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.

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.