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Popken, Jens (2016): Stage-dependent changes of the nuclear architecture, envelope and lamina during mammalian early embryonic development studied with a novel 3D structured illumination microscopy protocol. Dissertation, LMU München: Faculty of Biology



Super-resolution fluorescence microscopy performed via 3D structured illumination microscopy (3D-SIM) features an 8-fold volumetric resolution improvement over conventional microscopy and is well established on flat, adherent cells. However, blastomeres in mammalian embryos are non-adherent, round and large. Scanning whole mount mammalian embryos with 3D-SIM is prone to failure due to non-adherent embryos moving during scanning and a large distance to the cover glass. The biggest challenge and achievement of this doctorate thesis was the development of a novel method to perform 3D-SIM on mammalian embryos (“3D structured illumination microscopy of mammalian embryos and spermatozoa” published in BMC Developmental Biology). The development and fine-tuning of this method took over two years due to the time-intense generation of embryos and the subsequent two day long embryo staining, embedding and scanning with steps that required novel techniques such as micromanipulation which was not associated with sample preparation prior to this protocol. Problem identification was time-intensive since each of the numerous steps necessary could negatively affect the image quality. This method was fine-tuned during three studies. The first study “Reprogramming of fibroblast nuclei in cloned bovine embryos involves major structural remodeling with both striking similarities and differences to nuclear phenotypes of in vitro fertilized embryos” (published in Nucleus) investigates the profound changes of nuclear architecture during cattle preimplantation development of embryos generated by somatic cell nuclear transfer (SCNT) and in vitro fertilization (IVF). Fibroblast nuclei in embryos generated by SCNT go through similar changes in nuclear architecture as embryos generated by IVF. In both embryo types the occurrence of a large, chromatin-free lacuna in the center of nuclei around major embryonic genome activation (EGA) was noted. Similarly, the chromosome territory-interchromatin compartment (CT-IC) model applied to both types of embryos, featuring a lacuna or not, with an enrichment of RNA polymerase II and H3K4me3, a histone modification for transcriptionally competent chromatin, in less concentrated chromatin and an enrichment of H3K9me3, a transcriptionally restrictive histone modification, in more concentrated chromatin. However, large, highly concentrated H3K4me3 and H3K9me3 clusters were noted in both embryo types at chromatin concentrations that did not fit to the model. The chromatin-free lacunas were highly enriched in newly synthesized mRNA. The second study “Remodeling of the Nuclear Envelope and Lamina during Bovine Preimplantation Development and Its Functional Implications” (published in PLOS ONE) presents the changes of the nuclear envelope and lamina during bovine preimplantation development. Before major EGA, chromatin-free areas of the nuclear periphery were also free of nuclear pore complexes (NPCs), whereas after major EGA, the entire nuclear periphery was equipped with at least a fine layer of chromatin and associated NPCs. Three types of nuclear invaginations were predominant at different stages. The most common invagination was lamin B and NUP153 positive and was most prominent between the 2-cell and 8-cell stages until the onset of major EGA. Lamin B positive, but NUP153 negative invaginations were most prominent during stages with large nuclear volume and surface reductions. The least common invagination was lamin B negative but NUP153 positive and occurred almost exclusively at the morula stage. RNA-Seq and 3D-SIM data showed large deposits of spliced NUP153 mRNA and cytoplasmic NUP153 protein clusters until shortly after major EGA. NUP153 association with chromatin was initiated at metaphase. The third study “Stage-dependent remodeling of the nuclear envelope and lamina during rabbit early embryonic development” (published in the Journal of Reproduction and Development) demonstrated that rabbit embryonic nuclei feature a nuclear invagination type containing a large volume of cytoplasm that provides cytoplasmic proximity to nucleoli in addition to the small volume invaginations that were previously observed in bovine nuclei. The underlying mechanism for these two invaginations must differ from each other since small volume invaginations were frequently emanating from large volume invaginations emanating from the nuclear border but large volume invaginations were never emanating from small volume invaginations emanating from the nuclear border. Abundance of import/export competent invaginations featuring NPCs peaked at the 4-cell stage, which is the last stage before a drastic nuclear volume decline and also the last stage before major EGA is initiated at the 8- to 16-cell stage. Import/export incompetent invaginations positive for lamin B but not NUP153 peaked at the 2-cell stage. This was the stage with the largest variability in nuclear volumes. This may hint at an interphase nuclear surface reduction mechanism. Additionally, previously generated but unpublished 3D-FISH data about the localization changes of a stably inserted reporter gene upon activation in cloned bovine embryos was analyzed and documented in the study “Positional changes of a pluripotency marker gene during structural reorganization of fibroblast nuclei in cloned early bovine embryos” (published in Nucleus). This study showed that the stably inserted OCT-4 reporter gene “GOF” in bovine fetal fibroblasts was initially moved towards the nuclear interior in day 2 bovine embryos generated by SCNT of bovine fetal fibroblasts. However, in day 4 SCNT embryos the localization of GOF had moved towards the periphery while it was still activated. Its carrier chromosome territory did not significantly move differently compared with the non-carrier homolog. Constant proximity of GOF to its carrier chromosome territory ruled out a movement by giant loops. In cooperation with the Department of Histology and Embryology of the Ege University (Izmir, Turkey) the destructive effects of cryopreservation on blastomere integrity were analyzed in the study “Ultra-Structural Alterations in In Vitro Produced Four-Cell Bovine Embryos Following Controlled Slow Freezing or Vitrification” (published in Anatomia, Histologia, Embryologia). The cryopreservation method slow freezing caused more damage to blastomeres and to the zona pellucida than its fast freezing alternative vitrification. This was most likely caused by ice crystal formation and the longer exposure to the toxic side effects of cryoprotectants before freezing was complete.