NPY- and CGRP-like immunoreactive nerve fibers in the testis and mesorchium of the toad (Bufo arenarum)

The presence and distribution of peptidergic nerve fibers were studied in the testis and mesorchium of the toad by means of immunohistochemistry. Cryostat sections of the testis and whole-mount preparations of mesorchia were immunostained with antisera to calcitonin gene-related peptide (CGRP) and neuropeptide tyrosine (NPY). After leaving the mesorchium CGRP-immunoreactive (IR) fibers were seen predominantly running in between the seminiferous tubules. In addition, a small population of CGRP-IR nerve fibers formed thin plexuses around blood vessels. Conversely, NPY-like immunoreactivity predominated in nerve fibers that formed dense plexuses around vessels both in the mesorchium and testis. Additionally, some single NPY-IR nerve fibers could be seen in both structures studied. The functional significance of these peptidergic systems in the testis of the toad remains to be analyzed.     

Apoptotic cell death in the central nervous system of Bufo arenarum tadpoles induced by cypermethrin

Tadpoles of the toad Bufo arenarum treated with cypermethrin (CY) at concentrations above 39 μg CY/L showed dose-dependent apoptotic cell death in immature cells of the central nervous system as demonstrated by morphometric analysis, the TUNEL method, and DNA fragmentation assay. Light-and electron-microscopic studies showed structural alterations in the intermediate and marginal layers of the brain. Immature cerebral tissue showed cellular shrinkage, nuclear fragmentation and increase of intercellular spaces. In this study we demonstrated high toxicity of CY to larval stages of Bufo arenarum. Our results show that doses lower than those used in routine insecticide applications can cause massive apoptosis in the immature cells of the central nervous system. These results coincide with our previous studies in Physalaemus biligonigerus, confirming the severe toxic effects of CY to the central nervous system of anuran species from Argentina. This may increase the mortality index in wild animals and contribute to the loss of biodiversity in our agroecosystems. We postulate that CY induces apoptosis in central nervous system cells of Bufo arenarum tadpoles by specific neurotoxic mechanisms.     

Membranes during yolk-platelet development in oocytes of the toad Bufo marinus

Summary Oocytes of the toad Bufo marinus have been studied by means of thin section and particularly freeze-fracture electron microscopy to characterize the cytoplasmic membranes around the yolk organelle, and the storage of yolk material in precursors and platelets. This appears to be a previously unknown type of yolk-platelet formation. During yolk-organelle development from the primordial precursor to the bi-partite fully grown yolk platelet, numerous lipoid droplets are attached to the periphery of the platelet, indicating an intense uptake of lipids. As is typical for amphibians, the fully grown yolk platelet has a crystalline internum covered by a dense osmiophilic externum, and the whole organelle is enveloped by a plasma membrane that shows no direct connection or fusion with endocytotic vesicles. The yolk membrane exhibits few intramembraneous particles (IMPs) at the core areas and some more where it borders fields of lipoid droplets. Here the IMPs show a net-like arrangement in the furrows between adjacent droplets.     

Chloride channel blockers activate an endogenous cationic current in oocytes of<Emphasis Type="Italic"> Bufo arenarum</Emphasis>

A two-electrode, voltage-clamp technique was used to measure the effect of the Cl^– channel blockers, 9-anthracene carboxylic acid and niflumic acid, upon the ionic currents of oocytes of the South American toad Bufo arenarum. The main results were: (1) both blockers produced a reversible increase of the outward currents on a dose-dependent manner; (2) the activated outward current was voltage dependent; (3) the 9-anthracene carboxylic acid-sensitive current was blocked with barium; and (4) the effect of 9-anthracene carboxylic acid was more pronounced in a zero-K⁺ solution than in standard (2 mmol l^–1) or high (20 mmol l^–1) K⁺ solutions, indicating that a K⁺ conductance is activated. The effect of the Cl^– channel blockers could be due to a direct interaction with endogenous cationic channels. Another possible explanation is that Cl^– that enter the cell during depolarizing steps in control solution inhibit this cationic conductance; thus, the blockade of Cl^– channels by 9-anthracene carboxylic acid and niflumic acid would remove this inhibition, allowing the cationic current to flow freely.Abbreviations 9-AC 9-anthracene carboxylic acid - E_r reversal potential - NA niflumic acid - NSC non-selective cation channel     

Perivitelline space of marsupial oocytes: Extracellular matrix of the unfertilized oocyte and formation of a cortical granule envelope following the cortical reaction

