[Comparative analysis of the serum proteins in 2 species of the genus Pleurodeles (urodele amphibia): Pleurodeles waltlii and Pleurodeles poireti].
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Preparations of metaphase chromosomes of the amphibian urodele Pleurodeles poireti were obtained by squashing cells from tailbuds of ten days old embryos which had been in 50% acetic acid. The Q-bands karyotype is described. Frequently the induced secondary constrictions exhibit a bright fluorescence, as do the centromeres, except for that of chromosome XII. In contrast, satellites on chromosomes III, IV, and XI exhibit little fluorescence. This pattern is compared with that in Pleurodeles waltlii. Differences are observable in centromeres of chromosomes III and XII and the proximal part of the long arm of chromosome VII, differences which can be used as chromosome marker in case of hybridization.
Our work was aimed at developing a simple and effective method of identification of most or all chromosomes of Pleurodeles newts. To this end, we used DAPI staining of the chromomeres of newt lampbrush chromosomes and immunochemical reactions between the ribonucleoprotein (RNP) marker loops and polyclonal antibodies against human zinc-finger protein Ro52 (52-kDa Ro/SS-A). A method has been developed to obtain newt lampbrush chromosome preparations. Cytological maps of P. waltl chromosomes (Spanish population/subspecies) showing distributions of chromomeres and marker loops along the chromosome length were constructed.
Presumptive ectoderm from young gastrulae of the Pleurodele and the Axolotl was treated with concanavalin A(5-25 micrograms/ml). Then, it was combined, in a sandwich, with the dorsal lip of the blastopore and cultured in vitro for 5-7 days. The results of the experiments show that the treated ectoderm becomes more fragile than untreated control ectoderm. Nevertheless, the neural induction and the histological differentiation of the neural tissue appeared in a rather normal fashion.
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As revealed in chondrogenetic cells the LDH activity that is very low during cellular migration, suddenly increases at the time of cytodifferentiation and reaches a peak at the onset of the first chondrocyte differentiation. A relationship is likely to exist between this increase in LDH-activity, chondroitin-sulfate synthesis and NAD metabolism.
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A light form of DNA ligase (EC 6.5.1.2), the only form present in oocytes of the axolotl (Ambystoma mexicanum), has been shown to be replaced by a heavy form of the enzyme when the egg enters cleavage. This early biochemical event has been assumed to rely on direct nuclear input. Sucrose gradient analysis permits discrimination between enzymes from axolotl and the sharp-ribbed salamander (Pleurodeles waltlii) for both heavy and light enzymatic forms of DNA ligase. Genetic activity of blastula nuclei transplanted in activated cytoplasm has been tested by determination of the enzymatic forms and specific types of DNA ligases when the implanted egg enters cleavage. A blastula nucleus of Pleurodeles in axolotl cytoplasm determines a heavy ligase of the Pleurodeles type. Conversely, a haploid androgenetic nucleus of Pleurodeles in axolotl cytoplasm controls a light ligase of the Pleurodeles type. Reciprocal experiments give homologous results. To our knowledge, this is the earliest nucleus-dependent synthesis revealed in development for any system. The heavy ligase of one species may coexist with the light form of the other species but not with the light form of its own specific type. Inhibition of the production of the heavy form for one genome results in the expression of the light form. We conclude that genetic control of DNA ligase in very early development involves structural genes for heavy and light forms of enzyme, with an exclusion process operating an alternative expression of corresponding genes. This exclusion relationship between nonallelic genes is species specific.
To obtain more insight into the vasotocinergic and mesotocinergic systems of amphibians and the evolution of these neuropeptidergic systems in vertebrates in general, the distribution of vasotocin (AVT) and mesotocin (MST) was studied immunohistochemically in the brains of the anuran Rana ridibunda and the urodele Pleurodeles waltlii. In Rana, AVT-immunoreactive cell bodies are located in the nucleus accumbens, the dorsal striatum, the lateral and medial part of the amygdala, an area adjacent to the anterior commissure, the magnocellular preoptic nucleus, the hypothalamus, the mesencephalic tegmentum, and in an area adjacent to the solitary tract. In Pleurodeles, AVT-immunoreactive somata are confined to the medial amygdala, the preoptic area, and an area lateral to the presumed locus coeruleus. In both species, the distribution of MST-immunoreactive cell bodies is more restricted: in the frog, MST-immunoreactive somata are present in the medial amygdala and the preoptic area, whereas, in the urodele, cell bodies are found only in the preoptic area. Both in Rana and Pleurodeles, AVT- and MST-immunoreactive fibers are distributed throughout the brain and spinal cord. A major difference is that in Rana the number of MST-immunoreactive fibers is evidently higher than that of AVT-immunoreactive fibers, whereas the opposite is found in Pleurodeles. This holds, in particular, for the forebrain and the brainstem. The presence of several extrahypothalamic AVT-immunoreactive cell groups and the existence of well-developed extrahypothalamic networks of AVT- and MST-immunoreactive fibers are features that amphibians share with amniotes. However, this study has revealed that major differences exist not only between species of different classes of vertebrates, but also within a single class. In order to determine whether features of these neuropeptidergic systems are primitive or derived, a broad selection of species of each class of vertebrates is needed.
