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
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.
Because low levels of DNA double strand breaks (DSBs) appear not to activate the ATM-mediated prophase I checkpoint in full-grown oocytes, there may exist mechanisms to protect chromosome integrity during meiotic maturation. Using live imaging we demonstrate that low levels of DSBs induced by the radiomimetic drug Neocarzinostatin (NCS) increase the incidence of chromosome fragments and lagging chromosomes but do not lead to APC/C activation and anaphase onset delay. The number of DSBs, represented by γH2AX foci, significantly decreases between prophase I and metaphase II in both control and NCS-treated oocytes. Transient treatment with NCS increases >2-fold the number of DSBs in prophase I oocytes, but less than 30% of these oocytes enter anaphase with segregation errors. MRE11, but not ATM, is essential to detect DSBs in prophase I and is involved in H2AX phosphorylation during metaphase I. Inhibiting MRE11 by mirin during meiotic maturation results in anaphase bridges and also increases the number of γH2AX foci in metaphase II. Compromised DNA integrity in mirin-treated oocytes indicates a role for MRE11 in chromosome integrity during meiotic maturation.
- MeSH
- ATM protein genetika MeSH
- DNA vazebné proteiny genetika MeSH
- dvouřetězcové zlomy DNA účinky léků MeSH
- enzymy opravy DNA genetika MeSH
- histony genetika MeSH
- meióza genetika MeSH
- metafáze genetika MeSH
- myši MeSH
- oocyty růst a vývoj metabolismus MeSH
- poškození DNA účinky léků genetika MeSH
- zinostatin aplikace a dávkování MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural 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
