Limits...
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.

Show MeSH
The effect of Bub1dn injection on meiosis I and II in mouse oocytes. (A) Time-lapse video microscopy. Uninjected, and Bub1dn-injected oocytes were kept 1 h in dbcAMP before they were released from the prophase block. 3 h after GVBD, the oocytes were placed under the microscope in a 37°C heating box. Pictures were taken every 45 min for 12 h. Red arrows indicate the extruded PB. Time points (h) after GVBD are indicated. Bar, 50 μm. (B) Bub1dn disrupts the SAC in meiosis II. Uninjected, and Bub1dn-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. Oocytes were fixed and stained with PI (red) and an anti-tubulin antibody, followed by an FITC-labeled secondary antibody (green; a and d), or with DAPI alone (b and e). The position of the PB is marked, and the oocyte chromosomes are boxed. (c and f) The DAPI stained chromosomes from b and e were enlarged by 2.6 times. Bar, 10 μm. (C) Single cell H1/MBP kinase assay. Total H1 and MBP kinase activities were assayed in whole cell lysates prepared from uninjected, GV-stage, and Bub1dn-injected oocytes. The autoradiograph is shown.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2172623&req=5

fig3: The effect of Bub1dn injection on meiosis I and II in mouse oocytes. (A) Time-lapse video microscopy. Uninjected, and Bub1dn-injected oocytes were kept 1 h in dbcAMP before they were released from the prophase block. 3 h after GVBD, the oocytes were placed under the microscope in a 37°C heating box. Pictures were taken every 45 min for 12 h. Red arrows indicate the extruded PB. Time points (h) after GVBD are indicated. Bar, 50 μm. (B) Bub1dn disrupts the SAC in meiosis II. Uninjected, and Bub1dn-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. Oocytes were fixed and stained with PI (red) and an anti-tubulin antibody, followed by an FITC-labeled secondary antibody (green; a and d), or with DAPI alone (b and e). The position of the PB is marked, and the oocyte chromosomes are boxed. (c and f) The DAPI stained chromosomes from b and e were enlarged by 2.6 times. Bar, 10 μm. (C) Single cell H1/MBP kinase assay. Total H1 and MBP kinase activities were assayed in whole cell lysates prepared from uninjected, GV-stage, and Bub1dn-injected oocytes. The autoradiograph is shown.

Mentions: Because the localization of Bub1dn-YFP to kinetochores might enable it to interfere with Bub1 function during meiosis, we microinjected the mRNA encoding Bub1dn into prophase oocytes, and monitored progression through meiosis. Time-lapse video microscopy (Fig. 3 A) revealed that most of the Bub1dn mRNA injected oocytes extruded their first PB 3 h earlier (i.e., 6–7 h after GVBD) than non- or control-injected oocytes (Table I). Thus, the Bub1dn mutant accelerated progression through meiosis I, probably by interfering with the SAC.


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 Bub1dn injection on meiosis I and II in mouse oocytes. (A) Time-lapse video microscopy. Uninjected, and Bub1dn-injected oocytes were kept 1 h in dbcAMP before they were released from the prophase block. 3 h after GVBD, the oocytes were placed under the microscope in a 37°C heating box. Pictures were taken every 45 min for 12 h. Red arrows indicate the extruded PB. Time points (h) after GVBD are indicated. Bar, 50 μm. (B) Bub1dn disrupts the SAC in meiosis II. Uninjected, and Bub1dn-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. Oocytes were fixed and stained with PI (red) and an anti-tubulin antibody, followed by an FITC-labeled secondary antibody (green; a and d), or with DAPI alone (b and e). The position of the PB is marked, and the oocyte chromosomes are boxed. (c and f) The DAPI stained chromosomes from b and e were enlarged by 2.6 times. Bar, 10 μm. (C) Single cell H1/MBP kinase assay. Total H1 and MBP kinase activities were assayed in whole cell lysates prepared from uninjected, GV-stage, and Bub1dn-injected oocytes. The autoradiograph is shown.
© Copyright Policy
Related In: Results  -  Collection

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

fig3: The effect of Bub1dn injection on meiosis I and II in mouse oocytes. (A) Time-lapse video microscopy. Uninjected, and Bub1dn-injected oocytes were kept 1 h in dbcAMP before they were released from the prophase block. 3 h after GVBD, the oocytes were placed under the microscope in a 37°C heating box. Pictures were taken every 45 min for 12 h. Red arrows indicate the extruded PB. Time points (h) after GVBD are indicated. Bar, 50 μm. (B) Bub1dn disrupts the SAC in meiosis II. Uninjected, and Bub1dn-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. Oocytes were fixed and stained with PI (red) and an anti-tubulin antibody, followed by an FITC-labeled secondary antibody (green; a and d), or with DAPI alone (b and e). The position of the PB is marked, and the oocyte chromosomes are boxed. (c and f) The DAPI stained chromosomes from b and e were enlarged by 2.6 times. Bar, 10 μm. (C) Single cell H1/MBP kinase assay. Total H1 and MBP kinase activities were assayed in whole cell lysates prepared from uninjected, GV-stage, and Bub1dn-injected oocytes. The autoradiograph is shown.
Mentions: Because the localization of Bub1dn-YFP to kinetochores might enable it to interfere with Bub1 function during meiosis, we microinjected the mRNA encoding Bub1dn into prophase oocytes, and monitored progression through meiosis. Time-lapse video microscopy (Fig. 3 A) revealed that most of the Bub1dn mRNA injected oocytes extruded their first PB 3 h earlier (i.e., 6–7 h after GVBD) than non- or control-injected oocytes (Table I). Thus, the Bub1dn mutant accelerated progression through meiosis I, probably by interfering with the SAC.

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