Adenylic nucleotides and energy charge during the embryonic development of Bufo arenarum

The contents of ATP, ADP and AMP were determined by HPLC and adenylic energy charge (AEC) was estimated during different stages of the embryonic development of Bufo arenarum up to the tailbud stage. All the developmental stages studied showed a high ATP content (about 1.04-1.48 nmol/emb.). The concentration of ADP was low (0.025-0.041 nmol/emb.) but rose slightly at the neural tube stage. AMP was undetectable before the tailbud stage. AEC values were almost constant (about 0.987-0.992) throughout the period studied. Only a fall at the tailbud stage could be detected which can be related to this more advanced cellular differentiation stage.     

Immunoidentification of CRF-like material in the intermaxillary glands during Bufo arenarum development

The presence of corticotropin-releasing factor-like material in the intermaxillary glands was studied by immunocytochemical techniques during the metamorphosis of Bufo arenarum. The intermaxillary glands appeared at stage XV (midprometamorphosis) with CRF-like material slightly immunoreactive. These glands are located posterior to the premaxillae and between the nasal capsules in the roof of the mouth and are formed of alveoli or tubules. During metamorphic climax, corticotropin-releasing factor-like material was identified strongly immunostained at the apices of the secretory cells. It was observed that collecting ducts of the gland open to the anterior palatal surface suggesting that the secretion could be ingested by tadpoles. Our results clearly showed that ir-CRF-like material present in the intermaxillary glands is ingested by tadpoles during metamorphosis and could play an important role during amphibian development.     

Expression of N-CAM-180 and N-cadherin during development in two South-American anuran species (Bufo arenarum and Hyla nana)

Cadherins and N-CAM are Ca++-dependent and Ca++-independent cell adhesion molecules respectively. These molecules play a key role in morphogenesis and histogenesis. We determined the spatiotemporal pattern of N-cadherin and N-CAM-180 kDa expression by immunohistochemistry during development in two South-American anuran species (Bufo arenarum, toad and Hyla nana, frog). Both N-cadherin and N-CAM were not detectable during early developmental stages. Expression of N-cadherin appeared between the inner and the outer ectoderm layers at stages 19-20. At stages 24-25, N-cadherin was expressed in the neural tube and the heart. In early tadpoles, N-cadherin expression increased along with the central nervous system (CNS) morphogenesis, and reached its maximum level at metamorphic climax stage. N-Cadherin expression was not uniformly distributed. At stage 42, olfactory placodes and retina expressed N-cadherin. Contrary to N-CAM, the strongly myelinated cranial nerves were not labeled. N-Cadherin was present in several mesoderm derivatives such as the notochord, heart and skeletal muscle. The non-neural ectoderm and the endoderm were always negative. Expression of N-CAM appeared first in the neural tube at stages 24-25 and the level of expression became uniform from pre-metamorphic to metamorphic climax tadpoles. At this latter stage, a clear N-CAM immunolabeling appeared in the nerve terminals of pharynx and heart. N-Cadherin and N-CAM were found mainly co-expressed in the CNS from early tadpole to metamorphic climax tadpole. Our results show that the expression of N-CAM and N-cadherin is evolutionary conserved. Their increased expression during late developmental stages suggests that N-CAM and N-cadherin are involved in cell contact stabilization during tissue formation.     

Studies of oogenesis in Bufo arenarum (author's transl)

Based on a series of macroscopic and histological observations, during an annual cycle, the main stages of oogenesis in Bufo arenarum (Hensel) have been recognized, pointing out the most significant features. The analysis has established five characteristic stages which permit the individualization of the maturation stage of the oocyte in the ovary. All the information obtained has provided the possibility of drawing up a synthetic table so that the oogenetic stages of this amphibian species, very much used in Argentine experimentation, could be easily recognized.     

Pregnenolone and progesterone metabolism by the testes of Bufo arenarum

Sliced testis tissue from Bufo arenarum was incubated in the presence of [³H]pregnenolone. Testis fragments were also used for double isotope experiments using [³H]pregnenolone and [¹⁴C]progesterone. Specific activities were equated with the addition of radioinert pregnenolone. When yields of radiometabolites were analysed, pregnenolone was found to be a good precursor for C₁₉ steroids such as dehydroepiandrosterone, 5-androsten-3β,17β diol, testosterone, 5α-dihydrotestosterone and a C₂₁ steroid, 5α-pregnan-3,20 dione. Progesterone mainly converts to 5α-pregnan-3,20 dione, a steroid with unknown function in amphibians. The 5-ene pathway, including 5-androsten-3β,17β diol as intermediate, could be predominant for androgen biosynthesis. Testes bypass not only progesterone but also androstenedione for testosterone biosynthesis. Accepted: 17 April 1998     

