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Epigenetics and the periconception environment in ruminants
A. Van Soom1, L. Vandaele
1,2, K. Goossens
3, S. Heras
1, E. Wydooghe
1,M.B. Rahman
1, M.M.
Kamal1, M. Van Eetvelde
1, G. Opsomer
1, L. Peelman
3
1Department of Reproduction, Obstetrics and Herd Health, Faculty of Veterinary Medicine, UGent, Salis-
burylaan 133, 9820 Merelbeke, Belgium.
2Institute for Agricultural and Fisheries Research (ILVO), Animal Sciences Unit, Scheldeweg 68, 9090 Melle,
Belgium.
3Department of Nutrition, Genetics and Ethology, Ghent University, Heidestraat 19, Merelbeke, Belgium.
*Correspondence to: A. Van Soom, Department of Reproduction, Obstetrics and Herd Health, Faculty of Veteri-
nary Medicine, UGent, Salisburylaan 133, 9820 Merelbeke, Belgium.
E-mail: [email protected]
Received: 29.02.2012 Accepted: 22.11.2012 Published: 26.02.2013
Abstract
Particularly in ruminant species, conclusive evidence has been accrued that environmental influences to which
gametes or embryos are exposed to before, during and after conception (i.e. the periconception period) can in-
duce epigenetic changes in the genome. Such epigenetic changes may adversely affect the future health,
development, productivity and fertility of those offspring, in some cases even leading to the so-called “Large
Offspring Syndrome”. Epigenetics are adjustments in gene expression which are established "on top" (i.e. “epi”
in Greek) of the genes. Earlier data obtained in ruminants have demonstrated changes in DNA-methylation of
imprinted genes in the placenta and in the organs of calves or lambs born after cloning or in vitro culture, but
more recently dietary changes have also been implicated in changes in the phenotype of resulting offspring,
causing insulin resistance and hypertension. In this review paper we will first explain how epigenetics can influ-
ence gene expression and why gametes and embryos are especially vulnerable to epigenetic changes caused by
environmental influences at periconception, next we will describe how placental function is affected by epigenet-
ic changes in “Large Offspring Syndrome” and finally we will discuss how maternal nutrition can affect in an
epigenetic way ruminant embryonic and fetal development.
Keywords: epigenetics, periconception environment, ruminants, large offspring syndrome, assisted reproduction
P Belg Roy Acad Med Vol. 2: 1-23 A. Van Soom et al.
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INTRODUCTION/BACKGROUND
Many things have evolved in the
field of assisted reproduction since the
birth of the first test-tube baby, Louise
Brown, in 1978. The techniques for in vitro
fertilization and in vitro production of em-
bryos have been refined both in human and
in several other mammalian species, lead-
ing to applications of these versatile
techniques for treatment of sterility, pre-
vention of genetic disease and fundamental
research on embryonic development. Cattle
were the first species, after the human, in
which transfers of in vitro produced em-
bryos were performed on a large scale
during the 1990s. Coinciding with the first
reports of the birth of the first cloned
calves and sheep, troubling anecdotical
data became available on congenital ab-
normalities associated with cloned animals
and later in a broader perspective, also of
abnormal offspring born after in vitro cul-
ture of ruminant embryos (1-3). The most
obvious characteristic of the abnormal off-
spring was an overgrowth phenotype, and
thus the syndrome was termed “Large Off-
spring Syndrome” or “Abnormal Offspring
Syndrome” (for review see 4).
How severe these congenital malfor-
mations of calves born after assisted
reproduction could be is strikingly illus-
trated in Fig. 1. Not only were these calves
and lambs many times heavier than nor-
mal, but also other congenital
abnormalities were obvious, such as cere-
bellar hypoplasia, ventricular septum
defects and contracted limbs; and cirrhosis
of the liver and fibrosis of the lungs (2, 5-
7). Enlarged hearts and livers were also
described (7).
Coinciding with the fetal oversize, placen-
tal enlargement was observed too, and it is
likely that some of the observed fetal ab-
normalities, such as enlarged heart,
enlarged umbilical cord, and abdominal
ascites, are most probably consequences of
placental dysfunction (8). Moreover, hy-
dro-allantois, or the presence of an
excessive amount of allantoic fluid, is
more frequently associated with pregnan-
cies established from in vitro produced
embryos (2,9). In some cases, exposure of
ruminant embryos to an unusual environ-
ment for about a week (from zygote to
blastocyst stage), was sufficient to initiate
the syndrome. It appeared that the severity
and incidence of the syndrome was influ-
enced by culture conditions (serum-
containing; coculture), animal species (ru-
minants) and levels of maternal nutrients
(high levels of urea) during pregnancy
(4).Already in 1998 it was hypothesized
that the mechanism was probably related
with changes in DNA-methylation of im-
printed genes, which were imposed upon
the embryo by its exposure during a critical
period to a perturbing environment (10).
The fact that no similar syndrome had been
described at that time in men or mice, born
after in vitro production of embryos, was
according to the authors related to the fact
that in those species the culture period is
shorter (a few days instead of a week),
without the use of in vitro maturation and
that commonly used media are serum-free
(10). In follow-up studies on the further
development of perturbed bovine off-
P Belg Roy Acad Med Vol. 2: 1-23 A. Van Soom et al.
