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.     

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.     

L'ultrastructure de l'adénohypophyse du crapaud Bufo bufo L.

Résumé Les cellules glycoprotidiques de type II (cyanophiles, palissadiques, périvasculaires) de l'adénohypophyse du Crapaud subissent d'importantes modifications après thyroïdectomie chirurgicale. Elles paraissent donc responsables de la fonction thyréotrope (TSH).Ces cellules se transforment en cellules chromophobes hypertrophiées (cellules de thyroïdectomie) à la suite d'une dégranulation massive, une hypertrophie importante de l'ergastoplasme qui envahit tout le cytoplasme et une extension appréciable de l'appareil de Golgi.La libération massive des produits de sécrétion des cellules glycoprotidiques de type II se ferait principalement par lyse intracytoplasmique des granules sans destruction de leur membrane limitante.

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The ultrastructure of the adenohypophysis in the toad, Bufo bufo L.II. On the formation of glycoprotein containing cells of type II (perivascular basophils) after thyroidectomy

Summary In the toad's adenohypophysis, the glycoprotein containing cells of type II (perivascular basophils) undergo important modifications after surgical thyroidectomy. They seem also to be responsible for the production of the thyroid-stimulating-hormone (TSH).These cells, which become chromophobic and hypertrophie (thyroidectomy cells) after an intense release of the secretory granules, show a great development of the ergastoplasm and a notable extension of the Golgi complex.The heavy release of the secretory products from the glycoprotein containing cells of type II may take place by intracytoplasmic lysis without destruction of the granule's limiting membrane.

Nous remercions tout particulièrement Mme Sidler-Ansermet, photographe, et Mme Estoppey, secrétaire, de l'aide qu'elles ont apportée à la réalisation de ce mémoire.     

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.     

Seasonal changes in the migratory behaviour of the toad Bufo bufo: direction and magnitude of movements

Summary The migratory behaviour of the toad Bufo bufo was studied from February 1985 to April 1986 in the submontane region of Bavaria, West Germany. Toads were fitted with a mechanical tracking device to record individual paths of migration. Three aspects of migratory behaviour were quantified: orientation in relation to the breeding site, straightness of path, and locomotory activity. The annual activity period began with migration from the hibernation sites to the breeding pond in April. The paths went straight towards the breeding pond independent of the distance (70–420 m). During the period of oviposition the preference for the breeding site direction vanished and toads moved away from the breeding pond, but in less straight paths than before. In summer migratory activity decreased considerably and was restricted to small areas, the home ranges, at distances of 55–1600 m from the natal breeding pond. The straightness of path was rather low, because toads often returned to their starting points. During rainy nights toads occasionally left their home ranges for extensive excursions. In autumn most toads again migrated towards the breeding pond, but paths were significantly less straight and direct than in spring. However, toads stopped before reaching the breeding pond and hibernated in holes or under the leaf layer. The mortality rate of tracked toads was about 45%. The relative influence of 17 environmental variables on locomotory activity was evaluated by principal component analysis and stepwise multiple regression. Temperature at night and rainfall variables accounted for significant amounts of variance, whereas temperature by day, air humidity, and atmospheric pressure showed no correlation. Activity decreased if temperature approached 0° C or after long periods without rainfall. Within a certain range of tolerance, however, the locomotory activity of the toads was widely independent of environmental factors, indicating that endogeneous factors are more important sources of variation in the migratory behaviour of these toads than commonly assumed.     

Vascularization of the pars intermedia of the hypophysis in the toad,Bufo bufo (L.) (Amphibia,Anura)

The vascularization of the pars intermedia of the hypophysis of the toad, Bufo bufo (L.) was studied by injection of a mixture of India-ink and gelatine into the circulatory system of the head via the arteria carotis communis. Further methyl-methacrylate corrosion casts of brains were made and the hypophysial region of the corrosion casts was examined with the scanning electron microscope. The results showed that the vascularization of the pars intermedia of the toad hypophysis consists of a single-layered vascular network, which is located on the ventral surface of the pars intermedia. The network is formed by capillaries, which primarily run caudally in a fan-like manner and which show only a few cross-connections. In the rostral region of the pars intermedia this network lies rather superficially, while in the caudal region it slightly penetrates the parenchyma. The vascular network originates from vessels of the neural stalk and from wide capillaries of the rostro-ventral region of the neurointermediate junction. The venous drainage of the pars intermedia is exerted by veins, which leave the caudal region and drain into the veins leaving the venous pole of the pars distalis. The flat, wide meshed vascular net on the ventral side of the pars intermedia, demonstrated in this study, fits into the concept that the pars intermedia of the anuran hypophysis is under the control of nerve fibers coming from the hypothalamus.     

Vascularization of the pars distalis of the hypophysis in the toad,Bufo bufo (L.) (Amphibia,Anura)

Summary The vascularization of the pars distalis of the hypophysis of the toad, Bufo bufo (L.), was studied by the traditional method of injecting a mixture of India-ink and gelatine into the circulatory system of the head via the arteria carotis communis. Further, methyl-methacrylate corrosion casts of the brains were made; the hypothalamo-adenohypophysial region of these corrosion casts was studied with the scanning electron microscope. The results showed that the portal vessels which arise from the median eminence do not supply distinct areas in the pars distalis as is supposed by the point-to-point-hypothesis. The portal vessels enter the ventro-median region of the pars distalis and branch off into a three-dimensional network of the secondary capillary plexus of the pars distalis. The plexus is made up mostly by four- to six-sided meshes. This angioarchitecture guarantees an optimal supply of the glandular cells of the pars distalis with nutritional factors and releasing hormones, on the one hand, and facilitates the removal of the hormones which are released by these cells, on the other hand. The venous drainage of the pars distalis is exerted mainly by two large veins, which bilaterally leave the dorso-lateral region (venous pole) of the pars distalis and by a few small veins, which drain into the wide, sinus-like vessel, which curves around the dorso-caudal region of the pars distalis and joins bilaterally the vena hypophysea transversa.This work was supported by the Fonds zur Förderung der wissenschaftlichen Forschung (Projekt Nr. 2183/N 39) and by the Stiftungs- und Förderungsgesellschaft der Paris-Lodron-Universität SalzburgThe authors wish to thank Mag. Ursula Albrecht for excellent technical assistance and Mr. Gerhard Sulzer for photographic work     

Visual performance of the toad (Bufo bufo) at low light levels: retinal ganglion cell responses and prey-catching accuracy

The accuracy of toad snapping towards moving worm dummies under various levels of dim illumination (from absolute threshold to moonlight) was videorecorded and related to spike responses of retinal ganglion cells exposed to equivalent stimuli. Some toads (at ca. 16 °C) successfully snapped at dummies that produced only one photoisomerization per 50 rods per second in the retina, in good agreement with thresholds of sensitive retinal ganglion cells. One factor underlying such high sensitivity is extensive temporal summation by the ganglion cells. This, however, is inevitably accompanied by very long response latencies (around 3 s near threshold), whereby the information reaching the brain shows the dummy in a position where it was several seconds earlier. Indeed, as the light was dimmed, snaps were displaced successively further to the rear of the dummy, finally missing it. The results in weak but clearly supra-threshold illumination indicate that snaps were aimed at the advancing head as seen by the brain, but landed further backwards in proportion to the retinal latency. Near absolute threshold, however, accuracy was too good, suggesting that the animal had recourse to a neural representation of the regularly moving dummies to correct for the slowness of vision.     

Quantitative histochemical studies of the effect of levamisole on splenic T-cells in thymectomized Bufo bufo larvae

Summary The ability of levamisole (LVS) to correct the effect of thymectomy on splenic T-cell population in Bufo bufo larvae was tested. Using the nonspecific acid -naphthyl acetate esterase (ANAE) technique, the T-lymphocytes displayed a characteristic reddish brown spot (T-pattern). The total number of white cells per spleen and the number of T-pattern cells per 100 splenic white cells were determined. In addition, differential counts of the T-patterns were made on the basis of the size of their spots.Thymectomized larvae in comparison to sham-operated larvae posessed a lower total number of splenic white cells with a similar proportion of T-pattern cells, but a lower proportion of cells with a large T-pattern. After seven days of LVS treatment, the thymectomized larvae showed an increased total number of splenic white cells and an increased proportion of cells with a large T-pattern, both data falling within normal limits. The effect of LVS appeared marked in thymectomized larvae, but was slight or absent in sham-operated larvae. Differential counts of the large T-pattern cells, based on the size of ANAE reaction product, showed the capacity of LVS to stimulate T-cell maturation. It was concluded that LVS induced proliferation and differentiation of splenic T-cells and that the different sizes of T-pattern reaction represent different maturational stages of T-cells.     

Effect of transection at different levels of hypothalamus on the hypothalamo-hypophysial system of the toad,Bufo bufo,with particular reference to the ultrastructure of the zona externa of the median eminence

Summary The ultrastructural changes taking place in the median eminence of Bufo bufo 3 hours to 4 months after transection of the brain at different levels, are described.5 types of neurons in the zona externa of the median eminence of normal toads are described. All types of neurons degenerate, and profound changes of the ultrastructure of the capillaries are observed after transection just behind, or immediately in front of the optic chiasma. A few neurons containing dense granules with a diameter of about 1,000–1,300 Å remain intact, however. The degeneration following denervation in front of the optic chiasma was considerably delayed compared to degeneration after denervation behind the optic chiasma.After transection more rostral to the optic chiasma, no significant degeneration of the median eminence was observed.The results are discussed with regard to degenerative dynamics and origin of the different nerve types. It is concluded, that all types of neurons terminating in the median eminence, originate at a level between the caudal and rostral parts of the preoptic nucleus, some fibres, however, containing dense, 1,000–1,300 Å granules, originate caudally to the optic chiasma, in the posterior hypothalamus.Part of this study was presented at the Vth International Symposium on Neurosecretion, Kiel, Germany, August 1969.     

The vascularization of the neural stalk and the pars nervosa of the hypophysis in the toad,Bufo bufo (L.) (amphibia,anura)

The angioarchitecture of the neural stalk and the encephaloposthypophysial portal system of the hypophysis of the toad, Bufo bufo (L.), was studied using three different methods. The neural stalk is mainly supplied by branches of the arteria infundibularis superficialis which form a widemeshed vascular network. Dorsally this network continues into the plexus of the pars nervosa. The vascularization of the pars nervosa is made up of the encephalo-posthypophysial portal system. This portal system consists of a hypothalamic branch (=portion), a mesencephalic and a mesencephalicbulbar branch (=portion). The hypothalamic branch was found to drain the pars ventralis of the tuber cinereum as well as more dorsal regions of the diencephalon. The mesencephalic-bulbar trunk enters the hypothalamic branch. The resulting common stem of the encephalo-posthypophysial portal vein the curves around the retroinfundibular communicating artery, crosses its ventral side and runs caudally. The secondary capillary plexus of the pars nervosa is characterized by well defined capillary plexus of the pars nervosa is characterized by well defined capillary networks which are located at the periphery of the parenchyma of the pars nervosa, thus forming a rostral, dorsal and ventro-caudal net. The central region of the parenchyma of the pars nervosa is supplied only by main branches of the encephalo-postpophysial portal vein. The venous drainage of the pars nervosa is via the vena hypophysea transversa.     

Epidermal tissue homeostasis: Apoptosis and cell emigration as mechanisms of controlled cell deletion in the epidermis,of the toad,Bufo bufo

Summary In normal, non-expanding toad epidermis more cells are produced than needed to replace cells lost by moulting. By implication, cell deletion additional to moulting must take place. This paper deals with the mechanisms by which the surplus of cells is deleted, taking advantage of the fact that the ratio between cell birth rate (K _b) and the rate of desquamation (K _d), which in normal toads is 2 to 3, can be manipulated. In toads deprived of the pars distalis of the pituitary gland it is decreased to 0.2 to 0.3, and in toads with hydrocortisone pellets implanted into the subcutaneous lymph space it is increased to 7 to 10. Thus, structures candidates for the morphological manifestation of the deletion process should occur rarely in toads in which the pars distalis has been removed and frequently in toads with hydrocortisone pellets implanted. Categorization and enumeration of such structures by light microscopy in the epidermis from operated, normal, and hormone-treated toads were performed. The incidence of structures referred to as dark cells and omega-figures were found to correlate relatively well with the K _b/K_d-ratio. A subsequent ultrastructural analysis — on a cell-by-cell basis — of dark cells showed these to reflect various stages of apoptosis. The duration of the apoptotic process was calculated to be approximately 7 h. Light- and electron microscopy of omega-figures combined with histochemical observations of PSA-lectin binding were interpreted as reflecting a release of cells from the basal epidermis and their final elimination within the dermis. It is concluded (i) that apoptosis is an important mechanism of controlled cell deletion, (ii) that emigration to, and elimination in, the dermis is a possible deletion mechanism, and (iii) that necrosis is unlikely to play a role in controlled cell deletion.Supported in part by the Danish National Science Research Council (grant no. 11-6498) (PB)Part of this work was presented at the XVth Meeting of the European Study Group for Cell Proliferation, Sundvollen, Norway, 16–20 September 1987     

Tissue kinetics,ion transport,and recruitment of mitochondria-rich cells in the skin of the toad (Bufo bufo) in response to exposure to distilled water

