The mechanism to maintain stem cell properties and to exit into differentiation pathways is a pivotal question in stem cell research. Spermatogonia are the adult stem cells of the male germ line, which are used in biomedical research as a source of undifferentiated cells. The communication between germ line stem cells and specialized somatic cells (Sertoli cells and Leydig cells) plays important roles in stem cell maintenance, germ cell proliferation, and differentiation. With regard to the biology of stem cells and spermatogenesis, the medaka (Oryzias latipes) is used as a teleost model organism, and it is also used to assess the effects of endocrine disruptors on reproductive phenomena. However, the lack of suitable molecular markers hampers the detection, isolation and analysis of different testis cells including gonial stem cells and Leydig cells. Therefore, oct4, sox2 and cyp11b were chosen to create transgenic reporter lines for the labeling of stem cells and Leydig cells, respectively. The present study had the aim to examine the temporal and spatial expression of the respective genes during embryonic development and in adult gonads of the medaka, and to describe the application of these transgenic lines in stem cell biology and reproductive biology. The mCherry expression in transgenic fish of the line FSI-Tg(sox2-mCherry)17 marks embryonic stem cells, Leydig cells and interstitial cells in adult testis. Faithful EGFP and DsRed expression in transgenic reporters strains for oct4 and cyp11b mimics the endogenous expression of oct4/pou2 and cyp11b-protein, respectively. The reporter gene expression in the strains FSI-Tg(oct4-EGFP)9 and FSI-Tg(oct4-EGFP)A allows the visualization of oct4 positive cells during embryonic development, PGCs, early germ cells and adult gonial cells. The Leydig cells express brightly green or red fluorescence in the medaka strains FSI-Tg(cyp11b-EGFP)20 and FSI-Tg(cyp11b-DsRed)1434, respectively, allowing the easy identification of Leydig cells in adult testis. The oct4-EGFP reporter labels medaka embryonic and spermatogonial stem cells, in which the spermatogonial stem cells at the ends of the testicular lobules show brightly green fluorescence. The transgenic expression in stem cells is also shown in the flow plot of primary testis cells. The spermatogonia are the largest cells and have the strongest fluorescence, which decreased upon differentiation. Therefore, the oct4-EGFP reporter strains will provide an opportunity to detect and to isolate the EGFP expressing cells for transplantation. These strains will also facilitate further experiments on the effects of drugs or hypoxia on these cells, because the strongest EGFP expressing cells can be easily detected in transgenic lines. Labeling of Leydig cells in cyp11b reporter lines opens a new area to study the seasonal variation of spermatogenesis. The medaka is a seasonal breeder in its natural habitat and the simulation of seasonal changes allows the simultaneous quantitative analysis of oct4-EGFP and cyp11b-DsRed expressing cells under such conditions.