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Fig. 1. An oversized calf at a gestational age of 7 months, born after elective caesarean section in a recipi-
ent presented with hydro-allantois. The calf weighed already over 60 kg, 2 months before its due date, and
showed several malformations, including abnormal placentation. The pregnancy was generated after
transfer of an in vitro produced embryo.
spring, it seems that affected surviving
calves achieved similar weights as control-
animals at 1 year after birth (11) but
enlarged organs were still present (12)
whereas reproductive parameters seemed
to be normal (13).However, also in humans
there were similar reports on the influence
of the intrauterine or perinatal environment
on fetal development. In the mid-1980s,
Gunther Dörner found that men born dur-
ing the world war, with a shortage of food
supply, had a lower prevalence of insulin-
treated diabetes mellitus in later life (14).
A few years later, professor Barker postu-
lated that a baby with a low birth weight
has a higher risk to suffer from cardiovas-
cular disease as an adult (15). This
hypothesis was later called the “develop-
mental origins of health and disease” or
DOHAD hypothesis, and by the mid-1990s
the concept that late-onset diseases are
related with earlier prenatal events, was
well established (9,16). Barker studied
mainly fetal fetal undergrowth,but also
fetal overgrowth has been reported in hu-
mans. Assisted reproduction, which is
currently accounting for 5-6 % of the live
birth rates in Belgium (17), has recently
indeed been associated with increased risk
of imprinting diseases such as Beckwith–
Wiedemann syndrome, which is a fetal
overgrowth syndrome (18). Both “Large
Offspring Syndrome” in cattle and the
“Developmental Origins Of Adult Health
And Disease” hypothesis in humans are
reflections of the fact that small changes in
the environment to which the embryo is
exposed can either lead to obvious pheno-
typical changes in the neonate (oversized
calf, Beckwith-Wiedemann baby) or to
P Belg Roy Acad Med Vol. 2: 1-23 A. Van Soom et al.
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4
more subtle, long-term programming ef-
fects, which can lead to impaired health
during adulthood (19) (Fig. 2). Such an
important concept (DOHAD) called for
more in depth research, and this has indeed
been performed during the last decade. The
field of environmental epigenetics, which
is closely related to the concept of Devel-
opmental Origins Of Health And Disease,
has been studied extensively by using vari-
ous animal models. These models provide
a means to understand how environmental
factors, which are present at periconcep-
tion, may induce heritable changes in gene
expression and as such, can cause diseases
that cannot be explained by conventional
genetic mechanisms (20).In this paper we
argue that, besides rodent models, rumi-
nant models are very valuable to
demonstrate consequences of epigenetic
influences exerted on gametes and embry-
os at periconception. Advantages of the
cattle/sheep model over the mouse model
are that they represent an outbred model,
that might mimic the human condition; that
embryos can be generated in vitro from the
abundant source of slaughterhouse ovaries;
that ruminant embryos can generate an
economical interest in animal production
and therefore may lead to long-term fol-
low-up of offspring; and the fact that the
ruminant embryo, just like the human em-
bryo, is more sensitive to adverse culture
conditions than mouse embryos are (21).
Environmentally induced epigenetic
changes in ruminant offspring can be in-
duced in vivo through exposure of embryos
to changes in maternal diet or metabolism,
or in vitro by manipulation of embryos
(nuclear transfer) or exposure to specific
components in embryo culture media. In
this review paper we will first explain how
epigenetics can influence gene expression
and why gametes and embryos are espe-
Figure 2. An unusual environment to which the embryo is exposed will lead to short and long term effects,
both of which are caused by epigenetic modifications and which in some cases can be transgenerational.
At present, these effects have been shown to be induced by in vitro embryo culture in mice, man and cattle.
P Belg Roy Acad Med Vol. 2: 1-23 A. Van Soom et al.
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5
cially vulnerable to epigenetic changes
caused by environmental influences at
periconception, next we will describe how
placental function is affected by epigenetic
changes in Large Offspring Syndrome and
finally we will discuss how maternal nutri-
tion can affect in an epigenetic way
embryonic and fetal development.
EPIGENETICS AND ENVIRON-
MENTAL INFLUENCES IN THE
PERICONCEPTION ENVIRONMENT
It has become increasingly evident
in recent years that the environment during
the earliest stages of embryo development
has a direct and profound influence on the
health and future developmental capacity
of the individual. Whereas the genotype is
determined at fertilization, there exists a
period during this early stage development,
when phenotypic expression of the organ-
isms genotype is adjusted to match
environmental cues. These adjustments in
gene expression are established "on top"
(i.e. “epi” in Greek) of the genes, and this
area of research is therefore referred to as
epigenetics. Understanding the epigenetic
mechanisms involved in embryonic devel-
opment will help to address such issues as
(a) the risks associated with stress, illness
or dietary restrictions and metabolic imbal-
ances during the peri-conceptional period;
(b) the effects of maternal and paternal
nutritional status/stress on epigenetic pro-
gramming through the germline; and (c)
transgenerational effects where, in future,
greater emphasis in livestock species
should be placed on traits of agricultural
importance.
Epigenetic changes may be less harmful
than genetic mutations since they are re-
versible. Understanding the healthy
settings of the periconception environment
that avoid deleterious epigenetic changes
will allow to potentially improve this envi-
ronment to attain the ideal conditions to
which breeding animals and embryos
should be exposed in order to prevent epi-
genetic mutations to occur. The
periconception environment encompasses
ontogenesis and the organs and tissues in
which gametogenesis, embryogenesis, im-
plantation and placentation take place.
