A temporal study of protease expression employing the technique of SDS-PAGE gelatin substrate zymography revealed a definitive appearance of proteases during early development in the sea urchins, Lytechinus pictus and Strongylocentrotus purpuratus. The levels of these proteases increase substantially during gastrulation in each species. The two major proteases with relative molecular masses of 57 and 50 kDa were found to be inhibited by the zinc chelator, 1,10-phenanthroline, the more nonspecific metal chelator, EDTA, and the reducing agent, dithiothreitol. The serine protease inhibitor, benzamidine, exerted no effects on the activities of these proteases, and both enzymes exhibited activity in the neutral to slightly basic pH range. Treatment of embryos with actinomycin D, an inhibitor of transcription, beginning up to 9 hr after fertilization, inhibited the subsequent appearances of the two proteases 48 hr after fertilization, as well as any morphological changes associated with gastrulation. Treatments beginning 15 and 21 hr after fertilization resulted in increased levels of proteases that correlate with arrests at successively more advanced stages of gastrulation. SDS-PAGE zymographic analyses of five different embryo fractions indicated that the 57- and 50-kDa proteases are localized in the blastocoel, and blastocoelic protease activity was further confirmed microscopically by in situ zymography. Hence, the 57- and 50-kDa proteases are characterized as metalloproteases. Their expression is dependent on transcription of the embryonic genome, and their spatiotemporal appearance suggests an involvement in blastocoelic matrix remodeling during gastrulation.
The synthesis of DNA in fertilized eggs of the American Gulf Coast sea urchin Lytechinus variegatus is 90% inhibited in the presence of 5.0 micrograms/ml aphidicolin. This inhibition may be imposed immediately upon addition of aphidicolin to the external medium when embryos are in "S" phase. Observations of living embryos with Nomarski optics and time-lapse video microscopy reveal that when eggs are fertilized and cultured in the continuous presence of aphidicolin, nuclear envelope breakdown, chromosome condensation, and cytokinesis are inhibited. All other post-fertilization events observable with this technique, including the assembly and disassembly of a bipolar spindle, proceed in the presence of aphidicolin. Antitubulin immunofluorescence microscopy of aphidicolin-arrested embryos demonstrates that microtubules attempt to assemble a mitotic apparatus at the first cell cycle; the arrested intact zygote nucleus is embedded within this bipolar structure. Subsequent cycles of microtubule assembly and disassembly proceed roughly on schedule with later division cycles, but the microtubule organizing centers (MTOC's) are unable to duplicate properly and irregular monasters are observed. If aphidicolin is added to embryos after the first DNA synthetic period, nuclear envelope breakdown, chromosome condensation, and cytokinesis proceed for that cycle and the embryos arrest at the two-cell stage. These results suggest that the direct inhibitory effects of aphidicolin may well be limited to the synthesis of DNA, which itself regulates nuclear cycles independently from the subsequent generation of mitotic poles, and that cytoplasmic clocks regulate microtubule assembly cycles but not the configuration of microtubule arrays.
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