Isolation of plasma membranes, Golgi bodies and mitochondria of Xenopus laevis morulae. Identification of plasma membrane proteins.

Homogenates of Xenopus morulae at the 16-32 cell stage were centrifuged on discontinuous sucrose gradients. Isolated fractions were identified by electron microscopy (EM) as mitochondria, a fraction enriched in Golgi vesicles, and plasma membranes. A special effort was made to prepare plasma membranes free of cytoplasmic contaminants. The resulting purified plasma membranes appeared morphologically identical to plasma membranes in situ. The external surface is covered with a fibrillar coat while vesicles are seen attached to their inner surface. O'Farell's method (1975) was used to obtain protein patterns of the various fractions on 2-dimensional gel electrophoresis. Each fraction displayed a specific pattern. By comparing the different patterns, it was possible to identify a group of proteins as belonging to the plasma membrane fractions. Labelling of cell surface with sulfo-N-hydroxysuccinimido-biotin together with differential extraction of proteins has allowed us to tentatively allocate these proteins in different structures of the plasma membrane fractions. The data presented in this paper corroborate and extend our ultrastructural studies on neogenesis of interblastomeric plasma membranes (Bieliavsky and Geuskens, 1990).

Autonomous mesoderm formation in blastocoelic roof explants from inverted Xenopus embryos.

Xenopus eggs, artificially fertilized, were prevented from undergoing equilibrium rotation by incubation in medium containing ficoll. Three orientations were selected: normal, with animal pole uppermost; inverted, with vegetal pole away from gravity; and an off-axis orientation, with embryos tilted approximately 90 degrees from the animal-vegetal axis. At blastula stage 8, cells forming the blastocoelic roof were cultured in isolation as explants. These cells are normally fated to from epidermis ventrally and neural derivatives dorsally. Unexpectedly, in the fragments originating from inverted or 90 degrees-off-axis embryos, axial structures were found: notochord, somites, neural cells, cement glands, and sometimes sensory organs. Inverted eggs could be exploited in studies of mesodermal specification.

Presence of high molecular weight glycans on the surface of germ and embryonic cells of Xenopus laevis.

Xenopus laevis male germ cells, fertilized eggs and gastrula cells were labelled with 3H labelled sodium borohydride reduction after galactose oxidase treatment. After pronase digestion, the bulk of the label is carried by high molecular weight glycans (greater than or equal to 6,000 D). The high molecular weight of these labelled glycans and their susceptibility to degradation by endo-beta-galactosidase suggest that they may be related to the polylactosaminoglycans.

Changes in patterns of protein synthesis in axolotl oocytes during progesterone-induced maturation.

Patterns of protein phosphorylation and synthesis during axolotl (Ambystoma mexicanum) oocyte maturation were studied by incorporation of [32P]orthophosphate and [35S]methionine into polypeptides, followed by two-dimensional gel electrophoresis. Various alterations were observed after progesterone treatment: de novo appearance of [35S]methionine-labelled polypeptides, a quantitative increase in previously synthesized proteins and a quantitative decrease in or disappearance of other previously synthesized proteins. Changes in 32P- and 35S-labelling were observed very early during maturation. Neither prior oocyte enucleation nor alpha-amanitin treatment had a significant effect on these changes. Stimulation with MPF provided the same final protein pattern as PG treatment. However, cholera toxin inhibited all the changes seen during maturation. Comparisons between the patterns of [35S]methionine- and [32P]phosphate-labelling provide further information on the biochemical events that take place during oocyte maturation.

Increased rate of capping of concanavalin A receptors during early Xenopus development is related to changes in protein and lipid mobility.

The mobility characteristics of plasma membrane constituents were studied in dissociated cells from embryos of Xenopus laevis at various stages of development from early blastula until neurulation. An increased rate of fluorescein isothiocyanate-concanavalin A induced patching and capping of Con A-binding proteins during this period of development was correlated with a threefold increase in the lateral mobility of the receptor molecules, as determined by the fluorescent photobleaching recovery (FPR) method, the major change occurring at the onset of gastrulation. Using the same method, it was demonstrated that the lateral mobility of plasma membrane lipids increases twofold during this period of development. The major change being detectable, however, at the late blastula stage. This is in coincidence with the initiation of cell motility in dissociated Xenopus embryo cells. It is concluded that the lateral mobility of membrane proteins and lipids increases significantly during early Xenopus development, but are at least in part subject to different control mechanisms. The results suggest that the initiation of morphogenetic movements is related to changes in the dynamic properties of plasma membrane constituents.

