Effect of steroid hormones on Bufo arenarum oviduct. Ultrastructural study.

The endocrine regulation of the mucosa of the oviductal pars convoluta was analyzed by ultrastructural studies demonstrating that ovariectomy, together with a decrease in ovarian steroids circulating levels, caused a marked regression in this portion of Bufo arenarum oviduct. Twenty-five days after ovariectomy, a decrease in the depth of the epithelial and glandular layers was observed due to the notable loss of secretory cells, whose number was clearly smaller than in nonovariectomized females. The remaining secretory cells showed involution signs, with few secretory granules in their cytoplasm, little endoplasmic reticulum near poorly developed Golgi complexes and a large amount of lipid droplets. Cells in an advanced autolysis state were found in the lumen. These characteristics evidence a nonfunctional state of the pars convoluta. Treatment with 5alpha-dihydrotestosterone (DHT) completely reversed the ovariectomy effect, inducing pars convoluta growths and restoring the characteristics of epithelial and glandular secretory cells in the whole pars convoluta, with micrographs similar to the control. These same effects were observed after treatment with estradiol-17beta (E2), progesterone (P) o E(2)+P in the glandular layer of the whole pars convoluta, but only in the epithelial layer of the most anterior region of this duct. In the secretory cells of other segments these treatments induced the formation of granules of high electron density and homogeneous aspect. Each steroid had a particular effect on the pars convoluta. Although E2 and DHT induced the development of the organoids involved in the proteins biosynthesis, P and DHT acted as secretagogues.

Spontaneous and LH-induced maturation in Bufo arenarum oocytes: importance of gap junctions.

It has been demonstrated in Bufo arenarum that fully grown oocytes are capable of meiotic resumption in the absence of a hormonal stimulus if they are deprived of their follicular envelopes. This event, called spontaneous maturation, only takes place in oocytes collected during the reproductive period, which have a metabolically mature cytoplasm. In Bufo arenarum, progesterone acts on the oocyte surface and causes modifications in the activities of important enzymes, such as a decrease in the activity of adenylate cyclase (AC) and the activation of phospholipase C (PLC). PLC activation leads to the formation of diacylglycerol (DAG) and inositol triphosphate (IP(3)), second messengers that activate protein kinase C (PKC) and cause an increase in intracellular Ca(2+). Recent data obtained from Bufo arenarum show that progesterone-induced maturation causes significant modifications in the level and composition of neutral lipids and phospholipids of whole fully grown ovarian oocytes and of enriched fractions in the plasma membrane. In amphibians, the luteinizing hormone (LH) is responsible for meiosis resumption through the induction of progesterone production by follicular cells. The aim of this work was to study the importance of gap junctions in the spontaneous and LH-induced maturation in Bufo arenarum oocytes. During the reproductive period, Bufo arenarum oocytes are capable of undergoing spontaneous maturation in a similar way to mammalian oocytes while, during the non-reproductive period, they exhibit the behaviour that is characteristic of amphibian oocytes, requiring progesterone stimulation for meiotic resumption (incapable oocytes). This different ability to mature spontaneously is coincident with differences in the amount and composition of the phospholipids in the oocyte membranes. Capable oocytes exhibit in their membranes higher quantities of phospholipids than incapable oocytes, especially of PC and PI, which are precursors of second messengers such as DAG and IP(3). The uncoupling of the gap junctions with 1-octanol or halothane fails to induce maturation in follicles from the non-reproductive period, whose oocytes are incapable of maturing spontaneously. However, if the treatment is performed during the reproductive period, with oocytes capable of undergoing spontaneous maturation, meiosis resumption occurs in high percentages, similar to those obtained by manual defolliculation. Interestingly, results show that LH is capable of inducing GVBD in both incapable oocytes and in oocytes capable of maturing spontaneously as long as follicle cells are present, which would imply the need for a communication pathway between the oocyte and the follicle cells. This possibility was analysed by combining LH treatment with uncoupling agents such as 1-octanol or halothane. Results show that maturation induction with LH requires a cell-cell coupling, as the uncoupling of the gap junctions decreases GVBD percentages. Experiments with LH in the presence of heparin, BAPTA/AM and theophylline suggest that the hormone could induce GVBD by means of the passage of IP(3) or Ca(2+) through the gap junctions, which would increase the Ca(2+) level in the oocyte cytoplasm and activate phosphodiesterase (PDE), thus contributing to the decrease in cAMP levels and allowing meiosis resumption.

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 mug 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.

Mitochondrial lipids in Bufo arenarum full-grown oocytes.

Both the content and composition of polar and neutral lipids from the mitochondrial fraction of ovarian full-grown Bufo arenarum oocytes were analysed in the present study. Triacylglycerols (TAG) represent 33% of the total lipids, followed by phosphatidylcholine (PC), free fatty acids (FFA) and phosphatidylethanolamine (PE). Diphosphatidylglycerol (DPG) or cardiolipin, a specific component of the inner mitochondrial membrane, represents about 4% of the total lipid content. Palmitic (16:0) and arachidonic (20:4n6) acids are the most abundant fatty acids in PC and PE, respectively. DPG is enriched in fatty acids with carbon chain lengths of 18, the principal component being linoleic acid. In phosphatidylinositol (PI), 20:4n6 and stearic acid (18:0) represent about 72 mol% of the total acyl group level. The main fatty acids in TAG are linoleic (18:2), oleic (18:1), and palmitic acids. The fatty acid composition of FFA and diacylglycerols (DAG) is similar, 16:0 being the most abundant acyl group. PE is the most unsaturated lipid and sphingomyelin (SM) has the lowest unsaturation index.

Structure of the kidney of Bufo arenarum: intermediate segment, distal tubule and collecting tubule.

The ultrastructure of the intermediate segment (IS), distal tubule and collecting tubule (CT) of the south american toad Bufo arenarum, was studied by light and transmission electron microscopy. The IS is composed of cubical ciliated cells which propel the urine along the renal tubule. The distal tubule is divided into two portions: the early distal tubule (EDT) and the late distal tubule (LDT). The EDT is characterized by only one type of cells with well developed basolateral interdigitations and numerous elongated mitochondria, which are oriented normal to the basal surface. The "macula densa--like" is a specialized zone of the EDT in contact with the vascular pole, where cells are more tightly packed than in the rest of the tubule. The LDT shows two types of cells called dark and light cells according to the appearance of their cytoplasm. Dark cells have microplicae and few but long microvilli at their luminal surface, and abundant mitochondria in their cytoplasm. Light cells show basal and lateral infoldings and few mitochondria. The CT, which is composed of dark and light cells, exhibits an enlarged lumen with an undulated surface and dilated spaces between neighbouring cells. This work is a contribution to the knowledge of the kidney of B. arenarum; frequently used as an experimental model for physiological and biochemical studies.

Antibacterial activity of lactose-binding lectins from Bufo arenarum skin.

Amphibians respond to microbial infection through cellular and humoral defense mechanisms such as antimicrobial protein secretion. Most humoral defense proteins are synthetized in the skin. In this study we isolated two beta-galactoside-binding lectins with molecular weights of 50 and 56 KDa from the skin of Bufo arenarum. These lectins have significant hemagglutination activity against trypsinized rabbit erythrocytes, which was inhibited by galactose-containing saccharides. They are water-soluble and independent of the presence of calcium. The antimicrobial analysis for each lectin was performed. At mumolar concentration lectins show strong bacteriostatic activity against Gram negative bacteria (Escherichia coli K12 4100 and wild strains of Escherichia coli and Proteus morganii) and Gram positive bacteria (Enterococcus faecalis). The antibacterial activity of these lectins may provide an effective defense against invading microbes in the amphibian Bufo arenarum.

