| Popis: |
In the present study, we investigated the changes of the nuclear envelope and its underlying lamina, as well as features of higher order chromatin organisation in bovine embryos generated by in vitro fertilization during pre-implantation development. We used super-resolution, 3-dimensional structured illumination microscopy combined with 2-colour immunostaining of the nucleoporin Nup153 and lamin B serving as markers for nuclear pore complexes (NPC) and the nuclear lamina, respectively. DNA was counterstained with 4′,6-diamidino-2-phenylindole (DAPI). We examined 20 nuclei for the zygote (10 male pronuclei and 10 female pronuclei; n = 10) and the blastocyst (10 trophectoderm and 10 inner cell mass nuclei; n = 1) stage, and 10 nuclei for each the 2-cell (n = 5), 4-cell (n = 3), 8-cell (n = 2), 19-cell (n = 1), and morula (n = 1) stages. We report 4 major findings: (1) At the onset of major genome activation (MGA) nuclei showed a peripheral location of chromosome territories (CT), separated by wide IC channels and surrounding a major lacuna depleted of chromatin. The NPC were exclusively present at sites where DAPI-stained DNA contacted the nuclear lamina, whereas extended lamina regions without such contacts lacked NPC. In post-MGA nuclei, the CT formed a higher order chromatin network distributed throughout the entire nuclear space and the major lacuna disappeared. In line with a switch to a ubiquitous lining of DNA at the lamina, NPC were also uniformly distributed throughout the entire nuclear envelope. These findings shed new light on the conditions that control the integration of NPC into the nuclear envelope. (2) The switch from maternal to embryonic production of mRNA was accompanied by an increased amount of nuclear lamina invaginations covered with NPC, which may serve the increased demands of mRNA export and protein import. (3) Other invaginations, as well as interior nuclear segments and vesicles without contact to the nuclear envelope, were exclusively positive for lamin B. Because an increase in these lamin B positive structures occurred in concert with a massive nuclear volume reduction, we suggest that they reflect a mechanism for fitting the nuclear envelope and its lamina to a shrinking nuclear size throughout bovine pre-implantation development. (4) Throughout the cytoplasm, randomly distributed extranuclear clusters of Nup153 without associated lamin B were frequently observed from the zygote stage up to MGA. These clusters may represent a deposit of maternal Nup153 and likely other nucleoporines not studied here. Corresponding RNA-Seq data revealed deposits of spliced, maternally provided NUP153 mRNA and little unspliced RNA before MGA, which increased strongly at the initiation of embryonic NUP153 expression at MGA. After MGA, these clusters were exclusively located at or near the nuclear border and were no longer present at the morula stage and later. In conclusion, our findings demonstrate the dynamic adaptation of the nuclear envelope to the special needs of bovine pre-implantation development and show the necessity of chromatin association for the integration of nuclear pores into the nuclear envelope. |