Captan (N-trichloromethylthio-4-cyclohexene-1,2-dicarboximide) is a fungicide used to inhibit the growth of many types of fungi on plants used as foodstuffs. The toxic and genotoxic potentials of captan were evaluated with the micronucleus test (MNT; AFNOR,2000) and the comet assay (CA) using amphibian larvae (Xenopus laevis and Pleurodeles waltl). Acute toxicity results showed that captan was toxic (1) to Xenopus larvae exposed to from 2 mg/L to 125 or 62.5 microg/L, depending on the nature of the water [reconstituted water containing mineral salts or mineral water (MW; Volvic, Danone, France)] and (2) to Pleurodeles exposed to from 2 mg/L to 125 microg/L in both types of water. The MNT results obtained in MW showed that captan (62.5 microg/L) was genotoxic to Xenopus but not genotoxic to Pleurodeles at all concentrations tested. CA established that the genotoxicity of captan to Xenopus and Pleurodeles larvae depended on the concentration, the exposure times, and the comet parameters (tail DNA, TEM, OTM, and TL). The CA and MNT results were compared for their ability to detect DNA damage at the concentrations of captan and the exposure times applied. CA showed captan to be genotoxic from the first day of exposure. In amphibians, CA appears to be a sensitive and suitable method for detecting genotoxicity such as that caused by captan.
We isolated and characterized a cDNA coding for heat-shock protein 70 of the amphibian Pleurodeles waltl. This 2212-bp sequence exhibited one open reading frame of 645 amino acids. The predicted amino acid sequence exhibited the three conserved elements of the HSC/HSP70 protein family. Comparison of nucleotide and amino acid sequences between this gene and other hsc/hsp-like genes revealed a high identity with the cognate form HSC70. By in vitro translation, this gene encoded a 70-kDa protein which was different than the inducible Pleurodeles waltl HSP70 protein. This translated protein was recognized by Pleurodeles waltl N1 anti-HSC/HSP70 antibody. Heat-inducibility tests showed that this gene was constitutively expressed during oogenesis and embryogenesis, and its expression was not increased after a heat-shock. These results led us to conclude that we recovered a Pleurodeles waltl cognate hsc70 gene.
In the urodele amphibian Pleurodeles waltl, sex differentiation is genetically controlled, that is, ZZ male vs ZW female, but may be influenced by temperature, which induces a female-to-male sex reversal. We investigated whether steroidogenic factor 1 (SF-1) could be involved in Pleurodeles sex differentiation or in temperature-dependent sex reversal by cloning a Pleurodeles SF-1 cDNA and examining its developmental expression. The 468-amino-acid deduced protein is highly conserved in comparison with other species. In ZZ and ZW control larvae, SF-1 mRNA is detected at the first stage of the thermosensitive period (TSP) in the gonad-mesonephros-interrenal complex (GMI). By the end of TSP at stage 55, SF-1 is expressed in the gonad (Gd) and in the mesonephros-interrenal (MI) both in ZZ and ZW larvae. During this stage, a transient, ZW-specific increase of SF-1 transcription occurs not only in Gd but also in MI, this increase starting earlier in Gd than in MI. Therefore, in P. waltl, an SF-1 upregulation occurs after the onset of the ovarian-specific increase of aromatase mRNA expression. At the end of metamorphosis, the SF-1 transcription level in Gd and MI is nearly the same in both ZZ and ZW larvae. Besides, after long-term heat treatment leading to sex reversal, SF-1 mRNA upregulation is not observed in ZW larvae, in either Gd or MI. However, SF-1 expression is not decreased after a 48-h heat shock applied at the end of the TSP, suggesting that temperature has no inhibitory effect by itself in long-term heat treatment. Estradiol benzoate treatments show that, at the end of the TSP, SF-1 gene transcription could be controlled by the estrogen level. This is in accordance with the female-enriched SF-1 expression and the decreased SF-1 expression following long-term, sex-reversing heat treatment, which is known to decrease aromatase expression and activity. Thus, it is unlikely that SF-1 is directly involved in Pleurodeles temperature-dependent sex reversal.
Thymectomy was performed on the urodele amphibian Pleurodeles waltlii Michah. from the 5th to the 20th week of larval development. Thymectomized animals were allografted at 7 months of age. The results obtained are as follows: (1) Pleurodeles thymectomized before 10 weeks always tolerated allografts: (2) Pleurodeles thymectomized within 11 to 13 weeks generally rejected allografts in a chronic manner, but sometimes tolerated them; (3) normal rejections occurred in Pleurodeles thymectomized at 14 weeks, or later. Stage 52 of the developmental table (10 weeks of age) is a critical "end stage" for the efficiency of thymectomy. The results are confirmed by the fate of second set allografts performed in all these animals.
