To date, studies into sex determination and gonadal development have focused on the regulatory mechanisms governing development of the male or female phenotype. However, the formation of the testis and ovary from the bipotential gonad also present a fascinating model of tissue organisation which has been largely overlooked. When seeking to understand tissue organisation during gonadal development, the formation of testis cords takes center stage. However, despite a growing understanding of the cellular events in testis development, a number of key questions about the formation of testis cords remain unanswered. Specifically, I aimed to investigate the role of cell migration in testis organization, and the structure and morphogenesis of testis cords in three dimensions. To address these aims experimentally, I investigated the early morphogenesis of testis cords and the dependence of cord formation on cell migration from the mesonephros. I found that virtually all of the migrating cells express endothelial markers, indicating that endothelial, not peritubular myoid cells underlie the dependence of cord formation on cell migration. Further, disruption of endothelial cell migration and vascular organisation using a blocking antibody to VE-cadherin, also disrupted the development of testis cords. These data reveal that migrating endothelial cells are required for testis cord formation, consistent with increasing evidence of a broader role for vasculature in establishing tissue architecture during organogenesis. To address the question of cord structure and morphogenesis, I developed and applied a novel fluorescence-based three-dimensional modeling approach to show that Sertoli cells coalesce into irregular groups surrounding germ cells, and that these groups are remodeled to form highly regular toroidal loops, joined by a flattened plexus at the dorsal side. This plexus is punctured by blood vessels as they ingress from the mesonephros, and contracts during maturation to form part of the rete testis. Variation in cord number and position demonstrates that cord establishment is not a stereotypic process. However, a tightly regulated modeling mechanism must contribute to uniformity on cord diameter and orientation as these parameters remain consistent across samples of the same age. These data clarify questions of cord structure and organisation, establish that cord formation is a variable process, and demonstrate novel structural features within the network of testis cords. Finally, to investigate an in vivo model where vascularisation and cord formation may be disrupted, I analysed gonads from embryos lacking Cited2. Consistent with a previous study, I found that testis development was delayed in Cited2-/- gonads, but found that despite the reported transcriptional recovery after the delay, testis vascular and cord structure was permanently disrupted. To investigate the defects in cord formation I assayed cell migration and found that migration was not disrupted in XY gonads, or mesonephroi lacking Cited2. However, ectopic cell migration was observed in the XX gonad in a dose-dependent response to loss of functional Cited2 alleles. Correspondingly, the female pathway was initially delayed but rallied for a late recovery, implicating Sf1 in the initiation of ovarian differentiation. These data underscore the fragility of the molecular control of sex determination as absence of Cited2 in the male permanently disrupts testis morphology, whereas in the female, promoters of the male pathway are not sufficiently suppressed. From these studies I construct an integrated model of testis cord formation and conclude that testis cord formation is a novel form of tubulogenesis. This morphogenesis is unique and offers insights into cell and tissue organisation, vascular interactions in organogenesis, and mechanisms of tube formation. Further study of cord formation is likely to lead to a broader understanding of tissue morphogenesis during development.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 5 uppsatser.

