Metaphase protein phosphorylation in Xenopus laevis eggs.

Cytoplasmic extracts of metaphase (M-phase)-arrested Xenopus laevis eggs support nuclear envelope breakdown and chromosome condensation in vitro. Induction of nuclear breakdown is inhibited by AMPP(NH)P, a nonhydrolyzable ATP analog, but not by ATP or gamma-S-ATP, a hydrolyzable ATP analog, suggesting that protein phosphorylation may be required for M-phase nuclear events in vitro. By addition of [gamma-32P]ATP, we have identified in cytoplasmic extracts and in intact eggs at least six phosphoproteins that are present during M-phase but absent in G1/S-phase. These phosphoproteins also appear in response to partially purified preparations of maturation-promoting factor. A subset of these proteins are thiophosphorylated by gamma-S-ATP under conditions that promote nuclear envelope breakdown and chromosome condensation. Each of these proteins is phosphorylated on serine and threonine, and one, a 42-kilodalton protein, is also phosphorylated on tyrosine both in extracts and in intact eggs. These results indicate that activation of protein kinases accounts for at least part of the increased phosphorylation in M-phase and that both protein-serine-threonine kinases and protein-tyrosine kinases may play a role in controlling M-phase nuclear behavior.     

Soluble cytokeratins in Xenopus laevis oocytes and eggs

Xenopus oocytes contain a radial network of cytokeratins which seems to fragment during meiosis reinitiation (maturation). The mature egg contains only a cortical network of cytokeratins. We have looked for the presence of soluble cytokeratins in oocytes and unfertilized eggs and have found them in both cases. However, the proportion of soluble to insoluble cytokeratins is slightly higher in the egg than in the oocyte. Soluble cytokeratins incorporate 35S-methionine at a high rate in the oocyte but to a lesser extent in the egg. This suggests that they are biosynthetic intermediates in the oocyte. In the egg, at least a fraction of the soluble cytokeratins may arise from the fragmentation of the polymer which seems to occur during the maturation process. Insoluble cytokeratins are strongly labeled with 32P both in oocytes and eggs. On the other hand only the soluble keratins of the egg incorporate 32P. Since the isoelectric point of soluble and insoluble cytokeratins is the same in oocytes and eggs, their absolute level of phosphorylation probably remains relatively constant. This suggests that: i) phosphate turnover is very slow in oocyte soluble cytokeratins, ii) phosphorylation is not a major way of changing the structural state of cytokeratins in amphibian oocytes and eggs.     

DNA ligase I from Xenopus laevis eggs.

We have purified the major DNA ligase from Xenopus laevis eggs and raised antibodies against it. Estimates from SDS PAGE indicate that this DNA ligase is a 180 kDa protein. This enzyme is similar to the mammalian type I DNA ligase which is presumed to be involved in DNA replication. We have also analysed DNA ligase activity during X. laevis early development. Unfertilized eggs contain the highest level of activity reflecting the requirement for a large amount of DNA replicative enzymes for the period of intense replication following fertilization. In contrast with previous studies on the amphibians axolotl and Pleurodeles, the major DNA ligase activity detected during X. laevis early development is catalysed by a single enzyme: DNA ligase I. And the presence of this DNA ligase I in Xenopus egg before fertilization clearly demonstrates that the exclusion process of two forms of DNA ligase does not occur during X. laevis early development.     

Microinjected Tetrahymena rDNA ends are not recognized as telomeres in Xenopus eggs

Telomeres are essential structures that stabilize the ends of eukaryotic chromosomes and allow complete replication of linear DNA molecules. We examined the structure and replication of telomeres by observing the fate of the linear extrachromosomal rDNA of Tetrahymena after injection into unfertilized Xenopus eggs. The rDNA replicated efficiently as a linear extrachromosomal molecule, increasing in mass 30-50-fold by 15-20 h after injection. In addition, the molecules increased in length by addition of up to several kilobases of DNA to their termini. Sequence analysis demonstrated that the added DNA bore no resemblance to known telomeres. The junction between the rDNA and added DNA was apparently random, indicating that the addition reaction did not involve a site-specific recombination or integration event. Surprisingly, Southern blot analysis showed that the added DNA did not derive from Xenopus DNA, but rather from co-purifying and therefore co-injected Tetrahymena DNA. The nonspecific ligation of random DNA fragments to the rDNA termini suggests that microinjected Tetrahymena rDNA ends are not recognized as telomeres in Xenopus eggs.     