Antibacterial activity of lactose-binding lectins from Bufo arenarum skin

Amphibians respond to microbial infection through cellular and humoral defense mechanisms such as antimicrobial protein secretion. Most humoral defense proteins are synthetized in the skin. In this study we isolated two beta-galactoside-binding lectins with molecular weights of 50 and 56 KDa from the skin of Bufo arenarum. These lectins have significant hemagglutination activity against trypsinized rabbit erythrocytes, which was inhibited by galactose-containing saccharides. They are water-soluble and independent of the presence of calcium. The antimicrobial analysis for each lectin was performed. At mumolar concentration lectins show strong bacteriostatic activity against Gram negative bacteria (Escherichia coli K12 4100 and wild strains of Escherichia coli and Proteus morganii) and Gram positive bacteria (Enterococcus faecalis). The antibacterial activity of these lectins may provide an effective defense against invading microbes in the amphibian Bufo arenarum.

Structure of the kidney of Bufo arenarum: intermediate segment, distal tubule and collecting tubule

The ultrastructure of the intermediate segment (IS), distal tubule and collecting tubule (CT) of the south american toad Bufo arenarum, was studied by light and transmission electron microscopy. The IS is composed of cubical ciliated cells which propel the urine along the renal tubule. The distal tubule is divided into two portions: the early distal tubule (EDT) and the late distal tubule (LDT). The EDT is characterized by only one type of cells with well developed basolateral interdigitations and numerous elongated mitochondria, which are oriented normal to the basal surface. The "macula densa--like" is a specialized zone of the EDT in contact with the vascular pole, where cells are more tightly packed than in the rest of the tubule. The LDT shows two types of cells called dark and light cells according to the appearance of their cytoplasm. Dark cells have microplicae and few but long microvilli at their luminal surface, and abundant mitochondria in their cytoplasm. Light cells show basal and lateral infoldings and few mitochondria. The CT, which is composed of dark and light cells, exhibits an enlarged lumen with an undulated surface and dilated spaces between neighbouring cells. This work is a contribution to the knowledge of the kidney of B. arenarum; frequently used as an experimental model for physiological and biochemical studies.

Antibacterial activity of lactose-binding lectins from Bufo arenarum skin

Amphibians respond to microbial infection through cellular and humoral defense mechanisms such as antimicrobial protein secretion. Most humoral defense proteins are synthetized in the skin. In this study we isolated two beta-galactoside-binding lectins with molecular weights of 50 and 56 KDa from the skin of Bufo arenarum. These lectins have significant hemagglutination activity against trypsinized rabbit erythrocytes, which was inhibited by galactose-containing saccharides. They are water-soluble and independent of the presence of calcium. The antimicrobial analysis for each lectin was performed. At mumolar concentration lectins show strong bacteriostatic activity against Gram negative bacteria (Escherichia coli K12 4100 and wild strains of Escherichia coli and Proteus morganii) and Gram positive bacteria (Enterococcus faecalis). The antibacterial activity of these lectins may provide an effective defense against invading microbes in the amphibian Bufo arenarum. (AU)

Structure of the kidney of Bufo arenarum: intermediate segment, distal tubule and collecting tubule

The ultrastructure of the intermediate segment (IS), distal tubule and collecting tubule (CT) of the south american toad Bufo arenarum, was studied by light and transmission electron microscopy. The IS is composed of cubical ciliated cells which propel the urine along the renal tubule. The distal tubule is divided into two portions: the early distal tubule (EDT) and the late distal tubule (LDT). The EDT is characterized by only one type of cells with well developed basolateral interdigitations and numerous elongated mitochondria, which are oriented normal to the basal surface. The "macula densa--like" is a specialized zone of the EDT in contact with the vascular pole, where cells are more tightly packed than in the rest of the tubule. The LDT shows two types of cells called dark and light cells according to the appearance of their cytoplasm. Dark cells have microplicae and few but long microvilli at their luminal surface, and abundant mitochondria in their cytoplasm. Light cells show basal and lateral infoldings and few mitochondria. The CT, which is composed of dark and light cells, exhibits an enlarged lumen with an undulated surface and dilated spaces between neighbouring cells. This work is a contribution to the knowledge of the kidney of B. arenarum; frequently used as an experimental model for physiological and biochemical studies. (AU)

Vitelline envelope of Bufo arenarum: biochemical and biological characterization.

