Orientador: Sergio Ricardo Batlouni / Banca: Renata Guimarães Moreira / Banca: Irene Bastos Franceschini Vicentini / Resumo: O desenvolvimento de técnicas para indução à reprodução de peixes migradores (espécies reofílicas) de água doce possibilitou uma produção consistente de larvas destas espécies, embora, seja comum deparar-se com relatos de produtores sobre fêmeas que não ovulam adequadamente ou simplesmente não ovulam após os procedimentos de desova induzida. Algumas evidências sugerem que a origem deste problema pode estar ligada ao manejo inapropriado das matrizes. Infelizmente pouco se sabe sobre o manejo correto de reprodutores de peixes reofílicos em cativeiro. Deste modo, a proposta deste estudo foi avaliar comparativamente o processo de desenvolvimento ovocitário de reprodutores de Prochilodus lineatus mantidos em dois sistemas de criação: viveiros escavados (VE) e tanques rede (TR), seguindo as densidades de 1 peixe.m2 e 17 peixes.m3 respectivamente. A escolha do TR se deu por ser um sistema que simula experimentalmente muitas das condições inapropriadas de manejo empregadas em diversas pisciculturas, tais como: elevadas densidades de estocagem e água de má qualidade. A cada dois meses foram feitas amostragens de 30 animais de cada tratamento, de onde 5 fêmeas foram escolhidas ao acaso e sacrificadas para remoção das gônadas, fígado e vísceras para determinação dos índices gonado (IGS), hepato e viscero-somático, fator K, e para as análises histomorfométricas dos ovários. Amostras de sangue foram coletadas para a determinação da variação anual dos níveis de esteróides gonadais (estradiol e 17-alfa-hidroxiprogesterona)... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The development of techniques of induced breeding of South American migratory fresh water fish (rheophilic species) has enabled a consistent production of fingerlings of these species, however nowadays it is very common to come across reports of fingerling producers about females that do not ovulate properly or just do not ovulate. In this context, some evidences suggest that the origin of such problem may be an inappropriate broodstock management. Unfortunately few is known about the proper management of migratory breeders in captivity, thus the main purpose of this study was to evaluate, comparatively, the process of oocyte production in Prochilodus lineatus breeders kept into two distinct production systems: earthen ponds (EP) and cages (C), with the following stocking densities 1fish.m2 and 17fish.m3 respectively. The reasons for the choice of C was because this system shares a number of inappropriate characteristics employed in diverse fish farms, such as high stocking densities and poor water quality. Every two months thirty specimens of one of the EP and C unities were randomly collected for biometric analysis. After that, five specimens of each treatment were randomly chosen to be sacrificed for removal of gonads, liver and viscera for determination of gonadosomatic index (GSI), hepato-and viscero-somatic indexes, K factor, and also for the histomorphometric analysis of the ovaries. Blood samples were also collected for determining the annual variation in plasma gonadal steroids levels (stradiol and 17 alpha-hydroxyprogesterone).The results showed that the process of ovarian maturation is completely compromised in females kept in C, with significant 30 reduction in the volume of vitellogenic oocytes, estradiol, hydroxyprogesterone, and consequently in the GSI values. Besides the reproductive period is shortened and starts late in relation to that of females of VE... (Complete abstract click electronic access below) / Mestre

Made available in DSpace on 2014-06-11T19:22:23Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-02-21Bitstream added on 2014-06-13T19:27:40Z : No. of bitstreams: 1 hainfellner_p_me_jabo.pdf: 1559702 bytes, checksum: 8fd03c9d0af341bc6a7cf1927ef7d4a9 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O desenvolvimento de técnicas para indução à reprodução de peixes migradores (espécies reofílicas) de água doce possibilitou uma produção consistente de larvas destas espécies, embora, seja comum deparar-se com relatos de produtores sobre fêmeas que não ovulam adequadamente ou simplesmente não ovulam após os procedimentos de desova induzida. Algumas evidências sugerem que a origem deste problema pode estar ligada ao manejo inapropriado das matrizes. Infelizmente pouco se sabe sobre o manejo correto de reprodutores de peixes reofílicos em cativeiro. Deste modo, a proposta deste estudo foi avaliar comparativamente o processo de desenvolvimento ovocitário de reprodutores de Prochilodus lineatus mantidos em dois sistemas de criação: viveiros escavados (VE) e tanques rede (TR), seguindo as densidades de 1 peixe.m2 e 17 peixes.m3 respectivamente. A escolha do TR se deu por ser um sistema que simula experimentalmente muitas das condições inapropriadas de manejo empregadas em diversas pisciculturas, tais como: elevadas densidades de estocagem e água de má qualidade. A cada dois meses foram feitas amostragens de 30 animais de cada tratamento, de