Nuclear architecture and gene expression in Caenorhabditis elegans Mgr. Jitka Bolková ABSTRACT The parental genomes are initially separated in each pronucleus after fertilization. During the first mitosis this spatial distribution is being disintegrated. In my thesis we used green-to-red phoroconversion of Dendra2-H2B-labeled pronuclei to distinguish maternal and paternal chromatin domains and to track their distribution in space in living Caenorhabditis elegans embryos starting shortly after fertilization. Both of the parental chromatin domains within the nucleus are separated in the zygote and at the 2-cell stage. Intermingling occurs first after chromatin decondensation at the beginning of the cell cycle at the 4-cell stage. To our knowledge, we report to the first live observation of the separation and subsequent mixing of parental chromatin during embryogenesis. Following of the photoconverted chromatin also allowed us to detect a reproducible 180ř rotation of the nuclei during cytokinesis of the zygote. Tracking of fluorescently-labelled P granules and polar bodies showed that the entire embryo rotates during the first cell division. In the second part of the thesis we used the C. elegans model to investigate relationship between nuclear architecture and gene expression. We focused on localization of...

O presente trabalho teve como objetivo avaliar possíveis alterações fisiológicas associadas à reprodução em machos e fêmeas de Arapaima gigas (pirarucu) em seu ambiente natural. O pirarucu (Arapaima gigas) é uma espécie de respiração aérea que pertence ao mais primitivo grupo dos teleósteos, que habita as águas do rio Amazonas. Os objetivos principais foram caracterizar o perfil plasmático dos esteróides gonadais plasmáticos (17β-Estradiol, Testosterona, 11ceto-Testosterona (11ceto-T) e 17α -hidroxiprogesterona (17α -OHP)); concentrações de lipídios e proteínas teciduais (gônadas, fígado, músculo branco, vermelho e ventrecha), e o perfil de ácidos graxos ao longo do ciclo reprodutivo de machos e fêmeas coletados em seu ambiente natural localizado na Ilha Mexiana no estado do Pará/Brasil. As coletas foram realizadas em Setembro/2007; Janeiro/2008; Julho/2008 e Setembro/2008 e foram determinados quatro estádios de maturação gonadal para as fêmeas: Repouso, Maturação, Maduro e Regressão. No presente estudo foram observados ainda animais imaturos. Os machos foram classificados em relação à quantidade de espermatozóides presente nos túbulos seminíferos, sendo que os animais que apresentavam poucos espermatozóides no lúmen são referidos como grupo 1 e os animais que apresentavam muitos espermatozóides no lúmen como grupo 2. As análises histológicas realizadas nas gônadas permitem concluir que o pirarucu possui ovário do tipo gymno-ovariano com desenvolvimento oocitário assincrônico e a estrutura testicular de A. gigas é do tipo não cística. As análises metabólicas em fêmeas mostraram que a ventrecha é o tecido de maior concentração de lipídios, seguido do músculo vermelho e branco, evidenciando que, dentre os diferentes tipos musculares, a ventrecha é a região preferencial de armazenamento de lipídios. As concentrações lipídicas teciduais , demonstraram um aumento no acúmulo gonadal e hepático à medida que o ciclo reprodutivo avança, alcançando a concentração máxima na fase madura. Já as proteínas hepáticas e gonadais não apresentaram diferença significativa entre os estádios reprodutivos. Nos machos, os lipídios e as proteínas não apresentaram diferença estatística significativa entre os grupos em nenhum tecido analisado. Os PUFAs (ácidos graxos polinsaturados) n3 na fração polar ovariana foram maiores na fase madura em relação às demais fases reprodutivas, inclusive no animal imaturo, sendo que essa alta porcentagem reflete na importância desses ácidos graxos no ovo, que serão utilizados para o futuro desenvolvimento da larva. De uma forma geral, foi observada uma característica de maior porcentagem de PUFAs n6 (fração polar) e PUFAs n3 (fração neutra) do tecido hepático em animais imaturos quando comparados às fêmeas adultas. Nos machos, os ácidos graxos monoinsaturados foram maiores no grupo 2 no músculo branco e ventrecha, devido à necessidade de um maior aporte energético na musculatura para o comportamento reprodutivo. Os resultados das análises plasmáticas dos esteróides gonadais mostraram que o perfil de 17β-Estradiol em fêmeas apresentou-se alto nas fases iniciais do ciclo reprodutivo, decaindo na fase madura e