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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 Cdc20-4AVGR injection into mouse oocytes. (A) Time-lapse video microscopy. Uninjected, or oocytes injected with the Cdc20-4AVGR mutant were treated as in Fig. 3 A. Red arrows indicate the extruded PB. The oocyte marked by the green box failed, despite several attempts, to extrude its PB. The pink arrow indicates the spot where the PB should have been extruded. The oocyte marked by the blue box, however, divided abnormally and extruded two PBs, indicated by the light blue arrow. Time points (h) after GVBD are indicated. Bar, 50 μm. (B) Immunofluorescence staining of uninjected, and Cdc20-4AVGR–injected oocytes. Oocytes were treated as in Fig. 4 B. (Left) The whole oocyte is shown in an overview. (Right) The area of the spindle is enlarged, and the DNA and tubulin staining are shown individually, as well as overlaid (merge). The uninjected oocyte extruded the PB, and the chromosomes are aligned on a metaphase plate with an intact spindle apparatus. Examples of the various phenotypes of Cdc20-4AVGR–injected oocytes are shown below. In b–d, the oocytes did not extrude their first PB. The abnormal spindles in c and d are marked with red arrows. In e, an oocyte with an interphase nucleus in the absence of any extruded PB is shown. In f, a typical example of an oocyte with one PB extruded is shown. In g, an oocyte is shown with two extruded PBs. Bars, 10 μm. (C) Activation of Cdc20-4AVGR–injected oocytes. The oocytes were treated as in Fig. 4 C. The position of the PB is marked. The oocyte nuclei are boxed, and shown enlarged in the insets. (D) Single cell H1/MBP kinase assay. Total H1 and MBP kinase activities were assayed in whole cell lysates prepared from uninjected, GV-stage, and Cdc20-, Cdc20-4AV–, and Cdc20-4AVGR–injected oocytes. The autoradiograph is shown.
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fig7: The effect of Cdc20-4AVGR injection into mouse oocytes. (A) Time-lapse video microscopy. Uninjected, or oocytes injected with the Cdc20-4AVGR mutant were treated as in Fig. 3 A. Red arrows indicate the extruded PB. The oocyte marked by the green box failed, despite several attempts, to extrude its PB. The pink arrow indicates the spot where the PB should have been extruded. The oocyte marked by the blue box, however, divided abnormally and extruded two PBs, indicated by the light blue arrow. Time points (h) after GVBD are indicated. Bar, 50 μm. (B) Immunofluorescence staining of uninjected, and Cdc20-4AVGR–injected oocytes. Oocytes were treated as in Fig. 4 B. (Left) The whole oocyte is shown in an overview. (Right) The area of the spindle is enlarged, and the DNA and tubulin staining are shown individually, as well as overlaid (merge). The uninjected oocyte extruded the PB, and the chromosomes are aligned on a metaphase plate with an intact spindle apparatus. Examples of the various phenotypes of Cdc20-4AVGR–injected oocytes are shown below. In b–d, the oocytes did not extrude their first PB. The abnormal spindles in c and d are marked with red arrows. In e, an oocyte with an interphase nucleus in the absence of any extruded PB is shown. In f, a typical example of an oocyte with one PB extruded is shown. In g, an oocyte is shown with two extruded PBs. Bars, 10 μm. (C) Activation of Cdc20-4AVGR–injected oocytes. The oocytes were treated as in Fig. 4 C. The position of the PB is marked. The oocyte nuclei are boxed, and shown enlarged in the insets. (D) Single cell H1/MBP kinase assay. Total H1 and MBP kinase activities were assayed in whole cell lysates prepared from uninjected, GV-stage, and Cdc20-, Cdc20-4AV–, and Cdc20-4AVGR–injected oocytes. The autoradiograph is shown.

Mentions: Because the above results suggested that the two Cdc20 mutants acted differently, we combined them to generate a double mutant, Cdc20-4AVGR. This indeed had a synergistic effect on the dominant behavior of Cdc20. When mRNA encoding a Cdc20-4AVGR double mutant was injected into mouse oocytes, the onset of anaphase of meiosis I was even earlier than with any of our other mutant proteins, starting at 5–6 h after GVBD (Fig. 7 A; Table I). However, despite the accelerated entry into anaphase, the exit from meiosis I generally lasted more than an hour (Fig. 7 A) and often could not be completed properly. Some oocytes failed to extrude their PB, despite multiple attempts (Fig. 7 A, green box; the place where the PB should be extruded is indicated by a yellow arrow), or produced two (Fig. 7 A, blue box; Table IV). When Cdc20-4AVGR–injected oocytes were fixed after overnight culture, and stained with PI and a tubulin-specific antibody, a variety of phenotypes was revealed.


