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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 Cdc20-YFP. Cdc20-YFP and histone H2B-RFP were coinjected into GV-stage oocytes, kept in dbcAMP for 1 h, released and fixed 6 h after GVBD (1st meiosis), 9–10 h after GVBD (1st anaphase and 1st telophase), or after overnight culture (2nd meiosis). (Left) The whole oocyte is shown in an overview, with chromosomes in red (histone H2B-RFP) and Cdc20-YFP in green. The position of the PB is marked in the metaphase II-arrested oocytes. (Right) The chromosomes are enlarged, and the signals from histone H2B-RFP and Cdc20-YFP are shown individually, as well as overlaid (merge). Bars, 10 μm.
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fig5: Localization of Cdc20-YFP. Cdc20-YFP and histone H2B-RFP were coinjected into GV-stage oocytes, kept in dbcAMP for 1 h, released and fixed 6 h after GVBD (1st meiosis), 9–10 h after GVBD (1st anaphase and 1st telophase), or after overnight culture (2nd meiosis). (Left) The whole oocyte is shown in an overview, with chromosomes in red (histone H2B-RFP) and Cdc20-YFP in green. The position of the PB is marked in the metaphase II-arrested oocytes. (Right) The chromosomes are enlarged, and the signals from histone H2B-RFP and Cdc20-YFP are shown individually, as well as overlaid (merge). Bars, 10 μm.

Mentions: First, we expressed YFP-tagged Cdc20 to monitor its subcellular localization in mouse oocytes. Cdc20-YFP localized strongly to kinetochores, and, more pronounced in meiosis II than in meiosis I, to spindles (Fig. 5). Other than in mitotic cells, Cdc20-YFP did not localize to the spindle poles.


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 Cdc20-YFP. Cdc20-YFP and histone H2B-RFP were coinjected into GV-stage oocytes, kept in dbcAMP for 1 h, released and fixed 6 h after GVBD (1st meiosis), 9–10 h after GVBD (1st anaphase and 1st telophase), or after overnight culture (2nd meiosis). (Left) The whole oocyte is shown in an overview, with chromosomes in red (histone H2B-RFP) and Cdc20-YFP in green. The position of the PB is marked in the metaphase II-arrested oocytes. (Right) The chromosomes are enlarged, and the signals from histone H2B-RFP and Cdc20-YFP are shown individually, as well as overlaid (merge). Bars, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172623&req=5

fig5: Localization of Cdc20-YFP. Cdc20-YFP and histone H2B-RFP were coinjected into GV-stage oocytes, kept in dbcAMP for 1 h, released and fixed 6 h after GVBD (1st meiosis), 9–10 h after GVBD (1st anaphase and 1st telophase), or after overnight culture (2nd meiosis). (Left) The whole oocyte is shown in an overview, with chromosomes in red (histone H2B-RFP) and Cdc20-YFP in green. The position of the PB is marked in the metaphase II-arrested oocytes. (Right) The chromosomes are enlarged, and the signals from histone H2B-RFP and Cdc20-YFP are shown individually, as well as overlaid (merge). Bars, 10 μm.
Mentions: First, we expressed YFP-tagged Cdc20 to monitor its subcellular localization in mouse oocytes. Cdc20-YFP localized strongly to kinetochores, and, more pronounced in meiosis II than in meiosis I, to spindles (Fig. 5). Other than in mitotic cells, Cdc20-YFP did not localize to the spindle poles.

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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