92 NUCLEAR INVAGINATIONS ADAPT TO RABBIT EARLY EMBRYONIC DEVELOPMENT

Abstract

Nuclear invaginations carrying nuclear pores may facilitate increased mRNA export and protein import to areas inside the nucleus remote from the nuclear border. In this study on rabbit embryos, we investigated whether large early embryonic nuclei and the increased import/export demands around major embryonic genome activation (MGA) at the 8-cell stage affected the quantity of nuclear invaginations carrying nuclear pores. To achieve this objective, we used super-resolution 3-dimensional structured illumination microscopy on 10 pronuclei or nuclei per stage of 23 in vivo-fertilized and in vitro-cultured embryos stained with antibodies for the nucleoporin NUP153 and lamin B and stained with 4′,6-diamidino-2-phenylindole (DAPI) for chromatin. Statistical comparisons between stages were performed using the Wilcoxon rank-sum test. At the zygote stage, the female pronucleus displayed on average 16.5 and the male pronucleus featured on average 15.25 wide and narrow nuclear envelope invaginations, carrying large or tiny amounts of cytoplasm. Subsequent stages showed predominantly wide invaginations targeting nucleoli. The contact areas between nucleoli and invaginations were free of nuclear pores. In contrast, narrow invaginations, which are the almost exclusive type of invaginations during cattle and mouse pre-implantation development, were not in contact with nucleoli. At the 2-cell stage, the number of invaginations increased to an average of 27.3 invaginations per nucleus (P < 0.05) and increased again to peak at the 4-cell stage with 51.2 invaginations per nucleus (P < 0.01). At the 8-cell stage (MGA), the amount of nuclear invaginations was reduced to 25.4 invaginations per nucleus (P < 0.01). The reduced number of nuclear invaginations at the 8-cell stage could be associated with a significant decrease in average nuclear volume from 2593 µm³ at the 4-cell stage to 960 µm³ at the 8-cell stage (P < 0.001) and a subsequent reduced average distance from areas inside the nucleus to the nuclear border. Nuclear invagination numbers continued their decline at the 21-cell stage with 5.2 invaginations per nucleus (P < 0.001), whereas nuclear volumes decreased to 618 µm³ (P < 0.001). The morula stage, with 6.9 invaginations per nucleus (P = 0.9), and the blastocyst stage, with 4.5 invaginations per nucleus (P = 0.5), showed no more significant changes. Large NUP153 cytoplasmic clusters present before MGA may represent a maternally provided NUP153 deposit. MGA may mark the switch from the use of a NUP153 deposit to on-demand production. Additionally, over- and under-representation analyses on mitotic blastomeres revealed that NUP153 association with chromatin is initiated during metaphase before the initiation of the regeneration of the lamina (P < 0.001; chi-squared goodness-of-fit test). In conclusion, rabbit embryonic development is accompanied by stage-dependent changes of the amount of nuclear invaginations carrying nuclear pores. Although cattle and mouse embryos almost exclusively feature narrow invaginations restricted to the nuclear periphery and not in contact with nucleoli, rabbit embryos feature additional wide invaginations that can reach across the nucleus and target nucleoli.