Analisou-se por métodos quantitativos e qualitativos o testículo de roedores histricomorfos nas fases, pré-púbere, púbere e adulto da Paca (Cuniculus paca), Cutia (Dasyprocta leporina) e Porquinho-da-índia (Cavia porcellus). Mediante as técnicas de microscopia de luz, microscopia eletrônica de varredura, microscopia eletrônica de transmissão e método estereológico. Verificou-se a influência do Letrozol um inibidor da enzima aromatase nas dosagens de 0,15mg\\kg e 1,0 mg\\kg, sobre a morfologia testicular de pacas macho. Este modelo animal apresenta importância, comercial e científica sendo excelente alternativa para a pesquisa, colaborando com o desenvolvimento de investigações vitais ao homem e aos próprios animais. Os resultados desta investigação demonstraram, que os aspectos qualitativos da morfologia testicular do Porquinho-da-índia, Cutia e de Paca foram semelhante nas fases pré-púbere, púbere e adulta. As avaliações quantitativas do Porquinho-da-índia e Cutia realizados mediante o método estereológico permitiram quantificar parâmetros, sem hipóteses de forma, tamanho, orientação ou distribuição das partículas contadas, demonstrando ser um método imparcial e confiável para ser aplicado em estudos de reprodução. Considerando o estrógeno ser essencial para a função da espermatogênese, o efeito do Letrozol no testículo da paca (Cuniculus paca), sugeriu que a inibição da enzima aromatase mediante a utilização de Letrozol na dosagem de 0,15mg \\kg, sugeriu em um aumento no número das células de Sertoli, refletindo sobre as demais células testiculares, comparando com o testículo basal (controle). O testículo tratado (Letrozol) demonstrou epitélio germinativo íntegro, sendo este dado importante para a reprodução. No animal púbere houve um aumento no número de células de- Sertoli, semelhante ao descrito anteriormente. O epitélio seminífero mostrou com alterações morfológicas, tais como proliferação celular, presença de células germinativas com características apoptóticas, e vacúolos e núcleos em picnose. Estes dados poderão ser úteis para o estudo da Síndrome da Disgenesia testicular / Analysis by quantitative and qualitative methods testicular histricomorfs rodents in phases, pre-pubertal, pubertal and adult paca (Cuniculus paca), agouti (Dasyprocta leporina) and guinea-pig (Cavia porcellus). By means of light microscopy techniques, scanning electron microscopy, transmission electron microscopy, stereological method and the influence of an aromatase inhibitor, the Letrozole in dosages of 0.15 mg\\kg and 1.0 mg\\kg on testicular male morphology paca. This animal model has importance, excellent commercial and scientific alternative to research, contributing to the development of vital research to humans and from animals themselves. The results of this research have shown that the qualitative aspects of testicular morphology guinea-pig, Agouti and paca were similar in pre-pubertal, pubertal and adult stages. Quantitative assessments of the guinea-pig and agouti performed by the stereological method allowed quantify parameters, no change of shape, size, orientation or distribution of particles counted, it is proving to be a partial and reliable method to be applied to reproduction studies. Considering the estrogen is essential for the function of spermatogenesis, the effect of Letrozole in the testis of paca (Cuniculus paca), suggested that inhibition of aromatase enzyme through the use of Letrozole in a dosage of 0.15 mg\\kg suggested in an increase in number of Sertoli cells, reflecting on the other testicular cells, comparing the baseline testis (control). The treaty testis (Letrozole) showed intact germinal epithelium, which is important data for reproduction. In pubertal animals there was an increase in the number of de Sertoli cells, similar to that described above. The seminiferous epithelium showed to morphological changes such as cell proliferation, the presence of germ cells with apoptotic features and vacuoles and nuclei picnosis. These data may be useful for the study of testicular dysgenesis syndrome

The domestic dog, Canis familiaris, has over 450 naturally occurring inherited diseases. Over half of these diseases are clinically similar to human diseases making the dog an excellent model in which to study human hereditary diseases. Alport syndrome (AS), a group of heterogeneous, hereditary renal diseases, is one example of such a human disease. The disease is transmitted in three fashions: X-linked, autosomal recessive, and autosomal dominant. AS is caused by mutations in COL4α3, COL4α4 or COL4α5, all members of the type IV collagen family. The proteins products of these genes along with those of the other type IV collagen family members (COL4α1, COL4α2, and COL4α6) are structural components of basement membranes throughout the body. This dissertation describes the measurement of mRNA transcripts in two canine models of AS: a mixed breed model of X-linked AS (XLAS) and the English Cocker Spaniel (ECS) model of autosomal recessive AS (ARAS). The work done revealed a decrease in COL4α4 transcripts. The similarity between the decrease of COL4α5 in the XLAS model and that for COL4α4 in the ARAS model lead to the investigation of COL4α4 as the gene harboring the mutation causative for ARAS in the ECS. Upon sequencing COL4α4, the causative mutation was determined to be an A to T transversion in exon 3. To provide an in vitro model to study type IV collagens, a protocol was designed and experimentally validated to isolate and culture canine Sertoli cells. Canine testes cells were isolated and cultured. Cells were verified as Sertoli cells through positive identification of both SOX9 and Clusterin B proteins, along with sequence verification of SOX9 transcripts. This in vitro model provides a tool to further study the type IV collagens. Overall, the research described herein lead to the identification of the mutation causative for ARAS in the ECS. With this knowledge a genetic test was developed to test for the disease. This research also provided valuable information about the transcript levels of type IV collagens in two models of AS, and provided a novel model in which to study the type IV collagens further.

