Q64681426
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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.
- 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
Sledování buněčného cyklu v živých buňkách je klíčové pro lepší pochopení molekulárních mechanismů regulujících buněčné dělení. Významným milníkem v monitorování buněčného cyklu v živých buňkách představuje objev fluorescenčních proteinů. Připojením sekvence kódující fluorescenční protein k vybranému buněčnému proteinu se vytvoří tzv. fluorescenční biosenzor. Mezi nejvýznamnější senzory v této oblasti patří senzor PCNA -GFP (proliferating -cell nuclear antigen, proliferační buněčný jaderný antigen) nebo DHB -mVenus (DNA helikasa B) a FUCCI systém (fluorescent ubiquitination -based cell cycle indicator, fluorescenční indikátor buněčného cyklu založený na ubikvitinaci). PCNA je replikační faktor, který při DNA replikaci tvoří v jádře zřetelná replikační ohniska. DHB -GFP nebo -mVenus byl použit na rozlišení fáze buněčného cyklu díky translokaci z jádra do cytoplazmy. FUCCI systém je založený na detekci exprese a následné degradace proteinů Cdt1 a gemininu, které hrají roli v licencování replikačních počátků. Zmíněné biosenzory jsou schopné rozlišit všechny fáze buněčného cyklu. Vhodným nástrojem k sledování buněčného cyklu v živých buňkách jsou také tzv. chromobodies, což jsou malé fragmenty protilátek spojených s chromoforem. Využít lze například PCNA -chromobody. Přestože fluorescenční biosenzory vykazují i jisté nevýhody, jsou zatím nejlepším nástrojem pro monitorování buněčného cyklu v živých buňkách.
Cell cycle monitoring in live cells is crucial for better understanding of molecular mechanisms regulating cell cycle. Important achievement in cell cycle monitoring in live cells represents discovery of fluorescent proteins. Fluorescent biosensor is created by attaching coding sequence of a fluorescent protein to the protein of interest. Among most important fluorescent biosensors in cell cycle monitoring are PCNA -GFP sensor (proliferating -cell nuclear antigen) or DHB -mVenus (DNA helicase B) and FUCCI system (fluorescent ubiquitination -based cell cycle indicator). PCNA is replication factor, which creates distinct replication foci during ongoing replication. DHB -GFP or mVenus was used for monitoring of cell cycle phases because of its translocation from the nucleus to the cytoplasm. FUCCI system is based on the detection of expression and following degradation of Cdt1 and geminin proteins that play an important role in licensing of replication origins. Above mentioned biosensors are able to distinguish all cell cycle phases. A suitable tool for cell cycle monitoring in live cells are also chromobodies (small antibodies fragments with an attached fluorophore). As an example, there is PCNA- -chromobody on market. Although fluorescent biosensors show also certain disadvantages, they are now the best solution for cell cycle monitoring in live cells
AID (Auxin Inducible Degron) systém bol objavený u rastlín ako degradačná dráhá regulovaná fytohormónom – auxínom. Tento sytém možno aplikovať aj u cicavcov a využiť pre rýchlu a špecifickú degradáciu exogénnych aj endogénnych proteínov. Aby v prítomnosti auxínu bol študovaný proteín u cicavcov degradovaný, musí obsahovať AID sekvenciu a bunky musia zároveň exprimovať receptor pre AID – t.j. OsTIR proteín (TIR1 proteín exprimovaný u Oryza Sativa – ryža siata). Vďaka svojej rýchlosti, efektívnosti degradácie a reverzibilnému účinku predstavuje AID systém ideálnu metódu pre štúdium funkcie proteínov u cicavcov.
AID (Auxin Inducible Degron) system was discovered in plants as a protein degradation pathway regulated by the phytohormone auxin. This system is applicable also in mammals and it can be used for rapid and specific degradation of exogenic as well as endogenic proteins. In order to be degraded in presence of auxin in mammals, the protein has to contain the AID sequence and the cells have to express the AID receptor – i.e. OsTIR. Thanks to a rapid, effective degradation and reversibility effect, AID system represents an ideal approach for studying protein functions in mammals.
Důležitou úlohou oocytů je zajištění přenosu genetické informace do dalších generací. Narušení integrity DNA oocytů může představovat riziko pro zrání oocytů a vývoj embrya, a aktivní mechanizmy detekce a opravy poškozené DNA jsou proto nezbytné pro přežití potomstva. Nejnovější studie však ukazují, že oocyty jsou schopné zrát i za přítomností poškozené DNA až do embryonálního stádia a reagují pouze na větší míru poškození. Nicméně oocyty mají kapacitu průběžně opravovat DNA během zrání, otázkou však zůstává efektivita této opravy.
