Ultrastructural and immunohistochemical characteristics of the olfactory organ cardinal tetra, Paracheirodon axelrodi (Characiformes: Characidae).

The cardinal tetra Paracheirodon axelrodi belongs to the family Characidae, an economically important and morphologically diverse family of fishes. Information on the olfactory system of this species is scattered and scarce. Among teleost fishes, differences exist in the shape, number, and arrangement of the olfactory lamellae, in the distribution of the sensory and nonsensory epithelium, as well as in the abundance of various receptor cell types. Here, an anatomical and morphological description of the olfactory system was carried out using light microscopic histology, immunohistochemistry, scanning electron microscopy, and transmission electron microscopy. P. axelrodi is a ditremous and isosmat species. It has an arrow-shaped olfactory rosette arrangement. The olfactory epithelium is covering the 12-14 lamellae of the olfactory rosette and, using scanning electron microscopy, we observed that the apical surface of the olfactory epithelium carries a dense layer of mucus. Based on the histological, immunohistochemical, and ultrastructural descriptions, all characteristic sensory and nonsensory cell types of the olfactory epithelium of teleost fish were identified. Three types of olfactory receptor neurons were identified: ciliated, microvilli, and crypt cells. The distribution of sensory and nonsensory cell types is like that described in Aphyocharax anisitsi, another species of the Characidae family. A. anisitsi inhabits slow-flowing water bodies with high-density vegetation such as P. axelrodi.

Effects of intraspecific chemical cues on the behaviour of the bloodfin tetra Aphyocharax anisitsi (Ostariophysi: Characidae).

Chemical communication can induce a multitude of behaviours when detected by fish olfactory systems, from parental care, predation and alarm signalling, to foraging, schooling, reproduction, and migration. Chemical cues provide information that visual traits cannot and fish can respond to chemical cues without any additional sensory cue. In this way, pheromones play an essential role in the fitness of fishes. Given that Aphyocharax anisitsi inhabits environments characterized by cloudy and highly vegetated waters, it is interesting to evaluate the olfactory contribution in their communication. Here, we investigated the relevance of chemical cues in the types of behaviours triggered in A anisitsi by two experimental contexts: 1) non-social and olfactory context (conspecific-chemical cues), and 2) social context (conspecific female or male presence). Non-social context experiments suggest that males of A. anisitsi respond to both male and female-chemical cues even in the absence of other sensory inputs. The high olfactory sensitivity of characids in general and of A. anisitsi, in particular, could facilitate vital functions, such as foraging and conspecific recognition in habitats that impose severe restrictions on the visual system.

Olfactory subsystems in the peripheral olfactory organ of anuran amphibians.

Anuran amphibians (frogs and toads) typically have a complex life cycle, involving aquatic larvae that metamorphose to semi-terrestrial juveniles and adults. However, the anuran olfactory system is best known in Xenopus laevis, an animal with secondarily aquatic adults. The larval olfactory organ contains two distinct sensory epithelia: the olfactory epithelium (OE) and vomeronasal organ (VNO). The adult organ contains three: the OE, the VNO, and a "middle cavity" epithelium (MCE), each in its own chamber. The sensory epithelia of Xenopus larvae have overlapping sensory neuron morphology (ciliated or microvillus) and olfactory receptor gene expression. The MCE of adults closely resembles the OE of larvae, and senses waterborne odorants; the adult OE is distinct and senses airborne odorants. Olfactory subsystems in other (non-pipid) anurans are diverse. Many anuran larvae show a patch of olfactory epithelium exposed in the buccal cavity (bOE), associated with a grazing feeding mode. And other anuran adults do not have a sensory MCE, but many have a distinct patch of epithelium adjacent to the OE, the recessus olfactorius (RO), which senses waterborne odorants. Olfaction plays a wide variety of roles in the life of larval and adult anurans, and some progress has been made in identifying relevant odorants, including pheromones and feeding cues. Increased knowledge of the diversity of olfactory structure, of odorant receptor expression patterns, and of factors that affect the access of odorants to sensory epithelia will enable us to better understand the adaptation of the anuran olfactory system to aquatic and terrestrial environments.

Conservation of Glomerular Organization in the Main Olfactory Bulb of Anuran Larvae.