The purpose of this study was to characterize the structure of the vestments surrounding unfertilized and cortical granule-reacted oocytes from a marsupial, the grey short-tailed opossum Monodelphis domestica and to determine if a cortical granule envelope (CGE) forms in the perivitelline space (PVS) following the cortical reaction. Unfertilized oocytes collected from mature ovarian follicles and oviducal oocytes that had undergone a cortical reaction were fixed for electron microscopy in the presence of ruthenium red which stabilizes extracellular matrices (ECM) and facilitates demonstration of a CGE. Unfertilized oocytes were surrounded by a zona pellucida and had a PVS which contained a thick ECM comprised of granules and filaments. This matrix appeared to attach to the oolemma and was structurally similar to matrices reported previously in the PVS of unfertilized oocytes from eutherian mammals and two other marsupials, the Virginia opossum and the fat-tailed dunnart. The cortex of unfertilized oocytes contained cortical granules which were absent in oocytes recovered from the oviducts of mated females. Oviducal oocytes which lacked cortical granules exhibited a new coat within the PVS between the zona pellucida and the tips of the oocyte microvilli. This coat, the CGE, appeared structurally similar to CGEs described previously around fertilized eutherian oocytes. The CGE of the grey short-tailed opossum is approximately 1 μm thick and is made up of numerous small dense granules. The coats of the opossum oocyte are compared to those present around other marsupial and eutherian oocytes. © 1995 Wiley-Liss, Inc.     

Isolation and characterization of granules of the toad bladder

Summary The electron-dense granules that lie just below the apical plasma membrane of granular epithelial cells of toad urinary bladder contribute glycoproteins to that apical membrane. Also, exocytosis of granules (and tubules) elicited by antidiuretic hormone potentially doubles that apical surface, during the same period the transport changes characteristic of the hormonal response occur.Granules separated from other membrane systems of the cells provide the material to assess the importance of the granules as glycocalyx precursors and in hormone action. We used isosmotic media to effect preliminary separations by differential centrifugation. Then granules were isolated by centrifugation on self-forming gradients of Percoll of decreasing hypertonicity.We find qualitative and quantitative changes in protein composition and enzymic activities in the isolated fractions. The primary criterion for granule purification was electron microscopic morphology. In addition, polypeptide species found in the granule fraction are limited in number and quantity. The granules are enzymically and morphologically not lysosomal in nature. Granules may provide the glycoproteins of the apical glycocalyx but they differ from the isolated plasma membrane fraction enzymically, in protein composition and in proportion of esterified cholesterol.We conclude that the granules are not average plasma membrane precursors. Their role in the membrane properties of the toad urinary bladder may now be evaluated by characterizing permeability and other properties of the isolated organelles.     

Reactivity of isolated toad aortic rings to angiotension II: the role of nitric oxide

Little is known about the vascular actions of angiotensin II (Ang II) and nitric oxide (NO) in Amphibia. This study investigated (1) Ang II contractility, (2) NO concentrations, and (3) correlations between Ang II contractility, NO concentration and mean arterial pressure (MAP) in isolated Bufo arenarum toad aortic rings. Contractility was measured in isometric conditions, NO concentrations were determined by the Griess reaction, and MAP was determined by a direct method. In isolated toad aortic rings, Ang II produced a contractile response (292.7 ± 89.2 mg; n = 20). Furthermore, a contractile response to norepinephrine (NE) was also obtained. A significant correlation between both the Ang II and NE contractile responses was found (r = 0.89; n = 11; P < 0.01). Administration of Ang II increased MAP values (Basal 16.8 ± 1.7; n = 19 vs. Ang II 28.4 ± 1.8 mmHg; n = 19; P < 0.001), and the increase of MAP by Ang II was positively correlated with the Ang II contractile response (P < 0.01). Administration of L-NAME also increased MAP values, and this effect was higher in those toads that presented a lower pressure response to Ang II (Pearson r = −0.68; P < 0.05). NO was present in all aortic rings, and its concentrations were negatively related to the Ang II contractile response (P < 0.036) and pressure response (Pearson r = −7.08; P < 0.001). These findings suggest that, in the B. arenarum toad, the NO system contra-regulates both the contractile and pressure Ang II responses, although its action could be different in each specimen.     