Subcellular distribution in Xenopus laevis oocytes of a microinjected poly(A)-binding protein from Artemia salina gastrulae.

The poly(A)-binding protein P38 of non-polysomal mRNP from Artemia salina gastrulae was labelled by reductive methylation and microinjected into the cytoplasm of Xenopus laevis oocytes. The labelled protein has a half-life of approximately 20 h and accumulated in the nucleus of the oocyte. The kinetics of accumulation reached a plateau at about 15 h after microinjection. P38 accumulates in the nucleus to a final concentration 3.15 times higher than that reached by free diffusion. This fact suggests that P38, a cytoplasmic poly(A)-binding protein, might also play some role in the nucleus of the cell.

An ultrastructural study of the effects of wheat germ agglutinin (WGA) on cell cortex organization during the first cleavage of Xenopus laevis eggs. I. Inhibition of furrow formation.

Xenopus laevis fertilized eggs have been treated with wheat germ agglutinin (WGA) before the onset of the first cleavage, at the stripe stage and during groove deepening. The ultrastructure of the animal cortex of the arrested embryos has been compared with that of the same region of control embryos at different stages of first furrow formation and of cytochalasin B-treated embryos. The outer side of the plasma membrane of WGA-treated embryos is covered with a coat which is different from the diffuse material observed in either control or cytochalasin B-treated embryos and which is distributed in patches in the groove region. Narrow indentations of the plasma membrane in the cortex of WGA-treated eggs have been observed, particularly in the blocked or regressed groove. In WGA-treated eggs, a few bundles of microfilaments are located under the plasma membrane at the animal pole, but they are never arrayed in a continuous layer as in the control eggs. In the latter, many microtubules are located in close proximity to the microfilament layer at the beginning of cleavage, but they are only occasionally observed in the same region of WGA-treated eggs. It is concluded that the binding of WGA molecules to their receptors on the surface of the Xenopus zygote interferes with the alignment of microfilaments in the furrow region and provokes the disorganization of the aligned microfilaments once the cleavage has begun. Internalization of portions of the nascent membrane in the groove could play an important part in the arrest of cleavage.

An ultrastructural study of the effects of wheat germ agglutinin (WGA) on cell cortex organization during the first cleavage of Xenopus laevis eggs. II. Cortical wound healing.

Uncleaved fertilized eggs of Xenopus laevis treated with wheat germ agglutinin (WGA) have been pricked at the animal pole both inside and outside the regressed furrow region. The wounded cortex of both regions has been studied with the electron microscope and compared with the same region of wounded, untreated eggs. In all 3 cases, filaments are organized in an annular zone in the damaged cortex. When the surface is pricked outside the regressed furrow of WGA-treated embryos, bundles of microfilaments radiate from the ring and extend in deep folds which form a 'star' around the wound at the surface of the embryo. However, when the surface is pricked in the new membrane of the regressed furrow, filaments are intermingled with internalized portions of the plasma membrane. It is suggested that, when the surface is pricked outside the furrow region, more filaments are mobilized to counteract the tangential retraction of the membrane which has acquired more rigidity after WGA binding.

Ribonucleotide reductase activity during amphibian development.

Uniformly labeled [3H] uridine is incorporated into DNA by dissociated Pleurodeles blastulae; the label is found in cytosine and to a much lesser extent in thymine. Ribonucleotide reductase activity cannot be detected in full grown oocytes of Xenopus and Pleurodeles, but is present in unfertilized egg. The enzyme is synthesized (or activated) when maturation is induced in Xenopus oocytes by in vitro hormonal treatment. The enzymatic activity increases after fertilization and reaches a peak at the 2--4 cell stage; it decreases at the blastula, gastrula and neurula stages to the low level initially present in unfertilized eggs. The enzyme is no longer detectable in swimming tadpoles. Addition of hydroxyurea (1 mg/ml) to fertilized eggs leads to complete loss of ribonucleotide reductase activity: cycloheximide (20 mug/ml) inhibits the rise in activity characteristic of early cleavage, while actinomycin D (20 mug/ml) has no effect. The significance of these results in discussed.