Stage-dependent changes of the nuclear architecture, envelope and lamina during mammalian early embryonic development studied with a novel 3D structured illumination microscopy protocol
Beschreibung
vor 8 Jahren
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.
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.
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