The distribution and colocalization of neuropeptides and catecholamines in nerves supplying the gall bladder of the toad,Bufo marinus

The indirect immunofluorescence technique was used to determine the distribution of peptide-containing axons in the gall bladder of the cane toad, Bufo marinus. In addition, the adrenergic innervation of the gall bladder was examined by use of immunoreactivity to the catecholamine-synthesizing enzyme, tyrosine hydroxylase, and glyoxylic acid-induced fluorescence. On the basis of peptide coexistence, two intrinsic populations of neurones and their projecting fibres could be distinguished substance P neurones and vasoactive intestine peptide neurones. Neither of these two types of neurones contained any other colocalized neuropeptides. Four populations of nerve fibres arising from cell bodies outside the gall bladder were identified: nerves containing colocalized galanin, somatostatin and vasoactive intestinal peptide; nerves containing colocalized calcitonin gene-related peptide and substance P; adrenergic nerves containing neuropeptide Y; and nerves containing only adrenaline.     

Innervation of the gastrointestinal canal of the toad Bufo marinus by neurons containing 5-hydroxytryptamine-like immunoreactivity

Summary The gut of the toad, Bufo marinus, was examined for evidence of enteric neurons containing 5-hydroxytryptamine-like immunoreactivity. Such neurons were absent from the stomach. They were present in the small intestine, with processes confined to the myenteric plexus. Immunoreactive nerve cell bodies lay on branches of the pelvic nerves supplying the large intestine; fibres were found in the submucosa of the posterior large intestine and in the muscularis externa of the anterior large intestine. It is concluded, on morphological grounds, that the neurons in the small intestine are interneurons, whereas those in the large intestine are postganglionic parasympathetic motoneurons.     

Temporal spacing of breeding activity in the natterjack toad,Bufo calamita

Summary The reproductive behaviour of the toad Bufo calamita was studied in a large population in the northern Rhineland, Germany. Toads bred at four spatially separated but neighbouring areas including 34 breeding ponds. Three methods were applied to monitor quantitatively the intensity of breeding and migratory activity: capture-mark-recapture, a mechanical tracking device, and radio-telemetry. In 1987 a twofold temporal spacing of breeding activity was observed: (1) division of the reproductive period into early, main, and late breeding periods; (2) subdivision of each breeding period into several short calling periods. Since weather conditions were favourable during most of the reproductive period, environmental factors contributed little (23.4%–41.0%) to the temporal variation in breeding intensity as shown by multivariate statistics. The period of vocalization (April to August) included the activity of three distinct male groups. The first group called from April to mid-May (2 calling periods), the second from May to early July (5 calling periods) and the third from mid-July to August (2 calling periods). Within a calling period most males stayed near their calling sites and migratory activity was low. Extensive movements were detected when a new group of males immigrated from the feeding regions to the breeding areas and males that had been calling previously emigrated, at least partly. However, exchange of males among the neighbouring breeding choruses were clearly audible. It is suggested that the energetic costs of vocalization impeded continuous calling activity in individuals and accounted for the periodical variations in chorus size. The short-time variations of breeding activity therefore probably reflect the physiological limits of toads in a situation where the weather permits continuous reproductive activity.     

Amphibian larvae and zinc sulphate: a suitable model to study the role of brain-derived neurotrophic factor (BDNF) in the neuronal turnover of the olfactory epithelium

The vertebrate olfactory system has fascinated neurobiologists over the last six decades because of its ability to replace its neurons and synaptic connections continuously throughout adult life, under both physiological and pathological conditions. Among the factors that are proposed to be involved in this regenerative potential, brain-derived neurotrophic factor (BDNF) is a candidate for having an important role in the neuronal turnover in the olfactory epithelium (OE) because of its well-documented neurogenic and trophic effects throughout the nervous system. The aim of the present study was to generate a suitable model to study the participation of BDNF in the recovery of the OE after injury in vivo. We developed an experimental design in which the OE of Rhinella arenarum tadpoles could be easily and selectively damaged by immersing the animals in ZnSO₄ solutions of various concentrations for differing time periods. Image analysis of histological sections showed that different combinations of each of these conditions produced statistically different degrees of injury to the olfactory tissue. We also observed that the morphology of the OE was restored within a few days of recovery after ZnSO₄ treatment. Immunohistochemical analysis of BDNF was performed with an antiserum whose specificity was confirmed by Western blotting, and which showed drastic changes in the abundance and distribution pattern of this neurotrophin in the damaged olfactory system. Our results thus suggest that BDNF is involved in the regeneration of the OE of amphibian larvae, and that our approach is suitable for further investigations of this topic. This work was supported by grants from CONICET (PIP 5842), Universidad de Buenos Aires (UBACYT X131) and ANPCYT (PICT 32219).     

