O alumínio e o pH ácido exercem efeitos tóxicos sobre a fauna íctica. O objetivo deste trabalho foi avaliar os efeitos do alumínio e do alumínio em pH ácido na fisiologia reprodutiva de Oreochromis niloticus. Para a condução deste experimento, fêmeas desta espécie foram expostas a concentração de 0,5 mg de Al L-1 em pH neutro (Al N), 0,5 mg de Al L-1 em pH ácido (Al - Ác), somente em pH neutro (CTR N) e somente em pH ácido (CTR Ac) por 96h. Após o período de exposição aguda, os animais foram sacrificados e tecidos como encéfalo, brânquias, fígado, gônadas e músculo, foram retirados para a determinação da concentração de alumínio por espectrofotometria de absorção atômica. A hipófise foi coletada e congelada para a quantificação da expressão da subunidade β dos genes das gonadotropinas, FSH (hormônio folículo estimulante) e LH (hormônio luteinizante) por qRT-PCR. Os resultados mostraram que animais expostos ao alumínio, tanto em pH ácido quanto neutro, acumularam mais alumínio no encéfalo e no músculo esquelético em relação aos grupos controles. Nas brânquias, apenas quando os animais são expostos ao alumínio em pH neutro, acúmulos diferenciados são encontrados e, adicionalmente, fêmeas expostas ao pH ácido, independente da concentração do alumínio na água, acumulam mais metal nos filamentos branquiais. Nos ovários, tanto a presença de maiores concentrações deste metal na água, quanto a acidez da mesma foram determinantes na incorporação do alumínio. Os dados de expressão gênica evidenciam que, animais submetidos ao alumínio em pH 5,5 apresentaram uma redução na expressão do gene do FSH, no entanto em pH neutro esta alteração na expressão deste gene não foi encontrada. Os animais expostos ao alumínio, tanto em pH ácido quanto neutro apresentaram diminuição na expressão de LH. A análise dos resultados de expressão do LH combinada com o perfil dos progestágenos plasmáticos previamente conhecidos para os mesmos animais, mostram que, em condições adversas de pH (ácido) a ação do alumínio como um disruptor endócrino foi traduzida em alterações na fisiologia gonadal, limitando a produção do 17αOHP (hidroxiprogesterona), importante hormônio na ovulação. Por outro lado, quando as condições de pH foram favoráveis (neutro), a diminuição da expressão de LH não levou ao desajuste na produção de 17αOHP, ou seja, os animais de alguma forma, compensaram esta disfunção. / Aluminum and acidic pH are known to be toxic to the ichthyofauna. The main goal of the present study was to evaluate the effects of aluminum and acidic pH in the reproductive physiology of Oreochromis niloticus. To conduct this experiment, females were exposed to aluminum at 0.5 mg of Al L-1 in neutral pH (Al N), 0.5 mg of Al L-1 in acidic pH (Al - Ac), a control group in neutral pH (CTR N) and acidic pH (CTR Ac) for 96h. After the acute exposition period, the animals were killed and the following tissues, brain, gills, liver, gonads and muscle, were frozen for aluminum determination by atomic absorption spectrophotometer. The pituitary was collected and also frozen to quantify the gene expression of the β subunit of the gonadotropins FSH (follicle stimulating hormone) and LH (luteinizing hormone) using qRT-PCR. The results showed that animals exposed to aluminum, even in acidic or neutral pH, accumulated more aluminum in brain and white muscle comparing with their control groups. In the gills, only when the animals were exposed to aluminum in neutral pH, different patterns of accumulation were found and, additionally, females exposed to acidic pH, independent of the water aluminum concentration, accumulated more metal in the gills filament. In the ovaries even the presence of higher aluminum concentration in water and the acidic pH were essential in aluminum deposition. The gene expression data showed that, animals exposed to aluminum in pH 5.5 reduce FSH gene expression, however in neutral pH this alteration was not observed. Animals exposed to aluminum, even in acidic or neutral pH, reduced LH expression. The data analyses of LH gene expression combined with the plasma progestagens, previously known for the same animals, showed that, in adverse pH conditions (acidic), the aluminum role as an endocrine disruptor was translated in alterations in gonad physiology, reducing the production of 17αOHP (hidroxy progesterone), an important hormone in ovulation. On the other hand, when the pH conditions were optimum (neutral), the reduced LH gene expression did not reflect in impairments in the 17αOHP production, which means the animals, somehow, compensated this dysfunction.

