ESA-SRB 2013* Localisation of DNA methyltransferase isoforms during preimplantation embryogenesis (#110)
During
early embryogenesis, temporal and spatial regulation of gene expression
controlling cell fate and differentiation is mediated by epigenetic
modification such as DNA methylation. DNA methyltransferase (DNMT) enzymes
mediate methylation of CpG dinucleotides through two processes: (i) maintenance
methylation (DNMT1); and (ii) de novo methylation
(DNMT3A and 3B). The accepted paradigm of epigenetic reprogramming in embryos holds
that there is preferential demethylation of the paternal pronucleus while the maternally-derived
DNA undergoes passive demethylation. Recent analyses show that the presumed loss
of methylation following fertilization is accounted for by a progressive onset
of antigenic masking during zygotic maturation (). Full
antigenic retrieval by tryptic digestion showed that high levels of DNA
methylation persisted throughout zygotic maturation and subsequent mitoses to
the 8-cell stage. Thereafter, cytosine methylation was lost from the inner
cells of the embryo. In the blastocyst, the trophectoderm remained
hypermethylated while the nuclei of the inner cell mass were substantially hypomethylated.
In order
to understand the mechanisms mediating the differential methylation patterns of
the inner cell mass and trophectoderm, protein localization of the various DNMT
isoforms were examined by immunolocalization. Each DNMT enzyme showed characteristic
patterns at each stage of development. DNMT1 was restricted to the cytoplasm of
blastomeres at each stage of preimplantation development. DNMT3a was expressed
in the nucleus of cleavage stage embryos but was progressively lost from the
8-cell stage, so that blastocysts were largely devoid of DNMT3A. DNMT3B was
absent from the early stage embryos but increased in the nuclei from the 8-cell
stage through to blastocysts. Interestingly, DNMT3B was only found to be
localized to the nuclei of the outer cells of the morula and the trophectoderm of
the blastocyst, little was detected in the inner cells at each stage. The
patterns of expression of the DNMT isoforms suggest a role for DNMT3A as the
dominant methylating enzyme in the cleavage stage embryo and DNMT3B as the
dominant enzyme after the 8-cell stage. The differential localisation of DNMT3B
in the inner and outer cells of the embryo may account for the lineage-specific
pattern of differential methylation in the early embryo.