Localization by immunofluorescence of a gastrin-like substance in the brain of the rainbow trout,Salmo gairdneri

Summary With the aid of an indirect immunofluorescence technique neurones containing a gastrin-like substance were identified in the brain of Salmo gairdneri. The perikarya of these neurones appear to be located along the periventricular part of the nucleus lateralis tuberis between the hypophysial stalk and the most rostral tip of the saccus vasculosus. The fibres of these perikarya run rostrally toward the hypophysis, where they can be followed in the protrusions of the neurohypophysis into the proximal pars distalis. Here the bundle of immunoreactive fibres divides into numerous smaller bundles and into single fibres. Immunohistochemical specificity tests have shown this immunoreactive substance to belong to the gastrin group, sharing an antigenic determinant with cholecystokinin (CCK) and pentagastrin (common aminoacid sequence Trp-Met-Asp-Phe). A possible function of these gastrin (or CCK)-containing neurones in the rainbow trout is discussed.     

Ultrastructural Identification of gastrin-like immunoreactive nerve fibres in the brain of Xenopus laevis by means of colloidal gold or ferritin immunocytochemical methods

Summary By use of an anti-gastrin serum and colloidal gold- or ferritin-labelled sheep anti-rabbit -globulins, nerve fibres and nerve terminals containing a gastrin-like substance were characterized at the ultrastructural level in the median eminence of Xenopus laevis. These immunoreactive fibres contain neurosecretory granules displaying medium to high electron density and a mean diameter of 75 nm. Labelling intensity varies from granule to granule. This is the first demonstration at the ultrastructural level of the precise location of a gastrin-like hormone in the median eminence of a vertebrate.Supported by the D.G.R.S.T., Contrat no 80.7.0242     

LHRH-like system in the brain of Xenopus laevis daud

Summary Rabbit antiserum to synthetic LHRH was used with the immunofluorescence technique to identify the LHRH-secreting neurons and their axonal pathways in the brain of Xenopus laevis. Three groups of immunoreactive neurons were identified: the first, in the telencephalon, is a paired group of cells scattered near the two telencephalic ventricles; the second group lies near the preoptic recess; the third group occurs in the ventral wall of the infundibulum. Two principal neuronal pathways were observed: Fibres originating from the dorsally located telencephalic neurons converge on the cephalic median plane where they form a single bundle behind the telencephalic furrow. This bundle descends towards the anterior border of the preoptic recess where it divides into two nerve bundles which pass on either side of the preoptic recess, run above the optic chiasma then cross the infundibular floor and finally terminate in the median eminence. The second pathway is more direct. The more ventrally located telencephalic LHRH cells give rise to this second pathway. Their axons converge with the other LHRH fibres near the lateral border of the preoptic recess. Most of the LHRH nerve fibres terminate in the median eminence although some terminate near the paired pars tuberalis. No reaction was observed after the use of antiserum absorbed with synthetic antigen.Equipe de Recherche associée C.N.R.S. n 492. This work was financed by the D.G.R.S.T., Contract n 7470046     

Immunohistochemical demonstration of TSR-, LH- and ACTH-cells in the hypophysis of tadpoles of Xenopus laevis D.

Summary By use of the immunofluorescence technique TSH-, LH- and ACTH-cells were localized in the hypophysis of tadpoles of Xenopus laevis. The first signs of the activity of these cells were observed in early stages of the development, i.e., stage 39 for ACTH, and stage 42 for TSH and LH.     

Immunocytochemical localization and spatial relation to the adenohypophysis of a somatostatin-like and a corticotropin-releasing factor-like peptide in the brain of four amphibian species

Summary The distribution of somatostatin (SRIF) — and corticotropin-releasing factor (CRF)-like — immunoreactive material was studied in the brain of four amphibian species (Ambystoma mexicanum, Pleurodeles waltlii, Xenopus laevis, Rana ridibunda) by use of immunocytochemistry. A wide network of SRIF-immunoreactive fibers and numerous perikarya were observed in all amphibians examined, with a dense accumulation of nerve endings in the external layer of the median eminence (ELME). In the representatives of the four amphibian species the CRF-like system was more circumscribed. Immunoreactive perikarya were present in the preoptic area, mainly in a ventrobasal position, and in the interpeduncular nucleus. The tract running along the ventral part of the tuber cinereum ends in the ELME facing the rostroventral lobe of the pars distalis that contains corticotrophs. CRF fibers were scarce or absent in the neural lobe. In all species studied in the present work, CRF fibers end in the area of the ELME close to the pituitary lobe containing corticotrophs. This correlation is similar to that reported for the Japanese quail and several teleosts.This work was supported by a grant from the Belgian Nationaal Fonds voor Geneeskundig Wetenschappelijk Onderzoek and the CNRS     

