Machos adultos de P. lineatus tiveram suas gônadas processadas de acordo com as rotinas de microscopia de luz e microscopia eletrônica de transmissão. Para a cultura de células, os testículos foram digeridos enzimaticamente, a suspensão testicular foi separada por gradiente descontínuo com Percoll seguido pelo plaqueamento diferencial por adesão e as células foram cultivadas. Foi realizado o método de enriquecimento das espermatogônias por citometria de fluxo. Os testículos apresentam as regiões anterior, média e posterior com distribuição semelhante dos tipos de células, e o diâmetro nuclear das células germinativas diminui significativamente durante a espermatogênese. As espermatogônias cultivadas por 15 dias com meio para proliferação celular resultaram em grandes aglomerados celulares que foram caracterizados com o anti-Vasa, anti-GFRa1 e anti-OCT4. As culturas que receberam o meio para diferenciação celular mostraram processo de proliferação lento das espermatogônias primárias comparado com a cultura que teve o meio indicado para proliferação celular. / Adult males of P. lineatus had their gonads used according routines of light microscopy and transmission electron microscopy. For cell culture, the testes were enzymatically digested; testicular suspension was separated by discontinuous gradient with Percoll followed by adhesion differential plating and the cells were cultured. The enrichment of the spermatogonia was carried by flow cytometry. The testes present three regions with similar distribution of cell types, and nuclear diameter of germ cells decreases significantly during spermatogenesis. The spermatogonia cultured for 15 days with medium for cell proliferation, resulted in large cell agglomerates which were characterized with the antibodies anti-Vasa, anti-GFRa1 and anti-anti-OCT4. The cultures that receiving medium for cell differentiation showed slow proliferation process of primary spermatogonia compared to cell culture medium suggestive for cell differentiation.

Les effets négatifs des produits pharmaceutiques (PPs) sur les écosystèmes aquatiques est une préoccupation de plus en plus sensible. Nous avons analysé les effets du 17α-éthinylestradiol (EE2), composant des pilules contraceptives, et de la fluoxétine (FLX), l'antidépresseur le plus vendu au monde, sur deux poissons. Le pejerrey (Odontesthes bonariensis) vit dans des lagunes qui sont des réservoirs d'eaux usées, où la présence d'EE2 est détectée. En utilisant l'expression des gènes cyp19a1b (aromatase cérébrale) et cyp19a1a (aromatase gonadique) comment marqueurs biologiques nous avons constaté que l'exposition à EE2 n'affectait pas l'expression du gène cyp19a1b. Cependant, augmente significativement l'expression du cyp19a1a, liée à la différenciation ovarienne, et diminue l'expression de hsd11b2, liée a la différenciation testiculaire, chez les larves et mâles juvéniles. Aussi, produit une déviation des ratios male/femelles en faveur des femelles chez les larves et l'apparition de caractéristiques typique du développement ovarien dans les testicules de mâles juvéniles. Chez le poisson zèbre (Danio rerio) nous avons évalué le rôle de la sérotonine (5-HT) comme modulateur de la prolifération; et l'effet de la FLX, inhibiteur sélectif de sa recapture. Tout d'abord, nous avons montré que les neurones sérotoninergiques de l'hypothalamus sont générés à partir de cellules gliales radiaires présente dans cette région. Ensuite, nous avons constaté une réduction significative du nombre de cellules en prolifération dans le noyau du recessus lateral (NRL) et posterior (NRP) de l'hypothalamus, après de l'inhibition de la synthèse de 5-HT ; et dans le NRL, après d'exposition a FLX. / Over the last 10-15 years risks of pharmaceuticals and personal care products (PPCPs) in the environment have acquired significant relevance. In the present work we studied the effects of 17α-ethinylestradiol (EE2), component of contraceptive pills, and fluoxetine (FLX), the world’s most widely prescribed antidepressant, in fishes. Pejerrey (Odontesthes bonariensis) a south American teleost fish that inhabit aquatic ecosystems where EE2 has been reported. Exposure to EE2 did not alter the expression of cyp19a1b (brain aromatase). In contrast, expression of cyp19a1a (gonadal aromatase), involved in ovarian differentiation, was up-regulated; while expression of hsd11b2, associated with testicular differentiation, was down-regulated in larvae or juvenile males. Also, EE2 was able to shift sex ratios toward the female in larvae and alter the gonadal morphology of testicles of juvenile males. In zebrafish, a model organism considered as a useful tool in EDs screening, we investigated the potential role of serotonin (5-HT) as modulators of brain cell proliferation; in particular, we also analyzed the effects of FLX, a selective 5-HT reuptake inhibitor. We showed that 5-HT neurons of the hypothalamus originate from proliferative radial glial cells presents in this area. Treatment 5-HT synthesis inhibitor and exposure to environmentally relevant concentrations of FLX caused a significant decrease in the number of proliferating cells in the hypothalamus, resulting in a further decline in the number of own 5-HT neurons. These results provide evidence for the detrimental effect in teleost fish induced by the exposure to two pharmaceuticals considered as endocrine disruptors.

