Post-metamorphic development of skin glands in a true toad: Parotoids versus dorsal skin.

Chemical defenses in amphibians are a common antipredatory and antimicrobial strategy related to the presence of dermal glands that synthesize and store toxic or unpalatable substances. Glands are either distributed throughout the skin or aggregated in multiglandular structures, being the parotoids the most ubiquitous macrogland in toads of Bufonidae. Even though dermal glands begin to develop during late-larval stages, many species, including Rhinella arenarum, have immature glands by the end of metamorphosis, and their post-metamorphic growth is unknown. Herein, we compared the post-metamorphic development of parotoids and dorsal glands by histological and allometric studies in a size series of R. arenarum. Histological and histochemical studies to detect proteins, acidic glycoconjugates, and catecholamines, showed that both, parotoids and dorsal glands, acquire characteristics of adults in individuals larger than 50 mm; that is, a moment in which the cryptic coloration disappears. Parotoid height increased allometrically as a function of body size, whereas the size of small dorsal glands decreased with body size. The number of glands in the dorsum was not linearly related to body size, appearing to be an individual characteristic. Only adult specimens had intraepithelial granular glands in the duct of the largest glands of the parotoids. Since toxic secretions accumulate in the central glands of parotoids, allometric growth of parotoids may translate into greater protection from predators in the largest animals. Conversely, large glands in the dorsum, which produce a proteinaceous secretion of unknown function, grow isometrically to body size. Some characteristics, like intraepithelial glands in the ducts and basophilic glands in the dorsum, are limited to adults.

Development and steroidogenic properties of the Bidder's organ of the tadpole of Rhinella arenarum (Amphibia, Anura).

Several studies suggested that in anuran amphibians steroidogenic enzymes are critical for gonadal differentiation, proposing that the amount of sex steroids would adjust this differentiation. Among anurans, bufonids are important for the study of sex differentiation due to the presence of Bidder's organ (BO) that differentiates as a rudimentary ovary in the cephalic portion of the genital ridge. Considering that in adult males of Rhinella arenarum, the BO synthesizes estradiol, the main purpose of this work is to examine, in this species, the morphogenesis of BO and the steroidogenic capacity of this organ during larval development. BO and the proper gonads are distinguished from Gosner stage 26. During metamorphosis, BO primary oogonia develop in oogonia in nests, early previtellogenic oocytes and late previtellogenic oocytes in follicles while proper gonads remain undifferentiated. Aromatase was detected by immunohistochemistry in almost all the largest follicles of the BOs while the cytochrome P450 side-chain cleavage was observed in only few oocytes. The proper gonad was not immunoreactive in any stage. The determination of aromatase and 5α-reductase activities showed that the population of tadpoles between stages 36-41 is not homogeneous in terms of aromatase activity. In addition, from stage 26 to the end of metamorphosis, all the stages were able to produce estradiol from endogenous substrate but stages 40-41, corresponding to the end of pro-metamorphosis, produced the highest values. In conclusion, BO is able to synthesize estradiol from endogenous precursors and proper gonad remains undifferentiated at least until the end of the metamorphosis.

EDA-containing fibronectin increases proliferation of embryonic stem cells.

Embryonic stem cells (ESC) need a set of specific factors to be propagated. They can also grow in conditioned medium (CM) derived from a bovine granulosa cell line BGC (BGC-CM), a medium that not only preserves their main features but also increases ESC´s proliferation rate. The mitogenic properties of this medium were previously reported, ascribing this effect to an alternative spliced generated fibronectin isoform that contains the extra domain A (FN EDA(+)). Here, we investigated if the FN EDA(+) isoform increased proliferation of mouse and human ES cells. We analyzed cell proliferation using conditioned media produced by different mouse embryonic fibroblast (MEF) lines genetically engineered to express FN constitutively including or excluding the EDA domain (FN EDA(-)), and in media supplemented with recombinant peptides containing or not the EDA. We found that the presence of EDA in the medium increased mouse and human ESC's proliferation rate. Here we showed for the first time that this FN isoform enhances ESC's proliferation. These findings suggest a possible conserved behavior for regulation of ES cells proliferation by this FN isoform and could contribute to improve their culturing conditions both for research and cell therapy.

Environmentally-relevant concentrations of atrazine induce non-monotonic acceleration of developmental rate and increased size at metamorphosis in Rhinella arenarum tadpoles.

