, that genes may be
regulated to affect phenotype by activity, chromatin structure, dosage compensation, and
epigenetic memory, without changes in the nucleic acid code per se (Martin & Zhang, 2005;
Wolffe & Matzke, 1999).
Epigenetic changes play a key role in normal cellular function, as well as the development and
differentiation of various cell types (Drake & Walker, 2004; Monk, 1998; Rahnama et al,
2006; Reik, 2007). Examples include X-chromosome inactivation in female mammals, and
genomic imprinting in which one parental allele is altered resulting in parent-of-origin, or
random modification of gene transcription (Willard et al, 1993). The epigenetic state can be
disrupted by maternal environmental influences such as hypoxia, protein deprivation, caloric
excess, and so forth which alter DNA methylation or modify histones. Also importantly, a wide
variety of environmental toxins, including low dose radiation and psychological stress, have
been demonstrated to be important in epigenetic mechanisms (Dolinoy et al, 2007; Feinberg,
2007; Hertz-Piccioto et al, 2008; Jirtle & Skinner, 2007; Pryce et al, 2002; Szyf et al, 2007).
Increasingly, epigenetic changes are being recognized to be of importance in ageing, and the
development of cancer and other diseases. Despite the general understanding that DNA and/
or histone modifications constitute a major factor in the pathogenesis of epigenesis, little is
known of the molecular mechanisms whereby these chemical reactions/changes are regulated,
and/or how they are transmitted between generations (Bird, 2007).
From an historical context, epigenetics has several facets. For the pioneer Edinburgh geneticist
Conrad Hal Waddington (1905-1975), who coined the term, epigenetics was the study of how
phenotypes arise from genotypes during development (Waddington, 1939; 1940; 1942;
1957). Epigenetics later was defined as heritable changes in gene expression not due to any
alteration in DNA sequence (Holliday, 1987). In the mid-1970s, the concept of covalent
chemical DNA modifications, including methylation was proposed to account for this
phenomenon (Holliday & Pugh, 1975; Riggs, 1975). More recently, others have defined
epigenesis as the study of mitotically and/or meiotically heritable changes in gene function
without a change in DNA sequence (Dolinoy et al, 2007; Russo et al, 1996). As defined by
Adrian Bird, epigenetics is “the structural adaptation of chromosomal regions so as to register,
signal or perpetuate altered activity states” (Bird, 2007). The latter a definition focuses on
chromosomes and genes, including those aspects such as DNA repair, cell-cycle phases, and
those stable changes maintained from generation to generation.
In terms of perspective, epigenetics has a history that antedates understanding the genome and
its regulation. The French biologist/zoologist and comparative anatomist who contributed
greatly to classification of life forms, Jean-Baptiste Pierre Antoine de Mont, Chevalier de
Lamarck (1774-1829), noted that organisms may inherit traits acquired during their parent’s
lifetime. Although discredited by many, in part, because of his view that simple life forms arose
from dead matter by spontaneous generation to become more complex as they were
transformed into new species, Lamarck held that organisms inherit characteristics acquired
during their parent’s lifetimes, evolving in a constant process of striving toward greater
complexity and “perfection” (Lamarck, 1801; 1809; 1815-1822). Lamarck’s “First Law ...”
stated that a change in the environment alters the needs of organisms in that environment,
resulting in a change in behavior. Such behavioral alteration leads to greater or lesser use of a
given structure with resultant increase or decrease in the size of that structure or organ. His
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“Second Law” stated that all such changes were heritable as a result (For instance, that a
giraffe’s neck elongated as it ate from the highest leaves on a tree, and this feature would be
seen in the next generation) (Haig, 2006; West-Eberhard, 2007). Of course, neo-Lamarckian
biologists soundly reject such an idea. In more contemporary times, the views of the Russian
biologist-agronomist, Trofim Denisovich Lysenko (1898-1976), gained considerable press for
the agricultural “revolution” he promoted, in concert with Soviet collectivization policies. In
essence, Lysenko held that acquired characteristics of a plant (or other organism) could be
inherited by succeeding generations. An ideological-political creation, Lysenkoism held the
study of classic genetics to be “bourgeois” or “fascist” pseudoscience. Lysenkoism invoked
by biological determinists, as with the eugenics and scientific racism adopted by social
constructivists, may be seen as the extremes to which political dogma can use science in
promoting its propaganda (Soyfer, 2001).
