Control mechanisms of spindle assembly and chromosome segregation are vital for preventing aneuploidy during cell division. The mammalian germ cells and embryos are prone to chromosome segregation errors, and the resulting aneuploidy is a major cause of termination of development or severe developmental disorders. Here we focused on early mouse embryos, and using combination of methods involving microinjection, immunodetection and confocal live cell imaging, we concentrated on the Spindle Assembly Checkpoint (SAC) and Anaphase Promoting Complex/Cyclosome (APC/C). These are two important mechanisms cooperating during mitosis to ensure accurate chromosome segregation, and assessed their activity during the first two mitoses after fertilization. Our results showed, that in zygotes and 2-cell embryos, the SAC core protein Mad1 shows very low levels on kinetochores in comparison to oocytes and its interaction with chromosomes is restricted to a short time interval after nuclear membrane disassembly (NEBD). Exposure of 2-cell embryos to low levels of spindle poison does not prevent anaphase, despite the spindle damage induced by the drug. Lastly, the APC/C is activated coincidentally with NEBD before the spindle assembly completion. This early onset of APC/C activity, together with precocious relocalization of Mad1 from chromosomes, prevents proper surveillance of spindle assembly by SAC. The results contribute to the understanding of the origin of aneuploidy in early embryos.

• Klíčová slova
• Mad1, anaphase, anaphase-promoting complex, chromosome segregation, embryo, spindle, spindle assembly checkpoint,
• Publikační typ
• časopisecké články MeSH

The onset of an early development is, in mammals, characterized by profound changes of multiple aspects of cellular morphology and behavior. These are including, but not limited to, fertilization and the merging of parental genomes with a subsequent transition from the meiotic into the mitotic cycle, followed by global changes of chromatin epigenetic modifications, a gradual decrease in cell size and the initiation of gene expression from the newly formed embryonic genome. Some of these important, and sometimes also dramatic, changes are executed within the period during which the gene transcription is globally silenced or not progressed, and the regulation of most cellular activities, including those mentioned above, relies on controlled translation. It is known that the blastomeres within an early embryo are prone to chromosome segregation errors, which might, when affecting a significant proportion of a cell within the embryo, compromise its further development. In this review, we discuss how the absence of transcription affects the transition from the oocyte to the embryo and what impact global transcriptional silencing might have on the basic cell cycle and chromosome segregation controlling mechanisms.

• Klíčová slova
• cell cycle, embryo, oocyte, transcriptional repression, translation,
• MeSH
• buněčný cyklus genetika   MeSH
• chromatin genetika   MeSH
• embryo savčí fyziologie   MeSH
• embryonální vývoj genetika   MeSH
• genetická transkripce genetika   MeSH
• lidé MeSH
• segregace chromozomů genetika   MeSH
• umlčování genů fyziologie   MeSH
• vývojová regulace genové exprese genetika   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• chromatin MeSH

Chromosome segregation during female meiosis is frequently incorrect with severe consequences including termination of further development or severe disorders, such as Down syndrome. Accurate chromosome segregation requires tight control of a protease called separase, which facilitates the separation of sister chromatids by cohesin cleavage. There are several control mechanisms in place, including the binding of specific protein inhibitor securin, phosphorylation by cyclin-dependent kinase 1 (CDK1), and complex with SGO2 and MAD2 proteins. All these mechanisms restrict the activation of separase for the time when all chromosomes are properly attached to the spindle. In our study, we focused on securin and compared the expression profile of endogenous protein with exogenous securin, which is widely used to study chromosome segregation. We also compared the dynamics of securin proteolysis in meiosis I and meiosis II. Our study revealed that the expression of both endogenous and exogenous securin in oocytes is compartmentalized and that this protein accumulates on the spindle during meiosis I. We believe that this might have a direct impact on the regulation of separase activity in the vicinity of the chromosomes.

• Klíčová slova
• meiosis, mouse, oocyte, securin, separase,
• Publikační typ
• časopisecké články MeSH

Aneuploidy is the most frequent single cause leading into the termination of early development in human and animal reproduction. Although the mouse is frequently used as a model organism for studying the aneuploidy, we have only incomplete information about the frequency of numerical chromosomal aberrations throughout development, usually limited to a particular stage or assumed from the occurrence of micronuclei. In our study, we systematically scored aneuploidy in in vivo mouse embryos, from zygotes up to 16-cell stage, using kinetochore counting assay. We show here that the frequency of aneuploidy per blastomere remains relatively similar from zygotes until 8-cell embryos and then increases in 16-cell embryos. Due to the accumulation of blastomeres, aneuploidy per embryo increases gradually during this developmental period. Our data also revealed that the aneuploidy from zygotes and 2-cell embryos does not propagate further into later developmental stages, suggesting that embryos suffering from aneuploidy are eliminated at this stage. Experiments with reconstituted live embryos revealed, that hyperploid blastomeres survive early development, although they exhibit slower cell cycle progression and suffer frequently from DNA fragmentation and cell cycle arrest.

