DNA methylation in the preimplantation embryo: the differing stories of the mouse and sheep
Introduction
This review will describe the recent evidence that major epigenetic reprogramming events previously thought to be key for reprogramming the sperm and egg genomes into a functional zygotic nucleus are not necessarily conserved between mammals. The paper will focus on the most comprehensively studied epigenetic modification to DNA, DNA methylation, and will describe the dynamics of this process in the contrasting mouse and sheep embryo, including descriptions of other mammalian species where available. The functional significance of these species differences in epigenetic reprogramming are then considered.
Section snippets
DNA methylation
The most characterized epigenetic modification of DNA in the nucleus associated with transcriptional silencing and heritability of this phenomenon through cell divisions is DNA methylation (reviewed by Bird, 2002). This DNA modification occurs post replication mainly on the CpG dinucleotides of vertebrate genomes (Jaenisch and Bird, 2003, Bird, 2002). DNA methyltransferase enzymes transfer methyl groups donated from s-adenosyl methionine onto the C5 positions of 70–80% of the cytosine residues
DNA methylation during development
The cells of the cleavage stage embryo are totipotent, i.e. they have the ability to form all of the cells of the body. However, nuclear transfer experiments using blastomeres from different stages of embryos have demonstrated the rapid loss or decline in totipotentiality with each progressive cell division post-fertilization (reviewed by Wilmut et al., 2002). A large scale sequencing experiment identifying genes expressed at each stage between fertilization and blastocyst from cDNA libraries
DNA methylation in the mouse embryo
The dramatic changes in DNA methylation that occur in the early mouse embryo were first documented almost 20 years ago when Sanford et al., 1984, Sanford et al., 1987 observed differences in levels of repeat sequence methylation between sperm and oocytes that were propagated into the early embryo, with the cleavage stages showing an intermediate level to the hypermethylated sperm and hypomethylated oocyte. By 7.5 days of mouse development, however, the genome was once again highly methylated
DNA methylation in the sheep embryo
To gain further insight into these aspects of global methylation changes during preimplantation development, we focused on another species, the sheep. We have analyzed the dynamics of DNA methylation changes after fertilization in in vivo derived and in vitro fertilised embryos (Beaujean et al., 2004). These studies uniquely included computer-assisted quantification of 5mC immunostaining on confocal images, using the same monoclonal antibodies recognising methylated cytosine for immunodetection
DNA methylation in cow, pig, rabbit and human
Akin to the situation in the mouse, Dean et al. (2001) have reported dramatic demethylation of one pronucleus in the pig and rat embryo and we have observed the same phenomenon in the human embryo (Beaujean et al., 2004). It is of note that while the paternal pronuclei of the mouse can be distinguished by a size differential, it has not yet been verified which of the equivalently-sized pronuclei are demethylated in the other species. However, the lack of observed asymmetric pronuclear
Mechanisms regulating preimplantation methylation dynamics
It is intriguing why in some species the paternal, but not maternal, nucleus is demethylated. Suggestions have included the differential binding of maternally-stored cytoplasmic factors to the two zygotic genomes (Arney et al., 2002), that a signal promoting active demethylation is associated with the sperm nucleus or that the sperm genomic composition facilitates demethylation (Bourc’his et al., 2001), that oocyte demethylation activity targets the naked DNA of the male pronucleus during the
Functional significance of embryonic methylation?
Since rapid, genome-wide demethylation of the male pronucleus does not occur in the sheep or rabbit, the previously suggested function of this process must now be questioned. The 5-methylcytosine antibody recognizes both heterochromatin and euchromatin (de Capoa et al., 1998) but the staining is most intense in heterochromatic foci and thus does not inform us about gene expression. Therefore very dynamic changes in individual gene loci (such as in imprinted genes) may occur but not be reflected
Unresolved issues
Our observations of non-conservation among mammals in the dynamics of methylation reprogramming leave a number of key issues requiring investigation. Firstly, the issue of whether asymmetric pronuclear methylation is an artifactual result, secondary to an asymmetric structural barrier to antibody accessibility, urgently needs to be resolved. Secondly, identifying the nature and source of the active demethylating activity will be important in designing functional studies to unravel the role of
Conclusion
Identification of the changes in epigenetic status induced both in normal fertilised embryos and in the reprogrammed somatic cell nucleus after nuclear transfer is likely to define the blueprint epigenetic state of totipotent cells and an indication of how to achieve this state at the mechanistic level. Ultimately this information may prove critical to developing novel means to transdifferentiate a somatic cell into a totipotent embryonic cell without the need for reprogramming in an oocyte
Acknowledgements
We are grateful to Richard Meehan for helpful discussions during this work, Jane Taylor and John Gardner for producing the sheep embryos, the Large Animal Unit staff (Roslin Institute) for expert assistance in sheep surgery and Michelle McGarry for collection of mouse embryos. This work was supported by BBSRC grants No. GTH14114/15.
References (79)
- et al.
Regulation of transcriptional activity during the first and second cell cycles in the preimplantation mouse embryo
Dev. Biol.
(1997) - et al.
Non-conservation of mammalian preimplantation methylation dynamics
Curr. Biol.
(2004) - et al.
Endogenous transcription occurs at the 1-cell stage in the mouse embryo
Exp. Cell Res.
(1995) - et al.
Dynamic organization of DNA replication in one-cell mouse embryos: relationship to transcriptional activation
Exp. Cell Res.
(1997) - et al.
Delayed and incomplete reprogramming of chromosome methylation patterns in bovine cloned embryos
Curr. Biol.
(2001) - et al.
Promoter-specific activation and demethylation by MBD2/demethylase
J. Biol. Chem.
(2002) - et al.
Epigenetic reprogramming: how now, cloned cow?
Curr. Biol.
(2002) - et al.
A unique developmental pattern of Oct-3/4 DNA methylation is controlled by a cis-demodification element
J. Biol. Chem.
(2002) - et al.
Analysis of DNA (cytosine 5) methyltransferase mRNA sequence and expression in bovine preimplantation embryos, fetal and adult tissues
Gene Exp. Patterns
(2003) - et al.
DNA global hypomethylation in EBV-transformed interphase nuclei
Exp. Cell Res.
(1999)