Mitochondria-rich cells (MRC) of the amphibian epidermis are responsible for active chloride uptake at low external salinity, and new MRCs are recruited in response to exposure to distilled (deionized) water. The time-course of this recruitment, the tissue kinetics and ion transport have been studied in toads (Bufo bufo) immediately before, and after 2,7, and 14 days exposure to distilled water. General epidermal structure was not affected. However, the numbers of MRCs per mm² (D_MRC) increased throughout the experiment as revealed by staining of epidermal sheets with AgNO₃ (Ag) or methylene blue (MB). Part of the increased D_MRC was accounted for by an increase in MRC subpopulation(s) that stained neither with Ag nor MB. The cell birth rate (K_b) decreased and cell loss by moulting (K_d) increased without any significant change in epidermal cell pool size, indicating a reduced apoptotic rate. The increase in D_MRC was accompanied by a 3-fold increase in Cl^- current (I_Cl). At day-2 there was a transient reduction in the I_Cl per MRC. H⁺ secretion was progressively reduced during prolonged exposure to distilled water. Thus, at day-2 MRCs appeared incompletely differentiated as indicated by decreased I_Cl and H⁺ flux per MRC, and by the increased proportion of MRCs unstained by Ag or MB. Full Cl^- (but not H⁺) transport capacity, was restored at day-7. We conclude that increased D_MRC following exposure to low external Cl^-, rather than being due to an increased K_b, is the combined effect of a decreased apoptotic rate and an increased rate of differentiation, where morphological differentiation precedes functional differentiation.Parts of this study have been presented at the 32th International Congress of Physiological Sciences, 1–6 August, 1993, Glasgow, Scotland, and the 19th meeting of the European Study Group for Cell Proliferation, 5–9 October, 1993, Bruges, Belgium     

Multilocus species tree analyses resolve the radiation of the widespread Bufo bufo species group (Anura, Bufonidae)

New analytical methods are improving our ability to reconstruct robust species trees from multilocus datasets, despite difficulties in phylogenetic reconstruction associated with recent, rapid divergence, incomplete lineage sorting and/or introgression. In this study, we applied these methods to resolve the radiation of toads in the Bufo bufo (Anura, Bufonidae) species group, ranging from the Iberian Peninsula and North Africa to Siberia, based on sequences from two mitochondrial and four nuclear DNA regions (3490 base pairs). We obtained a fully-resolved topology, with the recently described Bufo eichwaldi from the Talysh Mountains in south Azerbaijan and Iran as the sister taxon to a clade including: (1) north African, Iberian, and most French populations, referred herein to Bufo spinosus based on the implied inclusion of populations from its type locality and (2) a second clade, sister to B. spinosus, including two sister subclades: one with all samples of Bufo verrucosissimus from the Caucasus and another one with samples of B. bufo from northern France to Russia, including the Apennine and Balkan peninsulas and most of Anatolia. Coalescent-based estimations of time to most recent common ancestors for each species and selected subclades allowed historical reconstruction of the diversification of the species group in the context of Mediterranean paleogeography and indicated a long evolutionary history in this region. Finally, we used our data to delimit the ranges of the four species, particularly the more widespread and historically confused B. spinosus and B. bufo, and identify potential contact zones, some of which show striking parallels with other co-distributed species.     

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.     

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.     

Types of supernumerary outgrowths produced after inverting the dorsoventral limb axis of the anuranBufo bufo

Summary Contralateral grafts were performed on the larval limb buds of the anuranBufo bufo. The dorsoventral axis of 80 buds at stages IV or V was inverted. Ten tadpoles were used as controls. Fifty-two supernumerary structures developed, all of them in dorsal or ventral locations on the host stump. The majority (32 out of the 44 outgrowths with more than 3 toes) were normal limbs of stump handedness. However, the following abnormal structures were also observed: 2 double-posterior, 3 mixed-symmetric, and 7 undetermined cases. These results are in agreement with the predictions of a hierarchical polar coordinate model for epimorphic regeneration.     

The morphology of the juxtaglomerular apparatus in the toad,Bufo bufo

Summary In a light microscopic study the course of the tubule in the kidney of the toadBufo bufo was studied. The distal tubule returning to the glomerulus of its origin appears to enclose the afferent arteriole. In that area, from which a three dimensional graphic reconstruction is made, there is an intimate contact between tubular and vascular wall. The latter contains granulated media cells. In the part of the tubule adjacent to the afferent arteriole an accumulation of nuclei is present. It is suggested that this structure is similar to the macula densa of the mammalian juxtaglomerular apparatus. The functional significance of a stricture in the tubule distally from the macula densa-like structure is discussed.The authors wish to thank Mrs. Ineke van de Mee-Wienen and Miss Ans Rouwenhorst for their technical assistance and Mr. J. J. M. de Bekker for the realization of the graphic reconstruction.     

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.