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Fetal germ cell development is a key stage of female reproductive life. The DAZ family proteins (DAZ, DAZL and BOLL) are RNA-binding proteins with critical roles in murine germ cell development but their expression and potential targets in the human are largely unknown. The studies in this Thesis investigated the expression and function of DAZL and BOLL in human fetal ovary. Both DAZL and BOLL mRNA are increased dramatically at the time of entry into meiosis. Immunohistochemical analysis with specific meiotic markers suggested that DAZL and BOLL have distinct spatial-temporal expression patterns, with minimal co-expression – BOLL expression was transient prior to follicle formation. This pattern was shown not to be present in the mouse fetal ovary, where Dazl and Boll are co-expressed, indicating a limitation of the mouse for exploring the function of Boll. Two human cell lines, embryonic kidney derived HEK293 cells and germ cell tumour derived TCam-2 cells were used as models to identify the mRNA targets of DAZL and BOLL after transfection of DAZL or BOLL vectors. In HEK293 cells, TEX19 and TEX14 were confirmed as potential targets of both DAZL and BOLL, and CDC25A as a potential DAZL target. Further experiments indicated that DAZL and BOLL did not increase target mRNA transcription but increased stabilisation. A DAZL/GFP co-transfection-FACS system for TCam-2 cells was established as this cell line has very low transfection efficiency. TEX14 and SYCP3 significantly increased in GFP+ve-DAZL+ve cells when compare to the GFP-ve-DAZL-ve cells, whilst SOX17 and DNMT3L significantly decreased in the GFP+ve-DAZL+ve cells. A 3'-UTR luciferase assay confirmed regulation of TEX14 and SOX17 by DAZL through their 3'-UTR. RNA immunoprecipitation further demonstrated direct binding between human TEX14, TEX19, SYCP3, SOX17 mRNA and DAZL protein, and that TEX14 binding is through its 3'-UTR. Dual fluorescence immunohistochemistry showed that SOX17 and DMNT3L are expressed in early germ cells with DAZL, and are later down-regulated co-incident with that of DAZL, consistent with the novel repressive effect of human DAZL on these two potential targets. These studies indicate that DAZL and BOLL are associated with different key meiotic stages of germ cell development in human fetal ovary. Several potential mRNA targets of DAZL and BOLL, and a novel repression function of human DAZL on its mRNA targets were identified giving further insight into the role of these factors in human ovarian development.

Skeletal muscle (SkM) possesses an impressive ability to regenerate in response to injury or chronic disease. This regenerative capacity is attributed to its resident mononuclear myogenic progenitors. Previous studies have identified several types of myogenic progenitors within SkM, some of which are isolated by fluorescence activated cell sorting (FACS) using cell surface markers. Studies in our laboratory have identified melanoma cell adhesion molecule (MCAM) as a cell surface marker expressed by myogenic progenitors in human fetal SkM. However, the relationship between MCAM expression and the degree of myogenic commitment of distinct MCAM+ populations has not been elucidated. In the present study, subpopulations of MCAM+ cells were purified by FACS on the basis of Hoechst 33342 dye uptake. Specifically, MCAM+ side population (SP) was isolated by Hoechst exclusion and MCAM+ main population (MP) on Hoechst incorporation. Sorted populations were first optimized for growth in vitro since SkM SP cells are difficult to maintain in culture. In particular, Invitrogen’s StemPro® MSC SFM medium was found to support propagation of human fetal SkM SP cells with minimal differentiation. Following this optimization, sorted populations were assessed for expression of myogenic markers before and after propagation and then for fusion potential in vitro and engraftment potential in vivo. The MCAM+ subpopulations were found to express myogenic markers to a significantly greater extent than MCAM- subpopulations. Furthermore, the MCAM+ subpopulations fused robustly into myotubes in vitro whereas the MCAM- subpopulations did not. Interestingly, the MCAM+ SP population exhibited the highest fusion potential in vitro and was the only MCAM+ subpopulation to engraft into dystrophic muscle in vivo following propagation. These results indicate that MCAM is associated with myogenicity and can be used to prospectively isolate a pure myogenic fraction from human fetal SkM tissue. Moreover, the MCAM+ SP retain its myogenic potential to a greater extent than MCAM+ MP after propagation. This suggests that the MCAM+ SP fraction contains a higher percentage of early myogenic progenitors compared to the MCAM+ MP fraction. Additional studies on MCAM-expressing populations in human fetal SkM may elucidate a potent population for use in cell-based therapeutic strategies for treating muscle diseases.

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.

No description available.

Neurological disorders affect more than 14% of the population worldwide and together with traumatic brain and spinal cord injuries represent major health, public and economic burden of the society. Incidence of inherited and idiopathic neurodegenerative disorders and acute CNS injuries is growing globally while neuroscience society is being challenged by numerous unanswered questions. Therefore, research of the CNS disorders is essential. Since animal models of the CNS diseases and injuries represent the key step in the conversion of the basic research to the clinics, we focused our work on generation of new animal models and on their use in pre-clinical research. We generated and characterized transgenic minipig model of Huntington's disease (HD) which represents the only successful establishment of a transgenic model of HD in minipig which should be valuable for testing of long term safety of HD therapeutics. Next, we crossed the well characterized R6/2 mouse HD model with the gad mouse model which lacks the expression of UCHL1 which led to results that support the theory of "protective" role of mutant huntingtin aggregates and suggest that UCHL1 function(s) may be affected in HD disturbing certain branches of Ubiquitin Proteasome System. Traumatic spinal cord injury and Amyotrophic Lateral...