Comparative study of the localization of incorporated 14C-labeled amino acids and 35SO 4 in the sea urchin ovary, egg and embryo

Abstract

1. 1. The incorporation of 14C-labeled amino acids and of 35SO 4 into the ovary, oocyte, egg and embryo of the sea urchin was studied by means of autoradiography combined with histochemical analysis. The species employed were Echinus esculentus, Paracentrotus lividus, Psammechinus miliaris and Sphaerechinus granularis. 2. 2. Precursors were injected into the body cavity of the adult female, and labeling was observed in sections of the ovary as well as in sections of eggs and embryos obtained from eggs removed from the ovary. The extent of incorporation depended on the degree of maturity of the eggs at incubation, and on the species. 3. 3. The first site of incorporation of 14C is the nucleolus of the oocyte. It is inferred that amino acids are complexed with low-polymer RNA, which accumulates in the outer rim of the nucleolus. From this site the low-polymer RNA-amino acids complex migrates into the germinal vesicle and thence, into the cytoplasm. 4. 4. Labeled sulfate is incorporated directly into the cytoplasm of the oocyte. The distribution is random at first, but as the maturation proceeds, the labeled area becomes limited to the cortex. 5. 5. Eggs labeled while in the ovary were fertilized and the origin and nature of the substances which constitute the fertilization membrane and the substance present in the perivitelline space were traced. In this respect, species differences were found. 6. 6. Precursors were added to a suspension of eggs or embryos for varying lengths of time, and at various stages. No incorporation is obtained in mature unfertilized eggs after 30 minutes' incubation. Incorporation of 14C, although weak, is obtained in eggs immediately after fertilization, whereas incorporation of 35SO 4 is first noticeable only at blastula stage. 7. 7. Added 35SO 4 was incorporated into the blastocoelic gel. Parallel staining with Hale reagent showed that in earlier stages the SO 4-group of the acid polysaccharides contained in this gel are unmasked. From 9–10 hours after fertilization, an incorporation of labeled amino acids into blastocoelic gel was found. At the same time the Hale staining disappeared, indicating masking of the SO 4-groups by proteins. 8. 8. It was demonstrated that, from a period immediately preceding the mesenchyme blastula stage, the incorporation of labeled amino acid becomes more intense in the vegetal region. At the same time the incorporation of 35SO 4 becomes demonstrable. The incorporation of both kinds of labeled precursors has, in the mesenchyme blastula and the early gastrula, its maximum in a region bordering the vegetal region of the blastocoel. 9. 9. The mesenchyme cells are imbedded in a matrix of sulfated polysaccharides combined with proteins. The importance of such a matrix for the formation of filopodial projections is pointed out. 10. 10. In the gastrula stage a parallel incorporation of labeled amino acids and of 35SO 4 was observed in mesenchyme and endomesoderm. The Hale staining indicated, however, that in the mesenchyme connected with the animal region of the ectoderm the sulfate of the acid polysaccharides is masked by proteins. In the mesenchyme connected with the vegetal region of the ectoderm the sulfate is unmasked, not being closely linked to proteins. This latter holds true also for the acid polysaccharides present in the exterior and interior border of the intestine. 11. 11. The RNA available to Hale and pyronin staining is considered to be a low-polymer RNA. This is formed in the outer rim of the nucleolus (cf. p. 361), but also in the most animal region of the developing late blastulae or gastrulae. The possibility is mentioned that the animal region of the larva may be the main site of production of low-polymer RNA. 12. 12. The parallel incorporation of 35SO 4 and 14C-labeled amino acids is considered to suggest that acid polysaccharides may have an acceptor function in protein synthesis. This function would involve a removal of proteins from their site of formation on RNP-particles, favoring the synthesis of new protein molecules. The suggestion is also made that unmasked acid polysaccharides may have an ion-exchange effect which would be important for the control of diffusion of certain substances.