Characterization of StAR protein of Rhinella arenarum (Amphibia, Anura).

The steroidogenic acute regulatory (StAR) protein performs the delivery of cholesterol from the outer to inner mitochondrial membrane. This is considered the rate-limiting step of acute steroid production, widely studied in mammals. However, there are only few reports regarding the characterization and expression of StAR protein in non-mammalian vertebrates. In this study, StAR protein sequence of Rhinella arenarum has been characterized and deduced from interrenal and testis cDNA sequences. StAR encodes a 285 amino acid protein with a conserved domain containing putative lipid binding sites. In vitro incubations showed that expression of StAR mRNA in testis, determined by qPCR, and testosterone synthesis determined by radioimmunoassay were stimulated after treatment with hCG and 8Br-cAMP. However, StAR mRNA expression results obtained with hCG show a higher stimulation than those obtained with 8Br-cAMP, even though steroidogenic production is the same with both treatments.

Estradiol and reproduction in the South American toad Rhinella arenarum (Amphibian, Anura).

Rhinella arenarum is a South American toad with wide geographic distribution. Testes of this toad produce high amount of androgens during the non reproductive season and shift steroid synthesis from androgens to 5α-pregnanedione during the breeding. In addition, plasma estradiol (E2) in males of this species shows seasonal variations but, since testes of R. arenarum do not express aromatase, the source of plasma E2 remained unknown for several years. However, the Bidder's organ (BO), a structure located at one pole of each testis, is proposed to be the main source of E2 in male's toads since it expresses several steroidogenic enzymes and is able to produce E2 from endogenous substrates throughout the year. In addition, there were significant correlations between plasma E2 and total activity of BO aromatase, and between plasma E2 and the amount of hormone produced by the BO in vitro. In the toad, apoptosis induced by in vitro treatment with E2 was mostly detected in spermatocytes during the breeding and in spermatids during the post-reproductive season, suggesting that this steroid has an important role in controlling spermatogenesis. However, in vitro treatment with E2 had no effect on proliferation. This evidence suggests that the mechanism of action of E2 on amphibian spermatogenesis is complex and more studies are necessary to fully understand the role of estrogens regulating the balance between cellular proliferation and apoptosis. In addition, in R. arenarum in vitro studies suggested that E2 has no effect on CypP450c17 protein levels or enzymatic activity, while it reduces 3ß-hydroxysteroid dehydrogenase/isomerase (3ß-HSD/I) activity during the post reproductive season. As well, E2 regulates FSHß mRNA expression all over the year suggesting a down regulation process carried out by this steroid. The effect on LHß mRNA is dual, since during the reproductive season estradiol increases the expression of LHß mRNA while in the non-reproductive season it has no effect. In conclusion, the effect of E2 on gonadotropins and testicular function is complex, not clearly understood and probably varies depending on the species. The aim of the current article is to review evidence on reproductive endocrinology and on the role of estradiol regulating reproduction in amphibians, with emphasis on the South American species Rhinella arenarum.

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.

Apoptosis, proliferation and presence of estradiol receptors in the testes and Bidder's organ of the toad Rhinella arenarum (Amphibia, Anura).

The dynamic equilibrium between spermatogonial proliferation and testicular apoptosis determines the progression of spermatogenesis in amphibians. Estrogens and their receptors play a central role in regulating spermatogenesis in vertebrates, and in some species of anurans, estradiol (E2 ) is involved in the regulation of spermatogonial proliferation and apoptosis of germ cells. Bidder's organ (BO) is a structure characteristic of Bufonidae that has historically been compared to an undeveloped ovary. In adult Rhinella arenarum males, BO is one of the main sources of plasma E2 . The aim of this study was 1) to describe the seasonal variations in testicular apoptosis, spermatogonial proliferation, and cellular proliferation in BO; and 2) to analyze the presence and localization of estrogen receptor ß (ERß) in the testes and BO of R. arenarum. Testicular fragments and BOs from animals collected during the year were labeled with 5-bromo-2'-deoxyuridine (BrdU) and BrdU incorporation was determined using immunohistochemistry. Apoptosis in testicular sections was detected using the TUNEL method, and ERß localization was assessed using immunohistochemistry in testes and BOs. The results indicate that spermatogonial proliferation is highest during the reproductive season and that cysts of spermatocytes and spermatids undergo apoptosis during the postreproductive season. Furthermore, the proliferation of follicular cells is highest during the reproductive and postreproductive seasons. ERß was primarily detected by immunolocalization in Sertoli cells, follicular cells, and oocytes. Taken together, these results suggest that cysts that do not form spermatozoa are removed from testes by apoptosis and that estrogens regulate both spermatogenesis and oogenesis in adult males of R. arenarum.

Effect of estradiol on apoptosis, proliferation and steroidogenic enzymes in the testes of the toad Rhinella arenarum (Amphibia, Anura).

Estrogens inhibit androgen production and this negative action on amphibian steroidogenesis could be related to the regulation of steroidogenic enzymes. Estrogens are also involved in the regulation of amphibian spermatogenesis by controlling testicular apoptosis and spermatogonial proliferation. The Bidder's organ (BO) is a structure characteristic from the Bufonidae family and in adult males of Rhinella arenarum it is one of the main sources of plasma estradiol (E2). The purpose of this study is to analyze the effect of E2 on testicular steroidogenic enzymes, apoptosis and proliferation in the toad R. arenarum. For this purpose, testicular fragments were treated during 24h with or without 2 or 20nM of E2. After treatments, the activities of cytochrome P450 17α-hydroxylase-C17-20 lyase (CypP450c17) and 3ß-hydroxysteroid dehydrogenase/isomerase (3ß-HSD/I) were measured by the transformation of radioactive substrates into products, and CypP450c17 expression was determined by Western blot analysis. Apoptosis in testicular sections was detected with a commercial fluorescent kit based on TUNEL method, and proliferation was evaluated by BrdU incorporation. Results indicate that E2 has no effect on CypP450c17 protein levels or enzymatic activity, while it reduces 3ß-HSD/I activity during the post reproductive season. Furthermore, although E2 has no effect on apoptosis during the pre and the post reproductive seasons, it stimulates testicular apoptosis during the reproductive season, mostly in spermatocytes. Finally, E2 has no effect on testicular proliferation all year long. Taken together, these results suggest that E2 is involved in the regulation of testicular steroidogenesis and spermatogenesis.