Development of an in vitro microenvironment for maturing oocytes

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 typ