regressão, provavelmente devido ao feedback negativo. Os animais imaturos apresentaram as maiores concentrações plasmáticas de 17β-Estradiol e 17α-OHP em relação aos adultos, já que esses hormônios estimulam o desenvolvimento de características secundárias, desenvolvimento neural, crescimento e maturação sexual nos animais imaturos, em machos e fêmeas. Foram observadas altas concentrações de 17α -OHP nos machos em ambos os grupos sugerindo a disponibilidade desse hormônio para a conversão de 17α,20β-DHP, que atua na motilidade dos espermatozóides, já que ambos os grupos apresentam espermatozóides no lúmen. As concentrações de testosterona excedem à de 11ceto-T em machos, sugerindo que essa espécie esteja utilizando outros andrógenos e não somente a 11ceto-T para a reprodução. O acompanhamento destes animais no ambiente natural evidencia que não é possível utilizar características secundárias como coloração da escama como indicativo do sexo, porém a sexagem é possível de se realizar utilizando a razão entre os hormônios plasmáticos 11ceto-T e 17β-Estradiol. / The aim of the present study was to investigate the possible physiological alterations related with reproduction in males and females of Arapaima gigas (pirarucu) in their natural environment. Pirarucu is an obligatory aerial respiration species belonging to a very ancient group of teleosts and inhabits Amazonian river. The main goals of this study were to describe the plasmatic gonad steroids profile (17β-Estradiol, Testosterone, 11keto-Testosterone (11keto-T) and 17α- hidroxyprogesterone (17α-OHP)), lipids and proteins concentrations of the main storage tissues (gonads, white muscle, red muscle and ventrecha), and fatty acid profile along the reproductive cycle of males and females collected in their natural environment at Ilha Mexiana (Pará/ Brazil). The animals were caught from the wild in September/2007, January/2008, July/2008 and September/2008 and four stages of gonad development were determined: Rest, Maturation, Mature and Regression. Immature animals were also observed. Males were classified according to spermatozoid amount inside the seminiferous tubes, and the animals having a few spermatozoids were referred as group1 and those having many spermatozoids were referred as group 2. Microscopic analyses allowed concluding that pirarucu has gymno-ovary type with assynchronic oocyte development and testis has non-cystic structure. The metabolic analyses in females showed that the tissue presenting the highest lipids concentration was the ventrecha, followed by the red and white muscle, suggesting that ventrecha is the preferred lipids storage among different muscles. Lipids concentrations increased in ovaries and liver along the reproductive cycle, getting the highest value at mature stage. Liver and gonads protein did not show any difference among the reproductive stages. Lipids and proteins in males did not present any difference between the groups of any analyzed tissue. In polar lipids ovary fraction, PUFA (polyunsaturated fatty acids) n3 were higher at mature stage, compared with the other stages of the reproductive cycle and immature animals, and these high amounts reflect the importance of those fatty acids, that will be used in the larval development. In general, the immature animals showed the highest percentage of PUFA n6 (polar fraction) and PUFA n3 (neutral fraction) in the liver when compared with adults females. Monounsaturated fatty acids in males in group 2 were higher in white muscle and ventrecha, because those muscles have an intense energetic demand for reproductive behavior. 17β-Estradiol was higher in females, at initial stages of the reproductive cycle, decreasing at mature and regression stages, probably due to a negative feedback. Immature animals showed the highest concentrations of 17β-Estradiol and 17α-OHP, probably because these steroids stimulate sexual secondary features, neural development, growth and sexual maturity in those animals. 17α-OHP concentrations were higher in both male groups, suggesting its availability for 17α, 20β-DHP conversion, an important steroid on spermatozoid motility. Testosterone concentrations in males exceed 11ceto-T, suggesting that other androgens were more effective and not just 11ceto-T, for reproduction. On this environment, features like scales coloration as secondary feature were not suggested to distinguish the sex of those animals, but it is possible when using 11ceto-T and 17β- Estradiol plasma ratio.