Radioiodination studies of the envelopes from Xenopus laevis eggs

To investigate the molecular basis of the observed morphological and biological characteristics of coelomic egg envelopes (CE), vitelline envelopes (VE), and fertilization envelopes (FE) of Xenopus laevis eggs, envelopes were radioiodinated under a variety of conditions: in situ, isolated and intact, or solubilized. The distribution of 125I in envelope components was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Each envelope type displayed unique profiles when iodinated in the intact state. A major constituent of VE, the 41,500 molecular weight component, was not labeled in the intact state, although the corresponding component of CE was heavily labeled. After dissociation of the envelope by guanidine-HCl or sodium dodecyl sulfate, all of the components could be radioiodinated. However, when the envelopes (VE and FE) were dissolved by heating and subsequently radioiodinated by lactoperoxidase, the resulting radioactivity profile was similar to that of the intact envelopes, suggesting that in the heat-dissolved envelope, the individual components retain similar structural relations as in the intact envelope. Quantitative but not qualitative differences were found between the inner and outer aspects of VE and FE. The significance of these findings is discussed in relation to what is known about the morphological, biological, and molecular properties of the envelopes.     

Lithium inhibits morphogenesis of the nervous system but not neuronal differentiation in Xenopus laevis

Xenopus embryos treated with 100 mM-lithium from the 2- to 4-cell stage to the early blastula stage (4h) failed to neurulate and developed without a discernible anteroposterior axis. The internal structure of defective embryos was grossly disorganized, but immunohistochemical staining with cell-type-specific antibodies revealed differentiated nerve and muscle cells. Quantitative assay in tissue cultures from control and acutely abnormal lithium-treated embryos showed that neural differentiation was enhanced and muscle differentiation unaffected. The embryos took up about 0.5 mM-lithium at threshold, maximal effects resulted at 2-3 mM. Most of the lithium was extruded from the cells into the blastocoel fluid, where lithium reached 17 mM. The threshold intracellular concentration was about 150 microM. Lithium uptake rose steeply as the osmotic/ionic strength of the bathing medium increased. Sodium, potassium and lithium were equally able to increase the permeability of the embryo. However, sodium ions enhanced, while potassium ions interfered with, the uptake of lithium. Treatment with lithium at progressively later stages reduced the developmental defects and neural differentiation returned to normal levels. The uptake of lithium did not decline concomitantly. We conclude that lithium does not inhibit neural induction, but interferes with dorsal patterning. The sensitivity of the embryo to lithium is determined by developmental stage. The very low, effective intracellular concentrations may be important in understanding the mechanism of lithium-generated defects.     

Neural inducing factors from Xenopus laevis eggs and embryos

Large foreheads can be induced by ribonucleoprotein particles from Xenopus laevis eggs and embryos. The host embryos develop only a rudimentary primary axis. A neural inducing factor from the cytosol of gastrula-neurula stages has been partially purified. The factors are associated with other proteins in larger complexes.     

DNA synthesis in a multi-enzyme system from Xenopus laevis eggs

Cytoplasm from unfertilized eggs of the frog Xenopus laevis was separated by DEAE-cellulose column chromatography into nine fractions. Supercoiled pXir 11 DNA molecules (pXir 11 is a Col El-based recombinant plasmid containing part of the Xenopus laevis 18S and 28S ribosomal genes and transcribed spacer region) were incubated with each fraction singly and in various combinations. After incubation for 4 hr at 26 degrees C, the pXir 11 DNA was reisolated and examined by electron microscopy. Using appropriate reaction conditions (pH 7.2, 10 mM Mg2+, 250 micron NTP, 50 50 micron dNTP, 50 MM KCl, fractions III and IV or VI), at least 5-10% of the input DNA was converted to theta structures (presumed intermediates in DNA replication).     

