Intrinsic de novo DNA methylation in early embryos enables the generation of semi-cloned mice from methylation-deficient androgenetic haploid stem cells

This SuperSeries is composed of the SubSeries listed below. Abstract Among all mammalian cell types, sperm cells exhibit one of the highest levels of DNA methylation, with ~80% of CpG sites being methylated. The role of this sperm hypermethylation in offspring development, aside from the methylation at the well-characterized H19-DMR and Dlk1-Dio3 intergenic germline DMR (IG-DMR) loci, remains largely unexplored. In mouse germ cells, removal of DNA methylation by deleting methyltransferase (Dnmt) genes causes meiotic catastrophe and infertility. To circumvent this limitation, we inactivated them instead in androgenetic haploid embryonic stem cells (AG-haESCs) lacking H19-DMR and IG-DMR to remove methylation prior to oocyte injection, and subsequently reactivated them in the resulting embryos during cleavage, to ensure Dnmt sufficiency. This strategy enabled the generation of viable offspring from unmethylated paternally-derived haploid cells. In the resulting embryos, the paternal genome rapidly reacquired methylation and was comparable to wild-type in post-implantation embryos. These semi-cloned embryos could develop to term and survived to adulthood, exhibiting near-normal morphological and physiological parameters. These findings indicate that the essential hereditary function of the paternal genome methylation is mainly restricted to the imprinted loci Igf2-H19 and Dlk1-Dio3, while methylation elsewhere appears dispensable for subsequent development, as de novo methylation activity intrinsic to the early embryo ensures epigenetic reprogramming for offspring development.