Multiple anti-predator mechanisms in the red-spotted Argentina Frog (Amphibia: Hylidae)

Anurans employ a wide variety of anti-predator mechanisms to defend themselves. In casque-headed hylids, defence is thought to be a complex combination of several anti-predator mechanisms. However, the defence traits of only a few species are known; some hypotheses have yet to be addressed, whereas others, already tested in some species, need to be tested in additional taxa. The anti-predator mechanism of the casque-headed frog, Argenteohyla siemersi, is described here. It is a complex mechanism consisting of (1) behavioural and ecological traits, including secretive and semi-phragmotic habits and posture; (2) morphological features, including cryptic and aposematic colourations, a skull covered with bony dermal spines and protuberances that are associated with two types of granular venom glands; and (3) physiological and chemical traits, such as a highly lethal skin secretion. Our results are compared with those of previous studies of defence mechanisms in casque-headed frogs in an effort to understand the mechanisms and evaluate their potential phylogenetic signal in this group of anurans.Fil: Cajade, Rodrigo. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional del Nordeste. Facultad de Ciencias Exactas, Naturales y Agrimensura. Departamento de Biología; ArgentinaFil: Hermida, Gladys Noemí. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Biodiversidad y Biología Experimental; ArgentinaFil: Piñeiro, Jose Miguel. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional del Nordeste. Facultad de Ciencias Exactas, Naturales y Agrimensura. Departamento de Biología; ArgentinaFil: Regueira, Eleonora. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Biodiversidad y Biología Experimental; ArgentinaFil: Alcalde, Leandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Limnología "Dr. Raúl A. Ringuelet". Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Instituto de Limnología; ArgentinaFil: Fusco, Luciano Sebastian. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional del Nordeste. Facultad de Ciencias Exactas Naturales y Agrimensura. Departamento de Bioquímica; ArgentinaFil: Marangoni, Federico. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional del Nordeste. Facultad de Ciencias Exactas, Naturales y Agrimensura. Departamento de Biología; Argentin

Aesculus hippocastanum L. seed extract shows virucidal and antiviral activities against respiratory syncytial virus (RSV) and reduces lung inflammation in vivo

Respiratory syncytial virus (RSV) is a leading cause of lower respiratory tract disease and bronchiolitis in children worldwide. No vaccine or specific, effective treatment is currently available. β-escin is one of the main bioactive constituents of Aesculus hippocastanum L. (Hippocastanaceae) seed extract (AH), and both β-escin and AH have demonstrated a beneficial role in clinical therapy because of their anti-edematous, anti-inflammatory and antioxidative effects. Besides, we have reported that β-escin and AH show virucidal, antiviral and immunomodulatory activities against the enveloped viruses HSV-1, VSV and Dengue virus in vitro. In this study, we demonstrate that β-escin and AH have virucidal and antiviral activities against RSV, as well as NF-κB, AP-1 and cytokine modulating activities in RSV infected epithelial and macrophage cell lines in vitro. Besides, in a murine model of pulmonary RSV infection, AH treatment improves the course of acute disease, evidenced by decreased weight loss, reduced RSV lung titers, and attenuated airway inflammation. In contrast, even though β-escin showed, similarly to AH, antiviral and immunomodulatory properties in vitro, it neither reduces viral titers nor attenuates lung injury in vivo. Thus, our data demonstrate that AH restrains RSV disease through antiviral and immunomodulatory effect.Fil: Salinas, Franco Maximiliano. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Vázquez, Luciana. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud "Dr. C. G. Malbrán"; ArgentinaFil: Gentilini, Maria Virginia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Medicina Traslacional, Trasplante y Bioingeniería. Fundación Favaloro. Instituto de Medicina Traslacional, Trasplante y Bioingeniería; ArgentinaFil: O´Donohoe, Ailin. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Biodiversidad y Biología Experimental. Laboratorio de Histología Animal; ArgentinaFil: Regueira, Eleonora. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Biodiversidad y Biología Experimental. Laboratorio de Histología Animal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Nabaes Jodar, Mercedes Soledad. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Viegas, Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez"; ArgentinaFil: Michelini, Flavia Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Hermida, Gladys Noemí. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Biodiversidad y Biología Experimental. Laboratorio de Histología Animal; ArgentinaFil: Alche, Laura Edith. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Bueno, Carlos Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentin

Regulation of seasonal variations in testicular sensitivity to glucocorticoids action in Rhinella arenarum (Amphibia, Anura). Interrelation between testis and interrenal

Los anfibios anuros tienen variaciones estacionales en los andrógenos y glucocorticoides (GCs) plasmáticos. En Rhinella arenarum la concentración plasmática de GCs aumenta durante el período reproductivo provocando una disminución en la síntesis de andrógenos vía un receptor citosólico (GR). El objetivo de esta tesis fue evaluar las variaciones estacionales en la sensibilidad testicular a la acción de los GCs. Para ello se estudiaron los cambios en la cantidad de GR así como el efecto de los andrógenos, los GCs y la melatonina en la regulación de dichos cambios. Los estudios de la interrelación entre la glándula interrenal y el testículo indicaron que la cantidad de proteína GR testicular varía estacionalmente y que los GCs regulan de manera positiva su propio receptor mientras que melatonina disminuye la unión del GC al GR y antagoniza el efecto de los GCs. En cambio, melatonina no disminuye la síntesis de andrógenos de manera directa. En la glándula interrenal, una concentración de andrógenos alta (período prerreproductivo) disminuye la síntesis de GCs. El estudio histológico de la glándula mostró que hay dos tipos de células esteroidogénicas que podrían producir distinta cantidad de GCs y que ambas presentan una regionalización antero-posterior. Se infiere que las variaciones estacionales de andrógenos y GCs están determinadas, en parte, por la regulación recíproca entre ambas hormonas y que la sensibilidad del testículo a la acción de los GCs, cuando se considera la cantidad de GR, varía de manera estacional y está regulada por los mismos GCs y la melatonina.Anuran amphibians are characterized by having seasonal changes in plasma androgens and glucocorticoids (GCs). In Rhinella arenarum, plasma GCs concentration is highest during the reproductive season and GCs produce a decrease in androgen synthesis via a testicular cytosolic glucocorticoid receptor (GR). The aim of this thesis was to evaluate seasonal variations in testicular sensitivity to GCs’ action by studying changes in the amount of testicular GR as well as the effect of androgens, GCs, and melatonin on its regulation. In addition, it was analyzed the interrelation between the interrenal gland and the testis. Results indicated that the amount of GR protein varies seasonally and GCs positively regulate its own receptor whereas melatonin decreases the binding of GC to the GR antagonizing GCs’ effect. However, melatonin has no a direct effect on androgen synthesis. In the interrenal gland, high concentrations of androgens - corresponding to the pre-reproductive period - decrease the synthesis of GCs. Histological examination of the interrenal showed two anteroposterior regionalized steroidogenic cell types that could produce different amounts of GCs. Taking together, these results suggest that the seasonal variations of androgens and GCs are determined, at least in part, by the interregulation between these hormones. In addition, the GR-dependent sensitivity of the testis to the action of GCs varies seasonally and is regulated by GCs and melatonin.Fil:Regueira, Eleonora. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina

Morphological Changes in Skin Glands During Development in Rhinella Arenarum (Anura: Bufonidae)

Avoiding predation is critical to survival of animals; chemical defenses represent a common strategy among amphibians. In this study, we examined histologically the morphology of skin glands and types of secretions related to chemical skin defense during ontogeny of Rhinella arenarum. Prior to metamorphic climax the epidermis contains typical bufonid giant cells producing a mucous substance supposedly involved in triggering a flight reaction of the tadpole school. An apical layer of alcianophilic mucus covers the epidermis, which could produce the unpleasant taste of bufonid tadpoles. Giant cells disappear by onset of metamorphic climax, when multicellular glands start developing, but the apical mucous layer remains. By the end of climax, neither the granular glands of the dorsum nor the parotoid regions are completely developed. Conversely, by the end of metamorphosis the mucous glands are partially developed and secrete mucus. Adults have at least three types of granular glands, which we designate type A (acidophilic), type B (basophilic) and ventral (mucous). Polymorphic granular glands distribute differently in the body: dorsal granular glands between warts and in the periphery of parotoids contain protein; granular glands of big warts and in the central region of parotoids contain catecholamines, lipids, and glycoconjugates, whereas ventral granular glands produce acidic glycoconjugates. Mucous glands produce both mucus and proteins. Results suggest that in early juveniles the chemical skin defense mechanisms are not functional. Topographical differences in adult skin secretions suggest that granular glands from the big warts in the skin produce similar toxins to the parotoid glands.Fil: Regueira, Eleonora. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Biodiversidad y Biología Experimental. Laboratorio de Biología de Anfibios; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Dávila, Camila. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Biodiversidad y Biología Experimental. Laboratorio de Biología de Anfibios; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Hermida, Gladys Noemí. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Biodiversidad y Biología Experimental. Laboratorio de Biología de Anfibios; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Characterization and seasonal changes in LHβ and FSHβ mRNA of Rhinella arenarum (Amphibia, Anura)

