Activation of Xenopus laevis eggs in the absence of intracellular Ca activity by the protein phosphorylation inhibitor, 6-dimethylaminopurine (6-DMAP).
Xenopus laevis eggs pricked or microinjected with water or saline in medium containing a limited quantity of free Ca (1.0 to 2.0 microM) remain unactivated for at least 6 hr, even after transfer to oocyte medium containing Ca at higher concentrations (0.5-1.0 mM). These injected eggs, when later pricked in oocyte medium or exposed to A23187 or urethane are fully capable of activation. This confirms the observations of Wangh ('89). However, eggs injected in this Ca-limited medium (CaLM) with 6-DMAP as well as those simply exposed to this drug undergo changes characteristic of activation, including cortical contraction, cortical granule breakdown, a loss of MPF and CSF activities, and pronuclear formation. The time required for 6-DMAP to induce egg activation is inversely correlated to its concentration. Interestingly, eggs that have been injected with EGTA, and thus are unable to respond to activation stimuli such as pricking and A23187 or urethane treatment, can also be activated by exposure to 6-DMAP. In contrast, eggs exposed to or injected with a 6-DMAP analogue (6-aminopurine or puromycin) or a protein synthesis inhibitor (cycloheximide or emetine or puromycin) are not activated. As well, eggs injected in CaLM with 6-DMAP simultaneously with a phosphatase inhibitor (NaF or ammonium molybdate) fail to become activated. Although 6-DMAP-activated eggs remain at the pronucleus stage so long as 6-DMAP is present, they resume cell cycle activities after the drug is withdrawn. They form cleavage furrows, disassemble pronuclear envelopes, and recondense chromosomes. Also, MPF activity reappears and cycles at least twice, peaking each time shortly before cleavage furrow formation. These results suggest that activation of Xenopus eggs arrested at metaphase II by inhibition of protein phosphorylation does not require intracellular Ca release and that maintenance of the egg at metaphase II depends upon continuous protein phosphorylation.