The glomerular array in the olfactory bulb of many vertebrates is segregated into molecularly and anatomically distinct clusters linked to different olfactory functions. In anurans, glomerular clustering is so far only described in Xenopus laevis. We traced olfactory projections to the bulb in tadpoles belonging to six distantly related anuran species in four families (Pipidae, Hylidae, Bufonidae, Dendrobatidae) and found that glomerular clustering is remarkably conserved. The general bauplan consists of four unequally sized glomerular clusters with minor inter-species variation. During metamorphosis, the olfactory system undergoes extensive remodeling. Tracings in metamorphotic and juvenile Dendrobates tinctorius and Xenopus tropicalis suggest a higher degree of variation in the glomerular organization after metamorphosis is complete. Our study highlights, that the anatomical organization of glomeruli in the main olfactory bulb (MOB) is highly conserved, despite an extensive ecomorphological diversification among anuran tadpoles, which suggests underlying developmental constraints.

Morphology and immunohistochemistry of the olfactory organ in the bloodfin tetra, Aphyocharax anisitsi (Ostariophysi: Characidae).

Among teleost fishes, differences exist in the shape, number, and arrangement of the olfactory lamellae, the distribution of the sensory and non-sensory epithelium, as well as, the abundance of various receptor cells. The objective of this work was to describe the morphology, immunohistochemistry, and scanning electron microscopy ultrastructure of the olfactory epithelium of the bloodfin tetra, Aphyocharax anisitsi. This is the first complete description including the anatomy, histology, and immunohistochemistry of the peripheral olfactory organ from a Characiformes. Based on the external morphology of the olfactory organ, A. anisitsi was classified as a ditermous species, with an olfactory cavity containing two openings divided by a skin flap that separates the anterior and posterior nostril. This species belongs to the group of isosmates, since the presence of accessory olfactory sacs was not observed, and non-sensory ciliated cells were identified. A. anisitsi has an olfactory rosette with an arrow-shaped arrangement, with differences in length between the anterior and posterior lamellae. In the olfactory epithelium, three types of olfactory receptor neurons were identified using histology and confirmed by immunohistochemistry, that is, ciliated olfactory receptor neurons in the basal region of the epithelium, microvillar olfactory receptor neurons in the middle region; and Crypt cells, in smaller numbers compared to the other neuronal types, present in the apical region. Sensory and non-sensory areas were scattered and mixed along the lamellar lateral surface but the nasal cavity and the midline raphe lacked olfactory receptor neurons. The presence of abundant kinocilia in the non-sensory cells could be related in A. anisitsi with ventilation and quality control of water entering the olfactory cavity. The spatial organization of the sensory and non-sensory areas in A. anisitsi was similar to that observed in other species that also inhabit still and slow-flowing bodies of water with high-density vegetation.

Multi-glomerular projection of single olfactory receptor neurons is conserved among amphibians.

Individual receptor neurons in the peripheral olfactory organ extend long axons into the olfactory bulb forming synapses with projection neurons in spherical neuropil regions, called glomeruli. Generally, odor map formation and odor processing in all vertebrates is based on the assumption that receptor neuron axons exclusively connect to a single glomerulus without any axonal branching. We comparatively tested this hypothesis in multiple fish and amphibian species (both sexes) by applying sparse cell electroporation to trace single olfactory receptor neuron axons. Sea lamprey (jawless fish) and zebrafish (bony fish) support the unbranched axon concept, with 94% of axons terminating in single glomeruli. Contrastingly, axonal projections of the axolotl (salamander) branch extensively before entering up to six distinct glomeruli. Receptor neuron axons labeled in frog species (Pipidae, Bufonidae, Hylidae, and Dendrobatidae) predominantly bifurcate before entering a glomerulus and 59 and 50% connect to multiple glomeruli in larval and postmetamorphotic animals, respectively. Independent of developmental stage, lifestyle, and adaptations to specific habitats, it seems to be a common feature of amphibian olfactory receptor neuron axons to frequently bifurcate and connect to multiple glomeruli. Our study challenges the unbranched axon concept as a universal vertebrate feature and it is conceivable that also later diverging vertebrates deviate from it. We propose that this unusual wiring logic evolved around the divergence of the terrestrial tetrapod lineage from its aquatic ancestors and could be the basis of an alternative way of odor processing.

Morphological and immunohistochemical comparison of the pituitary gland between a tropical Paracheirodon axelrodi and a subtropical Aphyocharax anisitsi characids (Characiformes: Characidae)

Cardinal tetra Paracheirodon axelrodi and bloodfin tetra Aphyocharax anisitsi are two species of characids with high trade value as ornamental fish in South America. Although both species inhabit middle water layers, cardinal neon exhibits a tropical distribution and bloodfin tetra a subtropical one. Generally, these species are difficult to grow, so it becomes essential to know some key components of the neuroendocrine system to achieve their reproduction in captivity. Considering the importance of deepening the knowledge of the reproductive physiology through functional morphology, for the first time in this work we performed an anatomical, morphological and immunohistochemical analysis of the pituitary gland of these two species. In both species, a leptobasic type pituitary is found in the ventral zone of the hypothalamus and it is characterized by a neurohypophysis which has a well-developed pituitary stalk and a globular adenohypophysis. The pituitary components, characterized by histochemistry and immunohistochemistry, shows a distribution pattern of cells types similar to other teleost species, with only slight differences in the distribution of βFSH and βLH for P. axelrodi.(AU)

El cardenal tetra Paracheirodon axelrodi y el tetra Aphyocharax anisitsi son dos especies de carácidos con alto valor comercial como peces ornamentales en América del Sur. Aunque ambas especies habitan en las capas medias de agua, el neón cardenal exhibe una distribución tropical, mientras que el tetra cola roja una distribución subtropical. En general estas especies son difíciles de cultivar, por lo que es esencial conocer algunos componentes clave de los sistemas neuroendocrinos para lograr su reproducción en cautiverio. Considerando la importancia de profundizar en el conocimiento de la fisiología reproductiva a través de la morfología funcional, en este trabajo realizamos, por primera vez, un análisis anatómico, morfológico e inmunohistoquímico de la glándula pituitaria de estas dos especies. En ambas especies, la hipófisis, del tipo leptobásica, se encontró en la zona ventral del hipotálamo y se caracteriza por una neurohipófisis con un tallo hipofisario bien desarrollado y una adenohipófisis globular. Los componentes hipofisarios, caracterizados por la histoquímica y la inmunohistoquímica, mostraron un patrón de distribución de tipos de células similares a otras especies de teleósteos, con solo pequeñas diferencias en la distribución de βFSH y βLH para P. axelrodi.(AU)

MALDI-MS argininyl bufadienolide esters fingerprint from parotoid gland secretions of Rhinella arenarum: Age, gender, and seasonal variation.

In many amphibians, the granular glands can be grouped in special regions forming macroglands. This is the case of toads, characterized by the presence of a pair of parotoid macroglands, strategically located to give protection by poison release in case of attacks. The product secreted consists of a wide variety of chemical compounds including proteins, peptides, biogenic amines, toxic steroidal bufadienolides, and various alkaloids, depending on the species. In this work, using Rhinella arenarum, we have performed, for the first time, the matrix assisted-ultraviolet laser desorption/ionization mass spectrometry and tandem mass spectrometry characterization of the components of the secretion used as crude material, just suspended in MeOH (or MeCN). The crude sample as a whole (whole suspension) was spotted on the matrix assisted-ultraviolet laser desorption plate for analysis. Electrospray ionization-Orbitrap was used for cross-checking experiments. The pattern of signals obtained at m/z ranges 600 to 800 and 1200 to 1600 could be assigned as the argininyl bufadienolide esters fingerprint characteristic of female and male. Variation patterns for gender (female, male), age (non-reproductive, reproductive), and season (non-reproductive, reproductive) are described.

Quantitative comparative analysis of the nasal chemosensory organs of anurans during larval development and metamorphosis highlights the relative importance of chemosensory subsystems in the group.

The anuran peripheral olfactory system is composed of a number of subsystems, represented by distinct neuroepithelia. These include the main olfactory epithelium and vomeronasal organ (found in most tetrapods) and three specialized epithelia of anurans: the buccal-exposed olfactory epithelium of larvae, and the olfactory recess and middle chamber epithelium of postmetamorphic animals. To better characterize the developmental changes in these subsystems across the life cycle, morphometric changes of the nasal chemosensory organs during larval development and metamorphosis were analyzed in three different anuran species (Rhinella arenarum, Hypsiboas pulchellus, and Xenopus laevis). We calculated the volume of the nasal chemosensory organs by measuring the neuroepithelial area from serial histological sections at four different stages. In larvae, the vomeronasal organ was relatively reduced in R. arenarum compared with the other two species; the buccal-exposed olfactory epithelium was absent in X. laevis, and best developed in H. pulchellus. In postmetamorphic animals, the olfactory epithelium (air-sensitive organ) was relatively bigger in terrestrial species (R. arenarum and H. pulchellus), whereas the vomeronasal and the middle chamber epithelia (water-sensitive organs) was best developed in X. laevis. A small olfactory recess (likely homologous with the middle chamber epithelium) was found in R. arenarum juveniles, but not in H. pulchellus. These results support the association of the vomeronasal and middle chamber epithelia with aquatic olfaction, as seen by their enhanced development in the secondarily aquatic juveniles of X. laevis. They also support a role for the larval buccal-exposed olfactory epithelium in assessment of oral contents: it was absent in X. laevis, an obligate suspension feeder, while present in the two grazing species. These initial quantitative results give, for the first time, insight into the functional importance of the peripheral olfactory subsystems across the anuran life cycle.

Overcrowding-mediated stress alters cell proliferation in key neuroendocrine areas during larval development in Rhinella arenarum.

Exposure to adverse environmental conditions can elicit a stress response, which results in an increase in endogenous corticosterone levels. In early life stages, it has been thoroughly demonstrated that amphibian larval growth and development is altered as a consequence of chronic stress by interfering with the metamorphic process, however, the underlying mechanisms involved have only been partially disentangled. We examined the effect of intraspecific competition on corticosterone levels during larval development of the toad Rhinella arenarum and its ultimate effects on cell proliferation in particular brain areas as well as the pituitary gland. While overcrowding altered the number of proliferating cells in the pituitary gland, hypothalamus, and third ventricle of the brain, no differences were observed in areas which are less associated with neuroendocrine processes, such as the first ventricle of the brain. Apoptosis was increased in hypothalamic regions but not in the pituitary. With regards to pituitary cell populations, thyrotrophs but not somatoatrophs and corticotrophs showed a decrease in the cell number in overcrowded larvae. Our study shows that alterations in growth and development, produced by stress, results from an imbalance in the neuroendocrine systems implicated in orchestrating the timing of metamorphosis.

AT1 receptor blockade delays postlactational mammary gland involution: a novel role for the renin angiotensin system.

Angiotensin II (AngII), the main effector peptide of the renin-angiotensin system (RAS), participates in multiple biological processes, including cell growth, apoptosis, and tissue remodeling. Since AngII activates, in different cell types, signal transducing pathways that are critical for mammary gland postlactational regression, we investigated the role of the RAS during this process. We found that exogenous administration of AngII in mammary glands of lactating Balb/c mice induced epithelium apoptosis [2.9±0.5% (control) vs. 9.6±1.1% (AngII); P < 0.001] and activation of the proapoptotic factor STAT3, an effect inhibited by irbesartan, an AT(1) receptor blocker. Subsequently, we studied the expression kinetics of RAS components during involution. We found that angiotensin-converting enzyme (ACE) mRNA expression peaked 6 h after weaning (5.7-fold; P<0.01), while induction of angiotensinogen and AT(1) and AT(2) receptors expression was detected 96 h after weaning (6.2-, 10-, and 6.2-fold increase, respectively; P<0.01). To assess the role of endogenously generated AngII, mice were treated with losartan, an AT(1) receptor blocker, during mammary involution. Mammary glands from losartan-treated mice showed activation of the survival factors AKT and BCL-(XL), significantly lower LIF and TNF-α mRNA expression (P<0.05), reduced apoptosis [12.1±2.1% (control) vs. 4.8±0.7% (losartan); P<0.001] and shedding of epithelial cells, inhibition of MMP-9 activity in a dose-dependent manner (80%; P<0.05; with losartan IC(50) value of 6.9 mg/kg/d] and lower collagen deposition and adipocyte invasion causing a delayed involution compared to vehicle-treated mice. Furthermore, mammary glands of forced weaned AT(1A)- and/or AT(1B)-deficient mice exhibited retarded apoptosis of epithelial cells [6.3±0.95% (WT) vs. 3.3±0.56% (AT(1A)/AT(1B) DKO); P<0.05] with remarkable delayed postlactational regression compared to wild-type animals. Taken together, these results strongly suggest that AngII, via the AT(1) receptor, plays a major role in mouse mammary gland involution identifying a novel role for the RAS. angiotensin system.

Glucocorticoid-induced impairment of mammary gland involution is associated with STAT5 and STAT3 signaling modulation.

