The production of viable germ cells during human embryonic development determines adult reproductive success. This is particularly true for females, as development of germ cells (GCs) into primordial follicles before birth is imperative for future fertility. During fetal development GCs migrate to the genital ridge to form the gonad, after which several tightly regulated events, including proliferation, differentiation, and association with somatic cells, must occur to form a functional gonad. In the ovary these processes also include the initiation and subsequent arrest of meiosis. These developmental processes are orchestrated by local autocrine and paracrine factors, many of which remain to be identified in the human. In order to decipher further the pathways by which the gonad and GCs develop, potential regulators including prostaglandin (PG) E2, the interleukin (IL)6-type cytokines, and the prokinetecins (PROKs), were examined in the human fetal ovary and PROKs in the human fetal testis. Patterns of gene expression, protein localisation, function, and interaction of the potential mediators throughout human development (8-20 weeks gestation) were determined. Primary fetal tissue was investigated, in addition to immortalized GCs (T-Cam2 cells) and a murine model of fetal ovarian development. PGE2 interacts with known regulators of GC development in non-reproductive organs. It was postulated PGE2 may regulate GC progression by modulating these factors. Examination of PGE2 receptors and precursor enzymes in the fetal ovary revealed that all were present and some were developmentally regulated, with mRNA expression increasing with gestation. These developmentally regulated components were localised to the GCs. The PGE2 receptors were among those differentially expressed, with one localised solely to mature GCs. Culture of human fetal ovary confirmed that PGE2 regulates known regulators of GC development, increasing expression of survival and anti-apoptotic factors. To test the hypothesis that PGE2 is necessary for female GC development, paracetamol, an inhibitor of PGE2 precursor enzymes, was utilised in a murine model of fetal exposure. Fetal ovaries from this experiment displayed disruption of normal development. The IL6-type cytokines are also postulated to be involved in early gonad development, and are known to regulate proliferation and differentiation of mouse embryonic stem and GCs in vitro. A significant increase in transcript levels of the shared receptor components was determined in second trimester human ovaries, as well as developmental increases of several of the IL6-type ligands. Both common receptor components were located specifically in the GCs identifying them as the target of IL6 action in the human fetal ovary. The PROKs regulate cell migration, proliferation and differentiation, and modulate secretion of PGE2 and expression of some IL6-type cytokines. To-date, PROKs have not been examined in the human fetal gonad. Transcript levels were higher in the fetal testis compared to the ovary, with receptor and ligand components increasing with gestation. Most components also increased with gestation in the ovary. However, location of PROK components was strikingly different between the two tissues, with GCs being the primary target of PROK action in the fetal ovary, and Leydig and interstitial cells being the target in the testis. PROKs interaction with other regulators of gonad development was examined utilising a GC line in the case of the ovary and primary interstitial cell cultures in the case of the testis. These studies have identified new factors involved in human fetal gonad development, and how they interact with known regulatory pathways of development.

Le choriocarcinome est une tumeur trophoblastique hautement maligne qui se développe souvent suite à des grossesses molaires dénommées moles hydatiformes (MH). La progression d’une MH vers un choriocarcinome reste à ce jour non caractérisée. L’implication de facteurs angiogéniques dans ce processus a été proposée. Nous avons étudié le rôle d'un nouveau facteur angiogénique spécifique du placenta, l’EG-VEGF (Endocrine Gland-Derived Endothelial Growth Factor) dans la pathogenèse du choriocarcinome. EG-VEGF agit via deux récepteurs (RCPG), PROKR-1 et PROKR-2. Trois approches ont été utilisées pour vérifier cette hypothèse. Une approche clinique, utilisant des échantillons placentaires et des sera collectés chez des patientes MH (n = 38) et avec choriocarcinome (n = 3) et chez des femmes normales (n = 18), tous prélevés au cours du premier trimestre de la grossesse. Une approche in vitro, utilisant les cellules JEG3, lignée humaine de cellules de choriocarcinome et des cellules trophoblastiques normales du premier trimestre (CTN). Une approche in vivo qui a visé à développer un modèle animal du choriocarcinome qui a servi à l’étude de la progression du choriocarcinome. Les niveaux circulants d’EG-VEGF étaient significativement plus élevés dans les MH et le choriocarcinome comparés aux niveaux chez les patientes normales. Au niveau placentaire, à la fois les niveaux d’expression de l’EG-VEGF et ses récepteurs ont été augmentés. Dans les cellules JEG3, EG-VEGF augmente i) l'expression de PROKR-1 et PROKR-2, ii). La migration, l'invasion, la prolifération et la formation de sphéroïdes dans des systèmes de culture 2 et 3D. Ces effets étaient significativement diminués en présence des antagonistes des deux récepteurs, iii) la phosphorylation de diverses protéines impliquées dans la progression tumorale, ainsi que la sécrétion de MMP-2 et MMP-9. Le modèle de choriocarcinome a été développé par injection de cellules JEG3 en orthotopie dans le placenta de la souris SCID (brevet en cours). En 12 jours, les souris gestantes injectées ont développés un choriocarcinome qui a métastasé dans différents organes. L'injection des antagonistes des récepteurs de l’EG-VEGF réduit significativement le développement et la progression de la tumeur. Par ailleurs, nous avons caractérisé le mécanisme par lequel EG-VEGF contribuerait à la progression tumorale. Ce mécanisme implique le clivage de la