As noted, environmental influences may have profound effects on gene regulation. This is
clearly evident in the cells of multicellular organisms; although being genetically
homogeneous, they are structurally and functionally heterogenesis. Many of these differences
in gene expression arise during development and are retained through mitosis. Such stable,
epigenetic changes, although heritable in the short term, are not a consequence of DNA
mutation. Rather, as recent studies are demonstrating, to a great degree epigenesis appears to
be a consequence of DNA methylation and histone modification. The term “epigenomics” has
been applied to the study of altered chromatin structure, such as complex folding, altered
nucleosome configuration, and related phenomena (Murrell et al, 2005). Importantly, several
lines of evidence indicate that, in addition to maternal to fetal transfer, epigenetic modifications
may be inherited across generations (Anway et al, 2005; 2006; Crews et al, 2007; Lane et al,
2003; Morgan et al, 1999; Pembray et al, 2006; Rakyan et al, 2003).
What are the Roles of DNA Methylation and Histone Modification in Placental Gene
Expression?
As noted above, both DNA methylation and histone modification play important roles in
development. These changes also may be important aspects of the ageing process and the
development of cancer. In this review, we concentrate on the epigenetic influences of maternal
diet, hypoxia, and related stress as observed in the placenta and fetal tissues, and that may have
long-term consequences in the fetal origins of adult health and disease.
As with most phenomena of biology and life, the molecular mechanisms whereby genes are
repressed or active in a stable manner are exceedingly complex. The best studied of these
epigenetic modifications is that of DNA methylation, which first was suggested in 1975 by
two groups (Holliday & Pugh, 1975; Riggs, 1975). This post-replication, covalent methylation
occurs predominantly in repetitive genomic regions, on the 5′-carbon of cysteine residues that
are followed by a guanine residue, i.e., “CpG methylation” which induces gene repression or
“a silent chromatin state”, (The “p” in CpG refers to the phosphodiester bond between cytosine
and guanine). Occurring at or around promoter regions, forming CpG islands, this can occur
directly by inhibiting the binding of specific transcription factors, and indirectly by recruiting
methyl-CPG binding proteins, with their associated repressive chromatin-remodeling activities
(Razin & Riggs, 1980). A seeming paradox in this scenario, is that methylation of some specific
DNA sequences may permit expression of neighboring genes. Both intrinsic factors and
environmental/nutritional factors can determine the activity of methyltransferases upon which
DNA methylation are dependent (Bestor, 2000). Because of the requirement for a high DNA
synthesis rate during both gametogenesis and early embryogenesis, considerable activity in
DNA methylation/demethylation patterning occurs during both this period of development, a
time during which the cells are vulnerable to abnormal environmental factors. Nonetheless,
nuances of molecular regulation of DNA methylation and histone modification during
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embryogenesis are beyond the scope of this synopsis, and a number of reviews on this topic
are available (Bird, 2007c; Jaenisch, 1997; Jaenisch & Bird, 2003; Jones & Takai, 2001; Santos
et al, 2002).
During the course of mammalian development, a wave of DNA demethylation occurs during
cleavage, followed by genome-wide de novo methylation following implantation (Jaenish,
1997). Although the male genome is widely demethylated shortly after fertilization (Mayer et
al, 2000; Oswald, 2000), the maternal genome is only partly demethylated with subsequent
cleavage divisions (Li, 2002). In the gastrulating embryo, the extent of methylation is high,
decreasing in various tissues during the course of differentiation (Ehrlich et al, 1982). For the
developing embryo and fetus, the methylation/demethylation patterns while being of great
significance, are enormously complex.