The mammary epithelium undergoes cyclical periods of cellular proliferation, differentiation, and regression. During lactation, the signal transducer and activator of transcription factor (STAT)-5A and the glucocorticoid receptor (GR) synergize to induce milk protein expression and also act as survival factors. During involution, STAT3 activation mediates epithelial cell apoptosis and mammary gland remodeling. It has been shown that the administration of glucocorticoids at weaning prevents epithelial cell death, probably by extracellular matrix breakdown prevention. Our results show that the synthetic glucocorticoid dexamethasone (DEX) modulates STAT5A and STAT3 signaling and inhibits apoptosis induction in postlactating mouse mammary glands, only when administered within the first 48 h upon cessation of suckling. DEX administration right after weaning delayed STAT5A inactivation and degradation, preserving gene expression of target genes as ß-casein (bcas) and prolactin induced protein (pip). Weaning-triggered GR down-regulation is also delayed by the hormone treatment. Moreover, DEX administration delayed STAT3 activation and translocation into epithelial cells nuclei. In particular, DEX treatment impaired the increment in gene expression of signal transducer subunit gp130, normally up-regulated from lactation to involution and responsible for STAT3 activation. Therefore, the data shown herein indicate that glucocorticoids are able to modulate early involution by controlling the strong cross talk that GR, STAT5, and STAT3 pathways maintains in the mammary epithelium.

Heterogeneous distribution of G protein alpha subunits in the main olfactory and vomeronasal systems of Rhinella (Bufo) arenarum tadpoles.

We evaluated the presence of G protein subtypes Galpha(o), Galpha(i2), and Galpha(olf) in the main olfactory system (MOS) and accessory or vomeronasal system (VNS) of Rhinella (Bufo) arenarum tadpoles, and here describe the fine structure of the sensory cells in the olfactory epithelium (OE) and vomeronasal organ (VNO). The OE shows olfactory receptor neurons (ORNs) with cilia in the apical surface, and the vomeronasal receptor neurons (VRNs) of the VNO are covered with microvilli. Immunohistochemistry detected the presence of at least two segregated populations of ORNs throughout the OE, coupled to Galpha(olf) and Galpha(o). An antiserum against Galpha(i2) was ineffective in staining the ORNs. In the VNO, Galpha(o) neurons stained strongly but lacked immunoreactivity to any other Galpha subunit in all larval stages analyzed. Western blot analyses and preabsorption experiments confirmed the specificity of the commercial antisera used. The functional significance of the heterogeneous G-protein distribution in R. arenarum tadpoles is not clear, but the study of G- protein distributions in various amphibian species is important, since this vertebrate group played a key role in the evolution of tetrapods. A more complete knowledge of the amphibian MOS and VNS would help to understand the functional organization and evolution of vertebrate chemosensory systems. This work demonstrates, for the first time, the existence of a segregated distribution of G-proteins in the OE of R. arenarum tadpoles.

Amphibian larvae and zinc sulphate: a suitable model to study the role of brain-derived neurotrophic factor (BDNF) in the neuronal turnover of the olfactory epithelium.

The vertebrate olfactory system has fascinated neurobiologists over the last six decades because of its ability to replace its neurons and synaptic connections continuously throughout adult life, under both physiological and pathological conditions. Among the factors that are proposed to be involved in this regenerative potential, brain-derived neurotrophic factor (BDNF) is a candidate for having an important role in the neuronal turnover in the olfactory epithelium (OE) because of its well-documented neurogenic and trophic effects throughout the nervous system. The aim of the present study was to generate a suitable model to study the participation of BDNF in the recovery of the OE after injury in vivo. We developed an experimental design in which the OE of Rhinella arenarum tadpoles could be easily and selectively damaged by immersing the animals in ZnSO(4) solutions of various concentrations for differing time periods. Image analysis of histological sections showed that different combinations of each of these conditions produced statistically different degrees of injury to the olfactory tissue. We also observed that the morphology of the OE was restored within a few days of recovery after ZnSO(4) treatment. Immunohistochemical analysis of BDNF was performed with an antiserum whose specificity was confirmed by Western blotting, and which showed drastic changes in the abundance and distribution pattern of this neurotrophin in the damaged olfactory system. Our results thus suggest that BDNF is involved in the regeneration of the OE of amphibian larvae, and that our approach is suitable for further investigations of this topic.