Larval development and metamorphosis of the olfactory and vomeronasal organs in the toad Rhinella (Bufo) arenarum (Hensel, 1867)

Jungblut, L.D., Pozzi, A.G. and Paz, D.A. 2010. Larval development and metamorphosis of the olfactory and vomeronasal organs in the toad Rhinella (Bufo) arenarum (Hensel, 1867). — Acta Zoologica (Stockholm) 92 : 305–315. The olfactory and the vomeronasal system are the two major chemosensory systems found in terrestrial vertebrates. Among tetrapods, amphibians are unique in having an aquatic larval stage, followed by metamorphosis to a terrestrial adult. In the present work, we studied the histological development of the olfactory and vomeronasal organ and associated multicellular glands of the toad Rhinella (Bufo) arenarum, from early poshatching larva to postmetamorphic toadlets. As in other bufonids, the olfactory epithelium of R. arenarum in larvae is divided into dorsal and ventral branches in the rostral and mid‐nasal regions. At metamorphic climax, the larval pattern changes drastically and the adult olfactory configuration develops. Bowman’s glands appear in the olfactory epithelium of R. arenarum at the onset of metamorphic climax. The vomeronasal epithelium develops early in larval development in R. arenarum, around the time of operculum development. Interestingly, a novel sensory epithelium develops in the floor of the principal chamber of R. arenarum at metamorphic climax. This novel sensory epithelium resembles larval sensory epithelium lacking Bowman’s glands, and suggests that these animals would be able to sense not only air‐borne, but also water‐borne odors during their adult terrestrial life.     

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.     

Delta-aminolevulinic acid dehydratase (ALAD) activity in blood of Bufo arenarum (Anura)

The aim of the present investigation was to standardize a method for measuring delta-aminolevulinic acid dehydratase (ALAD) activity in circulating red blood cells of adult Bufo arenarum kept in controlled environmental conditions, and to obtain reference basal values suitable for environmental monitoring of lead exposure. The normal ALAD activity for B. arenarum was 131.86 +/- 14.47 U per liter of red blood cells (n = 38, mean +/- SEM; interval 72.98-236.33). In animals exposed to lead, ALAD activity decreased as lead dose increased.     

Basal and amiloride-induced short-circuit current across isolated toad skin (Bufo arenarum)

We have previously demonstrated that amiloride (amil) addition to the isolated ventral pelvic (VPel) skin of Bufo arenarum toad induces negative short-circuit current values, which are equivalent to the isotopically measured net chloride transport. In the present work, we found that exposure of various regions of toad skin to amil yielded different values of short-circuit current (aSCC): negative aSCC was found in the VPel and ventral pectoral skin, while those of the dorsal one were not different from zero. The distinct values of aSCC found show a regional difference in the active chloride absorption, probably related to postural adaptations. A possible role of this adaptation would be related to chloride participation in the saline balance of the animals, or the maintenance of epithelial integrity.     

Identification of mRNA-binding proteins during development: characterization of Bufo arenarum cellular nucleic acid binding protein

Ultraviolet irradiation was used to covalently cross-link poly(A)+RNA and associated proteins in eggs and embryos of the toad Bufo arenarum. Four major proteins with apparent sizes of 60, 57, 45 and 30-24 kDa were identified. It was observed that the same mRNA-binding proteins were isolated from eggs to gastrula and neural stages of development. The 30 kDa polypeptide, p30, appeared as the main ultraviolet (UV) cross-linked protein in the developmental stages analyzed. By means of polyclonal antibodies, it was determined that this polypeptide has a cytoplasmic localization and it was detected in liver, eggs and embryos. The presence of p30 was also analyzed by western blot during oogenesis and development. The 30 kDa polypeptide was present in all stages analyzed but it could not be detected in stages I-II of oogenesis. At the neural stage, the relative amount of p30 began to decrease, reaching its lowest levels after stages 26-30 (tail-bud in Bufo arenarum). On the basis of purification, immunoprecipitation and western blot assays the 30 kDa protein was identified as the Bufo arenarum cellular nucleic acid binding protein.     

