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.

Cellular and molecular mechanisms of postembryonic muscle fibre recruitment were investigated in zebrafish (Danio rerio L.), a standard animal model for developmental and genetic studies. Distinct cellular mechanisms of postembryonic muscle fibre recruitment in fast and slow myotomal muscles were found. In slow muscle, three overlapping waves of stratified hyperplasia (SH) from distinct germinal zones sequentially contributed to a slow and steady increase in fibre number (FN) through the life span. In fast muscle, SH only contributed to an initial increase of FN in early larvae. Strikingly, mosaic hyperplasia (MH) appeared in late larvae and early juveniles and remained active until early adult stages, accounting for >70% of the final fibre number (FFN). The molecular regulation of postembryonic muscle fibre recruitment was then studied by characterising myospryn and cee, two strong candidate genes previously identified from a large scale screen for genes differentially expressed during the transition from hyperplastic to hypertrophic muscle phenotypes. Zebrafish myospryn contained very similar functional domains to its mammalian orthologues, which function to bind to other proteins known to regulate muscle dystrophy. Zebrafish myospryn also shared a highly conserved syntenic genomic neighbourhood with other vertebrate orthologues. As in mammals, zebrafish myospryn were specifically expressed in striated muscles. Zebrafish cee was a single-copy gene, highly conserved among metazoans, ubiquitously expressed across tissues, and did not form part of any wider gene family. Its protein encompassed a single conserved domain (DUF410) of unknown function although knock-down of cee in C. elegans and yeast have suggested a role in regulating growth patterns. Both myospyrn and cee transcripts were up-regulated concomitant with the cessation of postembryonic muscle fibre recruitment in zebrafish, indicating a potential role in regulating muscle growth. Furthermore, a genome-wide screen of genes involved in the regulation of postembryonic muscle fibre recruitment was performed using microarray. 85 genes were found to be consistently and differentially expressed between growth stages where muscle hyperplasia was active or inactive, including genes associated with muscle contraction, metabolism, and immunity. Further bioinformatic annotation indicated these genes comprised a complex transcriptional network with molecular functions, including catalytic activity and protein binding as well as pathways associated with metabolism, tight junctions, and human diseases. Finally, developmental plasticity of postembryonic muscle fibre recruitment to embryonic temperature was characterised. It involved transient effects including the relative timing and contribution of SH and MH, plus the rate and duration of fibre production, as well as a persistent alteration to FFN. Further investigation of FFN of fish over a broader range of embryonic temperature treatments (22, 26, 28, 31, 35°C) indicated that 26°C produced the highest FFN that was approximately 17% greater than at other temperatures. This finding implies the existence of an optimal embryonic temperature range for maximising FFN across a reaction norm. Additionally, a small but significant effect of parental temperature on FFN (up to 6% greater at 24 and 26°C than at 31°C) was evident, suggesting some parental mechanisms can affect muscle fibre recruitment patterns of progeny. This work provides a comprehensive investigation of mechanisms underlying postembryonic muscle fibre recruitment and demonstrates the power of zebrafish as an ideal teleost model for addressing mechanistic and practical aspects of postembryonic muscle recruitment, especially the presence of all major phases of muscle fibre production in larger commercially important teleost species.

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No. of bitstreams: 2 Dissertação - Lara Barroso Brito - 2016.pdf: 19284674 bytes, checksum: 73aa3e2fc34b886b960712220045839d (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2016-03-23 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / Pharmaceuticals can be introduced directly into the environmental by household disposal or pharmaceutical industry waste and indirectly through the excretion of humans and animals. Antibiotics are considered emerging contaminants because they are typically present at very low levels in the environmental and their human or ecological health effects are unclear. β-lactams represent more than 70% of antibiotics consumed in Brazil. Thus, in this context, this work evaluated the environmental impact of antibiotic amoxicillin (AMX) in different organisms, considering its presence in water. For that, we used the phytotoxicity test with seeds of tomato (Lycopersicon esculentum), cucumber (Cucumis sativus) and lettuce (Lactuca sativa), brine shrimp toxicity assay (Artemia salina), and embryo-larval toxicity test zebrafish (D. rerio), considering the lethal and sublethal effects and biomarkers determinations. AMX showed no toxicity to seeds of tomato, cucumber and lettuce in relation to seed germination and root elongation endpoints. For microcrustacean A. salina, AMX did not induce significant mortality after 24 h and 48 h exposure (LC50 > 100 mg/L) and it was classified as non-toxic (not categorized) according to the Globally Harmonized System (GHS). AMX also did not cause significant mortality in embryos and larvae zebrafish during 168 h of exposure. It had no significant effect on embryos hatching and larvae equilibrium. However, AMX significantly increased the larvae size at 6.25, 12.5 and 25 mg/L. Catalase (CAT), glutathione S-transferase (GST) and lactate dehydrogenase (LDH) activities in embryos and larvae of zebrafish were inhibited at 12.5 mg/L of AMX, 6.25, 12.5, 25, 50 and 100 mg/L of AMX and 1.5, 3.0, 6.25 and 12.5 mg/L of AMX, respectively. Therefore, AMX showed no significant acute toxicity to tested organisms, but it induced sublethal effects on larvae zebrafish in concentrations greater than those found in the aquatic environment, indicating that long-term chronic exposures must be investigated. / Os fármacos e insumos farmacêuticos podem ser introduzidos no ambiente de forma direta, ou seja, através do descarte doméstico ou por efluentes da indústria farmacêutica, e ainda indiretamente, por meio da excreção humana e animal. Os antibióticos são considerados contaminantes emergentes, uma vez que são detectados em concentrações muito baixas no ambiente e seus efeitos sobre o ambiente e saúde humana permanecem incertos. Os β- lactâmicos representam mais de 70% dos antibióticos consumidos no Brasil. Assim, dentro deste contexto, este trabalho propôs avaliar o impacto ambiental do antibiótico amoxicilina (AMX) em diferentes organismos, considerando a sua presença nos recursos hídricos. Para tanto foram utilizados os ensaios de fitotoxicidade com sementes de tomate (Lycopersicon esculentum), pepino (Cucumis sativus) e alface (Lactuca sativa) e de toxicidade aguda com Artemia salina, assim como o teste com o estágio embriolarval de zebrafish (Danio rerio), considerando os efeitos letais, subletais e atividade de biomarcadores enzimáticos. A AMX não foi tóxica para as sementes de tomate, pepino e alface, não apresentando diferenças estatísticas significativas para os parâmetros de germinação das sementes e do desenvolvimento das raízes. Para o microcrustáceo A. salina, a AMX não induziu mortalidade significativa com valores de CL50 maior que 100 mg/L para 24 h e 48 h de exposição, sendo classificada como não tóxica (não categorizada) de acordo com o Globally Harmonized System (GHS). A AMX não provocou mortalidade significante nos embriões e larvas de zebrafish durante as 168 h de exposição, assim como não alterou significativamente a eclosão dos embriões e o equilíbrio de larvas de zebrafish. Entretanto, o antibiótico aumentou significativamente o tamanho das larvas desse peixe nas concentrações de 6,25; 12,5 e 25 mg/L. As atividades da catalase (CAT), da glutationa Stransferase (GST) e da lactato desidrogenase (LDH) de embriões e larvas de peixezebra foram inibidas significativamente nas exposições à 12,5 mg/L de AMX, 6,25; 12,5; 25; 50 e 100 mg/L de AMX e 1,5; 3,0; 6,25 e 12,5 mg/L de AMX, respectivamente. Portanto, o antibiótico AMX não apresenta toxicidade aguda relevante para os organismos testados, mas causa alguns efeitos subletais em larvas de zebrafish em concentrações superiores às encontradas no ambiente aquático, o que indica a necessidade de se investigar exposições a esse composto a longo prazo.

