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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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Overview of the oocyte injection experiments. MI, AI, and TI indicate metaphase, anaphase, and telophase of meiosis I, respectively. IK, interkinasis; MII, metaphase of meosis II. Time after GVBD is indicated in hours.
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fig1: Overview of the oocyte injection experiments. MI, AI, and TI indicate metaphase, anaphase, and telophase of meiosis I, respectively. IK, interkinasis; MII, metaphase of meosis II. Time after GVBD is indicated in hours.

Mentions: To check whether the SAC is involved in the CSF-mediated metaphase II arrest in mouse oocytes, we chose to microinject interfering mutants of SAC components into oocytes. GV stage (prophase I) oocytes were injected with in vitro transcribed and capped mRNAs encoding the mutant protein, kept in prophase for 1 h by medium containing dbcAMP, and subsequently released for completion of meiosis by exchanging the medium. Resumption of meiotic maturation can be monitored because the oocytes dissolve their nuclear membrane (GVBD) 60–90 min after the release. The first meiotic division, which results in the extrusion of a PB, takes place 9–10 h after GVBD. Oocytes then enter meiosis II without an intermittent S phase, and become arrested at metaphase of meiosis II, by the activity of CSF. This metaphase II arrest is stable for more than 15 h in our in vitro culture system (see Fig. 1 for a schematical representation of our experimental set up).


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)

Overview of the oocyte injection experiments. MI, AI, and TI indicate metaphase, anaphase, and telophase of meiosis I, respectively. IK, interkinasis; MII, metaphase of meosis II. Time after GVBD is indicated in hours.
© Copyright Policy
Related In: Results  -  Collection

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

fig1: Overview of the oocyte injection experiments. MI, AI, and TI indicate metaphase, anaphase, and telophase of meiosis I, respectively. IK, interkinasis; MII, metaphase of meosis II. Time after GVBD is indicated in hours.
Mentions: To check whether the SAC is involved in the CSF-mediated metaphase II arrest in mouse oocytes, we chose to microinject interfering mutants of SAC components into oocytes. GV stage (prophase I) oocytes were injected with in vitro transcribed and capped mRNAs encoding the mutant protein, kept in prophase for 1 h by medium containing dbcAMP, and subsequently released for completion of meiosis by exchanging the medium. Resumption of meiotic maturation can be monitored because the oocytes dissolve their nuclear membrane (GVBD) 60–90 min after the release. The first meiotic division, which results in the extrusion of a PB, takes place 9–10 h after GVBD. Oocytes then enter meiosis II without an intermittent S phase, and become arrested at metaphase of meiosis II, by the activity of CSF. This metaphase II arrest is stable for more than 15 h in our in vitro culture system (see Fig. 1 for a schematical representation of our experimental set up).

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.

Show MeSH