Introduction: The mechanisms that dictate nucleus size are largely mysterious. Experiments in fission yeast suggest that nuclear size is dictated by the size of the cell, whereas studies in Xenopus indicate that nuclear import rates control nuclear volume. Here we set out to exploit the reductive divisions of early development present to what regulates nucleus size in mammalian embryos.
Materials and methods: We generated three-dimensional confocal images of preimplantation mouse embryos to determine nuclear/cytoplasmic volume ratio. Fluorescent recovery after photobleaching (FRAP) of GFP-tagged nuclear localization signal (GFP-NLS) was used to establish nuclear-import rates. Micromanipulation was used to probe the influence of cell volume upon nuclear size.
Results and discussion: Nuclear size decreases with successive cleavage divisions from one-cell stage to blastocyst, and nuclear/cytoplasmic ratio was relatively constant at any given developmental stage, suggesting a direct relationship between nuclear and cell size. Experimental cytoplasmic removal reduced significantly nuclear size (P<0.01), further suggesting that cell size directly influences nuclear volume. However, intriguingly, the nuclear/cytoplasmic ratio set-point changes progressively through development (from 0.057 in two-cells to 0.141 in morulae), revealing that factors other than cell volume influence nuclear size. Nuclei in experimentally-generated embryos with double-sized blastomeres were of normal size, also suggesting that nuclear size is controlled by a developmental programme. The rate of nuclear import was identical in two-, four-, and eight-cell stage embryos (P>0.1), revealing that in contrast to other systems, nuclear import does not explain the developmental programme. This nuclear volume in mammalian embryos is determined cooperatively by cell size and developmentally-regulated cytoplasmic factors.
02 Sep 2014 - 04 Sep 2014