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The development of in vitro culture systems, comparable to the in vivo microenvironment in terms of effect on the oocyte growth and development could provide a valuable experimental tool for studying the mechanisms governing folliculogenesis. This tool might serve as well for practical clinical, agricultural, zoological, or biotechnological applications. This thesis reports on the importance of the microenvironment for the ovarian folliculogenesis process. The complexity of such a microenvironment was approached with a strategy based on functionalized PEG-hydrogels. The PEG matrix not only served as a scaffold, but it was also used a "reservoir" of immobilized cues. Tethered integrin-binding peptides in combination with other signaling factors aimed at better understanding the interactions of the oocyte and its surrounding granulosa cells that, most probably, determine the efficiency of the actual and the future in vitro mature oocyte production strategies. This work led also to investigating the lymph angiogenesis and the lymphatic transport in the context of oocyte maturation and their impact on mice fertility. In a first step, the mechanical properties of PEG-hydrogel were optimized for culturing secondary follicles. This report shows that the growth of the follicles was highly dependent on the mechanical properties of the surrounding environment. The optimal elastic modulus was found to be close to 900 Pa. In a second step, the effect of Arg-Gly-Asp (RGD) peptide, the minimal integrin-binding sequence, was studied. RGD presence did not influence the follicle growth rate but it significantly improved the quality of the produced oocytes. These findings demonstrated that approaching, biochemically and mechanically, the complexity of the ovarian extracellular matrix could be a winning strategy. The effect of key soluble factors was also investigated in order 1- to confirm their compatibility with the established 3-D culture system and 2- to further improve qualitatively and quantitatively the produced mature oocytes. Various combinations of gonadotropins such as the follicle-stimulating hormone (FSH) or the luteinizing hormone (LH) were tested. Interestingly, the effects of the gonadotropins in the 3-D PEG system were close to their known effects in vivo. Here the aberrant effects of these hormones in the used 2-D systems appeared clearly. c-Kit ligand (KL) is suspected to be one of the most important factors for the activation of primordial follicles and thus for controlling the exit from the resting pool. Previously, studying the effects of the two forms of KL (soluble and membrane-attached) had to cope with the lack of biologically-relevant immobilization strategies. For overcoming this problem, KL constructs were designed to include a substrate sequence for Factor XIIIa (NQEQVSPL or NQEQVSPLRCG). Thus the produced recombinant KL proteins could be enzymatically crosslinked to the PEG matrix. The different constructs of KL, including a wild type extracellular domain, were successfully cloned and produced from mammalian HEK-293 cells. The identity and the activity of the produced proteins were confirmed. Ovarian tissues from four days-old mice were cultured in PEG-hydrogels functionalized with KL. The results showed that the primordial follicles grew and were activated in the PEG-hydrogels where KL was immobilized but not when the soluble form of KL was preset in the medium. This experiment showed that the membrane-attached and the soluble form of KL play a different role in the rodent folliculogenesis. In parallel it was observed that blocking vascular endothelial growth factor receptor-3 (VEGFR-3) signaling had a critical but still unsuspected role in reproduction. This study demonstrates that variation in lymphangiogenesis is a regular, non-pathological event during folliculogenesis in the ovary; blocking lymphangiogenesis, might have an effect on hormone transport and thus on pregnancy. The reported results demonstrates that the blockade of lymphangiogenesis decreases the progesterone and estradioal levels during pregnancy and in fine results in failed fetal development. In conclusion, this study demonstrates the efficiency and the flexibility of a novel 3-D culture system. Circumventing problems inherent to the "on-plastic" standard culture, such as the loss of the granulosa-oocyte interactions, allowed the emergence of a culture system tailored for investigating fundamental folliculogenesis-related questions. Furthermore, the reported culture system might serve as a platform for developing clinical and biotechnology applications.