DYNAGAME

Infertility is a major challenge in all occidental and developing countries since 16 % of genitally active couples consult at least once for an infertility problem, and 10% of them will use an assisted reproductive program to achieve their child project. Sperm-oocyte fusion is a major biological event of fertilization. Research in this area has so far been limited to classical approaches, and very few teams have addressed this problem because of the difficulty in obtaining mammalian oocytes in large numbers and in imaging molecular events during sperm-oocyte interaction with sufficiently high resolution. The key questions reside in the processes that take place between the onset contact of sperm and oocyte till fusion regarding the membrane proteins necessary for fertilization, the oocyte membrane organization, its attachments to the cytoskeleton and its dynamics. The present project aims to answer some of the most pertinent questions thanks to the complementarity of three research teams in reproductive medicine, biology/biochemistry and physics. The objective is to investigate the oocyte-sperm contact by a panel of innovating techniques, among which a biomembrane force probe that allows intercellular force measurement at single molecular level and a new microfluidic development that allows achieving a guided fertilization under confocal microscope and therefore to image with high resolution in space and time of the sperm-egg contact area. These approaches will be combined with the production of molecular constructs and gene knock-out cells and functional assays. One oocyte membrane protein, the tetraspanin CD9, known as necessary for fertilization, was shown by us to organize the oocyte membrane in pro-fusional domains strongly linked to the cytoskeleton while the rest of the membrane only produces weak links. On the sperm side, one protein, Izumo 1, has been shown to be mandatory for fertilization. In this project, recombinant Izumo proteins generated by Partners 2 and 3 and cells transfected with Izumo 1 by Partner 1 will be used to study the specific action of this essential protein in the gamete adhesion and fusion process, to determine possible sperm partner and to identify a putative receptor for Izumo or its patners on egg membrane. Several recent results suggest that the egg membrane is attached to the cytoskeleton indirectly through EWI and ERM proteins. The inhibition of these molecules is expected to produce the disappearance of pro-fusional strong adhesion similarly to CD9 deletion in oocytes. A membrane remodeling is known to take place along with gamete interaction, but its role is unclear. This remodeling will be observed through an original setup for guided fertilization experiments: a microfluidic system brings one sperm to adhere on one chosen spot of an oocyte surface. With the proper fluorescence labelling, the diffusion of egg proteins, their association, and membrane reorganization during the transition from sperm-egg adhesion to fusion will be followed through high resolution in time and space confocal imaging. Partner 1 has already obtained the proof of concept of this guided fertilization. Most of the challenging stages of this project have already received a proof that they are achievable. The whole project is nevertheless difficult, but the deep expertise of the three complementary partners, each in his discipline, gives fair chances of success.