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

SOUSA, Solange Damasceno. Proteômica do fluido da Rete Testis de carneiros Morada Nova. 2015. 133 f. : Dissertação (Mestrado) - Universidade Federal do Ceará, Centro de Ciências, Departamento de Zootecnia, Programa de Pós-Graduação em Zootecnia. Fortaleza-CE, 2015. / Submitted by Eric Santiago (erichhcl@gmail.com) on 2016-08-10T12:26:45Z No. of bitstreams: 1 2015_dis_sdsousa.pdf: 3355993 bytes, checksum: 3f9c9ea989725756a91c3d6679b0614a (MD5) / Approved for entry into archive by Nádja Goes (nmoraissoares@gmail.com) on 2016-08-10T15:18:50Z (GMT) No. of bitstreams: 1 2015_dis_sdsousa.pdf: 3355993 bytes, checksum: 3f9c9ea989725756a91c3d6679b0614a (MD5) / Made available in DSpace on 2016-08-10T15:18:50Z (GMT). No. of bitstreams: 1 2015_dis_sdsousa.pdf: 3355993 bytes, checksum: 3f9c9ea989725756a91c3d6679b0614a (MD5) Previous issue date: 2015 / The aim of the study was to identify and characterize the proteins of the rete testis fluid from Morada Nova rams. The testicles, obtained from six slaughtered Morada Nova rams, were immediately dissected. The head of the epididymis was separated to gain access to the efferent ducts. The fluid from the efferent ducts was obtained by testis massage. Thereafter, the fluid was centrifuged to remove cell debris and sperm. Proteins were precipitated with acetone at -20°C and quantified by the Bradford assay. Each sample (400 µg) was focused in strips of 13 cm (pH 4-7) and the second dimension was conducted on SDS-PAGE 15%. The gels were scanned with an ImageScanner II (GE Lifesciences, USA) and analyzed using the PDQuest® version 8.0.1 (Bio-Rad Laboratories, USA). Spots detected after PDQuest analysis of 2-D maps were cut from gels and submitted to trypsin digestion. Proteins were identified using tandem mass spectrometry. Protein information obtained by MASCOT was analyzed using the software tool for searching annotations of proteins (STRAP). Protein-protein interaction networks were obtained from STRING version 9.0 database. In the gels were detected 227 ± 32.1 spots (mean ± SD), where 51% of the proteins were found above 40 kDa, corresponding to 65% of the intensity of all spots detected. Based on gene ontology analysis, the most common biological processes associated with proteins from rete testis fluid were regulation (24.28%) and cellular process (23.27%). Binding (27.42%) and catalytic activity (19.30%) corresponded to the most frequent molecular functions for those proteins. The most intensely expressed proteins were: albumin, clusterin, serotransferrin, immunoglobulin gamma-1 chain and alpha-2-HS-glycoprotein. The rete testis fluid has a large quantity of proteins related to the spermatogenesis. This feature is important in view of the fact that these molecules contribute to the development of the germ cells, as a result of the transport and conversion of substances required to the production of male gametes. / O objetivo do estudo foi identificar e caracterizar as proteínas do fluido da rete testis de carneiros Morada Nova. Os testículos, obtidos de seis carneiros Morada Nova abatidos, foram imediatamente dissecados. A cabeça do epidídimo foi separada para ter acesso aos ductos eferentes. O fluido oriundo dos ductos eferentes foi obtido por massagem do testículo. Posteriormente, o fluido foi centrifugado para remoção dos debris celulares e espermatozoides. As proteínas foram precipitadas com acetona a -20°C e quantificadas pelo método de Bradford. Cada amostra (400 μg) foi focalizada em tiras de 13 cm (pH 4-7) e a segunda dimensão foi realizada em SDS-PAGE a 15%. Os géis obtidos foram escaneados com um ImageScanner II (GE Lifesciences, EUA) e analisados utilizando o aplicativo PDQuest® versão 8.0.1 (Bio-Rad Laboratories, EUA). Os spots detectados após a análise dos mapas 2-D no PDQuest foram cortados dos géis e submetidos a digestão com tripsina. As proteínas foram identificadas por espectrometria de massa em tandem. A informação sobre a proteína adquirida pela busca no MASCOT foi analisada através da utilização do programa para anotações de proteínas (STRAP). Redes de interação proteína-proteína foram obtidas a partir do banco de dados STRING versão 9.0. Nos géis foram detectados em média 227 ± 32,1 spots, onde 51% das proteínas se encontraram acima de 40 kDa representando 65% da intensidade de todos os spots detectados nos géis. Com base na análise da ontologia gênica, os processos biológicos mais comuns associados às proteínas do fluido rete testis foram regulação (24,28%) e processos celulares (23,27%). Ligação (27,42%) e a atividade catalítica (19,30%) corresponderam às funções moleculares mais frequentes. As proteínas mais intensamente expressas foram: albumina, clusterina, serotransferrina, imunoglobulina gama-1 e alfa-2-HS-glicoproteína. A maioria das proteínas identificadas desempenha importantes papéis nos processos de espermatogênese, proteção espermática, motilidade, capacitação e reação acrossômica. O fluido da rete testis possui várias proteínas envolvidas na espermatogênese, o que representa um importante fator, uma vez que estas moléculas contribuem para o desenvolvimento das células germinativas, participando no transporte e conversão de substâncias requeridas para a produção dos gametas masculinos.