Temperature and humidity alter prolactin receptor expression in the skin of toad (Bufo bankorensis and Bufo melanostictus)

Bufo bankorensis and Bufo melanostictus, the only two species of Bufonidae genus in Taiwan, live in habitats that differ in altitude and humidity. This study tested the hypothesis that prolactin receptor (PRLR) expression responds to environmental change. Western blot analysis showed that the PRLR protein was widely distributed in brain, lung, liver, kidney, dorsal skin and ventral skin of toads. The level PRLR protein was elevated in the dorsal skin of the two toad species treated with dry or wet conditions for 14 days. The increase in PRLR of dorsal skin in B. bankorensis was higher than that in B. melanostictus. This experimental result suggests that B. bankorensis secretes more mucus to reduce water evaporation from its thinner cuticle than B. melanostictus. The expression of PRLR protein was increased in the lung of B. bankorensis and decreased in the lung of B. melanostictus. Moreover, PRLR protein levels were increased in the kidneys in the two species toad, likely due to reduction in water lost through lung and urine. The two toad species were subjected to varying temperatures (25 degrees C, 15 degrees C and 10 degrees C) for 14 days. The lowest PRLR protein expression was observed at 10 degrees C. Comparison of the decreasing trend in PRLR protein levels demonstrated that the variation in B. bankorensis was significantly higher than that in B. melanostictus. Comparisons of variation in PRLR protein expression in the two species under different environments suggest that B. bankorensis is more adaptable to different environments than B. melanostictus.     

Effects of the ecological conditions on morphological variations of the green toad, Bufo viridis, in Turkey

Ten populations of the green toad, Bufo viridis, from three areas having different climatic regimes were compared. Multivariate analyses of morphological characteristics revealed three distinct populations of green toads: the first group inhabiting the lowland and humid localities was characterized by a small body size, the second group occurred at higher altitudes and in desert areas had a significantly larger body size, while the third group from the Mediterranean localities was characterized by a large tympanum, a longer tarsus and short first toes. Interestingly, the specimens from Giresun Island were clustered with the second group with a similar pattern of body size. Differences in the morphological characteristics among populations were positively associated with ecological conditions, and not with geographic distance. Some characteristics previously used for taxonomic purposes in B. viridis were shown to be associated with local climatic conditions.     

中华大蟾蜍卵母细胞质膜存在钙依赖性的氯离子通道

本文采用双微电极电压钳方法研究了中华大蟾蜍卵母细胞内源性电压门控型离子通道的成分及其生理特性。卵母细胞去极化至 -30 mV 及更正电压时,有一持续的电压依赖性外向电流出现。钾离子通道拮抗剂四乙基氯化氨(tetraethy-lammonium chloride, TEA, 10 mmol/L)和 4- 氨基吡啶(4-aminopyridine, 4-AP, 10 mmol/L)协同作用时,该电流只能被抑制到最大电流幅度的(23.4±0.72)%。但是,上述浓度的TEA和4-AP 与氯离子通道拮抗剂5- 硝基-2, 3- 苯酚丙胺苯甲酸盐 (5-nitro-2,3-phenypropylamino benzoate, NPPB, 30 μmol/L)、无钙 Ringer 氏液或钙离子通道拮抗剂维拉帕米(40 μmol/L)协同作用时,可分别将此外向电流抑制到最大电流幅度的(2.1±0.08)%、(2.2±0.04)% 和(3.1±0.15)%。结果表明,中华大蟾蜍卵母细胞质膜上除有钾离子电流之外,还存在钙依赖性的氯离子电流。     

The distribution and colocalization of neuropeptides and 5-hydroxytryptamine in pelvic nerves supplying the posterior large intestine of the toad,Bufo marinus

The distribution and colocalization of neuropeptides and 5-hydroxytryptamine in the posterior portion of the large intestine of the toad was studied using single- and dual-label immunohistochemistry. Neurons containing colocalized galanin/somatostatin or vasoactive intestinal peptide alone were observed along intramural pelvic nerves. Some of the galanin/somatostatin neurons also contained 5-hydroxytryptamine. Synaptic boutons containing colocalized calcitonin gene-related peptide/vasoactive intestinal peptide were associated with the galanin/somatostatin neurons. The muscle of the large intestine was also innervated by axons containing galamin/somatostatin, vasoactive intestinal peptide/calcitonin gene-related peptide or vasoactive intestinal peptide alone. Nerve fibres containing calcitonin gene-related peptide/substance P, probably representing primary afferent nerves, were also associated with muscle bundles. Submucosal blood vessels carried dense plexuses of fibres containing vasoactive intestinal peptide alone or and calcitonin gene-related peptide/substance P. Adrenergic perivascular nerves also contained galanin and neuropeptide Y.     