Submitted by (edna.saturno@ufrpe.br) on 2017-02-09T15:50:20Z No. of bitstreams: 1 Draussio Pinheiro Veras.pdf: 2415488 bytes, checksum: c1bef0097caba696278592343621305f (MD5) / Made available in DSpace on 2017-02-09T15:50:20Z (GMT). No. of bitstreams: 1 Draussio Pinheiro Veras.pdf: 2415488 bytes, checksum: c1bef0097caba696278592343621305f (MD5) Previous issue date: 2008-02-08 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / The parrotfishes, Sparisoma axillare and S. frondosum (Family: Scaridae), are reef fish species frequently caught as bycatch on the trap fishery for lobster and goatfishes in the State of Pernambuco. Between 2002 and 2005, the total of parrotfish caught by the marine fisheries in Pernambuco State, varied from 136.5 to 280.5 t. These numbers show a growing fishing pressure on this important zoological group, with a significant impact on coral reef ecosystems, highlighting the importance of biological and ecological studies on these species. In this context, the present study intends to generate information regarding the main reproductive parameters of S. axillare e S. frondosum, with the purpose of supporting the management of their fisheries in the region. For the study of the reproductive biology, the stages of gonad maturation were defined through microscopic analysis of 283 specimes of S. axillare and 185 specimes of S. frondosum. The standard length (SL) varied between 13.8 and 33.4 cm, for S. axillare, and from 12.6 to 29.5 cm, for S. frondosum. The sex ratio between male and female was 1:6.4, for S. axillare, and 1:1.2, for S. frondosum. The average values of Gonad Index (G.I.) for S. axillare were higher during the second semester of the year, with the largest values for females occurring in July, August, September and November and for males, in September. For S. frondosum, females presented the highest average GI in February and males in April. The size of first sexual maturity (L50) for females of S. axillare was estimated at 20.2 cm of SL. It was not possible to determine the L50 for males. The L50 for females of S. frondosum was estimated at 17.0 cm of SL and for males at 17.7 cm. The average fecundity was 50.075 oocytes for S. axillare and 80.276 oocytes for S. frondosum. These results indicate that both species present multiple or batch spawning, with reproduction occurring all year. / Os budiões batata, Sparisoma axillare e S. frondosum (Família: Scaridae), são espécies de peixes recifais que vêm sendo bastante capturadas como fauna acompanhante na pescaria de covo voltada para lagosta e saramunete no Estado de Pernambuco. Entre os anos de 2002 e 2005, a pesca extrativa marinha obteve capturas de escarídeos que variaram entre 136,5 e 280,5 t. Esses dados evidenciam uma elevada e crescente pressão pesqueira sobre esse importante grupo zoológico, com um significativo impacto sobre os ecossistemas recifais em que habitam. Desta forma, é de fundamental importância o desenvolvimento de estudos que possam aportar informações biológicas sobre as espécies capturadas, muitas das quais são ainda pouco conhecidas, como é o caso das espécies aqui estudadas. Nesse contexto, o presente estudo pretendeu gerar informações acerca dos principais parâmetros reprodutivos de S. axillare e S. frondosum, com o objetivo de subsidiar o ordenamento da sua pesca na região. Para o estudo da biologia reprodutiva foram determinados os estágios de maturação gonadal através de análises microscópicas de cortes histológicos do aparelho reprodutor de 283 indivíduos de S. axillare e 185 indivíduos de S. frondosum. O comprimento padrão (CP) dos exemplares variou entre 13,8 e 33,4 cm, para S. axillare, e entre 12,6 a 29,5 cm, para S. frondosum. A proporção sexual, entre macho e fêmea, foi de 1:6,4, para S. axillare, e de 1:1,2, para S. frondosum. Os valores médios do índice gonadal para as fêmeas e machos de S. axillare foram mais altos nos meses do segundo semestre do ano. Para S. frondosum, as fêmeas apresentaram maior valor médio mensal em fevereiro e os machos em abril. O tamanho de primeira maturação sexual (L50) para as fêmeas de S. axillare foi estimado em 20,2 cm de CP, não tendo sido possível determinar a curva de maturação para os machos. O L50 das fêmeas de S. frondosum foi estimado em 17,0 cm de CP e o dos machos em 17,7 cm. A fecundidade média estimada foi de 50.075 ovócitos/gônada para S. axillare e de 80.276 ovócitos/gônada para S. frondosum. Os resultados indicam que ambas as espécies apresentam desova múltipla ou parcelada, reproduzindo-se durante o ano todo.