The domestic dog, Canis familiaris, has over 450 naturally occurring inherited diseases. Over half of these diseases are clinically similar to human diseases making the dog an excellent model in which to study human hereditary diseases. Alport syndrome (AS), a group of heterogeneous, hereditary renal diseases, is one example of such a human disease. The disease is transmitted in three fashions: X-linked, autosomal recessive, and autosomal dominant. AS is caused by mutations in COL4α3, COL4α4 or COL4α5, all members of the type IV collagen family. The proteins products of these genes along with those of the other type IV collagen family members (COL4α1, COL4α2, and COL4α6) are structural components of basement membranes throughout the body. This dissertation describes the measurement of mRNA transcripts in two canine models of AS: a mixed breed model of X-linked AS (XLAS) and the English Cocker Spaniel (ECS) model of autosomal recessive AS (ARAS). The work done revealed a decrease in COL4α4 transcripts. The similarity between the decrease of COL4α5 in the XLAS model and that for COL4α4 in the ARAS model lead to the investigation of COL4α4 as the gene harboring the mutation causative for ARAS in the ECS. Upon sequencing COL4α4, the causative mutation was determined to be an A to T transversion in exon 3. To provide an in vitro model to study type IV collagens, a protocol was designed and experimentally validated to isolate and culture canine Sertoli cells. Canine testes cells were isolated and cultured. Cells were verified as Sertoli cells through positive identification of both SOX9 and Clusterin B proteins, along with sequence verification of SOX9 transcripts. This in vitro model provides a tool to further study the type IV collagens. Overall, the research described herein lead to the identification of the mutation causative for ARAS in the ECS. With this knowledge a genetic test was developed to test for the disease. This research also provided valuable information about the transcript levels of type IV collagens in two models of AS, and provided a novel model in which to study the type IV collagens further.