Chez les vertébrés gnathostomes, le système immunitaire repose en grande partie sur les lymphocytes T qui se développent dans un organe conservé évolutivement : le thymus. Chez les mammifères, cet organe constitue une cible privilégiée pour les œstrogènes. La question soulevée ici est donc de savoir si c’est également le cas chez les poissons téléostéens. Dans ce but, la distribution des différents sous-types de récepteurs aux œstrogènes a d’abord été étudiée dans le contexte d’une description de l’anatomie fonctionnelle du microenvironnement thymique. Par la suite, l’expression de gènes relatifs à la fonction thymique et aux différents sous-types de lymphocyte T a été analysée dans le thymus, le rein-antérieur et la rate de bars exposés au 17ß-œstradiol. De plus, la capacité de flambée oxydative a été évaluée sur des leucocytes du rein-antérieur et de rate à la suite d’expositions in vivo et in vitro. Finalement, la variation du nombre de thymocytes a été examinée sur des bars capturés durant trois ans. La thèse fournit de nouvelles connaissances concernant l’évolution des fonctions immunomodulatrices des œstrogènes sur la différenciation des cellules T. En effet, en plus d’une organisation morpho-fonctionnelle fortement conservée, la distribution des sous-types de récepteurs aux œstrogènes ainsi que les effets œstrogéniques apparaissent conservés au cours de l’évolution. Nos résultats suggèrent que, chez le bar comme chez les mammifères, les œstrogènes (1) stimulent une voie alternative de maturation des lymphocytes T ayant des propriétés similaires aux cellules immunitaires innées, (2) augmentent la tolérance immunitaire et (3) régulent la plasticité du thymus. / Jawed vertebrates have developed an efficient adaptive immune system partly based on T lymphocytes. They develop in an evolutionarily conserved organ, the thymus. In mammals, endogenous oestrogens are well known to regulate thymus function and plasticity. The question is, therefore, whether this is also the case in lower vertebrates, such as teleosts. To achieve these aims, firstly the distribution of oestrogen receptor subtypes was investigated on the background of a detailed description of the functional anatomy of the thymic microenvironment. Secondly, thymic function- and T cell-related gene expression was analysed in the thymus, the head-kidney and the spleen of sea bass exposed to 17ß-oestradiol. Moreover, the oxidative burst capacity in the two latter organs was evaluated in vivo and in vitro in leucocytes of the head-kidney and spleen following exposure to oestrogen. Eventually, age- and size-dependent variations in thymocyte number were examined in sea bass caught at various time points over three years. The thesis provides new insights into the evolution of the immunomodulatory function of oestrogen with respect to the thymic and peripheral T cell differentiation in vertebrates. As a matter of fact, in addition to a highly conserved morpho-functional organisation, the distribution of oestrogen receptor subtypes as well as the oestrogenic effects appear to be evolutionarily conserved. Our results suggest that in sea bass, similar to mammals, oestrogen (1) stimulates a thymic alternative pathway of T cell maturation with innate-like properties, (2) enhances immune tolerance by promoting Treg differentiation, and (3) actively regulate thymic plasticity.

Tesis por compendio de publicaciones / La luz y los patrones de alimentación influyeron sobre el comportamiento, fisiología y reloj molecular de dos peces teleósteos. Se clonaron dos criptocromos de lubina (cry1 y cry2), expresándose en todos los tejidos estudiados y mostrando ritmos diarios de expresión en cerebro, hígado y corazón (excepto cry2). La fase (día/noche) de alimentación a demanda en lubina afecta a los patrones diarios de actividad amilasa intestinal y expresión de period1 en tejidos periféricos (i.e. hígado), pero no en cerebro. Durante las inversiones estacionales de alimentación de la lubina, mostraron mayores valores de glucosa sanguínea durante la fase de alimentación, revelando también los mayores valores medios en lubinas nocturnas invierno. El pez cebra utilizó un nuevo comedero a demanda, mostrando independencia de fase entre ritmos circadianos de alimentación (nocturno) y locomoción (diurno), con diferentes periodicidades. Además la actividad y posición del pez cebra resultó espectro-dependiente, provocando mayores efectos las longitudes de onda cortas. / Thesis summary in English Light and feeding patterns influence on the behaviour, physiology and molecular clock of two teleost fish. Two cryptochromes (cry1 and cry2) were cloned, being expressed in all studied tissues and showing daily rhythm of expression in brain, liver and heart (except cry2). The self-seabass feeding phase (day/night) affected on daily patterns of intestinal amylase activity and on the period1 expression in peripheral tissues (i.e. liver), but not in brain. During the seasonal feeding inversions in seabass, the highest blood glucose levels were shown during their feeding phase, revealing also the highest values in nocturnal seabass in winter. Zebrafish used a new self-feeding system, displaying phase-independence between feeding (nocturnal) and locomotor (diurnal) circadian rhythms, with different periodicities. Moreover, the zebrafish activity and vertical position were spectrum-dependent, being the main effects provoked by short-wavelengths.

