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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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Cdc20-4AVGR mutant induces sister chromatid separation. (A) Chromosome spreads were prepared from noninjected, or Cdc20-4AVGR–injected oocytes after overnight culture. The chromosomes were stained using a 2% Giemsa solution. In the insets, chromosomes are shown that were found outside the main field. a, uninjected in 1st meiosis (6 h after GVBD); b, uninjected in 2nd meiosis; c and d, Cdc20-4AVGR–injected oocytes with the PB extruded; e and f, Cdc20-4AVGR–injected oocytes with no PB extruded. Bar, 10 μm. (B) Quantification of A. On the x axis are the numbers of chromosomes counted per oocyte, on the y axis the number of oocytes. a, control; b, Cdc20-4AVGR–injected oocytes with PB extruded; c, Cdc20-4AVGR–injected oocytes with no PB extruded. (C) Time-lapse video microscopy. The Cdc20-4AVGR construct was coinjected into oocytes either with (a) cyclin B1N170-RFP (a 170–amino acid NH2-terminal fragment of cyclin B1 fused to RFP) or with (b) Pds1-RFP. The oocytes were kept in dbcAMP for 1 h, released 4 h after GVBD, and observed by differential interference contrast, and fluorescence time-lapse video microscopy. Pictures were taken every hour (BN170-RFP) or every 45 min (Pds1-RFP). The complete series is shown. Time points (h) after GVBD are indicated. Bars, 50 μm.
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fig8: Cdc20-4AVGR mutant induces sister chromatid separation. (A) Chromosome spreads were prepared from noninjected, or Cdc20-4AVGR–injected oocytes after overnight culture. The chromosomes were stained using a 2% Giemsa solution. In the insets, chromosomes are shown that were found outside the main field. a, uninjected in 1st meiosis (6 h after GVBD); b, uninjected in 2nd meiosis; c and d, Cdc20-4AVGR–injected oocytes with the PB extruded; e and f, Cdc20-4AVGR–injected oocytes with no PB extruded. Bar, 10 μm. (B) Quantification of A. On the x axis are the numbers of chromosomes counted per oocyte, on the y axis the number of oocytes. a, control; b, Cdc20-4AVGR–injected oocytes with PB extruded; c, Cdc20-4AVGR–injected oocytes with no PB extruded. (C) Time-lapse video microscopy. The Cdc20-4AVGR construct was coinjected into oocytes either with (a) cyclin B1N170-RFP (a 170–amino acid NH2-terminal fragment of cyclin B1 fused to RFP) or with (b) Pds1-RFP. The oocytes were kept in dbcAMP for 1 h, released 4 h after GVBD, and observed by differential interference contrast, and fluorescence time-lapse video microscopy. Pictures were taken every hour (BN170-RFP) or every 45 min (Pds1-RFP). The complete series is shown. Time points (h) after GVBD are indicated. Bars, 50 μm.

Mentions: The numbers of chromosomes from Cdc20-4AVGR–injected oocytes clearly differed from those of their uninjected counterparts (compare Fig. 8 A, b with c, d; a quantification is shown in Fig. 8 B). In uninjected metaphase II oocytes, we counted 18–24 chromosomes with 20 being the expected number (Fig. 8 A b; Fig. 8 B). In Cdc20-4AVGR–injected oocytes that had extruded a PB, chromosome counts were between 20 and 50 (Fig. 8 A, c and d; Fig. 8 B). The increase in the number of chromosomes indicated that the sister chromatids were at least partially separated in the Cdc20-4AVGR–injected oocytes. The wide variation of counts, and the fact that some were above the expected maximum of 40 chromatids, suggested that chromosome mis-segregation had occurred during meiosis I. Chromosomes prepared from Cdc20-4AVGR–injected oocytes that had failed to extrude their PB (Fig. 8 A, e and f) were not bivalent like those from control oocytes in the first meiosis (Fig. 8 A, a). Chromosome counts from these oocytes were higher than 40, ranging up to the expected maximum of 80 (Fig. 8 B).


