Mammalian oocytes are arrested at meiotic prophase I. The dual-specificity phosphatase CDC25B is essential for cyclin-dependent kinase 1 (CDK1) activation that drives resumption of meiosis. CDC25B reverses the inhibitory effect of the protein kinases WEE1 and MYT1 on CDK1 activation. Cdc25b-/- female mice are infertile because oocytes cannot activate CDK1. To identify a role for CDC25B following resumption of meiosis, we restored CDK1 activation in Cdc25b-/- oocytes by inhibiting WEE1 and MYT1, or expressing EGFP-CDC25A or constitutively active EGFP-CDK1 from microinjected complementary RNAs. Forced CDK1 activation in Cdc25b-/- oocytes allowed resumption of meiosis, but oocytes mostly arrested at metaphase I (MI) with intact spindles. Similarly, approximately a third of Cdc25b+/- oocytes with a reduced amount of CDC25B arrested in MI. MI-arrested Cdc25b-/- oocytes also displayed a transient decrease in CDK1 activity similar to Cdc25b+/+ oocytes during the MI-MII transition, whereas Cdc25b+/- oocytes exhibited only a partial anaphase-promoting complex/cyclosome activation and anaphase I entry. Thus, CDC25B is necessary for the resumption of meiosis and the MI-MII transition.

• Klíčová slova
• Anaphase I, CDC25B, Meiotic maturation, Mouse oocytes, Resumption of meiosis,
• MeSH
• anafáze MeSH
• anafázi podporující komplex metabolismus   MeSH
• fosfatasy cdc25 MeSH
• meióza * MeSH
• metafáze MeSH
• myši MeSH
• oocyty * metabolismus   MeSH
• savci MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• anafázi podporující komplex MeSH
• Cdc25b protein, mouse MeSH Prohlížeč
• fosfatasy cdc25 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

Optimal culture conditions are essential for successful IVM of mammalian oocytes and for their further development into an embryo. In the present study we used live cell imaging microscopy to assess the effects of suboptimal culture temperature on various aspects of IVM, including duration of meiosis I, dynamics of polar body extrusion, chromosome congression, anaphase-promoting complex/cyclosome (APC/C) activation and aneuploidy. The data showed that even a small deviation from the optimal incubation temperature causes marked changes in the duration and synchronicity of meiosis, APC/C activity and the frequency of chromosome congression and segregation errors. In vitro manipulation and maturation of germ cells is widely used in both human and animal artificial reproduction techniques. Mammalian oocytes are naturally prone to chromosomal segregation errors, which are responsible for severe mental and developmental disorders. The data presented herein demonstrate that exposure of mouse oocytes to suboptimal temperature during manipulation and maturation could further increase the frequency of chromosome segregation defects in these cells.

• MeSH
• anafázi podporující komplex metabolismus   MeSH
• aneuploidie * MeSH
• buněčné kultury metody   MeSH
• chromozomální aberace * MeSH
• meióza fyziologie   MeSH
• myši MeSH
• oocyty cytologie   metabolismus   MeSH
• segregace chromozomů * MeSH
• teplota * MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• anafázi podporující komplex MeSH

