Gonadal morphogenesis in the South American toad Rhinella Arenarum (Anura, Bufonidae) unveils an extremely delayed rate of sex differentiation.

Among anurans, Bufonids are recognized for their retarded sex differentiation. However, few studies have addressed gonadal morphogenesis in this family. Here, we analyzed the early gonadogenesis in laboratory-reared Rhinella arenarum. Few germ cells were identified in the genital ridge at Gosner stage 26. At metamorphosis, somatic cells and germ cells were observed in the outer region of the undifferentiated gonad, whereas the central region was occupied by stromal tissue. A cortico-medullary organization was first recognized on Day 7 postmetamorphosis. The cortex was composed of germ cells and encompassing epithelial cells, whereas the medulla contained cells presumptively derived from the coelomic epithelium. Medullary somatic cells formed metameric knots along the length of the undifferentiated gonad. Consequently, a series of 12-14 gonomeres became recognizable externally. The first sign of ovarian differentiation was observed on Day 15 postmetamorphosis, when a cavity was formed within each gonomere. In contrast, testes were recognized by a uniform distribution of germ cells and intermingled somatic cells, as the division into cortex and medulla was lost. By Day 50 postmetamorphosis, the gonadal metameric organization was still apparent both in the ovaries and testes. Follicles containing diplotene oocytes were observed within the ovary. In the testis, an incipient lobular architecture was recognized without initiation of meiosis within the seminiferous cords. These observations reveal an extremely delayed gonadal development in R. arenarum, not reported previously for other anuran species. In addition, the late differentiation of the gonads contrasted with the early appearance of follicles in the Bidder's organ. Lastly, we observed that delayed metamorphs exhibited an undifferentiated gonad, demonstrating that gonadogenesis in this species is more dependent on somatic development than on age.

Effects of waterborne exposure to the antidepressant fluoxetine on swimming, shoaling and anxiety behaviours of the mosquitofish Gambusia holbrooki.

Chemical pollution from pharmaceuticals is increasingly recognized as a major hazard to the aquatic biota. Among the wide variety of pharmaceuticals, fluoxetine (FLX) is one of the most widely prescribed antidepressants, and therefore, it is frequently identified in the aquatic environment. As FLX is designed to alter human behaviour and many physiological pathways are conserved across vertebrates, this drug may affect the behaviour of fish living in FLX-polluted environments. Here, we exposed groups of female mosquitofish Gambusia holbrooki to waterborne FLX for 14 days, under semi-static conditions with daily renewal of test solutions. Following exposure, we conducted a set of behavioural assays in individual fish, aimed at assessing the effects of FLX on their locomotor activity and behavioural responses. We found that FLX impaired swimming behaviour at high concentrations (25 µg/L and 50 µg/L) but not at low concentrations close to environmental levels (1 µg/L and 5 µg/L). When swimming activity was assessed 5 min after transfer of the focal fish to the testing tank, 50 µg/L FLX was the only concentration showing significant effects. However, when the same trials were performed 24 h later, 25 µg/L FLX turned out to be an effect concentration in addition to 50 µg/L. Interestingly, these concentrations would elicit fish plasma concentrations comprised within the range of human therapeutic doses. When subjected to a light/dark preference test, fish showed tendency to remain less time in the dark area at high FLX concentrations, thus suggesting an anti-anxiety response. Shoaling behaviour was not affected by FLX exposure. Our study contributes to the growing body of literature evaluating the effects of FLX on animal behaviour. Regarding the experimental design used in behavioural testing, our findings suggest that focal fish should be subjected to long habituation periods, namely of at least a few hours, in order to better assess the effects of drug exposure.

Growth and reproduction in captivity unveils remarkable life-history plasticity in the smallnose fanskate, Sympterygia bonapartii (Chondrichthyes: Rajiformes)

We analyzed growth and reproduction of captive-born smallnose fanskates Sympterygia bonapartii. Egg cases were obtained from oviposition of two females caught in the wild and held at Temaikèn Aquarium. Following hatching, growth was analyzed in 13 females and 21 males until sexual maturity. Pattern of oviposition activity and reproductive performance were evaluated in six of the captive-reared females. Four models were fitted to growth data, among which the logistic function was the one attaining the best fit. The highest growth rate for both sexes was recorded during the first year of life, whereas growth was significantly higher in females than in males during the second year. Size at first oviposition was 61.7 ± 3.5 cm TL, similar to wild specimens. However, captive-reared females reached maturity before two years of age, i.e. much earlier than wild skates, implying a significant phenotypic plasticity in this species. The similarity in size at maturity and the difference in age at maturity between captive and wild specimens indicate that there is a decoupling of both parameters mediated through growth rates. Captive-born skates reproduced successfully and yielded viable offspring, indicating that the environment at Temaikèn Aquarium is suitable for S. bonapartii to attain its full life cycle.(AU)

Analizamos el crecimiento y la reproducción de ejemplares de raya marmorada, Sympterygia bonapartii nacidas en cautiverio. Se obtuvieron ovicápsulas a partir de la oviposición de dos hembras capturadas en la naturaleza y mantenidas en el Acuario Temaikèn. Posteriormente a la eclosión, se analizó el crecimiento en 13 hembras y 21 machos hasta la madurez sexual. Se evaluó el patrón de oviposición y la performance reproductiva en seis de las hembras criadas en cautiverio. Se ajustaron cuatro modelos a los datos de crecimiento, entre los cuales la función logística fue la que logró el mejor ajuste. Para ambos sexos, la mayor tasa de crecimiento se registró durante el primer año de vida. Durante el segundo año, la tasa de crecimiento fue significativamente mayor en hembras que en machos. La talla de primera oviposición fue de 61,7 ± 3,5 cm LT, similar a la estimada en rayas silvestres. Sin embargo, las hembras criadas en cautiverio alcanzaron la madurez antes de los dos años de vida, es decir mucho antes que en el ambiente natural, lo que implica una plasticidad fenotípica significativa en esta especie. La similitud en la talla de madurez y la diferencia en la edad de madurez entre individuos cautivos y silvestres indican que hay un desacople de ambos parámetros mediado por la tasa de crecimiento. Los individuos nacidos en cautiverio se reprodujeron exitosamente y dejaron descendencia viable, lo que indica que el ambiente del Acuario Temaikèn es adecuado para que S. bonapartii complete su ciclo de vida.(AU)

Effects of a fungicide formulation on embryo-larval development, metamorphosis, and gonadogenesis of the South American toad Rhinella arenarum.

Sublethal toxicity of the formulated fungicide Maxim(®) XL on embryonic, larval and juvenile development of Rhinella arenarum was evaluated by means of standardized bioassays. Maxim(®) XL, one of the most used fungicides in Argentina, is based on a mixture of two active ingredients: Fludioxonil and Metalaxyl-M. Maxim(®) XL exposure induced severe sublethal effects on the embryos, expressed as general underdevelopment, axial flexures, microcephaly, cellular dissociation, abnormal pigmentation, underdeveloped gills, marked edema and wavy tail. As the embryo development advanced, alterations in behavior as spasmodic contractions, general weakness and inanition were observed. Maxim(®) XL did not affect neither the time required to complete metamorphosis nor sex proportions, but gonadal development and differentiation were impaired. Gross gonadal analysis revealed a significant proportion of exposed individuals with underdevelopment of one or both gonads. Histological analysis confirmed that 18% and 10% of the individuals exposed to 0.25 and 2mg/L Maxim(®) XL, respectively, exhibited undifferentiated gonads characterized by a reduced number (or absence) of germ cells. Taking into account the risk evaluation performed by means of Hazard Quotients, this fungicide could be a threat to R. arenarum populations under chronic exposure. This study represents the first evidence of toxic effects exerted by Maxim(®) XL on amphibians. Finally, our findings highlight the properties of this fungicide that might jeopardize non-target living species exposed to it in agricultural environments.