Additionally, gene expression is determined by the biochemical organization of the histones
in the nucleosomes around which the DNA is wrapped. Several post-translational covalent
modifications occur on the amino acids that constitute the histone N-terminal tails that modify
their interaction with DNA and/or other nuclear proteins. Acetylation, methylation,
phosphorylation and/or ubiquitination alone, or in combination play a key role in the regulation
by repression or expression of contiguous genes (Jenuwein & Allis, 2001; Strahl & Allis,
2000; Turner, 2000). Again, the regulation of histone modification by acetylation and/or
methylation is highly complex, has been shown to be specific for essentially every cell type,
and may act with DNA methylation to constitute a system of cellular memory (Bird, 2007).
The combination of the several epigenetic modifications of genes as well as non-coding
sequences, the so-called “epigenome” or “epigenotype”, determine the extent to which a given
gene is maintained repressed or active, and influences the phenotype at birth.
What is the Role of MicroRNAs in Placental Gene Expression?
MicroRNAs (miRNA) have emerged as important players in DNA methylation and post-
transcriptional gene regulation (Lujambio et al, 2007; Saito et al, 2006). These are subtypes
of small, non-coding RNA, which are 21-25 nucleotides in length. These miRNAs are capable
of base pairing with mRNA, and fine-tuning gene expression during development and
differentiation, by suppressing their expression in sequence specific manner. Following the
discovery of first miRNA “lin4” in 1993, as a small temporal RNA (Lee et al, 1993), there has
been enormous growth in this family, and identification of their targets. Although miRNAs are
similar to small interfering RNA (siRNA) in their generation pathway and molecular
characteristics, unlike siRNA, miRNA this does not degrade the target mRNA. Rather, they
target the 3′ untranslated regions of mRNAs with which they share partial sequence
complementarily, thereby silencing post-transcriptional gene translation. In this way, the
biological system increases or decreases miRNA production to up- or down-regulate gene
expression according to the developmental need, producing desired morphologic and
physiological changes. Moreover, placental miRNA (miR-141, miR-149, miR-229-5p, and
miR135b) are secreted in maternal plasma, and their concentration decreases significantly after
parturition (Chim et al, 2008). This suggests that placental miRNA, in addition to regulating
gene expression in placenta, may be playing an important role in maternal conditions with
obscure etiology, such as preeclampsia or related hypertensive disorders. Studies reveal
differential expression of miRNA (miR-210 and miR-182) in placenta from patients with
preeclampsia and with small for gestational age newborn infants (Pineles et al, 2007). As must
be evident, additional studies will be vital to examine and understand the complexity of
placental genetic regulation, and their contribution to fetal and maternal health and disease.
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What are the Human Correlates?
In several human population studies, it has been reported that the nutritional state of individuals
may have phenotypic consequences for their grandchildren (Kaati et al, 2002; Lumley, 1992).
An example of the role of diet in progeny DNA methylation status and phenotype is evident
in patients with hyper-homocysteinemia (Ingrosso et al, 2003). This disorder is characterized
by excess cellular adenosylhomocysteine, a potent inhibitor of S-adenosylmethionine-
dependent methyltransferases. This suggests the possibility of significantly altered DNA
methylation. In these patients, dietary supplementation with folate restored global methylation
levels, as well as that of the imprinted IGF2-H19 locus (Ingrosso et al, 2003). Several earlier
studies have indicated the developmental importance of folic acid as a dietary factory in
utero, and the manner in which it modulates disease risks later in life (Torrens et al, 2006). It
remains to be determined whether, as in the case of hyper-homocysteinaemia, these phenotypic
effects occur through altered DNA methylation (McKay et al, 2004).