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

Chromosome segregation errors are highly frequent in mammalian female meiosis, and their incidence gradually increases with maternal age. The fate of aneuploid eggs is obviously dependent on the stringency of mechanisms for detecting unattached or repairing incorrectly attached kinetochores. In case of their failure, the newly formed embryo will inherit the impaired set of chromosomes, which will have severe consequences for its further development. Whether spindle assembly checkpoint (SAC) in oocytes is capable of arresting cell cycle progression in response to unaligned kinetochores was discussed for a long time. It is known that abolishing SAC increases frequency of chromosome segregation errors and causes precocious entry into anaphase; SAC, therefore, seems to be essential for normal chromosome segregation in meiosis I. However, it was also reported that for anaphase-promoting complex (APC) activation, which is a prerequisite for entering anaphase; alignment of only a critical mass of kinetochores on equatorial plane is sufficient. This indicates that the function of SAC and of cooperating chromosome attachment correction mechanisms in oocytes is different from somatic cells. To analyze this phenomenon, we used live cell confocal microscopy to monitor chromosome movements, spindle formation, APC activation and polar body extrusion (PBE) simultaneously in individual oocytes at various time points during first meiotic division. Our results, using oocytes from aged animals and interspecific crosses, demonstrate that multiple unaligned kinetochores and severe congression defects are tolerated at the metaphase to anaphase transition, although such cells retain sensitivity to nocodazole. This indicates that checkpoint mechanisms, operating in oocytes at this point, are essential for accurate timing of APC activation in meiosis I, but they are insufficient in detection or correction of unaligned chromosomes, preparing thus conditions for propagation of the aneuploidy to the embryo.

• MeSH
• anafáze MeSH
• anafázi podporující komplex MeSH
• aneuploidie MeSH
• časosběrné zobrazování metody   MeSH
• histony genetika   metabolismus   MeSH
• kinetochory metabolismus   MeSH
• komplexy ubikvitinligas genetika   metabolismus   MeSH
• konfokální mikroskopie metody   MeSH
• kontrolní body M fáze buněčného cyklu MeSH
• metafáze MeSH
• mikroinjekce MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• oocyty cytologie   metabolismus   MeSH
• párování chromozomů * MeSH
• proteolýza MeSH
• savčí chromozomy genetika   metabolismus   MeSH
• savci MeSH
• segregace chromozomů * MeSH
• sekurin MeSH
• transportní proteiny genetika   metabolismus   MeSH
• tubulin genetika   metabolismus   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
• anafázi podporující komplex MeSH
• histony MeSH
• komplexy ubikvitinligas MeSH
• sekurin MeSH
• transportní proteiny MeSH
• tubulin MeSH

Cell cycle control must be modified at meiosis to allow two divisions to follow a single round of DNA replication, resulting in ploidy reduction. The mechanisms that ensure meiosis termination at the end of the second and not at the end of first division are poorly understood. We show here that Arabidopsis thaliana TDM1, which has been previously shown to be essential for meiotic termination, interacts directly with the Anaphase-Promoting Complex. Further, mutations in TDM1 in a conserved putative Cyclin-Dependant Kinase (CDK) phosphorylation site (T16-P17) dominantly provoked premature meiosis termination after the first division, and the production of diploid spores and gametes. The CDKA;1-CYCA1.2/TAM complex, which is required to prevent premature meiotic exit, phosphorylated TDM1 at T16 in vitro. Finally, while CYCA1;2/TAM was previously shown to be expressed only at meiosis I, TDM1 is present throughout meiosis. These data, together with epistasis analysis, lead us to propose that TDM1 is an APC/C component whose function is to ensure meiosis termination at the end of meiosis II, and whose activity is inhibited at meiosis I by CDKA;1-TAM-mediated phosphorylation to prevent premature meiotic exit. This provides a molecular mechanism for the differential decision of performing an additional round of division, or not, at the end of meiosis I and II, respectively.

• MeSH
• anafázi podporující komplex metabolismus   MeSH
• Arabidopsis cytologie   genetika   MeSH
• biologické modely MeSH
• chromozomy rostlin genetika   MeSH
• cykliny genetika   metabolismus   MeSH
• dominantní geny MeSH
• fosforylace MeSH
• fosfothreonin metabolismus   MeSH
• genetická epistáze MeSH
• genetické testování MeSH
• meióza * MeSH
• mutace genetika   MeSH
• podjednotky proteinů metabolismus   MeSH
• proteiny huseníčku genetika   metabolismus   MeSH
• tetraploidie MeSH
• tubulin metabolismus   MeSH
• vazba proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• anafázi podporující komplex MeSH
• cykliny MeSH
• fosfothreonin MeSH
• podjednotky proteinů MeSH
• proteiny huseníčku MeSH
• TDM1 protein, Arabidopsis MeSH Prohlížeč
• tubulin 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.

• Klíčová slova
• APC/C, BI2536, Live cell imaging, Mouse zygote, PLK1, Securin, Spindle assembly,
• 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
• polo-like kinasa 1 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
• Názvy látek
• anafázi podporující komplex MeSH
• BI 2536 MeSH Prohlížeč
• protein-serin-threoninkinasy MeSH
• proteiny buněčného cyklu MeSH
• protoonkogenní proteiny MeSH
• pteridiny MeSH

Mammalian oocytes are arrested at prophase I until puberty when luteinizing hormone (LH) induces resumption of meiosis of follicle-enclosed oocytes. Resumption of meiosis is tightly coupled with regulating cyclin-dependent kinase 1 (CDK1) activity. Prophase I arrest depends on inhibitory phosphorylation of CDK1 and anaphase-promoting complex-(APC-CDH1)-mediated regulation of cyclin B levels. Prophase I arrest is maintained by endogenously produced cyclic adenosine monophosphate (cAMP), which activates protein kinase A (PKA) that in turn phosphorylates (and activates) the nuclear kinase WEE2. In addition, PKA-mediated phosphorylation of the phosphatase CDC25B results in its cytoplasmic retention. The combined effect maintains low levels of CDK1 activity that are not sufficient to initiate resumption of meiosis. LH triggers synthesis of epidermal growth factor-like factors in mural granulosa cells and leads to reduced cGMP transfer from cumulus cells to oocytes via gap junctions that couple the two cell types. cGMP inhibits oocyte phosphodiesterase 3A (PDE3A) and a decline in oocyte cGMP results in increased PDE3A activity. The ensuing decrease in oocyte cAMP triggers maturation by alleviating the aforementioned phosphorylations of WEE2 and CDC25B. As a direct consequence CDC25B translocates into the nucleus. The resulting activation of CDK1 also promotes extrusion of WEE2 from the nucleus thereby providing a positive amplification mechanism for CDK1 activation. Other kinases, e.g. protein kinase B, Aurora kinase A and polo-like kinase 1, also participate in resumption of meiosis. Mechanisms governing meiotic prophase I arrest and resumption of meiosis share common features with DNA damage-induced mitotic G2-checkpoint arrest and checkpoint recovery, respectively. These common features include CDC14B-dependent activation of APC-CDH1 in prophase I arrested oocytes or G2-arrested somatic cells, and CDC25B-dependent cell cycle resumption in both oocytes and somatic cells.

• MeSH
• AMP cyklický metabolismus   MeSH
• anafázi podporující komplex MeSH
• Cdh1 proteiny MeSH
• cyklin B1 metabolismus   MeSH
• epidermální růstový faktor metabolismus   MeSH
• G2 fáze * MeSH
• inhibiční proteiny cyklin-dependentních kinas metabolismus   MeSH
• komplexy ubikvitinligas metabolismus   MeSH
• meióza * MeSH
• metafáze MeSH
• myši MeSH
• oocyty metabolismus   MeSH
• oogeneze * MeSH
• profáze meiózy I MeSH
• proliferace buněk * MeSH
• proteiny buněčného cyklu metabolismus   MeSH
• signální transdukce * MeSH
• věkové faktory MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• AMP cyklický MeSH
• anafázi podporující komplex MeSH
• Ccnb1 protein, mouse MeSH Prohlížeč
• Cdh1 proteiny MeSH
• cyklin B1 MeSH
• epidermální růstový faktor MeSH
• Fzr1 protein, mouse MeSH Prohlížeč
• inhibiční proteiny cyklin-dependentních kinas MeSH
• komplexy ubikvitinligas MeSH
• proteiny buněčného cyklu MeSH

Polo-like kinase 1 (PLK1) orchestrates multiple events of cell division. Although PLK1 function has been intensively studied in centriole-containing and rapidly cycling somatic cells, much less is known about its function in the meiotic divisions of mammalian oocytes, which arrest for a long period of time in prophase before meiotic resumption and lack centrioles for spindle assembly. Here, using specific small molecule inhibition combined with live mouse oocyte imaging, we comprehensively characterize meiotic PLK1's functions. We show that PLK1 becomes activated at meiotic resumption on microtubule organizing centers (MTOCs) and later at kinetochores. PLK1 is required for efficient meiotic resumption by promoting nuclear envelope breakdown. PLK1 is also needed to recruit centrosomal proteins to acentriolar MTOCs to promote normal spindle formation, as well as for stable kinetochore-microtubule attachment. Consequently, PLK1 inhibition leads to metaphase I arrest with misaligned chromosomes activating the spindle assembly checkpoint (SAC). Unlike in mitosis, the metaphase I arrest is not bypassed by the inactivation of the SAC. We show that PLK1 is required for the full activation of the anaphase promoting complex/cyclosome (APC/C) by promoting the degradation of the APC/C inhibitor EMI1 and is therefore essential for entry into anaphase I. Moreover, our data suggest that PLK1 is required for proper chromosome segregation and the maintenance of chromosome condensation during the meiosis I-II transition, independently of the APC/C. Thus, our results define the meiotic roles of PLK1 in oocytes and reveal interesting differential requirements of PLK1 between mitosis and oocyte meiosis in mammals.

• MeSH
• anafázi podporující komplex metabolismus   MeSH
• jaderný obal metabolismus   MeSH
• kinetochory metabolismus   MeSH
• konfokální mikroskopie MeSH
• meióza fyziologie   MeSH
• myši MeSH
• oocyty růst a vývoj   MeSH
• organizační centrum mikrotubulů metabolismus   MeSH
• počítačové zpracování obrazu MeSH
• polo-like kinasa 1 MeSH
• protein-serin-threoninkinasy metabolismus   MeSH
• proteiny buněčného cyklu metabolismus   MeSH
• protoonkogenní proteiny metabolismus   MeSH
• segregace chromozomů fyziologie   MeSH
• western blotting MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• anafázi podporující komplex MeSH
• protein-serin-threoninkinasy MeSH
• proteiny buněčného cyklu MeSH
• protoonkogenní proteiny MeSH

Progression through the cell cycle is driven by cyclin-dependent kinases that control gene expression, orchestration of mitotic spindle, and cell division. To identify new regulators of the cell cycle, we performed transcriptomic analysis of human non-transformed cells expressing a fluorescent ubiquitination-based cell cycle indicator and identified 701 transcripts differentially expressed in G1 and G2 cells. Family with sequence similarity 110 member A (FAM110A) protein is highly expressed in G2 cells and localized at mitotic spindle and spindle poles during mitosis. Depletion of FAM110A impairs chromosomal alignment, delays metaphase-to-anaphase transition, and affects spindle positioning. Using mass spectrometry and immunoprecipitation, we identified casein kinase I (CK1) in complex with FAM110A during mitosis. CK1 phosphorylates the C-terminal domain of FAM110A in vitro, and inhibition of CK1 reduces phosphorylation of mitotic FAM110A. Wild-type FAM110A, but not the FAM110A-S252-S255A mutant deficient in CK1 phosphorylation, rescues the chromosomal alignment, duration of mitosis, and orientation of the mitotic spindle after depletion of endogenous FAM110A. We propose that CK1 regulates chromosomal alignment by phosphorylating FAM110A and promoting its interaction with mitotic spindle.

• Klíčová slova
• G2 phase, casein kinase 1, cell cycle, chromosomal alignment, mitosis,
• MeSH
• anafáze MeSH
• aparát dělícího vřeténka * metabolismus   MeSH
• fosforylace MeSH
• HeLa buňky MeSH
• lidé MeSH
• mitóza genetika   MeSH
• proteiny buněčného cyklu * metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• proteiny buněčného cyklu * MeSH