Histochemistry of the juxtaglomerular apparatus in the toad Bufo bufo

Summary An investigation regarding the question of whether there exists a macula densa as part of the juxtaglomerular apparatus in the kidney of amphibians has been carried out. With the aid of a histochemical reaction for glucose-6-phosphate dehydrogenase activity, the presence of a macula densa zone as a specialized part of the distal tubule in the toad Bufo bufo was demonstrated. The functional significance of the high glucose-6-phosphate dehydrogenase activity in the macula densa cells is discussed.     

Radial astrocytes and ependymocytes in the spinal cord of the adult toad (Bufo bufo L.)

Summary Immunohistochemical and ultrastructural techniques have been used to demonstrate glial fibrillary acidic protein (GFAP) immuno-positive cells in the adult toad spinal cord. Two types of GFAP-immunoreactive cells were observed: ependymocytes and radial astrocytes. GFAP-positive ependymocytes were scarce and contained the immunoreactive product in their processes. They showed intermediate filaments in the basal pole and in their processes when studied with the electron microscope. These immuno-positive ependymocytes represent the tanycytic form of ependymal cells because their processes ended at the subpial zone. The radial astrocytes showed a more intensive immunoreactive product in somata and processes when they were located far away from the ependymal layer. Cell bodies and processes were also associated with blood vessels, but most of the processes ended at the subpial zone forming a continuous subpial glia limitans. The GFAP-positive processes, which form this subpial glia limitans in the toad spinal cord, belong to both tanycytic ependymocytes and radial astrocytes, whose somata are located in the grey matter. These findings lead us to suggest that both types of GFAP-immunopositive cells might be the functional equivalents of mammalian astrocytes.     

Organogenesis and differentiation of the pancreas in the toad Bufo bufo L.

Summary In Bufo bufo at stage III₆ the first endocrine islets appear in the part of the pancreas corresponding to the dorsal anlage. At stage IV₂, 5 days later, the pancreatic duct develops and new islets arise by budding off from the ductal epithelium. The ultrastructural study of the secretory granules morphology of endocrine cells has distinguished four different cell types: B-cells (stage III₉), A-cells (stage IV₃), D-cells (stage IV₃) and a fourth type not yet identified (stage IV₃). By immunocytology insulin and corticotropin-releasing factor (CRF) cells have been demonstrated at stage III₉, and glucagon and somatostatin cells at stage IV₁. Lastly, endocrine islets can be homogeneous (predominantly containing insulin cells, rarely glucagon cells) or heterogeneous (insulin cells at the centre and glucagon or somatostatin cells at the periphery). Hypotheses are put forward for the origin and the constitution of the different generations of endocrine islets and isolated cells.     

Induction from posterior hypothalamus is essential for the development of the pituitary proopiomelacortin (POMC) cells of the toad (Bufo japonicus)

The role of the posterior hypothalamus in the development of the epithelial hypophysis was studied in Bufo embryos. In animals from which the central part of the neural plate (NP) had been surgically removed at the open neurula stage, the infundibulum did not develop, and the epithelial hypophysis was formed away from the normal site without morphological connection with the brain. Immunoreactive MSH cells and ACTH cells, i.e, the pituitary POMC cells, were not detected in any of the surgically treated animals while other types of secretory cells (PRL, GH, TSH and GTH cells) were invariably present. In view of the fact that POMC cells originate in the anterior neural ridge, and not in the neural plate, the embryonic brain seems to exert an inductive influence upon the primordial pituitary POMC cells. Since these cells differentiate in a tail graft, isolated from the brain at a later stage (tail-bud stage), the inductive stimuli must be conveyed from/via the posterior hypothalamus to the pituitary anlage between the open neurula and the tail-bud stages.Abbreviations ACTH Adrenocorticotropic hormone - ANR anterior neural ridge - GH growth hormone - GTH gonadotropic hormone - MSH metanocyte stimulating hormone - NP central part of anterior neural plate - POMC proopiomelanocortin - PRL prolactin - TSH thyroid-stimulating hormone