An optimal uterine environment has been shown to be essential for establishment and
maintenance of embryonic epigenetic patterns (Vickaryous & Whitelaw, 2005). Because
embryo culture and manipulation are employed in contemporary assisted reproductive
technologies (ART), the question arises as to the extent to which ART or related procedures
alter DNA methylation patterns, thereby inducing epigenetic changes in the developing
organism (Brar et al, 2001; Feil, 2006; Khosla et al, 2001a; 2001b; Vickaryous & Whitelaw,
2005). Normally DNA methylation is confined to only one of the two parent alleles, thus
imprinted gene loci allow minor alterations to be detected. An issue of great importance is the
extent to which the chemical composition of culture medium, the duration of culture, or other
factors, play a role in effecting changes in DNA methylation or histone modification (Doherty
et al, 2000; Khosla et al, 2001a; 2001b; Mann et al, 2004; Young et al, 2001).
An additional consideration of importance, is the role of environmental toxins in producing
alterations in the nucleosome with epigenetic consequences. An obvious example from mid-
twentieth century is the ingestion of the estrogen-receptor agonist diethylstilbestrol (DES) by
women in an attempt to reduce the risk of spontaneous abortion. This was followed by vaginal
clear cell carcinoma (Swan, 2000), and altered limb development in the first generation, and
deafness in the second generation (Stoll et al, 2003). Anticancer drugs and other environmental
compounds may alter expression of specific genes, as well as the stress-related chaperone
protein heat shock protein (HSP)-90, which may play a role in histone modification (Feil,
2006; Rutherford & Lindquist, 1998). A host of environmental contaminants including
endocrine-disrupting chemicals are now known to demonstrate epigenetic effects on the germ
line, and promote disease across several generations (Crews et al, 2007).
In humans, a number of factors, genetic and epigenetic, can influence placental/fetal growth,
development, and long-term sequelae. Several hypotheses have been proposed to account for
these phenomena. The “thrifty genotype” hypothesis, proposes the existence of genes that
influence birthweight, and determining whether an infant will experience intrauterine growth
restriction (Ong & Dunger, 2000; Prentice et al, 2005; Stöger, 2008). The “thrifty phenotype”
hypothesis postulates that impairment of nutritional supply in early life results in permanent
changes in tissue/organ function to conserve glucose, and prioritize development of the brain,
heart, and other vital organs (Hales & Barker, 2001). A third hypothesis proposes that
epigenetic alterations in gene expression, in the absence of altered DNA sequence, can be
heritable, and may be reversible (Holness & Sugden, 2006). A challenge for our future is to
develop strategies to negate the long-term consequences of these molecular alterations. A
related issue of consequence is the epigenetic basis of dysregulation of gene expression in
cancer. Rather than isolated instances, this may be a major factor in the seemingly increasing
and intractable pandemic of this classes of diseases (for instance see Esteller, 2008; Gil-Yam
et al, 2008; Palii & Robertson, 2007). Recognizing the importance of these vital issues, a recent
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National Institutes of Health initiative, as part of its “Roadmap” program, seeks applications
to study the “Epigenomics of Human Health and Disease” (RFA-RM-08-017).
What are the Overall Perspectives, and Critical Questions to be Explored?
For the long-term well-being of an individual, both placental and fetal growth are essential.
Thus, one would anticipate that profound inhibition of cellular growth at key time points during
development would have grave long-term consequences for the embryo/fetus. This suggests
that the timing of the treatment is a key determinant in the effect on the organism. Since their
development, cDNA and oligo microarrays have proven to be powerful tools in the elucidation
of gene expression patterns and discovery. In addition to examining cellular processes at the
global gene expression level, these instruments have allowed analysis of numerous facets of
normal growth and differentiation, as well as that occurring as a consequence of stress or
malignant transformation. Of particular value, such studies allow analysis of gene expression
by functional classes, as an aid in understanding pathways of cell metabolism, proliferation,
senescence, and death.