In anurans, two types of gonadotropins were described in several species of Ranidae and Pipidae families but only in one of the Bufonidae family. Rhinella arenarum is a bufonid that have the lowest concentration of plasma androgens during the breeding. The objective of this paper was to characterize the cDNA sequence of β subunit of LH and FSH from toad pituitary and study seasonal variation in gonadotropins mRNA using quantitative real-time RT-PCR. The LHβ cDNA is a 636 bp sequence containing an open reading frame (ORF), 45 bp of 5′-untranslated region (UTR) and 174 bp of 3′-UTR. The ORF encodes for a signal peptide of 26 amino acids and a mature protein of 113 amino acids with one N-glycosylation site at the 34th position. The FSHβ cDNA sequence is a 535 bp fragment containing an ORF, 8 bp of 5′-UTR and 152 bp of 3′-UTR. The ORF encodes for a signal peptide of 20 amino acids and a mature protein of 104 amino acids with two N-glycosylation sites at 25th and 42nd positions. Multiple alignments of aminoacid deduced sequences of LHβ and FSHβ (teleosts, amphibians, birds, mammals) showed that all the tetrapods studied conserve 12 cysteins and one (LH) or two (FSH) N-Glycosylation sites. LHβ is closer to teleosts than to mammals and birds while FSHβ is closer to mammals. The analysis of seasonal changes in LHβ and FSHβ mRNA indicates that transcript levels have seasonal variations and that the profile of androgens is opposite to that of the gonadotropins mRNA.Fil: Volonteri, María Clara. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Biodiversidad y Biología Experimental. Laboratorio de Endocrinología Comparada; ArgentinaFil: Regueira, Eleonora. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Biodiversidad y Biología Experimental. Laboratorio de Endocrinología Comparada; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Scaia, María Florencia. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Biodiversidad y Biología Experimental. Laboratorio de Endocrinología Comparada; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Ceballos, Nora Raquel. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Biodiversidad y Biología Experimental. Laboratorio de Endocrinología Comparada; Argentin

5alpha-reductase, an enzyme regulating glucocorticoid action in the testis of Rhinella arenartum (Amphibia, Anura)

The reduction of A-ring of glucocorticoids to produce 5a-dihydro-derivatives by 5a-reductases has been considered as a pathway of irreversible inactivation. However, 5a-reduced metabolites of corticosterone and testosterone have significant biological activity. In this paper, we investigated whether toad testicular 5a-reductase (5a-Red) is able to transform corticosterone into 5a-dihydrocorticosterone. Furthermore, we studied the role of 5a-reduced metabolite of corticosterone as a glucocorticoid receptor (GR) agonist. The activity of 5a-Red was assayed in subcellular fractions with [3H]corticosterone or [3H]testosterone as substrate. The enzyme localises in microsomes and its optimal pH is between 7 and 8. The activity is not inhibited by finasteride. These results support the conclusion that toad 5a-Red resembles mammalian type 1 isoenzyme. Kinetic studies indicate that neither Km nor Vmax for both corticosterone and testosterone were significantly different among reproductive periods. The Km value for testosterone was significantly higher than that for corticosterone, indicating that the C-21 steroid is the preferred substrate for the enzyme. Studies of the binding capacity of 5a-dihydrocorticosterone (5aDHB) to the testicular GR show that 5aDHB is able to displace the binding of [3H]dexamethasone to testicular cytosol with a similar potency than corticosterone. The inhibition constant (Ki) values for corticosterone and 5aDHB were similar, 31.33 ± 2.9 nM and 35.24 ± 2.3 nM, respectively. In vitro experiments suggest that 5aDHB is an agonist of toad testicular GR, decreasing the activity of the key enzyme for androgen synthesis, the cytochrome P450 17-hydroxylase, C17,20-lyase.Fil: Tesone, Amelia J.. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Biodiversidad y Biología Experimental. Laboratorio de Endocrinología Comparada; ArgentinaFil: Regueira, Eleonora. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Biodiversidad y Biología Experimental. Laboratorio de Endocrinología Comparada; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria; ArgentinaFil: Canosa, Luis Fabian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); ArgentinaFil: Ceballos, Nora Raquel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Biodiversidad y Biología Experimental. Laboratorio de Endocrinología Comparada; Argentin

Estradiol production by the Bidder's organ of the toad Rhinella arenarum (Amphibia, Anura): Seasonal variations in plasma estradiol

In bufonids, the Bidder´s organ (BO), located in the anterior pole of the testis, is sometimes referred to as a rudimentary ovary because of the presence of previtellogenic follicles. In males of Rhinella arenarum it has been demonstrated that some follicles are vitellogenic and also express several steroidogenic enzymes in follicular cells. The purpose of this study is to describe seasonal variations in plasma estradiol (E2) and in aromatase activity of the BO, and to determine the capacity of the BO to synthesize E2 from cholesterol in males of R. arenarum. E2 was determined by radioimmunoassay and aromatase activity was measured by transformation of radioactive substrates into products. Results indicate that plasma E2 reached the highest concentration in April and the lowest one in animals captured in June, showing a progressive increase to the end of the year. Plasma E2 and total activity of aromatase in the BO were significantly lower during the pre‐reproductive season than during the reproductive and post‐reproductive seasons. It was also demonstrated that the BO is able to produce E2 from endogenous substrates throughout the year. No correlation was found between plasma E2 and total BO weight, while there was a significant correlation between plasma E2 and total activity of aromatase, and between plasma E2 and E2 produced in vitro. Taken together, these results demonstrate seasonal variations in plasma E2, in bidderian total activity of aromatase and, that the BO of adult males of R. arenarum is able to produce E2 from endogenous substrates.Fil: Scaia, María Florencia. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Biodiversidad y Biología Experimental. Laboratorio de Endocrinología Comparada; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Regueira, Eleonora. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Biodiversidad y Biología Experimental. Laboratorio de Endocrinología Comparada; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Volonteri, María Clara. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Biodiversidad y Biología Experimental. Laboratorio de Endocrinología Comparada; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Ceballos, Nora Raquel. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Biodiversidad y Biología Experimental. Laboratorio de Endocrinología Comparada; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Anteroposterior variation of the cell types in the interrenal gland of the male toad Rhinella arenarum (Amphibia, Anura)

The interrenal gland of anurans synthesizes the steroids aldosterone and corticosterone, but it is unknown whether these hormones are synthesized by the same cell type. In this work, we aim to elucidate whether there are different steroidogenic cell types and whether they have specific regionalization in the interrenal gland of the male toad Rhinella arenarum. We characterized all cell types using histological, immuhistochemical, and histochemical methods as well as transmission electron microscopy. Furthermore, we evaluated the organization of the cell types in the gland and anteroposterior variations in the synthesis of the steroids. We found evidence of five cell types: two morphologically different steroidogenic cells, type 1: polyhedral cells tightly attached to each other that have spherical euchromatic nuclei and type 2: retracted cells loosely attached to each other that have oval heterochromatic nuclei. Cell type 2 is mainly observed in the inner zone of the gland. In addition, we observed two types of chromaffin cells, called type 3 and 4 cells, randomly distributed throughout the interrenal gland, as well as type 5 cells, recognized as summer cells. Morphometric analyses of the cell types in the anterior and posterior zones of the interrenal showed that the ratio "area of type 2 cells/total interrenal area" is significantly lower in the posterior zone. In vitro incubations showed that the posterior portion of the gland produces significantly higher amounts of both corticosterone and aldosterone. Overall, our results suggest that the type 2 cells are less active to synthesize both aldosterone and corticosterone, compared to type 1 cells. Unlike most previous reports on the interrenal gland of anurans, in R. arenarum there is a zonation of the steroidogenic cell types, which implies that the organ is not anteroposterior or dorsoventrally homogeneous. © 2012 Wiley Periodicals, Inc.Fil: Regueira, Eleonora. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Biodiversidad y Biología Experimental. Laboratorio de Endocrinología Comparada; ArgentinaFil: Scaia, María Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Biodiversidad y Biología Experimental. Laboratorio de Endocrinología Comparada; ArgentinaFil: Volonteri, María Clara. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Biodiversidad y Biología Experimental. Laboratorio de Endocrinología Comparada; ArgentinaFil: Ceballos, Nora Raquel. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Biodiversidad y Biología Experimental. Laboratorio de Endocrinología Comparada; Argentin

Delayed osteogenesis and calcification in a large true toad with a comparative survey of the timing of skeletal ossification in anurans?

Postembryonic skeletogenesis in anuran amphibians has been widely studied, yet less than one percent of the extant diversity has been covered and relatively few comparative studies exist. Here we document the sequence and timing of ossification of the Common Toad Rhinella arenarum, a large true toad (Bufonidae) from South America that is a model organism for varied ongoing research. We study histological sections and cleared-and-stained specimens of an ontogenetic series ranging from early larval stages to juveniles,documenting the ossification sequence of the entire skeleton. To diminish potential environmental biases we also study the skeletogenesis of the frog Leptodactylus latinasus (Leptodactylidae) from the same pond and season. We summarize comparative data from numerous anuran species to contextualize our results in a broad phylogenetic context. Histological data shows that skeletal calcification in R. arenarum is temporally dissociated from osteoid matrix formation and occurs later than in most other anurans, whichis unexpected given its generalized pond-type larva and heavily ossified adult skeleton. At the onset of metamorphosis, exoccipitals, parasphenoid, and frontoparietals are the only ossified skull elements, whereas most of the postcranium has already started ossification. This pattern is rare among anurans but is shared by other bufonids, in which it has been previously linked to rapid development. Our comparative survey, however, suggests that the delayed bufonid pattern is not related to fast-developing larvae but instead might be a distinctive feature of true toads.Fil: Gómez, Raúl O.. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Geociencias Básicas, Aplicadas y Ambientales de Buenos Aires. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Geociencias Básicas, Aplicadas y Ambientales de Buenos Aires; ArgentinaFil: Regueira, Eleonora. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Biodiversidad y Biología Experimental; ArgentinaFil: O'donohoe, María Elena Ailin. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Biodiversidad y Biología Experimental; ArgentinaFil: Hermida, Gladys Noemí. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Biodiversidad y Biología Experimental; Argentin

Seasonal Changes and Regulation of the Glucocorticoid Receptor in the Testis of the Toad Rhinella arenarum

Several studies indicate that wild free‐living vertebrates seasonally regulate plasma glucocorticoids. However, not only glucocorticoids but also the amount of receptors is important in determining biological responses. In this context, seasonal regulation of glucocorticoid receptor (GR) is crucial to modulate the response to glucocorticoids. Rhinella arenarum is an anuran exhibiting seasonal variations in plasma glucocorticoids and also in the number of binding sites (Bmax) of the testicular cytosolic GR. In this work, we evaluated if the annual pattern of GR protein in the testis varies seasonally and, by an in vitro approach, the role of glucocorticoids, androgens, and melatonin in the regulation of the GR Bmax and protein level. For this purpose, testes were treated with two physiological concentrations of melatonin (40 and 200 pg/ml), with or without luzindole (melatonin‐receptor antagonist); with testosterone, cyanoketone (inhibitor of steroidogenesis) or casodex (androgen‐receptor antagonist); or with dexamethasone or RU486 (GR antagonist). After treatments, Bmax and protein level were determined by the binding of [3 H]dexamethasone and Western blot, respectively. Results showed that GR protein decreases in the winter. The in vitro treatment with melatonin produced a biphasic effect on the Bmax with the lowest concentration decreasing this parameter by a receptor‐mediated mechanism. However, melatonin had no effect on the GR protein level. Conversely, a high concentration of dexamethasone up‐regulated the GR protein and androgens neither changed the Bmax nor the protein level. These findings suggest that seasonal changes in plasma melatonin and glucocorticoids modulate the effect of glucocorticoids in the testis of R. arenarum.Fil: Regueira, Eleonora. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Biodiversidad y Biología Experimental. Laboratorio de Endocrinología Comparada; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Sassone, Alina Grisel. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Biodiversidad y Biología Experimental. Laboratorio de Endocrinología Comparada; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Scaia, María Florencia. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Biodiversidad y Biología Experimental. Laboratorio de Endocrinología Comparada; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Volonteri, María Clara. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Biodiversidad y Biología Experimental. Laboratorio de Endocrinología Comparada; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Ceballos, Nora Raquel. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Biodiversidad y Biología Experimental. Laboratorio de Endocrinología Comparada; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin