Estrogens, such as the biologically active 17-b estradiol (E2) have many actions in the male and female brain. Not only does E2 regulate reproductive behavior in adults, it organizes and activates the brains of younger animals in a sex-specific manner. In addition, many human studies have shown E2 to provide protection against a variety of neurological disorders, including stoke. These studies have been controversial and depend largely on the type and timing of hormone replacement. Animal studies are much less controversial and clearly demonstrate a neuroprotective role for E2 following ischemic brain injury. Because much of E2 neuroprotection requires sex steroid hormone receptors, it is essential to understand expression patterns of these receptors. For the current studies, I evaluated estrogen receptor alpha (ER α), estrogen receptor beta (ER β) and androgen receptor (AR) expression in the cortex. It is known that these receptors change in expression at several times in an animal’s life span including during early postnatal development and following ischemic brain injury. Here I used an in vitro cortical explant model to further examine how these receptors change both during development and following injury. This in vitro model is important because it provides a way to investigate changes in receptor expression pattern in the cortex without input from other brain regions. In addition to characterizing this model, I also evaluated the contribution of E2 to changes in receptor expression and on cell death following injury in the explants. To begin to decipher mechanisms for E2 mediated neuroprotection, I added antagonist for each of the receptors before and after injury. In each these experiments, I also examined potential sex differences by separating the female and male brains before I cultured the explants. Overall, these experiments showed that cortical explants are a good in vitro model. Here we found that E2 was protective in female, but not male cortical explants following injury. However, the exact mechanisms of E2-mediated neuroprotection are still to be deciphered.

Les activités d'élevage sont responsables de la dissémination dans l'environnement d'un grand nombre de polluants, dont les perturbateurs endocriniens (PE) qui affectent le système endocrinien des organismes vivants. La disruption endocrinienne se fait par la liaison des PE sur des récepteurs nucléaires spécifiques dont le récepteur aux strogènes ERα, le récepteur à la dioxine AhR, le récepteur X aux pregnanes PXR, le récepteur aux androgènes AR et le récepteur régulant la prolifération des peroxysomes, PPARγ. Les hormones stéroïdes sont particulièrement actives sur le récepteur ERα. Elles sont naturellement produites par les animaux et concentrées dans les effluents d'élevage épandus sur les sols agricoles. Ce travail de thèse s'est focalisé sur l'étude du devenir des hormones dans des systèmes de gestion des lisiers porcins, à échelle réelle ou contrôliée, en conditions anaérobies ou aérobies. Au préalable, la méthodologie pour l'analyse des hormones dans le lisier a été validée. Les résultats montrent que les hormones sont principalement contenues dans la fraction solide; dans cette fraction les mesures d'activité strogénique (ERα) ont révélé une activité non explicable par les concentrations en hormones, suggérant la présence d'autres composés ayant une activité sur ERα. La quantification des nonylphénols, actifs sur ERα n'a pas permis d'expliquer cette activité. L'étude d'autres voies de perturbation endocrinienne via l'activation des récepteurs AhR, PXR, PPARγ et AR a permis de détecter une forte activité sur le récepteur AhR. L'analyse chimique de cette fraction a montré l'absence de dioxines et d'hydrocarbures aromatiques polycycliques, suggérant l'implication d'autres PE qui restent à être identifiés. Dans les systèmes de traitement sous conditions réelles ou contrôlées, les conditions aérobies sont plus efficaces pour éliminer les hormones et les activités strogéniques et « dioxin-like » associées, bien que cette dernière semble plus récalcitrante. / Livestock activities are responsible for the release of a wide range of pollutants in the environment. Among these pollutants, the endocrine disruptors (ED) can affect the endocrine functions of living organism. The endocrine disruption occurs via the binding of ED to specific nuclear receptors such as the estrogen receptor ERα, dioxin receptor AhR, pregnane-X-receptor (xenobiotics), androgen receptor AR and peroxysome proliferator-activated receptor γ, PPARγ. Steroid hormones are particularly active on ERα receptor. Animals naturally produce high amounts of hormones which are concentrated in manure used for land spreading. This study deals with the fate of hormones in swine waste management facilities at full or pilot scale, under both anaerobic and aerobic conditions. Firstly, the analysis of hormones in such a complex matrix was validated. Hormones are mainly present in the solid fraction of the manure. At the same time, estrogenic activity measurements showed that in this solid fraction, the estrogenic activity cannot be explained by hormones; which suggest the presence of other compounds active on ERα. Nonylphenols concentrations cannot explain this additional activity. The study of the other pathways of endocrine disruption through the activation of AhR, PXR, PPARγ, and AR allowed to measure high AhR activity in the solid fraction of manure. The chemical analysis of this fraction did not show the presence of dioxins and polycyclic aromatic hydrocarbons traditionally involved in such kind of activity; it suggests the presence of other AhR ligands that remain to be identified. In swine manure treatment systems, aerobic conditions were more efficient to remove hormones and the associated estrogenic and AhR activities, even if this last seemed to be more recalcitrant.

