Abstract
The present study demonstrates a major remodeling of the nuclear envelope and its underlying lamina during bovine preimplantation development. Up to the onset of major embryonic genome activation (MGA) at the 8-cell stage nuclei showed a non-uniform distribution of nuclear pore complexes (NPCs). NPCs were exclusively present at sites where DNA contacted the nuclear lamina. Extended regions of the lamina, which were not contacted by DNA, lacked NPCs. In post-MGA nuclei the whole lamina was contacted rather uniformly by DNA. Accordingly, NPCs became uniformly distributed throughout the entire nuclear envelope. These findings shed new light on the conditions which control the integration of NPCs into the nuclear envelope. The switch from maternal to embryonic production of mRNAs was accompanied by multiple invaginations covered with NPCs, which may serve the increased demands of mRNA export and protein import. Other invaginations, as well as interior nuclear segments and vesicles without contact to the nuclear envelope, were exclusively positive for lamin B. Since the abundance of these invaginations and vesicles increased 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 during bovine preimplantation development. In addition, a deposit of extranuclear clusters of NUP153 (a marker for NPCs) without associated lamin B was frequently observed from the zygote stage up to MGA. Corresponding RNA-Seq data revealed deposits of spliced, maternally provided NUP153 mRNA and little unspliced, newly synthesized RNA prior to MGA, which increased strongly at the initiation of embryonic expression of NUP153 at MGA.
Highlights
We observed a massive reorganization of nuclear architecture in bovine preimplantation embryos generated either by in vitro fertilization (IVF) or by somatic cell nuclear transfer (SCNT) [1,2]
In our study we combined DAPI staining of DNA with indirect immunostaining of NUP153 and lamin B
Nuclear pore complexes (NPCs) clusters, which were indicated by confluence of NUP153 signals, appeared more prominent in the large pre-major embryonic genome activation (MGA) nuclei compared with the smaller post-MGA nuclei (S2 Fig)
Summary
We observed a massive reorganization of nuclear architecture in bovine preimplantation embryos generated either by in vitro fertilization (IVF) or by somatic cell nuclear transfer (SCNT) [1,2] These changes were most prominent during the transit through minor and major genome activation (mGA and MGA) which occur at the 2-cell stage [3] and at the 8-cell stage [4], respectively. After MGA, CTs were redistributed throughout the nucleus and showed features of nuclear architecture typical for somatic cell types This conventional nuclear phenotype is characterized by a layer of densely DAPI-stained chromatin beneath the nuclear envelope and around nucleoli, a non-random radial chromatin arrangement with gene-poor chromatin enriched in the nuclear periphery and gene-rich chromatin towards the nuclear center [5,6,7,8]. Thereafter fixation of antibodies was performed with 4% PFA in PBS for 10 min, followed by washing twice in PBS. Embryos or individual blastomeres were attached to precision cover glasses (18 mm x 18 mm; 170 ± 5 μm; Carl Roth, Germany; LH22.1) in PBS and embedded in Vectashield (Vector Laboratories)
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