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.

A plasticidade do desenvolvimento gonadal em peixes, que contrasta com os padrões mais estáveis encontrados nos demais vertebrados, deu origem a várias questões intrigantes relativas tanto ao seu significado adaptativo quanto aos fatores genéticos e fisiológicos que modulam o processo. A inversão do sexo em peixes hermafroditas seqüenciais (protândricos ou protogínicos) ocorre em decorrência de diversos fatores, dentre eles, fisiológicos, genéticos e comportamentais podendo ser ainda decorrente do comportamento social. A garoupa verdadeira, Epinephelus marginatus é um peixe teleósteo, hermafrodita protogínico característico do litoral rochoso, maturando primeiramente como fêmeas, e posteriormente, na vida adulta, os ovários são substituídos por testículos, transformando-os em machos reprodutivamente ativos. No presente trabalho foram realizados três experimentos de inversão sexual em condições assistidas, em diferentes estações do ano, utilizando a mesma dose do inibidor de aromatase (IA) letrozole (100μg/Kg) para promover a inversão sexual de E. marginatus visando contribuir e aprimorar o conhecimento dos mecanismos fisiológicos envolvidos nesse processo. O primeiro e o terceiro experimentos foram iniciados na primavera e início da primavera respectivamente e promoveram a inversão sexual com uma diminuição no índice gonadossomático (IGS) nos animais do grupo experimental que apresentaram uma desorganização gonadal peculiar, com lamelas características de ovário, no entanto em seu interior observou-se o compartimento intersticial desenvolvido com cistos de células germinativas masculinas em estágios avançados da espermatogênese, (espermatócitos, espermátides) células de Sertoli e grande quantidade de cistos rompidos com a liberação de espermatozoides no lúmen. Após trinta dias do início do experimento obteve-se um aumento significativo de 11 ceto-testosterona (11KT) e a concentração de estradiol (E2) permaneceu inalterada; com setenta e sete dias após o início do experimento constatou-se uma elevação na concentração de testosterona (T), uma queda nos níveis de 11KT, persistindo a manutenção na concentração de E2. Neste cenário, principalmente as concentrações de andrógenos podem ter ativado a alça de feedback negativo e juntamente com a ativação do sistema da kisspeptina podem ter promovido a ação do GnRH desviando a produção de FSH para LH. Nesse momento o número de cópias obtidas de βFSH na hipófise foi significativamente menor que o número encontrado no grupo controle. Ao observarmos o aumento significativo da concentração plasmática de 17-alfaOHprogesterona podemos sugerir que a ação do GnRH tenha aumentado a secreção do LH, principal gonadotropina que estimula a produção de progestágenos. Os machos invertidos produziram um volume médio de sêmen de 118,20 ± 24,83 μL com motilidade de 90% e densidade espermática de 8,94 ± 4,34 x 109 células mL-1. Não foram obtidas diferenças nas concentrações de proteínas totais plasmática, hepática e musculares. No experimento implantado no verão de 2010 o tratamento não promoveu a inversão sexual, o IGS não apresentou diferença significativa entre os animais tratados com inibidor de aromatase e os animais do grupo controle. Nas gônadas dos animais do grupo experimental foi observada uma ligeira desorganização da arquitetura gonadal com cistos em formação e um número grande de ovócitos em degeneração demonstrando que ao longo do processo de inversão sexual pode haver uma fase de intersexo. Após trinta dias do início do experimento obteve-se um aumento significativo de T e a concentração de E2 permaneceu inalterada. Não foram observadas diferenças significativas entre o número de cópias de βFSH e βLH ao longo do experimento. O resultado obtido para a concentração de proteínas totais do músculo dos animais do grupo experimental apresentou um aumento significativo em relação à concentração encontrada nos animais do grupo controle. Não ocorreu espermiação e a inversão se deu de forma incompleta promovendo indivíduos intersexo. De uma forma geral podemos concluir que o uso de inibidor de aromatase é um método eficaz para promover a inversão sexual em hermafroditas protogínicos como a garoupa verdadeira. O aumento dos andrógenos pode ser o gatilho da inversão sexual com papel fundamental na reestruturação gonadal e formação do tecido germinativo masculino neste processo / The plasticity of gonadal development in fish, which contrasts with the more stable patterns found in other vertebrates, has given rise to several thrilling questions concerning both, its adaptive significance regarding the genetic and physiological factors that modulate the process. The sex inversion in sequential hermaphrodites fish (protandrous or protogynuos) occurs due to several factors, including, physiological, genetic and behavioral, and may also be due to social behavior. The dusky grouper, Epinephelus marginatus is a teleost fish, protogynous hermaphrodite characteristic of rocky bottoms, maturing first as females and later in adult life, the ovaries are replaced by the testes, turning them into reproductively males. In this work three sex inversion experiments were performed in captivity in different seasons of the year, using the same dose of aromatase inhibitor (AI) letrozole (100μg/Kg) to promote sexual inversion in E. marginatus, to contribute and improve the knowledge of the physiological mechanisms involved in this process. The first and third experiments started in the spring and early spring, respectively and promoted sexual inversion with a decrease in the gonadosomatic index (GSI) in the animals from the experimental group, that showed a peculiar disorganized gonadal, with lamellae, characteristic from ovaries, however with an interstitial compartment developed with cysts of male germ cells in advanced stages of spermatogenesis (spermatocytes, spermatids), Sertoli cells and a large amount of cysts ruptured with the release of sperm in the lumen. After thirty days from the beginning of the experiment, there was an increase in 11- ketotestosterone (11KT) levels and an unchanged concentration of estradiol (E2); after seventy-seven days, there was a significant increase in testosterone (T) levels, a decrease in the 11KT levels and the maintenance of E2 plasma concentration. In this point of view, mainly the concentration of androgens may have activated the negative feedback loop and with the activation of the kisspeptin system may have promoted the action of GnRH diverting the production of FSH to LH. At this moment the number of copies of pituitary βFSH produced was ignificantly lower than the number of control group copies. The significant increase in plasma 17- alfaOHprogesterona concentration observed could suggest that the action of GnRH has increased the secretion of LH, the main gonadotropin that stimulates the production of progestogen. Inverted males produced an average volume of semen of 118.20 ± 24.83 μl with 90% motility and sperm density of 8.94 ± 4.34 x 109 cells.ml-1. No differences were obtained at the concentrations of total protein in plasma, liver and muscle. In the experiment established in the summer of 2010, the treatment did not promote sexual inversion, the GSI was not significantly different between animals treated with aromatase inhibitor and the control group. In the gonads of the animals from the experimental group there was a slight disruption of gonadal architecture with cysts in formation and a large number of degenerating oocytes showing that during the process of sex inversion may be a phase of intersex. Thirty days after the beginning of the experiment there was a significant increase of T and E2 concentration remained unchanged. No significant differences were observed between the number of copies of βFSH and βLH throughout this experiment. The result obtained for the total protein concentration of the muscle in experimental group showed a significant increase compared with the control animals. There was no spermiation and the sex inversion occurred incompletely, with the presence of intersex individuals. In general we could conclude that the use of an aromatase inhibitor is an effective method to promote the sex inversion in hermaphroditic protogynous as the groupers. The increase of androgens could trigger the sex inversion exerting an important role in gonadal restructuring and the development of male germ tissue in the process

A inversão do sexo em peixes hermafroditas sequenciais (protândricos ou protogínicos) ocorre em decorrência de diversos fatores, dentre eles, fisiológicos, genéticos, podendo ser ainda decorrência do comportamento social. Nos peixes hermafroditas marinhos as alterações sexuais são acompanhadas por alterações anátomo-funcionais das gônadas, coloração e comportamento dos animais. A espécie Epinephelus marginatus (garoupa verdadeira), serranídeo da fauna nativa e amplamente difundida no litoral brasileiro, é um hermafrodita protogínico que apresenta dificuldade na manutenção de indivíduos do sexo masculino em seus cardumes e, além disso, encontra-se na lista de espécies sobreexplotadas. Utilizando-se técnicas de inversão sexual induzida, em cativeiro, para a obtenção de machos, este trabalho teve como objetivo estudar as alterações de alguns tecidos endócrinos durante o processo de inversão sexual, utilizando tratamentos com inibidores de aromatase (AI) e metiltestosterona (MT) em animais jovens. Ao longo de 90 dias de experimento a utilização do AI e do MT promoveu uma aparente desorganização da arquitetura gonadal padrão, massiva degeneração das células germinativas femininas, surgimento de centros melanomacrofágicos, proliferação de estruturas associadas ao sexo masculino (tecido intersticial e células germinativas masculinas) e a maturação das células germinativas masculinas. A utilização do andrógeno sintético MT (sozinho ou combinado com AI) possibilitou a obtenção de gônadas em fases mais avançadas de espermatogênese, inclusive a fase de espermiogênese inicial, com redução significativa dos valores do índice gonadossomático (IGS). Já com a utilização do AI foram encontradas gônadas em processos de intersexo mais clássicos após 90 dias experimentais. As análises de imunomarcação das células gonadotrópicas hipofisárias foram viáveis utilizando-se anticorpos de salmão e os resultados mostraram que as células produtoras de FSH (hormônio folículo-estimulante) e LH (hormônio luteinizante) distribuem-se pela proximal pars distalis e pars intermedia da adenohipófise. Os resultados obtidos com as análises imunohistoquímicas da hipófise demonstraram uma marcação de pouca intensidade utilizando-se o anticorpo anti-βFSH no grupo tratado com AI após 90 dias. A análise dos esteróides gonadais mostrou que nos animais tratados com AI, não houve diminuição na concentração do estradiol aos 90 dias, desta forma a alça de feedback negativo provavelmente foi acionada, e a produção de FSH diminuiu, causando o aumento da concentração plasmática de 11 cetotestosterona (11 KT) (via ação do LH). Já nos grupos onde o MT foi administrado, houve uma diminuição na concentração de estradiol após o tratamento, causando possivelmente uma menor intensidade na alça de feedback negativo na hipófise, sendo a ação do andrógeno sintético mais efetiva na espermiogênese nestes animais. As análises das gônadas e da hipófise sugerem a atuação do FSH no início da transição do sexo, sendo proposta a liberação desta glicoproteína como o gatilho para a inversão sexual em hermafroditas protogínicos. Estudos mais detalhados ao longo dos 90 dias de transição gonadal em E. marginatus precisam ser realizados para constatar o real envolvimento do FSH na inversão do sexo e verificar se a liberação do LH estaria envolvida com a produção de 11-ceto-testosterona, a espermiogênese completa e espermiação / The sex inversion in sequential hermaphrodite fish (protandrous or protogynous) occurs due to several factors, including physiological, genetics, and also can be associated with the social behavior. In marine hermaphrodite fish, the sex alterations are followed by anatomic and functional alterations in the gonads, color patterns and animal behavior. The species Epinephelus marginatus (dusky grouper), a native serranid and broadly distributed in the Brazilian coast, is a protogynous with problems in maintaining males in the shoal and, additionally, this species is part of the overexploited marine fish red list. Induction of sex change, in captivity, using aromatase inhibitors (AI) and 17α-methyltestosterone (MT) has been conducted with the purpose to study the morphophysiological alteration in some endocrine tissues in juveniles. During 90 experimental days, the use of AI and MT promoted an apparent disorganization of the standard gonad architecture, a massive degeneration of the female germ cells, the appearance of melanomacrophagic centers, a proliferation of the structures associated with the male sex (interstitial tissue and male germ cells) and the maturation of male germ cells. The use of the synthetic androgen, MT (alone or combined with AI) allowed the obtaining of gonads in a more advanced spermatogenesis phase, including an initial spermiogenesis phase, with a significant reduction of gonadossomatic index (GSI). With the use of AI, the gonads were found in a more classic intersex process after 90 days of experiment. The immunostaining analysis of the gonadotropic cells were viable using salmon antibodies and the results showed that FSH and LH cells were distributed in proximal pars distalis and pars intermedia in adenohypophysis. The immunohistochemistry analysis of the pituitary showed a low intensity of staining with the anti-βFSH in the AI group, after 90 experimental days. The analysis of the gonad steroids showed that in the animals from the AI group, there was no decrease in estradiol levels after 90 days, so the negative feedback loop probably was activated, the FSH secretion decreased, allowing the increase of plasma 11- ketotestosterone (11KT) (through LH action). In the MT groups there was a decrease in plasma estradiol, what probably decreased the intensity of the negative feedback loop in the pituitary, and in this case, the action of the synthetic androgen was predominant in spermiogenesis. The analysis of the gonads and pituitary suggest the action of FSH in the beginning of the sex transition, with the proposal that this glycoprotein acts triggering the sex inversion in protogynous hermaphrodites. We suggest that more detailed studies during the 90 days of gonad transition in E. marginatus must be done in order to find the real involvement of FSH in sex inversion and to elucidate the actual role of LH with 11KT production, the full spermiogenesis and spermiation

A inversão do sexo em peixes hermafroditas sequenciais (protândricos ou protogínicos) ocorre em decorrência de diversos fatores, dentre eles, fisiológicos, genéticos, podendo ser ainda decorrência do comportamento social. Nos peixes hermafroditas marinhos as alterações sexuais são acompanhadas por alterações anátomo-funcionais das gônadas, coloração e comportamento dos animais. A espécie Epinephelus marginatus (garoupa verdadeira), serranídeo da fauna nativa e amplamente difundida no litoral brasileiro, é um hermafrodita protogínico que apresenta dificuldade na manutenção de indivíduos do sexo masculino em seus cardumes e, além disso, encontra-se na lista de espécies sobreexplotadas. Utilizando-se técnicas de inversão sexual induzida, em cativeiro, para a obtenção de machos, este trabalho teve como objetivo estudar as alterações de alguns tecidos endócrinos durante o processo de inversão sexual, utilizando tratamentos com inibidores de aromatase (AI) e metiltestosterona (MT) em animais jovens. Ao longo de 90 dias de experimento a utilização do AI e do MT promoveu uma aparente desorganização da arquitetura gonadal padrão, massiva degeneração das células germinativas femininas, surgimento de centros melanomacrofágicos, proliferação de estruturas associadas ao sexo masculino (tecido intersticial e células germinativas masculinas) e a maturação das células germinativas masculinas. A utilização do andrógeno sintético MT (sozinho ou combinado com AI) possibilitou a obtenção de gônadas em fases mais avançadas de espermatogênese, inclusive a fase de espermiogênese inicial, com redução significativa dos valores do índice gonadossomático (IGS). Já com a utilização do AI foram encontradas gônadas em processos de intersexo mais clássicos após 90 dias experimentais. As análises de imunomarcação das células gonadotrópicas hipofisárias foram viáveis utilizando-se anticorpos de salmão e os resultados mostraram que as células produtoras de FSH (hormônio folículo-estimulante) e LH (hormônio luteinizante) distribuem-se pela proximal pars distalis e pars intermedia da adenohipófise. Os resultados obtidos com as análises imunohistoquímicas da hipófise demonstraram uma marcação de pouca intensidade utilizando-se o anticorpo anti-βFSH no grupo tratado com AI após 90 dias. A análise dos esteróides gonadais mostrou que nos animais tratados com AI, não houve diminuição na concentração do estradiol aos 90 dias, desta forma a alça de feedback negativo provavelmente foi acionada, e a produção de FSH diminuiu, causando o aumento da concentração plasmática de 11 cetotestosterona (11 KT) (via ação do LH). Já nos grupos onde o MT foi administrado, houve uma diminuição na concentração de estradiol após o tratamento, causando possivelmente uma menor intensidade na alça de feedback negativo na hipófise, sendo a ação do andrógeno sintético mais efetiva na espermiogênese nestes animais. As análises das gônadas e da hipófise sugerem a atuação do FSH no início da transição do sexo, sendo proposta a liberação desta glicoproteína como o gatilho para a inversão sexual em hermafroditas protogínicos. Estudos mais detalhados ao longo dos 90 dias de transição gonadal em E. marginatus precisam ser realizados para constatar o real envolvimento do FSH na inversão do sexo e verificar se a liberação do LH estaria envolvida com a produção de 11-ceto-testosterona, a espermiogênese completa e espermiação / The sex inversion in sequential hermaphrodite fish (protandrous or protogynous) occurs due to several factors, including physiological, genetics, and also can be associated with the social behavior. In marine hermaphrodite fish, the sex alterations are followed by anatomic and functional alterations in the gonads, color patterns and animal behavior. The species Epinephelus marginatus (dusky grouper), a native serranid and broadly distributed in the Brazilian coast, is a protogynous with problems in maintaining males in the shoal and, additionally, this species is part of the overexploited marine fish red list. Induction of sex change, in captivity, using aromatase inhibitors (AI) and 17α-methyltestosterone (MT) has been conducted with the purpose to study the morphophysiological alteration in some endocrine tissues in juveniles. During 90 experimental days, the use of AI and MT promoted an apparent disorganization of the standard gonad architecture, a massive degeneration of the female germ cells, the appearance of melanomacrophagic centers, a proliferation of the structures associated with the male sex (interstitial tissue and male germ cells) and the maturation of male germ cells. The use of the synthetic androgen, MT (alone or combined with AI) allowed the obtaining of gonads in a more advanced spermatogenesis phase, including an initial spermiogenesis phase, with a significant reduction of gonadossomatic index (GSI). With the use of AI, the gonads were found in a more classic intersex process after 90 days of experiment. The immunostaining analysis of the gonadotropic cells were viable using salmon antibodies and the results showed that FSH and LH cells were distributed in proximal pars distalis and pars intermedia in adenohypophysis. The immunohistochemistry analysis of the pituitary showed a low intensity of staining with the anti-βFSH in the AI group, after 90 experimental days. The analysis of the gonad steroids showed that in the animals from the AI group, there was no decrease in estradiol levels after 90 days, so the negative feedback loop probably was activated, the FSH secretion decreased, allowing the increase of plasma 11- ketotestosterone (11KT) (through LH action). In the MT groups there was a decrease in plasma estradiol, what probably decreased the intensity of the negative feedback loop in the pituitary, and in this case, the action of the synthetic androgen was predominant in spermiogenesis. The analysis of the gonads and pituitary suggest the action of FSH in the beginning of the sex transition, with the proposal that this glycoprotein acts triggering the sex inversion in protogynous hermaphrodites. We suggest that more detailed studies during the 90 days of gonad transition in E. marginatus must be done in order to find the real involvement of FSH in sex inversion and to elucidate the actual role of LH with 11KT production, the full spermiogenesis and spermiation

A plasticidade do desenvolvimento gonadal em peixes, que contrasta com os padrões mais estáveis encontrados nos demais vertebrados, deu origem a várias questões intrigantes relativas tanto ao seu significado adaptativo quanto aos fatores genéticos e fisiológicos que modulam o processo. A inversão do sexo em peixes hermafroditas seqüenciais (protândricos ou protogínicos) ocorre em decorrência de diversos fatores, dentre eles, fisiológicos, genéticos e comportamentais podendo ser ainda decorrente do comportamento social. A garoupa verdadeira, Epinephelus marginatus é um peixe teleósteo, hermafrodita protogínico característico do litoral rochoso, maturando primeiramente como fêmeas, e posteriormente, na vida adulta, os ovários são substituídos por testículos, transformando-os em machos reprodutivamente ativos. No presente trabalho foram realizados três experimentos de inversão sexual em condições assistidas, em diferentes estações do ano, utilizando a mesma dose do inibidor de aromatase (IA) letrozole (100μg/Kg) para promover a inversão sexual de E. marginatus visando contribuir e aprimorar o conhecimento dos mecanismos fisiológicos envolvidos nesse processo. O primeiro e o terceiro experimentos foram iniciados na primavera e início da primavera respectivamente e promoveram a inversão sexual com uma diminuição no índice gonadossomático (IGS) nos animais do grupo experimental que apresentaram uma desorganização gonadal peculiar, com lamelas características de ovário, no entanto em seu interior observou-se o compartimento intersticial desenvolvido com cistos de células germinativas masculinas em estágios avançados da espermatogênese, (espermatócitos, espermátides) células de Sertoli e grande quantidade de cistos rompidos com a liberação de espermatozoides no lúmen. Após trinta dias do início do experimento obteve-se um aumento significativo de 11 ceto-testosterona (11KT) e a concentração de estradiol (E2) permaneceu inalterada; com setenta e sete dias após o início do experimento constatou-se uma elevação na concentração de testosterona (T), uma queda nos níveis de 11KT, persistindo a manutenção na concentração de E2. Neste cenário, principalmente as concentrações de andrógenos podem ter ativado a alça de feedback negativo e juntamente com a ativação do sistema da kisspeptina podem ter promovido a ação do GnRH desviando a produção de FSH para LH. Nesse momento o número de cópias obtidas de βFSH na hipófise foi significativamente menor que o número encontrado no grupo controle. Ao observarmos o aumento significativo da concentração plasmática de 17-alfaOHprogesterona podemos sugerir que a ação do GnRH tenha aumentado a secreção do LH, principal gonadotropina que estimula a produção de progestágenos. Os machos invertidos produziram um volume médio de sêmen de 118,20 ± 24,83 μL com motilidade de 90% e densidade espermática de 8,94 ± 4,34 x 109 células mL-1. Não foram obtidas diferenças nas concentrações de proteínas totais plasmática, hepática e musculares. No experimento implantado no verão de 2010 o tratamento não promoveu a inversão sexual, o IGS não apresentou diferença significativa entre os animais tratados com inibidor de aromatase e os animais do grupo controle. Nas gônadas dos animais do grupo experimental foi observada uma ligeira desorganização da arquitetura gonadal com cistos em formação e um número grande de ovócitos em degeneração demonstrando que ao longo do processo de inversão sexual pode haver uma fase de intersexo. Após trinta dias do início do experimento obteve-se um aumento significativo de T e a concentração de E2 permaneceu inalterada. Não foram observadas diferenças significativas entre o número de cópias de βFSH e βLH ao longo do experimento. O resultado obtido para a concentração de proteínas totais do músculo dos animais do grupo experimental apresentou um aumento significativo em relação à concentração encontrada nos animais do grupo controle. Não ocorreu espermiação e a inversão se deu de forma incompleta promovendo indivíduos intersexo. De uma forma geral podemos concluir que o uso de inibidor de aromatase é um método eficaz para promover a inversão sexual em hermafroditas protogínicos como a garoupa verdadeira. O aumento dos andrógenos pode ser o gatilho da inversão sexual com papel fundamental na reestruturação gonadal e formação do tecido germinativo masculino neste processo / The plasticity of gonadal development in fish, which contrasts with the more stable patterns found in other vertebrates, has given rise to several thrilling questions concerning both, its adaptive significance regarding the genetic and physiological factors that modulate the process. The sex inversion in sequential hermaphrodites fish (protandrous or protogynuos) occurs due to several factors, including, physiological, genetic and behavioral, and may also be due to social behavior. The dusky grouper, Epinephelus marginatus is a teleost fish, protogynous hermaphrodite characteristic of rocky bottoms, maturing first as females and later in adult life, the ovaries are replaced by the testes, turning them into reproductively males. In this work three sex inversion experiments were performed in captivity in different seasons of the year, using the same dose of aromatase inhibitor (AI) letrozole (100μg/Kg) to promote sexual inversion in E. marginatus, to contribute and improve the knowledge of the physiological mechanisms involved in this process. The first and third experiments started in the spring and early spring, respectively and promoted sexual inversion with a decrease in the gonadosomatic index (GSI) in the animals from the experimental group, that showed a peculiar disorganized gonadal, with lamellae, characteristic from ovaries, however with an interstitial compartment developed with cysts of male germ cells in advanced stages of spermatogenesis (spermatocytes, spermatids), Sertoli cells and a large amount of cysts ruptured with the release of sperm in the lumen. After thirty days from the beginning of the experiment, there was an increase in 11- ketotestosterone (11KT) levels and an unchanged concentration of estradiol (E2); after seventy-seven days, there was a significant increase in testosterone (T) levels, a decrease in the 11KT levels and the maintenance of E2 plasma concentration. In this point of view, mainly the concentration of androgens may have activated the negative feedback loop and with the activation of the kisspeptin system may have promoted the action of GnRH diverting the production of FSH to LH. At this moment the number of copies of pituitary βFSH produced was ignificantly lower than the number of control group copies. The significant increase in plasma 17- alfaOHprogesterona concentration observed could suggest that the action of GnRH has increased the secretion of LH, the main gonadotropin that stimulates the production of progestogen. Inverted males produced an average volume of semen of 118.20 ± 24.83 μl with 90% motility and sperm density of 8.94 ± 4.34 x 109 cells.ml-1. No differences were obtained at the concentrations of total protein in plasma, liver and muscle. In the experiment established in the summer of 2010, the treatment did not promote sexual inversion, the GSI was not significantly different between animals treated with aromatase inhibitor and the control group. In the gonads of the animals from the experimental group there was a slight disruption of gonadal architecture with cysts in formation and a large number of degenerating oocytes showing that during the process of sex inversion may be a phase of intersex. Thirty days after the beginning of the experiment there was a significant increase of T and E2 concentration remained unchanged. No significant differences were observed between the number of copies of βFSH and βLH throughout this experiment. The result obtained for the total protein concentration of the muscle in experimental group showed a significant increase compared with the control animals. There was no spermiation and the sex inversion occurred incompletely, with the presence of intersex individuals. In general we could conclude that the use of an aromatase inhibitor is an effective method to promote the sex inversion in hermaphroditic protogynous as the groupers. The increase of androgens could trigger the sex inversion exerting an important role in gonadal restructuring and the development of male germ tissue in the process

<p> This thesis focuses on an examination of cortical and trabecular bone density and geometry at the radius and tibia in postmenopausal women, primarily women with history of breast carcinoma, while also assessing musculoskeletal changes in postmenopausal breast cancer patients after treatment with the Aromatase Inhibitor, Anastrozole. The first sub-study is an investigation of the reproducibility of the pQCT measurement parameters at the radius and tibia in healthy pre-and postmenopausal women. Results indicated that the reproducibility was good at the radius and even better at the tibia for all parameters measured. The second study is an appraisal of the level of osteoporosis knowledge in a cohort of postmenopausal women. The participants were assessed via the Facts on Osteoporosis Quiz, a well validated questionnaire, and the data revealed significantly lower test scores among the breast cancer subjects in comparison with healthy postmenopausal women. In the remaining group of studies, pQCT technology was utilized to describe trabecular and cortical bone at the radius and tibia in postmenopausal women and women with a history of breast carcinoma whom had been prescribed Anastrozole. The following measurement sites were significantly lower in the breast cancer subjects: TOT_DEN and TOT_CNT at the 4% radius; CRT_DEN, TOT_CNT, and CRT_CNT at the 20% radius; TOT_DEN at the 4% tibia; and CRT_DEN at the 38% tibia. With respect to time on Anastrozole, TOT_CNT at the 4% radius (r=-0.36); TOT_CNT (r=-0.33), CRT_CNT (r=-0.34) and CRT_DEN (r=-0.44) at the 20% radius; and CRT_DEN (r=-0.39) and CRT_CNT (r=-0.27) at the 38% tibia were significantly negatively correlated with days on Anastrozole. Furthermore, after two years of Anastrozole treatment in a small cohort of breast cancer subjects, there was a significant decrease in CRT_DEN (p=0.025) at the 20% diaphyseal radius and also at the 38% diaphyseal tibia (p=0.051). Together, the sub-studies that comprise this thesis demonstrate that there are noteworthy deficiencies in osteoporosis knowledge among postmenopausal women, particularly those with a history of breast carcinoma, and yet, these are the same women that have an increased need to understand the preventative and treatment options regarding this disease as they demonstrate reduced bone density at all measurement sites. It also appears that time on Anastrozole primarily affects cortical bone density in these women. In summary, this thesis provides novel details regarding cortical bone in breast cancer subjects and emphasizes the need for a normative database of bone quality parameters at different skeletal sites in order to gain a better understanding of the utility of each skeletal site with regard to fracture risk prediction. </p> / Thesis / Doctor of Philosophy (PhD)

La résistance aux anti-aromatases (AAs) constitue un obstacle thérapeutique majeur dans le traitement des cancers du sein RE+. Les objectifs de ce travail étaient : (i) de caractériser les événements moléculaires associés à la résistance acquise aux AAs ; (ii) d’identifier de manière globale de nouveaux profils de miRNAs spécifiquement associés à la résistance aux AAs. Notre étude a mis en évidence le rôle central de la voie Akt/mTOR dans la résistance acquise et de novo aux AAs dans des modèles cellulaires, mais également dans des échantillons de patientes ayant récidivé sous anastrozole. La combinaison d’un AA avec le MK-2206, inhibiteur d’Akt ou avec la rapamycine, inhibiteur de mTOR, augmente la sensibilité à l’AA dans les cellules contrôles et est suffisante pour surmonter la résistance et restaurer la sensibilité à l'hormonothérapie dans les cellules résistantes. Notre travail propose également un modèle de résistance acquise aux AAs basé sur la sélection de cellules « cancer-initiating-like » dotées de propriétés d'auto-renouvellement, d’une résistance intrinsèque aux AAs et d’une sensibilité au MK-2206. Notre étude à grande échelle des miRNAs a identifié la voie Akt/mTOR comme une des cibles privilégiées de ces miRNAs. Nous avons identifié et validé trois miRNAs dérégulés capables de moduler le statut d’activation de la voie Akt/mTOR, qui représentent des cibles potentielles. En conclusion, notre projet a mis en évidence de nouvelles voies de signalisations ciblées par ces miRNAs et de nouveaux évènements moléculaires, qui représentent des candidats potentiels dans la résistance aux AAs / Resistance to aromatase inhibitors (AIs) remains a major drawback in the treatment of ER+ breast cancers. Our objectives were (i) to characterize molecular events associated with acquired AI resistance (ii) to capture a global view of the miRNA expression profiles associated with AI resistance. Our results showed the major role of the Akt/mTOR pathway in both de novo and acquired resistance to AI in cellular models and also in breast tumors of patients who relapsed under anastrozole. Combining AI with the Akt inhibitor MK-2206 or with the mTOR inhibitor rapamycin increased sensitivity to this AI in the control cells and was sufficient to overcome resistance and restore sensitivity to endocrine therapy in the resistant cells. Our findings propose a model of AI-acquired resistance based on the selection of cancer-initiating-like cells possessing self-renewing properties, intrinsic resistance to AI and sensitivity to MK-2206. Our large-scale study identified the Akt/mTOR pathway as one of the main targets of the deregulated miRNAs. We identified and validated three miRNAs able to modulate the Akt/mTOR activation status, suggesting these miRNAs as potential targets. To conclude, our project identified new miRNA-targeted signaling pathways and new molecular events, representing strong candidates in the mediation of AI resistance

No description available.

Indiana University-Purdue University Indianapolis (IUPUI) / Aromatase inhibitors (AIs) are the current standard of care for the treatment of hormone receptor positive breast cancer in postmenopausal women. Nearly one-half of patients receiving AI therapy develop musculoskeletal toxicity that is characterized by joint and/or muscle pain and approximately one-fourth of patients discontinue their therapy as a result of musculoskeletal pain. Since there are no effective strategies for prevention or treatment, insight into the mechanisms of AI-induced pain is critical to improve treatment. However, there are few studies of AI effects in animal models of nociception. To determine whether AIs produce hypersensitivity in animal models of pain, I examined the effects of AI administration on mechanical, thermal, and chemical sensitivity in rats. The results demonstrate that (1) repeated injection of 5 mg/kg letrozole in male rats produces mechanical, but not thermal, hypersensitivity that extinguishes when drug dosing is stopped; (2) administering a single dose of 1 or 5 mg/kg letrozole in ovariectomized (OVX) rats also induces mechanical hypersensitivity, without altering thermal sensitivity and (3) a single dose of 5 mg/kg letrozole or daily dosing of letrozole or exemestane in male rats augments flinching behavior induced by intraplantar ATP injection. To determine whether the effects of AIs on nociceptive behaviors are mediated by activation or sensitization of peptidergic sensory neurons, I determined whether letrozole exposure alters release of calcitonin gene-related peptide (CGRP) from isolated rat sensory neurons and from sensory nerve endings in rat spinal cord slices. No changes in basal, capsaicin-evoked or high extracellular potassium-evoked CGRP release were observed in sensory neuronal cultures acutely or chronically exposed to letrozole. Furthermore, letrozole exposure did not alter the ability of ATP to augment CGRP release from sensory neurons in culture. Finally, chronic letrozole treatment did not augment neuropeptide release from spinal cord slices. Taken together, these results do not support altered release of this neuropeptide into the spinal cord as mediator of letrozole-induced mechanical hypersensitivity and suggest the involvement of other mechanisms. Results from this dissertation provide a new experimental model for AI-induced hypersensitivity that could be beneficial in delineating mechanisms mediating pain during AI therapy.