Although sexual reproduction is globally
robust, it is also a vulnerable process.
Gametes and embryos are especially vul-
nerable to epigenetic changes. Most
epigenetic marks are systematically erased
in the preimplantation embryo and in the
primordial germ cells in order to down-
regulate the inheritance of epigenetic (ac-
quired) information between generations,
and appear again later on. Likewise, epige-
netic processes are responsible for laying
down the gender-specific imprinting that
allows for gender-specific gene expression,
which is of paramount importance for em-
bryonic development and placentation.
EPIGENETIC MODIFICATIONS
INFLUENCE GENE EXPRESSION
The genetic code is formed by the
DNA sequence inside the chromosome,
and expression of particular genes from
this code in an undifferentiated or differen-
tiated cell will affect subsequent cellular
phenotype (Fig. 3). Epigenetics is the study
of heritable changes in gene expression or
P Belg Roy Acad Med Vol. 2: 1-23 A. Van Soom et al.
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6
Figure 3. A chromosome is a single piece of coiled DNA containing many genes, regulatory elements and
other nucleotide sequences. The chromosomal DNA is wound around histones (highly alkaline DNA-
bound proteins) which serve as a spool to package the DNA and control its functions. Acetylation and
methylation and vice versa of the histone tails can influence gene function. Also direct methylation (Me) of
certain DNA bases can repress gene activity. As such, epigenetic influences sustained during early devel-
opment can influence adult phenotype and traits of domestic animals.
cellular phenotype caused by mechanisms
other than changes in the underlying DNA
sequence. As such one can state that the
genetic information of a DNA sequence is
complemented by epigenetic modifica-
tions, and these epigenetic patterns are
imposed on the cellular genome as a natu-
ral part of cellular differentiation, in a
predetermined way (22). Epigenetic pro-
gramming of gametes and embryos is
essential for the development of a new
individual (23). To ensure proper gene
expression in the embryo, the epigenetic
code comprises two basic ways of interac-
tion: the DNA methylation code, and the
histone code (histone methylation, acetyla-
tion and phosphorylation (24,25). In
general, cells with high plasticity, such as
embryonic cells, have more loosely packed
euchromatin, which permits active tran-
scription whereas cells with limited
developmental potential have more con-
dense packed heterochromatin which
constrains transcription (Fig. 4). As such,
P Belg Roy Acad Med Vol. 2: 1-23 A. Van Soom et al.
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7
Fig. 4. Euchromatin represents the open and transcriptionally active part of the gene/chromosome and
heterochromatin is the highly condensed part which is transciptionally silent. The blue cylinders represent
the histones around which the DNA (black line) is entangled. The green circles and hexagons depict his-
tone acetylation and methylation respectively, which makes gene transcription feasible, leading to
euchromatin. The burgundy hexagons represent DNA methylation of CpG islands which is silencing gene
transcription (heterochromatin). The enzymes responsible for these respective actions are DNA methyl-
transferase (DNMT), Histone Deacetylase (HDAC), Histone Actetylase (HAC) and Histone
methyltransferase (HMT).
modifications of the DNA and histone tails
that influence the density of the chromatin,
for instance methylation, acetylation and
phosphorylation, ubiquitination, ADP-
ribosylation (for review 24, 26), are all
considered to be epigenetic regulators of
gene expression at the level of transcrip-
tion (27). The best-studied examples of
epigenetic modifications so far are DNA
methylation and histone deacetylation,
both of which serve to suppress gene ex-
pression without altering the sequence of
the silenced genes (Fig. 4). DNA methyla-
tion, the first epigenetic mark identified in
general, is referring to the conversion of a
cytosine residue into a 5-methyl cytosine
and is mediated by DNA methyltransferase
enzymes (23).
These DNA methyltransferases (DNMTs)
are responsible for establishing and main-
taining methylation patterns (28). They
consist of five members, DNMT1,
DNMT2, DNMT3A, DNMT3B and
DNMT3L. DNMT1 is responsible for
P Belg Roy Acad Med Vol. 2: 1-23 A. Van Soom et al.
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8
maintenaning the methylation patterns dur-
ing the replication of DNA. DNMT3A and
DNM3B are essential for de novo methyla-
tion, and DNMT3L potentiates DNA
methylation by interacting with DNMT3A
and DNMT3B (29). DNA methylation
changes have been studied in ruminants to
clarify the relation between nutrition, epi-
genetics and disease (20). In sheep, diets
deficient in specific micronutrients (i.e. B
vitamins such as B12 and folate, and ami-
no acids such as methionine) fed at the
time of conception led to adult offspring
that were insulin resistant and hyperten-
sive, and this was associated with global
alterations to DNA methylation (30). Other
dietary factors which can be linked with
DNA methylation are riboflavin (vitamin
B2), pyridoxine (vitamin B6), cholin and
alcohol (31).
IMPRINTING: A SPECIAL CASE OF
EPIGENETICS?