La pression toxique induite par les perturbateurs endocriniens sur l’environnement ainsi que sur la santé de l’homme a augmenté de manière significative durant les dernières décennies. Il est devenu urgent de mettre en place des outils pour détecter et surveiller les perturbateurs endocriniens et pour déterminer la possibilité de cause de perturbations endocriniennes pour les produits chimiques nouvellement introduits. Le développement de nouveaux tests biologiques in vivo en utilisant les larves d'organismes aquatiques tels que medaka (Oryzias latipes) ou Xenopus laevis semble être une stratégie adéquate pour identifier les perturbateurs endocriniens. Durant cette thèse, trois nouveaux modèles transgéniques à base de l’embryon de medaka ont été développés. Le modèle transgénique de medaka ChgH -gfp permet la détection rapide des œstrogènes et des inhibiteurs de l'aromatase en seulement 24 heures avec une sensibilité de 15 ng / L d’éthinylestradiol. Le modèle transgénique de medaka 42sp50-gfp_CgH-gfp montre une fluorescence dans le foie en réponse aux œstrogènes, en plus de la fluorescence émise dans les œufs en développement. C’est donc un système approprié pour étudier le lien entre la signalisation de l'axe ostrogénique et des aberrations de la détermination du sexe chez les poissons. Un embryon transgénique de medaka spiggin - GFP a aussi été développé pour détecter les androgènes et anti-androgènes dans un test de 96 heures avec une sensibilité de 1,5 ug / L de 17α–méthyl testostérone et 276 ug / L de flutamide. Les nouveaux modèles de medaka transgéniques rapporteurs développés dans cette thèse permettent une détection rapide, simple et fiable des perturbateurs d'axe ostrogénique, androgénique et de l’aberration de détermination du sexe chez le medaka. Ils ont été appliqués pour détecter les perturbateurs endocriniens dans les eaux de surface de l'environnement et pour évaluer le rôle potentiel des produits chimiques dans les perturbations endocriniennes. Cela a permis de démontrer l’applicabilité de ces embryons fluorescents comme outils biologiques dans la procédure de détection et la caractérisation des perturbateurs endocriniens. / The toxic pressure of endocrine disruptors on biodiversity and human health has increased significantly over recent decades. As a consequence tools are needed to detect and monitor endocrine disruptors in surface water and to determine the endocrine disrupting potential of newly introduced chemicals. Fish and amphibian larvae, notably the medaka (Oryzias latipes) and Xenopus laevis, offer multiple advantages in this context. In the research carried out in the context of this thesis, different novel medaka-based transgenic models were developed. First, transgenic ChgH-gfp medaka model was designed and optimized for the rapid detection of estrogens and aromatase inhibitors. The model shows significant response within 24 hours with a sensitivity of 15 ng/L ethinylestradiol. Second, a double transgenic 42sp50-gfp_ChgH-gfp medaka which exhibits fluorescence both in the liver in response to estrogens and in developing oocytes as a function of phenotypic sex. It is therefore a suitable model for studying the link between estrogen axis signalling and aberrations of sex determination in fish. Third, a novel spiggin-gfp medaka model was developed to detect androgens and anti-androgens. This model can be exploited in a 96-hour assay with a sensitivity of 1.5 μg/L 17α- methyltestosterone and 276 μg/L flutamide. The novel transgenic medaka models developed in this thesis allow rapid, simple and reliable detection of estrogen and androgen axis disruption and aberrations in medaka sex determination. They have been successfully applied to detect endocrine disruptors in environmental surface water and to assess chemicals with unknown endocrine disrupting potential. Taken together these results demonstrate the applicability of medaka reporter larvae as biological tools in the procedure of detection and characterization of endocrine disruptors.

The mechanism to maintain stem cell properties and to exit into differentiation pathways is a pivotal question in stem cell research. Spermatogonia are the adult stem cells of the male germ line, which are used in biomedical research as a source of undifferentiated cells. The communication between germ line stem cells and specialized somatic cells (Sertoli cells and Leydig cells) plays important roles in stem cell maintenance, germ cell proliferation, and differentiation. With regard to the biology of stem cells and spermatogenesis, the medaka (Oryzias latipes) is used as a teleost model organism, and it is also used to assess the effects of endocrine disruptors on reproductive phenomena. However, the lack of suitable molecular markers hampers the detection, isolation and analysis of different testis cells including gonial stem cells and Leydig cells. Therefore, oct4, sox2 and cyp11b were chosen to create transgenic reporter lines for the labeling of stem cells and Leydig cells, respectively. The present study had the aim to examine the temporal and spatial expression of the respective genes during embryonic development and in adult gonads of the medaka, and to describe the application of these transgenic lines in stem cell biology and reproductive biology. The mCherry expression in transgenic fish of the line FSI-Tg(sox2-mCherry)17 marks embryonic stem cells, Leydig cells and interstitial cells in adult testis. Faithful EGFP and DsRed expression in transgenic reporters strains for oct4 and cyp11b mimics the endogenous expression of oct4/pou2 and cyp11b-protein, respectively. The reporter gene expression in the strains FSI-Tg(oct4-EGFP)9 and FSI-Tg(oct4-EGFP)A allows the visualization of oct4 positive cells during embryonic development, PGCs, early germ cells and adult gonial cells. The Leydig cells express brightly green or red fluorescence in the medaka strains FSI-Tg(cyp11b-EGFP)20 and FSI-Tg(cyp11b-DsRed)1434, respectively, allowing the easy identification of Leydig cells in adult testis. The oct4-EGFP reporter labels medaka embryonic and spermatogonial stem cells, in which the spermatogonial stem cells at the ends of the testicular lobules show brightly green fluorescence. The transgenic expression in stem cells is also shown in the flow plot of primary testis cells. The spermatogonia are the largest cells and have the strongest fluorescence, which decreased upon differentiation. Therefore, the oct4-EGFP reporter strains will provide an opportunity to detect and to isolate the EGFP expressing cells for transplantation. These strains will also facilitate further experiments on the effects of drugs or hypoxia on these cells, because the strongest EGFP expressing cells can be easily detected in transgenic lines. Labeling of Leydig cells in cyp11b reporter lines opens a new area to study the seasonal variation of spermatogenesis. The medaka is a seasonal breeder in its natural habitat and the simulation of seasonal changes allows the simultaneous quantitative analysis of oct4-EGFP and cyp11b-DsRed expressing cells under such conditions.