Effects of low concentrations of ethanol on the embryonic development of Bufo arenarum

The effects of low concentrations of ethanol on embryos of Bufo arenarum were studied. Embryos maintained continuously (from stage 3 on) in 0.84-3.34 microM ethanol showed concentration dependent effects: delay in the development rate and on eclosion, loss of equilibrium, arrhythmic contractions, swimming in atypical positions and high death rate. At lower concentrations the anomalies were concentration independent. Embryos treated continuously with 0.002 and 0.21 microM ethanol until stages 19, 20 and 21 showed an important and selective increase in their Na content. When the embryos were incubated discontinuously in 0.002 and 0.21 microM ethanol, from stage 3 to stages 12-13 and then transferred to Holtfreter solution, only a few of the above mentioned anomalies were observed; they were transient and the embryos recovered rapidly. Treated embryos did not exhibit ultrastructural changes in their ectodermal cells.     

Detailed lipid analysis of yolk platelets of amphibian (Bufo arenarum) oocytes

Yolk platelets, the principal components of amphibian oocytes, have been generally considered as material reservoirs. Their biochemical composition and function during oogenesis and early development have not been fully elucidated. The aim of this study was to carry out a lipidic characterization of yolk platelets from full-grown Bufo arenarum oocytes. Ovarian oocytes were manually obtained and the subcellular fraction was isolated by centrifugation at low velocity. Lipids were separated by thin-layer chromatography. For compositional analysis, they were derived by methanolysis, being identified and quantified in a gas-liquid chromatograph. Phospholipid content indicates that phosphatidylcholine and phosphatidylethanolamine are the main phospholipids followed by phosphatidylinositol, sphingomyelin, phosphatidylserine, and phosphatidic acid. Phospholipidic profile is similar to that in whole oocytes except for the absence of diphosphatidylglycerol in yolk platelets. Oleic, palmitic, and linoleic acids are the main fatty acids in phosphatidylcholine, and oleic acid is the principal one in phosphatidylethanolamine. In phosphatidic acid, palmitic, estearic, palmitoleic, and oleic acids represent 68 mol% of the total acyl groups. Phosphatidylinositol, enriched in arachidonic acid, is the most unsaturated phospholipid while sphingomyelin shows the lowest unsaturation index. The acyl group distribution in triacylglycerols is similar when yolk platelets and whole oocytes are compared. Polar and neutral lipids of yolk platelets determine the lipidic profile of the whole oocyte. The presence of unusual fatty acids as 14:0, 15:0, 15:1, 17:0, and 17:1 in phospholipids and triacylglycerols may indicate an oxidation mechanism different from beta-oxidation in yolk platelets and/or a structural and functional relation with mitochondria. Given that yolk platelets in amphibian oocytes may act in a dynamic fashion in development, their role should be reconsidered.     

Incorporation and intraparticulate hydrolysis of 131 I-albumin by liver and kidney of an amphibian (Bufo arenarum)

After a single injection of formaldehyde-treated 131 I-albumin into the heart, the incorporation of the labelled protein by liver (% of total injected radioactivity/% of body weight of the organ) was far greater than in other organs. In kidney and spleen it was respectively six and three times greater than in lungs, intestine, testis and fatty body. No radioactivity was found in brain. The radioactivity in liver and kidney reached a peak 30 minutes after the injection, and quickly decreased during the following four hours. In the 27,000 g × ten minute particles recovered from liver homogenates of animals sacrificed at various times after injection, the rate of 131 I-albumin hydrolysis in vitro and the percentage of trichloroacetic acid soluble radioactivity at zero time of incubation showed different stages of intraparticulate hydrolytic activity. The incorporation and intraparticulate hydrolysis in toad kidney was very low if compared with that of toad liver or mouse kidney; however the catheptic specific activity in toad kidney doubles that of mouse kidney. Isolated toad liver was perfused with total blood, containing 131 I-albumin, for five hours at 22°C in a special chamber. In this conditions, 16% of the labelled albumin was hydrolyzed by the liver.     

An Intra-Axonal Protozoon in the Spinal Cord of the Toad Bufo arenarum (Hensel)

SYNOPSIS. A protozoon was found in myelinated axons of the spinal cord and brain of the toad, Bufo arenarum. Examination with the light microscope using Giemsa, Feulgen, PAS and methylene blue technics revealed a primary cell as large as 30 μ in diameter and containing up to 80 nuclei. Electron micrographs showed that the protozoon ranged from 2 μ to 30 μ in diameter and that larger specimens contained numerous secondary cells (2 μ) in addition to multiple nuclei. A few specimens were found in which the secondary cells had long processes with microtubules. Multiple nuclei together with secondary cells suggest that it may be a schizont form of a sporozoon.
The protozoon was found most frequently in axons of the perimedullary plexus just beneath the pia. These axons are without degenerative changes, are up to 3 times the diameter of the largest normal myelinated fibers. The myelin appears normal altho there are fewer laminae than in myelin of other large nerve fibers. The protozoon apparently causes axonal swelling but does not block the fibers completely.
Light microscopic attempts to locate similar forms or other stages in the life cycle by examining blood, skin lesions, spleen, liver, small intestine, dorsal and ventral roots, or sensory ganglia were unsuccessful.
Examination of spinal cords which had been mechanically severed excluded the possibility of confusing the protozoa with multinucleated macrophages. Altho observations do not prove their mode of entrance to the nervous system, the preponderance of protozoa in the peripherally located perimedullary plexus suggests that the path may be by way of the cerebrospinal fluid or along the endoneurium.     

