Abstract
In cells containing disrupted spindles, the spindle assembly checkpoint arrests the cell cycle in metaphase. The budding uninhibited by benzimidazole (Bub) 1, mitotic arrest-deficient (Mad) 1, and Mad2 proteins promote this checkpoint through sustained inhibition of the anaphase-promoting complex/cyclosome. Vertebrate oocytes undergoing meiotic maturation arrest in metaphase of meiosis II due to a cytoplasmic activity termed cytostatic factor (CSF), which appears not to be regulated by spindle dynamics. Here, we show that microinjection of Mad1 or Mad2 protein into early Xenopus laevis embryos causes metaphase arrest like that caused by Mos. Microinjection of antibodies to either Mad1 or Mad2 into maturing oocytes blocks the establishment of CSF arrest in meiosis II, and immunodepletion of either protein blocked the establishment of CSF arrest by Mos in egg extracts. A Mad2 mutant unable to oligomerize (Mad2 R133A) did not cause cell cycle arrest in blastomeres or in egg extracts. Once CSF arrest has been established, maintenance of metaphase arrest requires Mad1, but not Mad2 or Bub1. These results suggest a model in which CSF arrest by Mos is mediated by the Mad1 and Mad2 proteins in a manner distinct from the spindle checkpoint.
Highlights
Vertebrate eggs undergo an arrest of the cell cycle with an intact spindle at metaphase of meiosis II while awaiting fertilization
Blastomeres injected with Mad1 arrested cell division with kinetics similar to that seen after Mos injection, and Mad1-arrested blastomeres arrested in metaphase, as witnessed by DNA and spindle morphology (Fig. 1 E)
The metaphase arrest mediated by cytostatic factor (CSF) activity occurs in the presence of a spindle that has kinetochores fully attached to microtubules
Summary
Vertebrate eggs undergo an arrest of the cell cycle with an intact spindle at metaphase of meiosis II while awaiting fertilization. This arrest is a result of inhibition of the anaphase-promoting complex/cyclosome (APC/C), and is mediated by an activity generated during oocyte maturation, termed cytostatic factor (CSF; Masui and Markert, 1971). One pathway arises from the synthesis of Cdk and cyclin E proteins during oocyte maturation. Cdk and cyclin E are present at very low levels in resting oocytes, and their levels increase dramatically during entry into meiosis II (Rempel et al, 1995). Injection of maturing oocytes with antisense oligonucleotides specific for Cdk attenuates CSF arrest in vivo (Gabrielli et al, 1993), and addition of a
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