Ce travail s’intègre dans le cadre du programme européen COMET (7ième PCRD EURATOM) et vise à évaluer les réponses épigénétiques (particulièrement la méthylation de l’ADN), lors d’expositions chroniques à de faibles niveaux de substances radioactives.Lors d’une première expérience, les poissons zèbres (Danio rerio) ont été exposés au laboratoire à des concentrations environnementales d’uranium appauvri : 2 et 20 µg L-1. Cette expérimentation a révélé un impact sur la méthylation de l’ADN génomique, majoritairement chez les mâles exposés, croissant avec la durée et le niveau d’exposition. Lors d’une seconde expérience, nous avons observé un impact sur les profils de méthylation de la descendance issue de parents exposés, ainsi qu’une perturbation du statut transcriptomique et des dommages histologiques dans le muscle squelettique des larves issues de parents exposés.Les outils développés ont pu être appliqués à la problématique d’étude des effets biologiques induits par les radionucléides émis lors de l’accident de la centrale nucléaire de Fukushima Daiichi. Les analyses réalisées sur la grenouille arboricole Japonaise (Hyla japonica) ont révélé une corrélation positive entre la dose totale de radioactivité absorbée par ces grenouilles, l’hyperméthylation de l’ADN génomique et l’augmentation des dommages à l’ADN mitochondrial.L’ensemble de ces travaux a permis de mettre en évidence la sensibilité des réponses épigénétiques chez différents organismes soumis à de faibles doses de radionucléides, qui pourraient ainsi permettre de mieux caractériser les mécanismes d’adaptation et les potentiels effets transgénérationnels induits par les radionucléides. / This work integrates within the general framework of the European program COMET (7th Framework Programme EURATOM) and aims to assess the epigenetic responses (particularly DNA methylation), during chronic exposure to low levels of radioactive materials. During a first experiment, zebrafish (Danio rerio) were exposed in laboratory controlled conditions to environmentally relevant concentrations of depleted uranium: 2 and 20 µg L-1. This experiment allowed an impact on the genomic DNA methylation to be demonstrated, mainly in exposed males, which increased with the duration and level of exposure. In a second experiment, we observed an impact on DNA methylation patterns in the progeny of exposed parents, as well as a perturbation of transcriptomic, and histological damage in larvae skeletal muscle from exposed parents.The methods developed were applied to the second context focusing on the study of biological effects induced by radionuclides emitted following the Fukushima Daiichi nuclear power plant accident. The analyses performed on the Japanese tree frog (Hyla japonica) revealed a positive correlation between the total dose of radiation absorbed by these frogs, hypermethylation of genomic DNA as well as increasing damage to mitochondrial DNA.This work highlighted the sensitivity of epigenetic responses in different biological models exposed to low levels of radionuclides, and could be used to further characterize adaptation mechanisms and potential transgenerational effects induced by radionuclides.

ABCB1, a member of the ATP binding cassette (ABC) transporter family, hydrolyses ATP as energy source for the translocation of substrate chemicals across the cell membrane. ABCB1-like transporters are found in all studied species. Typically, these transporters are abundant in tissues that separate compartments of the body such as the blood-brain barrier. Among the ABC transporters the ABCB1-like transporter proteins are of particular interest because they accept a broad variety of substrates and are therefore able to confer multidrug resistance (MDR) and multixenobiotic resistance (MXR) in wildlife, respectively. Inhibitors of the ABCB1-like transporter function can cause chemosensitisation, i.e. accumulation and increased sensitivity of organisms towards potentially harmful (natural/man-made) ABCB1-like substrate chemicals. In zebrafish (Danio rerio) Abcb4 was identified as functionally homologous to ABCB1. The aim of this study was to further characterise Danio rerio Abcb4 and to provide a database to approach the question to what extent ABCB1-like transporter related functions/effects are of ecotoxicological relevance. Main objectives are whether and how known ABCB1 ATPase stimulators and inhibitors interact with Abcb4 ATPase activity; to what extent ABCB1 ATPase assay data are transferable to Abcb4 ATPase assay data; and whether and how environmental chemicals interact with Danio rerio Abcb4 ATPase activity. In this study we established a test system – the ATPase assay with recombinant Danio rerio Abcb4 – to study the interaction of chemicals with the ATPase activity of the transporter protein. To relate obtained data to data for the well-known Homo sapiens ABCB1 and because available data for Homo sapiens ABCB1 were not in all cases suitable for a comparison, the ATPase assay with recombinant ABCB1 was adapted accordingly. Chemicals were tested up to concentrations in the range of their water solubilities to modulate basal and stimulator co-treated Abcb4 and/or ABCB1 ATPase activities. ATPase stimulators are often transported substrates. However, lipophilic compounds stimulating the transporter ATPase activity are not or little transported by transporter