Despite of the various studies reporting on the subject, anticipating the impacts of the widely-used herbicide atrazine on anuran tadpoles metamorphosis remains complex as increases or decreases of larval period duration are almost as frequently reported as an absence of effect. The aim of the present study was to examine the effects of environmentally-relevant concentrations of atrazine (0.1, 1, 10, 100, and 1000µg/L) on the timings of metamorphosis and body size at metamorphosis in the common South American toad, Rhinella arenarum (Anura: bufonidae). None of the atrazine concentrations tested significantly altered survival. Low atrazine concentrations in the range of 1-100µg/L were found to accelerate developmental rate in a non-monotonic U-shaped concentration-response relationship. This observed acceleration of the metamorphic process occurred entirely between stages 25 and 39; treated tadpoles proceeding through metamorphosis as control animals beyond this point. Together with proceeding through metamorphosis at a faster rate, tadpoles exposed to atrazine concentrations in the range of 1-100µg/L furthermore transformed into significantly larger metamorphs than controls, the concentration-response curve taking the form of an inverted U in this case. The no observed effect concentration (NOEC) was 0.1µg atrazine/L for both size at metamorphosis and timings of metamorphosis. Tadpoles exposed to 100µg/L 17ß-estradiol presented the exact same alterations of developmental rate and body size as those treated with 1, 10 and 100µg/L of atrazine. Elements of the experimental design that facilitated the detection of alterations of metamorphosis at low concentrations of atrazine are discussed, together with the ecological significance of those findings.

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 (B(max)) 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 B(max) 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, B(max) 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 B(max) 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 B(max) 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.

The Bidder's organ of the toad Rhinella arenarum (Amphibia, Anura). Presence of steroidogenic enzymes.

The Bidder's organ (BO) of male true toads of Bufonidae family is located in the anterior pole of the testis and it has been compared to a rudimentary ovary because of the presence of previtellogenic follicles. In some species, BO remains in both sexes, while in others only adult males preserve the structure. Several studies suggest that the development of BO is inhibited by the differentiation of the corresponding gonad. The purpose of this study is to describe morphological and histological variability of the BO of Rhinella arenarum and also analyze its steroidogenic capacity. Observations indicate that although most bidderian follicles are in pre vitellogenesis, there are others in early or late vitellogenesis. Moreover, we found that BOs weight was significantly lower in males during the pre-reproductive period and that there is no significant correlation between the weights of BO and the adjacent testis. We also analyzed the presence of steroidogenic enzymes using immunohistochemistry. Results indicate that all the follicles were immunoreactive with the antibody against aromatase, while only few of them were positive for the cytochrome P450 side-chain cleavage. Furthermore, activities of 3ß-hydroxysteroid dehydrogenase/isomerase, cytochrome P450 17-hydroxylase, C17,20-lyase and aromatase were detected by the transformation of radioactive substrates into products. Taken together, these results confirm the steroidogenic capacity of the BO in adult males of R. arenarum.

Production of the main surface antigen of Toxoplasma gondii in tobacco leaves and analysis of its antigenicity and immunogenicity.

We adapted a previously described Agrobacterium-mediated transient expression system to test the expression level of three constructs carrying the surface antigen 1 (SAG1) of Toxoplasma gondii. Two constructs were based in a Potato virus X (PVX) amplicon. In one of them, the PVX movement protein genes were replaced by the sag1 gene. In the other, the sag1 gene was placed under the control of an additional coat protein subgenomic promoter. In the third construct, the sag1 gene was fused to an apoplastic peptide signal under the CaMV 35S promoter. Western blot analysis of leaf extracts infiltrated with each construct revealed a protein of 35 kDa. SAG1 accumulation in leaves ranged from 0.1 to 0.06% of total soluble protein (equivalent to 10 microg and 6 microg of SAG1 per gram of fresh leaf tissue, respectively). Three of five human seropositive samples reacted with tobacco-expressed SAG1 in Western blot analysis. The C3H mice were immunized with SAG-expressing leaf extracts and perorally challenged with a nonlethal dose of the T. gondii Me49 strain. Mice vaccinated with SAG1 showed significantly lower brain cyst burdens compared to those from the control group. Immunization with SAG1-expressing leaves elicited a specific humoral response with predominant participation of type IgG2a. In conclusion, a functional SAG1 version could be transiently expressed in tobacco leaves.