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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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The effect of Mad2ΔC and BubR1d injection on meiosis I and II in mouse oocytes. (A) Time-lapse video microscopy. Mad2ΔC or BubR1d injected oocytes were treated as in Fig. 3 A. Pictures were taken every 30 min for 12 h. Red arrows indicate the extruded PB. Time points (h) after GVBD are indicated. Bar, 50 μm. (B) Immunofluorescence staining of Mad2ΔC (left) or BubR1d-injected (right) oocytes. Mad2ΔC or BubR1d-injected oocytes were kept 1 h in dbcAMP before they were released from the prophase block. 20 h after GVBD, the oocytes were fixed, chromosomes stained with PI (red), and the meiotic spindles visualized using an anti-tubulin antibody, followed by an FITC-labeled secondary antibody (green). Note the extruded PB, and the chromosomes aligned on a metaphase plate with an intact spindle apparatus, indicating a CSF-arrest. Bar, 10 μm. (C) Activation of Mad2ΔC and BubR1d-injected oocytes. Uninjected, and Mad2ΔC- or BubR1d-injected oocytes were cultured for 14–15 h after GVBD and transferred into individual drops of medium containing nocodazole, or strontium, or both, and then cultured for an additional 3–4 h. Chromosomes were stained with DAPI. The position of the PB is marked. The oocyte nuclei are boxed, and shown enlarged in the insets. Bar, 10 μm.
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fig4: The effect of Mad2ΔC and BubR1d injection on meiosis I and II in mouse oocytes. (A) Time-lapse video microscopy. Mad2ΔC or BubR1d injected oocytes were treated as in Fig. 3 A. Pictures were taken every 30 min for 12 h. Red arrows indicate the extruded PB. Time points (h) after GVBD are indicated. Bar, 50 μm. (B) Immunofluorescence staining of Mad2ΔC (left) or BubR1d-injected (right) oocytes. Mad2ΔC or BubR1d-injected oocytes were kept 1 h in dbcAMP before they were released from the prophase block. 20 h after GVBD, the oocytes were fixed, chromosomes stained with PI (red), and the meiotic spindles visualized using an anti-tubulin antibody, followed by an FITC-labeled secondary antibody (green). Note the extruded PB, and the chromosomes aligned on a metaphase plate with an intact spindle apparatus, indicating a CSF-arrest. Bar, 10 μm. (C) Activation of Mad2ΔC and BubR1d-injected oocytes. Uninjected, and Mad2ΔC- or BubR1d-injected oocytes were cultured for 14–15 h after GVBD and transferred into individual drops of medium containing nocodazole, or strontium, or both, and then cultured for an additional 3–4 h. Chromosomes were stained with DAPI. The position of the PB is marked. The oocyte nuclei are boxed, and shown enlarged in the insets. Bar, 10 μm.

Mentions: In time course experiments, overexpression of either Mad2ΔC or BubR1d accelerated passage of the oocytes through meiosis I, when compared with uninjected controls (Fig. 4 A; Table I). But, as with the Bub1dn mutant, neither construct was able to overcome CSF and induce exit from the metaphase II arrest. The spindle apparatus as well as the condensed metaphase chromosomes were similar to the control CSF-arrested oocytes (Fig. 4 B). Again, as with Bub1dn, the injected proteins were functional in abolishing the SAC, because they allowed metaphase II-arrested oocytes to proceed to interphase when released from the CSF in the presence of nocodazole (Fig. 4 C; Table III). Together, these results strongly argue against an essential role of the SAC in the CSF-mediated metaphase II arrest in mouse oocytes.


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)

The effect of Mad2ΔC and BubR1d injection on meiosis I and II in mouse oocytes. (A) Time-lapse video microscopy. Mad2ΔC or BubR1d injected oocytes were treated as in Fig. 3 A. Pictures were taken every 30 min for 12 h. Red arrows indicate the extruded PB. Time points (h) after GVBD are indicated. Bar, 50 μm. (B) Immunofluorescence staining of Mad2ΔC (left) or BubR1d-injected (right) oocytes. Mad2ΔC or BubR1d-injected oocytes were kept 1 h in dbcAMP before they were released from the prophase block. 20 h after GVBD, the oocytes were fixed, chromosomes stained with PI (red), and the meiotic spindles visualized using an anti-tubulin antibody, followed by an FITC-labeled secondary antibody (green). Note the extruded PB, and the chromosomes aligned on a metaphase plate with an intact spindle apparatus, indicating a CSF-arrest. Bar, 10 μm. (C) Activation of Mad2ΔC and BubR1d-injected oocytes. Uninjected, and Mad2ΔC- or BubR1d-injected oocytes were cultured for 14–15 h after GVBD and transferred into individual drops of medium containing nocodazole, or strontium, or both, and then cultured for an additional 3–4 h. Chromosomes were stained with DAPI. The position of the PB is marked. The oocyte nuclei are boxed, and shown enlarged in the insets. Bar, 10 μm.
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fig4: The effect of Mad2ΔC and BubR1d injection on meiosis I and II in mouse oocytes. (A) Time-lapse video microscopy. Mad2ΔC or BubR1d injected oocytes were treated as in Fig. 3 A. Pictures were taken every 30 min for 12 h. Red arrows indicate the extruded PB. Time points (h) after GVBD are indicated. Bar, 50 μm. (B) Immunofluorescence staining of Mad2ΔC (left) or BubR1d-injected (right) oocytes. Mad2ΔC or BubR1d-injected oocytes were kept 1 h in dbcAMP before they were released from the prophase block. 20 h after GVBD, the oocytes were fixed, chromosomes stained with PI (red), and the meiotic spindles visualized using an anti-tubulin antibody, followed by an FITC-labeled secondary antibody (green). Note the extruded PB, and the chromosomes aligned on a metaphase plate with an intact spindle apparatus, indicating a CSF-arrest. Bar, 10 μm. (C) Activation of Mad2ΔC and BubR1d-injected oocytes. Uninjected, and Mad2ΔC- or BubR1d-injected oocytes were cultured for 14–15 h after GVBD and transferred into individual drops of medium containing nocodazole, or strontium, or both, and then cultured for an additional 3–4 h. Chromosomes were stained with DAPI. The position of the PB is marked. The oocyte nuclei are boxed, and shown enlarged in the insets. Bar, 10 μm.
Mentions: In time course experiments, overexpression of either Mad2ΔC or BubR1d accelerated passage of the oocytes through meiosis I, when compared with uninjected controls (Fig. 4 A; Table I). But, as with the Bub1dn mutant, neither construct was able to overcome CSF and induce exit from the metaphase II arrest. The spindle apparatus as well as the condensed metaphase chromosomes were similar to the control CSF-arrested oocytes (Fig. 4 B). Again, as with Bub1dn, the injected proteins were functional in abolishing the SAC, because they allowed metaphase II-arrested oocytes to proceed to interphase when released from the CSF in the presence of nocodazole (Fig. 4 C; Table III). Together, these results strongly argue against an essential role of the SAC in the CSF-mediated metaphase II arrest in mouse oocytes.

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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