Test of the DNMT1o imprinted maintenance methylation model reveals progressive methylation loss between 8-cell and blastocyst stages.

DNMT1o, the maternal oocyte-derived isoform of DNA methyltransferase 1, is a key protein to maintain imprinted methylation. A model was proposed in 2001, and refined in 2008, where DNMT1o acts to maintain imprinted methylation exclusively at the 8-cell stage during preimplantation development. Nearly 20 years later, this is the first comprehensive analysis testing the maternal DNMT1o imprinted maintenance methylation model. We analyzed Snrpn and H19 imprinted methylation in individual control and maternal Dnmt1o-deficient mouse embryos across five preimplantation stages. Our findings challenged assumptions that DNMT1o acted solely at the 8-cell stage to produce ∼50% methylation loss in all embryos. Compared to controls, maternal Dnmt1o-deficient embryos experienced (1) progressive Snrpn and H19 imprinted methylation loss between the 8-cell to blastocyst stages, with inter-embryo variability extending beyond the predicted 50% loss; cleavage stage embryos exhibited similar methylation levels; (2) some embryos maintained maternal Snrpn and paternal H19 methylation, bypassing imprinted methylation loss; and (3) at the DNA strand level, Snrpn and H19 methylation loss was sporadic rather than complete. These findings point to potential compensation mechanisms during early preimplantation development.