In fish, maternal stress prior to spawn has been reported to have effects on offspring phenotype. Cortisol, the main glucocorticoid (GC) stress hormone, has been proposed as a potential mediator of such effects because of its organizational role in early teleost development. The present thesis tested whether maternal social stress or treatment with cortisol (as a proxy for maternal stress) prior to spawn affects the cortisol response to stress and anxiety-related behaviours in offspring. In zebrafish (Danio rerio), offspring of dominant females exhibited greater boldness at 6 days post-fertilization (DPF). Interestingly, offspring of females that engaged in social interactions, regardless of the resulting social status of the two females, exhibited greater survival at 1 DPF, a greater fear-related decrease in activity in response to bright light at 6 DPF, and decreased baseline whole-body cortisol content at 0 and 30 DPF. A field experiment with wild largemouth bass (Micropterus salmoides) revealed that maternal cortisol treatment prior to spawn also affected offspring phenotype; offspring of cortisol-treated females had higher masses right after hatch, had greater fear responses, were less bold and less anxious, and exhibited an attenuated cortisol response to an acute stressor. Together, the results of the present thesis suggest that effects of maternal stress prior to spawn on offspring survival, growth, responses to stress, and anxiety-related behaviours are mediated, at least in part, by elevated maternal cortisol but not likely via increased deposition of maternal cortisol into eggs. The effects of maternal stress and cortisol treatment on offspring reported in the present thesis also suggest that maternal stress may prime offspring with adaptive traits to better survive in a stressful environment.

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.

Discerning the extent and patterns of genetic connectivity and understanding population demographic processes is essential for framing proper management and conservation measures for species of concern. Although genetic connectivity may be influenced by numerous biotic and abiotic factors, habitat utilization and dispersal potential are often key factors driving connectivity, especially in marine fishes. While dispersal potential is of key importance with respect to shaping connectivity, other extrinsic (e.g., oceanographic processes) and intrinsic (e.g., reproductive behavior) factors may also influence connectivity; however, the relative influence of such factors is immensely variable across species and life-stages. This dissertation explores genetic connectivity and demographic history in marine fishes with diverse dispersal potentials to determine which processes, in addition to the known dispersal potential of the species, may be shaping connectivity. Genetic connectivity and demographic history is assessed for four marine fishes: two shark species with juxtaposing dispersal potentials, the highly migratory tiger shark (Galeocerdo cuvier) and the reef associated Caribbean reef shark (Carcharhinus perezi), which possess high and low dispersal potentials, respectively, and two teleost species, the pelagic roundscale spearfish (Tetrapturus georgii) and the Nassau grouper (Epinephelus striatus), which possess high and low adult dispersal potentials, respectively. This work demonstrates that dispersal potential does, in fact, play a key role in delineating genetic structure for these species; however, other factors, such as contemporary oceanographic currents (e.g., upwelling and temperature), habitat availability (e.g., coral cover), and historical events, such as cyclical glacial cycles, also influence genetic connectivity across variable spatial scales, thereby creating complex patterns of genetic population structure, requiring composite management strategies to ensure the persistence of these species.

Osmoregulation is an essential process to maintain water and ionic balance and when euryhaline fish move between freshwater and seawater environments as part of their life cycle this presents additional osmoregulatory challenges. Migrating fish can be exposed in both environments to pollutants such as endocrine disrupting chemicals (EDCs) that include natural hormones (e.g. 17β-estradiol; E2), synthetic hormones (e.g. 17α-ethinylestradiol; EE2), and industrial chemicals (e.g. nonylphenol). The focus of this thesis was to study the effects of different categories of EDCs on the osmoregulatory functions of euryhaline fish such as three-spined sticklebacks (Gasterosteus aculeatus) and rainbow trout (Oncorhynchus mykiss). Osmoregulatory variables (such as osmolality, water and ionic content) were compared in plasma and tissues (white muscle and carcass) of rainbow trout. This validated the use of specific tissue parameters as a surrogate of plasma responses to various osmoregulatory challenges. Waterborne exposure to 17α-ethinylestradiol revealed differential sensitivity of vitellogenesis in the three-spined sticklebacks (no induction) and rainbow trout, but had a significant effect on calcium homeostasis in both species. Intraperitoneal implants of 17β-estradiol reduced CaCO3 production and apparent water absorption in the intestine and increased in tissue calcium stores of seawater-acclimated trout, but fish were able to compensate and showed no overall osmoregulatory disturbance. Waterborne exposure to nonylphenol in freshwater trout was also investigated, but no effects on osmoregulation were found up to 2 ng/l. Overall, estrogens can affect osmoregulation differentially in euryhaline fish species, and sometimes at EDC levels lower than the threshold for reproductive effects (i.e. vitellogenin induction).

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.

