Gut-Associated lymphoid tissue (GALT) in the amphibian urodele Pleurodeles waltl

Even though salamanders are supposed to lack intestinal lymphoid tissue, Pleurodeles waltl shows lymphoid aggregates throughout the gut, especially in the small intestine, as cell infiltrates in the lamina propia. They have a phylogenetic significance in relation to the existence of an intestinal immunologic barrier in vertebrates.     

Glycan residues of N- and O-linked oligosaccharides in the premeiotic spermatogenetic cells of the urodele amphibian Pleurodeles waltl characterize by means of lectin histochemistry

The aim of this work was the characterization of the glycoconjugates of the premeiotic spermatogenetic cells of the testis of an urodele amphibian, Pleurodeles waltl, by means of lectins in combination with several chemical and enzymatic procedures, in order to establish the distribution of N- and O-linked oligosaccharides in these cells. In the cytoplasm of the primordial germ cells, primary and secondary spermatogonia and primary spermatocytes, a granular structure can be observed close to the nucleus. These granules contain four types of sugar chains according to their appearance during the differentiation process: 1. some oligosaccharides that are identified in all the four cell types above mentioned, which include N-linked oligosaccharides with Fuc, Gal beta1,4GlcNAc and Neu5Ac alpha2,3Gal beta1,4GlcNAc and O-linked oligosaccharides with Gal beta1,4GlcNAc and Neu5Ac alpha2,3Gal beta1,4GlcNAc; 2. other glycan chains that are not present in the primary spermatocytes (N-linked oligosaccharides with DBA-positive GalNAc, GlcNAc, and a slight amount of Neu5Ac alpha2,6Gal/GalNAc and O-linked oligosaccharides with WGA-positive GlcNAc); 3. the sugar chains that are not in the earliest step of spermatogenesis (formed by both N-linked and O-linked oligosaccharides with Glc); and 4. other that appear at the earliest and latest stages, but not in the intermediate ones, (N-linked oligosaccharides with Man and O-linked oligosaccharides with SBA- and HPA-positive GalNAc and PNA-positive Gal beta1,3GalNAc). This structure could be related with the Drosophila spectrosome and fusome, unusual cytoplasmic organelles implicated in cystic germ cell development. Data from the present work, as compared with those from mammals and other vertebrates, suggest that, although no dramatic changes in the glycosylation pattern are observed, some cell glycoconjugates are modified in a predetermined way during the early steps of the spermatogenetic differentiation process.     

Identification of the vitellogenin proteins in the newt Pleurodeles waltl (urodele amphibian)

Vitellogenin derived from the blood of estrogen-treated Pleurodeles waltl was identified by immunochemical and electrophoretic analyses, using an antiserum against plasma vitellogenin isolated by dimethylformamide precipitation. Pleurodeles vitellogenin migrates as four bands on native PAGE, designated alpha-, beta-, gamma- and delta- VTG, with apparent mol. wts of 250,000, 270,000, 280,000 and 520,000 respectively. In the plasma, from estrogen-treated males like from ovariectomized estrogen-treated females, an additional band (mu-VTG) was found by native PAGE, never observed in estrogen-treated female plasma. It has a mol. wt of about 380,000 and shows complete immunological cross-reactivity with the vitellogenin antiserum. At least two polypeptides, termed VTG-I and VTG-II (mol. wt = 180,000 and 210,000) were identified by SDS-PAGE. Rocket immunoelectrophoresis displays three distinct precipitate lines indicating major immunological differences between the plasma vitellogenins.     

