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The spindle assembly checkpoint is not essential for CSF arrest of mouse oocytes.

Tsurumi C, Hoffmann S, Geley S, Graeser R, Polanski Z - J. Cell Biol. (2004)

Bottom Line: Passage through meiosis I was accelerated, but even though the SAC was disrupted, injected oocytes still arrested at metaphase II.Bub1dn-injected oocytes released from CSF and treated with nocodazole to disrupt the second meiotic spindle proceeded into interphase, whereas noninjected control oocytes remained arrested at metaphase.Similar results were obtained using dominant-negative forms of Mad2 and BubR1, as well as checkpoint resistant dominant APC/C activating forms of Cdc20.

View Article: PubMed Central - PubMed

Affiliation: Max-Planck-Institut fuer Immunbiologie, Developmental Biology, Freiburg, Germany. tsurumi@immunbio.mpg.de

ABSTRACT
In Xenopus oocytes, the spindle assembly checkpoint (SAC) kinase Bub1 is required for cytostatic factor (CSF)-induced metaphase arrest in meiosis II. To investigate whether matured mouse oocytes are kept in metaphase by a SAC-mediated inhibition of the anaphase-promoting complex/cyclosome (APC/C) complex, we injected a dominant-negative Bub1 mutant (Bub1dn) into mouse oocytes undergoing meiosis in vitro. Passage through meiosis I was accelerated, but even though the SAC was disrupted, injected oocytes still arrested at metaphase II. Bub1dn-injected oocytes released from CSF and treated with nocodazole to disrupt the second meiotic spindle proceeded into interphase, whereas noninjected control oocytes remained arrested at metaphase. Similar results were obtained using dominant-negative forms of Mad2 and BubR1, as well as checkpoint resistant dominant APC/C activating forms of Cdc20. Thus, SAC proteins are required for checkpoint functions in meiosis I and II, but, in contrast to frog eggs, the SAC is not required for establishing or maintaining the CSF arrest in mouse oocytes.

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Localization of Bub1dn-YFP on kinetochores. Bub1dn-YFP and histone H2B-RFP mRNAs were coinjected into GV-stage oocytes, kept in dbcAMP for 1 h, released, and fixed 5–6 h after GVBD (1st meiosis) or overnight culture (2nd meiosis). (Left) The whole oocyte is shown in an overview, with chromosomes in red (histone H2B-RFP) and Bub1dn-YFP in green. The position of the PB is marked. (Right) Chromosomes are enlarged, and the signals from histone H2B-RFP and Bub1dn-YFP individually, as well as overlaid (merge). Note the dotted appearance of the Bub1dn-YFP signal around the chromosomes suggesting a kinetochore localization. Bar, 10 μm.
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fig2: Localization of Bub1dn-YFP on kinetochores. Bub1dn-YFP and histone H2B-RFP mRNAs were coinjected into GV-stage oocytes, kept in dbcAMP for 1 h, released, and fixed 5–6 h after GVBD (1st meiosis) or overnight culture (2nd meiosis). (Left) The whole oocyte is shown in an overview, with chromosomes in red (histone H2B-RFP) and Bub1dn-YFP in green. The position of the PB is marked. (Right) Chromosomes are enlarged, and the signals from histone H2B-RFP and Bub1dn-YFP individually, as well as overlaid (merge). Note the dotted appearance of the Bub1dn-YFP signal around the chromosomes suggesting a kinetochore localization. Bar, 10 μm.

Mentions: A YFP-fusion protein of a dominant-negative version of Bub1 (Bub1dn, amino acids 1–331) was used to test whether Bub1 is required for the metaphase II arrest in mouse oocytes. This mutant lacks the kinase domain, and disrupts the SAC in somatic cells by competing with the endogenous kinase for kinetochore localization. When microinjected into mouse oocytes, the fusion protein localized to kinetochores during meiosis (Fig. 2).


The spindle assembly checkpoint is not essential for CSF arrest of mouse oocytes.

Tsurumi C, Hoffmann S, Geley S, Graeser R, Polanski Z - J. Cell Biol. (2004)

Localization of Bub1dn-YFP on kinetochores. Bub1dn-YFP and histone H2B-RFP mRNAs were coinjected into GV-stage oocytes, kept in dbcAMP for 1 h, released, and fixed 5–6 h after GVBD (1st meiosis) or overnight culture (2nd meiosis). (Left) The whole oocyte is shown in an overview, with chromosomes in red (histone H2B-RFP) and Bub1dn-YFP in green. The position of the PB is marked. (Right) Chromosomes are enlarged, and the signals from histone H2B-RFP and Bub1dn-YFP individually, as well as overlaid (merge). Note the dotted appearance of the Bub1dn-YFP signal around the chromosomes suggesting a kinetochore localization. Bar, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172623&req=5

fig2: Localization of Bub1dn-YFP on kinetochores. Bub1dn-YFP and histone H2B-RFP mRNAs were coinjected into GV-stage oocytes, kept in dbcAMP for 1 h, released, and fixed 5–6 h after GVBD (1st meiosis) or overnight culture (2nd meiosis). (Left) The whole oocyte is shown in an overview, with chromosomes in red (histone H2B-RFP) and Bub1dn-YFP in green. The position of the PB is marked. (Right) Chromosomes are enlarged, and the signals from histone H2B-RFP and Bub1dn-YFP individually, as well as overlaid (merge). Note the dotted appearance of the Bub1dn-YFP signal around the chromosomes suggesting a kinetochore localization. Bar, 10 μm.
Mentions: A YFP-fusion protein of a dominant-negative version of Bub1 (Bub1dn, amino acids 1–331) was used to test whether Bub1 is required for the metaphase II arrest in mouse oocytes. This mutant lacks the kinase domain, and disrupts the SAC in somatic cells by competing with the endogenous kinase for kinetochore localization. When microinjected into mouse oocytes, the fusion protein localized to kinetochores during meiosis (Fig. 2).

Bottom Line: Passage through meiosis I was accelerated, but even though the SAC was disrupted, injected oocytes still arrested at metaphase II.Bub1dn-injected oocytes released from CSF and treated with nocodazole to disrupt the second meiotic spindle proceeded into interphase, whereas noninjected control oocytes remained arrested at metaphase.Similar results were obtained using dominant-negative forms of Mad2 and BubR1, as well as checkpoint resistant dominant APC/C activating forms of Cdc20.

View Article: PubMed Central - PubMed

Affiliation: Max-Planck-Institut fuer Immunbiologie, Developmental Biology, Freiburg, Germany. tsurumi@immunbio.mpg.de

ABSTRACT
In Xenopus oocytes, the spindle assembly checkpoint (SAC) kinase Bub1 is required for cytostatic factor (CSF)-induced metaphase arrest in meiosis II. To investigate whether matured mouse oocytes are kept in metaphase by a SAC-mediated inhibition of the anaphase-promoting complex/cyclosome (APC/C) complex, we injected a dominant-negative Bub1 mutant (Bub1dn) into mouse oocytes undergoing meiosis in vitro. Passage through meiosis I was accelerated, but even though the SAC was disrupted, injected oocytes still arrested at metaphase II. Bub1dn-injected oocytes released from CSF and treated with nocodazole to disrupt the second meiotic spindle proceeded into interphase, whereas noninjected control oocytes remained arrested at metaphase. Similar results were obtained using dominant-negative forms of Mad2 and BubR1, as well as checkpoint resistant dominant APC/C activating forms of Cdc20. Thus, SAC proteins are required for checkpoint functions in meiosis I and II, but, in contrast to frog eggs, the SAC is not required for establishing or maintaining the CSF arrest in mouse oocytes.

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