Aurora-A kinase (AURKA), a member of the serine/threonine protein kinase family, is involved in multiple steps of mitotic progression. It regulates centrosome maturation, mitotic spindle formation, and cytokinesis. While studied extensively in somatic cells, little information is known about AURKA in the early cleavage mouse embryo with respect to acentrosomal spindle assembly. In vitro experiments in which AURKA was inactivated with specific inhibitor MLN8237 during the early stages of embryogenesis documented gradual arrest in the cleavage ability of the mouse embryo. In the AURKA-inhibited 1-cell embryos, spindle formation and anaphase onset were delayed and chromosome segregation was defective. AURKA inhibition increased apoptosis during early embryonic development. In conclusion these data suggest that AURKA is essential for the correct chromosome segregation in the first mitosis as a prerequisite for normal later development after first cleavage.

• MeSH
• aurora kinasa A antagonisté a inhibitory   metabolismus   MeSH
• azepiny farmakologie   MeSH
• časosběrné zobrazování MeSH
• fluorescenční mikroskopie MeSH
• fosforylace účinky léků   MeSH
• inhibitory proteinkinas farmakologie   MeSH
• konfokální mikroskopie MeSH
• kultivace embrya MeSH
• mitóza účinky léků   fyziologie   MeSH
• myši inbrední BALB C MeSH
• myši inbrední C57BL MeSH
• pyrimidiny farmakologie   MeSH
• segregace chromozomů účinky léků   fyziologie   MeSH
• zvířata MeSH
• zygota účinky léků   fyziologie   MeSH
• Check Tag
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Polo-like kinase 1 (PLK1) is involved in essential events of cell cycle including mitosis in which it participates in centrosomal microtubule nucleation, spindle bipolarity establishment and cytokinesis. Although PLK1 function has been studied in cycling cancer cells, only limited data are known about its role in the first mitosis of mammalian zygotes. During the 1-cell stage of mouse embryo development, the acentriolar spindle is formed and the shift from acentriolar to centrosomal spindle formation progresses gradually throughout the preimplantation stage, thus providing a unique possibility to study acentriolar spindle formation. We have shown previously that PLK1 activity is not essential for entry into first mitosis, but is required for correct spindle formation and anaphase onset in 1-cell mouse embryos. In the present study, we extend this knowledge by employing quantitative confocal live cell imaging to determine spindle formation kinetics in the absence of PLK1 activity and answer the question whether metaphase arrest at PLK1-inhibited embryos is associated with low anaphase-promoting complex/cyclosome (APC/C) activity and consequently high securin level. We have shown that inhibition of PLK1 activity induces a delay in onset of acentriolar spindle formation during first mitosis. Although these PLK1-inhibited 1-cell embryos were finally able to form a bipolar spindle, not all chromosomes were aligned at the metaphase equator. PLK1-inhibited embryos were arrested in metaphase without any sign of APC/C activation with high securin levels. Our results document that PLK1 controls the onset of spindle assembly and spindle formation, and is essential for APC/C activation before anaphase onset in mouse zygotes.

• MeSH
• anafáze MeSH
• anafázi podporující komplex metabolismus   MeSH
• aparát dělícího vřeténka metabolismus   MeSH
• blastocysta MeSH
• časosběrné zobrazování MeSH
• centrozom metabolismus   MeSH
• kinetika MeSH
• kinetochory metabolismus   MeSH
• konfokální mikroskopie MeSH
• mitóza MeSH
• myši MeSH
• protein-serin-threoninkinasy antagonisté a inhibitory   metabolismus   MeSH
• proteiny buněčného cyklu antagonisté a inhibitory   metabolismus   MeSH
• protoonkogenní proteiny antagonisté a inhibitory   metabolismus   MeSH
• pteridiny farmakologie   MeSH
• zvířata MeSH
• zygota účinky léků   metabolismus   MeSH
• Check Tag
• mužské pohlaví MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Mammalian oocytes/zygotes contain atypical nucleoli that are composed exclusively of a dense fibrillar material. It has been commonly accepted that these nucleoli serve as a repository of components that are used later on, as the embryo develops, for the construction of typical tripartite nucleoli. Indeed, when nucleoli were removed from immature oocytes (enucleolation) and these oocytes were then matured, fertilized or parthenogenetically activated, development of the produced embryos ceased after one or two cleavages with no detectable nucleoli in nuclei. This indicated that zygotic nucleoli originate exclusively from oocytes, i.e. are maternally inherited. Recently published results, however, do not support this developmental biology dogma and demonstrate that maternal nucleoli in one-cell stage embryos are necessary only during a very short time period after fertilization when they serve as a major heterochromatin organizing structures. Nevertheless, it still remains to be determined, which other functions/roles the atypical oocyte/zygote nucleoli eventually have.