It is generally thought that in amphibians, thyroid hormones (THs) regulate metamorphosis, while sex steroids (estrogens and androgens) regulate gonadal differentiation. However, inhibition of TH synthesis in frogs alters gonadal differentiation, suggesting instead that these two endocrine axes interact during development. Specifically, THs may be involved in male development, while estrogens may inhibit tadpole metamorphosis. However, we do not currently know the mechanisms that account for these interactions, let alone how such mechanisms may differ between species. To develop and test new hypotheses on the roles of sex steroids and THs, I first examined transcriptional profiles (mRNA) of enzymes and receptors related to sex steroids and THs during embryogenesis and metamorphosis in Silurana tropicalis. Tadpoles were exposed to either an estrogen synthesis inhibitor (fadrozole) or TH (triiodothyronine, T3) during early larval or tadpole development. Acute exposures of S. tropicalis to fadrozole or T3 during early development resulted in increased expression of androgen- and TH-related genes in whole body larvae, while chronic exposure to fadrozole during metamorphosis affected gonadal differentiation but did not affect tadpole development. On the other hand, acute exposure to T3 during metamorphosis increased the expression of androgen-related transcripts both in the brain and gonad. In S. tropicalis, the results suggested that cross-talk is primarily in one direction (i.e., effect of THs on the reproductive axis) with a strong relationship between TH and androgen status. Lastly, I established developmental transcript profiles and investigated T3 regulation of brain and gonad transcripts in Engystomops pustulosus. I then compared these results with S. tropicalis and an earlier study in Lithobates pipiens. While each species developed with similar profiles, they differed in their response to T3. Exposure to T3 resulted in either an increase in androgen-related genes (S. tropicalis) or a decrease in estrogen-related genes (E. pustulosus and L. pipiens). In conclusion, these data demonstrated that cross-talk mechanisms differ among these three evolutionary separate species, but in all cases, T3 appears to affect the balance of sex steroids, stimulating the androgen system and providing potential mechanisms of the masculinising effects of THs. These results will contribute to understanding the mechanisms of hormone interactions and their evolutionary basis in frogs.

It is generally thought that in amphibians, thyroid hormones (THs) regulate metamorphosis, while sex steroids (estrogens and androgens) regulate gonadal differentiation. However, inhibition of TH synthesis in frogs alters gonadal differentiation, suggesting instead that these two endocrine axes interact during development. Specifically, THs may be involved in male development, while estrogens may inhibit tadpole metamorphosis. However, we do not currently know the mechanisms that account for these interactions, let alone how such mechanisms may differ between species. To develop and test new hypotheses on the roles of sex steroids and THs, I first examined transcriptional profiles (mRNA) of enzymes and receptors related to sex steroids and THs during embryogenesis and metamorphosis in Silurana tropicalis. Tadpoles were exposed to either an estrogen synthesis inhibitor (fadrozole) or TH (triiodothyronine, T3) during early larval or tadpole development. Acute exposures of S. tropicalis to fadrozole or T3 during early development resulted in increased expression of androgen- and TH-related genes in whole body larvae, while chronic exposure to fadrozole during metamorphosis affected gonadal differentiation but did not affect tadpole development. On the other hand, acute exposure to T3 during metamorphosis increased the expression of androgen-related transcripts both in the brain and gonad. In S. tropicalis, the results suggested that cross-talk is primarily in one direction (i.e., effect of THs on the reproductive axis) with a strong relationship between TH and androgen status. Lastly, I established developmental transcript profiles and investigated T3 regulation of brain and gonad transcripts in Engystomops pustulosus. I then compared these results with S. tropicalis and an earlier study in Lithobates pipiens. While each species developed with similar profiles, they differed in their response to T3. Exposure to T3 resulted in either an increase in androgen-related genes (S. tropicalis) or a decrease in estrogen-related genes (E. pustulosus and L. pipiens). In conclusion, these data demonstrated that cross-talk mechanisms differ among these three evolutionary separate species, but in all cases, T3 appears to affect the balance of sex steroids, stimulating the androgen system and providing potential mechanisms of the masculinising effects of THs. These results will contribute to understanding the mechanisms of hormone interactions and their evolutionary basis in frogs.