Presence of beta-follicle-stimulating hormone and beta-luteinizing hormone transcripts in the brain of Cichlasoma dimerus (Perciformes: Cichlidae): effect of brain-derived gonadotropins on pituitary hormone release.

Follicle-stimulating hormone (FSH) and luteinizing hormone (LH) play key roles in vertebrate gametogenesis and steroidogenesis. They are mainly synthesized in the pituitary gland. While investigating the ontogeny of FSH and LH cells in the cichlid fish Cichlasoma dimerus by immunohistochemistry (IHC), we unexpectedly found immunoreactive neurons in the preoptic area, sending their projections through different brain areas and neurohypophysis. Our previous work using Western blot and IHC techniques applied to the adult brain confirmed these findings. To further demonstrate the extrapituitary expression of these hormones, we performed RT-PCR detecting sequences coding for beta-FSH and beta-LH subunits in the C. dimerus pituitary and brain (preoptic-hypothalamic area). The expression of these transcripts in both organs was consistent with their peptide expression showing a high sequence homology when compared with other phylogenetically related fish. An individual pituitary in vitro culture system was utilized to study the possible modulatory effect of brain-derived gonadotropins on pituitary hormone secretion. Pituitary explants were cultured with different concentrations of LH or FSH, and the culture media were analyzed by Western blot. Exogenous LH produced a dose-dependent increase in pituitary beta-LH, beta-FSH and somatolactin (SL) releases. No effect was observed on growth hormone (GH). The effect on prolactin (PRL) was not consistent among treatments. Exogenous FSH produced an inhibition in beta-LH release, dose-dependent increases in beta-FSH and SL releases, and no effect on PRL and GH releases. These findings support the concept of regulation of pituitary trophic hormones by brain-derived gonadotropins.

Immunohistochemical localization of vascular endothelial growth factor and its receptor Flk-1 in the amphibian developing principal and accessory olfactory system.

In the last years several studies have shown that vascular endothelial growth factor (VEGF) is present in neural stem cells and mature neurons from different neural tissues where it may play an important role as a neuroproliferative and/or antiapoptotic factor. The olfactory neuroepithelium has the capability to replace dying neurons with new neurons formed by cell division from stem cells in the basal region of the epithelium. The present study demonstrates, for the first time, that VEGF is present in the olfactory epithelium, nerves and bulbs (both main and accessory) during the development of the toad Bufo arenarum. In this report, we detected VEGF immunoreactivity in mature olfactory neurons from early larval stages until the beginning of the metamorphic climax. VEGF expression decreases dramatically after metamorphosis. VEGF receptor Flk-1 was localized by immunohistochemistry, from premetamorphic larval stages until the climax in the neurons of the olfactory epithelium with a more intense labeling in the basal cell layer. Double-label immunofluorescence studies localized VEGF to the cytoplasm and the nucleus of mature neurons whereas Flk-1 was expressed in cell membranes. Flk-1 was present in neurons of both the main and accessory olfactory bulbs. After the end of metamorphosis, Flk-1 expression was limited to basal cells in the olfactory epithelium and Bowman's glands. The main and accessory olfactory bulbs showed the same pattern of Flk-1 immunostaining before and after the end of metamorphosis. The presence of VEGF and its receptor in the olfactory system suggests that VEGF may play an important role during neural development.

Identification of immunoreactive FSH and LH cells in the cichlid fish Cichlasoma dimerus during the ontogeny and sexual differentiation.

Follicle-stimulating hormone (FSH) and luteinizing hormone (LH) expressing cells were detected in pituitary, brain and ovary of the Perciform cichlid fish Cichlasoma dimerus. This detection was carried out by immunohistochemistry (IHC) and Western blot techniques using antisera of the Cyprinodontiform Fundulus heteroclitus raised against the conservative region of the teleost betaFSH and the betaLH subunits. The estimated molecular weights were 24 kDa for LH and 19 and 15 kDa for FSH. In the adult pituitary, both cell types were distributed along mid and ventral zones of the proximal pars distalis (PPD, mid-immunoreactive cells), and along the ventral and dorsal external border of the pars intermedia (PI, high-immunoreactive cells). Double IHC showed that FSH and LH are mainly expressed in different pituitary cells. FSH cells were detected in the pituitary around day 21 after hatching (ah) (prior to sex differentiation), while LH cells were detected by day 60 ah (during the sexual differentiation period). A correlation between gonadal sex differentiation and FSH was demonstrated in a 15 days organ culture system. FSH and LH neurons were localized in the nucleus lateralis tuberis and their fibers project through the ventral hypothalamus, preoptic area and neurohypophysis. FSH neurons differentiated on day 21 ah, while LH neurons appeared on day 15 ah. In the ovary, the immunoreactivity for both FSH and LH was restricted to the cytoplasm of previtellogenic and early vitellogenic oocytes.

