Balanced DNA-to-Cytoplasm Ratio at the 2-Cell Stage Is Critical for Mouse Preimplantation Development

Abstract

Vertebrate development typically begins with a diploid fertilized egg but can also be initiated parthenogenetically with a haploid maternal genome, though such development fails to reach completion. Among vertebrates, mammalian haploid embryos are particularly prone to arrest during early preimplantation stages for reasons that remain unclear. Here, using mouse embryos with varying cell sizes and genome ploidy, we show that developmental arrest is primarily caused not by haploidy itself but an imbalance between genome ploidy and cytoplasmic volume. Haploid embryos exhibit cytokinesis failure, frequently accompanied by chromosome segregation failure due to malformed spindles beginning at the second mitosis, with about half of the affected blastomeres subsequently arresting before the morula stage. Strikingly, restoring the DNA-to- cytoplasm (D/C) ratio by halving the cytoplasmic volume alleviates these defects. Similarly, diploid embryos with doubled cytoplasmic volume show mitotic and developmental abnormalities resembling those of haploid embryos, whereas tetraploid embryos with the same volume develop normally. These findings demonstrate that a proper D/C ratio, rather than genome ploidy, is critical for mouse embryos to reach the morula stage. Moreover, halving the D/C ratio at later developmental stages, such as the late G2 phase of the 1-cell stage or during the 2-cell stage, progressively improves developmental outcomes. When the D/C ratio is reduced at the end of the 2-cell stage, development proceeds normally. Our findings identify a critical temporal window during which a balanced D/C ratio is essential for mitotic fidelity and successful preimplantation development. Graphic Abstract O_FIG O_LINKSMALLFIG WIDTH=198 HEIGHT=200 SRC="FIGDIR/small/651589v1_ufig1.gif" ALT="Figure 1"> View larger version (49K): org.highwire.dtl.DTLVardef@72a664org.highwire.dtl.DTLVardef@10a22c1org.highwire.dtl.DTLVardef@6fbedforg.highwire.dtl.DTLVardef@5c5b1d_HPS_FORMAT_FIGEXP M_FIG C_FIG O_LIHalved D/C ratio impairs preimplantation development in mouse embryos C_LIO_LIHalved D/C ratio causes cytokinesis failure from the second mitosis C_LIO_LIDelaying D/C ratio halving to late 2-cell stage alleviates developmental defects C_LI