<p>The gonad is a unique primordial organ that retains the ability to adopt one of two morphological fates through much of mammalian embryonic development. Previous work in our lab found that dimorphic vascular remodeling was one of the earliest steps during sex-specific morphogenesis. In particular, vessels in XY gonads display highly ordered behavior that coincides with testis cord formation. It was unknown how the vasculature may influence testis cord morphogenesis and, if so, how this was mechanistically related to sex determination. The work in this thesis addresses a single over-arching hypothesis: Male-specific vascular remodeling is required for testis morphogenesis and orchestrates differentiation of the XY gonad. </p><p>To address this question we have modified and developed techniques that allow us to isolate aspects of vascular behavior, gene expression, and endothelial influence on surrounding cells. In particular, the application of live imaging was instrumental to understanding the behavior of various gonadal cell-types in relation to remodeling vessels. It is difficult to grasp the complexity of an organ without understanding the dynamics of its constituents. A critical aim of my work was to identify specific inhibitors of the vasculature that do not affect the early stages of sex determination. Combining inhibitors, live imaging, cell sorting, qRT-PCR, mouse models, and whole organ culture has led to a far richer understanding of how the vasculature behaves and the cell-types that mediate its influence on organ morphogenesis. The beauty of our system is that we do not have to settle for a snapshot of the fate of cells in vivo, but can document their journeys and their acquaintances along the way. </p><p>Vascular migration is required for testis cord morphogenesis. Specific inhibitors revealed that in the absence of vessels, testis cords do not form. The work below shows that vessels establish a feedback loop with mesenchymal cells that results in both endothelial migration and subsequent mesenchymal proliferation. Interstitial control of testis morphogenesis is a new model within the field. The mechanisms regulating this process include Vegf mediated vascular remodeling, Pdgf induced proliferation, and Wnt repression of coordinated endothelial-mesenchymal dynamics. Our work also suggests that vascular patterning underlies testis patterning and, again, is mediated by signals within the interstitial space not within testis cords themselves. </p><p>A final aspect of my work has been focused on how vessels continue to influence morphology of the testis and the fate of surrounding cells. Jennifer Brennan, a graduate student in our lab, previously showed that loss of Pdgfr&#945; antagonizes cord formation and development of male-specific lineages. The mechanisms and cell-types related to this defect were not clear. I began to reanalyze Pdgfr&#945; mutants after finding remarkable similarity to gonads after vascular inhibition. This work is providing data suggesting that vessels are not simply responsible for testis morphology but also for the fate of specialized cells within the testis. On the whole, this thesis describes specific roles for endothelial cells during gonad development and mechanisms by which they are regulated.</p> / Dissertation

Aquaculture is an essential developing sector for world food production however one of the major bottlenecks for the sustainability of the aquaculture industry is the ability to control fish reproduction in captivity and to produce high quality seeds. European sea bass is a one of most commercially important species for the European fish farming industry. If broodstock management under captivity is well established, problems remain in hatcheries where survival can be low and deformity prevalence high as well as in on growing sites where fish reach puberty early especially with skewed sex ratio towards males. Sea bass displays strong seasonality in its physiology and is therefore an excellent candidate for the study of the photo-neuroendocrine control of reproduction and growth. The overall aims of this thesis were to better understand the molecular and endocrine drivers that control the Brain-Pituitary-Gonad axis in repeat spawner sea bass, and expand our knowledge of sea bass light and temperature regulation of melatonin production. First, this PhD project investigated the seasonal expression of kisspeptin, GnRH and gonadotropin genes in relation to the gonadal development throughout a reproductive cycle in male repeat spawning sea bass (Chapter 3). A partial sequence for the receptor kissr4 was isolated and described showing similarity to all other teleost species sequences available to date. QPCR molecular assays were validated to mesure the expression of a suite of genes along the BPG axis including kisspeptin related genes (Kiss1 and Kiss2 and its receptor kissr4) over a full reproductive cycle (12 months) in adult male European sea bass. Brain Kisspeptin mRNA expression levels (kiss1, 2 and kissr4) showed clear seasonal profiles and correlated well to other BPG markers (GnRHs, fshβ and lhβ), supporting a possible involvement of kisspeptin genes in the seasonal control of reproduction in repeat spawning sea bass. Moreover, clear seasonal patterns were observed for expression of the genes encoding for pituitary mRNA expression of lhβ and fshβ, with a significant correlation between expression of both subunits and GSI and steroids levels. However, no clear seasonal profiles in brain GnRHs