Unresponsive, a new behavioral mutant in Xenopus laevis

Abstract. A new recessive nonlethal behavioral mutant, unresponsive (ur), was recovered from a wild-caught Xenopus laevis female by gynogenesis and inbreeding. Mutant embryos do not move until they are three days old, just before feeding begins, in contrast to normal embryos which begin movements at one day, during tailbud stage. Recovery of mutant embryos is complete but slow, requiring another nine days. Grafting analysis suggests that sensory neuron function is normal in homozygous mutant embryos, but that both motorneurons and their target muscles derived from somites are affected by the mutation. Either muscle or motorneurons of unresponsive embryos can participate in normal movements during early development, prior to the stage at which intact mutant embryos recover, in chimerae with normal tissue. Failure of mutant muscle to respond normally to acetylcholine, along with the behavior of chimerae, suggests that mutant embryos do not move because they do not form functional neuromuscular junctions during early development and that the component process of neuromuscular junction formation affected by this mutation is normally performed by both nerve and muscle. during embryogenesis due to a defect in the muscle cells [5]. Armstrong and collaborators [I] have used the immobile mutant to demonstrate that the cholinergic stimulation- induced loss of gap junctions during development does not depend on muscle contraction. In this report, we examine a new mutant of Xenopus laevis, unresponsive (ur), which does not move voluntarily or in response to stimulation until just prior to the feeding stage, after which it recovers.     

Biochemical characterization of lysosomes in unfertilized eggs of Xenopus laevis.

Relations between lysosomes and yolk platelets of amphibian eggs have been suggested. This work demonstrates the presence of acid hydrolases in oocytes induced to ovulate in vitro. About 40% of the acid hydrolases are found in a sedimentable fraction, and, in accordance with the lysosomal concept, they display structural latency. Biochemical data did not indicate any association between lysosomal enzymes and yolk platelets. The mechanism of yolk resorption is discussed and it is suggested that the fusion of lysosomes and yolk platelets might be one of the mechanisms involved in yolk digestion.     

Regulated replication of DNA microinjected into eggs of Xenopus laevis

Purified circular DNA of SV40 or polyoma virus has been injected into unfertilized eggs of Xenopus laevis. Injected DNA initiates and completes multiple rounds of semiconservative replication while observing cellular regulatory signals. Thus replication initiation of double-stranded templates is induced after the oocyte is matured in vitro by progesterone. Only one round of replication of injected DNA is observed in a single cell cycle. When protein synthesis is inhibited unreplicated molecules continue to initiate replication at an undiminished rate, but reinitiation on previously replicated molecules is completely and selectively abolished. The DNA sequence requirements for the replication of injected DNA have been investigated. A variety of procaryotic DNA molecules and circularized fragments of SV40 or polyoma DNA replicate, regardless of whether they contain the viral origin of DNA replication. These results suggest that a specialized DNA sequence is not essential for the initiation of semiconservative DNA replication in the Xenopus embryo, nor is a specialized sequence essential for the mechanism which prevents reinitiation on a molecule which has already replicated within a cell cycle. The possibility is discussed that viral origins of replication are not valid models for the eucaryotic chromosome but are adaptations for uncoupling viral replication from the mechanism which prevents reinitiation within a cell cycle.     

Identification of novel microRNAs in Xenopus laevis metaphase II arrested eggs

Using a combination of deep sequencing and bioinformatics approach, we for the first time identify miRNAs and their relative abundance in mature, metaphase II arrested eggs in Xenopus laevis. We characterize 115 miRNAs that have been described either in Xenopus tropicalis (85), X. laevis (9), or other vertebrate species (21) that also map to known Xenopus pre-miRNAs and to the X. tropicalis genome. In addition, 72 new X. laevis putative candidate miRNAs are identified based on mapping to X. tropicalis genome within regions that have the propensity to form hairpin loops. These data expand on the availability of genetic information in X. laevis and identify target miRNAs for future functional studies.     

