Culture of Mouse Zygotes Induces Epigenetic Changes in the Expression of the agouti viable yellow Allele in the Resulting Progeny.

Abstract

It is well recognised that perturbation of fetal growth may influence postnatal homeostasis. Recent evidence indicates that the growth environment of the preimplantation embryo may also have long-term consequences for progeny. Epigenetic modification of the genome is one molecular mechanism that could explain a persistence of altered gene expression. This study aimed to assess whether culture of zygotes through to the blastocyst stage, followed by their transfer to recipient females, induced defined epigenetic changes in gene expression that persisted through to adulthood. The agouti viable yellow (Avy) allele is epigenetically sensitive, and is a widely used model for studying epigenetic phenomena. It has active and inactive epialleles that are hypo- and hyper-methylated, respectively. Progeny inheriting the allele in its active state have yellow fur; those with the inactive epiallele have an agouti coat. Some progeny are mosaics for the two epigenetic states and have a mottled coat color. Males (Avy/a C57BL/6) carrying the epiallele were mated to wildtype females (A/A FVB). The following treatments were applied: (1) zygotes were collected and cultured for 94 hours and the resulting blastocysts transferred to recipient females; (2) blastocysts were collected from females and then transferred to recipients without being subjected to embryo culture; and (3) pregnancy was allowed to proceed without intervention. Zygotes were cultured in Sydney IVF Media suite (Cook Womens Health, Australia), a widely used commercial sequential human embryo culture media formulation. Embryos were transferred to day 3 (post coitum) pseudopregnant white Swiss recipient females. The number, sex, genotype, and epi-phenotype of the resulting progeny were recorded. The culture of zygotes caused a significant shift in the expression of the epialleles in progeny compared to embryos that were transferred without culture (P=0.014), or those born following natural matings (P<0.001). Offspring that resulted from embryo culture are more likely to have active (hypomethyalted) epialleles, resulting in a higher proportion of adult progeny with yellow fur. The distribution of epialleles was not different (P>0.05) for the two control treatments not subjected to embryo culture. Embryo culture had no effect on the survival of embryos carrying the Avy allele compared to the wildtype allele. The study shows that culture of mouse zygotes through to the blastocyst stage changed the expected post-natal expression of an epigenetically sensitive allele. Embryo culture has been shown to cause many acute changes to the pattern of gene expression, and there are case reports of human ART progeny with abnormalities with an epigenetic basis. The results of this study provide evidence for the epigenetic basis for the long-term memory of preimplantation embryos growth environment, and for robust and readily accessible methodology for addressing the underlying molecular bases for this phenomenon.