gene expression were observed with the exception to some peaks in GnRH1 and GnRH2. The second part of this PhD project investigated the potential direct effect of the two kisspeptin core peptides (kiss1 and kiss2) on the pituitary gonadotropin gene expression (Chapter 4). The aim of this work was to better understand the mechanism by which kisspeptin acts on the BPG axis. This was done by testing the kisspeptin decapeptide core sequences on the lhβ and fshβ transcript expression in primary culture of sea bass pituitary cells using QPCR technique. The findings, as a whole, provided evidence that kisspeptin can act directly on the pituitary gonadotroph cells and modulate fshß and lhß mRNA expression in sea bass although effects were limited and not uniform. Of note, kissr4 gene expression was also detected in the sea bass pituitary. The third part of this PhD project focused on the effects of environmental signals (photoperiod and temperature) on melatonin production (Chapter 5). Environmental manipulation is routinely used in the aquaculture industry with the purpose of enhancing growth and manipulating the timing of reproduction in seasonal fish species like sea bass. Melatonin, known as the light perception and time keeping hormone, has been suggested to play key roles in the synchronisation of most physiological functions in vertebrates, although the mechanisms by which melatonin controls reproduction, growth and behaviour are still not fully understood in fish. The studies performed aimed .to determine the synergistic effects of both temperature and photoperiod on the daily phase and amplitudinal changes in melatonin production through both in vivo and in vitro trials. The results confirmed the diel melatonin rhythm in sea bass as previously reported in many teleost species with “high at night” and “low at day” melatonin profiles. Temperature showed clear effects on the amplitude of the melatonin production under both in vivo and in vitro conditions for both long day and short day photoperiods. Furthermore, no endogenous melatonin production was found under constant darkness in both in vivo and in vitro conditions. These results suggested a lack of intrapineal (or located elsewhere such as retina and/or deep brain) oscillators in sea bass, contrasting with previous reports. These results further enhance our knowledge of light perception and circadian rhythmicity in sea bass, while the circadian system remains to be characterised in sea bass and teleosts as a whole. Overall, this doctoral work broadened our understanding on the photoneuroendocrine control of reproduction in a seasonal fish species, sea bass. New knowledge gained and tools developed from this work should help to develop/optimise husbandry techniques for the sea bass farming industry with the view to increase production and profitability and thus promoting the sustainable expansion of the sea bass aquaculture in Europe. It has also the potential to help the fishery sector in the modelling of wild sea bass populations.

It is generally thought that in amphibians, thyroid hormones (THs) regulate metamorphosis, while sex steroids (estrogens and androgens) regulate gonadal differentiation. However, inhibition of TH synthesis in frogs alters gonadal differentiation, suggesting instead that these two endocrine axes interact during development. Specifically, THs may be involved in male development, while estrogens may inhibit tadpole metamorphosis. However, we do not currently know the mechanisms that account for these interactions, let alone how such mechanisms may differ between species. To develop and test new hypotheses on the roles of sex steroids and THs, I first examined transcriptional profiles (mRNA) of enzymes and receptors related to sex steroids and THs during embryogenesis and metamorphosis in Silurana tropicalis. Tadpoles were exposed to either an estrogen synthesis inhibitor (fadrozole) or TH (triiodothyronine, T3) during early larval or tadpole development. Acute exposures of S. tropicalis to fadrozole or T3 during early development resulted in increased expression of androgen- and TH-related genes in whole body larvae, while chronic exposure to fadrozole during metamorphosis affected gonadal differentiation but did not affect tadpole development. On the other hand, acute exposure to T3 during metamorphosis increased the expression of androgen-related transcripts both in the brain and gonad. In S. tropicalis, the results suggested that cross-talk is primarily in one direction (i.e., effect of THs on the reproductive axis) with a strong relationship between TH and androgen status. Lastly, I established developmental transcript profiles and investigated T3 regulation of brain and gonad transcripts in Engystomops pustulosus. I then compared these results with S. tropicalis and an earlier study in Lithobates pipiens. While each species developed with similar profiles, they differed in their response to T3. Exposure to T3 resulted in either an increase in androgen-related genes (S. tropicalis) or a decrease in estrogen-related genes (E. pustulosus and L. pipiens). In conclusion, these data demonstrated that cross-talk mechanisms differ among these three evolutionary separate species, but in all cases, T3 appears to affect the balance of sex steroids, stimulating the androgen system and providing potential mechanisms of the masculinising effects of THs. These results will contribute to understanding the mechanisms of hormone interactions and their evolutionary basis in frogs.