FundaÃÃo Cearense de Apoio ao Desenvolvimento Cientifico e TecnolÃgico / O objetivo do estudo foi identificar e caracterizar as proteÃnas do fluido da rete testis de carneiros Morada Nova. Os testÃculos, obtidos de seis carneiros Morada Nova abatidos, foram imediatamente dissecados. A cabeÃa do epidÃdimo foi separada para ter acesso aos ductos eferentes. O fluido oriundo dos ductos eferentes foi obtido por massagem do testÃculo. Posteriormente, o fluido foi centrifugado para remoÃÃo dos debris celulares e espermatozoides. As proteÃnas foram precipitadas com acetona a -20ÂC e quantificadas pelo mÃtodo de Bradford. Cada amostra (400 μg) foi focalizada em tiras de 13 cm (pH 4-7) e a segunda dimensÃo foi realizada em SDS-PAGE a 15%. Os gÃis obtidos foram escaneados com um ImageScanner II (GE Lifesciences, EUA) e analisados utilizando o aplicativo PDQuest versÃo 8.0.1 (Bio-Rad Laboratories, EUA). Os spots detectados apÃs a anÃlise dos mapas 2-D no PDQuest foram cortados dos gÃis e submetidos a digestÃo com tripsina. As proteÃnas foram identificadas por espectrometria de massa em tandem. A informaÃÃo sobre a proteÃna adquirida pela busca no MASCOT foi analisada atravÃs da utilizaÃÃo do programa para anotaÃÃes de proteÃnas (STRAP). Redes de interaÃÃo proteÃna-proteÃna foram obtidas a partir do banco de dados STRING versÃo 9.0. Nos gÃis foram detectados em mÃdia 227  32,1 spots, onde 51% das proteÃnas se encontraram acima de 40 kDa representando 65% da intensidade de todos os spots detectados nos gÃis. Com base na anÃlise da ontologia gÃnica, os processos biolÃgicos mais comuns associados Ãs proteÃnas do fluido rete testis foram regulaÃÃo (24,28%) e processos celulares (23,27%). LigaÃÃo (27,42%) e a atividade catalÃtica (19,30%) corresponderam Ãs funÃÃes moleculares mais frequentes. As proteÃnas mais intensamente expressas foram: albumina, clusterina, serotransferrina, imunoglobulina gama-1 e alfa-2-HS-glicoproteÃna. A maioria das proteÃnas identificadas desempenha importantes papÃis nos processos de espermatogÃnese, proteÃÃo espermÃtica, motilidade, capacitaÃÃo e reaÃÃo acrossÃmica. O fluido da rete testis possui vÃrias proteÃnas envolvidas na espermatogÃnese, o que representa um importante fator, uma vez que estas molÃculas contribuem para o desenvolvimento das cÃlulas germinativas, participando no transporte e conversÃo de substÃncias requeridas para a produÃÃo dos gametas masculinos. / The aim of the study was to identify and characterize the proteins of the rete testis fluid from Morada Nova rams. The testicles, obtained from six slaughtered Morada Nova rams, were immediately dissected. The head of the epididymis was separated to gain access to the efferent ducts. The fluid from the efferent ducts was obtained by testis massage. Thereafter, the fluid was centrifuged to remove cell debris and sperm. Proteins were precipitated with acetone at -20ÂC and quantified by the Bradford assay. Each sample (400 Âg) was focused in strips of 13 cm (pH 4-7) and the second dimension was conducted on SDS-PAGE 15%. The gels were scanned with an ImageScanner II (GE Lifesciences, USA) and analyzed using the PDQuest version 8.0.1 (Bio-Rad Laboratories, USA). Spots detected after PDQuest analysis of 2-D maps were cut from gels and submitted to trypsin digestion. Proteins were identified using tandem mass spectrometry. Protein information obtained by MASCOT was analyzed using the software tool for searching annotations of proteins (STRAP). Protein-protein interaction networks were obtained from STRING version 9.0 database. In the gels were detected 227  32.1 spots (mean  SD), where 51% of the proteins were found above 40 kDa, corresponding to 65% of the intensity of all spots detected. Based on gene ontology analysis, the most common biological processes associated with proteins from rete testis fluid were regulation (24.28%) and cellular process (23.27%). Binding (27.42%) and catalytic activity (19.30%) corresponded to the most frequent molecular functions for those proteins. The most intensely expressed proteins were: albumin, clusterin, serotransferrin, immunoglobulin gamma-1 chain and alpha-2-HS-glycoprotein. The rete testis fluid has a large quantity of proteins related to the spermatogenesis. This feature is important in view of the fact that these molecules contribute to the development of the germ cells, as a result of the transport and conversion of substances required to the production of male gametes.

Au cours des dernières décennies, nous avons progressivement vu augmenter un certain nombre d’anomalies de la fonction de reproduction masculine dans les pays industrialisés. Ces constatations ont fait émerger l’hypothèse selon laquelle certains polluants de notre environnement pourraient altérer le développement du testicule fœtal et ainsi être responsables de ces anomalies. Parmi les composants incriminés se trouvent les phtalates, largement répandus dans l’environnement. Ces composés ont été décrits comme reprotoxiques, ils altèrent le développement de la lignée germinale dans différentes espèces et entraînent une diminution de la production de testostérone chez le rat. Toutefois, très peu de données sont disponibles quant à leurs effets chez l’Homme. Dans cette étude, nous avons analysé les effets d’un phtalate, le MEHP, sur le développement du testicule fœtal humain au premier trimestre de la grossesse, dans un modèle de culture organotypique qui permet le maintien des différentes structures de l’organe. Nous avons tout d’abord démontré que le MEHP (10-4M) n’altère pas la production de testostérone du testicule fœtal humain, contrairement aux résultats décrits chez le rat. En revanche, nous avons montré que l’exposition au MEHP entraîne une rapide diminution du nombre de cellules germinales par apoptose. A la suite de ces résultats, nous avons testé l’effet de doses plus faibles de MEHP afin de se placer à des concentrations de phtalates ayant été mesurées dans les liquides biologiques. Nous avons ainsi démontré que les cellules germinales du testicule fœtal humain sont altérées suite à l’exposition à des doses de MEHP de 10-5M. Enfin, dans la 3ème partie de ce travail, nous nous sommes intéressés aux mécanismes d’action des phtalates. Différentes études, notamment dans le foie, démontrent l’implication des récepteurs nucléaires dans les effets de ces composés. Il nous a donc semblé important de rechercher leur implication dans les effets des phtalates sur le testicule fœtal. Nous avons démontré que LXRα est très certainement impliqué ces effets puisque l’expression des ARNm de ce récepteur est augmentée. Par ailleurs, ce récepteur nucléaire contrôle deux voies métaboliques, la synthèse de cholestérol et la synthèse des acides gras qui semblent toutes deux modulées par les phtalates dans le testicule fœtal humain. Enfin, nous avons montré que l’implication de ces voies métaboliques est commune entre la gonade mâle et la gonade femelle, sans pour autant que l’effet sur les cellules germinales mâles ai pu être mis en évidence dans l’ovaire fœtal. En conclusion, cette étude a contribué à caractériser les effets des phtalates sur la mise en place des fonctions de reproduction chez le fœtus humain. Nous avons également pu mettre en évidence un nouveau mécanisme de ces composés, impliquant la superfamille des récepteurs nucléaires ainsi que la synthèse du cholestérol et des acides gras. / Since the last decades, an increase in several abnormalities of the male reproductive function has been progressively evidenced in industrialized countries. According to these observations, it was hypothesized that exposure to some environmental pollutants may impair the fetal testis development, and therefore be at the origins of those abnormalities. Among incriminated compounds, phthalates are molecules highly produced worldwide. These compound are classified as reprotoxic molecules, as they disrupt the development of the germ cell lineage in different species and lead to a decrease in testosterone production in rat. Nevertheless, very few data are available concerning their effects in human. In this study we analyzed the effects of one phthalate, the MEHP, on the human fetal testis development during the first trimester of pregnancy. It was performed using an organotypic culture system that allows the preservation of the different testis structures. We first demonstrated that MEHP (10-4M) does not affect testosterone production of the human fetal testis, in opposition to the results described in rat. We also have demonstrated that MEHP exposure triggers apoptosis in the fetal germ cells, leading to a quick decrease in the total number of these cells. Following those results, we tested the effects of lower doses of MEHP that are close to the highest doses measured in human biological fluids. We therefore demonstrated that fetal germ cells are altered by exposure to this dose of MEHP (10-5M). Finally, in the third part of this work, we focused on the mechanisms of action of phthalate toxicity. Different studies, mostly in the liver, report the involvement of the nuclear receptor superfamilly in the effect of those compounds. Thus, it seemed important to investigate their implication in the effect of phthalates on the human fetal testis. We demonstrated that LXRα is certainly implicated in these effects as its transcriptional level is increased. Moreover, this nuclear receptor regulates two metabolic pathways: Cholesterol and fatty acid synthesis pathways, that seemed to by both modulated by phthalate exposure in the human fetal testis. We also showed that the modulation of these two metabolic pathways is a common process to both the male and female gonads. Nevertheless, the germ cell decrease we evidenced in the human fetal testis was never observable in the fetal ovary. In conclusion, this work contributed to improve our knowledge about the effects of phthalate exposure on the establishment and the development of the human fetal reproductive system. We also have evidenced a new mechanism of these compounds that involves members of the nuclear receptors superfamilly, as well as cholesterol and fatty acid synthesis.

Mad1 est une protéine clé du point de contrôle du fuseau en mitose. Associée à Mad2,elle est recrutée aux kinétochores non-attachés où elle y catalyse la production du complexe inhibiteur d’anaphase. La protéine Mad1 a longtemps été décrite comme étant un simple récepteur de Mad2 aux kinétochores. Certaines études laissaient toutefois entrevoir des rôles additionnels de cette protéine en mitose comme en interphase.Afin d’explorer ces fonctions additionnelles de Mad1, j’ai étudié le phénotype mitotique associé à une déplétion de la protéine par ARN interférence dans des lignées cellulaires S2 de drosophile. J’ai également analysé des mutations et délétions du domaine N-terminal de Mad1, celui-ci présentant certaines particularités de structures primaires et secondaires tels que des sites de phosphorylation, un putatif NLS et des hélices amphipathiques. J’ai ainsi montré que le recrutement de Mad1 aux kinétochores en mitose nécessitait la phosphorylation de son domaine N-terminal. Mes analyses cytologiques ont de plus permis de déterminer que le NLS, situé dans ce même domaine N-terminal, est non seulement fonctionnel mais également essentiel à la localisation de Mad1 à l’enveloppe nucléaire et dans le nucléoplasme en interphase.J’ai finalement étudié une protéine Mad1 déplétée de son domaine N-terminal (Mad1Δ71) en spermatocytes de drosophile. Notre laboratoire a récemment montré que dans ces cellules, la protéine Mad1 fait partie d’un territoire nucléaire associé à la chromatine, appelé MINT (Mad1 containing Intranuclear Territory). Ce nouveau territoire, comportant au moins quatre autres protéines (Mad2, Mtor/Tpr, Ulp1 et Raf2), est impliqué dans la régulation de la conformation de la chromatine. Mes analyses ont révélé que la protéine Mad1 Δ71 se localisait anormalement dans le noyau, restant accolée à l’enveloppe nucléaire, et entraînait avec elle l’ensemble de ses partenaires. Ceci suggère que Mad1 est essentielle à l’organisation de ces protéines dans le nucléoplasme, mais également qu’elle pilote la mise en place du territoire MINT. / Mad1 is a key component of the spindle assembly checkpoint in mitosis. Recruitedwith Mad2 to unattached kinetochores, they catalyze the formation of the anaphase inhibitor.Mad1 has long been described as a simple receptor for Mad2 at kinetochores. However,studies are pointing toward additional roles of this protein in mitosis as well as in interphase. To explore these additional functions of Mad1, I studied the mitotic phenotypeassociated with a depletion of the protein by RNA interference in Drosophia S2 cell lines. Ialso analyzed mutations and deletions of the N-terminal domain of Mad1, this one havinginteresting features in its primary and secondary structures, namely phosphorylation sites, aputative NLS and amphipathic helices. I have shown that Mad1 recruitment to kinetochore inmitosis depends on phosphorylations of its N-terminal domain. Moreover, my cytologicalanalyses allowed me to determine that the N-terminal NLS was not only functional but alsoessential for the localization of Mad1 into the nucleus in interphase. Finally, I studied a Mad1 mutant depleted for its N-terminal region (Mad1 Δ71) indrosophila spermatocytes. Our laboratory recently showed that in these cells, Mad1 is part ofa nuclear territory associated with chromatin, named MINT (Mad1 containg IntranuclearTerritory). This new territory, composed of at least four other proteins (Mad2, Mtor/Tpr,Ulp1, Raf2), is involved in chromatin conformation regulation. My studies revealed that inthese cells, Mad1 Δ71 is abnormally localized in the nucleus, staying closed to the nuclearenvelope, and carry with it all its partners. This suggests that Mad1 is essential for the nuclearorganization of these proteins, but also that it pilots the establishment of the MINT territory.

Tao Kin Pong. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 144-152). / Abstracts in English and Chinese. / Chapter i. --- Cover page --- p.1 / Chapter ii. --- Table of contents --- p.2 / Chapter iii. --- Abstract of thesis (English version and Chinese Version) --- p.4 / Chapter iv. --- Acknowledgements --- p.8 / Chapter v. --- Abbreviations --- p.9 / Chapter 1. --- Introduction / Interstitial tissue and Leydig cells --- p.11 / Seminiferous tubules --- p.11 / "Male germ cell line and spermatogenesis (Mitotic, Meiotic and Post-meiotic)" --- p.12 / Sertoli cells --- p.14 / Specialized organizations of junction present in testis --- p.15 / Dynamic nature of Sertoli-Sertoli & Sertoli-germ cell junctions --- p.16 / Role of proteases and protease inhibitors in male gametogenesis --- p.17 / Proteases and Proteases Inhibitors expressed in testis --- p.18 / Hormonal control of spermatogenesis --- p.19 / Hypothyroidism and testis development --- p.21 / Genes to be studied: / Proteases --- p.22 / Proteases Inhibitors --- p.27 / Other spermatogenesis related genes --- p.30 / Chapter 2. --- Objectives --- p.32 / Chapter 3. --- Materials and Methods / Animal treatments and tissue collection --- p.33 / RNA preparation --- p.34 / RT-PCRs --- p.35 / Real-time PCRs --- p.35 / Data manipulations and Statistics --- p.36 / Primer sequences used in this experiment --- p.37 / Chapter 4. --- Results / "Effect of neonatal hypothyroidism on developmental profile of body weight, absolute and relative testicular weight" --- p.40 / Developmental transcription profiles of genes under normal and hypothyroidism --- p.43 / Screening Data --- p.78 / Expression of non-spermatogenic genes at neonatal age --- p.88 / Responsiveness of gene transcription after thyroxin replacement --- p.89 / Changes of gene expression under different hypothyroid regimens --- p.97 / Chapter 5. --- Discussion / Changes in testicular size under hypothyroidism --- p.107 / Five patterns of transcription profile --- p.107 / "Suggestion on the role of ""MEIOTIC"" proteases and inhibitors" --- p.111 / "Suggestion on the role of ""POST-MEIOTIC"" proteases and inhibitors" --- p.113 / "Explanations on ""SOMATIC"" genes" --- p.114 / "Explanations on ""MITOTIC"" genes" --- p.115 / Explanations on the un-clustered pattern --- p.116 / Explanations on the age down-regulated group --- p.116 / Proposed clustering of genes according to their transcription profile --- p.117 / "Expression of some ""non-spermatogenic"" genes before puberty" --- p.123 / Neonatal hypothyroidism as a model for studying reproductive physiology --- p.125 / Different components of spermatogenesis --- p.127 / Chapter I. --- Roles of nuclear and chromatin related genes in assisting meiosis --- p.128 / Chapter II. --- Roles of specific transcription regulators in assisting gene selection --- p.129 / Chapter III. --- Role of signal transduction molecules for translation and activation --- p.131 / Chapter IV. --- Role of proteases and inhibitors for matrix and junctions dynamics --- p.132 / The somatic proteases and inhibitors system in the testis --- p.133 / Spermatogenic proteases and inhibitors system --- p.134 / Chapter V. --- Role of matrix and junctional molecules in intercellular interactions --- p.137 / Chapter VI. --- Role of cytoplasmic motors in cellular movement --- p.139 / Chapter 6. --- Conclusion / Proposed story of spermatogenesis - involvement of proteases and inhibitors --- p.140 / Future Direction --- p.141 / Chapter 7. --- References --- p.144

Non-clinical safety assessment is essential during the drug development process in the pharmaceutical industry, and involves numerous, detailed in vitro and in vivo toxicology tests (general, reproductive and genetic), and safety pharmacology studies. The testis is a common organ for adverse drug effects leading to attrition of potential compounds. It would, therefore, be useful to detect testicular toxicity as early as possible in the drug development process. Histopathology is the standard method for assessing testis toxicity, but a biomarker for ‘early warning’ detection of testicular toxicity would be far more useful in non-clinical toxicology studies. The aim of this thesis was to evaluate the feasibility of this approach. It is thought that proteins can leak from seminiferous tubules into testicular interstitial fluid following testicular damage, due to either loss of integrity of the blood-testis barrier (BTB) or germ cell damage. A potential biomarker protein could, therefore, leak out of seminiferous tubules into interstitial fluid and then into blood following toxicological insult to the testis. A suitable biomarker protein must be testis specific, abundant, and not normally be present in blood. It may also need to have a low molecular weight. To investigate if proteins do leak out of seminiferous tubules following testicular damage, three known testicular toxicants which affect different aspects of the testis were used; cadmium chloride causes disruption to the blood-testis barrier and spermatogenesis, methoxyacetic acid (MAA) specifically causes a loss of pachytene spermatocytes, and 1,3-dinitrobenzene (DNB) causes Sertoli cell vacuolation and subsequent germ cell disruption. Adult male Wistar rats were treated with various doses of these toxicants to give mild and moderate responses. Samples were collected 24 hours later. Testicular damage was investigated by immunohistochemistry for well-known germ cell markers (DAZL, VASA) and using a general antibody to seminiferous tubule proteins. The integrity of the BTB was evaluated using immunofluorescent co-localisation of occludin, ZO-1, claudin-11, N-cadherin and β-catenin, and a biotin tracer. Protein leakage was investigated using analysis of interstitial fluid samples by 1D gel electrophoresis and staining with Coomassie-based dye or Western blotting for germ cell proteins and with the general antibody to seminiferous tubule proteins. Protein leakage from seminiferous tubules into interstitial fluid was observed with high dose cadmium chloride treatment. This was coincident with a loss of integrity of the BTB. No leakage was observed with MAA treatment which caused a specific loss of pachytene spermatocytes, or DNB which caused Sertoli cell vacuolation. With both treatments the BTB did not appear to be damaged suggesting that protein leakage occurs only following loss