protéine de la jonction endothéliale, la VE-Cadhérine, suite à sa phosphorylation sur tyrosine 685 par l’EG-VEGF. Au total, mon projet de thèse i) démontre l’implication directe de l’EG-VEGF dans le développement et la progression du choriocarcinome ii) élucide le mécanisme de cette progression et iii) propose une piste thérapeutique via l’antagonisation de ses récepteurs. / Choriocarcinoma is a highly malignant trophoblastic tumor that often develop from molar pregnancies also called hydatidiform mole (HM). Nevertheless, HM progression towards choriocarcinoma remains uncharacterized. Involvement of angiogenic factors in this process is proposed. Here, we investigated the role of a new placental angiogenic factor, EG-VEGF (Endocrine Gland-Derived Endothelial Growth Factor) in choricarcinoma pathogenesis. EG-VEGF acts via two GPCR receptors PROKR-1 and PROKR-2. Three approaches were used to verify this hypothesis. A clinical approach using sera and placental samples collected from HM (n=38) and Choriocarcinoma patients (n=3) and from normal pregnant women (n=18); all collected during the first trimester of pregnancy. An In vitro approach using JEG3 cells, a human choriocarcinoma cell line and normal first trimester trophoblast cells (NTC). An in vivo approach that aimed at developing an animal model of choriocarcinoma in which therapeutic agents have been tested. Circulating EG-VEGF levels were significantly higher in HM and choriocarcinoma compared to normal patients. Placental EG-VEGF, PROKR1 and PROKR2 expression exhibited the same pattern. In JEG3 cells, EG-VEGF increased i) the expression of PROKR-1 and PROKR-2, ii). Their migration, proliferation invasion and spheroid formation using both 2 and 3D culture systems. These effects were abolished using PROKR1 and PROKR2 antagonists, iii) phosphorylation of different proteins involved in tumor progression as well as secretion of MMP-2 and MMP-9. Choriocarcinoma model has been developed by injection of JEG3 cells orthotopically within the placenta of SCID mice (Patent in progress). Within 12 days, injected gravid mice developed a choricarcinoma that metastasis in multiple organs. Importantly, injection of EG-VEGF receptors antagonists significantly reduced tumor development and its progression. Also, we have characterized the mechanism by which EG-VEGF contributes to tumor progression. This mechanism involves the cleavage of the key junctional protein, the VE-cadherin, following its phosphorylation at the tyrosine 685, by EG-VEGF. In total my thesis project i) demonstrated the direct involvement of the EG-VEGF in the development and progression of choriocarcinoma ii) elucidated the mechanism of this progression and iii) proposes a potential therapeutic for choriocarcinoma through the antagonisation of its receptors.

L’endothélium est considéré comme le plus grand organe endocrine, et est impliqué dans plusieurs processus physiologiques tels que l’inflammation et l’angiogenèse. Toute altération fonctionnelle/structurale peut induire de nombreuses maladies (des maladies cardiovasculaires jusqu’aux maladies métaboliques). L’identification de nouveaux facteurs moléculaires permettant de faire un lien entre les maladies cardiaques et les désordres métaboliques est de grande importance. Les prokinéticines sont des hormones peptidiques largement distribuées dans les tissus des mammifères. Elles agissent comme des facteurs de survie/mitogéniques dans plusieurs types cellulaires, notamment les cellules endothéliales, et sont impliquées dans la régulation des activités biologiques tels que l’angiogenèse et la physiopathologie cardiaque et rénale. Les prokinéticines agissent via deux récepteurs couplés aux protéines G: PKR1 et PKR2. Récemment il a été montré que l’activation de PKR1 induit l’angiogenèse tandis que l’activation de PKR2 induit la fenestration des cellules endothéliales, et que l’inactivation de PKR1 entraîne des anomalies cardiaques et rénales.Le but de cette étude est de comprendre à quel point la signalisation endothéliale de PKR1 peut affecter la fonction des organes adultes in vivo. Pour cela, nous avons généré une lignée de souris dans laquelle le PKR1 est spécifiquement inactivé dans les cellules endothéliales. Les souris mutantes présentent des dysfonctionnements endothéliaux avec des anomalies cardiaques, rénales et métaboliques. Les résultats montrent que PKR1 pourrait être considéré comme une cible pour le traitement des maladies cardiaques, rénales et métaboliques. / Endothelium is the largest living organ which has important role in many biological functions such as maintaining vascular smooth muscle tone, angiogenesis, cell proliferation, tissue growth and regulating lipid oxidation. Abnormalities in endothelium cause a large wide range of diseases from cardiovascular diseases to metabolic disorders. This emphasizes the important need for in-depth study of molecular and cellular mechanisms involved in endothelial dysfunction. Prokineticins are peptidic hormones widely distributed in peripheral and various endocrine mammalian tissues. Prokineticins are involved in many biological functions including angiogenesis, gastro-intestinal motility and regulation of heart and kidney physiopathology. These regulatory peptides act via two G protein couple receptor, PKR1 and PKR1. Recent studies show that PKR1 is involved in angiogenesis however PKR2 induces fenestration in coronary endothelial cells. However the role of PKR1 regulation in endothelial cells in vivo has not been studied yet.The aim of this study is to understand to what extent endothelial-prokineticin signaling affect adult organ functions in vivo. We had generated mutant mice lacking PKR1 in endothelium, by Cre-loxP technology, and studied how inactivation of PKR1 in endothelial cells affects different peripheral organ functions. These mice exhibit endothelial dysfunction as observed by abnormal endothelial cell proliferation and endothelial dependent relaxations. Moreover these mice exhibit cardiac, renal and metabolic disorders. Results indicate that PKR1 can be target for treatment of cardiovascular and metabolic disorders.