<p>In mammals sex chromosomes direct and initiate the development of male and female gonads and subsequently secondary sex characteristics. In most vertebrates each individual is pre-destined to either become male or female. The process by which this genetic decision is carried out takes place during the embryonic development and involves a wide range of genes. The <i>fushi tarazu</i> factor-1 (FTZ-F1) is a nuclear receptor and transcription factor, which in mammals has proven to be essential for gonad development and directs the differentiation of testicular Sertoli cells. A mammalian FTZ-F1 homologue subtype, steroidogenic factor-1 (SF-1), is a member of the nuclear receptor 5A1 (NR5A1) group and regulate several enzymes involved in steroid hormone synthesis. It also regulates the expression of the gonadotropin releasing hormone receptor GnRHr and the β-subunit of the luteinizing hormone (LH), indicating that it functions at all levels of the reproductive axis. Another mammalian FTZ-F1 subtype, NR5A2, is in contrast to SF-1, not linked to steroidogenesis or sex determination. Rather, NR5A2 is involved in cholesterol metabolism and bile acid synthesis in liver. Hormones and environmental factors such as temperature and pH can influence teleost development and reproductive traits, rendering them vulnerable to pollutants and climate changes. Very little is known about teleost FTZ-F1 expression, regulation and function. In this thesis, expression patterns of four zebrafish FTZ-F1 genes (ff1a, b, c and d) and two Arctic char genes (acFF1α and β) were studied during development, displaying complex embryonic expression patterns. Ff1a expression was in part congruent with expression of both mammalian NR5A1 and NR5A2 genes but also displayed novel expression domains. The complexity of the expression pattern of ff1a led to the conclusion that the gene may be involved in several developmental processes, including gonad development, which also was indicated by its transcriptional regulation via Sox9a. Two ff1a homologues were also cloned in Arctic char and were shown to be involved in the reproductive cycle, as the expression displayed seasonal cyclicity and preceded that of the down stream steroidogenic genes StAR and CYP11A. High levels were correlated to elevated plasma levels of 11-ketotestosterone (11KT) in males and 17β-estradiol (E2) in females respectively. Treatment with 11KT did not affect FTZ-F1 expression directly but was indicated to alter expression of CYP11A and 3β-hydroxysteroid dehydrogenase. E2 treatment was indicated to down-regulate the expression of testicular FTZ-F1, which may contribute to the feminising effect previously observed in E2 treated salmonids. Ff1d is a novel FTZ-F1 gene, expressed in pituitary and interrenal cells during development, suggesting steroidogenic functions. In adult testis and ovary ff1d was co-expressed with anti-Mullerian hormone (AMH), a gene connected to sex determination in mammals and previously not characterised in teleost fish. The co-expression between ff1d and AMH was found in Sertoli and granulosa cells, which is congruent with the co-expression of mammalian SF-1 and AMH. This suggests that ff1d and AMH may have similar functions in teleost sex differentiation and reproduction, as their mammalian homologues. In conclusion, this study present data that connects members of the teleost FTZ-F1 family to reproduction, cholesterol metabolism and sex determination and differentiation.</p>

In mammals sex chromosomes direct and initiate the development of male and female gonads and subsequently secondary sex characteristics. In most vertebrates each individual is pre-destined to either become male or female. The process by which this genetic decision is carried out takes place during the embryonic development and involves a wide range of genes. The fushi tarazu factor-1 (FTZ-F1) is a nuclear receptor and transcription factor, which in mammals has proven to be essential for gonad development and directs the differentiation of testicular Sertoli cells. A mammalian FTZ-F1 homologue subtype, steroidogenic factor-1 (SF-1), is a member of the nuclear receptor 5A1 (NR5A1) group and regulate several enzymes involved in steroid hormone synthesis. It also regulates the expression of the gonadotropin releasing hormone receptor GnRHr and the β-subunit of the luteinizing hormone (LH), indicating that it functions at all levels of the reproductive axis. Another mammalian FTZ-F1 subtype, NR5A2, is in contrast to SF-1, not linked to steroidogenesis or sex determination. Rather, NR5A2 is involved in cholesterol metabolism and bile acid synthesis in liver. Hormones and environmental factors such as temperature and pH can influence teleost development and reproductive traits, rendering them vulnerable to pollutants and climate changes. Very little is known about teleost FTZ-F1 expression, regulation and function. In this thesis, expression patterns of four zebrafish FTZ-F1 genes (ff1a, b, c and d) and two Arctic char genes (acFF1α and β) were studied during development, displaying complex embryonic expression patterns. Ff1a expression was in part congruent with expression of both mammalian NR5A1 and NR5A2 genes but also displayed novel expression domains. The complexity of the expression pattern of ff1a led to the conclusion that the gene may be involved in several developmental processes, including gonad development, which also was indicated by its transcriptional regulation via Sox9a. Two ff1a homologues were also cloned in Arctic char and were shown to be involved in the reproductive cycle, as the expression displayed seasonal cyclicity and preceded that of the down stream steroidogenic genes StAR and CYP11A. High levels were correlated to elevated plasma levels of 11-ketotestosterone (11KT) in males and 17β-estradiol (E2) in females respectively. Treatment with 11KT did not affect FTZ-F1 expression directly but was indicated to alter expression of CYP11A and 3β-hydroxysteroid dehydrogenase. E2 treatment was indicated to down-regulate the expression of testicular FTZ-F1, which may contribute to the feminising effect previously observed in E2 treated salmonids. Ff1d is a novel FTZ-F1 gene, expressed in pituitary and interrenal cells during development, suggesting steroidogenic functions. In adult testis and ovary ff1d was co-expressed with anti-Mullerian hormone (AMH), a gene connected to sex determination in mammals and previously not characterised in teleost fish. The co-expression between ff1d and AMH was found in Sertoli and granulosa cells, which is congruent with the co-expression of mammalian SF-1 and AMH. This suggests that ff1d and AMH may have similar functions in teleost sex differentiation and reproduction, as their mammalian homologues. In conclusion, this study present data that connects members of the teleost FTZ-F1 family to reproduction, cholesterol metabolism and sex determination and differentiation.

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.

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.