When Y-1 mouse adrenal tumor cells were treated with sodium orthovanadate, an intermediate filament (IF) inhibitor in BHK21-F cells, there was no change in the amount of 20α-dihydroprogesterone produced. A neurofilament inhibitor, β, β'-iminodipropionitrile (IDPN), enhanced the ability of Y-1 cells to produce steroid in response to ACTH by acting on the plasma membrane. Electron microscopy of Y-1 cells extracted with Triton X-100 revealed that both vanadate and IDPN caused the aggregation of cytoskeletal and granular structures in the perinuclear area. The steroidogenic effects of IDPN suggest that the perinuclear aggrergation of cytoskeletal structures may result from the detachment of IF from the plasma membrane, while the reason for the cytoskeletal changes by vanadate is unknown.

The thesis focuses on selected, yet unsolved question of the role of less common steroids and SHBG as a junction of three endocrine systems. Answering these questions may help to understand the complex mechanism of action of these hormones on the human organism. This thesis is based on five author and co-written studies mostly published in foreign scientific journals. In the case of studies of metabolites 16α-OH-DHEA and 7-oxo-DHEA (Zamrazilová et al., 2007; Kazihnitková et al., 2007) were focused on the development of appropriate methodological approach because recently used methods showed a low sensitivity and specificity. We developed and statistically evaluated new RIA methods which are rapid, sensitive and inexpensive. Both of them can be used in other research even in routine practice. New methods were also used to determine the metabolites in a statistically significant sample of healthy human population. In another study (Zamrazilová et al., 2010) we examined the relationships of selected steroid metabolites and SHBG in patients with CAH. We assumed that SHBG may act as a non-steroidal laboratory parameter reflecting the effectiveness of substitution therapy in these patients. Our assumption was not confirmed. We observed lower levels of SHBG which at least in women reflect effects of...

A exibição do comportamento vocal em muitas espécies de anfíbios anuros é relacionada aos níveis de hormônios esteroides gonadais e interrenais. Esses hormônios poderiam mediar a relação entre intensidade de sinais e imunidade, pois estão envolvidos no desenvolvimento das características sexuais secundárias, comportamento de corte e mobilização de reservas energéticas durante a atividade reprodutiva, enquanto apresentam também efeitos imunomoduladores. Nesse sentido, o objetivo deste trabalho foi explorar as relações entre comportamento reprodutivo, imunidade e níveis plasmáticos de testosterona e corticosterona em machos do sapo do semiárido brasileiro, Rhinella granulosa, em atividade reprodutiva e após manipulação hormonal. A precipitação foi o principal determinante ambiental para o aumento dos níveis de testosterona e corticosterona circulantes em machos de R. granulosa, estimulando o comportamento de vocalização. As relações fisiológicas encontradas indicam que os altos níveis plasmáticos de testosterona nos primeiros dias após a chuva devem promover o início do turno vocal, porém a corticosterona deve modular o esforço de vocalização. De forma geral, a exacerbação do comportamento vocal de R. granulosa tem efeitos negativos sobre a imunocompetência, porém alguns indivíduos que apresentam maiores concentrações plasmáticas de corticosterona apresentam concomitantemente alto esforço vocal e alta imunidade. De acordo, a aplicação transdérmica de corticosterona promoveu elevação aguda dos níveis plasmáticos deste glicocorticoide, bem como um aumento da função imune. Assim, apesar de a atenção principal ser comumente colocada no papel da testosterona na mediação de sinais honestos, nossos resultados corroboram a importância da corticosterona na mediação da expressão do comportamento de corte e imunocompetência em machos R. granulosa / In most anuran species, vocal behavior is usually related to plasmatic levels of gonadal and interrenal steroid hormones. These hormones could mediate the relationship between signal intensity and immunocompetence, since they are involved in the development of sexual traits, courtship behavior and energetic mobilization during reproductive activity and also exert immunomodulatory effects. The aim of this study was to explore relations among vocal behavior, immunity and plasmatic levels of testosterone and corticosterone in males of a toad species from Brazilian semi-arid, Rhinella granulosa, during reproductive activity and after hormonal manipulation. Precipitation was the main environmental determinant to increase circulating levels of testosterone and corticosterone in R. granulosa males, stimulating vocal behavior. Physiological relations indicate that high circulating testosterone levels in the days right after rain may promote the initiation of vocal behavior, while corticosterone may modulate vocal effort. In general, exacerbation of vocal behavior in R. granulosa has negative effects on immunocompetence, however, some individuals with higher plasmatic levels of corticosterone can present both high vocal effort and good immunocompetence. Corroborating this interpretation, acute increase in glucocorticoid plasmatic levels after hormonal manipulation was accompanied by raised immune function. Despite the emphasis on androgens influence on sexual selected male traits, our results corroborate greater influence of corticosterone in the mediation of expression of courtship behavior and immunocompetence in R. granulosa

A síntese dos hormônios esteroides é feita através da ação de um complexo enzimático chamado citocromo P450. As enzimas 3β-HSD (3β-hidroxiesteroide desidrogenase), P450c17 (citocromo P450 17α-hidroxilase/17,20-liase) e P450arom (citocromo P450 aromatase), fazem parte desse complexo e são responsáveis pela síntese dos hormônios progestágenos, andrógenos e estrógenos, espectivamente. Essas enzimas já foram identificadas em inúmeros tecidos, inclusive em embriões em período pré-implantação. Por isso, acredita-se que o blastocisto seja uma fonte de produção de progesterona e estrógeno, hormônios essenciais para implantação e manutenção da gestação. Em roedores, as informações sobre a capacidade esteroidogênica dos embriões são controversas e também restritas apenas a estudos com espécies laboratoriais. Para trazer mais informações sobre a capacidade esteroidogênica em blastocistos de roedores utilizamos o preá (Galea spixii), uma espécie de roedor silvestre encontrada nas regiões de Caatinga e Semiárido do Nordeste brasileiro e muito utilizado na alimentação de famílias de baixa renda como fonte proteica. Diante o exposto, o objetivo deste trabalho é fazer um apanhado histórico sobre os dados publicados a cerca da capacidade dos embriões de diversas espécies de produzir os hormônios esteroides além de identificar e quantificar a expressão das enzimas esteroidogênicas em embriões de preá através das técnicas de imunohistoquímica e PCR em tempo real. As análises morfológicas indicam que o desenvolvimento inicial do preá se assemelha com os demais roedores, porém o tempo de desenvolvimento difere das espécies usadas em laboratório. As marcações da imunohistoquímica foram positivas nos tecidos maternos, mas os embriões não tiveram marcação para nenhuma das três enzimas do estudo. No entanto, os dados obtidos pelo PCR indicam que os genes CYP11, CYP17 e CYP19 foram expressos nos embriões e, portanto, o concepto de G. spixii tem capacidade de produzir hormônios esteroides, assim como visto em várias outras espécies que já foram estudadas / The synthesis of steroid hormones is made through the action of an enzyme complex called cytochrome P450. 3β-HSD (3β-hydroxysteroid dehydrogenase), P450c17 (cytochrome P450 17 α-hydroxylase/17,20-lyase) and P450arom (cytochrome P450 aromatase), are present in this complex and are are responsible for the synthesis of progestins, androgens and estrogens hormones, respectively. These enzymes have been identified in numerous tissues, including embryos in pre-implantation period. Therefore, it is believed that the blastocyst is a source of production of progesterone and estrogen, hormones essential for the implementation and maintenance of pregnancy. In rodents, the information about the steroidogenic capacity of embryos are controversial and confined to studies with laboratory species. To get more information about the steroidogenic capacity in blastocysts of rodents we used the spix yellow-toothed cavy (Galea spixii), a species of wild rodent found in the northeastern of Brazil, and used in the feeding of low-income families as a source of protein. On the above, the aim of this work is to make a historic about the data published about the ability of embryos of various species to produce steroid hormones and identify and quantify the expression of steroidogenics enzymes in embryos of spix yellow-toothed cavy by immunohistochemical and real-time PCR techniques. The morphological analyses indicate that the G. spixii early development resembles other rodents, but development time differs from the species used in laboratory. The immunohistochemical stainning was positive in maternal tissues, but the embryos did not have stainnig for any of the three enzymes of the study. However, the data obtained by PCR indicate that CYP11, CYP17 and CYP19 genes were expressed in embryos and, therefore, the concept of G. spixii has capacity to produce steroid hormones, as seen in several other species that have been studied

Orientador: Sergio Ricardo Batlouni / Resumo: Nosso objetivo foi avaliar o efeito das diferentes densidades de estocagem e proporções sexuais sobre o desempenho reprodutivo da espécie Astyanax altiparanae em sistema semi-natural. Foram realizados dois experimentos distintos, para o primeiro objetivo testamos quatro densidades de estocagem (3; 6; 12; 24 indivíduos) com quatro réplicas, mantendo sempre a proporção sexual de dois machos para uma fêmea. Quando o efeito da proporção sexual foi avaliado, utilizamos cinco proporções diferentes (1:1; 2:1; 3:1; 1;2; 1:3; macho: fêmea) e dois controles com três repetições, respeitando a densidade de 12 indivíduos em cada unidade experimental. Em ambos os experimentos os tratamentos foram comparados pelo desempenho reprodutivo por meio de variáveis como: a sobrevivência dos reprodutores, a proporção de réplicas com desovas, o volume de desova, percentual médio de fêmeas desovadas por réplica, taxas de fertilidade e eclosão, número de embriões obtidos por tratamento, histologia dos ovários e concentrações plasmáticas dos esteroides sexuais. A densidade não influenciou de forma negativa no desempenho reprodutivo da espécie, sendo observados os melhores desempenhos para as variáveis: volume médio de desova considerando apenas as réplicas onde houve desova 131,50±33,17ml e no número médio de embriões viáveis 73819,2±20462,1, nas unidades com maior densidade (24) animais. Notamos desempenhos muito semelhantes quando as diferentes proporções foram testadas, sendo a variável volume relati... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Our objective was to evaluate the effect of different storage densities and sexual proportions on the reproductive performance of the species Astyanax altiparanae in a semi-natural system. Two different experiments were carried out. For the first objective, we tested four stocking densities (3; 6; 12; 24 individuals) with four replicates, always maintaining the sex ratio of two males to one female. When the sex ratio effect was evaluated, we used five different ratios (1: 1, 2: 1, 3: 1, 1, 2, 1: 3, male: female) and two controls with three replicates, respecting the density of 12 individuals in each experimental unit. In both experiments the treatments were compared by reproductive performance using variables such as: reproductive survival, proportion of replicates with spawning, spawning volume, mean percentage of spawned females per replicate, fertility and hatching rates, number treatment embryos, ovarian histology, and plasma steroid plasma concentrations. The density did not negatively influence the reproductive performance of the species, with the best performances for the variables: average spawning volume considering only spawning replicates of 131.50 ± 33.17 ml and the average number of viable embryos 73819.2 ± 20462.1, in the units with higher density (24) animals. We observed very similar performances when the different proportions were tested, being the relative variable spawning volume in which the proportion of (3 males: 1 female) was more efficient than the oth... (Complete abstract click electronic access below) / Mestre

Desde as primeiras dosagens de hormônios esteróides efetuadas em fezes por Adlercreuetz em 1976, os estudos baseados no uso desta técnica se multiplicaram com animais selvagens, como também em algumas espécies domésticas e, mais recentemente, em animais de laboratório. Diversos protocolos para a extração dos esteróides fecais foram elaborados e estão disponíveis na literatura. A eficiência dos mesmos pode ser avaliada medindo a sua capacidade de recuperar uma quantidade conhecida de hormônio marcado radioativamente e incorporado a algumas amostras fecais antes de submetê-las ao processo de extração. O presente trabalho teve por objetivo a determinação da capacidade de recuperação de três protocolos de extração de esteróides fecais dentre os mais freqüentemente empregados no nosso meio. São eles os protocolos descritos respectivamente por Brown et al. (1994), Graham et al. (2001) e Schwarzenberger et al. (1991). O estudo abrange sete espécies: hamster Sírio (Mesocricetus auratus), cutia (Dasyprocta aguti), lobo guará (Chrysocyon brachyurus), cabra doméstica (Capra hircus), ovelha doméstica (Ovis aries), chimpanzé (Pan troglodytes), gorila (Gorilla gorilla) e orangotango (Pongo pygmaeus) e três hormônios: progesterona, testosterona e cortisol. O protocolo de Graham alcançou maiores índices de recuperação da progesterona e da testosterona triciada. O protocolo de Schwarzenberger mostrou-se mais eficiente, do ponto de vista numérico, na recuperação do cortisol triciado. Os resultados obtidos sugerem, porém, que a metodologia empregada para avaliação da capacidade de recuperação dos três protocolos subestimou a eficiência do protocolo de Brown. / Adlercreutz performed the first quantifications of steroid hormones in human feces in 1976. Since then, studies based on this technique have been applied to wildlife, domestic animals, and recently to laboratory animals. Several procedures for extracting fecal steroids were established and are available in the literature. It is possible to assess the efficiency of these techniques measuring how they recover known amounts of radio-labelled hormones added to some fecal samples just before extraction. The aim of this study was to determine the recovery of three procedures for fecal steroid extraction that are commonly used in our laboratory. We assessed, then, how the techniques described by Brown et al. (1994), Graham et al. (2001) and Schwarzenberger et al. (1991) recover 3H-progesterone, 3H-testosterone and 3H-cortisol in the feces from eight animal species: Syrian hamster (Mesocricetus auratus), agouti (Dasyprocta aguti), maned wolf (Chrysocyon brachyurus), goat (Capra hircus), sheep (Ovis aries), chimpanzee (Pan troglodytes), gorilla (Gorilla gorilla) e orangutan (Pongo pygmaeus). Average recovery of Graham et al. (2001) procedure was higher for progesterone and testosterone and Schwarzenberger et al. (1991) procedure recovered more cortisol. However, our results suggested that the efficiency of the method established by Brown et al. (1994) was underestimated.

Tsang Suk Ying. / "August 2003." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (p. 196-226). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

Lau, Pui Ling. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 84-94). / Abstracts in English and Chinese. / Abstract (English) --- p.i / Abstract (Chinese) --- p.v / Acknowledgements --- p.ix / Contents --- p.x / List of Figures --- p.xiv / Chapter Chapter 1 --- General Introduction / Chapter 1.1 --- Regulations of gametogenesis and mass spawning in corals --- p.1 / Chapter 1.1.1 --- Endogenous cues --- p.2 / Chapter 1.1.2 --- Environmental cues --- p.3 / Chapter 1.2 --- Studies on black corals --- p.5 / Chapter 1.2.1 --- Introduction of black corals --- p.5 / Chapter 1.2.2 --- Black corals harvesting --- p.6 / Chapter 1.2.3 --- Biodiversity and distribution of black corals in Chinese and Hong Kong waters --- p.8 / Chapter 1.2.4 --- Threats to black corals --- p.9 / Chapter 1.3 --- Significance and objectives of the present study --- p.11 / Chapter 1.3.1 --- Objectives --- p.12 / Chapter 1.3.2 --- "The targeted species, Antipathes curvata and the study site" --- p.13 / Chapter 1.4 --- Thesis outline --- p.14 / Chapter Chapter 2 --- "Reproductive Biology of Antipatharian Black Coral, Antipathes curvata in Lan Giio Shui, Hong Kong" / Chapter 2.1 --- Introduction --- p.18 / Chapter 2.1.1 --- Coral Reproduction --- p.20 / Chapter 2.1.2 --- Sexual reproduction in black corals --- p.21 / Chapter 2.2 --- Materials and methods --- p.24 / Chapter 2.2.1 --- Sample collections and pre-treatment --- p.24 / Chapter 2.2.2 --- Histological processing --- p.25 / Chapter 2.2.3 --- Light microscopy --- p.26 / Chapter 2.2.4 --- Gametogenesis --- p.27 / Chapter 2.2.5 --- Environmental and statistical analysis --- p.27 / Chapter 2.3 --- Results --- p.28 / Chapter 2.3.1 --- General reproductive mode --- p.28 / Chapter 2.3.2 --- "Sex ratio, size at sexual maturity and density of gamete" --- p.28 / Chapter 2.3.3 --- Characteristics of polyps and gametes of A. curvata --- p.29 / Chapter 2.3.4 --- Changes in geometric diameter of gametes overtime --- p.30 / Chapter 2.3.5 --- Developmental stages of gametogenesis --- p.31 / Chapter 2.3.5.1 --- Oogenesis --- p.31 / Chapter 2.3.5.2 --- Spermatogenesis --- p.32 / Chapter 2.3.6 --- Development of oocytes and spermaries over time --- p.34 / Chapter 2.3.7 --- Correlation of black coral reproduction with seawater temperature --- p.35 / Chapter 2.3.8 --- Gametogenesis in individual colonies --- p.36 / Chapter 2.3.8.1 --- Female colonies --- p.37 / Chapter 2.3.8.2 --- Male colonies --- p.39 / Chapter 2.4 --- Discussion --- p.40 / Chapter 2.4.1 --- Asynchronization of gametogenic cycle --- p.40 / Chapter 2.4.2 --- Possible effect of seawater temperature on reproduction of A. curvata --- p.43 / Chapter Chapter 3 --- Detection of the Sex Steroid 17β-estradiol and its Possible Roles on Gametogenesis in Black Corals Antipathes curvata from Hong Kong / Chapter 3.1 --- Introduction --- p.55 / Chapter 3.1.1 --- "Roles of sex hormone, 17(3-estradiol (E2) in the reproduction of vertebrates" --- p.58 / Chapter 3.1.2 --- Roles of vertebrate-type sex steroids in Cnidaria --- p.59 / Chapter 3.2 --- Materials and methods --- p.63 / Chapter 3.2.1 --- Study site --- p.63 / Chapter 3.2.2 --- 17β-estradiol (E2) extraction --- p.63 / Chapter 3.2.3 --- 17β-estradiol (E2) assay --- p.65 / Chapter 3.2.4 --- Calculation and assay validation --- p.65 / Chapter 3.2.5 --- Gametogenesis of A. curvata --- p.66 / Chapter 3.2.6 --- Seawater temperature and statistical analysis --- p.67 / Chapter 3.3 --- Results --- p.67 / Chapter 3.3.1 --- Seasonal profile of E2 --- p.67 / Chapter 3.3.2 --- Gametogenesis --- p.68 / Chapter 3.3.3 --- Correlation with seawater temperature --- p.69 / Chapter 3.4 --- Discussion --- p.70 / Chapter Chapter 4 --- Summary and Perspectives --- p.78 / References --- p.84