Relationship between steroidogenesis and spermiation in Rana catesbeiana and Leptodactylus ocellatus.

The present study employs an in vitro system to analyse the role of steroid hormones in hCG-induced spermiation in two species of anuran amphibian: Rana catesbeiana and Leptodactylus ocellatus. In vitro spermiation was induced with 10 IU hCG and the effect of different steroid-biosynthesis inhibitors was analysed. Cyanoketone (10(-5)M), an inhibitor of 3-oxo-4-ene steroid biosynthesis, did not block hCG-inducing activity even when biosynthesis of androgen was significantly reduced. These results clearly showed that, in both species, spermiation-inducing action of hCG does not depend on the biosynthesis of 3-oxo-4-ene steroids. Moreover, when combined inhibitors, aminoglutethimide (10(-5)M) plus cyanoketone (10(-5)M), were employed, spermiation evoked by hCG was not modified while hCG-induced androgen secretion significantly decreased. Additionally, none of the steroids used, progesterone, 17, 20 alpha-dihydroxy-4-pregnen-3-one, testosterone and 5 alpha-dihydrotestosterone, were able to induce spermiation in the absence of hCG, confirming that steroids are not involved in that process. In conclusion, as previously described in Bufo arenarum, in L. ocellatus and R. catesbeiana hCG-induced spermiation does not depend on steroid biosynthesis.

Steroid production in toads.

In Bufo arenarum, androgen biosynthesis occurs through a complete 5-ene pathway, including 5-androstane-3beta,17beta-diol as the immediate precursor of testosterone. Besides, steroidogenesis changes during the breeding period, turning from androgens to C(21)-steroids such as 5alpha-pregnan-3alpha,20alpha-diol, 3alpha-hydroxy-5alpha-pregnan-20-one and 5alpha-pregnan-3,20-dione. In B. arenarum, steroid hormones are not involved in hCG-induced spermiation, suggesting that the steroidogenic shift to C(21)-steroids during the breeding be not related to spermiation. The activity of 17-hydroxylase-C(17-20) lyase (CypP450(c17)) decreases during the reproductive season, suggesting that this enzyme would represent a key enzyme in the regulation of seasonal changes. However, the increase in the affinity for pregnenolone of 3beta-hydroxysteroid dehydrogenase (3alphaHSD)/isomerase could also be involved. Moreover, the reduction in CypP450(c17) leading to a reduction in C(19)-steroids, among them dehydroepiandrosterone (DHE), would contribute to the conversion of pregnenolone into progesterone, avoiding the non-competitive inhibition exerted by DHE on this transformation. Additionally, CypP450(c17) possesses a higher affinity for pregnenolone than for progesterone, explaining the predominance of the 5-ene pathway for testosterone biosynthesis. Animals in reproductive condition showed a significant reduction in circulating androgens, enhancing the physiological relevance of all the in vitro results. The in vitro effects of mGnRH and hrFSH on testicular steroidogenesis revealed that both hormones inhibited CypP450(c17) activity. In summary, these results demonstrate that, in B. arenarum, the change in testicular steroidogenesis during the reproductive period could be partially due to an FSH and GnRH-induced decrease in CypP450(c17) activity.

Effects of mGnRH on testicular steroidogenesis in the toad Bufo arenarum.

GnRH controls vertebrate reproduction in several ways. This hormone not only affects the secretion of gonadotropins from the pituitary gland but also has a direct influence on several gonadal functions such as steroidogenesis, spermatogenesis, and spermiation. In the present paper we have studied the in vitro effects of GnRH on the testicular steroidogenesis of Bufo arenarum to ascertain the role of this peptide in the control of the steroidogenic pathway previously described in this species. It was found that GnRH is able to reduce basal as well as hCG-stimulated testosterone release, having an inhibitory effect on P450(c17) activity. Thus, GnRH could be involved in the mechanism that regulates the metabolic change in the testicular steroidogenesis. Additionally, testicular GnRH binding site has been characterised, showing a K(d) of 34 nM and a maximum binding of 4.7 pmol/mg protein.