Changes in mitochondrial respiration during the development of Xenopus laevis

Mitochondria isolated from Xenopus laevis embryos at various developmental stages show a good oxidative capacity and an acceptable respiratory control provided that certain requirements are fulfilled. The rates of respiration with pyruvate and Krebs' cycle intermediates, especially with citrate and isocitrate, are very low during cleavage stages and increase after gastrulation. Glutamate in the presence of malate is the only substrate to be readily oxidized during early development and its rate of oxidation decreases after gastrulation. These results, together with the altered sensitivity of embryonic mitochondria towards azide, support the view that the oxidative metabolism undergoes important changes around gastrulation and is associated with mitochondrial differentiation.     

Changes in energy metabolism during the early development of Xenopus laevis

Xenopus embryos at various development stages were incubated in the presence of labelled substrates and the 14CO2 production determined. From the rates of oxidation of glucose labelled in positions 1 and 6 and from that of radioactive acetate, pyruvate and glutamate, it was concluded that the Embden-Meyerhof pathway and the Krebs cycle are functional during early embryogenesis, but that their relative participation in the metabolic processes is limited and increases from gastrulation onwards. Early development is characterized by the predominance of the pentose cycle and the glutamate-aspartate cycle. Furthermore, it was shown that glutamate may be the main energy source up to gastrulation.     

Changes in polyamine content during limb regeneration in adult Xenopus laevis

Polyamine contents in the regenerates were determined at various stages after amputation of the forelimbs of the adult female Xenopus laevis. Putrescine, spermidine, spermine, and sym-homospermidine were detected in all the specimens examined. Cadaverine was detected only in a limited number of samples. At 5 days after amputation of forelimbs, well before the formation of regenerates, the putrescine content in the stump tissues increased, followed by the increase in spermidine content. The putrescine level in the forelimb regenerates was highest between 30 and 50 days after amputation, and then decreased. The spermidine concentration in the regenerates was about 20 times greater than that in intact forelimbs all throughout the experiments. The concentration of spermine was initially lower than that of both putrescine and spermidine and further decreased soon after amputation. The concentration of sym-homospermidine was originally very low and increased slightly during regeneration. The significance of these results, with respect to the function of polyamines in forelimb regeneration of Xenopus laevis, is discussed.     

Cathepsin D Activity in the Vitellogenesis of Xenopus laevis

An ovarian extract of Xenopus laevis exhibited in SDS-PAGE analyses an activity cleaving vitellogenin to lipovitellins under mildly acidic conditions. This activity was pepstatin-sensitive and inhibited by monospecific anti-rat liver cathepsin D antibody and thus identified as cathepsin D. Immunoblot analysis showed that two proteins of 43 kDa and 36 kDa immunoreacted with the antibody.
Immunocytochemical staining revealed that the enzyme was located in the cortical cytoplasm of stage I and II oocytes and in small yolk platelets and nascent forms of large yolk platelets in the cortical cytoplasm of stage III oocytes. In stage IV and V oocytes, small yolk platelets retained the immuno-staining but large yolk platelets decreased it. No immuno-positive signals were observed in oocytes at stage VI. When examined by immunoelectron microscopy, gold particles indicated that cathepsin D was located on dense lamellar bodies in the cortical cytoplasm of stage I and II oocytes. The particles were located on primordial yolk platelets and on the superficial layer of small yolk platelets in stage III oocytes, while they were sparse or not present at all on large yolk platelets in stage IV and V oocytes. These results indicate that cathepsin D plays a key role in vitellogenesis by cleaving endocytosed vitellogenin to yolk proteins in developing oocytes.     

Protein Changes in Regressing Tail Tissue of Xenopus laevis during Natural Metamorphosis

Sodium dodecyl sulfate (SDS) gel electrophoresis was used to study the soluble protein fraction of Xenopus laevis tail tissue during in vivo metamorphosis. Prior to morphological signs of tail regression stage 45, a new subunit protein was resolved. At stage 64 three additional subunit proteins were resolved at the end of tail resorption. Results indicate that the altered balance between protein synthesis and degradation has little effect on the protein subunit population prior to morphological signs of tail regression.     