As with most tissues and organ systems, placental development and its response to stress
remains a poorly understood process. Placental malfunction or failure accounts for numerous
instances of fetal mortality (Cross et al, 2003), and may play an important role in the genesis
of intrauterine growth restriction (Kingdom et al, 2000), as well as some maternal disease
(Newstead et al, 2007). Numerous genes have been shown to be essential for placental function
as an organ of respiratory gas and nutrient exchange, hormonal synthesis, immune function,
and so forth. As is evident, maternal stresses, whether hypoxia, protein deprivation, caloric
excess, or other, can result in profound alterations in placental gene expression patterns, and
their consequences for growth, differentiation, and metabolism. Figure 4 presents in summary
fashion established and potential pathways by which stress to the mother, whether hypoxia,
protein deprivation, caloric excess, or others, can trigger changes in DNA methylation patterns
and/or histone modification to effect alterations in the patterns of gene expression in the
placenta and/or fetal organs.
Far from presenting a complete picture, the present review depicts but a fraction of what we
need to know to understand more completely the molecular regulation of placental growth and
development, and to lessen the ravages of placental dysfunction. A major challenge for the
future will be to identify those portions of the genome particularly vulnerable to epigenetic
modification which underlie states of health and diseases, and to understand the molecular
mechanisms by which these changes occur. A few of the most obvious questions follow. How
are the demonstrated gene expression profiles regulated? In terms of stress, what determines
the individual patterns of expression, as opposed to the up- or down-regulation of those genes
common to all stressors? What are the developmental stages/times of vulnerability to
environmental, nutritional, or other stress? What environmental factors alter the epigenome in
a deleterious manner, and what are their dose-response relations? What are the mechanisms
by which DNA methylation and/or histone acetylation/methylation are regulated? To what
extent do patterns of gene expression alterations in the placenta influence gene expression in
the several fetal tissues/organs? To what extent can we use the findings of gene expression
responses to stress, to gain an understanding of the phenomenon of epigenesis and its various
manifestations. What is the role of epigenesis in normal development, and in the etiology of
disease? How is it that epigenetic changes evident at the molecular level during embryonic/
fetal life, do not become manifest in the adult organism for many years or decades? What is
the relative importance of epigenetic, as opposed to genetic, changes for long-term sequelae?
To what extent can we develop systems using molecular signatures/adducts to detect invidious
interactions in early life? To what extent can an understanding of these issues provide us
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effective means to contain or counteract their influence and consequences? Can epigenetic
biomarkers be identified that will allow disease detection at an early stage?
These are but a few of the vital questions that must be addressed in our pursuit to improve the
lives and well being of mothers and infants, and the latter’s life as an adult. As biomedical
scientists dedicated to betterment of the human condition, can we do less?
Summary
Successful placental development is crucial for optimal growth, development, maturation, and
survival of the embryo/fetus into adulthood. Numerous epidemiologic and experimental studies
demonstrate the profound influence of intrauterine environment on life, and the diseases to
which one is subject as an adult. For the most part, these invidious influences, whether maternal
hypoxia, protein or caloric deficiency or excess, and others, represent types of maternal stress.
In the present review, we examine certain aspects of gene expression in the placenta as a
consequence of maternal stressors. To examine these issues in a controlled manner, and in a
species in which the genome has been sequenced, most of these reported studies have been
performed in the mouse. Although each individual maternal stress is characterized by up- or
down-regulation of specific genes in the placenta, functional analysis reveals some patterns of
gene expression common to the several forms of stress. Of critical importance, these genes
include those involved in DNA methylation and histone modification, cell cycle regulation,
and related global pathways of great relevance to epigenesis and the developmental origins of
adult health and disease.
Acknowledgments
We thank Brenda Kreutzer and Jimin Suh for their assistance in the preparation of this manuscript. This work was
supported, in part, by USPHS grant HD-03807 to LDL.
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Figure 1.
Over-represented gene functional classes throughout mouse placental development. Listed are
the major groups of upregulated placental genes expressed from Embryonic day 10.5 to 17.5.
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Figure 2.
Proposed mechanism of hypoxia-mediated epigenetic changes with long-term programming.
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Figure 3.
A: p53 related genes up-regulated by protein restriction in the mouse placenta. + stands for
induced
B: Proposed mechanisms of protein restriction induced long-term changes in gene expression.
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Figure 4.
Proposed model of stress-induced long-term epigenetic changes in gene expression, adaptation,
and phenotype.
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