Persistent organic pollutants (POPs) in fish with different feeding habits inhabiting a shallow lake ecosystem.

The occurrence of persistent organic pollutants (POPs) in the environment can affect organisms inhabiting aquatic systems, in particular shallow lakes that are vulnerable to environmental stressors. This study aimed to assess POPs accumulation and changes at histological and physiological levels in tissues of three fish species with different trophic habits. Gills, brain, muscle, liver and gonads of Odontesthes bonariensis, Oligosarcus jenynsii and Cyphocharax voga were collected from the shallow lake La Peregrina, located in an agricultural area from Argentina. In addition, contaminant levels in surface water (SW), suspended particulate matter (SPM) and bottom sediments (BS) were assessed. Histological lesions were evaluated in fish tissues and levels of vitellogenin (VTG) were assessed in plasma of male fish in order to correlate these alterations with the presence of POPs in the environment. Organochlorine pesticides (OCPs), polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers (PBDEs) were determined by GC-ECD. Biotic and abiotic samples showed the same POPs distribution pattern: OCPs>PCBs>PBDEs. Although tissue distribution of OCPs was species-specific, muscle showed the lowest levels in all species. The most abundant contaminants were endosulfans, suggesting their widespread use in the area. O. bonariensis showed the highest endosulfans levels in liver (184.2-219ngg(-1)wet w), which was associated with the high SPM levels considering this species is a filter feeder. The occurrence of PCBs and PBDEs shows the ubiquity of these pollutants in the area. Histological lesions in gills and liver of O. bonariensis and O. jenynsii, might be related with the high levels of endosulfans in these organs. The detection of VTG in males warns about a possible exposure to estrogenic compounds in the environment. In conclusion, the simultaneous exposure of fish to multiple environmental pollutants leads to different alterations, so measures should be taken in order to prevent their occurrence and toxic effects.

Effects of waterborne exposure to 17ß-estradiol and 4-tert-octylphenol on early life stages of the South American cichlid fish Cichlasoma dimerus.

Estrogenic chemicals are often detected in the aquatic environment and can negatively affect animal development and reproduction. In teleost fishes, the hormonal regulation during a critical period of larval development has a strong influence on gonadal sex differentiation; thus this process may be affected by the exposure to environmental estrogens. In this study, we first assessed the lethal acute toxicity of the natural estrogen 17ß-estradiol (E2) and the weaker estrogen mimics 4-tert-octylphenol (OP) and 4-nonylphenol (NP) on larval stages of the South American cichlid fish Cichlasoma dimerus. In a further experiment, we analyzed the effects of chronic waterborne exposure to E2 and OP on gonad development and sex differentiation. Exposure to high concentrations of E2 had a pronounced feminizing effect directing sex differentiation towards ovarian development, while testis development was inhibited at a lower, environmentally relevant concentration. Among OP-exposed fish, 15-38.5% of the males exhibited testicular oocytes (TOs), a commonly reported biomarker of estrogenic exposure. However, since TOs were also recorded in control males and the proportion of males with TOs was not significantly higher in OP treatments, their occurrence could not be attributed to OP exposure. In addition, TOs did not seem to impair male gonad development and functionality since normal spermatogenesis was observed in testes of OP-treated fish. These results indicate that E2 occurring in the South American aquatic environment may affect male reproductive development and pose a risk for wild C. dimerus, especially under prolonged exposure, while the effects of weaker xenoestrogens such as OP would be negligible for gonad development in this species. As illustrated by this study, the natural occurrence of TOs indicates that conclusions concerning the causes of this phenomenon must be drawn with care.

Studies on the reproductive and developmental biology of Cichlasoma dimerus (Percifomes, Cichlidae).

