Parent-specific methylation reprogramming in early embryos: implications for mammalian development and embryo biotechnology
Final Report Abstract
There are considerable species differences in postzygotic methylation reprogramming. In mouse, the paternal genome is actively and drastically demethylated within a few hours after fertilization. Methycytosine (mC) staining of in vivo fertilized rabbit embryos revealed equally high methylation levels of the paternal and maternal genomes that were largely maintained from the zygote up to the 8-16-cell stage. The methylation patterns of rabbit embryos derived from somatic cell nuclear transfer were similar to those of in vivo fertilized embryos. FISH with chromosome-specific BACs demonstrated significantly increased chromosomal aneuploidy rates in cloned rabbit embryos, compared to in vivo fertilized embryos. Thus, the cloning procedure itself has detrimental effects on chromosome stability. The temporally and spatially highly co-ordinated reprogramming process is susceptible to failures affecting embryonal development. Genome-wide methylation alterations, which can be detected at the light-microscopic level, lead to early embryo loss. Following mC staining, mouse embryos derived from superovulated matings showed an approximately twofold increased frequency (14-20%) of genome-wide methylation disturbances and preimplantation developmental arrest, compared to non-superovulated females (5-10%). The dramatically differing rates (10-80%) of methylation reprogramming and developmental defects of in vitro fertilized mouse embryos that were cultured under different conditions demonstrated the influences of a more or less suboptimal environment at and/or shortly after fertilization. In addition, we have analysed the effects of ovarian stimulation on the methylation patterns of the differentially methylated imprinting control regions (DMRs) of Igf2 H19 and "Snrpn" in mouse 4-cell, 8-cell, and morula stages. Bisulphite sequencing showed a significant loss of DNA methylation in the DMRs of superovulated embryos, compared to embryos from non-superovulated matings. Asynchronous replication is a cytogenetic hallmark of imprinted chromosome regions. To reveal at which time during pre-implantation development non-imprinted regions switch from asynchronous to synchronous replication, we performed a comparative replication timing analysis of two imprinted and two non-imprinted control regions in mouse one-cell, 2-cell, 4 cell, 8-cell, and morula embryos, using two colour interphase FISH. Non-imprinted regions exhibited asynchronous replication patterns in one-cell and 2-cell embryos, however a transition from asynchronous to synchronous replication occurred between the 2-cell and morula stages. Imprinted genes maintained their asynchronous behaviour throughout pre-implantation development.