onde 5 fêmeas foram escolhidas ao acaso e sacrificadas para remoção das gônadas, fígado e vísceras para determinação dos índices gonado (IGS), hepato e viscero-somático, fator K, e para as análises histomorfométricas dos ovários. Amostras de sangue foram coletadas para a determinação da variação anual dos níveis de esteróides gonadais (estradiol e 17-alfa-hidroxiprogesterona)... / The development of techniques of induced breeding of South American migratory fresh water fish (rheophilic species) has enabled a consistent production of fingerlings of these species, however nowadays it is very common to come across reports of fingerling producers about females that do not ovulate properly or just do not ovulate. In this context, some evidences suggest that the origin of such problem may be an inappropriate broodstock management. Unfortunately few is known about the proper management of migratory breeders in captivity, thus the main purpose of this study was to evaluate, comparatively, the process of oocyte production in Prochilodus lineatus breeders kept into two distinct production systems: earthen ponds (EP) and cages (C), with the following stocking densities 1fish.m2 and 17fish.m3 respectively. The reasons for the choice of C was because this system shares a number of inappropriate characteristics employed in diverse fish farms, such as high stocking densities and poor water quality. Every two months thirty specimens of one of the EP and C unities were randomly collected for biometric analysis. After that, five specimens of each treatment were randomly chosen to be sacrificed for removal of gonads, liver and viscera for determination of gonadosomatic index (GSI), hepato-and viscero-somatic indexes, K factor, and also for the histomorphometric analysis of the ovaries. Blood samples were also collected for determining the annual variation in plasma gonadal steroids levels (stradiol and 17 alpha-hydroxyprogesterone).The results showed that the process of ovarian maturation is completely compromised in females kept in C, with significant 30 reduction in the volume of vitellogenic oocytes, estradiol, hydroxyprogesterone, and consequently in the GSI values. Besides the reproductive period is shortened and starts late in relation to that of females of VE... (Complete abstract click electronic access below)

Submitted by Automa??o e Estat?stica (sst@bczm.ufrn.br) on 2017-04-04T19:56:26Z No. of bitstreams: 1 MariliaAnielleDaSilvaFabricio_DISSERT.pdf: 3688508 bytes, checksum: 5f01a70876dd9ae37f19f323e534b5a3 (MD5) / Approved for entry into archive by Arlan Eloi Leite Silva (eloihistoriador@yahoo.com.br) on 2017-04-12T23:55:10Z (GMT) No. of bitstreams: 1 MariliaAnielleDaSilvaFabricio_DISSERT.pdf: 3688508 bytes, checksum: 5f01a70876dd9ae37f19f323e534b5a3 (MD5) / Made available in DSpace on 2017-04-12T23:55:10Z (GMT). No. of bitstreams: 1 MariliaAnielleDaSilvaFabricio_DISSERT.pdf: 3688508 bytes, checksum: 5f01a70876dd9ae37f19f323e534b5a3 (MD5) Previous issue date: 2016-12-19 / Existem no mundo apenas sete esp?cies de tartarugas marinhas. Dentre essas, h? registros de ocorr?ncia de cinco esp?cies no litoral do Brasil: Dermochelys coriacea, Chelonia mydas, Caretta caretta, Eretmochelys imbricata e Lepidochelys olivacea. De acordo com a Lista Vermelha da Uni?o Internacional para a Conserva??o da Natureza, todas as esp?cies de tartarugas marinhas existentes no Brasil se encontram amea?adas de extin??o. A maioria dos trabalhos relacionados a propor??o sexual desses r?pteis indicam um desequil?brio populacional, havendo um n?mero excessivo de f?meas. Assim, pesquisas sobre a biologia reprodutiva desses animais s?o extremamente necess?rias e importantes para conserva??o de gera??es futuras dessas esp?cies. Este trabalho tem como objetivo estudar diferentes aspectos morfohistol?gicos das g?nadas de machos e f?meas de tartarugas marinhas na Bacia Potiguar. Foram avaliados animais vivos e mortos, provenientes de encalhes entre as praias de Icapu?/CE e Cai?ara do Norte/RN, totalizando aproximadamente 300 km. Os animais encalhados mortos ou que vieram a ?bito na Base de Reabilita??o do Projeto Cet?ceos da Costa Branca-UERN foram necropsiados por uma equipe veterin?ria. As g?nadas foram coletadas e fixadas em formol a 10%, e posteriormente submetidas a prepara??o histol?gica, atrav?s das t?cnicas de Hematoxilina-Eosina, sendo analisadas em microsc?pio ?ptico. No per?odo de janeiro de 2011 a dezembro de 2015 foram registrados 3.960 encalhes de tartarugas marinhas na ?rea estudada. A amostra apresentou propor??o sexual de 3:1, com predom?nio de f?meas. Oitenta e cinco por cento dos animais registrados foram classificados como pertencentes a fase de desenvolvimento "juvenil". Foram analisadas microscopicamente 86 amostras de tecido gonadal, sendo 53 f?meas e 25 machos da esp?cie Chelonia mydas, 3 f?meas e 3 machos de Eretmochelys imbricata e 2 f?meas de Caretta caretta. Foi poss?vel estabelecer tr?s est?gios de matura??o gonadal: pr? pubescente, pubescente e maduro. O predom?nio das g?nadas analisadas foi de indiv?duos pr? pubescentes, com f?meas apresentando ov?citos homog?neos e machos com t?bulos semin?feros de pequeno di?metro com aus?ncia de espermatozoides. Os esp?cimes pr? pubescentes f?meas apresentaram comprimento curvil?neo da carapa?a (CCC) m?dio de 37,07 cm e os machos 38,68 cm; Pubescentes f?meas 77,04 cm e machos 89,92 cm, F?meas maduras com 101,35 cm e um indiv?duo macho maduro com 105 cm. Pesquisas sobre aspectos histol?gicos relacionados ao desenvolvimento ovariano e testicular de tartarugas marinhas s?o escassas, mas, os resultados obtidos est?o em concord?ncia com o que j? foi descrito. Considera-se a necessidade de continuidade de trabalhos como esse, associando os dados morfol?gicos e biom?tricos ?s an?lises histol?gicas para o melhor entendimento sobre a matura??o sexual das tartarugas marinhas e implementa??o de propostas de conserva??o para essas esp?cies. / Among the seven species of sea turtles, five occurs on the Brazilian coast: Dermochelys coriacea, Chelonia mydas, Caretta caretta, Eretmochelys imbricata e Lepidochelys olivacea. According to the Red List of the IUCN, all species of sea turtles in Brazil are endangered. The majority of works related that the sexual proportion of these animals indicate a population imbalance, having a number excessive of females. Thus, studies of reproductive biology of sea turtles are extremely necessary and important for the conservation of these species for future generations. This work aims to study different morphohistological aspects of male and female's gonads of sea turtle in Potiguar Basin. For this, were evaluated dead and living animals, from strandings between the beaches of Icapu?/CE and Cai?ara do Norte/RN, totaling approximately 300 km. The dead animals were necropsied. During the procedure, the gonads were collected and fixed in formalin 10%, then submitted to histological process, through the techniques of hematoxylin-eosin, according Tolosa (2005), being analyzed in an optical microscope. Between January 2011 and December 2015 were recorded 3.960 stranding of sea turtles in the study area. The sample showed sex ratio of 3:1, with a predominance of females. . Eighty-five percent of the animals were classified juvenile. 86 samples of gonadal tissue wereanalized microscopically, being 58 females and 28 males, of Chelonia mydas, Eretmochelys imbricata e Caretta caretta. It was possible to establish three stages of maturation: pre-pubertal, pubertal and mature. The predominance was of pre-pubertal individuals, females presenting homogeneous oocytes and males with seminiferous tubules with a small diameter with absence of sperm. The pre-pubertal females specimens exhibit an average of 37,07 cm (CCC) and the males 38,68 cm (CCC); Pubertal females with an average of 77,04 cm and males 89,92 cm; mature females with 101,9 cm (CCC) and a single mature male with 105 cm. Researches on histological aspects related to ovarian and testicular development of sea turtles are scarce, but, the obtained results are in agreement with what has already been described. Researches like this, associating morphological and biometric data to histological analyzes, are necessary for the best knowledge about sexual maturity of these animals and implementation of conservation proposals for these species.

Orientador: Irani Quagio-Grassiotto / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-14T18:28:03Z (GMT). No. of bitstreams: 1 Mazzoni_TalitaSarah_M.pdf: 22131608 bytes, checksum: df7814a1f55f5088400f2b70e4520216 (MD5) Previous issue date: 2009 / Resumo: Numa nova visão da morfogênese gonadal, sua descrição em Cyprinus carpio, mostra como a proliferação e diferenciação de células germinativas e somáticas a partir do primórdio gonadal levam à formação das diferentes estruturas ovarianas e testiculares e à constituição do epitélio germinativo que margeia as lamelas ovígeras e os túbulos testiculares. Em C. carpio, o primórdio gonadal é formado por células germinativas primordiais (CGPs) rodeadas por células somáticas. Após sucessivas divisões mitóticas das células somáticas, o tecido gonadal aumenta em comprimento e espessura. As CGPs isoladas entre células somáticas se dividem mitoticamente formando grupos de células germinativas, que se organizam em cordões contínuos, os quais são invadidos por células somáticas, levando à uma reorganização estrutural e diferenciação gonadal. Nas gônadas femininas, as oogônias são envolvidas por expansões citoplasmáticas das agora células pré-foliculares, formando cistos, delimitados por uma membrana basal em formação. Cada oogônia divide-se por mitose, formando novas oogônias ou entra em meiose originando os oócitos. Com a entrada e permanência em diplóteno, os oócitos, ainda no interior dos cistos, iniciam seu desenvolvimento que completar-se-á no interior dos folículos ovarianos. As células pré-foliculares progressivamente interpenetram nos cistos e envolvem cada oócito individualizando-os. Estas gradativamente sintetizam a membrana basal envolvendo progressivamente o folículo em formação. As células foliculares assentadas em parte na membrana basal do próprio epitélio germinativo mantêm uma região de contato entre o folículo ovariano e o epitélio germinativo, que compartilham uma mesma membrana basal. Ao término da foliculogênese, o oócito inicia seu crescimento primário. No tecido gonadal, células mesenquimais se interconectam e se diferenciam, dando origem ao estroma ovariano, isolado do compartimento germinativo pela membrana basal. Células indiferenciadas do estroma emitem prolongamentos que contatam os folículos em formação, constituindo a teca. Células somáticas periféricas por migração e invaginação no tecido gonadal, formam as lamelas ovígeras. Lâminas teciduais de células somáticas formam-se em ambos os lados do ovário, projetando-se até se contatarem, formando o lúmen ovariano. Nas gônadas masculinas, as células somáticas, pré- Sertoli, invadem os cordões contínuos de CGPs. Estas, agora espermatogônias, são envolvidas por expansões citoplasmáticas das células de Sertoli, formando cistos. Estes formam conjuntos celulares que se distribuem ao longo da gônada, cada qual circundado por células somáticas. Ao redor de cada conjunto celular, inicia-se a formação da membrana basal, porém de forma incompleta. Gradativamente, os cistos de espermatogônias que constituem um mesmo conjunto, afastam-se uns dos outros, criando um espaço central. Células somáticas de conjuntos celulares adjacentes afastam-se, permitindo fusão entre os dois conjuntos celulares e aumento do espaço central formando um único compartimento luminal, delimitado por cistos de espermatogônias, estes, apoiados na membrana basal. Formam-se os túbulos testiculares e o epitélio germinativo masculino é estabelecido. O compartimento germinativo encontra-se agora separado pela membrana basal dos demais componentes celulares, que irão se diferenciar no compartimento intersticial. Inicia-se a espermatogênese no interior de cada cisto, de forma sincrônica. Após espermiogênese, os espermatozóides são liberados no lúmen, e os túbulos testiculares se anastomosam, culminando com a formação do ducto espermático na porção dorsal do testículo / Abstract: The description of gonadal morphogenesis in Cyprinus carpio provides a new vision of proliferation and differentiation of germ and somatic cells from the gonadal primordium leading to the formation of different ovarian and testicular structures, and the constitution of germinal epithelium which borders the ovigerous lamellae and the seminiferous tubules. In C. carpio the gonadal primordium is an elongated structure with individual PGCs scattered among somatic cells. The PGCs divide and organize into continuous cords that are delimited by the somatic cells. Then, in female gonad, somatic cells move into the cords, wrap around and individualize the PGCs that subsequently differentiate in oogonia. Each oogonium is wrapped by the now prefollicle cells giving rise to a cyst. Prefollicle cells rest upon a forming basement membrane. Inside the cysts oogonium proliferates by mitosis, originating new oogonia. Or, they enter into meiosis, becoming oocytes. Oocytes advance to diplotene where meiosis is arrested. Still inside the cysts oocytes enter primary growth, they are subsequently surrounded by prefollicle cells, and they become ovarian follicles. The differentiating gonad maintains a compact structure, continues elongating and becomes larger. Invaginations appear in the ventral region of the ovary; these form the ovigerous lamellae. Mesenchymal cells scattered inside the lamellae move away from one another giving rise to the extra-vascular space in the developing stroma where the cellular processes of these cells connect to one another and form a cellular net. Meanwhile, epithelial cells coming from the gonad periphery, and present in the ventral invaginations, associate with oogonia forming germinal epithelium. These also interact with the follicles that become connected to the epithelium sharing some extension of the basement membrane. Mesenchymal cells surround the ovarian follicles, becoming theca and, include the follicle, forms the follicle complex. On either side of the developing ovary, a coelomic epithelial cell proliferation forms a laminar tissue that grows ventrally, then extending beneath the developing ovary and fusing to form the central lumen of the carp cystovarian ovary. In male gonad, somatic cells move into the cords, wrap around and individualize the PGCs that subsequently differentiate in spermagonia. Each spermatogonium wrapped by the now pre-Sertoli cells giving rise to a cyst. The cysts join one another forming clusters. A basement membrane is synthesized around each cluster. Pre-Sertoli cells rest upon the forming basement membrane. In the center of the clusters a space is created when pre-Sertoli cells move away from one another. Then, nearby clusters fuse to one another that become connected by the same luminal space. The progressive fusion of the clusters gives rise to the seminiferous tubules that are bordered by the new formed germinal epithelium constituted by the cysts that rest upon the basement membrane. Mesenchymal cells surround the seminiferous tubules give rise to the cellular components of the interstitial compartment. Inside the cysts spermatogenesis starts. As the final spermatic cells are released into the luminal compartment, the anostomosis of the testicular tubules occurs forming the spermatic duct on the dorsal region of the testis / Mestrado / Biologia Celular / Mestre em Biologia Celular e Estrutural

Orientador: Irani Quagio-Grassiotto / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-23T21:20:24Z (GMT). No. of bitstreams: 1 Mazzoni_TalitaSarah_D.pdf: 26204829 bytes, checksum: b3ecbe44f1a0367d6194fb7c297136c2 (MD5) Previous issue date: 2013 / Resumo: Considerando o status atual de conhecimento da morfogênese e diferenciação gonadal nos Teleostei frente à restrição de informações, especialmente em aspectos tangentes ao estabelecimento do epitélio germinativo e sua relação com a formação da estrutura gonadal tomou-se aqui como modelos biológicos Tanichthys albonubes, Gymnocorymbus ternetzi, Corydoras schwartzi, Amatitlania nigrofasciata e Poecilia reticulata, representando as séries Otophysi, Percomorpha e Atherinomorpha, visando estabelecer uma análise comparativa da diferenciação gonadal entre as espécies, considerando suas posições na escala filogenética. A proliferação e diferenciação de células germinativas e somáticas a partir do primórdio gonadal em T. albonubes, G. ternetzi, C. schwartzi, A. nigrofasciata e P. reticulata levam à formação das diferentes estruturas ovarianas e testiculares e à constituição do epitélio germinativo que margeia as lamelas ovígeras e os túbulos/lóbulos testiculares. Nesses animais, o primórdio gonadal é formado por células germinativas primordiais (CGPs) rodeadas por células somáticas. Após sucessivas divisões mitóticas das células somáticas, o tecido gonadal aumenta, originando uma gônada indiferenciada, com as mesmas características morfológicas entre machos e fêmeas de T. albonubes e C. schwartzi. Em A. nigrofasciata e P. reticulata grupos de células germinativas e somáticas organizam-se de maneira distintas em machos e fêmeas. Nas gônadas femininas, as CGPs estão distribuídas por todo o tecido gonadal, enquanto que nas masculinas, as CGPs localizam-se na periferia. Na região dorsal das gônadas masculinas de A. nigrofasciata e P. reticulata, células somáticas organizam-se formando o ducto testicular, enquanto que em T. albonubes, G. ternetzi e C. schwartzi esse é a última estrutura a se formar. Nas gônadas femininas das cinco espécies, os processos envolvidos na diferenciação gonadal, como a foliculogênese, o estabelecimento do epitélio germinativo e a formação da cavidade ovariana são bastante semelhantes. Nas gônadas masculinas, as células somáticas, pré-Sertoli, associam-se às CGPs, formando cistos de espermatogônias; estas proliferam formando conjuntos celulares. Em T. albonubes e A. nigrofasciata os cistos de espermatogônias que constituem um mesmo conjunto, afastam-se uns dos outros, criando um espaço central, que se torna maior, originando um compartimento luminal, delimitado por cistos de espermatogônias. Formam-se os túbulos e lóbulos testiculares e o epitélio 2 germinativo masculino é estabelecido. Em G. ternetzi o tecido gonadal masculino é estabelecido e organizado em túbulos após a diferenciação gonadal feminina, sobre um ovário previamente desenvolvido, constituindo uma diferenciação gonocorística do tipo indireta. C. schwartzi apresenta um tecido gonadal masculino compacto, que sofre degenerações no interior de estruturas acinares compostas por cistos, para constituir os túbulos testiculares. Em P. reticulata, os conjuntos de espermatogônias conectam-se ao ducto em formação, originando os primeiros lóbulos testiculares. Ao final da diferenciação gonadal, os testículos de T. albonubes, G. ternetzi e C. schwartzi apresentam espermatogônias distribuídas aleatoriamente no túbulo. Estes anastomosam-se, caracterizando o testículo como tubular anastomosado. Em P. reticulata, as espermatogônias ficam restritas na região periférica dos lóbulos, os quais não apresentam lúmen testicular, características estas de um testículo lobular restrito. A mesma distribuição aleatória de cistos nos túbulos dos Otophysi ocorre nos lóbulos testiculares de A. nigrofasciata, caracterizando um testículo com organização lobular do tipo irrestrita / Abstract: Considering the lack of information about the gonadal morphogenesis and differentiation in Teleostei, especially in tangent aspects concerning the establishment of the germinal epithelium and its relation with the formation of the gonadal structure, in the present study, it was taken Tanichthys albonubes, Gymnocorymbus ternetzi, Corydoras schwartzi, Amatitlania nigrofasciata and Poecilia reticulata as biological models, representing the series Otophysi, Percomorpha and Atherinomorpha, to establish a comparative analysis of the gonadal differentiation among the species, taking into account their position in the phylogenetic scale. The proliferation and differentiation of germ and somatic cells from the gonadal primordium of the T. albonubes, G. ternetzi, C. schwartzi, A. nigrofasciata and P. reticulata lead to the formation of different testicular and ovarian structures and to the formation of the germinal epithelium of the ovigerous lamellae and the testicular lobules/tubules. In these animals, the gonadal primordium is formed by primordial germ cells (PGCs) surrounded by somatic cell. After successive mitotic divisions of the somatic cells, the gonadal tissue increases, resulting in a undifferentiated gonad, with the same morphological characteristics between males and females of T. albonubes and C. schwartzi. In A. nigrofasciata and P. reticulata groups of germ and somatic cells organize distinctly in males and females. In the female gonads, the PGCs are distributed throughout the gonadal tissue. In the male, the PGCs are located in the periphery. In the dorsal region of the male gonads of A. nigrofasciata and P. reticulata somatic cells are organized forming the testicular duct, whereas at T. albonubes, G. ternetzi and C. schwartzi this is the last structure formed. In female gonads of the five species, the processes involved in gonadal differentiation, such as folliculogenesis, the establishment of the germinal epithelium and the formation of the ovarian cavity are quite similar. In male gonads, the somatic cells, pre-Sertoli, associate to the PGCs forming cysts of spermatogonia; these proliferate and form clusters. In T. albonubes and A. Nigrofasciata, the cysts of the spermatogonia, which form a single cluster, move away from each other, creating a central space, which becomes greater, originating a luminal compartment, delimited by cysts of spermatogonia. Thus, testicular tubules and lobules are formed and male germinal epithelium is established. In G. Ternetzi, the male gonadal tissue is established and organized in tubules after the female gonadal 4 differentiation on an ovary previously developed, constituting an indirect gonochoristic differentiation. C. schwartzi presents a compact male gonadal tissue, which undergoes degeneration within acinar structures composed of cysts, forming the testicular tubules. In P. reticulata, the clusters of spermatogonia connect to the duct in formation, originating the first testicular lobules. At the end of gonadal differentiation, the testis from T. albonubes, G. ternetzi and C. schwartzi present spermatogonia, distributed along the tubule. These anastomose, characterizing the testis as anastomosing tubular. In P. reticulata, the spermatogonia are restricted at the periphery of the lobules, which do not have lumen, features of a restricted lobular testis. The same random distribution of cysts in the tubules of the Otophysi occurs in testicular lobules of A. nigrofasciata, featuring a testis with unrestricted lobular organization / Doutorado / Biologia Celular / Doutora em Biologia Celular e Estrutural

Abstract The stem cell is a primitive cell that is capable of dividing to reproduce itself and can give rise to a selection of differentiated progeny. Stem cells are thought to be involved in or even main factors in many diseases. In postnatal humans, mesenchymal tissues have the capacity to regenerate from stem cells called mesenchymal stem cells (MSC). It is currently thought that these cells will become the basis of therapy for many diseases. In the present study, a novel in vitro method was developed to examine human bone marrow derived MSC differentiation into osteoblast lineage, and to study the role of MSC in a variety of mesenchymal diseases. The ability of MSCs to differentiate into osteoblasts was investigated during aging. In addition, the interindividual variability in the osteogenesis of MSCs and in the osteoblastic response of MSC to estrogen and testosterone was studied. Furthermore, an ex vivo model using a human aortic valve microenvironment was developed to explore whether the extracellular matrix influences the osteoblastic differentiation of the MSC. Finally, the role of MSC in neurofibromatosis type 1 (NF1) related congenital pseudarthrosis of the tibia (CPT) was studied. It was found that after menopause the osteogenic potential of MSCs does not decrease. It was also found that estrogen receptor (ER) alpha genotype confers interindividual variability of response to estrogen and testosterone in MSC derived osteoblasts. In addition, it was found that the non-calcified valves with living valve cells inhibited osteogenesis of co-cultured MSCs, whereas the calcified and devitalised valves promoted differentiation towards an osteoblastic lineage. Finally, MSCs from NF1-related pseudarthrosis showed altered NF1 gene expression, poor osteoblastic differentiation and bone formation. In conclusion, MSC can be easily isolated from the bone marrow and MSC has the capacity to regenerate tissue even at later stages of life. These results could help explain the contradictory effects of 17β-estradiol (E2) on osteoblasts in vitro and might also provide new insights into understanding the differences in responses to hormone replacement therapy. It seems that adult stem cells from bone marrow undergo milieu-dependent differentiation to express phenotypes that are similar to cells in the local microenvironment. Finally, the NF1 gene was shown to have a role in bone development and remodelling.

Les anomalies du développement sexuel recouvrent un spectre phénotypique large. Les hommes XX présentent dans la majorité des cas un développement testiculaire normal, lié à la présence SRY sur un des chromosomes X. Dans 10% des cas, aucune cause n’est retrouvée. Chez les femmes, l’origine de l’insuffisance ovarienne prématurée (IOP) n’est identifiée que dans 20% des cas. L’objectif de cette thèse a été d’identifier de nouveaux mécanismes moléculaires impliqués dans le développement gonadique, testiculaire et ovarien, ainsi que dans son fonctionnement. L’étude d’une cohorte de patients DSD 46,XX SRY négatifs a mis en évidence une duplication de la région RevSex dans un désert génique en amont de SOX9 chez quatre patients. Ceci a permis de redéfinir la région minimale impliquée dans l'activation de l'expression de SOX9 à une taille maximale de 41.9 kb et de proposer un mécanisme permettant cette expression chez des hommes XX. Le séquençage d’exome chez dix patients de la cohorte n’a pas mis en évidence de mutations dans des gènes d’intérêts. L’ensemble de ces résultats pose la question du rôle des régions régulatrices dans la survenue des DSD.L’analyse d’une cohorte de patientes ayant une IOP a permis d'identifier des délétions incluant le gène CPEB1. Des études précédentes chez la souris ont montré son implication dans le développement folliculaire. Le séquençage du gène CPEB1 dans la cohorte n'a pas mis en évidence de mutation pathogène. Ce travail a permis de montrer que la délétion impliquant le gène CPEB1est une cause rare mais récurrente d'IOP et concerne environ 1% des patientes. Une microdélétion contenant le gène CASP3 un gène de la voie des caspases impliquée dans la régulation du pool folliculaire a également été identifiée chez une patiente. L'ensemble de ces résultats montre l'intérêt de l'étude génétique des patients présentant une anomalie du développement de la gonade ou de son fonctionnement par des techniques d'étude globale du génome. / Disorders of Sex Development (DSD) can be identified in new-born and during infancy but also in adults because of infertility. Most 46,XX testicular DSD have a normal testicular development due to the presence of the SRY gene at the tip of one of their X chromosome. However, the genetic causes of 46,XX-SRY negative testicular DSD remain poorly defined. In women, disorders of gonadal development can be responsible for primary ovarian insufficiency (POI) and genetic causes are identify in only 20% of cases. The aim of this thesis was to identify molecular mechanisms involved in gonadal development and in its functioning. The cohort study of 46,XX testicular DSD identified four patients with a duplication in the previously reported RevSex region located about 550 kb upstream of SOX9. One duplication allowed us to refine the minimal region associated with 46,XX-SRY negative DSD to a 40.7–41.9 kb element. Exome sequencing of 10 patients from the cohort did not show any mutation in genes implicated in DSD or in new candidate genes. These results raise questions about the role of the regulatory sequences in the onset of DSD.The cohort study of POI patients identified three patients carrying a microdeletion including CPEB1 a good candidate gene for POI as study in mice showed the implication of CPEB1 in follicular development. Sequencing CPEB1 gene did not identified any mutation. Therefore, heterozygous deletion of CPEB1 gene leading to haploinsufficiency could be responsible for POI in humans. This microdeletion is rare but recurrent and was identified in about 1% of patients with POI. Another microdeletion containing CASP3 gene that belongs to the caspase family, which is implicated in the regulation of the follicular pool, was identified in a patient. Further studies are needed to confirm the role of CASP3 in POI. These results demonstrate the importance of genetic study of patients presenting with DSD or POI using whole genome techniques.

Chez les mammifères, la détermination du sexe est un processus moléculaire complexe impliquant une balance de gènes finement régulée entre la voie mâle et femelle. La formation de testicules est initiée par SRY en synergie avec SF1 par sur-activation de l'expression de SOX9 au delà d'un seuil critique, ce qui entraîne l'activation du programme de la voie mâle et la répression de la développement ovarien. Des erreurs dans ce processus peuvent entraîner des pathologies de dysgénésie gonadique (DSD). Bien que de nombreux gènes impliqués dans le développement des gonades, sont à présent identifiés, les mutations de ces gènes n'expliquent qu'une minorité de cas de DSD, et les mécanismes conduisant à un développement sexuel inapproprié restent encore largement méconnus.Nous avons identifié, pour la première fois, une mutation ponctuelle dans le gène DMRT1 humain, facteur crucial de la détermination du sexe chez différentes espèces, associée à une absence de développement testiculaire. Une série d'analyses fonctionnelles démontrent le mécanisme par lequel cette mutation pourrait être lie à un DSD et que, différemment de chez la souris, DMRT1 humain, est impliqué dans la détermination testiculaire primaire.Le séquençage d’exomes sur des patients présentant un DSD de type 46,XY ont permis d’identifier des mutations délétères chez SOX7 & SOX8, suggérant une possible redondance fonctionnelle parmi les gènes SOX dans la détermination du sexe; mais également des mutations chez GATA4 établissant un rôle pour ce gène comme cause de DSD. Nos résultats d’analyses fonctionnelles indiquent des changements dans l'activité biologique des protéines mutées, mais dans certains cas, ne révèlent pas les mécanismes impliqués dans l’apparition de DSD. Par conséquent, le développement de nouveaux modèles cellulaires in-vitro pourrait permettre d’élucider ces mécanismes. Notamment, l’utilisation de cellules souches embryonnaires de souris, nous permettra de développer un nouveau modèle cellulaire pour mieux comprendre l'effet biologique de ces mutations. / In mammals, sex determination and development is a complex process that involves genetic networks acting synergistically or antagonistically. Testis formation is initiated by SRY+SF1 up-regulation of SOX9 expression beyond a critical level, resulting in the activation of male-specific program and ovaries repression. In females, SRY absence results in the activation of ovarian development and testis repression. Errors in the process result in gonadal dysgenesis (DSD). Although, we begin to know the genes involved in gonad development, mutations in these genes explain only few DSD cases, and the mechanism leading to abnormal sexual development remain misunderstood. DMRT1 regulates sex-determination in different species. Its role in mammalian sex-determination is unclear. We identified the first point mutation in human DMRT1 associated with a lack of testis-determination. Functional analyses revealed the mechanism by which this mutation could lead to DSD. Showing that, differently to mouse, human DMRT1 is involved in primary testis-determination and that at molecular level sex-determination a very conserved process. Further studies using exome sequencing on patients with 46,XY DSD identify pathogenic mutations in 2 SOX genes: SOX7 & SOX8. Suggesting a possible functional redundancy amongst SOX genes in sex-determination. We identified mutations in GATA4 associated with 46,XY DSD, establishing a role for this gene as a DSD cause. The functional consequences of these mutations on their biological activity were assessed using classical approaches. Our results indicate changes in biological activity of the mutated proteins but in some cases don’t reveal the mechanisms involved in DSD. Therefore, the development of novel in-situ cellular models may provide a tool to identify these mechanisms. Using mouse embryonic stem cells we’re developing novel cellular models to understand the biological effect of these mutations in the appropriate environment.

Felplacering av gonadskydd på barn:En litteraturstudie om förekomst och konsekvenser Abstrakt: Bakgrund: Röntgenstrålning är en betydelsefull uppfinning inom medicinsk diagnostik,men kan ge biologiska skador vid exponering. Gonadskydd är väsentlig för unga patienterför att skydda strålkänsliga könsorgan som utsätts för röntgenstrålning. Sedan mångadecennier tillbaka har användandet av gonadskydd diskuterats på grund av frekventförekommande felanvändning vilket leder till ökad stråldos för patienter. Syfte: Syftet med litteraturstudien var att undersöka förekomsten och konsekvenser avfelplacerade gonadskydd vid röntgenundersökning bland barn. Metod: En litteraturstudie av nio kvantitativa studier. Dessa artiklar söktes via PubMed,Cinahl samt Scopus och kvalitetsgranskades. Analysen gjordes i enlighet med Fribergsanalysmetod. Resultat: Resultatet visar att tillämpning av gonadskydd utesluts i hög omfattning.Förekomsten av felplacering överstiger antalet korrekt applicerade gonadskydd ochkonsekvensen blir att patienterna utsätts för ökad stråldosen i ung ålder. Konklusion: Felplacerade gonadskydd förekommer frekvent och medför konsekvensersom kan bli skadliga för patienten. Genom att införa tydligare riktlinjer och utökakompetensen hos röntgensjuksköterskor kan optimal användning av gonadskydd uppnås.Dock är det fortfarande brist på studier som undersöker användandet av gonadskydd, merforskning skulle behövas för att få bättre förståelse kring fenomenet. Nyckelord: Barn, felplacering, Gonadskydd, Strålskydd / Misplacement of gonadal shields in children: A literature review on occurrence and consequences   Abstract:  Background: X-ray imaging is an essential invention in medical diagnostics but can cause biological damage upon exposure. Gonadal shields are necessary for patients in fertile age to protect the radiosensitive reproductive organs from X-ray radiations. The use of gonadal shields has been debated for decades due to frequent misuse, which leads to multiple consequence. Aim: The aim of this literature review was to investigate the occurrence and consequences of misplaced gonad shields in X-ray examinations among children. Method: A literature review of nine quantitative studies was conducted. These articles were searched for via PubMed, Cinahl and Scopus and were quality assessed. The analysis was conducted using Friberg analysis methos.   Results: The results show that the application of gonad shields is excluded on many occasions. The misplacing of gonadal shields occurs more frequently than correctly applied gonad shields which leads to increased radiation doses from young age as consequence. Conclusion: Misplaced gonadal shielding is a frequent occurrence that can have harmful consequences for patients. Updating the guidlines and enchanting radiographers’ competence can optimize the use of gonad shields. However, there is still a lack of studies on the subject and further research is needed for a better understanding of the phenomenon. Keywords: Children, Gonadal shielding, Misplacement, Radiation protection

Hartsfield syndrome is a rare clinical entity characterized by holoprosencephaly and ectrodactyly with the variable feature of cleft lip/palate. In addition to these symptoms patients with Hartsfield syndrome can show developmental delay of variable severity, isolated hypogonadotropic hypogonadism, central diabetes insipidus, vertebral anomalies, eye anomalies, and cardiac malformations. Pathogenic variants in FGFR1 have been described to cause phenotypically different FGFR1-related disorders such as Hartsfield syndrome, hypogonadotropic hypogonadism with or without anosmia, Jackson–Weiss syndrome, osteoglophonic dysplasia, Pfeiffer syndrome, and trigonocephaly Type 1. Here, we report three patients with Hartsfield syndrome from two unrelated families. Exome sequencing revealed two siblings harboring a novel de novo heterozygous synonymous variant c.1029G>A, p.Ala343Ala causing a cryptic splice donor site in exon 8 of FGFR1 likely due to gonadal mosaicism in one parent. The third case was a sporadic patient with a novel de novo heterozygous missense variant c.1868A>G, p.(Asp623Gly).