Genomic imprinting is defined as
parent-of-origin specific gene expression,
and takes place in a small cohort of genes
which are exclusively expressed from a
single (maternal or paternal) allele, and of
which the expression is regulated by epi-
genetic modifications of these imprinted
alleles (32). Thus the expression of im-
printed genes is dependent on whether they
are derived from the sperm (paternally
derived) or the egg (maternally derived)
(Box 1). Over 90 imprinted genes have
been identified (25), and many of them
play a role in embryonic development,
fetal growth regulation and placental func-
tion. Mechanisms which relate with
epigenetics such as DNA methylation play
an important role in imprinting. Equally
important for imprinting are the differen-
tially methylated regions or DMRs, which
BOX 1 : Why has imprinting evolved in some animal taxa?
Two hypotheses are at present proposed to explain the evolutionary benefit of imprinting (32):
the parental investment (or genetic conflict) theory (36) and the evolvability model (37). The
first theory, parental investment, is based on the observation that many imprinted genes are
involved in fetal or placental growth. It is of interest for a female to restrict fetal growth,
since then she can make sure that she has enough energy left for subsequent pregnancies and
in the end, she can generate more small but healthy offspring. Maternally expressed genes
such as Igf2R and Gnas, tend to curb fetal growth (38,39). On the other hand, paternally ex-
pressed genes such as Igf2 and Peg3, promote fetal growth and nutrient uptake, and this is
beneficial for the male since in that case he can ensure that his offspring will grow large (even
at the detriment of their mother), and will have better chances to survive. The evolvability
model is equally appealing: since imprinted genes are epigenetically regulated, they can easily
respond to environmental changes (and this is more advantageous in terms of evolutionary
fitness). For example an animal can carry an allele which promotes growth that, while im-
printed, has no phenotypic effects (32). When the embryo is exposed to an environment in
which increased growth becomes advantageous, the relevant allele is already present in the
genetic code and by rapid reversal of the imprinting, the allele can be expressed.
P Belg Roy Acad Med Vol. 2: 1-23 A. Van Soom et al.
___________________________________________________________________________
9
are methylated (or silenced) in accordance
to their paternal or maternal germline
transmission. Imprinted alleles are silenced
such that the genes are either expressed
only from the non-imprinted allele coming
from the mother (e.g. H19) or in other cas-
es from the non-imprinted allele inherited
from the father (e.g. IGF-2). It has become
increasingly evident that especially im-
printed genes appear to be sensitive to
culture conditions and to epigenetic modi-
fications (33). In mice, normal maternal
monoallelic expression of the H19 imprint-
ed gene was observed in optimized
medium, whereas aberrant biallelic expres-
sion was found in suboptimal medium
(34). Later, it has been demonstrated that
mouse embryo culture in five commercial
media systems resulted in imprinted meth-
ylation loss compared to in vivo-derived
embryos, although some media systems
were able to maintain imprinted methyla-
tion levels more similar to those of in vivo-
derived embryos (35).
WHY ARE GAMETES AND
EMBRYOS ESPECIALLY VULNE-
RABLE AT PERICONCEPTION?
The so-called “epigenome” of a ruminant
embryo can be influenced by tiny changes
in the maternal diet (in utero) or in the cul-
ture medium (in vitro). The heritability of
such an environmentally altered epige-
nome will depend largely on the ability of
these alterations to escape germline repro-
gramming. Under normal circumstances,
epigenetic reprogramming is necessary to
downregulate acquired information be-
tween generations. In the life of a mammal,
there are at least two critical periods in
which epigenetic reprogramming occurs:
the first during the formation of the gam-
etes (oogonia and spermatogonia) and the
second during preimplantation embryo
development (38).
Epigenetic reprogramming during gam-
etogenesis
Our understanding with respect to what is
happening with the epigenetic marks dur-
ing gametogenesis in mice is growing (see
reviews of 40;41), but the precise timing of
de novo DNA methylation during gameto-
genesis is still poorly understood; and is
mainly limited to data concerning the
remethylation of a group of imprinted
genes, a few single copy genes, and repeat
sequences in the mouse (42). Moreover,
the timing of de novo DNA methylation
happens to differ between the male and
female germlines.
Most studies on epigenetic reprogramming
of the germline have been performed in the
mouse. Developing primordial germ cells
are migrating from the yolk sac and hind-
gut at day 10.5 pc in the mouse fetus
towards the forming genital ridges. At that
time, imprinted and non-imprinted genes
as well as DNA repeats are heavily meth-
ylated in primordial germ cells (PGCs),
showing the same methylation pattern as in
somatic cells. At 11.5 dpc, most primordial
germ cells have reached their final destina-
tion and are undergoing reprogramming,
perhaps in response to a specific signal(s)
from the genital ridge. By 12.5 dpc, meth-
ylation of single copy genes is erased and
only some methylation on repetitive ele-
P Belg Roy Acad Med Vol. 2: 1-23 A. Van Soom et al.
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10
ments remains by the time PGCs enter mi-
totic/meiotic arrest at day 13.5 pc (43).
These findings suggests that early PGCs
initially possess a high level of methylation
which is similar to that of somatic cells,
but which is rapidly erased at all single
copy sequences shortly after entry into the
genital ridge. The observed demethylation
takes place despite the presence of Dnmt1
(but not Dnmt3a and 3b) in the nuclei of
PGCs (43). Unfortunately, similar data on
the timing of epigenetic reprogramming of
the primordial germ cell genome in rumi-
nants are lacking, but the arrival of
primordial germ cells in the genital ridge is
known to take place at day 22 in sheep and
day 25 in cattle (44), so we can assume by
extrapolation that the DNA of the primor-
dial germ cells will be largely devoid of
methylation at that time. It is not before
oocytes and spermatozoa are being formed
from the primordial germ cells that de novo
methylation of the DNA will occur (44).