action. Therefore, experiments revealing whether compounds are translocated by transporters chemical interference with the transporter protein will not be indicated. Chemicals inhibiting the stimulator (here verapamil) co-treated ATPase activity compete with the verapamil to stimulate ATPase activity or are non-competitive inhibitors. When tested individually, these chemicals can be stimulators or inhibitors of basal ATPase activity, or do not interact with basal ATPase activity. ATPase inhibitors mitigate ATPase activity and ABCB1-like transporter mediated translocation of substrate chemicals. Obtained ATPase assay data were analysed with regard to concentrations at half-maximal effects (EC50s) and effect strengths (percent modulation). ATPase assays with recombinant Abcb4 (at 27 °C) are comparable to ABCB1 ATPase assay data obtained at 37 °C. Danio rerio Abcb4 seems less temperature-sensitive than ABCB1. Calculated activation energies for Abcb4 ATPase activities (40.75 kJ/mol for basal ATPase activity) were up to half as high as those for ABCB1 ATPase activities (81.61 kJ/mol for basal ATPase activity). Larger activation energies were previously proposed to be indicative for larger conformational rearrangements and hence possibly smaller rearrangements take place in Abcb4 compared to ABCB1. Known standard modulators of Homo sapiens ABCB1 ATPase activity interacted specifically with Danio rerio Abcb4 ATPase actitiy. The EC50s of the tested chemicals – 16 of 17 tested chemiacals interacted with the ABCB1 and the Abcb4 ATPase activity – ranged from 0.09 to 296 µM for ABCB1 and from 0.14 to 171 µM for Abcb4. Qualitative ATPase assay data for ABCB1, as interaction or not, seems transferable to Danio rerio Abcb4. Furthermore, when aligning amino acid sequences of mammalian ABCB1 transporter proteins and Danio rerio Abcb4 and comparing ABCB1 residues known to bind to (lipophilic) chemicals no obvious hints were found that chemical binding to Abcb4 is certainly different from ABCB1. Twenty-five of 33 studied environmental chemicals modulated the Abcb4 ATPase activity as stimulators and/or inhibitors. Stimulation of basal Abcb4 ATPase activity was lower for environmental chemicals than for known standard modulators. EC50s of environmental chemicals ranged from below 10 to 357 µM. Effects by environmental chemicals on Abcb4 ATPase activity with EC50s close to their water solubilities may be rather unspecific. The results of this work underline that Abcb4 function is of ecotoxicological importance as on the one hand several environmental chemicals were identified to inhibit Abcb4 ATPase activity – likely acting as chemosensitisers, while on the other hand chemicals stimulating basal ATPase activity suggest that these chemicals are possibly transported. A number of environmental chemicals also inhibited the basal Abcb4 ATPase activity. Especially non-transported inhibitors of the basal Abcb4 ATPase activity would be of ecotoxicological relevance as organisms (here Danio rerio) exposed to these chemicals would not be protected by Abcb4 mediated multixenobiotic resistance and were moreover threatened by chemosensitisation. Future studies should systematically elucidate under which circumstances chemicals are apparently net transported by ABCB1-like transporters and relate these findings to concentrations of environmental chemicals and ABCB1-like transporter protein abundance in wildlife.

Pharmaka werden nach ihrer Einnahme bzw. Verabreichung über verschiedene Pfade in die Umwelt eingetragen. Obwohl Arzneimittel zu den toxikologisch best-untersuchten und -charakterisierten Stoffen gehören, ist ihre Wirkung auf die Umwelt und die darin lebenden Organismen weit weniger gut untersucht. Wenn in der Literatur Daten zur Ökotoxizität vorhanden sind, so beziehen sich diese meist nur auf die Wirkung von Einzelstoffen. In der Umwelt sind die Organismen jedoch gegenüber Mischungen exponiert. Aufgrund der geschilderten Problematik wurden eine Reihe von Arzneimitteln unterschiedlicher Indikationsgruppen einzeln und in Kombination mit dem Embryotest mit dem Zebrabärbling (Danio rerio, DarT) untersucht. Dieses Testsystem wurde durch Schulte & Nagel (1994) als Alternativmethode zum akuten Fischtest nach OECD 203 entwickelt und bietet den Vorteil neben letalen auch eine Reihe von subletalen Endpunkten erfassen zu können. Es handelt sich zudem nach dem deutschen Tierschutzgesetz nicht um einen Tierversuch. Die generelle Vergleichbarkeit der ermittelten Werte mit Daten aus akuten Fischtests nach OECD 203 sowie die Anwendbarkeit für verschiedenste Fragestellungen konnten in einer Reihe von Studien gezeigt werden (Nagel, 2002). Für die hier vorgestellten Untersuchungen wurden zunächst 32 Pharmaka und drei Pflanzenschutzmittel als Einzelstoffe mit dem DarT untersucht. Basierend auf den Ergebnissen der Einzelstofftests wurden Mischungen sowohl aus Substanzen mit ähnlichen als auch unähnlichen Wirkmechanismen getestet. Es zeigte sich, dass unabhängig vom Wirkmechanismus die Mischungstoxizität durch das Konzept der Konzentrationsadditivität gut vorhergesagt wurde, während das Konzept der Unabhängigen Wirkung die Mischungstoxizität unterschätzte. Ebenfalls konnte gezeigt werden, dass die Kombination der Stoffe auf Basis der NOEC, die im DarT anhand der Herzschlagfrequenz nach 48 Stunden ermittelt wird, zu deutlichen Mischungseffekten führt.

