Integrating morphology and physiology of the key endocrine organ during tadpole development: The interrenal gland.

Indirect development is widespread in anurans and is considered an ancestral condition. The metamorphosis of larvae into juveniles involves highly coordinated morphological, physiological, biochemical, and behavioral changes, promoted by the thyroid hormone and interrenal corticosteroids. Stress response to environmental changes is also mediated by corticosteroids, affecting the timing and rate of metamorphosis and leading to great developmental plasticity in tadpoles. Given the potential effect of interrenal gland ontogeny alterations on metamorphosis and the lack of studies addressing both the morphology and endocrinology of this gland in tadpoles, we present corticosterone (CORT) production and histological changes through the ontogeny of interrenal gland in the generalized pond-type tadpole of Rhinella arenarum (Anura, Bufonidae). This species shows the highest concentration of whole-body CORT by the early climax when drastic metamorphic changes begin. This is coincident with the morphological differentiation of steroidogenic cells and the formation of interrenal cords. By this stage, steroidogenic cells have a shrunken cytoplasm, with a significantly higher nucleus-to-cell diameter ratio. The lowest CORT concentration during premetamorphosis and late climax is associated with small undifferentiated cells with lipid inclusions surrounding large blood vessels between kidneys, and with cords of differentiated steroidogenic cells with a significantly lower nucleus-to-cell diameter ratio, respectively. Our study characterizes the morphological and physiological pattern of interrenal gland development, showing an association between certain histological and morphometric characteristics and CORT levels. Variations in this morpho-physiological pattern should be considered when studying the phenotypic plasticity or variable growth rates of tadpoles.

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. ß-escin is one of the main bioactive constituents of Aesculus hippocastanum L. (Hippocastanaceae) seed extract (AH), and both ß-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 ß-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 ß-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 ß-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.

Effect of glucocorticoids on androgen biosynthesis in the testes of the toad Rhinella arenarum (Amphibia, Anura).

In rat Leydig cells, glucocorticoids (GCs) inhibit testosterone production through the interaction with the glucocorticoid receptor (GR). However, the sensitivity of those cells to GCs is regulated by the enzyme 11ß-hydroxysteroid dehydrogenase Type 1 (11ß-HSD1). In the testes of the toad Rhinella arenarum, the presence of an 11ß-HSD similar to type 2 and a cytosolic GR has also been described. However, there is a lack of information regarding the effects of GCs on amphibian testicular steroidogenesis. In this study, the effects of corticosterone on androgen production, and the activity of two steroidogenic enzymes in toad testes were reported. Corticosterone inhibits androgen production via the GR because the GR antagonist RU486 prevents corticosterone-induced inhibition of testosterone. Corticosterone also reduced the activity of the cytochrome P450 17-hydroxylase, C17,20-lyase (Cyp450 c17 ) without affecting the 3ß-hydroxysteroid dehydrogenase/isomerase activity. This effect on Cyp450 c17 was likewise inhibited by RU486. On the other hand, corticosterone had no effect on the amount of steroidogenic acute regulator protein. These results suggest that GCs inhibit steroidogenesis in toad testes by reducing of Cyp450 c17 activity via a GR-mediated mechanism.

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.

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.

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.

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 (E(2)) and in aromatase activity of the BO, and to determine the capacity of the BO to synthesize E(2) from cholesterol in males of R. arenarum. E(2) was determined by radioimmunoassay and aromatase activity was measured by transformation of radioactive substrates into products. Results indicate that plasma E(2) 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 E(2) 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 E(2) from endogenous substrates throughout the year. No correlation was found between plasma E(2) and total BO weight, while there was a significant correlation between plasma E(2) and total activity of aromatase, and between plasma E(2) and E(2) produced in vitro. Taken together, these results demonstrate seasonal variations in plasma E(2), in bidderian total activity of aromatase and, that the BO of adult males of R. arenarum is able to produce E(2) from endogenous substrates.

Characterization and seasonal changes in LHß and FSHß 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 ß subunit of LH and FSH from toad pituitary and study seasonal variation in gonadotropins mRNA using quantitative real-time RT-PCR. The LHß 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 FSHß 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 LHß and FSHß (teleosts, amphibians, birds, mammals) showed that all the tetrapods studied conserve 12 cysteins and one (LH) or two (FSH) N-Glycosylation sites. LHß is closer to teleosts than to mammals and birds while FSHß is closer to mammals. The analysis of seasonal changes in LHß and FSHß mRNA indicates that transcript levels have seasonal variations and that the profile of androgens is opposite to that of the gonadotropins mRNA.

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.

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.

5α-Reductase, an enzyme regulating glucocorticoid action in the testis of Rhinella arenarum (Amphibia: Anura).

The reduction of A-ring of glucocorticoids to produce 5α-dihydro-derivatives by 5α-reductases has been considered as a pathway of irreversible inactivation. However, 5α-reduced metabolites of corticosterone and testosterone have significant biological activity. In this paper, we investigated whether toad testicular 5α-reductase (5α-Red) is able to transform corticosterone into 5α-dihydrocorticosterone. Furthermore, we studied the role of 5α-reduced metabolite of corticosterone as a glucocorticoid receptor (GR) agonist. The activity of 5α-Red was assayed in subcellular fractions with [(3)H]corticosterone or [(3)H]testosterone as substrate. The enzyme localizes 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 5α-Red resembles mammalian type 1 isoenzyme. Kinetic studies indicate that neither K(m) nor V(max) for both corticosterone and testosterone were significantly different among reproductive periods. The K(m) 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 5α-dihydrocorticosterone (5α-DHB) to the testicular GR show that 5α-DHB is able to displace the binding of [(3)H]dexamethasone to testicular cytosol with a similar potency than corticosterone. The inhibition constant (Ki) values for corticosterone and 5α-DHB were similar, 31.33±2.9 nM and 35.24±2.3 nM, respectively. In vitro experiments suggest that 5α-DHB 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.

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.

Further evidence on the role of heparan sulfate as protamine acceptor during the decondensation of human spermatozoa.

BACKGROUND: Human spermatozoa decondense in vitro upon exposure to heparin and glutathione. Glutathione is also the disulfide bond reducer in vivo, and heparan sulfate, a functional analogue of heparin, has been proposed as the protamine acceptor. The aim of this study was to evaluate the decondensing ability of chemically modified heparins and different glycosaminoglycans (GAGs) on isolated sperm nuclei in vitro, and to analyse the possible role of different GAGs as protamine acceptors. METHODS: Capacitated spermatozoa and isolated sperm nuclei from normospermic semen samples were decondensed in the presence of heparin (or its equivalent) and glutathione. After fixation with glutaraldehyde, the percentage of decondensed spermatozoa and nuclei was determined under phase-contrast. Proteins were extracted from sperm nuclei previously incubated in the presence of gluhathione and different GAGs by incubation with urea-beta-meracptoethanol-NaCl, and analysed by acid polyacrylamide gel electrophoresis. RESULTS: The ability of desulfated heparins and other GAGs to decondense isolated nuclei mirrored exactly the decondensation of capacitated spermatozoa, the only difference being the level of maximum decondensation achieved. Heparan sulfate and heparin, but not other GAGs, were able to release protamines from sperm chromatin. CONCLUSIONS: Heparan sulfate could be functioning as protamine acceptor in vivo during human sperm nuclear decondensation.