Development of the primary mouth in Xenopus laevis

The initial opening between the gut and the outside of the deuterostome embryo breaks through at the extreme anterior. This region is unique in that ectoderm and endoderm are directly juxtaposed, without intervening mesoderm. This opening has been called the stomodeum, buccopharyngeal membrane or oral cavity at various stages of its formation, however, in order to clarify its function, we have termed this the "primary mouth". In vertebrates, the neural crest grows around the primary mouth to form the face and a "secondary mouth" forms. The primary mouth then becomes the pharyngeal opening. In order to establish a molecular understanding of primary mouth formation, we have begun to examine this process during Xenopus laevis development. An early step during this process occurs at tailbud and involves dissolution of the basement membrane between the ectoderm and endoderm. This is followed by ectodermal invagination to create the stomodeum. A subsequent step involves localized cell death in the ectoderm, which may lead to ectodermal thinning. Subsequently, ectoderm and endoderm apparently intercalate to generate one to two cell layers. The final step is perforation, where (after hatching) the primary mouth opens. Fate mapping has defined the ectodermal and endodermal regions that will form the primary mouth. Extirpations and transplants of these and adjacent regions indicate that, at tailbud, the oral ectoderm is not specifically required for primary mouth formation. In contrast, underlying endoderm and surrounding regions are crucial, presumably sources of necessary signals. This study indicates the complexity of primary mouth formation, and lays the groundwork for future molecular analyses of this important structure.     

Cellular Commitment for Post-Gastrular Increase in Alkaline Phosphatase Activity in Xenopus laevis Development

Xenopus embryos were dissociated into cells and cultured in Ca²⁺-free medium to study the relationship between the cell-to-cell interaction and macromolecular synthesis. Under the conditions, cells did not aggregate at all, and remained isolated even while they were dividing actively. Synthesis of DNA and protein as studied by the incorporation of (³H)thymidine and (³H)leucine proceeded as in the aggregating cells. Also, the activity to synthesize rRNA, 5S RNA, and heterogeneous RNA as determined by the incorporation of (³H)uridine was not impaired. Such an increase in the activity of alkaline phosphatase, as occurs in embryos after the gastrula stage, was found to be inhibited greatly when early-blastula cells were cultured in the non-aggregating conditions. However, we found here that the inhibition was not observed with cells isolated from late-blastulae. Therefore, it appears that the increase in the activity of alkaline phosphatase during post-gastrular stages is dependent on some cellular commitment which may be established by cell-to-cell contact during the blastula stage.     

Changes in the polysome content of developing Xenopus laevis embryos

A method for preparing polysomes from all embryonic stages of Xenopus laevis is described. In the oocyte only about 1–2% of the total ribosomes are present in polysomes, the remainder being a developmental reserve. Upon conversion to an egg the polysome content rises by up to 3-fold, and by about a further 2-fold after fertilization. There is only a small further increase during cleavage, but by the tailbud stage, when organogenesis begins, there is a more rapid rise. Most of the ribosomes are incorporated into polysomes by stage 42, shortly before feeding begins.At very early stages, the changes in polysome content seem to mirror the changes in protein synthesis. At later stages the polysome contents reported here provide the only available guide to changes in the rate of protein synthesis. Judged by polysome content, the stage 42 tadpole seems to make protein about 20 times faster than the unfertilized egg, though it contains very few more ribosomes. The relationship between polysome content and the synthesis of various types of RNA is discussed.     

Mitochondrial DNA synthesis in large and mature oocytes of Xenopus laevis

Deoxynucleosides are incorporated into mitochondrial DNA (mtDNA) of large oocytes; the rate of incorporation is about 2% of the mtDNA amount per 24 hr. When oocytes have been induced to mature in vitro with human chorionic gonadotropin (HCG), uptake and actual incorporation of thymidine decrease, although phosphorylation is enhanced. An examination of mtDNA replication shows that HCG treatment induces an increase in the relative synthesis of E-strands and an accumulation of D-loops. A similar effect is obtained by ethidium bromide treatment. Thus, gonadotropin appears to delay E-strand elongation and to synchronize mtDNA molecules at the begining of their replication cycle.     

Regulated Formation of Extrachromosomal Circular DNA Molecules during Development in Xenopus laevis

Extrachromosomal circular DNA molecules of chromosomal origin have been detected in many organisms and are thought to reflect genomic plasticity in eukaryotic cells. Here we report a developmentally regulated formation of extrachromosomal circular DNA that occurs de novo in preblastula Xenopus embryos. This specific DNA population is not detected in the male or female germ cells and is dramatically reduced in later developmental stages and in adult tissues. The activity responsible for the de novo production of extrachromosomal circles is maternally inherited, is stored in the unfertilized egg, and requires genomic DNA as a template. The formation of circular molecules does not require genomic DNA replication but both processes can occur simultaneously in the early development. The production of extrachromosomal circular DNA does not proceed at random since multimers of the tandemly repeated sequence satellite 1 were over-represented in the circle population, while other sequences (such as ribosomal DNA and JCC31 repeated sequence) were not detected. This phenomenon reveals an unexpected plasticity of the embryonic genome which is restricted to the early developmental stage.