Submitted by (edna.saturno@ufrpe.br) on 2016-07-21T15:16:08Z No. of bitstreams: 1 Gyl Everson de Souza Maciel.pdf: 1066870 bytes, checksum: 5d3e6cb058a19d66e3669721aeb1a30a (MD5) / Made available in DSpace on 2016-07-21T15:16:08Z (GMT). No. of bitstreams: 1 Gyl Everson de Souza Maciel.pdf: 1066870 bytes, checksum: 5d3e6cb058a19d66e3669721aeb1a30a (MD5) Previous issue date: 2013-02-26 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Pomacea lineata (Spix, 1827) during the dry season buries itself in the ground and survives thanks to its ability to aestivation. Studies have shown that during aestivation levels of carbohydrates and uric acid, may provide more accurate information about the activity of the gonads in response to environmental stress. Thus, we tested the hypothesis that variations in the levels of uric acid and carbohydrate during aestivation induced, can affect gonadal histology and gametogenesis in P. lineata. The snails underwent aestivation of three, five and 10 months. Testes and ovaries were collected and processed for histological analysis. The results showed significant increase in the levels of uric acid and carbohydrates in proportion to the period of aestivation, and histological changes in the gonads. Thus, we conclude that the metabolism of proteins is proportional to the period of aestivation for carbohydrate synthesis and excretion of uric acid, thereby ensuring the integrity of the reproductive tract. / Pomacea lineata (Spix, 1827)durante a época de estiagem se enterra no solo e sobrevive graças a sua capacidade de estivação. Estudos têm mostrado que durante a estivação os níveis de carboidratos e ácido úrico, podem fornecer informações mais precisas sobre a atividade das gônadas em resposta ao estresse ambiental. Assim, testou-se a hipótese de que as variações nos níveis de carboidratos e acido úrico, durante a estivação induzida, pode afetar a histologia gonadal e gametogênese em P. lineata. Os caramujos foram submetidos à estivação por três, cinco e 10 meses. Testículos e ovários foram coletados e processados para análises histológicas. Os resultados mostraram aumento significativo dos níveis de carboidratos e ácido úrico proporcionalmente ao período de estivação, além de alterações histológicas nas gônadas. Assim, concluímos que a metabolização de proteínas é proporcional ao período de estivação, para síntese de carboidratos e excreção de ácido úrico, garantindo assim, a integridade do aparelho reprodutor.

O presente trabalho teve como objetivo avaliar possíveis alterações fisiológicas associadas à reprodução em machos e fêmeas de Arapaima gigas (pirarucu) em seu ambiente natural. O pirarucu (Arapaima gigas) é uma espécie de respiração aérea que pertence ao mais primitivo grupo dos teleósteos, que habita as águas do rio Amazonas. Os objetivos principais foram caracterizar o perfil plasmático dos esteróides gonadais plasmáticos (17&#946;-Estradiol, Testosterona, 11ceto-Testosterona (11ceto-T) e 17&#945; -hidroxiprogesterona (17&#945; -OHP)); concentrações de lipídios e proteínas teciduais (gônadas, fígado, músculo branco, vermelho e ventrecha), e o perfil de ácidos graxos ao longo do ciclo reprodutivo de machos e fêmeas coletados em seu ambiente natural localizado na Ilha Mexiana no estado do Pará/Brasil. As coletas foram realizadas em Setembro/2007; Janeiro/2008; Julho/2008 e Setembro/2008 e foram determinados quatro estádios de maturação gonadal para as fêmeas: Repouso, Maturação, Maduro e Regressão. No presente estudo foram observados ainda animais imaturos. Os machos foram classificados em relação à quantidade de espermatozóides presente nos túbulos seminíferos, sendo que os animais que apresentavam poucos espermatozóides no lúmen são referidos como grupo 1 e os animais que apresentavam muitos espermatozóides no lúmen como grupo 2. As análises histológicas realizadas nas gônadas permitem concluir que o pirarucu possui ovário do tipo gymno-ovariano com desenvolvimento oocitário assincrônico e a estrutura testicular de A. gigas é do tipo não cística. As análises metabólicas em fêmeas mostraram que a ventrecha é o tecido de maior concentração de lipídios, seguido do músculo vermelho e branco, evidenciando que, dentre os diferentes tipos musculares, a ventrecha é a região preferencial de armazenamento de lipídios. As concentrações lipídicas teciduais , demonstraram um aumento no acúmulo gonadal e hepático à medida que o ciclo reprodutivo avança, alcançando a concentração máxima na fase madura. Já as proteínas hepáticas e gonadais não apresentaram diferença significativa entre os estádios reprodutivos. Nos machos, os lipídios e as proteínas não apresentaram diferença estatística significativa entre os grupos em nenhum tecido analisado. Os PUFAs (ácidos graxos polinsaturados) n3 na fração polar ovariana foram maiores na fase madura em relação às demais fases reprodutivas, inclusive no animal imaturo, sendo que essa alta porcentagem reflete na importância desses ácidos graxos no ovo, que serão utilizados para o futuro desenvolvimento da larva. De uma forma geral, foi observada uma característica de maior porcentagem de PUFAs n6 (fração polar) e PUFAs n3 (fração neutra) do tecido hepático em animais imaturos quando comparados às fêmeas adultas. Nos machos, os ácidos graxos monoinsaturados foram maiores no grupo 2 no músculo branco e ventrecha, devido à necessidade de um maior aporte energético na musculatura para o comportamento reprodutivo. Os resultados das análises plasmáticas dos esteróides gonadais mostraram que o perfil de 17&#946;-Estradiol em fêmeas apresentou-se alto nas fases iniciais do ciclo reprodutivo, decaindo na fase madura e regressão, provavelmente devido ao feedback negativo. Os animais imaturos apresentaram as maiores concentrações plasmáticas de 17&#946;-Estradiol e 17&#945;-OHP em relação aos adultos, já que esses hormônios estimulam o desenvolvimento de características secundárias, desenvolvimento neural, crescimento e maturação sexual nos animais imaturos, em machos e fêmeas. Foram observadas altas concentrações de 17&#945; -OHP nos machos em ambos os grupos sugerindo a disponibilidade desse hormônio para a conversão de 17&#945;,20&#946;-DHP, que atua na motilidade dos espermatozóides, já que ambos os grupos apresentam espermatozóides no lúmen. As concentrações de testosterona excedem à de 11ceto-T em machos, sugerindo que essa espécie esteja utilizando outros andrógenos e não somente a 11ceto-T para a reprodução. O acompanhamento destes animais no ambiente natural evidencia que não é possível utilizar características secundárias como coloração da escama como indicativo do sexo, porém a sexagem é possível de se realizar utilizando a razão entre os hormônios plasmáticos 11ceto-T e 17&#946;-Estradiol. / The aim of the present study was to investigate the possible physiological alterations related with reproduction in males and females of Arapaima gigas (pirarucu) in their natural environment. Pirarucu is an obligatory aerial respiration species belonging to a very ancient group of teleosts and inhabits Amazonian river. The main goals of this study were to describe the plasmatic gonad steroids profile (17&#946;-Estradiol, Testosterone, 11keto-Testosterone (11keto-T) and 17&#945;- hidroxyprogesterone (17&#945;-OHP)), lipids and proteins concentrations of the main storage tissues (gonads, white muscle, red muscle and ventrecha), and fatty acid profile along the reproductive cycle of males and females collected in their natural environment at Ilha Mexiana (Pará/ Brazil). The animals were caught from the wild in September/2007, January/2008, July/2008 and September/2008 and four stages of gonad development were determined: Rest, Maturation, Mature and Regression. Immature animals were also observed. Males were classified according to spermatozoid amount inside the seminiferous tubes, and the animals having a few spermatozoids were referred as group1 and those having many spermatozoids were referred as group 2. Microscopic analyses allowed concluding that pirarucu has gymno-ovary type with assynchronic oocyte development and testis has non-cystic structure. The metabolic analyses in females showed that the tissue presenting the highest lipids concentration was the ventrecha, followed by the red and white muscle, suggesting that ventrecha is the preferred lipids storage among different muscles. Lipids concentrations increased in ovaries and liver along the reproductive cycle, getting the highest value at mature stage. Liver and gonads protein did not show any difference among the reproductive stages. Lipids and proteins in males did not present any difference between the groups of any analyzed tissue. In polar lipids ovary fraction, PUFA (polyunsaturated fatty acids) n3 were higher at mature stage, compared with the other stages of the reproductive cycle and immature animals, and these high amounts reflect the importance of those fatty acids, that will be used in the larval development. In general, the immature animals showed the highest percentage of PUFA n6 (polar fraction) and PUFA n3 (neutral fraction) in the liver when compared with adults females. Monounsaturated fatty acids in males in group 2 were higher in white muscle and ventrecha, because those muscles have an intense energetic demand for reproductive behavior. 17&#946;-Estradiol was higher in females, at initial stages of the reproductive cycle, decreasing at mature and regression stages, probably due to a negative feedback. Immature animals showed the highest concentrations of 17&#946;-Estradiol and 17&#945;-OHP, probably because these steroids stimulate sexual secondary features, neural development, growth and sexual maturity in those animals. 17&#945;-OHP concentrations were higher in both male groups, suggesting its availability for 17&#945;, 20&#946;-DHP conversion, an important steroid on spermatozoid motility. Testosterone concentrations in males exceed 11ceto-T, suggesting that other androgens were more effective and not just 11ceto-T, for reproduction. On this environment, features like scales coloration as secondary feature were not suggested to distinguish the sex of those animals, but it is possible when using 11ceto-T and 17&#946;- Estradiol plasma ratio.