Die Körpergröße ist von großer Bedeutung für die Fitness vieler Tiere, weil sie positiv mit Überleben und Reproduktionserfolg korreliert ist. Große Rogner vieler Fischarten sind fruchtbarer und produzieren Nachkommen von höherer Qualität verglichen mit kleineren Weibchen. Auch für Milchner einiger Fischarten wurde ein Einfluss der Körpergröße auf frühe Lebensstadien nachgewiesen. Der größenabhängige paternale Effekt verglichen mit maternalen Effekten ist weniger gut untersucht. Das Verständnis der Variation im Reproduktionserfolg als Funktion der Körpergröße der Laicher ist wichtig, weil die Fischerei die Durchschnittsgröße des Laicherbestands reduziert. In vorliegender Dissertation wurden in Laborversuchen an Zebrafischen (Danio rerio) größenabhängige paternale und maternale Effekte auf den Reproduktionserfolg und die Auswirkungen größenselektiver Entnahme auf Körperlänge, Reifung und Reproduktionserfolg untersucht. Die Köperlänge und Kondition waren wichtige Determinanten der Reifung bei Zebrafischen. Größere Rogner zeigten höheren Reproduktionserfolg als kleinere Fische und ein signifikanter Einfluss der Milchnerkörperlänge auf die frühen Lebensstadien ihrer Nachkommen wurde dokumentiert. Längere Männchen wurden von Rognern auch bei der Paarung bevorzugt. Die größenabhängigen maternalen und paternalen Effekte waren ausschlaggebend für den erhöhten Reproduktionserfolg von Zebrafischlaichbeständen, die, verglichen mit kleinen Laichern, aus großen oder zufällig zusammengesetzten Individuen zusammengesetzt waren. Die größenselektive Entnahme führte zu phänotypischen und genetischen Veränderungen, die nach Einstellung der experimentellen Befischung persistierten. Das deutet an, dass die durch die Fischerei ausgelöste Evolution schwierig umkehrbar sein könnte. Die Köpergröße ist von überragender Bedeutung in der Reproduktionsbiologie des Zebrafisches und der Schutz großer Laichfische kann wichtig für den Erhalt der Reproduktionskapazität von befischten Beständen sein. / Body size is a fundamentally important trait for fitness in many animal species because it correlates positively with survival and reproductive success. In many fish species, large females exhibit higher fecundity and produce higher quality offspring compared to small females. Similarly, male body size can affect offspring quality and early life-history traits but the importance of these effects to the reproductive biology of fish is poorly studied. The extent to which variation in reproductive success is explained by parental body size is an important research topic because size-selective fishing usually reduces the average size of reproducing adults in a population. In my dissertation, I studied the parental size effects on reproductive success in a model species (zebrafish, Danio rerio). I also studied the effects of size-selective harvesting on body size, maturation and reproductive output. Body size and condition factor were important determinants of the initiation of maturation in zebrafish. Large females were found to have higher reproductive success compared to small females and a significant effect of male body size on early life-history traits was documented. I found that large males were also favored by the females resulting to differential allocation of reproductive resources toward large males. The maternal- and paternal-size effects ultimately led to elevated reproductive success of experimental spawning stocks consisting of large or random-sized individuals compared to spawning stocks consisting of small individuals. Size-selective harvesting induced rapid phenotypic and genetic changes, which persisted after selection was halted. This suggests that fishing-induced changes might be hard to reverse. My results emphasize the importance of body size to the reproductive biology of zebrafish and suggest that protecting large fish might be important to maintain the reproductive potential of exploited fish stocks.