De novo methylation is happening much
later during oogenesis when compared to
spermatogenesis ; only after the primary
oocyte, which is arrested at the diplotene
stage of prophase I, is induced to grow, de
novo methylation is taking place (i.e. in the
adult cyclic animal). At least in sheep and
mouse, the most rapid phase of global
methylation of the oocyte is during antrum
formation, which is exactly the time point
at which the oocytes are collected for in
vitro maturation or are induced to ovulate
by means of hormonal stimulation (9). In
livestock we do not know much about
which stages of oogenesis are most suscep-
tible to epigenetic changes. Studies on this
topic are still scarce: an analysis of the
methylation profiles of individual alleles in
bovine oocytes has shown that critical epi-
genetic marks of imprinted alleles such as
H19/IGF2, PEG3, and SNRPN, were not
affected by maturation medium and were
not different between in vivo and in vitro
matured bovine oocytes (45). Gene expres-
sion was however different between the
three groups, suggesting that other regula-
tory mechanisms than DNA methylation
play a part in this changed gene expres-
sion. Another study showed a similar
tendency in cattle as compared to mice :
O‟Doherty characterized the establishment
of methylation at maternally imprinted
genes in bovine growing oocytes and
demonstrated for the first time that an in-
crease in bovine imprinted gene
methylation occurs in differentially meth-
ylated regions during oocyte growth, as has
been observed in mouse (28), and also in
sheep oocytes.
Only few studies have addressed the pat-
tern of DNA methylation during
spermatogenesis. In sexually mature mice,
in the early phases of spermatogenesis,
both gain and loss of methylation occur
during the transition from spermatogonia
to spermatocyte (46). These events are
completed by the end of the pachytene
stage. This study raises the possibility that
male germ cells may be especially sensi-
tive to potential „epimutations‟. At
fertilization, the spermatozoon is heavily
methylated in repeat regions and in pater-
nally imprinted genes but its global
methylation is less when compared to so-
matic cells (47). Most promoters in mouse
spermatozoa are hypomethylated, which is
similar to the situation in embryos and thus
P Belg Roy Acad Med Vol. 2: 1-23 A. Van Soom et al.
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11
maybe refers to a pluripotent programming
of the sperm cell prior to fertilization. Not
only DNA methylation patterns are chang-
ing during spermatogenesis : another
dramatic change in the structure of the
sperm chromatin is the replacement of the
majority of histones by protamines, in or-
der to increase the compaction of the
genome. This extreme compaction of the
sperm nucleus is reversed during fertiliza-
tion when protamines are replaced again
by histones, followed by histone H4K4
methylation (48). No similar data on epi-
genetic alteration during spermatogenesis
are available in ruminants although it has
recently been demonstrated that bull sper-
matozoa showed a decrease in chromatin
protamination when the bulls are exposed
to heat stress during epididymal transit and
during spermiogenesis (49). It remains to
be determined whether also differences in
epigenetic marks can be detected in the
heat-stressed spermatozoa.
Epigenetic reprogramming during em-
bryogenesis
From the zygote to the blastocyst stage, the
epigenome undergoes dramatic changes
(50,51) and the different epigenetic marks
in blastomeres at various embryonic stages
orchestrate the developmental potential of
these blastomeres. Prior to fertilization, the
maternal and paternal genomes are highly
methylated and transcriptionally inert. A
few hours after fertilization, the protamines
associated with the paternal chromatine are
rapidly replaced by acetylated histones.
Immediately after protamine-histone ex-
changes, these histones become
deacetylated and mono-methylated and the
paternal chromatin is demethylated rapidly
(except for some imprinted loci and repeat
elements) (50, 52-55). It is suggested that
this demethylation is an active enzymatic
reaction. The maternal chromatin main-
tains all types of histone H3 methylation
throughout zygotic development and un-
dergoes a slower, passive DNA
demethylation during the first cleavage
divisions (absence of Dnmt1) (50). These
epigenetic modifications render the DNA
accessible to the regulatory and transcrip-
tional machinery (for example
transcription factors) and occur during the
transition from a transcriptionally silent to
a transcriptionally active genome. Thus
embryonic genome activation is facilitated
and early embryonic development is safe-
guarded (Fig. 5). The paternal and
maternal DNA methylation reaches a low
level in the morula (56, 57), but from the
morula to the blastocyst stage, global lev-
els of methylation increase, yet the first
differentiation event is marked by epige-
netic differences such as differences in
DNA methylation between the trophecto-
derm (TE) and the inner cell mass (ICM)
(57). DNA methylation is gradually in-
creased in the ICM, while TE cells remain
relatively unmethylated (54). Proper DNA
methylation levels in ICM and TE are crit-
ical for embryonic development.
Hypomethylation predominantly affects
embryonic regulation and development
rather than extra-embyonic tissues, while
hypermethylation significantly impairs
epigenetic regulation and development of
extra-embryonic tissues (59). At the same
time, the distinct TE and ICM lineages
P Belg Roy Acad Med Vol. 2: 1-23 A. Van Soom et al.
