Reproductive Epigenetics: Preparing the Epigenome for the Next Generation
Jacquetta Trasler, MD, PhD, Departments of Pediatrics, Human Genetics and
Pharmacology & Therapeutics, McGill University; Developmental Genetics Laboratory,
Research Institute at the Montreal Children’s Hospital of the McGill University Health
Centre
Introduction
The phenotype of an individual is a consequence of the complex interactions
between the genome, epigenome, and the present and ancestral environments. Parental
exposures, such as diet and toxicants, can lead to altered offspring development including
changes to post-natal growth and an increased risk for disease in later life. Epigenetic
changes in parents or their developing offspring have been suggested as a mechanism
underlying phenotypic outcomes. Embryo and germline development represent key times
when epigenomic patterns are acquired and reprogrammed. Recent evidence indicates
that developmental epigenomic programming events, occurring in the prenatal and early
postnatal periods and during gametogenesis, can be associated with adult disease and
transgenerational effects. Thus, there is increasing interest in understanding how the
genes we inherit from our parents are influenced by diet, the environment (including
toxins and endocrine disruptors), and the experiences we encounter during prenatal life
and early childhood. These early events are particularly important as a number of
common diseases, such as diabetes, heart disease, cancer and neurobehavioral disorders,
can originate in very early life, a phenomenon referred to as the Developmental Origins
of Adult Health and Disease. It is largely unknown how conditions during pregnancy can
affect the health of offspring years or decades/generations later, but it has been proposed
that the fetal tissues become “programmed” through alterations in the epigenome during
development. The epigenome provides a way for our genes to respond to maternal underor over-nutrition, toxicant exposures, fetal growth restriction, or other stressful
conditions, by permanently altering cellular growth characteristics, metabolism and
physiology. The specific disease states that can be induced depend not only on the type of
in utero stress but also on the specific period during development when the exposure
occurs. Here, the timing and mechanisms underlying normal epigenomic reprogramming
during gametogenesis and embryogenesis will be reviewed. Recent examples of animal
and human exposures that can alter normal epigenomic programming will be discussed
along with the consequences for postnatal life.
Dynamic Epigenetic Programming Events in the Germline and Embryo
Epigenetic mechanisms include DNA methylation, covalent histone
modifications, and noncoding RNAs (ncRNA), each of which is able to induce changes
in transcriptional activation and repression of particular genes, and when perturbed,
increase genomic instability and structural defects in heterochromatic domains (1).
Epigenomic reprogramming, or the erasure of epigenetic information across the genome,
occurs between generations; it is thought that this occurs for the purpose of resetting the
control of gene expression patterns and in order to prohibit the inheritance of aberrant
epigenetic information prior to the start of embryo or germ cell development. The two
major epigenomic reprogramming periods in the lifetime of an individual occur in utero
during embryonic development. In the first, the epigenome is reprogrammed at the
beginning of embryo development in the preimplantation embryo. For instance, as
embryos develop from the 1 cell to blastocyst stage, DNA methylation is lost at most of
the 20-30 million CpG dinucleotides in the genome, with the exception of a minority of
sequences such as imprinted genes that maintain their male or female germ-cell specific
methylation patterns and certain repeat sequences (2,3). DNA methylation is then reacquired in a sex-, cell- and tissue-specific manner in the peri-implantation period (e.g.
days 5.5-7.5 of mouse gestation which normally lasts 20 days). The second period of
epigenomic reprogramming occurring in utero takes place in the germ cells of the
developing embryo between mid-gestation and term. At mid-gestation, in germ cell
precursors (the primordial germ cells), epigenetic information is for the most part erased
and reset at sex-specific times, in male germ cells at the end of gestation prior to birth and
in the female germ cells only after birth at the time of oocyte growth (4,5). The fact that
the major periods of epigenomic programming in somatic tissue (important for the F1
offspring) and germline tissue (important for the F2 offspring) occur during gestation
indicates that in utero development is a key time in life of particular susceptibility to the
induction of epigenetic defects with the potential to affect the health of two generations
of individuals.
Unique Epigenome of Oocytes and Sperm
Following the in utero phase of reprogramming, male and female germ cell
epigenomes are further remodeled to prepare for their sex-specific roles in fertilization
and embryo development. The sperm epigenome has been best studied since millions of
sperm are made each day from a pool of stem cells, the spermatogonial stem cells,
throughout the life of mammals. We and others have shown that the DNA methylation
patterns in sperm differ markedly from those of somatic cells (6,7,8). In addition,
although only 1-15% (species-dependent) of histones are retained in sperm, their
positions and modifications appear to be essential in preparing or ‘poising’ the
epigenome to contribute to early embryo development in mouse and human (9,10).