No description available.

Thesis (Ph. D.)--Michigan State University. Dept. of Zoology & Environmental Toxicology, 2008. / Title from PDF t.p. (viewed on Mar. 30, 2009) Includes bibliographical references. Also issued in print.

Nutritional efficiency of deeply frosted and repeatedly defrosted zooplankton has not been totally clarified, and it is often questioned, however, the nutrients composition and effect of such deteriorated feed are missing. The aim of this study was to assess nutritional eddiciency of repeatedly defrosted nauplia Artemis salina, as well as changes in their chemical composition on the basis of the growth, survival and presence of the deformations in early ontogeny of Japanese medaka ( Oryzias latipes). The samples of fresh hatched artemia nauplii served as a control group. The experiment was carried out with samples of live nauplii, permanently frosted at -14°C, defrosted to 4°C and again frosted and defrosted to 25°C and frosted again. The larvae of medaka in the period of one week after shift to the exogenous nutrition were examined. Before the commencement of the experiment the composition of samples in terms of nutrient, amino acids and fatty acid contents was determined. The results proved that Oryzias latipes fed with live artemia not only grew better but also survived more that other fis in other treatments. No deformations were recorded Fish fed frozen food survived better than those fed with live Artemia. It was also found that fish fed live Artemia seems to be the highest however from the amino acid point of view it seems to be more appropriate to freeze it to -14°C, defreeze to 25°C and refreezing to -4°C. as for the fatty acids Artemia adjusted by the permanently freezing to -14°C. The processes of thawing and repeated freezing of artemia nauplii result in leaching of some nutrients but in the thickening of some others.

This thesis has two parts. In the first part there is described an optimal methodological process for screening tests used in subsequently. Fertilized fish eggs of Oryzias latipes were incubated in 6 tests with different numbers (from 1 to 6) with standard conditions in ISO water. In the tests, hatching performance and duration of embryonic development were investigated and the results were applied on screening tests. The other part of the thesis is aimed on the proof of potential toxic effects of water with cyanobacterial water bloom. The fertilized eggs of Oryzias latipes were embedded into the test in stage 6 to 8. Three samples of cyanobacterial biomass from free waterbodies with known species composition and microcystin {--} LR, YR and RR contents were tested. The hatching performance, duration of embryonic development, lethal and sublethal effects were monitored during the tests. The tests were performed according to the OECD 212. There were detected significant differences in hatching performance, duration of embryonic development and in some cases also in induction of deformities between the control group and the tested groups.

La perte de biodiversité induite par les humains touche plus particulièrement les espèces de grande taille corporelle et génère des pressions de sélection contre les grands individus. Un nombre croissant d’études montrent que la diminution des abondances de ces espèces s’accompagne d’une évolution rapide vers des tailles corporelles réduites. La problématique générale de la thèse était d’étudier expérimentalement, de manière intégrée, « des gènes à l’écosystème », les implications évolutives et écologiques de la perte rapide de biodiversité.À l’aide d’une expérience de sélection artificielle sur la taille corporelle de médaka (Oryzias latipes) pendant 6 générations, j’ai montré que (1) un ensemble de traits d’histoire de vie corrélé génétiquement à la taille (e.g. croissance somatique, âge et taille à maturité sexuelle, fécondité) pouvaient évoluer rapidement, (2) la réponse des traits des médakas dépend de la direction de sélection, et que (3) les changements phénotypiques peuvent être reliés à des changements d’expressions d’hormones hypophysaires impliqués dans la régulation de la croissance (i.e. hormone de croissance) et la reproduction (i.e. hormone de croissance et gonadotropines). Ces résultats suggèrent que les mécanismes d’adaptations des populations aux pressions anthropiques pourraient fortement modifier des traits clés pour leur maintien dans les écosystèmes. Par ailleurs, dans une autre expérience, j’ai montré que les variations phénotypiques (taille et de forme d’un médaka), peuvent avoir autant d’importance sur l’intensité de la cascade trophique qu’elles génèrent, que les variations démographiques (présence-absence du médaka). Ce résultat révèle l’importance des traits des organismes dans l’écosystème et suggère que des changements micro-évolutifs pourraient se répercuter dans les réseaux d’interactions biotiques. Cette thèse souligne la nécessité de mieux comprendre les mécanismes adaptatifs afin d’appréhender au mieux les conséquences écologiques des pressions de sélection d’origine anthropique qui s’exercent sur les populations naturelles. Elle ouvre des perspectives sur la compréhension jointe des mécanismes évolutifs et écologiques qui peuvent agir en retour l’un sur l’autre dans des boucles de rétrocontrôle éco-évolutives. / Biodiversity loss induced by humans particularly affects species which large body size and generates selection pressures against large individuals. A growing number of studies show that the decline in abundance of these species is accompanied by a rapid shift towards smaller body sizes. The aim of the thesis was to study experimentally, in an integrative approach, "from genes to ecosystem," the evolutionary and ecological implications of the rapid loss of biodiversity. I applied an artificial selection on body size of medaka (Oryzias latipes) during 6 generations, and I showed that (1) a set of life history traits genetically correlated to the size (e.g. somatic growth, age and size at sexual maturity, fecundity) could evolve rapidly, (2) the response of medaka traits depend on the selection direction, and (3) the phenotypic changes can be related to changes of pituitary hormones expressions involved in the regulation of growth (i.e. growth hormone) and reproduction (i.e. growth hormone and gonadotropins). These results suggest that the adaptation mechanisms of populations to anthropogenic pressures could significantly alter key features for keeping them in ecosystems. Furthermore, in another experiment, I showed that phenotypic variations (size and shape of a medaka), may have as much importance on the intensity of the trophic cascade they generate, as demographic variations (presence-absence of medaka). This result shows the importance of the traits of organisms