published_or_final_version / Zoology / Master / Master of Philosophy

Die Transkriptionsfaktoren der p53 Familie besitzen diverse Aufgaben sowohl während der Tumorentstehung als auch während der Entwicklung. In seiner ursprünglichen, evolutionär konservierten Rolle hat p63, ein Mitglied der p53 Familie, die Aufgabe die genetische Stabilität von Keimzellen zu gewährleisten und beeinflusst zudem die Keimbahnentwicklung. Mit Hilfe von „knockout“ (KO) Mausmodellen wurde bereits der Einfluss der 3 Transkriptionsfaktoren p53, p63 und p73 auf die weibliche und männliche Keimbahn untersucht. Während p53KO und p63KO Mäuse fruchtbar sind, führt der gemeinsame Verlust aller p73 Isoformen oder der Verlust der transkriptionell aktiven Isoform TAp73 hingegen zur Infertilität der Tiere. Weibliche TAp73KO Tiere sind deshalb infertil, da es bei ihnen zu einer Inhibition der Ovulation kommt, die Blastozystenentwicklung gestört ist und Spindeldefekte in den Oozyten auftreten. Eine Erklärung für die Infertilität von männlichen TAp73KO Mäusen wurde hingegen noch nicht gegeben. In der vorliegenden wissenschaftlichen Studie beschreiben wir eine bisher unbekannte Funktion des Transkriptionsfaktors TAp73: seinen Einfluss auf die Entwicklung und den Erhalt der adulten männlichen Keimbahn. Untersuchungen an Mausmodellen zeigen, dass „total“ p73KO und Isoform-spezifische TAp73KO Mäuse im Alter von 6 Wochen oder älter einen starken Verlust von sich entwickelnden Keimzellen in den testikulären Tubuli aufweisen. ΔNp73KO Tiere zeigen diesen Phänotyp nicht und ihre Testis Morphologie entspricht der der Wildtyp (WT) Tiere. Der Phänotyp der p73KO und TAp73KO Testes lässt sich durch eine starke Reduktion in der Zahl der späten Stadien der Spermatozyten und Spermatiden charakterisieren, wohingegen die Zahl der basalen Spermatogonien und pachytänen Spermatozyten des Keimepithels nicht betroffen ist (reguläre Zellzahl und Proliferation). Die Depletion von p73 oder TAp73 führt zu einer Anhäufung von apoptotischen und unreifen Keimzellen im Lumen des Epididymis, was auf eine atypische, verfrühte Ablösung der Keimzellen aus dem Keimepithel schließen lässt. Diese Beobachtung wird dadurch bestätigt, dass die Sertoli-Zellen, welche die Keimzellen stützen und ernähren, im Elektronenmikroskop verkürzte cytoplasmatische Arme sowie eine verstärkte Vakuolisierung aufweisen. Außerdem zeigt das Keimepithel große strukturelle Veränderungen, die dadurch gekennzeichnet sind, dass die Keimzellen nicht mehr dicht zu einem Epithel gepackt sind und die Sertoli-Keimzell-Verbindung gestört ist. Der Verlust der Epithelstruktur begleitet von der Reduktion der Keimzellen scheint das Ergebnis eines Defekts der Blut-Testis-Schranke (BTS) zu sein. Dies wird durch einen in vivo BTS Permeabilitätstest und die gestörte Morphologie der Sertoli-Sertoli „tight junctions“ deutlich. Der funktionelle Verlust der BTS führt zur Zerstörung der epithelialen Polarität und der Umgebung der sich entwickelnden Keimzellen. Somit wird auch die basal-luminal gerichtete Migration der Keimzellen entlang der Keimzelltaschen der Sertoli-Zellen verhindert. Der molekulare Hintergrund für das Ungleichgewicht in der Reorganisation der Zell-Zell Verbindungen wurde mit Hilfe der quantitativen Expressionsanalyse des Gesamtgenoms untersucht. Dabei wurde Testis Gewebe von TAp73KO mit WT Mäusen verglichen, um TAp73-Zielgene zu finden. Die Depletion von TAp73 führt zur Induktion von Genen, die an der Adhäsion und Zellmigration beteiligt sind; Integrine und Proteaseinhibitoren wie Timp1 und die Serpine zeigen eine Hochregulation in der Expression. Es ist bekannt, dass diese Proteine bei der Reorganisation von Zell-Zell Verbindungen im Testis involviert sind. Die Studie zeigt, dass TAp73 vorwiegend in den Keimzellen exprimiert wird und dass diese parakrin auf die Sertoli-Zellen zu wirken scheinen, da die Überexpression von TAp73 Zielgenen in isolierten Sertoli-Zellen in der Langzeitkultur zurückgeht. Zusammenfassend konnte zum ersten Mal gezeigt werden, dass die Funktion von TAp73 für die adulte Spermatogenese unerlässlich ist. TAp73 reguliert im Testis ein Transkriptionsprogramm von Genen, die an Adhäsion und Migration beteiligt sind, sichert den Zusammenhalt des Keimepithels und verhindert den frühzeitigen Verlust von Keimzellen. Somit wird die ungestörte Keimzellentwicklung ermöglicht. Umgekehrt führt die Depletion von TAp73 zu starken Defekten in Zell-Zell Verbindungen von sich entwickelnden Keimzellen im Keimepithel, was die Infertilität von p73KO und TAp73KO Mäusen erklärt.

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 5 uppsatser.

Thesis (M.Sc.)--University of Alberta, 2009. / A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Master of Science in Experimental Surgery, Department of Surgery, University of Alberta. Title from pdf file main screen (viewed on July 28, 2009). Includes bibliographical references.

Thesis (Ph. D.)--University of Hong Kong, 2000. / Includes bibliographical references (leaves 97-146).