Comparative study of vitellogenesis in the anuran amphibians Ceratophrys cranwelli (Leptodactilidae) and Bufo arenarum (Bufonidae)

The present study analyses, by transmission electron microscopy, vitellogenesis in two anuran amphibian families: Leptodactilidae (Ceratophrys cranwelli) and Bufonidae (Bufo arenarum). These differ in the type of stimulus that sets off their reproductive period, pluvial changes being the trigger in C. cranwelli and temperature increase in B. arenarum. We found that vitellogenesis follows an endocytic pathway that involves membranous structures (coated pits, coated vesicles, endosomes and multivesicular bodies). This process results in a fully grown yolk platelet of similar structure in both species. Despite the above similarity, a distinctive feature in B. arenarum was that the multivesicular bodies exhibited condensed proteins together with lipid droplets, the latter remaining as such even in the primordial yolk platelet. In C. cranwelli, however, lipids droplets were only found attached to the primordial yolk platelet. The coexistence of lipid droplets together with proteins in the nascent precursor yolk platelets observed in B. arenarum is similar to that found in B. marinus. This fact might constitute a characteristic feature of the Bufonidae family.     

Inhibition of the synthesis of glycosphingolipid by a ceramide analogue (PPMP) in the gastrulation of Bufo arenarum

In the present study the role of glycosphingolipids (GSL) in amphibian development was investigated. We analysed the de novo synthesis of neutral GSL and gangliosides through the initial stages of Bufo arenarum embryo development and their participation during gastrulation using 1-phenyl-2-palmitoyl-3-morpholino-1-propanol (PPMP), a potent inhibitor of glucosylceramide synthase. Ganglioside synthesis began at the blastula stage and reached a maximum during gastrulation (stages 10-12) while neutral GSL synthesis showed a slight gradual increase, the former being quantitatively more significant than the latter. Ganglioside synthesis was reduced by 90% while neutral GSL synthesis was inhibited by 65% when embryos at blastula stage were cultured for 24 h in 20 microM PPMP. The depletion of GSL from amphibian embryos induced an abnormal gastrulation in a dose-dependent manner. We found that PPMP had a pronounced effect on development since no embryos exhibited normal gastrulation; their developmental rate either slowed down or, more often, became totally arrested. Morphological analysis of arrested embryos revealed inhibition of the gastrulation morphogenetic movements. Analysis of mesodermal cell morphology in those embryos showed a severe decrease in the number and complexity of cellular extensions such as filopodia and lamellipodia. Mesodermal cells isolated from PPMP-treated embryos had very low adhesion percentages. Our results suggest that glycosphingolipids participate in Bufo arenarum gastrulation, probably through their involvement in cell adhesion events.     

Inhibition of Bufo arenarum oocyte maturation induced by cholesterol depletion by methyl-beta-cyclodextrin. Role of low-density caveolae-like membranes

The invaginated structure of caveolae seems to provide an optimal environment for hormone binding leading to oocyte meiotic maturation. We conducted a quantitative analysis of lipids and proteins of detergent-free low-density membranes isolated from Bufo arenarum oocytes and we modulated cellular cholesterol to further understand how these domains perform their regulatory functions in the amphibian system. Light membranes derive from the plasma membrane as suggested by the enrichment in the activity of 5'nucleotidase. Lipid analysis by chromatography techniques revealed that this fraction is enriched in phosphatidylserine and cholesterol and that it evidences an important level of sphingomyelin. The finding of a single 21 kDa caveolin in light membranes indicates the presence of caveolae-like structures in B. arenarum oocytes. In support of this finding, c-Src is significantly associated to this fraction. Cholesterol content of oocytes treated with methyl-beta-cyclodextrin (MbetaCD) decreased when compared to control oocytes. Drug treatment inhibited meiotic maturation in a dose-dependent manner and affected the localization of caveolin and c-Src among membrane fractions. Repletion of cholesterol showed a recovery of the ability of MbetaCD-treated oocytes to mature, particularly at the 25 mM concentration in which reversibility was close to the control level. Results highlight the importance of caveolae-like microdomains for maturation signaling in Bufo oocytes.