Ultraweak emissions of the developing Xenopus laevis eggs and embryos

We measured ultraweak emissions of the Xenopus laevis eggs and embryos during normal development and under the influence of stress factors in a spectral range of 250 to 800 nm using a photomultiplier. The registered emissions were analyzed by several basic characteristics: mean intensity, histograms, kurtosis, linear trends, and Fourier spectra. We followed relationships between these parameters and developmental stage, as well as the number of individuals in optic contact with each other. The ultraweak emissions did not differ from the background at all developmental stages according to the mean intensity. But Fourier analysis revealed the reliable presence of a number of spectral lines of ultraweak emission, predominantly in the ranges of 10-20 and 30-40 Hz, in the embryos at developmental stages 2 to 11. The intensity of ultraweak emissions reliably decreased within the first 10 min after egg activation and fertilization, as well as in the case of optic interaction between groups of embryos. Sharp cooling, increase in osmotic medium pressure, and transfer in a Ca(2+)- and Mg(2+)-free medium induced a short term (approximately 1-5 min) increase in the mean intensity of ultraweak emission. We studied specific features of ultraweak emissions from different parts of the embryo. The intensity of emission from the animal part of early blastula exceeded those from the vegetal area and entire embryo. Separated fragments of the lateral ectoderm at the neurula stage had higher mean intensities of ultraweak emission than intact embryos at the same developmental stages.     

Properties of a cytostatic factor from Xenopus laevis eggs.

The cytoplasm of mature eggs of Xenopus laevis was found to contain a cytostatic factor (CSF) which induces cleavage arrest at metaphase when microinjected into one blastomere of a two-cell embryo of Xenopus laevis or Rana pipiens. The Rana CSF was found to be incapable of arresting mitosis in Xenopus embryos. Both Xenopus and Rana CSF were stabilized during the transfer procedure by Ca²⁺-chelation in the donor egg. The Xenopus CSF was not present in the germinal vesicle of immature oocytes, but arose in the cytoplasm at the time of germinal vesicle breakdown and subsequently disappeared at the time of fertilization or egg activation.     

Cytostatic factor (CSF) in the eggs of Xenopus laevis

Cytostatic factor (CSF) in unfertilized egg cytoplasm causes metaphase arrest when microinjected into zygotes. This was originally described in Rana pipiens eggs In Xenopus laevis, CSF has also been demonstrated. but only when the calcium-chelating agent, EGTA, was injected into the egg cytoplasm. In the present study, however, CSF was demonstrated in Xenopus eggs when donor egg activation was prevented by treatment with CO2 and Mg2+ instead of by EGTA, and recipient blastomere degeneration was prevented by increasing the KCl in the surrounding medium.     

Occurrence of nonlymphoid leukocytes that are not derived from blood islands in Xenopus laevis larvae

Previous immunohistochemical observations using the monoclonal antibody (XL-1) which recognizes all types of leukocytes in Xenopus laevis revealed the occurrence of XL-1+ cells in the mesenchyme throughout the early larval body, before the appearance of any lymphocytes. The present experiments were performed to determine whether these leukocytes originate, like lymphocytes and red blood cells (RBCs), in the ventral blood islands (VBI) or the dorsolateral plate (DLP). For tracing the derivation of cells, a specific staining by quinacrine to nuclei of X. laevis and Xenopus borealis hybrid (LB) cells was used to distinguish them from X. laevis (LL) cells. Orthotopic graftings of VBI tissue from st.22-23 LB embryos to the stage-matched LL embryos and examinations at st.44-45 before differentiation of the lymphocytes showed that the proportion of XL-1+ LB cells was always significantly lower than that of RBCs with the same marker in all experimental larvae. The head (LB)-body (LL) chimeras from st.22-23 embryos and culture of the head-portions as VBI- and DLP-free explants from st.14-23 embryos both demonstrated that a significant number of XL-1+ cells which had originated in the head portions had begun to differentiate by st.42-43. These results indicate that there is a significant population of larval nonlymphoid leukocytes (mostly macrophages) that do not originate from either the VBI or DLP region, and are distributed in the mesenchyme throughout the body.