Splenic white pulp as a thymus-independent area in the African clawed toad,Xenopus laevis

Summary An indirect immunofluorescence study of the frozen sections of the spleen of an anuran amphibian, Xenopus laevis, showed that lymphocytes bearing a small amount of immunoglobulin (Ig) were localized mostly in the white pulp of non-immunized toads. There were fewer fluorescent cells in the red pulp. In the toads hyperimmunized with human gamma globulin (HGG), cells with strong cytoplasmic fluorescence increased significantly in the outer part of the white pulp. Electron microscopy of spleens from these toads showed that plasma cells at different stages of maturation were abundant in the white pulp, whereas in the red pulp, a smaller number of maturer plasma cells were observed. These results indicate that, in contrast with its mammalian counterpart, the splenic white pulp of this anuran is the site where thymusin-dependent lymphocytes commence blast formation and transformation into plasma cells.     

Localisation of somatostatin- and gastrin-like immunoreactivity in the gastrointestinal tract of Ciona intestinalis L.

Summary Somatostatin- and gastrin-like immunoreactivity has been found by immunofluorescence in cells of the stomach and intestinal epithelia of Ciona intestinalis L. The cells containing the peptide immunoreactive to mammalian anti-gastrin can be restained with the Grimelius' technique for argyrophilia.     

Circus movements and blebbing locomotion in dissociated embryonic cells of an amphibian, Xenopus laevis.

Circus movements, which involve the circumferential rotation of a hyaline cytoplasmic protrusion, occur in cells obtained by EDTA dissociation of gastrula-stage Xenopus laevis embryos. Only a few dissociated blastula-stage cells show circus movements, more early gastrula-stage cells show them, and nearly all late gastrula-stage cells show them. Circus movements cease in cells prior to mitosis and begin again in daughter cells after mitosis is completed. In early gastrulae, only 17% of prospective endodermal cells show circus movements while 79% of prospective mesodern, archenteric roof, and posterior neural ectoderm do so. Isolated cells as well as groups of cells in vitro are often propelled by circus movements. There is an obvious antagonism between cell contact and circus movements. The morphogenetic significance of circus movements and blebbing locomotion is discussed.     

Expression of ras-like proteins in embryonic and adult cells of Xenopus laevis

A polyclonal antibody raised against v-Ha-ras p21 was purified and its specificity was checked on Ha-ras transformed cell lines. It was used to immunoprecipitate p21 from different Xenopus laevis cell types: brain cells, blood cells, and embryonic material. By one-dimensional Western blot analysis, we show that ras p21 is synthesized very early in oogenesis and accumulates throughout vitellogenesis. The ras p21 content, estimated to be 1.1 ng in the full-grown oocyte, remains constant during oocyte maturation and egg cleavage. Increase in the amount of ras p21 occurs at the beginning of neurulation. Two-dimensional Western blot patterns reveal the presence of multiple molecular forms of p21 in all Xenopus cell types studied. The numerous resolved polypeptides were ascribed to the expression of at least two different ras genes. Furthermore, specific charge modifications of the ras polypeptides are observed in brain, blood, and embryonic cells. During oogenesis and early embryonic development, differences in two-dimensional patterns mainly concern variations in the relative amounts of the different polypeptides. The results are discussed in relation to the well documented synthesis activities of the growing oocyte and of the early developing embryo.     

Effects of alpha-amanitin on the development of Xenopus (Xenopus laevis Daud.) heart in vitro]

The differentiation of Xenopus heart is studied in vitro, in the presence of alpha-amanitin. The results obtained depend on the concentration of the inhibitor, the length of treatment and the stage of primordium. The mRNA pool assures the differentiation of explants, removed from young stages, for 12 hours. This time is half for the primordia removed from stages greater than 3,5 mm.     

Epiboly connected with cleavage in morula and early blastula stages of Xenopus laevis,a study using time-lapse photography

Summary Measurements were made of the external surface areas occupied by animal and vegetal blastomeres and their daughter cells at successive cleavage cycles in 15 embyros of Xenopus laevis. On the animal side, after each cleavage a general area increase (epiboly) occurs from cycle 4 (16-cell stage, stage 5) to cycle 10 (stage 8 1/2), while on the vegetal side there is a slight general area decrease after each cleavage from cycle 6 to cycle 10. The comparison between the external surface areas of individual animal blastomeres and those of their daughter cells, visible at the next cycle, shows a significantly larger increase after radial than after tangential cleavage, a difference that may be connected with the insertion of new membrane into the external surface at radial cleavage.     

