Altered imprinted gene methylation and expression in completely ES cell-derived mouse fetuses: association with aberrant phenotypes

Abstract

In vitro manipulation of preimplantation mammalian embryos can influence differentiation and growth at later stages of development. In the mouse, culture of embryonic stem (ES) cells affects their totipotency and may give rise to fetal abnormalities. To investigate whether this is associated with epigenetic alterations in imprinted genes, we analysed two maternally expressed genes (Igf2r, H19) and two paternally expressed genes (Igf2, U2af1-rs1) in ES cells and in completely ES cell-derived fetuses. Altered allelic methylation patterns were detected in all four genes, and these were consistently associated with allelic changes in gene expression. All the methylation changes that had arisen in the ES cells persisted on in vivo differentiation to fetal stages. Alterations included loss of methylation with biallelic expression of U2af1-rs1, maternal methylation and predominantly maternal expression of Igf2, and biallelic methylation and expression of Igf2r. In many of the ES fetuses, the levels of H19 expression were strongly reduced, and this biallelic repression was associated with biallellic methylation of the H19 upstream region. Surprisingly, biallelic H19 repression was not associated with equal levels of Igf2 expression from both parental chromosomes, but rather with a strong activation of the maternal Igf2 allele. ES fetuses derived from two of the four ES lines appeared developmentally compromised, with polyhydramnios, poor mandible development and interstitial bleeding and, in chimeric fetuses, the degree of chimerism correlated with increased fetal mass. Our study establishes a model for how early embryonic epigenetic alterations in imprinted genes persist to later developmental stages, and are associated with aberrant phenotypes.

DNA methylation and gene expression changes in mouse pre- and post-implantation embryos generated by intracytoplasmic sperm injection with artificial oocyte activation

“…Some non-physiological embryo culture environments or manipulation in vitro may influence the epigenetic modification of imprinted genes during early embryo-genesis [ 35 ]. Some epigenetic changes in pre-implantation embryos may further affect gene expression during fetal development stage [ 36 ].Therefore, we focused the study to explore the effect of AOA on expression of imprinted genes. In the mouse, there are about 150 known imprinted genes, many of which occur in imprinted gene clusters that are regulated together.…”

Stem cells and regenerative medicine: principles, prospects and problems

“…Since, this monoclonal line showed no genomic changes when analysed by representational oligonucleotide microarray analysis at a resolution of ca. 30 000 base pairs (Ira Hall, personal communication of unpublished observations) a likely, but as yet unproven, explanation for its altered behaviour is that it has indeed undergone stable deleterious epigenetic change [38]. Since this is not seen with all monoclonal lines at high passage, we are at present attempting to ascertain whether such adverse change correlates with restriction in the capacity of the cells to differentiate in vitro.…”

Child health, developmental plasticity, and epigenetic programming

“…Different types of environmental stress, particularly those that result from transferring cells and embryos from their natural environment to in vitro conditions, can interfere with the somatic maintenance of imprints. Assisted reproductive technology (ART) is a stressor of special in-terest because in vitro culture of embryonic cells and embryos can perturb the imprints at ICRs in different model systems, thereby affecting fetal growth and development (235,(277)(278)(279)(280)(281)(282)(283)(284). Such findings have implications in humans because of the association between ART and increased incidence of BWS (231,281,(285)(286)(287), SRS (288), and Angelman syndrome, which is a severe neurodevelopmental condition characterized by microcephalus, absence of speech, severe mental retardation, and frequent laughing (279,289,290).…”