Many characteristics of the South American teleost fish Cichlasoma dimerus (body size, easy breeding, undemanding maintenance) make it amenable to laboratory studies. In the last years, many of the fundamental aspects of its reproductive and developmental biology have been addressed in our laboratory. Rather recently, the immunohistochemical localization of pituitary hormones involved in reproduction and in background color adaptation has been described in both adult and developing individuals, and the role of FSH in ovarian differentiation has been established. These findings have been correlated with mapping of some of their brain-derived controlling hormones. The latter include brain-derived gonadotropins which were shown to be active in vitro in the control of pituitary hormone secretions. The emerging picture shows C. dimerus as an interesting species in which many of their basic features have already been investigated and which conform a solid platform for comparative studies correlating neurohormones, pituitary hormones and behavior, from the molecular to the organismic level.

Studies on the reproductive and developmental biology of Cichlasoma dimerus (Percifomes, Cichlidae): [review]

Many characteristics of the South American teleost fish Cichlasoma dimerus (body size, easy breeding, undemanding maintenance) make it amenable to laboratory studies. In the last years, many of the fundamental aspects of its reproductive and developmental biology have been addressed in our laboratory. Rather recently, the immunohistochemical localization of pituitary hormones involved in reproduction and in background color adaptation has been described in both adult and developing individuals, and the role of FSH in ovarian differentiation has been established. These findings have been correlated with mapping of some of their brain-derived controlling hormones. The latter include brain-derived gonadotropins which were shown to be active in vitro in the control of pituitary hormone secretions. The emerging picture shows C. dimerus as an interesting species in which many of their basic features have already been investigated and which conform a solid platform for comparative studies correlating neurohormones, pituitary hormones and behavior, from the molecular to the organismic level.

Studies on the reproductive and developmental biology of Cichlasoma dimerus (Percifomes, Cichlidae): [review]

Many characteristics of the South American teleost fish Cichlasoma dimerus (body size, easy breeding, undemanding maintenance) make it amenable to laboratory studies. In the last years, many of the fundamental aspects of its reproductive and developmental biology have been addressed in our laboratory. Rather recently, the immunohistochemical localization of pituitary hormones involved in reproduction and in background color adaptation has been described in both adult and developing individuals, and the role of FSH in ovarian differentiation has been established. These findings have been correlated with mapping of some of their brain-derived controlling hormones. The latter include brain-derived gonadotropins which were shown to be active in vitro in the control of pituitary hormone secretions. The emerging picture shows C. dimerus as an interesting species in which many of their basic features have already been investigated and which conform a solid platform for comparative studies correlating neurohormones, pituitary hormones and behavior, from the molecular to the organismic level.(AU)

Efficacy of vardenafil and influence on self-esteem and self-confidence in patients with severe erectile dysfunction.

AIM: To assess the efficacy of vardenafil in a population of Spanish men with erectile dysfunction (ED), its influence on patients' self-esteem and self-confidence, and its effect on their quality of life. MAIN OUTCOME MEASURES: Efficacy was assessed by the International Index of Erectile Function-Erectile Function (IIEF-EF) domain, the Rosenberg Self-Esteem scale, the Johnson and McCoy Self-Confidence scale, the Medical Outcome Short Form (SF-36) scale, items 2 and 3 of the Sexual Encounter Profile questionnaire, and the Global Assessment Question (GAQ). Safety assessments included laboratory tests, physical exam, electrocardiogram, vital signs, and adverse events. METHODS: This was a randomized, double-blind, multicenter, placebo-controlled study. After a 4-week treatment-free period, patients received flexible-dose vardenafil or placebo for 12 weeks. The initial dose was 10 mg, which could be titrated up to 20 mg or down to 5 mg at weeks 4 and 8. RESULTS: A total of 121 patients were included in the intention-to-treat analysis (61 on vardenafil and 60 on placebo). Of these, 16 in the vardenafil group and 14 in the placebo group had severe ED. There was a greater improvement in IIEF-EF domain score with vardenafil vs. placebo for all patients (score change of 10.9 vs. 1.6, respectively, P < 0.001) and for patients with severe ED (score change of 13.4 vs. 2.2, respectively, P = 0.011). A significant difference in favor of vardenafil was also observed for positive responses to the GAQ (73.8% vs. 25.0%, P < 0.001). After 12 weeks, vardenafil-treated patients with severe ED showed a significant improvement in their self-esteem compared with patients receiving placebo (change from baseline -1.51 vs. 3.54, respectively, P = 0.036). Vardenafil treatment was well tolerated. CONCLUSIONS: Vardenafil was highly effective for improving EF in all patients with ED, and resulted in significant improvements in self-esteem in patients with severe ED.