Vitelline envelopes (VEs) of Bufo arenarum were isolated in order to study their composition and their role in fertilization. VEs are composed of four glycoproteins, with molecular masses of 120, 75, 41, and 38 kDa. To characterize its biological properties, we quantitatively determined sperm-VE binding and the induction of the acrosome reaction. Heterologous binding of B. arenarum sperm to Xenopus laevis VE components was observed with about one-third the efficiency of homologous binding. Equivalent binding of X. laevis sperm to the B. arenarum VE was observed. When B. arenarum sperm were incubated with fluorescein isothiocyanate-labeled VE, the labeled glycoproteins bound to the anterior end of the sperm head, showing a lateral distribution. Induction of the acrosome reaction was evaluated by incubating sperm in hypotonic saline media with VE glycoproteins. VEs induced the acrosome reaction in a time- and concentration-dependent manner. The acrosome reaction was maximal after 10 min. The half-maximal effect was obtained at a glycoprotein concentration of 1 microg/ml. Specificity was determined using fertilization envelope glycoproteins, which failed to induce the acrosome reaction. The B. arenarum VE is biochemically similar to other egg envelopes. It also seems that its biological properties are similar to other species in regard to sperm binding and induction of the acrosome reaction. However, as far as we are aware, this is the first observation of the VE inducing the sperm acrosome reaction in amphibians. The relatively small differences observed in heterologous sperm-VE binding in X. laevis and B. arenarum are inconsistent with the current paradigm that species specificity in fertilization is regulated at the sperm-VE binding step.

Immunohistochemical localisation of a galectin from Bufo arenarum ovary.

Galectins are a group of soluble animal lectins that exhibit specificity for beta-galactosides and conserve sequence homology in the carbohydrate-recognition domain. The galectin from Bufo arenarum ovary showed a strong cross-reaction with the lectin of 14.5 kDa purified from embryos at early blastula stage. In this paper, we studied the immunohistochemical localisation of the galectin of 14.5 kDa from ovary of the toad B. arenarum in adult ovary sections. We also analysed the immunohistochemical localisation of the embryonic lectin during early development using the antiserum anti-ovary galectin. In the ovary, oocytes in the previtellogenic stage showed strong reactivity in the nucleus and the cortex but not in the cytoplasm. Oocytes in the stage of primary vitellogenesis exhibited a similar pattern in the nuclear and cortical areas but showed immunostaining in the cytoplasm. Intense nuclear staining was detected in oocytes in the stage of late vitellogenesis and in mature oocytes, which also presented strong reactions in the yolk platelets that completely covered the cytoplasm. In blastula embryos the staining was found in the blastomeres, the yolk platelets and the blastocoele. Each lectin localisation is discussed in relation to potential biological roles in the corresponding tissues.

The lung of the common toad, Bufo arenarum (Anura: Bufonidae). A light and electron microscopy study

The present study describes the lung morphology of the common toad, Bufo arenarum, as observed by light and electron microscopy. The lung wall consists of four layers: mesothelium, dense connective tissue with thin elastic fibers, loose connective tissue containing smooth muscle fibers and an internal respiratory epithelium. The lung presents three types of folds defined by their epithelia. First order folds are coated by ciliated epithelium containing numerous goblet cells. Second order folds present the same type of epithelium but devoid of goblet cells, while third order folds are only lined by respiratory epithelium. The respiratory surface of the lung is lined by a single cell type, the pneumocyte, which presents characteristics of both type I and type II alveolar cells of higher vertebrates. The pneumocytes are prismatic in shape and possess attenuated cytoplasmatic processes which spread over the pulmonary capillaries to form the outer layer of the air-blood barrier. These cells present microvilli in the apical extreme and contain different types of cytoplasmic bodies: electron dense, multivesicular and lamellar

The lung of the common toad, Bufo arenarum (Anura: Bufonidae). A light and electron microscopy study.