• MeSH
• aneuploidie * MeSH
• blastomery cytologie   metabolismus   MeSH
• buněčný cyklus MeSH
• embryo savčí cytologie   metabolismus   MeSH
• embryonální vývoj * MeSH
• fertilizace in vitro MeSH
• myši MeSH
• těhotenství MeSH
• zvířata MeSH
• zygota cytologie   metabolismus   MeSH
• Check Tag
• myši MeSH
• těhotenství MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

In several species, including Xenopus, mouse and human, two members of cyclin A family were identified. Cyclin A2, which is ubiquitously expressed in dividing cells and plays role in DNA replication, entry into mitosis and spindle assembly, and cyclin A1, whose function is less clear and which is expressed in spermatocytes, leukemia cells and in postmitotic multiciliated cells. Deletion of the gene showed that cyclin A1 is essential for male meiosis, but nonessential for female meiosis. Our results revealed, that the cyclin A1 is not only dispensable in oocytes, we show here that its expression is in fact undesirable in these cells. Our data demonstrate that the APC/C and proteasome in oocytes are unable to target sufficiently cyclin A1 before anaphase, which leads into anaphase arrest and direct inhibition of separase. The cyclin A1-induced cell cycle arrest is oocyte-specific and the presence of cyclin A1 in early embryos has no effect on cell cycle progression or chromosome division. Cyclin A1 is therefore not only an important cell cycle regulator with biased expression in germline, being essential for male and damaging for female meiosis, its persistent expression during anaphase in oocytes shows fundamental differences between APC/C function in oocytes and in early embryos.

• MeSH
• anafáze * MeSH
• cyklin A1 fyziologie   MeSH
• cyklin A2 fyziologie   MeSH
• fluorescenční mikroskopie MeSH
• meióza MeSH
• metafáze MeSH
• mikroinjekce MeSH
• myši MeSH
• oocyty cytologie   MeSH
• proteasomový endopeptidasový komplex fyziologie   MeSH
• segregace chromozomů * MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• Ccna1 protein, mouse MeSH Prohlížeč
• CCNA2 protein, mouse MeSH Prohlížeč
• cyklin A1 MeSH
• cyklin A2 MeSH
• proteasomový endopeptidasový komplex MeSH

In both mitosis and meiosis, metaphase to anaphase transition requires the activity of a ubiquitin ligase known as anaphase promoting complex/cyclosome (APC/C). The activation of APC/C in metaphase is under the control of the checkpoint mechanism, called the spindle assembly checkpoint (SAC), which monitors the correct attachment of all kinetochores to the spindle. It has been shown previously in somatic cells that exposure to a small molecule inhibitor, prodrug tosyl-l-arginine methyl ester (proTAME), resulted in cell cycle arrest in metaphase, with low APC/C activity. Interestingly, some reports have also suggested that the activity of SAC is required for this arrest. We focused on the characterization of proTAME inhibition of cell cycle progression in mammalian oocytes and embryos. Our results show that mammalian oocytes and early cleavage embryos show dose-dependent metaphase arrest after exposure to proTAME. However, in comparison to the somatic cells, we show here that the proTAME-induced arrest in these cells does not require SAC activity. Our results revealed important differences between mammalian oocytes and early embryos and somatic cells in their requirements of SAC for APC/C inhibition. In comparison to the somatic cells, oocytes and embryos show much higher frequency of aneuploidy. Our results are therefore important for understanding chromosome segregation control mechanisms, which might contribute to the premature termination of development or severe developmental and mental disorders of newborns.

• Klíčová slova
• anaphase promoting complex, cell cycle, meiosis, oocytes, proTAME, spindle assembly checkpoint,
• MeSH
• anafázi podporující komplex metabolismus   MeSH
• embryo savčí účinky léků   metabolismus   MeSH
• embryonální vývoj účinky léků   MeSH
• kontrolní body M fáze buněčného cyklu * MeSH
• myši MeSH
• oocyty účinky léků   růst a vývoj   metabolismus   MeSH
• prekurzory léčiv MeSH
• skot MeSH
• tosylargininmethylester aplikace a dávkování   farmakologie   MeSH
• vztah mezi dávkou a účinkem léčiva MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• skot MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• anafázi podporující komplex MeSH
• prekurzory léčiv MeSH
• tosylargininmethylester MeSH

Early embryonic development is characterized by a plethora of very complex and simultaneously operating processes, which are constantly changing cellular morphology and behaviour. After fertilization, blastomeres of the newly created embryo undergo global epigenetic changes and simultaneously initiate transcription from the zygotic genome and differentiation forming separate cell lineages. Some of these mechanisms were extensively studied during the last several decades and valuable insight was gained into how these processes are regulated at the molecular level. We have, however, a still very limited understanding of how multiple events are coordinated during rapid development of an early mammalian embryo. In this review, we discuss some aspects of early embryonic development in mammals, namely the fidelity of chromosome segregation and occurrence of aneuploidy, as well as the clinical applications of cell cycle monitoring in human embryos.

• MeSH
• aneuploidie MeSH
• aparát dělícího vřeténka metabolismus   MeSH
• blastomery metabolismus   MeSH
• buněčný cyklus genetika   MeSH
• embryo savčí cytologie   metabolismus   MeSH
• embryonální vývoj genetika   MeSH
• lidé MeSH
• segregace chromozomů genetika   MeSH
• těhotenství MeSH
• zvířata MeSH
• zygota cytologie   metabolismus   MeSH
• Check Tag
• lidé MeSH
• těhotenství MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH