Protein synthesis requirements during resumption of meiosis in the hamster oocyte: early nuclear and microtubule configurations

Abstract

The organization of chromatin and cytoplasmic microtubules changes abruptly at M-phase entry in both mitotic and meiotic cell cycles. To determine whether the early nuclear and cytoplasmic events associated with meiotic resumption are dependent on protein synthesis, cumulus-enclosed hamster oocytes were cultured in the presence of 100 micrograms/ml puromycin or cycloheximide for 5 hr. Both control (untreated) and treated oocytes were analyzed by fluorescence microscopy after staining with Hoechst 33258 and tubulin antibodies. Freshly isolated oocytes exhibit prominent nucleoli and diffuse chromatin within the germinal vesicle as well as an interphase network of cytoplasmic microtubules. After 4-4.5 hr in culture, most oocytes were in prometaphase I of meiosis as characterized by a prominent spindle with fully condensed chromosomes and numerous cytoplasmic asters. After 5-5.5 hr in culture, microtubule asters are no longer detected in most cells, and the spindle is the only tubulin-positive structure. Incubation for 5 hr in the presence of inhibitors does not impair germinal vesicle breakdown, chromatin condensation, kinetochore microtubule assembly, or cytoplasmic aster formation in the majority of oocytes examined; however, under these conditions, a population of oocytes retains a germinal vesicle, exhibiting variable degrees of chromatin condensation and cytoplasmic aster formation. Meiotic spindle formation is inhibited in all oocytes. These effects are fully reversible upon culture of treated oocytes in drug-free medium for 5 hr. The data indicate that meiotic spindle assembly is dependent on ongoing protein synthesis in the cumulus-enclosed hamster oocyte; in contrast, chromatin condensation and aster formation are not as sensitive to protein synthesis inhibitors during meiotic resumption.