The present study describes the lung morphology of the common toad, Bufo arenarum, as observed by light and electron microscopy. The lung wall consists of four layers: mesothelium, dense connective tissue with thin elastic fibers, loose connective tissue containing smooth muscle fibers and an internal respiratory epithelium. The lung presents three types of folds defined by their epithelia. First order folds are coated by ciliated epithelium containing numerous goblet cells. Second order folds present the same type of epithelium but devoid of goblet cells, while third order folds are only lined by respiratory epithelium. The respiratory surface of the lung is lined by a single cell type, the pneumocyte, which presents characteristics of both type I and type II alveolar cells of higher vertebrates. The pneumocytes are prismatic in shape and possess attenuated cytoplasmatic processes which spread over the pulmonary capillaries to form the outer layer of the air-blood barrier. These cells present microvilli in the apical extreme and contain different types of cytoplasmic bodies: electron dense, multivesicular and lamellar.

Topographic differences in cell populations and insulin secretion in the endocrine pancreas of the toad Bufo arenarum.

We analyzed the endocrine cell topography within the amphibian pancreas and the relationship of this distribution to lobular variation in insulin content and secretion. Pancreases from adult male toad Bufo arenarum were separated into their five lobes: free, gastric, hepatic, duodenal, and jejunal. Pieces of each lobe were incubated with glucose, arginine, and K+ and the insulin concentration in the medium was measured by radioimmunoassay. In the presence of 2 or 8 mM glucose, 10 mM arginine, and 10 mM K+ the free lobe released a significantly greater amount of insulin than the other lobes, while the output of the gastric lobe was greater than that of the duodenal, hepatic, and jejunal. At 8 mM glucose, every pancreatic lobe released a significantly higher amount of insulin than at 2 mM. The insulin content of the free lobe was significantly higher than that of the others, whereas this parameter was comparable among the latter. These pancreases contained islets of variable size and irregular shape. B and non-B cells, detected by immunoperoxidase staining, were located at the central and peripheral zones of the islets, respectively. A large number of non-B cells were also scattered over the exocrine component. Morphometrical analyses revealed the following sequence of endocrine cell percentage: free lobe > gastric lobe = duodenal lobe > jejunal lobe = hepatic lobe. Some 48% of the endocrine cells were present in the islets, while the remaining 52% were found throughout the exocrine pancreas. In the free lobe, more endocrine cells were located within the islets (65%) than outside and in the gastric lobe the proportion was almost equal (48% within, 52% outside), but in the hepatic, duodenal, and jejunal lobes the majority lay outside the islets (61, 63, and 70% extrainsular, respectively). The area covered by B and D cells was far larger within the islets than outside, with the relative magnitude of this difference being free lobe > gastric lobe > duodenal lobe > hepatic lobe = jejunal lobe. In the free lobe, this relative distribution was more skewed than in the remaining lobes. PP and A cells occupied a more extensive area outside the islets than inside in every lobe. There were no significant differences among the extrainsular areas occupied by each type of endocrine cell within a given pancreatic lobe. These results constitute the first demonstration of the heterogeneity in morphology, insulin content, and secretory function among the pancreatic lobes in B. arenarum. The data further suggest that the nonuniform secretory capacities of amphibian pancreatic lobes reflect localized differences in their insulin content, which heterogeneity in turn stems from the dissimilar distribution and organization of their constituent endocrine-cell populations.