Amphibian oocytes accumulate a large pool of mRNA molecules for future embryonic development. Due to their association with specific proteins the stored maternal RNAs are translationally repressed. The identification of these RNA-binding proteins and the characterization of their functional domains may contribute to the understanding of the translational repression mechanisms and the subsequent activation processes during early embryogenesis. Here we present the complete Pleurodeles cDNA sequence of a cytoplasmic protein which is present in oocytes, eggs, and very early cleavage stage embryos but undetectable in postcleavage embryo and adult tissues. The predicted molecular mass of the protein is 55 kDa and the apparent molecular mass as determined by SDS-PAGE, 68 kDa. The deduced amino acid sequence reveals proline- and serine-rich domains in the aminoterminal part as well as two RGG boxes which represent characteristic motifs of several RNA-binding proteins. No distinct homologies to the consensus RNA recognition motif were found. The 55-kDa protein was recovered in cytoplasmic ribonucleoprotein (RNP) particles containing poly(A)+ RNA. It was therefore termed RAP55 for mRNA-associated protein of 55 kDa. However, a direct interaction of RAP55 with mRNA could not be demonstrated by UV-crosslinking experiments, indicating that it is bound to mRNP complexes via protein-protein interactions. RAP55 is evolutionarily conserved since antibodies raised against a recombinant Pleurodeles RAP55 fragment recognize the protein from Pleurodeles and Xenopus. The expression pattern and intracellular distribution of RAP55 suggest that it is part of those mRNP particles which are translationally repressed during oogenesis and become activated upon progesterone-induced oocyte maturation.
By immunizing Balb/c mice with oocyte nuclei of Pleurodeles waltl we obtained a monoclonal antibody, mAb 4A6, that labels distinct globular domains of the lampbrush chromosomal axes of Pleurodeles. These domains are found at corresponding sites of homologous chromosomes, often at telomeric and putative centromeric regions, and appear to be devoid of DNA. Because of these characteristic features it is most likely that the mAb 4A6-positive domains correspond to the central part of the "axial granules" of urodelan lampbrush chromosomes. In immunoblotting analyses mAb 4A6 reacts with a nuclear antigen of approximately Mr 180000 and a structurally nonrelated cytoplasmic protein of Mr 98000, which was not characterized any further. Comparative immunofluorescence and immunoblotting studies with mAb 4A6 and an antiserum against DNA topoisomerase II (topo II) as well as immunodepletion experiments demonstrated that the nuclear 4A6 antigen is topo II. Our results indicate that topo II is not a constituent of a continuous, loop-anchoring scaffold in lampbrush chromosomes of Pleurodeles but, rather, is restricted to the axial granules.
The effects of hormonal changes on the male-specific, middle-affinity, estrogen-binding component (MEBC) were investigated in the Pleurodele. Induction of MEBC was shown to be under androgen control, similar to that observed for the cytoplasmic middle-affinity estrogen-binding sites in rat liver and human hepatoma cells. But, in contrast to the male-specific middle-affinity estrogen-binding sites identified in the rat, the administration of estrogen to male Pleurodeles did not lead to the disappearance of MEBC but raised levels significantly. The MEBC displays the properties of type II middle-affinity estrogen-binding sites, which are characterized by an oestrogen-dependent rise, a sensitivity to reducing agents, a specificity for diethylstilbestrol, and a binding capacity enhanced by increasing dilutions of cytosol. In female Pleurodeles, MEBC can be induced by treatment with androgens. This induction appears to be modulated by the estrogen/androgen ratio. The induction of MEBC and the estrogen-dependent increase in the male were not found to be correlated with hepatocyte proliferation.
Previous work on Xenopus laevis suggests a temporal coincidence between inactivation of the M-phase promoting factor (MPF) and intracellular pH (pHi) increase during egg activation. In addition, we recently showed that during the early cell cycle of Xenopus eggs, MPF activity cycling and pHi oscillations were temporally and functionally related. In the present work, using eggs of another amphibian, Pleurodeles waltlii, which has a natural cell cycle considerably longer than that of Xenopus laevis, we show a temporal coincidence between MPF activity and pHi changes, both at the time of egg activation and at each of the following cell cycles. Egg activation-induced pHi changes in Pleurodeles did not involve classical plasma membrane ion exchangers, and were not due to the activation of a H+ conductance. On the other hand, the pHi oscillations intervening at each cell cycle were suppressed by inhibitors of protein synthesis or phosphorylation, as were their counterparts in Xenopus eggs. We propose that physiological pHi changes in Pleurodeles and Xenopus eggs might have a metabolic origin, in direct relation with the cascade of phosphorylations-dephosphorylations of proteins implicated in the control of the cell cycle.