L’uranium (U) est naturellement présent à l’état de trace dans l’eau (µg.L-1), sa concentration pouvant atteindre localement quelques mg.L-1 du fait des activités anthropiques. Plusieurs études écotoxicologiques sur Danio rerio ont mis en évidence la toxicité, e.g. le stress oxydant, la génotoxicité mais aussi la reprotoxicité (i.e. moins de pontes et d’œufs pondus chez les poissons contaminés) de l’U dont les mécanismes ne sont pas connus.L’objectif de cette étude est de contribuer à la compréhension de la reprotoxicité de l’U par l’élucidation de mécanismes moléculaires perturbés après contamination. Pour cela, des investigations ont été menées sur les ovaires de poissons zèbre Danio rerio, reproduits (R) ou non (NR), après exposition par voie directe en condition de laboratoire à des concentrations représentatives d’environnements contaminés.Ce travail de thèse a été divisé en deux volets. Un premier volet analytique avait pour but la poursuite des développements de méthodes pour l’identification des complexes U-protéines en condition non dénaturante, autour du couplage de techniques de séparation (chromatographie d’exclusion stérique SEC, électrophorèse hors gel OGE) et de détection sensible par spectrométrie de masse élémentaire (ICP MS) et moléculaire (ESI MS). Le second volet a été dédié à l’étude de la reprotoxicité de l’U à l’échelle moléculaire, avec i) l’étude des complexes natifs U-protéines (approche métallomique), et ii) l’analyse différentielle de l’expression des protéines (approche protéomique).Les développements analytiques ont permis de garder le tampon physiologique et non dénaturant d’extraction pour l’étape de séparation OGE, améliorant le taux de recouvrement en U. En écotoxicologie, les principaux résultats montrent que l’ovaire est un organe accumulateur de l’U et que le statut de reproduction a une influence sur le niveau d’accumulation (R<NR). En revanche, cet état a peu d’influence sur sa distribution protéique pour laquelle 4 fractions (dont 1 principale) ont été identifiées, toutes contenant aussi du phosphore. L’identification des cibles potentielles de l’U et des protéines exprimées différentiellement (vtg, GST, GAPDH,…) a montré que les processus biologiques perturbés suite à la contamination sont de deux niveaux : 1/ générique (stress oxydant) et 2/ plus spécifique de la gonade (développement et maturation des ovocytes). En conclusion, ces deux approches complémentaires ont permis de mettre en évidence un effet direct (complexation) et indirect (expression protéique modulée) de l’U, et de proposer l’hypothèse d’un défaut de maturation des ovocytes après contamination. Ce défaut pourrait impacter le développement embryonnaire et in fine expliquer la reprotoxicité observée lors d’études écotoxicologiques précédentes. / Uranium (U) is naturally presents at trace level (µg.L-1) in aquatic environment; its concentration can increase up to a few mg.L-1 due to human activities. Several ecotoxicological studies have shown uranium toxicity in contaminated zebrafishes Danio rerio, e.g. oxidative stress, genotoxicity and reprotoxicity (i.e. lower number of spawn and eggs laid) but mechanisms are not well known.The objective of this study is to contribute to the understanding of uranium reprotoxicity by elucidating the disrupted molecular mechanisms after contamination. Therefore, investigations have been carried out on ovaries from reproduced (R) and non-reproduced (NR) zebrafishes after waterborne exposure in laboratory conditions at environmentally relevant concentrations.This project was divided into two parts. Firstly, analytical investigations were carried out to continue the development of non-denaturing methods for U-protein identification by coupling separative techniques (size exclusion chromatography SEC, off gel electrophoresis OGE) with elemental (ICP MS) and molecular (ESI MS) sensitive mass spectrometry detection. Secondly, studies of U reprotoxicity were investigated by studying i) native U-protein complexes (metallomics approach) and ii) differential analysis of protein expression (proteomics approach)Analytical developments allowed keeping the physiological and non-denaturing extraction buffer for OGE separation step, improving U recovery. In ecotoxicology, the major results showed that ovary is an U accumulating organ and that the reproduction status modifies the accumulation level (R<NR). However, this status is of little influence on its distribution on proteins with 4 fractions (including a major one) determined, all of them coeluting with phosphorus. The identification of U potential targets and of protein expression differences (vtg, GST, GAPDH…) showed that biological processes disrupted after contamination are at two levels: 1/ generic (oxidative stress) and 2/ more specific to gonad (oocyte development and maturation).As a conclusion, these two complementary approaches showed a direct (complexation) and indirect (modification of protein expression) effects of U, and enabled to hypothesize a lack of oocyte maturation after contamination. This defect could impact embryo development and in fine explain the reprotoxicity observed in previous ecotoxicological studies.