Balanced DNA-to-cytoplasm ratio at the 2-cell stage is critical for mouse preimplantation development.

Among vertebrates, mammalian haploid embryos are highly prone to early developmental arrest for reasons that remain unclear. Using mouse preimplantation embryos with altered cell size and ploidy, we show that arrest primarily results from an imbalance between ploidy and cytoplasmic volume, rather than from haploidy itself. Haploid and double-sized diploid embryos exhibit spindle and contractile ring defects, resulting in chromosome nondisjunction and cytokinesis failure beginning at the second mitosis. Reducing cytoplasmic volume to restore the DNA-to-cytoplasm ratio rescues these abnormalities. Delaying the reduction of the DNA-to-cytoplasm ratio until the late 2-cell stage prevents developmental defects, revealing a critical temporal window for this ratio. Moreover, moderate transcriptional inhibition at this stage in normal diploid embryos induces similar errors. Our findings highlight that in mammalian embryos, where major zygotic genome activation occurs in large cells, balance between ploidy and cytoplasmic volume is necessary to sustain sufficient protein levels for mitosis and development.