La FSH est une hormone clé dans la fonction de reproduction. La compréhension de ces mécanismes moléculaires est essentielle pour comprendre ses effets biologiques. Nous montrons ici pour la première fois que non seulement la FSH induit le recrutement de polysomes dans la cellule de Sertoli, mais qu’en plus, elle stimule la traduction de deux ARNm sélectivement, c-fos et vegfa. La p70S6K est impliquée dans ce mécanisme. La FSH active et induit le recrutement de la p70S6K sur la coiffe des ARNm où elle active ses cibles traductionnelles. Les β-arrestines, des protéines d’échafaudage qui régulent la signalisation du RFSH, semblent participer à l’activation de la p70S6K. La déplétion des β-arrestines augmente massivement le recrutement de la p70S6K à la coiffe et diminue son activité enzymatique. De plus, la p70S6K et les β-arrestines interagissent mais cette interaction ne semble pas modulée par la FSH. Nos résultats suggèrent que les β-arrestines séquestreraient la p70S6K inactive pour permettre son activation et son recrutement à la coiffe m7GTP en réponse à la FSH. Ce travail apporte de nouvelles connaissances sur le rôle de la FSH dans la traduction et les mécanismes de signalisation impliqués. / FSH is a key hormone of the reproductive function. A clear understanding of its molecular mechanism is essential to fully understand its biological effects. Here, we show for the first time that FSH not only enhances the assembly of polysomes but also stimulates the translation of at least two mRNA selectively, c-fos and vegfa, in Sertoli cells. p70S6K participates in this mechanism. FSH activates and enhaced p70S6K recruitment to the m7GTP cap structure of mRNA where this kinase phosphorylates its targets. β-arrestins, which are scaffolding proteins that regulate FSH signalling, seem to participate in p70S6K activation. Accordingly, β-arrestins depletion increased p70S6K recruitment to the cap and reduced its enzymatic activity. Importantly, p70S6K and β-arrestins interact but the interaction is not FSH-dependent. We assume that β-arrestins sequester inactive p70S6K to activate it locally and then p70S6K translocates to the cap in response to FSH. In conclusion, this work brings new knowledge about FSH function in translational control and the signaling mechanisms involved.

Orientador: Wellerson Rodrigo Scarano / Banca: Ana Paula Alves Favareto / Banca: Patricia Fernanda Felipe Pinheiro / Resumo: As células de Sertoli, encontradas no epitélio seminífero, formam a barreira hematotesticular através de suas junções comunicantes e de oclusão, responsável por proporcionar um ambiente protegido e coordenar o processo espermatogênico, fundamental para a manutenção da fertilidade masculina. A dinâmica de formação da barreira hematotesticular está diretamente relacionada com o equilíbrio entre síntese e degradação de moléculas especializadas, e sofre influência através de regulação androgênica e de fatores de crescimento específicos como o TGF-β2, sendo particularmente sensível aos fatores ambientais. A exposição à desreguladores endócrinos (DEs), capazes de interferir na modulação da barreira, pode levar a infertilidade. Atualmente o fator masculino pode ser responsável por 30-50% dos casos de infertilidade, sendo de grande importância o estabelecimento da relação entre a exposição aos DEs e o processo de formação dos gametas. Este estudo objetivou correlacionar a ação de dois desreguladores endócrinos, o Monobutil ftalato (MBP) e o Bisfenol A (BPA), em diferentes períodos de exposição, a partir de uma dose de baixa toxicidade, sobre a linhagem HSec de células de Sertoli humanas (in vitro) e sua possível ação sobre proteínas da barreira hematotesticular e proteínas regulatórias envolvidas na modulação da degradação e síntese dessa barreira. Para tal, as células da linhagem HSec foram cultivadas em meio específico para a expansão e submetidas à exposição ao MBP e ao BPA em baixas doses por 6 e 48 horas. Em seguida, as células foram isoladas e submetidas à extração de proteínas. Foram analisadas as proteínas: Ocludina, Ncaderina, β-catenina e Receptor de Andrógeno (AR) por Western Blot. Os resultados foram comparados para os dois agentes nos diferentes períodos de exposição em relação às células não expostas, mostrando que os dois agentes foram capazes de reduzir a... / Abstract: Sertoli cells, found in the seminiferous epithelium, form the blood-testis barrier via their gap and tight junctions, responsible for providing a protected environment and coordinate the spermatogenic process, essential for male fertility maintenance. The dynamics of the blood-testis barrier formation is directly related to the balance between synthesis and degradation of specialized molecules, and is influenced by androgen regulation and specific growth factors such as TGF-β2, being particularly sensitive to environmental factors. Exposure to endocrine disruptors (EDs), which interfere in the barrier modulation, can lead to infertility. Currently the male factor may be responsible for 30-50% of infertility cases, being the establishment of the relationship between exposure to EDs and the process of gametes formation of great importance. This study aimed to correlate the action of two EDs, monobutyl phthalate (MBP) and bisphenol A (BPA) in different periods of exposure, from a dose of low toxicity on the HSec lineage of human Sertoli cells (in vitro) and its possible action on proteins of blood-testis barrier and regulatory proteins involved in modulation of degradation and synthesis of this barrier. To this end, cells were cultured in HSec lineage specific medium and exposed to MBP and BPA in low doses for 6 and 48 hours. Then, cells were isolated and subjected to protein extraction. Proteins analyzed were: occludin, Ncadherin, β-catenin and Androgen Receptor (AR) by Western blot. The results were compared for the two agents in different exposure times to non-treated cells, showing that both agents were able to reduce the expression of important BTB proteins, presenting potential for disrupting the structure and maintenance of the barrier and, consequently, sperm production and male fertility / Mestre