Microgravity and hypergravity effects on fertilization of the salamander Pleurodeles waltl (urodele amphibian)

Effects of microgravity (microG) on fertilization were studied in the urodele amphibian Pleurodeles waltl on board the MIR space station. Genetic and cytomorphologic analyses ruled out parthenogenesis or gynogenesis and proved that fertilization did occur in microG. Actual fertilization was demonstrated by the analysis of the distribution of peptidase-1 genes, a polymorphic sex-linked enzyme, in progenies obtained in microG. Further evidence of fertilization was provided by the presence of spermatozoa in the perivitelline space and in the fertilization layer of the microG eggs and by the presence of a female pronucleus and male pronuclei in the egg cytoplasm. Experiments in microG and in 1.4G, 2G, and 3G hypergravity showed for the first time that, compared to eggs in 1G, several characteristics of the fertilization process including the cortical reaction and the microvillus transformations were altered depending on the gravitational force applied to the eggs. Microvillus elevation, the most evident feature, was reduced on microG-eggs and amplified on eggs submitted to 2G and 3G. No lethal consequences of these alterations on the early development of microG-eggs were observed.     

Experimental teratomas from xenogenic embryonic implants in the urodele amphibian, Pleurodeles waltlii Michah

Ambystoma mexicanum embryos in stages 5 to 32 were implanted into the coelomic cavity of larvae or metamorphosing larvae of Pleurodeles waltlii. Out of 76 experimental animals, 43 presented teratomas a few months later. In another series of experiments, Ambystoma embryos together with Pleurodeles embryos were implanted in 18 Pleurodeles larvae. Teratomas developed in 13 cases. Considering the two series, tumors evolved slowly and remained benign in 31 animals, whereas in 25 others, the tumor growth was faster and uncontrolled. Histological examination of these destabilized tumors indicates they are certainly xenogenic teratocarcinomas.     

Effects of microgravity on the larval development, metamorphosis and reproduction of the urodele amphibian Pleurodeles waltl

The FERTILE experiment was twice performed onboard the Mir space station during the Cassiopée and Pégase French space missions. The goal was to analyze the effects of microgravity on fertilization and embryonic development, and then on further development on the ground in the amphibian Pleurodeles waltl. The present paper reports development that occurred in the laboratory after landing. Recovered on the ground at the hatching stage, young larvae reared at room temperature underwent metamorphosis and became adults without obvious abnormalities. Of particular interest was the rearing temperature that induced a delayed metamorphosis for animals from the Cassiopée space mission, but not for animals from the Pégase mission. The rate of development and the morphology were analogous in these animals and in ground controls reared in a similar annual period. Analysis of offspring was performed using these animals. Males born in space were first mated with control ground-born females and then with females born in space. The mating gave progeny that developed normally. Depending on the methods used and on the limits of the analyses, the results clearly demonstrated that animals born in space were able to live and reproduce after return to the ground.     

Sex-linked differences in activity of enzymes in the blood of the urodele amphibian Pleurodeles waltl.

Few data are available on enzyme activity in amphibian plasma or erythrocytes. We measured the activity of several blood enzymes in the urodele amphibian Pleurodeles waltl reared under standard laboratory conditions. In subsequent experiments, we will estimate and compare the physiological and biochemical conditions of P. waltl when reared under extreme temperature or microgravity conditions. The enzymes selected were glutamate dehydrogenase, aspartate aminotransferase, alanine aminotransferase, superoxide dismutase, catalase, isocitrate dehydrogenase and glucose-6-phosphate dehydrogenase. In fresh plasma samples, enzyme activity in females was higher than in males, except for aspartate and alanine aminotransferases, which were equivalent in females and males. Glutamate dehydrogenase activity was higher in males than in females. In female erythrocytes, the activity of all enzymes was higher than in male erythrocytes. We have also studied the storage conditions of samples and observed that for most enzymes, the activity in freshly isolated plasma and erythrocyte preparations decreased after storage at -18 or +4 degrees C.     

