Studies on nucleoside and amino acid uptake and on RNA and protein synthesis by growing oocytes, unfertilized and fertilized
The principal theme of these investigations concerns the inhibited state of mature unfertilized sea urchin eggs with respect to uridine uptake and protein synthesis. Part I demonstrates that unfertilized-eggs are relatively impermeable to uridine. Fertilized eggs, however, develop during the...
| Summary: | The principal theme of these investigations concerns the inhibited state of mature unfertilized sea urchin eggs with respect to uridine uptake and protein synthesis.
Part I demonstrates that unfertilized-eggs are relatively impermeable to uridine. Fertilized eggs, however, develop during the first hour an energy-dependent, uptake mechanism for uridine accumulation. Labeled uridine assimilated by fertilized eggs is recovered as phosphorylated nucleosides, primarily triphosphates. Experiments support the idea that uridine penetration into sea urchin eggs depends upon the phosphorylation of the 5' carbon atom at the cell surface. Tests with puromycin show that protein synthesis is unnecessary for the generation of uridine uptake after fertilization. The evidence favors the view that uridine kinase is sequestered within the unfertilized egg and thus incapable of activity at the cell surface until after fertilization.
Parts II and III use biochemical and autoradiographic methods to show that growing oocytes of sea urchins, in contrast to many other organisms, undergo considerable RNA and protein synthesis. Protein synthesis in isolated oocytes occurs throughout the germinal vesicle and cytoplasm and takes place on polyribosomes. RNA synthesis is localized in the nucleolus. Mature eggs, however, synthesize only little protein even in mixed suspensions with oocytes. Long-term maintenance of spawned female sea urchins, after but one injection of labeled uridine, produces ripe unfertilized eggs possessing highly radioactive RNA. The distribution of label in the extracted RNAs is 70-80% ribosomal, 10-20% heterogeneous, and 5-10% soluble.
Part IV is an electron microscopic and biochemical examination of RNA-labeled mature unfertilized and fertilized eggs. The findings are correlated with the difference in protein synthesizing activity before and after fertilization. The results show that unfertilized eggs synthesize protein upon RNase-sensitive polyribosomes. The large increase in protein synthesis after fertilization occurs in association with the assembly of additional polyribosomes. Homogenates of unfertilized eggs also possess synthetically inactive, RNase-resistant, ribosomal aggregates. Evidence suggests that trypsin followed by RNase disperses the aggregates. Homogenates of fertilized eggs, however, contain very few RNase-resistant ribosomal aggregates. By forty minutes after fertilization, about 70% of the new protein synthesis can be attributed to the new polyribosomes. The weight of the evidence indicates that the remaining 30% of the stimulation of protein synthesis is due to the activation of "masked" polyribosomes.
Appendix 1 shows that, for unfertilized and fertilized eggs, competition for uptake of amino acids occurs primarily among those belonging to the same charge group. Appendix 2 demonstrates that one amino acid can displace another of the same category from intact eggs both before and after fertilization. By combination of these facts, then, it is possible to achieve greater labeling of egg-proteins than has been previously realized.
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