Orientador : Irani Quagio-Grassiotto / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-07T07:43:09Z (GMT). No. of bitstreams: 1 Antoneli_FernandaNatalia_D.pdf: 38673941 bytes, checksum: b5d1b651382a852a4a58b296f16a2d96 (MD5) Previous issue date: 2006 / Resumo: Os peixes teleósteos exibem vários padrões sexuais durante sua ontogenia, nos quais as gõnadas se transformam em ovários ou testículos funcionais. Nas distintas Ordens podem ser encontradas espécies gonocóricas, assim como hermafroditas, tanto simultâneas quanto seqüenciais ou consecutivas. No hermafroditismo seqüencial (protândrico ou protogínico), a mudança de sexo nos indivíduos adultos envolve a degeneração do tecido gonadal do primeiro sexo e o crescimento e maturação do tecido do sexo oposto, passando por uma fase de intersexo. Nas espécies hermafroditas protogínicas, mais frequentemente observada nos teleósteos, os individuos desenvolvem-se como fêmeas e, posteriormente, na vida adulta, seus ovários funcionais são substituídos por testículos, transformando-as em machos reprodutivamente ativos. Fatores internos e externos (ambientais) parecem controlar a mudança de sexo nas espécies hermafroditas seqüenciais. Nos vertebrados, o funcionamento do sistema reprodutor envolve o sistema nervoso e complexas vias de feedback hormonais, ambos integrados no chamado eixo hipotalâmico-hipofisário-gonadal. Nos peixes teleósteos, os esteróides sexuais, assim como os hormônios hipotalâmicos e adenohipofisários são considerados os principais fatores que controlam o desenvolvimento sexual gonadal, e podem estar envolvidos na inversão de sexo em espécies hermafroditas. Synbranchus marmoratus é um peixe hermafrodita protogínico diândrico e, na busca das possíveis correlações entre as alterações morfológicas gonadais e variações plasmáticas dos esteróides sexuais, observou-se que, na dependência do grau de decréscimo da oogênese e aumento da atividade do tecido masculino, o processo de inversão de sexo foi dividido em três fases: inicial, intermediária e final. A gõnada transicional de S. marmoratusfoi caracterizada pela a) desorganização da estrutura típica ovariana; b) massiva degeneração de células germinativas femininas; c) surgimento das células germinativas masculinas iniciais nas bordas das lamelas ovigeras, em associação com prováveis células de Sertoli; d) intensa proliferação e migração de células mióides; e) intensa atividade macrofágica, com a presença de melanomacrófagos e centros melanomacrofágicos; e f) aparecimento de grupos de células de Leydig no compartimento intersticial. As células de Leydig constituíram estruturas masculinas pioneiras a surgirem no ovário, então considerada gônada transicional inicial. Assim, estas células foram consideradas marcadores mortológicos e funcionais do processo de inversão sexual nesta espécie. As células de Leydig apresentaram as típicas características ultraestruturais associadas à esteroidogênese, bem como alta atividade esteroidogênica (3j3-HSD),provavelmente, produzindo andrógenos necessários ao desenvolvimento do tecido masculino gonadal e à espermatogênese. Na fase intermediária, a arquitetura masculina gonadal começa a ser definida, com a proliferação de numerosos cistos com células germinativas em diferentes fases de desenvolvimento. Nas gônadas transicionais finais, apenas poucos oócitos em degeneração e centros melanomacrofágicos permanecem. A mortologia da gônada transicional nesta fase é caracterizada pela completa formação das "lamelas testiculares" e dos túbulos seminíferos, os quais podem ser classificados como do tipo lobular irrestrito. Em nível hormonal, o início da transição sexual está associado à diminuição dos níveis plasmáticos de 17'beta'-estradiol, e o aumento da testosterona circulante, sugerindo a implicação destas circunstâncias para dar início à inversão de sexo natural em S. marmoratus. Os níveis plasmáticos de 11-cetotestosterona apenas sofrem baixas variações durante o início e progresso do referido processo, indicando um papel secundário deste andrógeno na inversão de sexo em S. marmoratus. A identificação das células adenohípofisárias de S. marmoratus durante a inversão de sexo não mostra qualquer alteração no padrão de imunomarcação das células produtoras dos diferentes hormônios ao longo do processo, excetuando-se as células gonadotrópicas, cuja atividade de síntese foi bastante alta nos intersexos iniciais. Provavelmente, estas células ofereçam suporte hormonal para a inversão de sexo nessa espécie. Assim, a inversão de sexo em S. marmoratus parece ocorrer sob controle do eixo hipotalâmico-hipofisário-gonadal. Por sua vez, as alterações morto lógicas necessárias à efetivação do processo propriamente dito - desestruturação do tecido gonadal feminino e desenvolvimento do tecido do sexo oposto - são gradualmente promovidas, seja pela falta ou escassez de estrógenos, e/ou pelo aumento dos níveis de andrógenos. Por conseguinte, S. marmoratus constitui um excelente modelo para o estudo dos eventos morto-funcionais da inversão de sexo / Abstract: Teleost fishes exhibit several sexual patterns during their ontogeny, in which the gonads change into functional ovaries or testes. In distinct Orders, both gonochoristic and hermaphrodite species, simultaneous as well as sequential or consecutive, can be found. In sequential hermaphroditism (protandrous or protogynous), sex change in adult individuais involves the degeneration of the primary sex gonadal tissue and the growth and maturation of the opposite sex, passing through an intersex phase. In protogynous hermaphrodite species, most frequently observed in teleosts, individuais first develop into females and later, at adulthood, their functional ovaries are gradually replaced by testes, becoming them into active reproductively