Lectin histochemical localization of N- and O-linked oligosaccharides during the spermiogenesis of the urodele amphibian Pleurodeles waltl

The aim of this work is the characterization of the glycoconjugates of the spermatids during the spermiogenesis of the testis of an urodele amphibian, Pleurodeles waltl, by means of lectins in combination with several chemical and enzymatic procedures, in order to establish the distribution of N- and O-linked oligosaccharides in these cells. The acrosome was the most relevant lectin-labeled structure. The O-linked oligosaccharides contained DBA- and SBA-positive GalNAc, AAA-positive Fuc and PNA-positive Gal1,3GalNAc. Sialic acid was scarcely observed, the Neu5Ac2,-3Gal1,4GlcNAc sequence was found in N-linked oligosaccharides. Additionally, N-linked oligosaccharides containing HPA-positive GalNAc and AAA-positive Fuc were found. Moreover, with some lectins the acrosome showed a variable composition of the oligosaccharides in the different steps of the sperm maturation. Some residues were found only in the early steps in maturating acrosome, while others were in the later steps, showing that acrosomal glycoconjugates are modified during acrosome development in spermiogenesis. The changes observed during acrosome maturation suggest the existence of a predetermined pattern of storage of the acrosome components and a progressive compression of them.     

Expression of N-CAM precedes neural induction in Pleurodeles waltl (urodele, amphibian)

The appearance and localization of N-CAM during neural induction were studied in Pleurodeles waltl embryos and compared with recent contradictory results reported in Xenopus laevis. A monoclonal antibody raised against mouse N-CAM was used. In the nervous system of Pleurodeles, it recognized two glycoproteins of 180 and 140x10(3) M(r) which are the Pleurodeles equivalent of N-CAM-180 and -140. Using this probe for immunohistochemistry and immunocytochemistry, we showed that N-CAM was already expressed in presumptive ectoderm at the early gastrula stage. In late gastrula embryos, a slight increase in staining was observed in the neurectoderm, whereas the labelling persisted in the noninduced ectoderm. When induced ectodermal cells were isolated at the late gastrula stage and cultured in vitro up to 14 days, a faint polarized labelling of cells was observed initially. During differentiation, the staining increased and became progressively restricted to differentiating neurons.     

Sequencing and expression of the CD3 gamma/delta mRNA in Pleurodeles waltl (urodele amphibian)

The CD3 complex is an essential component of the T-cell receptor (TCR) implicated in T-cell maturation and activation. This TCR has been identified in both cartilaginous and bony vertebrates. In different studies where the CD3 chains were cloned and sequenced, it appeared that the CD3 complex is composed of several chains, all susceptible to phosphorylation and able to transduce signals. Here, by an approach combining degenerative oligonucleotide primers and RACE-PCR, we report the cloning and sequencing of a CD3 cDNA from the salamander Pleurodeles waltl, highly homologous to the Xenopus and chicken CD3 gamma/delta cDNAs. Using semi-quantitative PCR and Northern blot analysis, we found the highest CD3 gamma/delta mRNA expression in the thymus; weaker expression was observed in the spleen and blood, followed by the intestine, therefore confirming the tissue and lymphoid specificities of this mRNA. The signals in the spleen, blood and intestine represented 55%, 33% and 16%, respectively, of the signal detected in the thymus. During the embryonic and larval stages of Pleurodeles waltl development, CD3 gamma/delta mRNA expression begins early at the neurula stage (stage 15, 69 h after laying), increases up to stage 33 (9 days after laying) and afterwards remains stable, at least until the larval stage 42 (28 days after laying). As the thymus primordium appears much later, the question of the formation and maturation of the first T-cell precursors outside this organ is posed.     

Autoradiographic study of the DNA synthesis process along metaphasic chromosomes of Pleurodeles waltlii Michah (urodele amphibian)]

Chromosomes from embryos (stage 34) of the amphibian urodele Pleurodeles waltlii were labeled by 3H-thymidine at different times of the DNA synthesis cycle and studied at metaphase. Terminal segments of the chromosomes, induced secondary constrictions, and satellites were found to be late-labeling. Therefore, they may be considered as formed at least partially by heterochromatin. However, after other treatments or stainings, they do not present other features considered characteristic of heterochromatin. The question of the heterogeneity of their structures is discussed and the ambiguity of the term heterochromatin is emphasized. The proximal part of the long arm of chromosome VII shows characteristics similar to satellites, which may indicate the presence of a previously unrecognized satellite.     