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)

Cdc20-4AVGR mutant induces sister chromatid separation. (A) Chromosome spreads were prepared from noninjected, or Cdc20-4AVGR–injected oocytes after overnight culture. The chromosomes were stained using a 2% Giemsa solution. In the insets, chromosomes are shown that were found outside the main field. a, uninjected in 1st meiosis (6 h after GVBD); b, uninjected in 2nd meiosis; c and d, Cdc20-4AVGR–injected oocytes with the PB extruded; e and f, Cdc20-4AVGR–injected oocytes with no PB extruded. Bar, 10 μm. (B) Quantification of A. On the x axis are the numbers of chromosomes counted per oocyte, on the y axis the number of oocytes. a, control; b, Cdc20-4AVGR–injected oocytes with PB extruded; c, Cdc20-4AVGR–injected oocytes with no PB extruded. (C) Time-lapse video microscopy. The Cdc20-4AVGR construct was coinjected into oocytes either with (a) cyclin B1N170-RFP (a 170–amino acid NH2-terminal fragment of cyclin B1 fused to RFP) or with (b) Pds1-RFP. The oocytes were kept in dbcAMP for 1 h, released 4 h after GVBD, and observed by differential interference contrast, and fluorescence time-lapse video microscopy. Pictures were taken every hour (BN170-RFP) or every 45 min (Pds1-RFP). The complete series is shown. Time points (h) after GVBD are indicated. Bars, 50 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172623&req=5

fig8: Cdc20-4AVGR mutant induces sister chromatid separation. (A) Chromosome spreads were prepared from noninjected, or Cdc20-4AVGR–injected oocytes after overnight culture. The chromosomes were stained using a 2% Giemsa solution. In the insets, chromosomes are shown that were found outside the main field. a, uninjected in 1st meiosis (6 h after GVBD); b, uninjected in 2nd meiosis; c and d, Cdc20-4AVGR–injected oocytes with the PB extruded; e and f, Cdc20-4AVGR–injected oocytes with no PB extruded. Bar, 10 μm. (B) Quantification of A. On the x axis are the numbers of chromosomes counted per oocyte, on the y axis the number of oocytes. a, control; b, Cdc20-4AVGR–injected oocytes with PB extruded; c, Cdc20-4AVGR–injected oocytes with no PB extruded. (C) Time-lapse video microscopy. The Cdc20-4AVGR construct was coinjected into oocytes either with (a) cyclin B1N170-RFP (a 170–amino acid NH2-terminal fragment of cyclin B1 fused to RFP) or with (b) Pds1-RFP. The oocytes were kept in dbcAMP for 1 h, released 4 h after GVBD, and observed by differential interference contrast, and fluorescence time-lapse video microscopy. Pictures were taken every hour (BN170-RFP) or every 45 min (Pds1-RFP). The complete series is shown. Time points (h) after GVBD are indicated. Bars, 50 μm.
Mentions: The numbers of chromosomes from Cdc20-4AVGR–injected oocytes clearly differed from those of their uninjected counterparts (compare Fig. 8 A, b with c, d; a quantification is shown in Fig. 8 B). In uninjected metaphase II oocytes, we counted 18–24 chromosomes with 20 being the expected number (Fig. 8 A b; Fig. 8 B). In Cdc20-4AVGR–injected oocytes that had extruded a PB, chromosome counts were between 20 and 50 (Fig. 8 A, c and d; Fig. 8 B). The increase in the number of chromosomes indicated that the sister chromatids were at least partially separated in the Cdc20-4AVGR–injected oocytes. The wide variation of counts, and the fact that some were above the expected maximum of 40 chromatids, suggested that chromosome mis-segregation had occurred during meiosis I. Chromosomes prepared from Cdc20-4AVGR–injected oocytes that had failed to extrude their PB (Fig. 8 A, e and f) were not bivalent like those from control oocytes in the first meiosis (Fig. 8 A, a). Chromosome counts from these oocytes were higher than 40, ranging up to the expected maximum of 80 (Fig. 8 B).

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