The crystalline yolk-platelet proteins and their soluble plasma precursor in an amphibian, Xenopus laevis

A single lipophosphoprotein complex, vitellogenin, was isolated and purified from the plasma of oestrogen-stimulated female toads by preparative ultracentrifugation and chromatography on TEAE-cellulose (triethylaminoethylcellulose). The protein contains 12% lipid, 1.5% phosphorus, 1.6% calcium and smaller amounts of carbohydrates and biliverdin. In amino acid composition it is identical with total yolk-platelet protein. The platelet protein, however, is fractionated on TEAE-cellulose into two components, a high-molecular-weight lipovitellin and a smaller phosvitin. Analyses of the soluble plasma vitellogenin suggest that it is a complex of two phosvitin molecules covalently bound to one lipovitellin dimer, and that it is the immediate precursor of the yolk proteins, into which it is converted by a molecular rearrangement. Uptake of vitellogenin from the plasma into the growing oocyte, and its subsequent crystallization as a yolk platelet, appear to be enhanced by gonadotrophic hormones.     

Isolation and characterization of the hatching enzyme from the amphibian,Xenopus laevis

The proteolytic activity released at the time of Xenopus laevis embryo hatching, termed the hatching enzyme, was purified and characterized in terms of its physical and enzymatic properties. Using predominantly isoelectric focusing and preparative ultracentrifugation, the enzyme was purified 2200-fold over the starting crude hatching media. From disc gel electrophoretic experiments, the most highly purified form of the enzyme had two enzymatically active charge isomers present with molecular weights of 62,500. With time, the purified enzyme gave rise to a family of enzymatically active charge isomeric proteins. The enzymatic activity of hatching enzyme toward its ¹²⁵I-labeled natural substrate, the fertilization envelope, was optimal at pH 7.7 and was ionic strength dependent. The enzyme was inhibited by Zn²⁺ and by EDTA. From inhibition by the site-specific reagents diisopropylfluorophosphate and phenylmethylsulfonylfluoride, we concluded that the enzyme was of the serine protease type, although its inhibition by Zn²⁺ and EDTA prevents a clear and unequivocal classification of the protease. This enzyme is different from the hatching enzymes reported in fish and echinoderms, on the basis of size, but it is similar to that described in Rana chensinensis on the basis of size and specificity.     

Sequence analysis of 28S ribosomal DNA from the amphibian Xenopus laevis.

We have determined the complete nucleotide sequence of Xenopus laevis 28S rDNA (4110 bp). In order to locate evolutionarily conserved regions within rDNA, we compared the Xenopus 28S sequence to homologous rDNA sequences from yeast, Physarum, and E. coli. Numerous regions of sequence homology are dispersed throughout the entire length of rDNA from all four organisms. These conserved regions have a higher A + T base composition than the remainder of the rDNA. The Xenopus 28S rDNA has nine major areas of sequence inserted when compared to E. coli 23S rDNA. The total base composition of these inserts in Xenopus is 83% G + C, and is generally responsible for the high (66%) G + C content of Xenopus 28S rDNA as a whole. Although the length of the inserted sequences varies, the inserts are found in the same relative positions in yeast 26S, Physarum 26S, and Xenopus 28S rDNAs. In one insert there are 25 bases completely conserved between the various eukaryotes, suggesting that this area is important for eukaryotic ribosomes. The other inserts differ in sequence between species and may or may not play a functional role.     

Levitide, a neurohormone-like peptide from the skin of Xenopus laevis. Peptide and peptide precursor cDNA sequences

A novel peptide, levitide, less than Glu-Gly-Met-Ile-Gly-Thr-Leu-Thr-Ser-Lys-Arg-Ile-Lys-Gln-NH2 has been isolated from skin secretions of the South African frog Xenopus laevis and sequenced by fast atom bombardment mass spectrometry. Synthetic oligonucleotides were used as probes to screen a X. laevis skin cDNA library for species coding for preprolevitide. Two such clones were detected and their sequences are reported here. Preprolevitide is 88 residues long, exhibits a putative signal sequence at the amino terminus, and contains the levitide peptide at the carboxyl terminus. The levitide precursor shows a striking nucleotide and amino acid (86%) sequence homology with the precursor of xenopsin, a biologically active octapeptide from Xenopus skin, and also encodes a 25-residue amphipathic peptide that is released by processing at a single arginine residue.