males. Internal and external (environmenta) factors seem to control the sex change in sequential hermaphrodite species. In vertebrates, the dynamic of reproductive system involves the nervous system and complex hormonal biosynthetic pathways, all of them integrated in the called brain-pituitary-gonad axis. In teleost fish, sex steroids, as well as brain and pituitary hormones, are considered to be the main factors controlling gonadal sex development, and may be involved in sex reversal in hermaphrodite species. Synbranchus marmoratus is a protogynous diandric fish. Depending on the degree of female gametogenesis decrease and male tissue activity increase, the sex reversal process was divided into three phases: early, intermediate, and final. S. marmoratus transitional gonad was characterized by the: a) disorganization of the typical ovarian structure; b) massive degeneration of the female germ cells; c) appearance of initial male germ cells . at the border of ovigerous lamellae in association with putative Sertoli cells; d) intense proliferation and migration of myoid cells; e) intense macrophage activity with the presence of melanomacrophages and melanomacrophage centers; and f) appearance of clusters of Leydig cells in the interstitial compartment. Leydig cells constitute the pioneer male structures to arise in the ovary, considered the early transitional gonad at this point. Thus, these cells were considered morphological and functional markers of the sex reversal process in this species. Leydig cells presented the typical fine structures associated to steroidogenesis, such as high steroidogenic activity (3'beta'-HSD), probably, producing androgens necessary to development of gonadal male tissue, and spermatogenesis. In the intermediate phase, the male gonadal architecture begins to be defined, with the proliferation of numerous spermatocysts containing germ cells at different developmental stages. In final transitional gonads, only few degenerating oocytes and melanomacrophage centers remain. The morphology of transitional gonad at this phase is characterized by the complete formation of the testicular lamellae and the seminiferous lobules, which can be classified as unrestricted lobular type. At hormonal level, the beginning of the sexual transition is associated with the decrease of plasma estradiol-17 'beta' levels and the increase of circulating testosterone, suggesting the implication of such circumstances to trigger the natural sex change in S. marmoratus. Plasma 11-ketotestosterone levels onlysuffered weak variations during the initiation and progress of such process, which may indicate the secondary role of this androgen in S. marmoratus sex reversal. The identification of the adenohypophyseal cells of Synbranchus marmoratus during sex reversal does not show any alteration in the immunomarcation pattern of secretory cells of different hormones during the process, except for gonadotropin-producing oolls, which synthesis activity was very high in the initial intersexes. Probably, these cells offer hormonal support for sex reversal in this species. Therefore, sex reversal in S. marmoratus seems to occur under hypothalamic-hypophyseal-gonadal axis control. Morphological alterations necessary to sex reversal process accomplishment - disorganization of female gonadal tissue and development of the tissue of opposite sex - are gradually promoted by the estrogens privation, and/or by the androgens levels increase. Therefore, S. marmoratus constitutes an excellent model for the studying of morpho-functional eventsof sex change / Doutorado / Biologia Celular / Doutor em Biologia Celular e Estrutural

Neste estudo foi verificado que a hipófise de S. brasiliensis era composta por dois tecidos, a neuro hipófise (NH) e a adeno-hipófise (AH). Nesta última, foram distinguidos sete tipos celulares. Na RPD foram detectadas as células adrenocorticotrópicas e prolactínicas, na PPD as células gonadotrópicas, somatotrópicas e tireotrópicas, e na PI, as células melanotrópicas e somatolactínicas. Foi evidenciada única célula gonadotrópica, produtora de LH e FSH. O primórdio da hipófise foi detectado 12 horas após a eclosão (hpe), a NH com 72 hpe e o início da formação do pedúnculo com 300 hpe. Nos juvenis (600 hpe), a hipófise apresentou uma morfologia semelhante àquela observada nos adultos. As células prolactínicas foram detectadas com 12 hpe, juntamente com as células adrenocorticotrópicas e melanotrópicas, seguidas das somatotrópicas e somatolactínicas, com 36 hpe. Por outro lado, nas larvas e juvenis foram detectadas duas populações distintas de células gonadotrópicas, as células produtoras de FSH foram detectadas com 600 hpe, enquanto as produtoras de LH com 120 hpe. / This study showed that the pituitary gland of S. brasiliensis was formed by two tissues, neurohypophysis and adenohypophysis. In the latter, seven cell types were distinguished. In RPD, prolactin and adrenocorticotropic cells were present. In PPD, gonadotropic, somatotrope and thyrotropic cells were detected, and in PI, somatolactin and melanotropic cells were found. Interestingly, was detected a single gonadotropic cell responsible for producing both gonadotropins. The primordium of the pituitary gland was detected 12 hours after hatching (hah), the neurohypophysis was detected 72 hah and formation of the stalk 300 hah. In juveniles, 600 hah, the pituitary showed a similar morphology to that observed in adults of this species. Prolactin cells were detected 12 hah together with adrenocorticotropic and melanotropic cells, followed by somatotropic and somatolactin cells 36 hah. Unlike adults, larvae and juveniles have shown two distinct populations of gonadotropic cells. FSH-producing cells were detected 600 hah, while LH-producing cells were detected 120 hah.

Neste estudo foi verificado que a hipófise de S. brasiliensis era composta por dois tecidos, a neuro hipófise (NH) e a adeno-hipófise (AH). Nesta última, foram distinguidos sete tipos celulares. Na RPD foram detectadas as células adrenocorticotrópicas e prolactínicas, na PPD as células gonadotrópicas, somatotrópicas e tireotrópicas, e na PI, as células melanotrópicas e somatolactínicas. Foi evidenciada única célula gonadotrópica, produtora de LH e FSH. O primórdio da hipófise foi detectado 12 horas após a eclosão (hpe), a NH com 72 hpe e o início da formação do pedúnculo com 300 hpe. Nos juvenis (600 hpe), a hipófise apresentou uma morfologia semelhante àquela observada nos adultos. As células prolactínicas foram detectadas com 12 hpe, juntamente com as células adrenocorticotrópicas e melanotrópicas, seguidas das somatotrópicas e somatolactínicas, com 36 hpe. Por outro lado, nas larvas e juvenis foram detectadas duas populações distintas de células gonadotrópicas, as células produtoras de FSH foram detectadas com 600 hpe, enquanto as produtoras de LH com 120 hpe. / This study showed that the pituitary gland of S. brasiliensis was formed by two tissues, neurohypophysis and adenohypophysis. In the latter, seven cell types were distinguished. In RPD, prolactin and adrenocorticotropic cells were present. In PPD, gonadotropic, somatotrope and thyrotropic cells were detected, and in PI, somatolactin and melanotropic cells were found. Interestingly, was detected a single gonadotropic cell responsible for producing both gonadotropins. The primordium of the pituitary gland was detected 12 hours after hatching (hah), the neurohypophysis was detected 72 hah and formation of the stalk 300 hah. In juveniles, 600 hah, the pituitary showed a similar morphology to that observed in adults of this species. Prolactin cells were detected 12 hah together with adrenocorticotropic and melanotropic cells, followed by somatotropic and somatolactin cells 36 hah. Unlike adults, larvae and juveniles have shown two distinct populations of gonadotropic cells. FSH-producing cells were detected 600 hah, while LH-producing cells were detected 120 hah.

A tabarana Salminus hilarii (Characiformes) é uma espécie reofílica de grande importância ecológica na Bacia do Alto Tietê (São Paulo/SP, Brasil). Atualmente esta espécie sofre com a poluição (industrial e doméstica) e a construção de barragens na região, o que pode prejudicar o comportamento migratório reprodutivo das espécies reofílicas, bloqueando a reprodução. O presente estudo tem como objetivo analisar os mecanismos fisiológicos envolvidos no controle endógeno da reprodução Salminus hilarii, analisando-se o eixo hipófise-gônadas. Adicionalmente, buscou-se avaliar se o bloqueio da migração reprodutiva (parcial ou total) altera o funcionamento deste eixo. Os animais foram coletados no ambiente natural (Bacia do Alto Tietê), na saída da Barragem da Barragem de Ponte Nova e na Piscicultura de Ponte Nova (Salesópolis/SP) durante o período de abril de 2004 e agosto de 2006. As análises macro e microscópicas das gônadas e os valores de IGS mostraram que o período reprodutivo da tabarana ocorreu entre os meses de outubro a fevereiro. As análises macroscópicas e microscópicas dos ovários e dos testículos permitiram classificar o desenvolvimento ovariano em 4 estádios: repouso, maturação inicial, maturação avançada (cativeiro), maduro (ambiente natural) e regressão/esgotado e o desenvolvimento testicular em 3 estádio: repouso, maduro e regressão. O desenvolvimento oocitário da tabarana é do tipo sincrônico em grupo e a desova é total; os testículos são do tipo tubular anastomosado e espermatogonial irrestrito. Foram identificados 5 tipos celulares no interior dos ovários: oogônias, oócito perinucleolar, oócito cortical-alveolar, oócito vitelogênico e oócito maduro, além de estruturas derivadas desse desenvolvimento, os folículos pós-ovulatórios e os oócitos atrésicos. Identificou-se também 6 tipos celulares no interior dos testículos: espermatogônias, espermatócitos, espermátides, espermatozóides, células de Sertoli e células de Leydig. A hipófise de S. hilarii é composta de dois tecidos: a adeno-hipófise (tecido glandular endócrino) e a neuro-hipófise (origem nervosa). Adicionalmente, foi verificado que a adeno-hipófise é subdividida em: “rostral pars distalis" (RPD), “proximal pars distalis" (PPD) e “pars intermedia" (PI). Seis tipos celulares foram identificados na adeno-hipófise: as células produtoras de prolactina (PRL) e as células produtoras de hormônio adrenocorticotrópico (ACTH) foram identificadas na região da RPD; as células produtoras de hormônio de crescimento (GH) e as células produtoras de gonadotropinas (GtHs) estão situadas na região da PPD; as células produtoras de melanotropina (MSH) e as células produtoras de somatolactina (SL) estão presentes na PI. As células PRL não apresentam variações ao longo do ciclo reprodutivo da tabarana, no entanto, as células produtoras de GH diminuem a porcentagem de imunomarcação com o avanço do ciclo reprodutivo. Já as células produtoras de SL aumentam a porcentagem de imunomarcação com o desenvolvimento das gônadas. Adicionalmente, foi verificada a presença de duas gonadotropinas na região da adeno-hipófise, o hormônio folículo estimulante (FSH) e o hormônio luteinizante (LH). Em relação ao perfil plasmático dos esteróides gonadais, foi verificado que o estradiol aumenta com o avanço do ciclo reprodutivo, diminuindo do estádio maduro para o estádio regressão/esgotado (ambiente natural). No cativeiro ocorreu um aumento da concentração de estradiol do estádio repouso até o estádio maturação média, diminuindo logo em seguida nos estádios maturação avançada e regressão/esgotado. A concentração de testosterona eleva-se com o avanço da maturação gonadal, atingindo os valores máximos no estádio maduro (ambiente natural) e maturação avançada (cativeiro). Paralelamente, o estradiol apresentou-se alto somente na maturação média (cativeiro) e maduro (ambiente natural), evidenciando uma relação entre esses dois hormônios. Valores mais elevados de 17α-hidroxiprogesterona no cativeiro sugerem uma deficiência na conversão deste esteróide para17α, 20β-dihidroxy-4-pregnen-3-one, considerado como o hormônio indutor da maturação final e ovulação na maioria dos teleósteos. Os resultados encontrados evidenciam que o bloqueio da migração altera a fisiologia do eixo hipófise-gônadas em Salminus hilarii. / The tabarana Salminus hilarii (Characiformes) is a reofilic species of great ecological importance in the Region of the Alto Tietê Basin (São Paulo/SP, Brazil). This fish reaches greater sizes in this Basin and as a carnivorous species, which occupies the top of the alimentary chain. Nowadays, the Tietê Basin is affected by pollution (industrial and domestic) and dam constructions in the upper reaches of the river, hindering the reproductive migratory behavior of the reofilic species. When the “piracema" fish migration is obstructed, the reproduction fails, making evident, that the dam construction in the Brazilian rivers causes great impacts in the life cycle of the reofilic teleost. The aim of this study was analyze the physiological mechanisms involved in Salminus hilarii reproduction, analyzing the pituitary-gonads axis and additionally, to evaluate if the migration impediment (partial or total) modifies this axis physiology, and consequently the endogenous control of tabarana reproduction. From April 2004 to August.2006, the animals were collected in the natural environment (upper Tietê Basin), in the water exit of Ponte Nova Dam (Department of Water and Electric Energy) and in the Ponte Nova Dam Fishfarm (Salesópolis/SP). The microscopical analyses of the gonads and analyses of the gonadossomatic Index showed that the reproductive period of the tabarana occurred between October and February. The macroscopic and microscopical analyses of the ovaries and testis allowed to classify the ovarial development in 4 stages: resting, initial maturation, advanced maturation (captivity) or maturity (natural environmental) and regression/spawned, and the testis development in 3 stages: resting, mature and regression. The tabarana oocyte development is group-synchronous and the spawning is total. The anastomosing tubular testis and unrestricted spermatogonia were observed. Five cellular types in the interior of the ovaries were identified: oogonia, perinucleolar oocyte, cortical alveolar oocyte, vitellogenic oocyte and ripe oocyte, and beyond structures derived from this evelopment, the post-ovulatory follicles and the atretic oocyte. In the testis, 6 cellular types were identified: spermatogonia, spermatocytes, spermatids, spermatozoa, Sertoli cell and Leydig cell. S. hilarii pituitary is composed of two different tissues: adenohypophysis (endocrine tissue) and neurohypophysis (nervous origin). Additionally, it was verified that adenohypophysis is subdivided in: “rostral pars distalis" (RPD), “proximal pars distalis" (PPD) and “pars intermedia" (PI). Six cellular types were identified in adenohypophysis: prolactin cells (PRL) and adenocorticotropin cells (ACTH) have been identified in the RPD region; growth hormone cells (GH) and the gonadotropinas cells (GtHs) were situated in the PPD region; melanotropin cells (MSH) and somatolactin cells (SL) were present in the PI. PRL cells did not present variations throughout the reproductive cycle of the tabarana. However, the GH reaction showed a decrease in the cells stained percentage with the advance of the reproductive cycle. Also the SL cells increased the stained percentage along the gonads development. Additionally, the presence of two gonadotropins in the region of adenohypophysis, the follicle-stimulating hormone (FSH) and the luteinizing hormone (LH) was identified. The plasma levels of sex steroids showed that estradiol increased with the advance of the reproductive cycle, diminishing from the mature to the regression/spawning stage (natural environmental). In captivity an increase of the estradiol concentration from the resting to the medium maturation stage was observed, diminishing immediately afterwards in advanced maturation and regression/spawning stage. The testosterone concentration increased with the advance of the gonadal maturation, reaching the maximum values in the mature stage (natural environmental) and advanced maturation stage (captivity). On the other hand, estradiol was high only in the medium maturation (captivity) and ripe stage (natural environmental), evidencing an interdependence between these two steroids. The progestagene analyses showed that, in all maturation stages, 17α OHP levels were higher in captivity than in the natural environment, suggesting that the conversion of this steroid to 17α20β DHP (MIS) is affected in impaired environments, and that the conversion enzyme (20β desidrogenase) fails when migration is blocked. The results allow to conclude that the migration impairment change negatively the pituitary-gonads axis in S. hilarii.

A tabarana Salminus hilarii (Characiformes) é uma espécie reofílica de grande importância ecológica na Bacia do Alto Tietê (São Paulo/SP, Brasil). Atualmente esta espécie sofre com a poluição (industrial e doméstica) e a construção de barragens na região, o que pode prejudicar o comportamento migratório reprodutivo das espécies reofílicas, bloqueando a reprodução. O presente estudo tem como objetivo analisar os mecanismos fisiológicos envolvidos no controle endógeno da reprodução Salminus hilarii, analisando-se o eixo hipófise-gônadas. Adicionalmente, buscou-se avaliar se o bloqueio da migração reprodutiva (parcial ou total) altera o funcionamento deste eixo. Os animais foram coletados no ambiente natural (Bacia do Alto Tietê), na saída da Barragem da Barragem de Ponte Nova e na Piscicultura de Ponte Nova (Salesópolis/SP) durante o período de abril de 2004 e agosto de 2006. As análises macro e microscópicas das gônadas e os valores de IGS mostraram que o período reprodutivo da tabarana ocorreu entre os meses de outubro a fevereiro. As análises macroscópicas e microscópicas dos ovários e dos testículos permitiram classificar o desenvolvimento ovariano em 4 estádios: repouso, maturação inicial, maturação avançada (cativeiro), maduro (ambiente natural) e regressão/esgotado e o desenvolvimento testicular em 3 estádio: repouso, maduro e regressão. O desenvolvimento oocitário da tabarana é do tipo sincrônico em grupo e a desova é total; os testículos são do tipo tubular anastomosado e espermatogonial irrestrito. Foram identificados 5 tipos celulares no interior dos ovários: oogônias, oócito perinucleolar, oócito cortical-alveolar, oócito vitelogênico e oócito maduro, além de estruturas derivadas desse desenvolvimento, os folículos pós-ovulatórios e os oócitos atrésicos. Identificou-se também 6 tipos celulares no interior dos testículos: espermatogônias, espermatócitos, espermátides, espermatozóides, células de Sertoli e células de Leydig. A hipófise de S. hilarii é composta de dois tecidos: a adeno-hipófise (tecido glandular endócrino) e a neuro-hipófise (origem nervosa). Adicionalmente, foi verificado que a adeno-hipófise é subdividida em: “rostral pars distalis” (RPD), “proximal pars distalis” (PPD) e “pars intermedia” (PI). Seis tipos celulares foram identificados na adeno-hipófise: as células produtoras de prolactina (PRL) e as células produtoras de hormônio adrenocorticotrópico (ACTH) foram identificadas na região da RPD; as células produtoras de hormônio de crescimento (GH) e as células produtoras de gonadotropinas (GtHs) estão situadas na região da PPD; as células produtoras de melanotropina (MSH) e as células produtoras de somatolactina (SL) estão presentes na PI. As células PRL não apresentam variações ao longo do ciclo reprodutivo da tabarana, no entanto, as células produtoras de GH diminuem a porcentagem de imunomarcação com o avanço do ciclo reprodutivo. Já as células produtoras de SL aumentam a porcentagem de imunomarcação com o desenvolvimento das gônadas. Adicionalmente, foi verificada a presença de duas gonadotropinas na região da adeno-hipófise, o hormônio folículo estimulante (FSH) e o hormônio luteinizante (LH). Em relação ao perfil plasmático dos esteróides gonadais, foi verificado que o estradiol aumenta com o avanço do ciclo reprodutivo, diminuindo do estádio maduro para o estádio regressão/esgotado (ambiente natural). No cativeiro ocorreu um aumento da concentração de estradiol do estádio repouso até o estádio maturação média, diminuindo logo em seguida nos estádios maturação avançada e regressão/esgotado. A concentração de testosterona eleva-se com o avanço da maturação gonadal, atingindo os valores máximos no estádio maduro (ambiente natural) e maturação avançada (cativeiro). Paralelamente, o estradiol apresentou-se alto somente na maturação média (cativeiro) e maduro (ambiente natural), evidenciando uma relação entre esses dois hormônios. Valores mais elevados de 17α-hidroxiprogesterona no cativeiro sugerem uma deficiência na conversão deste esteróide para17α, 20β-dihidroxy-4-pregnen-3-one, considerado como o hormônio indutor da maturação final e ovulação na maioria dos teleósteos. Os resultados encontrados evidenciam que o bloqueio da migração altera a fisiologia do eixo hipófise-gônadas em Salminus hilarii. / The tabarana Salminus hilarii (Characiformes) is a reofilic species of great ecological importance in the Region of the Alto Tietê Basin (São Paulo/SP, Brazil). This fish reaches greater sizes in this Basin and as a carnivorous species, which occupies the top of the alimentary chain. Nowadays, the Tietê Basin is affected by pollution (industrial and domestic) and dam constructions in the upper reaches of the river, hindering the reproductive migratory behavior of the reofilic species. When the “piracema” fish migration is obstructed, the reproduction fails, making evident, that the dam construction in the Brazilian rivers causes great impacts in the life cycle of the reofilic teleost. The aim of this study was analyze the physiological mechanisms involved in Salminus hilarii reproduction, analyzing the pituitary-gonads axis and additionally, to evaluate if the migration impediment (partial or total) modifies this axis physiology, and consequently the endogenous control of tabarana reproduction. From April 2004 to August.2006, the animals were collected in the natural environment (upper Tietê Basin), in the water exit of Ponte Nova Dam (Department of Water and Electric Energy) and in the Ponte Nova Dam Fishfarm (Salesópolis/SP). The microscopical analyses of the gonads and analyses of the gonadossomatic Index showed that the reproductive period of the tabarana occurred between October and February. The macroscopic and microscopical analyses of the ovaries and testis allowed to classify the ovarial development in 4 stages: resting, initial maturation, advanced maturation (captivity) or maturity (natural environmental) and regression/spawned, and the testis development in 3 stages: resting, mature and regression. The tabarana oocyte development is group-synchronous and the spawning is total. The anastomosing tubular testis and unrestricted spermatogonia were observed. Five cellular types in the interior of the ovaries were identified: oogonia, perinucleolar oocyte, cortical alveolar oocyte, vitellogenic oocyte and ripe oocyte, and beyond structures derived from this evelopment, the post-ovulatory follicles and the atretic oocyte. In the testis, 6 cellular types were identified: spermatogonia, spermatocytes, spermatids, spermatozoa, Sertoli cell and Leydig cell. S. hilarii pituitary is composed of two different tissues: adenohypophysis (endocrine tissue) and neurohypophysis (nervous origin). Additionally, it was verified that adenohypophysis is subdivided in: “rostral pars distalis” (RPD), “proximal pars distalis” (PPD) and “pars intermedia” (PI). Six cellular types were identified in adenohypophysis: prolactin cells (PRL) and adenocorticotropin cells (ACTH) have been identified in the RPD region; growth hormone cells (GH) and the gonadotropinas cells (GtHs) were situated in the PPD region; melanotropin cells (MSH) and somatolactin cells (SL) were present in the PI. PRL cells did not present variations throughout the reproductive cycle of the tabarana. However, the GH reaction showed a decrease in the cells stained percentage with the advance of the reproductive cycle. Also the SL cells increased the stained percentage along the gonads development. Additionally, the presence of two gonadotropins in the region of adenohypophysis, the follicle-stimulating hormone (FSH) and the luteinizing hormone (LH) was identified. The plasma levels of sex steroids showed that estradiol increased with the advance of the reproductive cycle, diminishing from the mature to the regression/spawning stage (natural environmental). In captivity an increase of the estradiol concentration from the resting to the medium maturation stage was observed, diminishing immediately afterwards in advanced maturation and regression/spawning stage. The testosterone concentration increased with the advance of the gonadal maturation, reaching the maximum values in the mature stage (natural environmental) and advanced maturation stage (captivity). On the other hand, estradiol was high only in the medium maturation (captivity) and ripe stage (natural environmental), evidencing an interdependence between these two steroids. The progestagene analyses showed that, in all maturation stages, 17α OHP levels were higher in captivity than in the natural environment, suggesting that the conversion of this steroid to 17α20β DHP (MIS) is affected in impaired environments, and that the conversion enzyme (20β desidrogenase) fails when migration is blocked. The results allow to conclude that the migration impairment change negatively the pituitary-gonads axis in S. hilarii.

Abstract not available

L’embranchement Hemichordata regroupe les classes Enteropneusta et Pterobranchia. Hemichordata constitue, avec l’embranchement Echinodermata, le groupe-frère des chordés. Les entéropneustes sont des organismes vermiformes solitaires qui vivent sous ou à la surface du substrat et s’alimentent généralement par déposivorie, alors que les ptérobranches sont des organismes coloniaux filtreurs habitant dans un réseau de tubes appelé coenecium. Ce mémoire présente trois études dont le point commun est l’utilisation des hémichordés actuels pour répondre à des questions concernant l’évolution des hémichordés, des chordés, et du super-embranchement qui les regroupe, Deuterostomia. Notre première étude démontre que les fentes pharyngiennes, l’organe pré-oral cilié (POCO) et le pharynx de l’entéropneuste Protoglossus graveolens sont utilisés pour l’alimentation par filtration. Le système de filtration de P. graveolens permet la capture de particules jusqu’à 1.3 um, à un débit de 4.05 mm.s-1, pour une demande énergétique de 0.009 uW. Les similarités structurales et fonctionnelles avec le système de filtration des céphalochordés suggèrent que la filtration pharyngienne est ancestrale aux deutérostomes. Lors de notre deuxième étude, nous avons exploré l’hypothèse selon laquelle le POCO des entéropneustes, une structure ciliée pré-buccale au rôle possiblement chémorécepteur, serait homologue au « wheel organ » des céphalochordés et à l’adénohypophyse des vertébrés. Pour cela, nous avons déterminé par immunohistochimie l’expression de Pit-1, un facteur de transcription spécifique à ces deux structures, chez l’entéropneuste Saccoglossus pusillus. Pit-1 est exprimé dans des cellules sensorielles du POCO, mais aussi dans des cellules épithéliales distribuées dans le proboscis, collet et tronc. Ce patron d’expression ne permet pas de confirmer ou rejeter l’homologie du POCO et de l’adénohypophyse des vertébrés. Lors de notre troisième étude, nous avons caractérisé l’ultrastructure du coenecium des ptérobranches Cephalodiscus hodgsoni, Cephalodiscus nigrescens et Cephalodiscus densus par microscopie électronique à transmisison et à balayage. Cephalodiscus est le groupe frère de Graptolithina, un groupe qui inclut les graptolithes éteints ainsi que les ptérobranches du genre Rhabdopleura. Nous avons décrit les types de fibrilles de collagène présents, leur taille et leur organisation, ainsi que l’organisation globale du coenecium. Nous avons ainsi démontré la présence chez Cephalodiscus d’une organisation similaire au paracortex, pseudocortex et eucortex des graptolithes. La présence chez Cephalodiscus de ce type d’organisation suggère que le cortex est ancestral à la classe Pterobranchia. Ces trois études illustrent plusieurs axes importants de la recherche sur les hémichordés, qui en intégrant des données morphologiques, fonctionnelles et moléculaires permet de reconstruire certains évènements clés de l’évolution des deutérostomes. / The phylum Hemichordata comprises the classes Enteropneusta and Pterobranchia. Together with echinoderms, hemichordates are the sister-group to chordates. Enteropneusts are worm-shaped solitary deposit feeders. Pterobranchs are colonial filter feeders that live in a secreted collagenous domicile called a coenecium. In this thesis, three studies are presented. These studies are based on observations of extant hemichordates, and adress a variety of issues relating to the evolution of hemichordates, chordates, and the super-phylum to which they belong: Deuterostomia. Our first study demonstrates that the gill slits, pre-oral ciliary organ (POCO), and lining of the pharynx of the enteropneust Protoglossus graveolens are used in filter feeding. The filter-feeding system of P. graveolens enables particle capture down to 1.3 um, at a rate up to 4.05 mm.s-1, with a power consumption of 0.009 uW. Structural and functional similarities with the cephalochordate filter-feeding system suggest that pharyngeal filter-feeding is ancestral to the deuterostomes. In our second study, we address the hypothesis that the enteropneust POCO, a putative chemosensory structure located anterior to the mouth, is homologous to the cephalochordate wheel organ and vertebrate adenohypophysis. We characterized the expression pattern of the adenohypophysis-specific transcription factor Pit-1 in the adult enteropneust Saccoglossus pusillus with immunohistochemistry. Pit-1 is expressed in sensory cells of the POCO and in scattered epithelial cells of the proboscis, collar and trunk. This expression pattern does not allow to confirm or reject the homology of the POCO with the vertebrate adenohypophysis. In our third study, we characterized the ultrastructure of the coenecium of the pterobranchs Cephalodiscus hodgsoni, Cephalodiscus nigrescens and Cephalodiscus densus using transmission and scanning electron microscopy. Cephalodiscus is the sister-group to the Graptolithina, which includes the extinct graptolites and the extant pterobranch genus Rhabdopleura. We described the fibril types, size and organization, as well as the general organization of the coenecium. We demonstrated that the coenecium of Cephalodiscus shows similarities with the graptolite eucortex, paracortex and pseudocortex. The cortical-like organization of the coenecium of Cephalodiscus suggests that the cortex is ancestral to the Pterobranchia. Together, these three studies illustrate different axes of hemichordate research, and show how integrating morphological, functional and molecular data allows us toinfer key events in the evolution of deuterostomes.

L’embranchement Hemichordata regroupe les classes Enteropneusta et Pterobranchia. Hemichordata constitue, avec l’embranchement Echinodermata, le groupe-frère des chordés. Les entéropneustes sont des organismes vermiformes solitaires qui vivent sous ou à la surface du substrat et s’alimentent généralement par déposivorie, alors que les ptérobranches sont des organismes coloniaux filtreurs habitant dans un réseau de tubes appelé coenecium. Ce mémoire présente trois études dont le point commun est l’utilisation des hémichordés actuels pour répondre à des questions concernant l’évolution des hémichordés, des chordés, et du super-embranchement qui les regroupe, Deuterostomia. Notre première étude démontre que les fentes pharyngiennes, l’organe pré-oral cilié (POCO) et le pharynx de l’entéropneuste Protoglossus graveolens sont utilisés pour l’alimentation par filtration. Le système de filtration de P. graveolens permet la capture de particules jusqu’à 1.3 um, à un débit de 4.05 mm.s-1, pour une demande énergétique de 0.009 uW. Les similarités structurales et fonctionnelles avec le système de filtration des céphalochordés suggèrent que la filtration pharyngienne est ancestrale aux deutérostomes. Lors de notre deuxième étude, nous avons exploré l’hypothèse selon laquelle le POCO des entéropneustes, une structure ciliée pré-buccale au rôle possiblement chémorécepteur, serait homologue au « wheel organ » des céphalochordés et à l’adénohypophyse des vertébrés. Pour cela, nous avons déterminé par immunohistochimie l’expression de Pit-1, un facteur de transcription spécifique à ces deux structures, chez l’entéropneuste Saccoglossus pusillus. Pit-1 est exprimé dans des cellules sensorielles du POCO, mais aussi dans des cellules épithéliales distribuées dans le proboscis, collet et tronc. Ce patron d’expression ne permet pas de confirmer ou rejeter l’homologie du POCO et de l’adénohypophyse des vertébrés. Lors de notre troisième étude, nous avons caractérisé l’ultrastructure du coenecium des ptérobranches Cephalodiscus hodgsoni, Cephalodiscus nigrescens et Cephalodiscus densus par microscopie électronique à transmisison et à balayage. Cephalodiscus est le groupe frère de Graptolithina, un groupe qui inclut les graptolithes éteints ainsi que les ptérobranches du genre Rhabdopleura. Nous avons décrit les types de fibrilles de collagène présents, leur taille et leur organisation, ainsi que l’organisation globale du coenecium. Nous avons ainsi démontré la présence chez Cephalodiscus d’une organisation similaire au paracortex, pseudocortex et eucortex des graptolithes. La présence chez Cephalodiscus de ce type d’organisation suggère que le cortex est ancestral à la classe Pterobranchia. Ces trois études illustrent plusieurs axes importants de la recherche sur les hémichordés, qui en intégrant des données morphologiques, fonctionnelles et moléculaires permet de reconstruire certains évènements clés de l’évolution des deutérostomes. / The phylum Hemichordata comprises the classes Enteropneusta and Pterobranchia. Together with echinoderms, hemichordates are the sister-group to chordates. Enteropneusts are worm-shaped solitary deposit feeders. Pterobranchs are colonial filter feeders that live in a secreted collagenous domicile called a coenecium. In this thesis, three studies are presented. These studies are based on observations of extant hemichordates, and adress a variety of issues relating to the evolution of hemichordates, chordates, and the super-phylum to which they belong: Deuterostomia. Our first study demonstrates that the gill slits, pre-oral ciliary organ (POCO), and lining of the pharynx of the enteropneust Protoglossus graveolens are used in filter feeding. The filter-feeding system of P. graveolens enables particle capture down to 1.3 um, at a rate up to 4.05 mm.s-1, with a power consumption of 0.009 uW. Structural and functional similarities with the cephalochordate filter-feeding system suggest that pharyngeal filter-feeding is ancestral to the deuterostomes. In our second study, we address the hypothesis that the enteropneust POCO, a putative chemosensory structure located anterior to the mouth, is homologous to the cephalochordate wheel organ and vertebrate adenohypophysis. We characterized the expression pattern of the adenohypophysis-specific transcription factor Pit-1 in the adult enteropneust Saccoglossus pusillus with immunohistochemistry. Pit-1 is expressed in sensory cells of the POCO and in scattered epithelial cells of the proboscis, collar and trunk. This expression pattern does not allow to confirm or reject the homology of the POCO with the vertebrate adenohypophysis. In our third study, we characterized the ultrastructure of the coenecium of the pterobranchs Cephalodiscus hodgsoni, Cephalodiscus nigrescens and Cephalodiscus densus using transmission and scanning electron microscopy. Cephalodiscus is the sister-group to the Graptolithina, which includes the extinct graptolites and the extant pterobranch genus Rhabdopleura. We described the fibril types, size and organization, as well as the general organization of the coenecium. We demonstrated that the coenecium of Cephalodiscus shows similarities with the graptolite eucortex, paracortex and pseudocortex. The cortical-like organization of the coenecium of Cephalodiscus suggests that the cortex is ancestral to the Pterobranchia. Together, these three studies illustrate different axes of hemichordate research, and show how integrating morphological, functional and molecular data allows us toinfer key events in the evolution of deuterostomes.

Indiana University-Purdue University Indianapolis (IUPUI) / The LHX3 transcription factor is essential for pituitary gland and nervous system development in mammals. In humans, mutations in the LHX3 gene underlie combined pituitary hormone deficiency (CPHD) disease featuring deficits in anterior pituitary hormones and defects in the nervous system. The mechanisms that control temporal and spatial expression of the LHX3 gene are poorly understood. The proximal promoters of the human LHX3 gene are insufficient to guide expression in vivo and downstream elements including a conserved 7.9 kilobase (kb) enhancer region appear to play a role in tissue-specific expression in the pituitary and nervous system. In this study, I characterized the activity of this downstream enhancer region in regulating gene expression at the cellular level during development. Human LHX3 enhancer-driven Cre reporter transgenic mice were generated to facilitate studies of enhancer actions. The downstream LHX3 enhancer primarily guides gene transcription in αGSU-expressing cells secreting the TSHβ, LHβ or FSHβ hormones and expressing the GATA2 and SF1 transcription factors. In the developing nervous system, the enhancer serves as a targeting module for expression specifically in V2a interneurons. These results demonstrate that the downstream LHX3 enhancer is important in specific endocrine and neural cell types but also indicate that additional regulatory elements are likely involved in LHX3 gene expression in other cell types. Further, these studies demonstrate significant gonadotrope cell heterogeneity during pituitary development, providing insights into the cellular physiology of this key reproductive regulatory cell. The human LHX3 enhancer-driven Cre reporter transgenic mice provide a valuable tool for further developmental studies of cell determination and differentiation in the pituitary and nervous system. Furthermore understanding the regulation of human LHX3 gene will help develop tools to better diagnose and treat pituitary CPHD disease.

Indiana University-Purdue University Indianapolis (IUPUI) / LIM homeodomain 3 (LHX3) is an important regulator of pituitary and nervous system development. To date, twelve LHX3 gene mutations have been identified in patients with combined pituitary hormone deficiency disease (CPHD). Understanding the molecular mechanisms governing LHX3/Lhx3 gene regulation will provide critical insights into organ development pathways and associated diseases. DNA methylation has been implicated in gene regulation in multiple physiological systems. This dissertation examines the role of DNA methylation in regulating the murine Lhx3 gene. To determine if demethylation of the Lhx3 gene promoter would induce its expression, murine pre-somatotrope pituitary cells that do not normally express Lhx3 (Pit-1/0 cells) were treated with the demethylating reagent, 5-Aza-2’-deoxycytidine. This treatment lead to activation of the Lhx3 gene and thus suggested that methylation contributes to Lhx3 gene regulation. Proteins that modify chromatin, such as histone deacetylases (HDACs) have also been shown to affect DNA methylation patterns and subsequent gene activation. Pit-1/0 pituitary cells treated with a combination of the demethylating reagent and the HDAC inhibitor, Trichostatin A led to activation of the Lhx3 gene, suggesting crosstalk between DNA methylation and histone modification processes. To assess DNA methylation levels, treated and untreated Pit-1/0 genomic DNA were subjected to bisulfite conversion and sequencing. Treated Pit-1/0 cells had decreased methylation compared to untreated cells. Chromatin immunoprecipitation assays demonstrated interactions between the methyl-binding protein, MeCP2 and the Lhx3 promoter regions in the Pit-1/0 cell line. Overall, the study demonstrates that DNA methylation patterns of the Lhx3 gene are associated with its expression status.