• MeSH
• buněčné jadérko fyziologie   MeSH
• časové faktory MeSH
• embryo savčí MeSH
• embryonální vývoj MeSH
• fertilizace MeSH
• heterochromatin fyziologie   MeSH
• lidé MeSH
• maternální dědičnost MeSH
• myši MeSH
• nukleoplasminy genetika   MeSH
• oocyty fyziologie   ultrastruktura   MeSH
• zvířata MeSH
• zygota fyziologie   MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH

• MeSH
• experimenty na zvířatech MeSH
• myši MeSH
• ovum růst a vývoj   MeSH
• techniky in vitro MeSH
• transplantace MeSH
• zmrazování MeSH
• zygota růst a vývoj   MeSH
• Check Tag
• myši MeSH

• MeSH
• fluorescence MeSH
• lipidy MeSH
• myši MeSH
• ovum analýza   MeSH
• zygota analýza   MeSH
• Check Tag
• myši MeSH

• MeSH
• buněčné dělení MeSH
• histocytochemie MeSH
• myši MeSH
• ovum MeSH
• zygota MeSH
• Check Tag
• myši MeSH

The large, compact oocyte nucleoli, sometimes referred to as nucleolus precursor bodies (NPBs), are essential for embryonic development in mammals; in their absence, the oocytes complete maturation and can be fertilized, but no nucleoli are formed in the zygote or embryo, leading to developmental failure. It has been convincingly documented that zygotes inherit the oocyte nucleolar material and form NPBs again in pronuclei. It is commonly accepted that during early embryonic development, the original compact zygote NPBs gradually transform into reticulated nucleoli of somatic cells. Here, we show that zygote NPBs are not required for embryonic and full-term development in the mouse. When NPBs were removed from late-stage zygotes by micromanipulation, the enucleolated zygotes developed to the blastocyst stage and, after transfer to recipients, live pups were obtained. We also describe de novo formation of nucleoli in developing embryos. After removal of NPBs from zygotes, they formed new nucleoli after several divisions. These results indicate that the zygote NPBs are not used in embryonic development and that the nucleoli in developing embryos originate from de novo synthesized materials.

• MeSH
• blastocysta cytologie   MeSH
• buněčné dělení MeSH
• buněčné jadérko fyziologie   MeSH
• buňky NIH 3T3 MeSH
• kultivace embrya MeSH
• kumulární buňky cytologie   MeSH
• mikromanipulace MeSH
• myši inbrední C57BL MeSH
• myši inbrední DBA MeSH
• myši inbrední ICR MeSH
• myši MeSH
• oocyty cytologie   MeSH
• přenos embrya MeSH
• těhotenství MeSH
• zvířata MeSH
• zygota růst a vývoj   MeSH
• Check Tag
• mužské pohlaví MeSH
• myši MeSH
• těhotenství MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Gene targeting in mice mainly employs homologous recombination (HR) in embryonic stem (ES) cells. Although it is a standard way for production of genetically modified mice, the procedure is laborious and time-consuming. This study describes targeting of the mouse Rosa26 locus by transcription activator-like effector nucleases (TALENs). We employed TALEN-assisted HR in zygotes to introduce constructs encoding TurboRFP and TagBFP fluorescent proteins into the first intron of the Rosa26 gene, and in this way generated two transgenic mice. We also demonstrated that these Rosa26-specific TALENs exhibit high targeting efficiency superior to that of zinc-finger nucleases (ZFNs) specific for the same targeting sequence. Moreover, we devised a reporter assay to assess TALENs activity and specificity to improve the quality of TALEN-assisted targeting.

• MeSH
• endonukleasy metabolismus   MeSH
• genetické lokusy genetika   MeSH
• genový targeting metody   MeSH
• homologní rekombinace MeSH
• myši transgenní MeSH
• myši MeSH
• sekvence nukleotidů MeSH
• zvířata MeSH
• zygota metabolismus   MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

• MeSH
• embryo savčí MeSH
• experimenty na zvířatech MeSH
• myši MeSH
• ovum růst a vývoj   ultrastruktura   MeSH
• zygota MeSH
• Check Tag
• myši MeSH

It is well known that nucleoli of fully grown mammalian oocytes are indispensable for embryonic development. Therefore, the embryos originated from previously enucleolated (ENL) oocytes undergo only one or two cleavages and then their development ceases. In our study the interspecies (mouse/pig) nucleolus transferred embryos (NuTE) were produced and their embryonic development was analyzed by autoradiography, transmission electron microscopy (TEM) and immunofluorescence (C23 and upstream binding factor (UBF)). Our results show that the re-injection of isolated oocyte nucleoli, either from the pig (P + P) or mouse (P + M), into previously enucleolated and subsequently matured porcine oocytes rescues their development after parthenogenetic activation and some of these develop up to the blastocyst stage (P + P, 11.8%; P + M, 13.5%). In nucleolus re-injected 8-cell and blastocyst stage embryos the number of nucleoli labeled with C23 in P + P and P + M groups was lower than in control (non-manipulated) group. UBF was localized in small foci within the nucleoli of blastocysts in control and P + P embryos, however, in P + M embryos the labeling was evenly distributed in the nucleoplasm. The TEM and autoradiographic evaluations showed the formation of functional nucleoli and de novo rRNA synthesis at the 8-cell stage in both, control and P + P group. In the P + M group the formation of comparable nucleoli was delayed. In conclusion, our results indicate that the mouse nucleolus can rescue embryonic development of enucleolated porcine oocytes, but the localization of selected nucleolar proteins, the timing of transcription activation and the formation of the functional nucleoli in NuTE compared with control group show evident aberrations.

• MeSH
• blastocysta cytologie   metabolismus   MeSH
• buněčné jadérko fyziologie   transplantace   MeSH
• embryo savčí cytologie   metabolismus   MeSH
• embryonální vývoj fyziologie   MeSH
• klonování organismů MeSH
• myši MeSH
• oocyty cytologie   fyziologie   MeSH
• oogeneze fyziologie   MeSH
• prasata MeSH
• přenos embrya MeSH
• těhotenství MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• těhotenství MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH