Low fertility is a major cause of lost productivity in the cattle industry. In addition, cattle provide a convenient model to study ovarian physiology in monovular species including humans. Our previous microarray studies in the bovine ovary showed the upregulation of two clusters, miR-212-132 and miR-183-96-182, in luteal relative to follicular tissues. The studies in this thesis were aimed at establishing the functional involvement of these miRNAs during the follicle-luteal transition using a bovine model as well as human tissues. The aim of the first study was to characterise the expression of miR-132 and miR-96 within luteal tissue using ISH and FACS. The expression of miR-132 was detected in most luteal compartments while miR-96 was not detectable using ISH. Further examination using FACS showed that miR-212-132 expression remained unchanged in sorted endothelia (CD144+) and steroidogenic (CD144-Nile Red (NR) +) cell fractions. In contrast, expression of miR-183 and miR-96 was significantly increased in CD144+ compared to CD144-NR+ fractions. To elucidate potential roles of these miRNAs in the CL, I used existing online databases to identify putative miRNA targets. I identified 3042 predicted bovine gene targets of these miRNAs as well as 174 miRNA targets that had been experimentally validated in human, mouse and/or rat. I also identified putatively targeted signalling pathways primarily involved in cell survival, proliferation and differentiation. For further investigation, I narrowed my list of targets to FOXO1 and ADCY6, the expression of which was naturally down- regulated during luteinisation. The second study used an in vitro model of bovine granulosa cell luteinisation. Levels of miR-183-96-182 and miR-212-132 increased significantly (P < 0.05) during the first 4 days of luteinisation in vitro. The function of miR-132 and miR-96 during luteinisation in vitro was studied. Transfection of bovine granulosa cells with specific miRNA inhibitors or mimics of miR-132 and miR-96 led, respectively, to abolished expression and a significant increase in the levels of these miRNAs (P < 0.01) within 4 days. These changes in miRNA levels did not have any effect on transcript levels of the predicted mRNA targets, FOXO1 and ADCY6, during luteinisation. However, progesterone production by luteinising granulosa cells decreased (P < 0.05) on day 2 after transfection with miR-132 inhibitor. The results demonstrated that putative miRNA target genes remained unchanged during in vitro luteinisation which was not consistent with in vivo results. The third study aimed to elucidate the effect of miRNA inhibition in bovine luteal cells in culture. The loss of miR-132 led to an increase (P < 0.05) in FOXO1 transcript but not protein levels. In contrast, inhibition of miR-96 increased protein but not transcript levels of FOXO1. Moreover, miR-96 inhibition induced an increase in the caspase 3/7 response of luteal cells to serum deprivation indicating an anti-apoptotic effect of this miRNA on these cells. In the fourth study, I investigated the role of miR-132 and miR-96 in human luteinised granulosa cells obtained from IVF patients. The levels of FOXO1 protein were significantly increased following depletion of miR-132 and miR-96, whereas caspase3/7 increased in response to miR-96 inhibition, regardless of whether cells had been serum deprived or not. Similarly, using Annexin V and Trypan blue staining an increase in numbers of apoptotic cells was observed in response to miR- 96 inhibition. In addition, reduction of FOXO1 with the siRNA inhibited the apoptotic effect of miR-96 inhibition. Interestingly, inhibition of pooled miR-132 and miR-96 reduced progesterone secretion. However, this effect was prevented by transfecting cells with FOXO1 siRNA. These results suggest that the effects of these miRNAs on cell survival and progesterone production are mediated through targeting FOXO1. In summary, my results identify miR-96 and miR-132 as potentially critical factors in ensuring luteal cell survival and steroidogenesis in both cattle and human.

La mise en place de la lignée germinale au cours du développement constitue une des étapes fondamentales conditionnant la fertilité de l’individu adulte. Au cours des dernières décennies, le nombre croissant de couples consultant pour une aide médicale à la procréation a fait émerger l’hypothèse d’une altération des fonctions de reproduction chez l’Homme qui pourrait trouver son origine dans la perturbation du développement précoce. Dans l’ovaire fœtal, les cellules germinales s’orienteront vers la voie de l’ovogénèse, caractérisée entre autres par l’entrée en méiose de ces cellules. La majorité des données actuelles relatives à ces évènements sont issues du modèle murin alors que le développement de l’ovaire humain est significativement diffèrent de celui de la souris. Il est donc nécessaire d’approfondir nos connaissances du développement ovarien humain et d’identifier ses éventuelles perturbations. L’objectif de mon travail a été de mettre au point un outil d’étude du développement ovarien et d’identifier de nouvelles voies impliquées dans la régulation de l’entrée en méiose des cellules germinales fœtales humaines et leurs perturbations éventuelles.Nous avons mis au point un nouveau modèle de xénogreffe d’ovaires fœtaux humains du premier trimestre de gestation (au moment de l’apparition des premières cellules méiotiques). Ce modèle nous a permis d’observer un développement de l’organe et une différenciation des cellules germinales similaires à ceux observés in vivo. Ce modèle permettra des travaux à des âges auxquels le matériel d’étude est peu accessible. En couplant ce modèle de xénogreffe à une stratégie d’ARN-interférence, il nous a été possible d’inhiber l’expression d’un gène spécifiquement exprimé dans les cellules germinales ovariennes, DMRTA2, et de mettre en évidence un potentiel rôle de ce gène dans leur différenciation pré-méiotique. Nous avons observé une diminution du nombre de cellules ayant initié la méiose après inhibition de l’expression de ce gène. Par ailleurs, nous avons également identifié la présence dans l’ovaire fœtal de nombreux marqueurs décrits comme testiculaires chez la souris (PLZF, DNMT3L, FGF9, NANOS2 ou CYP26B1). L’expression de ces marqueurs pourrait expliquer la présence de cellules mitotiques tardives dans l’ovaire fœtal humain que nous avons pu observer jusqu’à 30 semaines de gestation. En parallèle de ces travaux, nous avons testé la sensibilité des cellules germinales à la dexaméthasone, glucocorticoïde pouvant être administré au cours de la grossesse. Il a été observé une augmentation de l’expression de PLZF, gène cible de l’activation des récepteurs aux glucocorticoïdes, pouvant expliquer la diminution du nombre de cellules germinales.En conclusion, ce travail de thèse a permis d’identifier un nouveau gène potentiellement régulateur de la transition mitose/méiose dans l’ovaire humain, et d’affiner nos connaissances sur le développement de l’ovaire humain et l’entrée en méiose des cellules germinales. Toutefois, de nombreuses questions restent posées ainsi de futures études devront clarifier si les cellules germinales mitotiques observées à des stades tardifs sont capables de se différencier en ovocytes compétents. / Woman fertility is partially dictated by the set up of the human female germ line. During the last ten years, which saw an increased number of couples consulting for assisted reproductive cares, the hypothesis of an early alteration in reproduction functions has emerged.In the fetal ovary, germ cells enter the path of oogenesis differentiation characterized by meiotic initiation. On this subject, vast majority of the scientific data are obtained from the mouse model, even if differences with human ovarian physiology are widely acknowledged. Therefore it is necessary to extend our knowledge on human ovarian development and identify its perturbations. The objective of my work was to assess a new model to study ovarian growth, studying regulation of meiotic entry and perturbation of germ line differentiation.We sat up a new xenograft model of early human fetal ovaries, when very early meiotic germ cells appear. Organ growth and germ cells differentiation were comparable with in vivo observations. Using this model with an RNA-interference strategy, we inhibited the expression of an oogonia germ cell gene, DMRTA2. This inhibition conducted to a significantly reduced number of germ cells gene that initiated meiosis and DMRTA2 seemed to be required for mitotic-meiotic transition. In another hand, we identified, in the ovary, the expression of germ cells markers described as specifically male in rodent (PLZF, DNMT3L, FGF9, NANOS2 ou CYP26B1). The expression of these markers in the human ovary could explain the observation of mitotic germ cells in late fetal ovaries (30 wpf).In parallel, we tested germ cells sensibility to a synthetic glucocorticoid, dexamethasone, administrated during pregnancy in some justified pathologies. We observed an increased expression of PLZF that could explain the decreased number of germ cells observed in treated ovaries.In conclusion, we identified a new gene expressed in human fetal ovaries, potentially involved in the meiotic entry, and we extended our knowledge to characterized human germ line development. However, many points have to be clarified, as the possible competence of late mitotic germ cells to form oocytes.