epigenetics and the periconception environment in...

1

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

mailto:[email protected]

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-


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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


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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.


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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


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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,


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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


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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.


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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-


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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


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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


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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


___________________________________________________________________________

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


___________________________________________________________________________

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


___________________________________________________________________________

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


___________________________________________________________________________

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-


___________________________________________________________________________

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).


___________________________________________________________________________

19

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