Yuen Chi Wai. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 84-106). / Abstracts in English and Chinese. / Abstract (in English) --- p.ii / Abstract (in Chinese) --- p.iv / Acknowledgement --- p.vi / Table of Contents --- p.vii / List of Figures --- p.xii / Symbols and Abbreviations --- p.xv / Scientific names --- p.xvii / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter 1.1 --- Pituitary --- p.1 / Chapter 1.2 --- Gonadotropins (GTHs) --- p.3 / Chapter 1.2.1 --- Structure --- p.3 / Chapter 1.2.2 --- Function --- p.5 / Chapter 1.2.3 --- Regulation --- p.7 / Chapter 1.2.3.1 --- Neuroendocrine hypothalamic regulators --- p.9 / Chapter 1.2.3.1.1 --- Gonadotropin-releasing hormone (GnRH) --- p.9 / Chapter 1.2.3.1.2 --- Dopamine (DA) --- p.11 / Chapter 1.2.3.2 --- Endocrine regulators from the gonads --- p.12 / Chapter 1.2.3.2.1 --- Gonadal steroids (T and E2) --- p.12 / Chapter 1.2.3.2.2 --- Negative steroid effect on pituitary GTH regulation --- p.12 / Chapter 1.2.3.2.3 --- Positive steroid effect on pituitary GTH regulation --- p.13 / Chapter 1.2.3.3 --- Paracrine regulators from within the pituitary --- p.14 / Chapter 1.3 --- Activin --- p.14 / Chapter 1.3.1 --- Structure --- p.14 / Chapter 1.3.2 --- Function --- p.16 / Chapter 1.4 --- Follistatin (FS) --- p.17 / Chapter 1.4.1 --- Structure --- p.17 / Chapter 1.4.2 --- Function --- p.19 / Chapter 1.5 --- Temporal Variations in the GTH Expressional and Releasing Profile and Sex Steroid Level in the Goldfish --- p.19 / Chapter 1.5.1 --- Hormone changes during annual cycle --- p.20 / Chapter 1.5.2 --- Hormone changes during ovulatory cycle --- p.21 / Chapter 1.6 --- Objectives of the Present Study --- p.23 / Chapter Chapter 2 --- "Effects of Dopamine on the Expression of Gonadotropin (GTH) Subunits in the Dispersed Pituitary Cells of the Goldfish, Carassius auratus" --- p.26 / Chapter 2.1 --- Introduction --- p.26 / Chapter 2.2 --- Materials and Methods --- p.27 / Chapter 2.2.1 --- Materials --- p.27 / Chapter 2.2.2 --- Primary culture of dispersed goldfish pituitary cells --- p.28 / Chapter 2.2.3 --- mRNA analysis --- p.29 / Chapter 2.2.4 --- Data analysis --- p.30 / Chapter 2.3 --- Results --- p.30 / Chapter 2.3.1 --- Effects of DA on GTH-Iβ and GTH-IIβ expression --- p.30 / Chapter 2.3.2 --- Effects of DA D1 and D2 agonists on GTH-Iβ expression --- p.33 / Chapter 2.3.3 --- Effects of DA D1 and D2 antagonists on DA- inhibited GTH-Iβ expression --- p.33 / Chapter 2.3.4 --- Effects of α-adrenergic agonists on GTH-Iβ expression --- p.33 / Chapter 2.4 --- Discussion --- p.37 / Chapter Chapter 3 --- Seasonal Variation of Activin-regulated Goldfish Pituitary GTH-Ip and GTH-IIβ Expression and Evidence for the Involvement of Gonadal Steroids --- p.40 / Chapter 3.1 --- Introduction --- p.40 / Chapter 3.2 --- Materials and Methods --- p.42 / Chapter 3.2.1 --- Materials --- p.42 / Chapter 3.2.2 --- Gonadectomy of the goldfish --- p.42 / Chapter 3.2.3 --- Primary culture of dispersed pituitary cells --- p.43 / Chapter 3.2.4 --- mRNA analysis --- p.43 / Chapter 3.2.5 --- Data analysis --- p.44 / Chapter 3.3 --- Results --- p.44 / Chapter 3.3.1 --- Effects of goldfish activin B on the expression of GTH-Iβ and GTH-IIβ --- p.44 / Chapter 3.3.2 --- Seasonal variation of activin-regulated expression of GTH-Iβ and GTH-IIβ --- p.45 / Chapter 3.3.3 --- Effects of gonadectomy on basal and activin- regulated expression of GTH-Iβ and GTH-IIβ --- p.45 / Chapter 3.3.4 --- Effects of sex steroids on basal and activin- regulated expression of GTH-Iβ and GTH-IIβ in vitro --- p.50 / Chapter 3.4 --- Discussion --- p.57 / Chapter Chapter 4 --- Evidence for the Autocrine/Paracrine