of integrity of the BTB. This was further investigated using treatments reported to specifically disrupt the BTB, namely intra-testicular administration of glycerol or transforming growth factor-β3, with samples collected 48 hours later. The damage caused was very localised, although BTB disruption with glycerol treatment caused some protein leakage. The presence of germ cell proteins in interstitial fluid samples before and after the development of the BTB during normal development was also evaluated, although most proteins of interest were not expressed in germ cells of the immature testis before BTB formation. Finally, five potential biomarker candidate proteins (ADAM3, Calpastatin, DAZL, FABP9, VASA) were selected and investigated using samples from the testicular toxicant studies. Smaller molecular weight proteins were thought to be more likely to leak out of seminiferous tubules, however, VASA, a large molecular protein (76kDa) was shown to leak into interstitial fluid following high dose cadmium chloride treatment. However, FABP9 (low molecular weight) was found to be the most promising biomarker for loss of BTB integrity. The results suggest that a biomarker could only be detected if there is a loss of integrity of the BTB and severe disruption of spermatogenesis, thus conferring no real advantage over present histopathology-based toxicity evaluations. Therefore, an automated immunohistochemistry and image analysis method was investigated as a refined method for detection of testicular toxicity at the end of a toxicology study, and shown to have promise.

Change of DNA methylation is a hallmark of cancer. It is frequently associated with cancer progression. Testicular germ cell tumor (TGCT) is the most common malignant tumor in young males. Currently, only a limited number of genes are known to be epigenetically changed in TGCT. Genome-wide analysis of differential methylation in a previously established testicular cell line is documented here. A total of 35,208 differentially methylated regions (DMR) were identified. However, only a small number of DMRs mapped to gene promoters. Genome-wide analysis of gene expression revealed a group of differentially expressed genes that were regulated by DNA methylation. Several candidate genes ( APOLD1, PCDH10 and RGAG1) were found to be dysregulated in TGCT patients. Surprisingly, APOLD1 was mapped to the TGCT susceptibility locus at 12p13.1, suggesting that it may be important in TGCT pathogenesis. / The majority of DMRs are located in introns or intergenic regions, but their functions are not well understood. Some of these DMRs were found to regulate non-coding RNAs (ncRNAs). In this study, differential methylation of 3 small nucleolar RNAs (snoRNA) and 3 microRNAs (miRNA) were identified. One of the miRNAs, miR-199a, is embedded in a conserved region in intron-14 of dynamin 3 at 1q24.3. Hypermethylation of miR-199a correlated with testicular cancer progression, and silencing of miR-199a. Re-expression of miR-199a in testicular cancer cells suppressed cell growth, cancer migration, invasion, and metastasis. miR-199a-5p, one of two mature miRNA species derived from miR-199a, is associated with cancer progression. An embryonal carcinoma antigen, podocalyxin-like protein 1 (PODXL), was identified to be a target of miR-199a-sp. PODXL is an anti-adhesive protein overexpressed in aggressive testicular cancer. Knockdown of PODXL suppressed cancer invasion. The inverse relationship between PODXL and miR-199a-5p expression suggests that PODXL is one of the downstream effectors mediating cancer invasion and metastasis. This study links DNA methylation, miR-199a dysregulation, and PODXL expression as a mechanism to explain testicular cancer progression. / Cheung, Hoi Hung. / Adviser: Woi-Yee Chan. / Source: Dissertation Abstracts International, Volume: 72-04, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 165-192). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. Ann Arbor, MI : ProQuest Information and Learning Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.