Recent empirical studies have demonstrated inter-generational morphological and life-history changes in fish stocks that have been impacted by size-selective harvest. Evolutionary processes in biological populations occur through differential survival and reproductive success based, in part, upon individual phenotypic variability. Fishing is a source of directional selection resulting in the directed removal of some phenotypes; however, many aspects of the evolutionary effects of fishing remain have yet to be described. In order to better understand the life-history and morphological changes that occur as a result of size-selective fishing, and their effect on fishery dynamics, I first determined the suitability of Japanese medaka (Oryzias latipes) for selection experiments. I performed selection experiments using Japanese medaka and report how morphology and life-history characteristics changed over multiple generations of selection. I then used these patterns of change in life-history and morphology to validate individual-based simulation candidate models to test general mechanisms of life-history relationships. Finally, I applied the individual-based simulation modeling approach in order to describe how biological and fishery characteristics change in a large, age-structured population exposed to size-selective fishing over multiple generations. I found that the Japanese medaka has attractive characteristics for biological investigation. The selection experiments indicated large changes in the age-atmaturity, including a nearly 50% decrease over four generations in the most intense sizeselective removal regimes. However, I did not observe significant changes in length-at-age or weight-at-age over the course of the experiment. Candidate simulation models were poor at predicting some aspects of the life-history characteristics of Japanese medaka. The simulation model to determine fishery characteristics predicted large decreases in yield and egg production as a result of decreases in length-at-age. Understanding the relationships of life-history characteristics and their role in determining population resilience is a step toward understanding the importance of evolutionary processes in fishery management. / Ph. D.

<div>Following the 2010 Deepwater Horizon oil spill, extensive research has been conducted on the toxicity of oil and polycyclic aromatic hydrocarbons (PAHs) in the aquatic environment. The location and timing of the Deepwater Horizon surface slick coincided with the spawning seasons of many important pelagic and estuarine fish species. As such, there has been particular emphasis placed on the effects of PAHs on sensitive life history stages in fish, such as the embryonic and larval periods. Additionally, the spill occurred throughout the spring and summer months which, in estuaries, are marked by regular fluctuations in abiotic environmental factors such as dissolved oxygen, salinity, and temperature. Until recently, there has been little work done to elucidate the combined effects that PAHs from oil spills and adverse environmental conditions (hypoxia, increased salinity, and elevated temperatures).</div><div>Work presented in this dissertation uses next generation sequencing technology (RNA Seq) to determine differential gene expression in larval estuarine teleosts following exposure to adverse environmental conditions and PAHs. Downstream canonical pathway and toxicological function analysis were then applied to the identified differentially expressed genes (DEGs) to predict cardiotoxic responses at the organismal level. To verify the predicted responses, a phenotypic anchoring study was conducted and identified a cardiotoxic phenotype (pericardial edema) and reduced cardiac output in embryos exposed to oil. Finally, the mechano-genetic interplay governing the morphological development of the teleost heart was investigated and correlations between developmental gene expression and blood flow forces within the cardiovascular system were identified.</div>