Noradrenergic and adrenergic systems in the brain of the urodele amphibian,Pleurodeles waltlii,as revealed by immunohistochemical methods

The distribution of noradrenaline and adrenaline in the brain of the urodele amphibian Pleurodeles waltlii has been studied with antibodies raised against noradrenaline and the enzymes dopamine--hydroxylase and phenylethanolamine-N-methyltransferase. Noradrenaline-containing cell bodies were found in the anterior preoptic area, the hypothalamic nucleus of the periventricular organ, the locus coeruleus and in the solitary tract/area postrema complex at the level of the obex. Noradrenergic fibers are widely distributed throughout the brain innervating particularly the ventrolateral forebrain, the medial amygdala, the lateral part of the posterior tubercle, the parabrachial region and the ventrolateral rhombencephalic tegmentum. Putative adrenergic cell bodies were found immediately rostral to the obex, ventral to the solitary tract. Whereas the cell bodies and their dendrites were Golgi-like stained, axons were more difficult to trace. Nevertheless, some weakly immunoreactive fibers could be traced to the basal forebrain. A comparison of these results with data previously obtained in anurans reveals not only several general features, but also some remarkable species differences.Abbreviations Acc Nucleus accumbens - AP area postrema - Apl amygdala, pars lateralis - Apm amygdala, pars medialis - ca commissura anterior - Cb cerebellum - cc central canal - Dp dorsal pallium - epl external plexiform layer - gl glomerular layer of the olfactory bulb - H ganglion habenulae - igl internal granular layer - Ip nucleus interpeduncularis - Lc locus coeruleus - Ll lateral line lobe - Lp lateral pallium - Ls lateral septum - ml mitral cell layer - Mp medial pallium - Ms medial septum - nPT nucleus pretectalis - NPv nucleus of the periventricular organ - nV nervus trigeminus - oc optic chiasm - Poa preoptic area - Ri nucleus reticularis inferior - SC nucleus suprachiasmaticus - sol solitary tract - Str striatum - thd thalamus dorsalis - thv thalamus ventralis - To tectum opticum - TP tuberculum posterius - V ventricle - VH ventral hypothalamic nucleus - III nucleus nervi oculomotorii - IXm nucleus motorius nervi glossopharyngei - Xm nucleus motorius nervi vagi     

Demonstration of Q bands on mitotic chromosomes of the amphibian urodele Pleurodeles poireti Gervais, after staining with quinacrine mustard. Comparison with Pleurodeles waltii]

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.     

Nucleocytoplasmic interactions during ontogenesis in individuals obtained by intra- and interspecific nuclear transplantation in the genus Pleurodeles (urodele amphibian)

Nuclei derived from a single blastula of Pleurodeles waltlii or Pleurodeles poireti are transplanted to the cytoplasm of eggs of both species (P. waltlii and P. poireti). Animals with identical genomes are obtained, some resulting from an intraspecific nucleocytoplasmic association, the others from an interspecific association (nucleocytoplasmic hybrids). A marked lethality results from the heterospecific association. Morphological and physiological effects, concomitant to initial egg conditioning, occur during the first stages of development. The study of lactic dehydrogenase (LDH) and malic dehydrogenase (MDH) zymograms reveals the genomic activity in the embryo from stage 28 (heart beat). A regionalization of this activity is evident; the nuclear activity can be shown in the ectomesodermic tissue sooner than in the endodermal cells. From stage 38 (feeding), the functional substitution of transplanted nucleus for effects of initial cytoplasmic conditioning appears to be total. No cytoplasmic heredity can be shown: the genetic effect of the transplanted nucleus is dominant. This is corroborated by the results of cutaneous cross-grafts between adult allocytoplasmic twins. Those animals present strictly identical immune reactions; their antigenic constitution seems to be independent from the specific origin of the egg cytoplasm.