Xenopus laevis vitellogenin is a plasma protein that contains a total of 5 mol of metal/440 kDa dimer, 2 mol of zinc, and 3 mol of calcium (Montorzi et al. (1994) Biochem. Biophys. Res. Commun. 200, 1407-1413]. There are no other group IIB or transition metals in the molecule. The zinc atoms are removed instantaneously by 1,10-phenanthroline (OP) (pK 4.8). Once internalized by receptor-mediated endocytosis, vitellogenin is cleaved into multiple polypeptides, i.e., the two lipovitellin subunits (1 and 2) plus phosvitin; these are then stored as microcrystals within yolk platelets. We here show by metal analysis of the individual proteins generated by vitellogenin processing that zinc and calcium occur in different domains of the vitellogenin polypeptide chain. All of the vitellogenin zinc is present in lipovitellin, in amounts equal to 1 mol of zinc/141 kDa. Calcium, in contrast, is detected exclusively in phosvitin which, in addition, contains 3 mol of magnesium/35 kDa, apparently acquired following vitellogenin entry into the oocyte. The zinc in lipovitellin is removed by OP in a concentration-dependent manner with a pK of 4.8, identical to that obtained for vitellogenin, and by exposure to acidic conditions (below pH 5). Following removal of zinc, the two lipovitellin subunits remain associated, suggesting that zinc is not involved in their interaction. On exposure to 1% SDS, lipovitellin does dissociate into 106 and 33 kDa subunits. The presence of stoichiometric quantities of zinc in both vitellogenin and lipovitellin calls for the study of the hitherto unrecognized biochemistry and functions of these proteins in zinc metabolism and development of the frog oocyte and embryo.

The determinants of Xenopus laevis embryos that act before their first cell division are mandatory for the formation of mRNas required to establish the dorsal axis. Although their chemical identities are unknown, a number of their properties have long been recognized. One of the determinants is present in the cytoplasm and is sensitive to UV light. Thus, exposing stage 1 embryos to either standard 254-nm or, as shown here, to 366-nm UV light during the 0.3-0.4 time fraction of their first cycle inactivates the cytoplasmic determinant. As a consequence, both types of irradiated embryos fail to express dorsal markers, e.g., goosecoid and chordin, without affecting formation of ventral markers, e.g., Vent-1. The developmental outcome is dorsal axis-deficient morphology. We report here that biliverdin IX␣, a normal constituent of cytoplasmic yolk platelets, is photo-transformed by irradiation with either 254-or 366-nm UV light and that the transformation triggers the dorsal axis deficiency. When the 254-or 366-nm UV-irradiated embryos, fated to dorsal axis deficiency, are incubated solely with M amounts of biliverdin, they recover and form the axis. In contrast, incubation with either in vitro photo-transformed biliverdin or biliverdin IX␣ dimethyl ester does not induce recovery. The results define an approach to produce dorsal axisdeficient embryos by photo-transforming its biliverdin by irradiation with 366-nm UV light and identify an unsuspected role for biliverdin IX␣ in X. laevis embryogenesis.

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The essential role of zinc in embryogenesis was identified through studies of its presence in eggs and embryos, the effects of its deficiency and its role in metallo proteins required for organ development and formation. The Xenopus laevis oocyte zinc content varies during oogenesis. It increases from 3 to 70 ng zinc/oocyte as it progresses from stage I to VI. The oocyte zinc is derived from the maternal liver as part of a metallo-complex with vitellogenin. The latter transports the metal in plasma and into the oocyte. Once internalized, most of the zinc is stored within yolk platelets bound to lipovitellin, one of the processed products of vitellogenin. About 90% of the total zinc is associated with the yolk platelet lipovitellin while the remaining I 0% is in a compartment associated with hitherto unknown molecule(s) or organelle(s) of the cytoplasm. The bi-compartmental distribution remains constant throughout embryogenesis since the embryo behaves as a closed system for zinc after fertilization. The yolk platelet zinc is used after the tadpole is hatched while we proposed that the I 0% of the zinc in the non-yolk platelet pool is the one used for embryogenesis. It provides zinc to newly synthesized molecules responsible for the development of zinc-dependent organ genesis. Interference with the availability of this zinc by the chelating agent I, I 0-phenanthroline results in the development of embryos that lack dorsal organs, including brain, eyes and spinal cord. The extensive teratology is proposed to be due to altered or absent zinc distribution between the cytosolic pool and zinc-transcription factors. The data identify the components of a zinc transport, storage and distribution system in a vertebrate organism.

Xenopus laevis vitellogenin contains 2 g-atoms (g-at) of Zn and 3 g-at of Ca/dimer, transports zinc in plasma, and plays a role in its distribution within the oocyte [Montorzi et al. (1994) Biochem. Biophys. Res. Commun. 200, 1407-1413; Montorzi et al. (1995) Biochemistry 34, 10851-10858]. We here report the dynamics and time course of Zn65-labeled vitellogenin uptake by and distribution within stages II and IV oocytes, the fate of the metal in oocytes as they progress from stages II to VI, as well as in the first two cleavage blastomeres, the blastula, and subsequent stages of the developing embryo and tadpole. Zn65 bound to vitellogenin is taken up within less than 30 min by either stage II or IV oocytes incubated under in vitro culture conditions whereas free Zn65 is not. Once internalized, Zn65 remains within the cytosol of stage II, whereas in stage IV oocytes, it is transferred within 4 h of its entry from the cytosol into yolk platelets. Nearly all of the transferred Zn65 is found within yolk platelets and their precursors where it is associated with the vitellogenin cleavage product, lipovitellin. Its distribution within the oocyte organelles differs at each stage of oogenesis. In the early stages (III-IV) most of the oocyte zinc is located first in the small endocytosed vesicles and then in multivesicular bodies. When the zinc transfer process is finalized in the late stages of oogenesis (V-VI), > 90% of the total oocyte zinc is within yolk platelets while the remainder is in the cytosol. In embryos and tadpoles, the larger of these two pools remain sequestered in yolk platelets and is inaccessible to cytosolic apoproteins throughout the entire period of embryo formation. Its redistribution to the cytosol does not begin until several days after the tadpole has hatched. The smaller pool, on the other hand, is already present in the cytosol and is, therefore, postulated to constitute the sole source of zinc required for embryogenesis.