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.

Gonadal development and sex differentiation in the cichlid fish Cichlasoma dimerus (Teleostei, Perciformes): a light- and electron-microscopic study.

Although the overall pattern and timing of gonadal sex differentiation have been established in a considerable number of teleosts, the ultrastructure of early stages of gonadal development is not well documented. In this study, gonads from larval and juvenile stages of laboratory-reared Cichlasoma dimerus were examined at the light-microscopic and ultrastructural levels. This freshwater species adapts easily to captivity and spawns with high frequency during 8 months of the year, providing an appropriate model for developmental studies. Larvae and juveniles were kept at a water temperature of 26.5 +/- 1 degrees C and a 12:12 hour photoperiod. Gonadal development was documented from 14-100 days postfertilization, covering the period of histologically discernible sex differentiation. Gonadal tissue was processed according to standard techniques for light and electron microscopy. C. dimerus, a perciform teleost, is classified as a differentiated gonochorist, in which an indifferent gonad develops directly into a testis or ovary. On day 14, the gonadal primordium consists of a few germ cells surrounded by enveloping somatic cells. Ovarian differentiation precedes testicular differentiation, as usual in teleost fishes. The earliest signs of differentiation, detected from day 42 onward, include the onset of meiotic activity in newly formed oocytes, which is soon accompanied by increased oogonial mitotic proliferation and the somatic reorganization of the presumptive ovary. The ovarian cavity is completely formed by day 65. Numerous follicles containing perinucleolar oocytes are observed by day 100. In contrast, signs of morphological differentiation in the presumptive testis are not observed until day 72. By day 100, the unrestricted lobular organization of the testis is evident. The latest stage of spermatogenesis observed by this time of testicular development is spermatocyte II.

Ontogeny and distribution of gonadotropin-releasing hormone (GnRH) neuronal systems in the brain of the cichlid fish Cichlasoma dimerus.

Using immunocytochemistry we have described the distribution and ontogeny of three distinct gonadotropin-releasing hormone (GnRH) neural systems, emphasizing the analysis during the period of sex differentiation in the South American cichlid fish Cichlasoma dimerus. In the forebrain a group of neurones immunoreactive to salmon GnRH that formed clusters in the nucleus olfacto retinalis (NOR), was located at the junction of the olfactory bulb and the telencephalon. These neurones differentiated 3 days after fertilization from the olfactory placodes. GnRH immunoreactive neurones along the olfactory nerves through the rostrobasal olfactory bulb were observed on day 4 and at the NOR on day 10. A group of neurones immunoreactive to chicken GnRH II was seen in the dorsal midbrain tegmentum. They originate from the ventricular ependyma between days 5 and 6. These neurones remained close to blood vessels throughout development. Between days 22 and 30 a group of neurones immunoreactive to seabream GnRH was detected in the anterior basal preoptic area. GnRH innervation of the pituitary was detected after the differentiation of preoptic neurones and in coincidence with gonadal differentiation. We hypothesize that the GnRH neural systems have three distinct embryonic origins. Furthermore, we show that the NOR and the midbrain GnRH neurones might have functions other than gonadal development, whereas the preoptic GnRH neurones in C. dimerus might be associated with gonadal sex differentiation.