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 Cdc20-4AVGR injection into mouse oocytes. (A) Time-lapse video microscopy. Uninjected, or oocytes injected with the Cdc20-4AVGR mutant were treated as in Fig. 3 A. Red arrows indicate the extruded PB. The oocyte marked by the green box failed, despite several attempts, to extrude its PB. The pink arrow indicates the spot where the PB should have been extruded. The oocyte marked by the blue box, however, divided abnormally and extruded two PBs, indicated by the light blue arrow. Time points (h) after GVBD are indicated. Bar, 50 μm. (B) Immunofluorescence staining of uninjected, and Cdc20-4AVGR–injected oocytes. Oocytes were treated as in Fig. 4 B. (Left) The whole oocyte is shown in an overview. (Right) The area of the spindle is enlarged, and the DNA and tubulin staining are shown individually, as well as overlaid (merge). The uninjected oocyte extruded the PB, and the chromosomes are aligned on a metaphase plate with an intact spindle apparatus. Examples of the various phenotypes of Cdc20-4AVGR–injected oocytes are shown below. In b–d, the oocytes did not extrude their first PB. The abnormal spindles in c and d are marked with red arrows. In e, an oocyte with an interphase nucleus in the absence of any extruded PB is shown. In f, a typical example of an oocyte with one PB extruded is shown. In g, an oocyte is shown with two extruded PBs. Bars, 10 μm. (C) Activation of Cdc20-4AVGR–injected oocytes. The oocytes were treated as in Fig. 4 C. The position of the PB is marked. The oocyte nuclei are boxed, and shown enlarged in the insets. (D) Single cell H1/MBP kinase assay. Total H1 and MBP kinase activities were assayed in whole cell lysates prepared from uninjected, GV-stage, and Cdc20-, Cdc20-4AV–, and Cdc20-4AVGR–injected oocytes. The autoradiograph is shown.
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Related In: Results  -  Collection

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

fig7: The effect of Cdc20-4AVGR injection into mouse oocytes. (A) Time-lapse video microscopy. Uninjected, or oocytes injected with the Cdc20-4AVGR mutant were treated as in Fig. 3 A. Red arrows indicate the extruded PB. The oocyte marked by the green box failed, despite several attempts, to extrude its PB. The pink arrow indicates the spot where the PB should have been extruded. The oocyte marked by the blue box, however, divided abnormally and extruded two PBs, indicated by the light blue arrow. Time points (h) after GVBD are indicated. Bar, 50 μm. (B) Immunofluorescence staining of uninjected, and Cdc20-4AVGR–injected oocytes. Oocytes were treated as in Fig. 4 B. (Left) The whole oocyte is shown in an overview. (Right) The area of the spindle is enlarged, and the DNA and tubulin staining are shown individually, as well as overlaid (merge). The uninjected oocyte extruded the PB, and the chromosomes are aligned on a metaphase plate with an intact spindle apparatus. Examples of the various phenotypes of Cdc20-4AVGR–injected oocytes are shown below. In b–d, the oocytes did not extrude their first PB. The abnormal spindles in c and d are marked with red arrows. In e, an oocyte with an interphase nucleus in the absence of any extruded PB is shown. In f, a typical example of an oocyte with one PB extruded is shown. In g, an oocyte is shown with two extruded PBs. Bars, 10 μm. (C) Activation of Cdc20-4AVGR–injected oocytes. The oocytes were treated as in Fig. 4 C. The position of the PB is marked. The oocyte nuclei are boxed, and shown enlarged in the insets. (D) Single cell H1/MBP kinase assay. Total H1 and MBP kinase activities were assayed in whole cell lysates prepared from uninjected, GV-stage, and Cdc20-, Cdc20-4AV–, and Cdc20-4AVGR–injected oocytes. The autoradiograph is shown.
Mentions: Because the above results suggested that the two Cdc20 mutants acted differently, we combined them to generate a double mutant, Cdc20-4AVGR. This indeed had a synergistic effect on the dominant behavior of Cdc20. When mRNA encoding a Cdc20-4AVGR double mutant was injected into mouse oocytes, the onset of anaphase of meiosis I was even earlier than with any of our other mutant proteins, starting at 5–6 h after GVBD (Fig. 7 A; Table I). However, despite the accelerated entry into anaphase, the exit from meiosis I generally lasted more than an hour (Fig. 7 A) and often could not be completed properly. Some oocytes failed to extrude their PB, despite multiple attempts (Fig. 7 A, green box; the place where the PB should be extruded is indicated by a yellow arrow), or produced two (Fig. 7 A, blue box; Table IV). When Cdc20-4AVGR–injected oocytes were fixed after overnight culture, and stained with PI and a tubulin-specific antibody, a variety of phenotypes was revealed.

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
Related in: MedlinePlus