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12
Figure 5. The epigenetic reprogramming cycle showing the two major waves of epigenetic reprogram-
ming. The first wave occurs during gametogenesis when the majority parental epigenetics marks are
erased and reestablished at time of oogenesis and spermatogenesis. A second epigenetic reprogramming
occurs soon after fertilization with a fast, active paternal demethylation and a slower, passive maternal
demethylation. At the blastocyst stage, novel methylation patterns are established in the ICM, whilst the
TE stays relatively unmethylated. Fluctuation in DNA methylation levels are presented by the arrows with
blue color indicating high methylation levels.
become marked by different histone modi-
fication profiles.
ABNORMAL PLACENTA FUNCTION
AND LARGE OFFSPRING
SYNDROME: AN EPIGENETIC
PROBLEM?
The high rate of fetal mortality in early
bovine pregnancies after somatic cloning
has been associated with poorly developed
placenta, characterized by very few placen-
tomes and little vascularization (60). Later
on in pregnancy, the placental dysfunction
is shown by the development of a hydro-
allantois (i.e. excessive accumulation of
allantoic fluid), placentomegaly with a
reduced number of placentomes (Fig. 6)
and/or increased birth weight or Large Off-
spring Syndrome (61). It has therefore
been postulated that instead of “Large Off-
spring Syndrome” one should use “Large
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___________________________________________________________________________
13
Figure 6. Enlarged oedematous placentomes in reduced numbers during placentation in an in vitro pro-
duced pregnancy in a cow.
Placenta Syndrome” (8). The underlying
causes of this placental dysfunction are
unclearDuring somatic cloning, the forced
reprogramming of the donor nucleus
makes the genetic material even more vul-
nerable to reprogramming errors, which
might be strongly related with the in-
creased placental and fetal developmental
anomalies (62,63). Under normal circum-
stances the trophectoderm of the
elongating conceptus is attaching gradually
to the endometrium of the cow (for review
see 64). Placentation in cattle is non-
invasive and the placenta is of the synepi-
theliochorial type. By gestational day 20, a
microvillous interdigitation of uterine and
trophoblastic epithalia starts to develop.
This local interdigitation forms later into
placentomes, which are mushroom-shaped
structures distributed along the uterine mu-
cosa, with fetal cotyledons and maternal
crypts or caruncles interconnecting. Each
bovine placenta consists normally of more
than hundred placentomes. In pregnancies
arising from nuclear transfer bovine em-
bryos, it was shown that the mean number
of placentomes at about day 220 of preg-
nancy was much lower i.e. 78 in nuclear
transfer vs 90 in the control, and the mean
weight of the placentomes was almost
double of controls (123 vs 68 g) (8). They
concluded from the results of morphomet-
ric analysis that probably the placental
abnormalities observed in SCNT result
from adaptation(s) to placental dysfunc-
tion, and that dysregulation of cellular
metabolism and cell signaling are respon-
sible for these underlying defects (65).
Gene expression comparison between
placentomes of SCNT, IVF and AI preg-
nancies revealed that all SCNT term and
preterm placentas, showed radically altered
P Belg Roy Acad Med Vol. 2: 1-23 A. Van Soom et al.
___________________________________________________________________________
14
gene expression profiles. These altered
expression profiles were irrespective of the
fact whether the investigated placentomes
were abnormal or not, and were associated
with epigenetic changes in SCNT fetuses
and placentas (65, 66). Which genes are
affected by these epigenetic modifications
is still under investigation. A loss of im-
printing and aberrant expression of the
gene encoding the type 2 insulin-like
growth factor receptor (IGF2R) has been
associated with Large offspring syndrome
in sheep (67). In humans, 50 % of the cases
of Beckwith-Wiedemann syndrome, which
is an overgrowth syndrome, are presented
with a loss of CpG methylation at
KvDMR1, which may function as a re-
gional imprinting center on the
Kcnq1ot1/Cdkn1c domain. Similar hypo-
methylation patterns in KvDMR1were
observed in 3/18 clinically normal children
conceived by ARTs (68). Moreover, it has
recently been reported that hypomethyla-
tion of KvDMR1 was present in mid-
gestation bovine fetuses produced by nu-
clear transfer (69), which may be
associated with the variable overgrowth
phenotypes seen in these fetuses. In anoth-
er study, it was demonstrated that loss of
CpG methylation of KvDMR1 and aber-
rant gene expression of Kcnq1ot1 and
Cdkn1c may indeed contribute to the large
offspring syndrome (70). These findings
further substantiate the hypothesis that
epigenetic modifications lie at the basis of
aberrant fetal phenotypes in cattle and hu-
mans produced after assisted reproduction.
EPIGENETICS AND IMPORTANCE
OF MATERNAL NUTRITION FOR
NORMAL EMBRYONIC
DEVELOPMENT IN CATTLE
The old adage “You are what you eat” has
lately not only been proven to be true but it
can also be extended to “You are also what
your mother and father ate”. This saying
directly refers to the fact that the pericon-
ception environment in its broadest sense
directs the health and characteristics of a
given individual.
As shown in humans, the intra-uterine en-
vironment and the nutrient supply in
particular, is a major determinant of fetal
growth. A compromised supply will result
in low birth weight neonates which suffer
an increased risk of perinatal mortality.
