Neural regeneration dynamics of Xenopus laevis olfactory epithelium after zinc sulfate-induced damage.

Neural stem cells (NSCs) of the olfactory epithelium (OE) are responsible for tissue maintenance and the neural regeneration after severe damage of the tissue. In the normal OE, NSCs are located in the basal layer, olfactory receptor neurons (ORNs) mainly in the middle layer, and sustentacular (SUS) cells in the most apical olfactory layer. In this work, we induced severe damage of the OE through treatment with a zinc sulfate (ZnSO4) solution directly in the medium, which resulted in the loss of ORNs and SUS cells, but retention of the basal layer. During recovery following injury, the OE exhibited increased proliferation of NSCs and rapid neural regeneration. After 24h of recovery, new ORNs and SUS cells were observed. Normal morphology and olfactory function were reached after 168h (7 days) of recovery after ZnSO4 treatment. Taken together, these data support the hypothesis that NSCs in the basal layer activate after OE injury and that these are sufficient for complete neural regeneration and olfactory function restoration. Our analysis provides histological and functional insights into the dynamics between olfactory neurogenesis and the neuronal integration into the neuronal circuitry of the olfactory bulb that restores the function of the olfactory system.

Distribution pattern of neuropeptide Y in the brain, pituitary and olfactory system during the larval development of the toad Rhinella arenarum (Amphibia: Anura).

The first NPY-immunoreactivity (ir) in the central nervous system of Rhinella arenarum was obtained just after hatching in the pre-optic area, ventral thalamus and rostral rhombencephalon. During pre-metamorphosis, new NPY-ir cells were observed in other brain areas such as pallium, septum and striatum, infundibulum and pars intermedia of the pituitary. Further maturation continued through pro-metamorphosis with the appearance of cell groups in the diagonal band, amygdala, pre-optic nucleus, dorsal nucleus of the habenula, anterior ventral and dorsal thalamus, suprachiasmatic nucleus, tuberculum posterior, tectum, torus semicircularis, inter-peduncular nucleus and median eminence. During the metamorphic climax and soon after, the relative abundance of NPY-ir fibres decreased in all hypothalamic areas and the staining intensity and number of NPY-ir cells in the pallium also decreased, whereas no cells were found in the striatum, dorsal nucleus of the habenula and tectum. In the olfactory epithelium, nerve or bulb, neither cells nor NPY-ir fibres were found during the stages of development analysed. The ontogeny pattern of the NPY-ir neuronal system in the brain of Rh. arenarum is more similar to the spatiotemporal appearance reported for Rana esculenta than to that reported for Xenopus laevis. Many NPY-ir fibres were found in the median eminence and in the pars intermedia of the pituitary, supporting the idea that this neuropeptide may play a role in the modulation of hypophyseal secretion during development.

Galanin: presence and distribution in the brain and pituitary of Rhinella arenarum (Amphibia: Anura) during development.

The immunohistochemical distribution of galanin (Gal) in the brain and pituitary of Rhinella arenarum was studied during development. Gal-immunoreactivity was first observed in the brain just after hatching in anterior preoptic area, infundibular area, median eminence and pars distalis of the pituitary as well as in the olfactory epithelium. At the beginning of prometamorphosis new Gal-immunoreactive (ir) cells were observed in the olfactory nerve and bulb. Later in prometamorphosis new Gal-ir cells were observed in the telencephalon, suprachiasmatic nucleus, rostral rhombencephalon and in the pars nervosa of the pituitary. The most numerous accumulations of Gal-ir neurons throughout the larval development were observed in the ventral hyphothalamus where numerous Gal-ir cells of cerebrospinal fluid-contacting type were found. During metamorphic climax and soon after we did not detect Gal-ir neurons in the pallium, medial or pretectal dorsal thalamus. In the median eminence and pars distalis of the pituitary many Gal-ir fibers were found during development indicating that Gal may play a role in the modulation of hypophyseal secretion. Furthermore, the distribution of Gal-ir elements observed throughout larvae development indicates that galaninergic system maturation continues until sexual maturity.

Seasonal changes in the activity of cytochrome P450(C17) from the testis of Bufo arenarum.

In Bufo arenarum, the biosynthesis of testosterone and 5alpha-dihydrotestosterone takes place through a complete 5-ene pathway, 5-androsten-3beta,17beta-diol being the immediate precursor of testosterone. Besides androgens, testes are able to synthesise 5alpha-pregnan-3,20-dione and several 3alpha and 20alpha reduced derivatives. During the breeding season, steroid biosynthesis turns from androgen to C21-steroid production. As a consequence, the cytochrome P450 17-hydroxylase, C17,20-lyase (CypP450(c17)) could be a key enzyme in that metabolic shift. The present study demonstrates that in testes of B. arenarum, CypP450(c17) co-localises with glucose-6-phosphatase in the microsomal fraction. CypP450(c17) possesses more affinity for pregnenolone than for progesterone in both non-reproductive (Km = 43.76 +/- 4.63 nM and 2,170 +/- 630 nM, respectively) and reproductive (Km = 37.46 +/- 4.19 nM and 3,060 +/- 190 nM, respectively) seasons. These results could explain the predominance of the 5-ene pathway for testosterone biosynthesis. Toad CypP450(c17) activity is higher in the non-reproductive period than the reproductive period, suggesting that this enzyme is an important factor in toad steroidogenic changes. Animals in reproductive conditions showed a significant reduction in circulating androgens. This is in agreement with the decrease in Vmax of cytochrome P450 17-hydroxylase activity, enhancing the physiological relevance of these in vitro results.

Kinetic properties of microsomal 3beta hydroxysteroid dehydrogenase-isomerase from the testis of Bufo arenarum H.

3beta-hydroxysteroid dehydrogenase 5-ene isomerase (3betaHSD/I) activity is necessary for the biosynthesis of hormonally active steroids. A dual distribution of the enzyme was described in toad testes. The present study demonstrates that in testicular tissue of Bufo arenarum H., microsomal 3betaHSD/I has more affinity for dehydroepiandrosterone (DHEA) than for pregnenolone (K(m)=0.17+/-0. 03 and 1.02 microM, respectively). The Hill coefficient for the conversion of DHEA and pregnenolone were 1.04 and 1.01, respectively. The inclusion of DHEA in the kinetic analysis of pregnenolone conversion affected V(max) while K(m) was not modified, suggesting a non-competitive inhibition of the conversion of pregnenolone. K(i) was calculated from replot of Dixon's slope for each substrate concentration. K(i) from the intercept and the slope of this replot were similar (0.276+/-0.01 and 0.263+/-0.02 microM) and higher than the K(m) for DHEA. The K(m) and K(i) values suggest the presence of two different binding sites. When pregnenolone was present in the assays with DHEA as substrate, no effect was observed on the V(max) while K(m) values slightly increased with pregnenolone concentration. Consequently, pregnenolone inhibited the transformation of DHEA in a competitive fashion. These studies suggest that, in this species, the microsomal biosyntheses of androgens and progesterone are catalysed by different active sites.

Human chorionic gonadotropin-induced spermiation in Bufo arenarum is not mediated by steroid biosynthesis.

This study employed an in vitro system to identify potential steroidal mediators of spermiation in Bufo arenarum. Testicular fragments were incubated for 2 h at 28 degrees. Spermiation was induced by 10 IU human chorionic gonadotropin (hCG) and the effect of different inhibitors of steroid biosynthesis was analyzed. Cyanoketone (10(-5)-10(-6) M), an inhibitor of 3-oxo-4-ene steroid biosynthesis, did not block hCG-inducing activity even when biosynthesis of 3-oxo-4-ene steroids and its reduced metabolites was inhibited by 95%. Aminogluthetimide at a concentration that inhibited testosterone biosynthesis (10(-4) and 10(-5) M) did not alter hCG actions. Similar results were obtained with spironolactone, an inhibitor of 17alpha-hydroxylase/17-20 lyase activity. No spermiation-inducing activity was found with different steroids (progesterone, 17-hydroxypregnenolone, 17, 20alpha/beta-dihydroxy-4-pregnene-3-one, estradiol, testosterone, etc.). It is concluded that spermiation induced by hCG is not steroid mediated in B. arenarum.

