Effects of in vivo heat treatment on lampbrush chromosome structure in amphibian oocytes

When Pleurodeles (Amphibian, Urodele) females were subjected to high temperatures (32-35 degrees C) for varying periods of time (45 min to 7 days), lampbrush chromosome structure underwent striking modifications. These changes included a numerical reduction in normal loops and progressive disorganization of RNP matrices of various loops. The degree of such disorganization was a function of the intensity and duration of the stress. These modifications were completely reversible when females or oocytes were returned to a normal breeding temperature (20 degrees C). Results are discussed in comparison with previous studies on morphological changes induced by heat shock in lampbrush chromosomes carried out in vitro.     

Cold-induced changes in amphibian oocytes

Female Pleurodeles waltl newts (Amphibia, urodele), usually raised at 20 °C, were submitted to low temperatures; oocytes responded to this cold stress by drastic changes both in lampbrush chromosome structure and in protein pattern. Preexisting lateral loops of lampbrush chromosomes were reduced in size and number, while cold-induced loops which were tremendously developed, occurred on defined bivalents of the oocyte at constant, reproducible sites. A comparison of protein patterns in control and stressed oocytes showed two main differences: in stressed oocytes, overall protein synthesis was reduced, except for a set of polypeptides, the “cold-stress proteins”; second, there was a striking inversion of the relative amount of β- and γ-actin found in the oocyte nucleus before and after cold stress. Whereas β-actin was the predominant form in control oocytes, γ-actin became the major form in stressed oocytes.     

Informosome formation in maturing amphibian oocytes

[14C]Uridine and/or RNA-binding [3H]protein preparations were microinjected into the oocytes of Rana temporaria frog. It was shown that both labelled compounds are incorporated into RNP particles with the buoyant density in CsCl characteristic of informosomes and with a homogeneous sedimentation distribution in sucrose gradients. Injection of actinomycin D into recipient oocyte leads to inhibition of synthesis of the informosome RNA component as well as to the incorporation of RNA-binding [3H] proteins into free informosomes. The results obtained provide experimental proofs for the assumption that the RNA-binding proteins under study form complexes predominantly with newly synthesized RNA.     

In vivo repair of the 3''terminus of transfer RNA injected into amphibian oocytes.

Yeast transfer RNA specific for phenylalanine has been treated chemically to remove either one or two nucleotides of its 3' terminus and has been injected into Xenopus laevis oocytes to test whether this RNA can be repaired in vivo. The results obtained showed that oocytes could aminoacylate and thus repair tRNAPhe that has lost both its terminal adenosine and 3' phosphate. A similar result was obtained with tRNAPhe that had undergone two full cycles of 3' terminal nucleotide removal. The oocytes cannot aminoacylate tRNAPhe whose 3' terminal ribose has been oxidized with periodate or the derivative that retains a 3' phosphate after adenosine removal. In vitro assays show that the Xenopus ovary contains a tRNA nucleotidyl transferase with the properties similar to enzymes obtained from other sources which may be responsible for the 3' terminal repair observed in vitro.     

Transcription of complementary repeat sequences in amphibian oocytes

Repeat sequences are transcribed in the germinal vesicles of amphibian oocytes. In the hnRNA population both complements of the repeats are found and can be readily detected because they form intermolecular duplex structures. The structure and formation of duplex regions have been studied in the hnRNA of Xenopus laevis, Triturus cristatus, Amphiuma means and Necturus maculosus, a series of amphibians of increasing genome size (C-value). In T. cristatus, the duplex structures are mostly 600-1200 bp in length, whereas in X. laevis they are shorter and in N. maculosus they tend to be longer. Although the proportion of RNA sequence capable of rapidly forming duplex structures is different in different organisms, this property bears no relationship to C-value. However the sequence complexity of complementary repeats, as estimated from the rate of duplex formation, does show an increasing trend with C-value. The complementary repeats found in oocyte hnRNA are transcribed from families of DNA sequence that are each represented in the genome by thousands of copies. The extent of cross-species hybridization is low, indicating that the repeat sequences transcribed in different amphibian genera are not the same. In situ hybridization experiments indicate that the repeat sequences are spread throughout the genome. The evolution and possible function of complementary repeats are considered.     

Activation of amphibian oocytes by sperm extracts

In the fertilization of most animals, egg activation is accompanied by an increase in cytoplasmatic Ca2+; however, the mechanism through which the fertilizing sperm induce this phenomenon is still controversial. An increase in intracellular free Ca2+ is required to trigger egg activation events, a process that includes cortical granule exocytosis, resumption and completion of meiosis and DNA replication, and culminates in the first mitotic cleavage. In this work, we investigated the effect of microinjection and incubation of different fractions of homologous sperm extract on the activation of Bufo arenarum oocytes matured in vitro. Two heat treatment-sensitive fractions obtained by chromatography were able to induce oocyte activation. The sperm fraction, which contained a 24 kDa protein, induced 90% activation when it was microinjected into the oocytes. Whilst the sperm fraction, which contained a 36 kDa protein, was able to induce about 70% activation only when it was applied on the oocyte surface.     

Conditions affecting protein synthesis in amphibian oocytes.

The endogenous protein synthesis of Xenopus laevis and Calyptocephalella caudiverbera oocytes was studied by measuring the incorporation into acid-precipitable material of radioactive amino acids placed in the extracellular medium. Large differences of incorporation into protein were observed by using different labeled amino acids. For example, it was found that radioactive aspartic acid or glutamic acid was very poorly incorporated at concentrations under 0.1 mm. These differences are due to differences in uptake constants and in the internal pools of free amino acids which are very large for the acidic amino acids. Both types of oocytes behaved similarly with respect to magnesium ion concentration, temperature optimum and inhibitors of protein synthesis. They differed however in sensitivity to pH since Xenopus laevis oocyte protein synthesis was twofold higher at pH 8.5 than at pH 7 while Calyptocephalella caudiverbera oocytes showed no difference. Isolation of oocyte germinal vesicles allowed a study of the entrance of newly synthesized protein into the cell nuclei.     

Lipid metabolic pathways operating in amphibian full-grown oocytes

The utilization of (2-3H)-glycerol in lipid biosynthesis was analyzed in Bufo arenarum and Xenopus laevis full-grown oocytes. The precursor was more actively incorporated in Xenopus laevis oocytes. Neutral glycerides were the lipids displaying the highest levels of radioactivity followed by phosphatidylcholine and phosphatidylethanolamine. After reincubation of prelabeled oocytes in a saline buffer solution, a net fall in labeled phosphatidic acid concomitant with an increase in phosphatidylcholine were detected. The present findings establish that glycerolipid biosynthesis is operative in full-grown oocytes. In addition, Xenopus laevis oocytes seem to be metabolically more active.     

Evidence for compartmentation of uridine nucleotide pools in rat hepatoma cells

Interaction between the de novo and salvage pathways of pyrimidine metabolism was studied in a line of rat hepatoma cells by co-labelling with [14C]-uridine and [3H]orotate. A difference in the ratio of 14C/3H between CTP and UTP in acid-soluble nucleotide pool was reflected in the corresponding ratios in CMP and UMP in RNA, with uridine labelling cytidine nucleotides relatively more effectively than orotate. These results are not compatible with the concept of a single UTP pool, and a new model for pyrimidine anabolic pathways, based on compartmentation of de novo from salvage pathways, is proposed.     

Interlocking and knotting of ring nucleoli in amphibian oocytes

Various configurations of interlocking and knotting of ring nucleoli from amphibian oocytes have been identified in material taken from 2 females of Eurycea bislineata and one female of Plethodon cinereus. The simplest configuration is a simple interlock between 2 rings of similar or different lengths. More complex interlocks have been seen, in which 3 rings are linked together in such a way that they cannot be extended into a chain. A third configuration involves complex knotting of single long rings. It is suggested that the interlocked configurations arise through the replication of rings of DNA that have low levels of supercoiling, and that the knotted rings arise by misjoining of the ends of linear molecules that have become wound around one another. Models are suggested in support of these proposals and the significance of interlocking of ring nucleoli in relation to the mechanism of gene amplification is discussed.     

Subcellular flux of potassium and rubidium in amphibian oocytes

To determine the net Rb+ influx and K+ efflux at a subcellular level, fully grown and a Ringer's solution where K+ was substituted for by Rb+ on a molar bases. For 40 hrs serial samples of the oocytes were cryofixed and cryosectioned for elemental analysis in mM per kg dry weight using electron probe x-ray microanalysis. Oocyte volume remained constant. Net Rb+ influx showed a slow exponential increase into the nucleus, the yolk-free cytoplasm and the yolk platelets. There was significant K+ efflux from the nucleus but not from the yolk-free cytoplasm or the yolk. The Na+ concentration remained unchanged in all compartments during the course of the experiment. There was however a slow but significant increase in the concentration of Cl- in each of the subcellular compartments but this increase was not sufficient to balance the observed increase in the sum of K+ plus Rb+. Thus Rb+ accumulates selectively in all three subcellular compartments indicating that Rb+ is not a good K+ surrogate in the oocytes. That K+ demonstrates efflux from the nucleus but not from the cytoplasmic compartments is interpreted to suggest that some of the nuclear K+ is lost in exchange for Rb+ but that essentially none of the cytoplasmic K+ is-lost in exchange for Rb+. The findings provide strong evidence for adsorption of Rb+ in the cell.