Not only is there an interest of the nutri-
tional effects on fetal growth and hence
birth weight, but also to effectors that may
programme changes within the foetus
which impinge on its neonatal viability and
its subsequent health and production poten-
tial through adulthood. Both fetal
undergrowth and overgrowth have been
related with important diseases later in life
(14-16).
In fact, in livestock, just as in humans,
compromised fetal or neonatal growth has
been shown to be associated with: (a) in-
creased neonatal morbidity and mortality;
(b) altered postnatal growth, including a
reduced average daily growth and weaning
weight; (c) poor body composition, includ-
ing increased fat, reduced muscle growth,
and reduced meat quality; (d) metabolic
disorders, such as a poor glucose tolerance
and insulin resistance; (e) cardiovascular
P Belg Roy Acad Med Vol. 2: 1-23 A. Van Soom et al.
___________________________________________________________________________
15
disease; and (f) dysfunction of specific
organs, including the ovaries, testes,
mammary gland, liver, and small intestine
(71).
Body systems other than those directly
linked to postnatal production traits, can
also be affected by undernutrition during
early pregnancy. Many studies illustrated
that with, or even without contemporary
effects on fetal growth, a diverse range of
fetal tissues, organs, neuroendocrine sys-
tems and enzymes are affected by
undernutrition in early to mid pregnancy.
Many of these studies are now searching
for the important molecular aspects which
involve the role of nutrition in gene ex-
pression. The nutritional environment
experienced by the embryo during its de-
velopment from a zygote to a blastocyst
can for example also influence the methyl-
ation and expression of genes important for
normal fetal growth and development.
Having this knowledge attributes very in-
teresting and innovative information for
application to practical ruminant produc-
tion. For example genetic selection for
enhanced production in the form of in-
creased ovulation rates, greater muscular
growth, higher milk yields or wool produc-
tion may place more stringent demands on
maternal nutrition during key periods of
embryonic and fetal development than
hitherto realized. It is clear now that abrupt
switching within a production system to
more muscular sires may impose additional
in utero demands for specific nutrients
during fetal myogenesis (71). Alternative-
ly, confronting animals that have been bred
for high levels of production with exten-
sive organic farming systems and lower
feed inputs may compromise fetal devel-
opment, neonatal viability and adult health
and production.
In sheep for example it has been shown
that maternal undernutrition during preg-
nancy and hence in utero undernutrition of
the fetuses, can affect adult reproductive
performance later on. Research has been
dedicated to identify critical periods during
which the fetal reproductive axis is most
vulnerable to maternal nutrition (72). Find-
ings show that there are significant effects
on the development of the fetal ovary be-
fore the onset of gonadotrophin secretion
from the fetal pituitary (Day 65 of gesta-
tion) and before gonadotrophin receptors
are present in the fetal ovaries (after day
135 of the 147 day gestation period). Also
female foetuses born out of overfed ado-
lescent sheep pregnancies were shown to
bear fewer ovarian follicles than normal
growing foetuses at mid- and late gestation
and hence only possess a limited pool for
follicular recruitment in adult life (73, 74).
Furthermore, low birth weight lambs pro-
duced by an in utero crowding model
induce fewer uterine caruncles than normal
birth weight lambs, and this may affect
subsequent placental growth and uterine
capacity (75). For male lambs, low birth
weight has been shown to be associated
with a delay in the onset of endocrine pu-
berty and attenuated testicular growth (76).
While most studies concerning ruminants
have been performed in sheep, hypotheses
on metabolic programming have recently
also been put forward in modern dairy cat-
tle (77-79). Despite the fact that modern
high yielding dairy cows are intensively
fed to reach top productions and hence do
P Belg Roy Acad Med Vol. 2: 1-23 A. Van Soom et al.
___________________________________________________________________________
16
not suffer from undernutrition, some of the
above mentioned clinical features may
nevertheless be relevant for them as during
lactation they have to go through a long
period of negative energy balance which
may extend into the early stages of the
subsequent pregnancy and may in this way
affect fetal development. In this regard, the
study of Leroy et al. (80) is very interesting
as it shows that in cattle, the physiological
status of „producing milk‟ was significant-
ly associated with a lower embryo quality
in cows from which Day 7 embryos were
harvested. Data from the dairy cow fur-
thermore confirm results from other
species in showing that maternal age dur-
ing pregnancy can affect birth size and also
suggest epigenetic effects of maternal milk
yield on calf development in utero. Whilst
very small calves are more likely to suffer
calf mortality, there have, to date, no ad-
verse effects been found of a relatively low
birth weight on subsequent fertility or
productivity in the first lactation. Indeed,
the trend in fertility was in the opposite
direction (77). However, restricting mater-
nal nutrition in cattle to 60% of
maintenance requirements (compared with
100% in controls) during the first third of
gestation resulted in female calves with a
60% lower antral follicle count compared
with calves born to mothers fed control
diets (81).
From the animal production point of view,
another important developmental phenom-
enon that can be adversely affected by
early-pregnancy undernutrition is fetal
myogenesis (82), leading to suboptimal
postnatal lean tissue growth. The latter
may have an impact on both the amount
and quality of the produced meat. While
the size of the newborn seems to be mostly
influenced by maternal nutrition during the
final stages of gestation, the uterine envi-
ronment and hence maternal nutrition
during early pregnancy may be of signifi-
cant importance for the amount of muscle
fibres and herewith also the fat/muscle and
connective tissue/muscular tissue ratios,
both of which are detrimental for meat
quality (82). Knowing this, nutritional
management of pregnant dams in the beef
industry will be an important and innova-
tive tool especially in breeds like the
Belgian Blue in which the size of the calf
is detrimental for the ease of calving and
the texture of the muscular tissue is of ma-
jor importance with regard to meat quality
(83).