Regulation of Gonadotropin Expression by Activin in the Goldfish Pituitary --- p.61 / Chapter 4.1 --- Introduction --- p.61 / Chapter 4.2 --- Materials and Methods --- p.63 / Chapter 4.2.1 --- RNA isolation --- p.63 / Chapter 4.2.2 --- Reverse transcription-polymerase chain reaction (RT-PCR) --- p.63 / Chapter 4.2.3 --- Primary culture of goldfish pituitary cells --- p.63 / Chapter 4.2.4 --- Slot-blot analysis --- p.64 / Chapter 4.2.5 --- Data analysis --- p.64 / Chapter 4.3 --- Results --- p.65 / Chapter 4.3.1 --- Expression of activin βB subunit and activin type IEB receptor in the goldfish pituitary --- p.65 / Chapter 4.3.2 --- Effects of human activin A on goldfish GTH-Iβ and GTH-IIβ expression --- p.65 / Chapter 4.3.3 --- Effects of follistatin on basal and activin- regulated GTH-Iβ and GTH-IIβ expression --- p.69 / Chapter 4.4 --- Discussion --- p.69 / Chapter Chapter 5 --- General Discussion --- p.77 / Chapter 5.1 --- Overview --- p.77 / Chapter 5.2 --- Contribution of the Present Study --- p.78 / Chapter 5.2.1 --- Dopamine as a potential neuroendocrine regulator in the differential regulation of GTH-Iβ and GTH-IIβ expression --- p.78 / Chapter 5.2.2 --- Seasonal variation of the effects of activin on GTH-Iβ and GTH-IIβ expression --- p.79 / Chapter 5.2.3 --- Autocrine/paracrine regulation of GTH expression by activin --- p.79 / Chapter 5.3 --- Future Prospects --- p.81 / References --- p.84

A qualidade do oócito é um fator limitante na fertilidade das fêmeas e reflete seu intrínseco potencial ao desenvolvimento embrionário subsequente. As alterações moleculares e bioquímicas no processo de maturação dos oócitos são necessárias para permitir a fecundação destes. Sob influência das gonadotrofinas, uma cascata de eventos é desencadeada, alterando a expressão gênica e a estrutura dos folículos. A maturação ocorre pelo intercâmbio entre o oócito e as células do cumulus que irão fornecer fatores para o desenvolvimento do oócito e criar o microambiente necessário para garantir o sucesso na maturação. A ação do FSH sobre a retomada da meiose ocorre, possivelmente, por ativação da proteína quinase C (PKC). A via de sinalização desta proteína parece estar envolvida na ativação da quinase ativada por mitógeno (MAPK) em oócitos e células do cumulus, na maturação induzida por FSH e LH, além de regular a síntese do Fator de Crescimento Epidermal (EGF). Deste modo, o objetivo do presente trabalho foi avaliar a ação da PKC na maturação de oócitos bovinos e se esta ativação envolve o EGF. Para tal foram realizados dois experimentos. Em ambos, a progressão do ciclo celular foi avaliada utilizando a sonda fluorescente Hoechst 33342. A expansão das células do cumulus foi avaliada utilizando-se o software Image Pro Plus 5.1 para análise das imagens dos oócitos geradas em microscópio Olympus IX81. O maior diâmetro de cada complexo cumulus oócito foi adotado como parâmetro de mensuração da expansão. A dosagem de progesterona do meio de cultivo foi realizada pela técnica de RIA. A ativação da PKC e da MAPK foi avaliada pela técnica de Western blot. Os dados foram avaliados pelo software SigmaPlot versão 12.2 e submetidos ao teste de normalidade (Shapiro-Wilk). Quando necessário, os dados foram transformados. Para comparação entre dois tratamentos, utilizou-se o teste t-student. Para mais de dois tratamentos foi realizada análise de variância e teste de comparação de médias (TUKEY), considerando-se 0,05 para rejeitar a hipótese de nulidade. No experimento 1 foi avaliado se a ativação da PKC foi estimulada por gonadotrofinas. Os oócitos foram maturados in vitro tratados com gonadotrofinas, na presença ou ausência do inibidor de PKC. A presença do inibidor de PKC diminuiu as taxas de quebra de vesícula germinativa e a expansão das células do cumulus, sem alterar a esteroidogênese. Estes resultados demonstram que a PKC participa da via de sinalização da retomada da meiose. No experimento 2 foi avaliado se o EGF está envolvido na via regulada pela PKC. Os oócitos foram maturados in vitro, na presença ou ausência de LH e FSH, do inibidor de PKC e do EGF. O EGF foi capaz de reverter os efeitos do inibidor de PKC, aumentando as taxas de quebra de vesícula germinativa e a expansão de células do cumulus. Não foi possível detectar, nas condições deste experimento, a ativação das proteínas PKC e MAPK através do Western Blot. Este trabalho permite concluir que a via de sinalização da maturação de oócitos bovinos envolve a PKC e sugere a participação do EGF nesta via. / Oocyte quality is a limiting factor in female fertility and reflects its potential to the subsequent embryonic development. Molecular and biochemical alterations during the oocyte maturation process are needed to allow fecundation. Under gonadotropin influence, cascade of events occurs changing gene expression and follicle structure. Maturation depends on the interaction between oocyte and cumulus cells interaction, which provides factors for oocyte development and create the ideal microenvironment for the success of the maturation process. The FSH stimulation of meiosis resumption probably occurs through PKC activation. The signaling pathway of PKC might be involved by the mitogen activated protein kinase (MAPK) in oocytes and cumulus cells during FSH-LH induced maturation. Furthermore, MAPK regulates the epidermal growth factor (EGF) synthesis. The aim of the present study was to evaluate PKC function during bovine oocyte maturation and if its activity involves EGF. Two experiments were performed. In both experiments, the cell cycle progression was analyzed by Hoechst 33342 fluorescent dye. The cumulus cells expansion was performed using software Image Pro Plus 5.1 by the analysis of oocyte images taken in Olympus IX81 microscope. The highest diameter of each cumulus oocyte complex was recorded as the expansion value. The RIA and Western Blot techniques were used to measure progesterone concentration in the culture media and the PKC and MAPK activity, respectively. Data was analyzed by SigmaPlot software, version 12.2. The Shapiro-Wilk test was used to assess for normality and, when needed, the data was transformed. Student t tests were carried out to compare two treatments. Differences between more than two means were assessed by analysis of variance followed by Tukey test, considering P-value lower than 0.05 as statistically significant. Experiment 1 studied whether PKC function was stimulated by gonadotropins. FSH and LH were used for oocyte maturation in vitro, with or without PKC inhibitor. The presence of PKC inhibitor decreased germinal vesicle breakdown and the cumulus cells expansion, but did not alter the steroidogenesis. These results show that PKC participates in the signaling pathway of meiosis resumption. The Experiment 2 evaluated whether EGF influences PKC signaling pathway. The oocytes were matured in vitro, in the presence or absence of LH and FSH, PKC inhibitor and EGF. Epidermal Growth Factor was able to reverse PKC inhibitor effects, increasing germinal vesicle breakdown rates and cumulus cells expansion. The Western Blot technique was not able to detect PKC and MAPK activity, considering the conditions of this study. In conclusion, PKC is involved in the signaling pathway of bovine oocytes maturation and its pathway is mediated by EGF.

O estradiol secretado pelo folículo dominante (DOM) desempenha importante papel na luteólise da vaca. Em adição, o reconhecimento materno da prenhez (MRP) requer um ambiente uterino otimizado, o qual por sua vez depende da função luteínica e de adequadas concentrações de progesterona circulante. Os objetivos do presente estudo foi testar diferentes estratégias para otimizar a função luteínica e prevenir a influência de um DOM durante o período crítico (CP) para o MRP (de D13 a D20 após o estro o estro). Diferentes abordagens foram testadas. No exp.1, 23 vacas Nelore foram tratadas com o protocolo ovsynch para induzir um cio sincronizado (D0). As vacas receberam: Gc (n=7) - nada mais; ThCG (n=5) - 3000 IU de hCG no D5; TE2 (n=6) - 5mg de 17b-estradiol (E2) no D12; ThCG/E2 (n=5) - hCG/D5 + E2/D12. Ultra-sonografias e dosagens de progesterona plasmática durante o ciclo estral permitiram determinar que o E2 reprogramou o ciclo ovariano ao prevenir a presença (P⁢0,05) de um DOM durante quase todo CP (0,6±0,7 dias entre D15 e D20), porém o E2 induziu a luteólise. As vacas que receberam a hCG desenvolveram corpo lúteo acessório e tiveram [P4] mais alta até o D13 (P⁢0,05). Portanto, a fase luteínica não foi prolongada No exp.2, os mesmos tratamentos foram impostos a 220 vacas Nelore (55 por grupo) após uma inseminação artificial em tempo fixo (TAI). As taxas de prenhez (PR) à TAI ou às inseminais de repasse durante uma estação com 64 dias de duração foram diminuídas (P⁢0,05) quando o E2 foi usado e a hCG não foi capaz de aumentar aquelas PR. No exp.3, vacas Red Angus no pós-parto tiveram seu estro sincronizado (D0) e receberam: nada mais (GCT, n=5) ou 200mg de gonadorrelina no D5 mais 3000 IU hCG no D13 (GRF, n=5); ou ablação de todos folículos ³7mm através de aspiração folicular em D14, D17 e D20 (GRM, n=5). GRF teve luteólise retardada (18,2±1,0b dias, 23,6&plusmn1,0a dias e 18,7&plusmn1,2b dias para GCT, GRF e GRM, respectivamente) e maior [P4] que os outros grupos. Folículos maiores que 7mm foram observados quando das aspirações. Foi possível concluir que: a) E2 permitiu consistentemente reprogramar o desenvolvimento folicular, porém causou luteólise e o seu uso trouxe efeitos negativos sobre as PR; b) a hCG melhorou a função luteínica mas não aumentou as PR; c) a ablação dos folículos ³7mm não preveniu a presença do DOM no CP para o MRP e d) a associação GnRH/hCG otimizou a função luteínica, retardou a luteólise e prolongou a fase luteínica de modo que todo o CP esteve sob influência da progesterona / Estradiol secreted from the dominant follicle (DOM) plays a key role in triggering luteolysis in the cow. In addition, maternal recognition of pregnancy (MRP) requires an optimum uterine environment, which directly depends on luteal function and adequate levels of circulating progesterone. The aim of this study was to test different strategies to optimize luteal function and prevent the influence of a DOM throughout the critical period (CP) for MRP (from D13 to D20 after estrus). Different approaches were tested. In exp.1, 23 Nelore cows were treated with the ovsync protocol to induce a synchronized estrus (D0). Cows received: Gc (n=7) - nothing else; ThCG (n=5) - 3000 IU of hCG five days (D5) after estrus; TE2 (n=6) - 5mg of 17b-estradiol (E2) on D12; ThCG/E2 (n=5) - hCG/D5 + E2/D12. Ultrasound evaluation and plasmatic progesterone concentration ([P4]) throughout estrous cycle allowed to conclude: E2 reprogrammed ovarian cycle by preventing the presence (P⁢.05) of a DOM during almost all CP (0.6±.7 days within the D15 to D20 interval) but induced luteolysis; cows receiving hCG developed accessory corpus luteum and had higher [P4] up to D13 (P⁢.05). Therefore, luteolysis was not delayed and luteal phase was not prolonged. In exp.2, same treatments were imposed to 220 Nelore cows (55 per group) after a timed artificial insemination (TAI). Pregnancy rates (PR) at TAI or at AIs thereafter over a 64-day period were reduced (P⁢0.05) by using E2 and hCG was not capable to improve those PR. In exp.3, postpartum Red Angus cows were estrus synchronized (D0) and received: nothing else (GCT, n=5) or 200mg of gonadorrelin on D5 plus 3000 IU hCG on D13 (GRF, n=5); or ablation of all follicles ³7mm through follicular aspiration on D14, D17 and D20 (GRM, n=5). GRF had delayed luteolysis (18.2±1.0b days, 23.6±1.0a days, 18.7±1.2b days for GCT, GRF, GRM, respectively) and higher [P4] than other groups. Follicles larger than 7mm were observed in all occasions of aspiration. In could be concluded that: a) E2 allowed to consistently reschedule follicular development but caused luteolysis and its use was detrimental to PR; b) hCG improved luteal function but did not increase PR; c) ablation of 7mm follicles did not prevent a DOM throughout CP for MRP and d) GnRH/hCG association optimized luteal function, delayed luteolysis and prolonged luteal phase in such a way that all CP was under progesterone influence

O objetivo, deste estudo foi investigar o comportamento dos hormônios esteróides 17β-Estradiol (E2), 17α-hidroxiprogesterona (17α-OHP), Testosterona (T) e 11-Ketotestosterona (11-KT), em piracanjuba Brycon orbignyanus de diferentes sexos e idades, na estação reprodutiva, e nas fêmeas submetidas à reprodução induzida. Os animais utilizados no trabalho, eram criados em piscicultura comercial, mantidos em 3 viveiros, separados por lotes de diferentes idades. A coleta dos animais, consistiu de quatro machos e cinco fêmeas (48 meses), identificados através do dimorfismo sexual da espécie, e as demais idades, (12 e 24 meses), coletaram-se, 20 peixes de cada idade, para identificação do sexo através de histologia. Já o experimento de caracterização dos esteróides sexuais na reprodução induzida, foram coletadas cinco fêmeas, selecionadas através das características com: abdome abaulado, papila urogenital, saliente e avermelhada. Após captura, os peixes foram transportados ao laboratório, onde houve coleta de sangue, para quantificação do perfil plasmático de E2, 17α-OHP, T e 11-KT. Posteriormente, os animais foram abatidos e suas gônadas coletadas e fixadas, a fim de que fosse realizada análise histológica para identificação do sexo. Na reprodução induzida, foi coletado sangue em dois momentos: pré-indução (PI) e pós-extrusão (PE). O nível plasmático de E2 nos machos de 12 meses destaca sua ação no processo de proliferação e renovação das espermatogônia observado em machos imaturos. Nas fêmeas o E2 apresentou os maiores níveis (P<0,05) nos animais de 48 meses, confirmando assim, sua principal função na estimulação do processo de vitelogênese, e maturação final do oócito. Quanto aos andrógenos T e 11-KT, os maiores níveis (p<0,05) foram observados nos peixes adultos (48 meses), permitindo afirmar que estes atuam como feedback negativo, do FSH e feedback positivo do LH, fundamental no processo de maturação final e liberação dos gametas, além de regular o comportamento reprodutivo. O resultado da 17α-OHP, sugere que, nas idades estudadas, é indispensável por participar como precursor dos principais esteróides (T, E2 e 11-KT), além da 17α,20β dihydroxy-4-pregnen-3-one (17α,20β-DHP), essencial no estágio final de maturação, e desova na reprodução induzida. / The objective of this study was to investigate the physiological behavior of steroid hormones 17β-Estradiol (E2), 17α-hydroxyprogesterone (17α-OHP), testosterone (T) and 11-Ketotestosterone (11-KT) in Brycon orbignyanus with different sex and ages on the reproductive season and in females submitted to induced reproduction. The animals used in the study were kept in three ponds on a commercial fish farming, separated by lots with different ages. The sampling of animals consisted of the collection of four males and five females (48 months) identified by the sexual dimorphism of the specie. In the other groups (12 and 24 months), 20 fish of each age were collected for identification of sex through histology. In the experiment with characterization of the sexual steroids in the induced reproduction, were collected five females selected through the following characteristics: bulging abdomen and prominent reddish genital papilla. After capture, the fish were transported to the laboratory, where blood was collected for quantification of the plasma profile of E2, 17α-OHP, T and 11-KT Subsequently, the animals were slaughtered and their gonads were collected and fixed for histological analysis. In the induced females, blood was collected at two moments: pre-induction (PI) and post-extrusion (PE). The plasma profile of E2 is fundamental in immature males, highlighting its action in the process of proliferation and renewal of spermatogonia, observed in males of 12 months. In females E2 presented the highest levels (P <0.05) in animals at 48 months, thus confirming its main function in the stimulation of the vitellogenesis process and final oocyte maturation. The highest levels (p <0.05) of T and 11-KT androgens were observed in adult fish (48 months), allowing to affirm that they are acting as FSH negative feedback and LH positive feedback, fundamental in the final maturation and release of the gametes, besides regulating the reproductive behavior of the fish. The results of 17α-OHP suggest this hormon is fundamental in the studied ages because it is a precursor of the main steroids (T, E2 and 11-KT) and 17α, 20β-dihydroxy-4-pregnen-3-one (17α, 20β-DHP), essential in the final stage of maturation and spawning in induced reproduction.

Les perturbateurs endocriniens (PE) sont une préoccupation majeure de par leurs effets sur les grandes fonctions physiologiques des organismes etparticulièrement sur la fonction de reproduction. Chez les vertébrés, la reproduction est sous le contrôle d’un axe neuroendocrinien nommé axegonadotrope. Celui-ci comprend différents acteurs cérébraux (gonadolibérines et kisspeptines), hypophysaires (gonadotropines) et gonadiques(stéroïdes sexuels) dont la mise en place et le fonctionnement sont régulés finement. Parmi les mécanismes de régulation, les stéroïdes vont jouer unrôle important en effectuant des boucles de rétrocontrôle. Les PE mimant ces stéroïdes sont donc potentiellement capables d’impacter ces régulations.L’objectif de ce travail était d’évaluer les effets d’un xénooestrogène sur les mises en place des acteurs cérébraux impliqués dans l’axe gonadotrope chezle bar européen (Dicentrarchus labrax). Nous avons d’abord décrit l’ontogénèse des systèmes à kisspeptine, à gonadolibérine et à gonadotropine aucours du développement larvaire. Nous avons mis en évidence une régulation différentielle de ces acteurs suggérant un rôle durant ce processusdéveloppemental. De plus, nous montrons que le 17α-éthinylestradiol (EE2) est capable de perturber l'ontogenèse des systèmes à GnRH2 et à GnRH3au cours des stades précoces du développement larvaire.Ces données constituent une base permettant l’évaluation des effets des PE sur l’ontogénèse des systèmes neuroendocriniens. / Endocrine disruptors (EDs) are a major concern because of their effects on the main physiological functions, particularly on the reproductive function.In vertebrates, reproduction is under the control of a neuroendocrine axis called hypothalamic-pituitary-gonadal (HPG) axis. It includes various actors atthe brain (gonadotropin releasing hormones (GnRH) and kisspeptins), pituitary (gonadotropic hormones) and gonadal (sex steroids) levels. Theirdévelopment and functioning are finely regulated. Among the regulators, steroids play an important role by performing feedback loops. EDs mimickingsteroids are therefore potentially able to impact these regulations. The objective of this work was to evaluate the effects of xenoestrogens on theimplementation of cerebral actors involved in the HPG axis in the European sea bass (Dicentrarchus labrax). We first described the ontogeny of thekisspeptin, GnRH and gonadotropin systems during larval development. We have highlighted a differential regulation of these actors suggesting a roleduring this developmental process. In addition, we show that 17α-ethinylestradiol (EE2) is able to disrupt the ontogeny of GnRH2 and GnRH3 systemsduring the early stages of larval development.These data provide a basis for evaluation of the EDs effects on the ontogeny of neuroendocrine systems.

Insulin-like growth factors (IGF) are known paracrine/autocrine regulators of ovarian development in teleosts. Initial studies investigated the hormonal and intracellular signal cascades involved in regulating the expression of ovarian-derived IGFs in zebrafish (Danio rerio). Quantitative real-time PCR was used to quantify the expression of igf3, igf2a, and igf2b in full grown immature (FG; 0.57-0.65 mm) and mid-vitellogenic (MV; 0.45-0.56 mm) follicles. Addition of the gonadotropin analogue human chorionic gonadotropin (hCG) and the adenylate cyclase activator forskolin increased igf3 expression in FG and MV follicles, but had no effect on igf2a or igf2b expression. The effects of hCG were blocked by the addition of the protein kinase A inhibitor H-89. Pituitary adenylate cyclase activating peptide stimulated a small increase in igf3 expression in FG follicles, while growth hormone and salmon gonadotropin releasing hormone had no effect on igf3, igf2a, or igf2b expression. Treatment with melittin, prostaglandin F2α, and prostaglandin E2 inhibited igf3 and igf2b expression in FG follicles whereas the protein kinase C activators, PMA and A23187, significantly inhibited igf3, igf2a, igf2b expression in FG and MV follicles. Secondary studies investigated the involvement of ovarian-derived IGFs in mediating the ovarian actions of gonadotropins on cell survival and steroidogenesis. Treatment of FG follicles with recombinant human IGF-I, hCG, or forskolin inhibited the induction of caspase-3/7 activity, which was used as a measure of apoptosis. The effects of hCG and forskolin on caspase-3/7 were attenuated by co-treatment with NVP-AEW54, an IGF-I receptor antagonist. hCG increased production of the maturation-inducing steroid 17α, 20β-dihydroxy-4-pregnen-3-one and co-treatment with NVP-AEW541 had no effect. These results suggest there is a high degree of hormonal specificity in regulating IGFs in the zebrafish ovary and the ovarian-derived IGFs, presumably IGF-III, are downstream mediators of gonadotropin-dependent cell survival, but are not involved in gonadotropin-induced steroidogenesis.

Diss. (sammanfattning). Uppsala : Sveriges lantbruksuniv., 2005. / Härtill 4 uppsatser.

Diss. (sammanfattning) Umeå : Sveriges lantbruksuniv. / Härtill 4 uppsatser. I publ. anges Department of Aquaculture som utgivande institution. Med sammanfattning på svenska och franska.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniversitet, 2006. / Härtill 5 uppsatser.