Teleost fishes incorporate renal ammonia excretion as part of a greater acid-base regulatory system. However, the transport mechanisms employed by the renal epithelium to excrete ammonia are relatively unknown. I hypothesized that, under metabolic acidosis, increased renal ammonia excretion would be the product of tubular secretion and involve a Na+/NH4+ exchange metabolon mediated through Rhesus (Rh) glycoproteins. To induce metabolic acidosis, goldfish (Carassius auratus) were exposed to a low pH environment (pH 4.0; 48-h). There was a clear signal of metabolic acidosis: a reduction in both plasma [HCO3-] and blood pH with no influence on plasma PCO2. Goldfish demonstrated an elevation in total plasma [ammonia] with a reduction in PNH3 under acidosis. Metabolic acidosis induced higher rates of urinary excretion of acidic equivalents in the form of both NH4+ and titratable acidity-HCO3- (TA-HCO3-) excretion. Urinary Na+ excretion was not affected by acidosis and urine [Na+] did not correlate with urinary [ammonia]. Alanine aminotransferase activity in the kidney was higher in acidotic goldfish. Glomerular filtration rate and urine flow rate were not affected by acidosis. Increased renal NH4+ excretion was due to increased secretion, and not increased filtration, of ammonia. There was a corresponding elevation in Rhcg1b mRNA expression but no change in renal Na+ reabsorption. My data support a secretion-based mechanism of teleost renal ammonia transport. This system is Na+ independent and is likely mediated by Rh glycoproteins and H+ ATPase, involving a parallel H+/NH3 secretion mechanism. To investigate effects of metabolic acidosis on elasmobranch fish, Pacific spiny dogfish (Squalus acanthias suckleyi) were infused with an acidic saline (125 mM HCl/375 mM NaCl; 3 ml/kg/h; 24-h). The results are preliminary, with no marked effects of HCl infusion on plasma acid-base or N-status, but increased branchial NHE2 and lower renal NHE3 protein expressions. These data are summarized in an Appendix. / Thesis / Master of Science (MSc)

Cerebellar organization and function have been studied in numerous species of fish. Fish models such as goldfish and weakly electric fish have led to important findings about the cerebellar architecture, cerebellar circuit physiology and brain evolution. However, most of the studied fish models are not well suited for developmental and genetic studies of the cerebellum. The rapid transparent ex utero development in zebrafish allows direct access and precise visualization of all the major events in cerebellar development. The superficial position of the cerebellar primordium and cerebellum further facilitates in vivo imaging of cerebellar structures and developmental events at single cell resolution. Furthermore, zebrafish is amenable to high-throughput screening techniques and forward genetics because of its fecundity and easy keeping. Forward genetics screens in zebrafish have resulted in several isolated cerebellar mutants and substantially contributed to the understanding of the genetic networks involved in hindbrain development (Bae et al. 2009; Brand et al. 1996). Recent developments in genetic tools, including the use of site specific recombinases, efficient transgenesis, inducible gene expression systems, and the targeted genome lesioning technologies TALEN and Cas9/CRISPR has opened up new avenues to manipulate and edit the genome of zebrafish (Hans et al. 2009; Scott 2009; Housden et al. 2016; Li et al. 2016)}. These tools enable the use of genome-wide genetic approaches, such as enhancer/exon traps and cell specific temporal control of gene expression in zebrafish. Several seminal papers have used these technologies to successfully elucidate mechanisms involved in the morphogenesis, neurogenesis and cell migration in the cerebellum (Bae et al. 2009; Chaplin et al. ; Hans et al. 2009; Volkmann et al. ; Volkmann et al. 2008). In addition, the use of genetically encoded sensors and probes that allows detection and manipulation of neuronal activity using optical methods have open up new means to study the physiology and function of the cerebellum (Simmich et al. 2012; Matsui et al. 2014). Taken together, these features have allowed zebrafish to emerge as a complete model for studies of molecular, cellular and physiological mechanisms involved in cerebellar development and function at both cell and circuit level.