Epigenetic studies in oocytes are limited by the small numbers of pure populations of
cells that can be isolated, but nevertheless, data on imprinted and repeat sequences (4,11)
and recent genome-wide reports indicate that the oocyte epigenome is also unique.
Sex-specific Germ Cell Epigenetic Events
To date, DNA methylation is the most well characterized epigenetic modulator
that has been shown to have essential functions in the germ line and embryo as well as in
genomic imprinting (variation in the expression of a subset of genes according to their
maternal or paternal origin, involves ~100 genes). DNA methylation takes place
predominantly at the 5-position of cytosine residues within CpG (where cytosine is 5’ to
a guanine) dinucleotides, with approximately 60-80% of CpG containing cytosines being
methylated. Demethylation can occur passively when methylation is not maintained
following DNA replication or it can be carried out actively, either through the Tet family
of enzymes and the conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine
(5-hmC), or through glycosylase/DNA repair pathways (12,13). De novo methylation
allows for the acquisition of new patterns of methylation in the genome whereas
maintenance methylation is required to ensure the propagation of DNA methylation
patterns following DNA replication. Methylation of DNA is catalyzed by a family of
DNA (cytosine-5)-methyltransferases (DNMT) enzymes and is linked to gene expression,
in that methylation of CpG sites within promoter regions of genes invariably silences
transcription.
DNA methylation patterns on repeat, single-copy and imprinted sequences are for
the most part erased in the founder cells of the germ line, the primordial germ cells
(PGCs), between 10-12 days of gestation in the mouse, and then re-acquired at genderspecific times during spermatogenesis and oogenesis (3). A second period of erasure
occurs in the preimplantation embryo, when methylation across much of the genome is
lost with the exception of imprinted genes, imprinted-gene-like sequences and some
repeat sequences. It is postulated that imprinted gene methylation patterns must be
maintained during preimplantation development since it is only in the germ line (male or
female depending on the gene) that imprinted genes acquire the allele-specific
methylation that will subsequently be responsible for monoallelic expression in the
postimplantation embryo and adult .
DNA methylation patterns are acquired at different developmental times in the
male and female germ lines. In the male, the major period of DNA methylation
acquisition (de novo methylation) occurs before birth in male germ cells of the fetal
testis; postnatally, the patterns must be maintained (maintenance methylation) during the
continuous, lifelong, cell divisions that occur in the male germ line stem cells
(spermatogonial stem cells) that produce sperm. In male germ cells a small amount of
additional methylation on a minority of sequences occurs as germ cells develop from
spermatogonia to spermatocytes, when meiotic recombination occurs. In contrast, in the
female germ line, DNA methylation in the developing oocytes is only acquired
postnatally, following meiotic recombination and during the oocyte growth phase. For
instance, in the male, we and others have shown that most de novo methylation on
imprinted (to date only 3 imprinted genes are methylated in male germ cells: H19,
Gtl2/Dlk, Rasgrf1), repeat (e.g. LINE1, IAP, minor satellite) and intergenic sequences is
acquired before birth between 15 and 18 days of gestation in the mouse and is then
completed at a minority of sequences after birth (14). Interestingly, the DNMTs that carry
out de novo methylation of DNA are expressed at their highest levels in the prenatal day
15-18 ‘window’ when male germ cell methylation occurs (15). Decreases in DNA
methylation in fetal male germ cells as a result of DNMT deficiency (DNMT3a or
DNMT3L) produces germ cells that cannot proceed through meiosis, resulting in a
complete lack of sperm and infertility (16,17). In the female germ line, germ cells in the
ovary remain unmethylated during fetal development and we and others have defined the
timing of imprinted gene and repeat sequence de novo methylation to occur during oocyte
growth in the postnatal period (4,11). As in the male, the DNMTs are only expressed at
the time of acquisition of methylation patterns, during postnatal oocyte growth in the
female; absence of these enzymes results in lack of methylation of female germ cells and
subsequent infertility dues to loss of embryos at mid-gestation as a result of imprinting
defects (18). Evidence to date indicates that similar timing (prenatal and prepubertal) of
DNA methylation and DNMT expression occur in human male and female germ cells
(19).
Disrupting DNA Methylation Programming in Germ Cells and Embryos
In animals and humans, several types of exposures (20-25), have been shown to
alter epigenomic patterns during development of germ cells and/or embryos resulting in
growth abnormalities, altered gene expression or disease phenotypes:
Diet (protein or calorie restricted, altered fat content)
Endocrine disruptors (vinclozolin, bisphenol A)
Ovulation induction
Intracytoplasmic Sperm Injection
Embryo culture
Somatic Cell Nuclear Transfer
The presentation will discuss the most well studied exposures where there are
comparative data across species, including the effects of assisted reproductive
technologies and somatic cell nuclear transfer.
.
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