As mentioned earlier, undernutrition dur-
ing early to mid pregnancy has been stated
to affect the early construction and devel-
opment of a diverse range of fetal tissues
and organs (84). In humans, the latter has
been illustrated by an intra-uterinely pro-
grammed pancreas leading to an impaired
insulin secretion and hence higher risk to
suffer from diabetes mellitus 2. Hence,
persisting effects of early malnutrition be-
come translated into pathology, thereby
determining chronic risk for developing
glucose intolerance and diabetes (85), but
also overnutrition or gestational diabetes
lead to abnormal fetal development and
increased proneness to disease (86). Earlier
studies at our department have shown that
insulin secretion is compromised in high
yielding dairy cows suffering from fertility
problems like cystic ovarian disease
(87,88). Impaired insulin secretions seem
P Belg Roy Acad Med Vol. 2: 1-23 A. Van Soom et al.
___________________________________________________________________________
17
to be at least partly caused by the elevated
blood levels of non esterified fatty acids
(NEFAs) in cows during the periparturient
period (89). At least in some cows, the
(endocrine) pancreas seems to be highly
sensitive for these elevated NEFAs, ren-
dering those cows at a higher risk to suffer
from lower peripheral insulin concentra-
tions. Hence, as in humans where an
impaired insulin secretion is a decisive
factor in the development of diabetes type
2 (90), also in modern dairy cows an im-
paired insulin secretion seems to be
associated with some of the so-called pro-
duction diseases (79), and may be related
to the existence of a negative energy bal-
ance during early pregnancy while in
utero.
Responses in fetal growth to maternal nu-
trition are characterized by their wide
diversity. For example, growth of the foe-
tus may remain unaffected by chronic
maternal undernutrition, it may also be
impaired or even enhanced by it. This di-
versity in responses also occurs with
overnutrition. Although the reasons for the
different responses are not always appar-
ent, there are often associations with age,
degree of maturity and body condition of
the dam and with the period of gestation
during which the under- or over-nutrition
is imposed. Knowledge of the important
influence of maternal age (degree of ma-
turity) and body condition and of protein
content of the diet on the partition of nutri-
ents between the gravid uterus and
maternal body will facilitate a qualitative
and increasingly quantitative resolution of
the diverse range of responses in fetal
growth to maternal nutrition (71).
CONCLUSIONS
The gametes and the embryo represent
particularly sensitive stages during which
the epigenome can be altered. The field of
environmental epigenetics around concep-
tion has gained considerable interest and
more research on this topic in the ruminant
model is very timely. Working and gener-
ating knowledge on this topic has been
presented as one of the most important
actions for the 5 coming years by the
FABRE TP group (Farm Animal Breeding
and Reproduction Technology Platform).
There is compelling evidence that assisted
reproduction and altered metabolism in
ruminants is associated with epigenetic
alterations and abnormal phenotypes, but
fine tuning of the environment to which the
embryos are exposed, will most probably
reduce the incidence of these offspring
phenotypes considerably. It would be ad-
visable that before implementation of a
new medium for culture of ruminant em-
bryos in practice, the epigenetic alterations
which this medium is causing specially in
imprinted genes need to be defined and
kept to a minimum. In vivo embryos
served in this regards as a gold standard.
One has to bear in mind that many factors
can influence the epigenetic profile of an
individual during the periconceptional pe-
riod. These factors can be very diverse
such as physical stressors like cold or heat,
high pressure, nutritional status of the ani-
mal, maternal health or sickness, feed
supplements or deficiencies, pharmaceuti-
cals, endocrine factors, and culture media.
Considerable progress has been made in
our understanding of the concept of epige-
P Belg Roy Acad Med Vol. 2: 1-23 A. Van Soom et al.
___________________________________________________________________________
18
netics, and through the rapid evolution in
molecular techniques such as Next Genera-
tion Sequencing, it has become feasible to
perform high-throughput screening to ana-
lyse environmental effects on samples
containing a limited number of cells, e.g.
embryos or gametes. In due time, the ru-
minant model may help to answer some of
the complex questions on how the envi-
ronment affect development and disease
through the epigenome.
ACKNOWLEDGEMENTS
This work was supported the Uni-
versity of Ghent (BOF12/GOA/011-
Pathways to pluripotency and differentia-
tion in embryos and embryonic stem cells),
by the Special Research Fund of the Uni-
versity of Ghent (01SF2010 –
Improvement of cattle fertility and fetal
programming guided by ultrasonography,
hormonal assay and metabolic profiling),
by the EU (COST Action FA0702-
GEMINI-Maternal interaction with gam-
etes and embryo and COST Action
FA1201-EPICONCEPT- Epigenetics and
Periconception Environment) and by the
Research Foundation Flanders (FWO
G.0355.11- Embryo-maternal interaction in
the horse).
P Belg Roy Acad Med Vol. 2: 1-23 A. Van Soom et al.
___________________________________________________________________________
19
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