Pregnenolone and progesterone metabolism by the testes of Bufo arenarum.

Sliced testis tissue from Bufo arenarum was incubated in the presence of [3H]pregnenolone. Testis fragments were also used for double isotope experiments using [3H]pregnenolone and [14C]progesterone. Specific activities were equated with the addition of radioinert pregnenolone. When yields of radiometabolites were analysed, pregnenolone was found to be a good precursor for C19 steroids such as dehydroepiandrosterone, 5-androsten-3 beta, 17 beta diol, testosterone, 5 alpha-dihydrotestosterone and a C21 steroid, 5 alpha-pregnan-3,20 dione. Progesterone mainly converts to 5 alpha-pregnan-3,20 dione, a steroid with unknown function in amphibians. The 5-ene pathway, including 5-androsten-3 beta, 17 beta diol as intermediate, could be predominant for androgen biosynthesis. Testes bypass not only progesterone but also androstenedione for testosterone biosynthesis.

Subcellular localization of 3 beta hydroxysteroid dehydrogenase isomerase in testis of Bufo arenarum H.

3 Beta-hydroxysteroid dehydrogenase 5-ene isomerase (3 beta HSD/I) catalyzes an essential step in the biosynthesis of steroid hormones and is usually considered to be mainly microsomal, although there is a dual distribution of the enzyme in toad interrenals. The present study demonstrates that in the testicular tissue, as in interrenals of Bufo arenarum H., 3 beta HSD/I is both mitochondrial and microsomal. The conversion of dehydroepiandrosterone to androstenedione takes place only in microsomes while pregnenolone is converted to progesterone in both microsomes and mitochondria. Kinetic constants of 3 beta HSD/I were determined by the oxidation of pregnenolone and dehydroepiandrosterone. The preferred substrate of the microsomal 3 beta HSD/I enzyme was dehydroepiandrosterone (K(m) = 0.17 microM and 0.53 microM for dehydroepiandrosterone and pregnenolone, respectively) not only during the breeding season but also in the non-breeding period (K(m) = 0.49 microM and 2.9 microM for dehydroepiandrosterone and pregnenolone, respectively).

Mitochondrial localization of 3 beta-hydroxysteroid dehydrogenase 5-ene isomerase in interrenals of the toad Bufo arenarum H.

The enzymatic activity of 3 beta-hydroxysteroid dehydrogenase 5-ene isomerase (3 beta HSD/I) catalyzes an essential step in the biosynthesis of steroid hormones including progesterone, mineralocorticoids, glucocorticoids, estrogens, and androgens. Its subcellular localization in steroidogenic tissues is usually considered to be mainly microsomal. The present study demonstrates that in the interrenal of Bufo aernarum H., 3 Beta HSD/I activity localizes in mitochondria and micromes. It also shows that the two distinct pathways to aldosterone previously demonstrated for interrenals of B. arenarum H. exhibited differential subcellular localizations, microsomal for the 4-ene route and mitochondrial for the 5-ene route. Kinetic constants of 3 Beta HSD/I were determined for the oxidation of pregnenolone and the recently described 3 Beta-hydroxy analogue of aldosterone (3 Beta AA). The preferred substrate of the mitochondrial 3 Beta HSD/I enzyme was 3 Beta AA (Km = 0.7 microM and 14.0 microM for 3 Beta AA and pregnenolone, respectively). However, the microsomal enzyme has a greater affinity for pregnenolone (Km = 0.8 microM) than for 3 Beta AA (Km = 17.0). Enzymes from both localizations have similar nucleotide (NAD+) requirements, activities being higher in summer. This dual localization opens novel possibilities for the regulation of interrenal functions.