An important role of oocytes is transmission of genetic information to the next generations. Disruption of DNA integrity may affect maturation of oocytes and embryo development and active mechanisms of DNA damage detection and repair are thus needed for the successful reproduction. However, the recent studies show that oocytes are able to mature in the presence of damaged DNA until the embryonic stage and respond only to higher levels of damage. Nevertheless, the oocytes have the capacity to repair DNA throughout meiotic maturation, however the effectivity of the process remains unclear.
- MeSH
- embryonální vývoj MeSH
- lidé MeSH
- modely u zvířat MeSH
- myši MeSH
- oocyty * MeSH
- oprava DNA MeSH
- poškození DNA * MeSH
- výzkum embrya MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- práce podpořená grantem MeSH
Meiotické delenie cicavčích oocytov je fyziologicky zastavené v profáze prvého meiotického delenia (blok v profáze I). Návrat do meiózy sa nazýva znovuzahájenie meiózy. U oocytov je kontrolované cyklín‑dependentnou kinázou 1 (CDK1). Aktivita CDK1 je negatívne regulovaná fosforyláciou na treoníne 14 a tyrozíne 15. Počas bloku v profáze I je CDK1 inaktivovaná, pretože tieto aminokyseliné zvyšky sú fosforylované prostredníctvom Wee1B/Myt1 kináz. Pre znovuzahájenie meiózy musí byť CDK1 aktivovaná a aminokyselinové zvyšky defosforylované pomocou CDC25 fosfatáz. Aktivácia/inaktivácia CDK1 závisí na aktivite regulačných kináz a fosfatáz a rovnako aj ich lokalizácii v oocyte.
Meiotic division in mammalian oocytes is physiologically arrested in prophase of the first meiotic division (prophase I arrest). The reactivation of meiosis is called meiosis resumption. In oocytes it is controlled by cyclin‑dependent kinase 1 (CDK1). Activity of CDK1 is negatively regulated by phosphorylation on threonine 14 and tyrosine 15. During prophase I arrest CDK1 is inactivated, because these amino residues are phosphorylated by Wee1B/Myt1 kinases. For meiosis resumption CDK1 has to be activated and amino residues dephosphorylated by the activation of CDC25 phosphatases. Activation/deactivation of CDK1 depends on activity as well as localization of these regulating kinases and phosphatases in oocytes
Aurora kinase A (AURKA), which is a centrosome-localized serine/threonine kinase crucial for cell cycle control, is critically involved in centrosome maturation and spindle assembly in somatic cells. Active T288 phosphorylated AURKA localizes to the centrosome in the late G(2) and also spreads to the minus ends of mitotic spindle microtubules. AURKA activates centrosomal CDC25B and recruits cyclin B1 to centrosomes. We report here functions for AURKA in meiotic maturation of mouse oocytes, which is a model system to study the G(2) to M transition. Whereas AURKA is present throughout the entire GV-stage oocyte with a clear accumulation on microtubule organizing centers (MTOC), active AURKA becomes entirely localized to MTOCs shortly before germinal vesicle breakdown. In contrast to somatic cells in which active AURKA is present at the centrosomes and minus ends of microtubules, active AURKA is mainly located on MTOCs at metaphase I (MI) in oocytes. Inhibitor studies using Roscovitine (CDK1 inhibitor), LY-294002 (PI3K inhibitor) and SH-6 (PKB inhibitor) reveal that activation of AURKA localized on MTOCs is independent on PI3K-PKB and CDK1 signaling pathways and MOTC amplification is observed in roscovitine- and SH-6-treated oocytes that fail to undergo nuclear envelope breakdown. Moreover, microinjection of Aurka mRNA into GV-stage oocytes cultured in 3-isobutyl-1-methyl xanthine (IBMX)-containing medium to prevent maturation also results in MOTC amplification in the absence of CDK1 activation. Overexpression of AURKA also leads to formation of an abnormal MI spindle, whereas RNAi-mediated reduction of AURKA interferes with resumption of meiosis and spindle assembly. Results of these experiments indicate that AURKA is a critical MTOC-associated component involved in resumption of meiosis, MTOC multiplication, proper spindle formation and the metaphase I-metaphase II transition.
- MeSH
- aparát dělícího vřeténka metabolismus MeSH
- blastodisk metabolismus MeSH
- buněčný cyklus fyziologie genetika MeSH
- buňky NIH 3T3 MeSH
- cyklin-dependentní kinasy fyziologie metabolismus MeSH
- financování organizované MeSH
- HeLa buňky MeSH
- lidé MeSH
- meióza fyziologie genetika MeSH
- myši inbrední BALB C MeSH
- myši inbrední C57BL MeSH
- myši MeSH
- oocyty enzymologie fyziologie MeSH
- organizační centrum mikrotubulů metabolismus MeSH
- protein-serin-threoninkinasy fyziologie genetika metabolismus MeSH
- protoonkogenní proteiny c-akt fyziologie metabolismus MeSH
- vazba proteinů MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
CDK1 is a pivotal regulator of resumption of meiosis and meiotic maturation of oocytes. CDC25A/B/C are dual-specificity phosphatases and activate cyclin-dependent kinases (CDKs). Although CDC25C is not essential for either mitotic or meiotic cell cycle regulation, CDC25B is essential for CDK1 activation during resumption of meiosis. Cdc25a -/- mice are embryonic lethal and therefore a role for CDC25A in meiosis is unknown. We report that activation of CDK1 results in a maturation-associated decrease in the amount of CDC25A protein, but not Cdc25a mRNA, such that little CDC25A is present by metaphase I. In addition, expression of exogenous CDC25A overcomes cAMP-mediated maintenance of meiotic arrest. Microinjection of Gfp-Cdc25a and Gpf-Cdc25b mRNAs constructs reveals that CDC25A is exclusively localized to the nucleus prior to nuclear envelope breakdown (NEBD). In contrast, CDC25B localizes to cytoplasm in GV-intact oocytes and translocates to the nucleus shortly before NEBD. Over-expressing GFP-CDC25A, which compensates for the normal maturation-associated decrease in CDC25A, blocks meiotic maturation at MI. This MI block is characterized by defects in chromosome congression and spindle formation and a transient reduction in both CDK1 and MAPK activities. Lastly, RNAi-mediated reduction of CDC25A results in fewer oocytes resuming meiosis and reaching MII. These data demonstrate that CDC25A behaves differently during female meiosis than during mitosis, and moreover, that CDC25A has a function in resumption of meiosis, MI spindle formation and the MI-MII transition. Thus, both CDC25A and CDC25B are critical for meiotic maturation of oocytes.
BACKGROUND INFORMATION: In fully grown mouse oocytes, a decrease in cAMP concentration precedes and is linked to CDK1 (cyclin-dependent kinase 1) activation. The molecular mechanism for this coupling, however, is not defined. PKB (protein kinase B, also called AKT) is implicated in CDK1 activation in lower species. During resumption of meiosis in starfish oocytes, MYT1, a negative regulator of CDK1, is phosphorylated by PKB in an inhibitory manner. It can imply that PKB is also involved in CDK1 activation in mammalian oocytes. RESULTS: We monitored activation of PKB and CDK1 during maturation of mouse oocytes. PKB phosphorylation and activation preceded GVBD (germinal vesicle breakdown) in oocytes maturing either in vitro or in vivo. Activation was transient and PKB activity was markedly reduced when virtually all of the oocytes had undergone GVBD. PKB activation was independent of CDK1 activity, because although butyrolactone I prevented CDK1 activation and GVBD, PKB was nevertheless transiently phosphorylated and activated. LY-294002, an inhibitor of phosphoinositide 3-kinase-PKB signalling, suppressed activation of PKB and CDK1 as well as resumption of meiosis. OA (okadaic acid)-sensitive phosphatases are involved in PKB-activity regulation, because OA induced PKB hyperphosphorylation. During resumption of meiosis, PKB phosphorylated on Ser(473) is associated with nuclear membrane and centrosome, whereas PKB phosphorylated on Thr(308) is localized on centrosome only. CONCLUSIONS: The results of the present paper indicate that PKB is involved in CDK1 activation and resumption of meiosis in mouse oocytes. The presence of phosphorylated PKB on centrosome at the time of GVBD suggests its important role for an initial CDK1 activation.
- MeSH
- aktivace enzymů MeSH
- centrozom metabolismus MeSH
- chromony farmakologie MeSH
- financování organizované MeSH
- fosforylace MeSH
- gama-butyrolakton analogy a deriváty farmakologie MeSH
- jaderný obal metabolismus MeSH
- kyselina okadaová farmakologie MeSH
- meióza MeSH
- morfoliny farmakologie MeSH
- myši MeSH
- oocyty fyziologie účinky léků MeSH
- proteinkinasa CDC2 metabolismus MeSH
- protoonkogenní proteiny c-akt antagonisté a inhibitory fyziologie MeSH
- serin metabolismus MeSH
- techniky in vitro MeSH
- threonin metabolismus MeSH
- transport proteinů MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