Papel de la resonancia magnética en la patología testicular y paratesticular / Role of magnetic resonance in testicular and paratesticular disease

Objetivo: Analizar y describir los hallazgos diagnósticos más relevantes en la patología testicular y paratesticular mediante Resonancia Magnética. Material y métodos: Se analizan 78 pacientes seleccionados ecográficamente, en los cuales se observó patología testicular o paratesticular, y en aquellos con sospecha clínica de enfermedad y ecografía no concluyente. Se utilizó una unidad de resonancia magnética (RM) de 0,5 T (Gyroscan T5, Philips) usando una bobina de superficie fijada sobre una sábana que cubría la región testicular. Se emplearon secuencias T1 eco del espín precontraste y poscontrate (TR/TE: 572/20, FOV: 180 mm, grosor: 6 mm, gap: 0,6 mm y matriz: 256 × 256) T2 FSE (TR/TE: 3257/120) y STIR. En todos los casos se llegó al diagnóstico definitivo mediante resultados anatomopatológicos o seguimiento. Resultados: Las secuencias potenciadas en T2 valoran el parénquima testicular por tener una señal alta, diferenciándose el epidídimo por su hipointensidad. En el escroto agudo la RM alcanzó una sensibilidad, especificidad e índice kappa del 100 por ciento. En los tumores no se obtuvo mayor rendimiento diagnóstico que la ecografía salvo en el tumor de Leydig el cual mostró un gran realce tras la administración de contraste. La capacidad multiplanar fue de especial importancia en la localización intratesticular o extratesticular de las lesiones. Conclusiones: Aunque la ecografía representa la primera técnica diagnóstica en las enfermedades escrotales hemos de considerar la RM como una técnica no sólo de apoyo en aquellos casos que no sean concluyentes, sino también de especial importancia en entidades como el escroto agudo y especialmente en los casos de torsión testicular (AU)

Immunocytochemical localization of different cell types in the adenohypophysis of the cichlid fish Cichlasoma dimerus (Heckel, 1840)

The adenohypophysis of the cichlid fish Cichlasoma dimerus was studied using the avidin-biotin-peroxidase method with antisera raised against piscine pituitary hormones and heterologous antisera against mammalian pituitary hormones. Antiserum raised against rabbit ACTH recognized a group of cells bordering the neurohypophysis (NH) in the rostral pars distalis (RPD). Anti-chum salmon prolactin (PRL) identified a compact group of cells in the periphery of the RPD. Gonadotropin II (GTH II), thyrotropin (TSH) and growth hormone (GH)-ir cells were localized in the proximal pars distalis. Ir-GTH II cells were also observed in the dorsal area of the pars intermedia (PI). Ir-GTH I cells could not be identified using anti-chum salmon GTH I, this may be due either to a failure of the antisera to recognize the gonadotropin or to a low expression of the hormone in adults of this species. PAS positive cells from the PI bound specifically with three different antisera raised against somatolactin (SL) of four different fish species. These cells surrounded deep branches of the NH in the PI.(AU)

Immunocytochemical localization of different cell types in the adenohypophysis of the cichlid fish Cichlasoma dimerus (Heckel, 1840)

The adenohypophysis of the cichlid fish Cichlasoma dimerus was studied using the avidin-biotin-peroxidase method with antisera raised against piscine pituitary hormones and heterologous antisera against mammalian pituitary hormones. Antiserum raised against rabbit ACTH recognized a group of cells bordering the neurohypophysis (NH) in the rostral pars distalis (RPD). Anti-chum salmon prolactin (PRL) identified a compact group of cells in the periphery of the RPD. Gonadotropin II (GTH II), thyrotropin (TSH) and growth hormone (GH)-ir cells were localized in the proximal pars distalis. Ir-GTH II cells were also observed in the dorsal area of the pars intermedia (PI). Ir-GTH I cells could not be identified using anti-chum salmon GTH I, this may be due either to a failure of the antisera to recognize the gonadotropin or to a low expression of the hormone in adults of this species. PAS positive cells from the PI bound specifically with three different antisera raised against somatolactin (SL) of four different fish species. These cells surrounded deep branches of the NH in the PI.