Adhesion and fusion between oocyte and sperm are crucial in the process of fertilization. Many questions about the molecular mechanisms that underlie these processes are still open today. During the last two decades, several proteins have been identified as playing an active role in mediating gametic interaction. However, the only transmembrane protein whose absence is known to cause a total defect of fertilization is the sperm protein Izumo1. Izumo1 belongs to the Izumo protein family, in turn belonging to the Immunoglobulin superfamily. Despite its essential role in mediating gametic fusion, it has not yet been determined whether the protein acts as an adhesion or fusion molecule, or as an organizer of proteins required for fusion or adhesion. This study has focused on the determination of the role of Izumo1 in the gamete interaction process. We have generated a transgenic construct, allowing the overexpression of Izumo1 on three different cell lines. We have then analyzed the interaction of Izumo1-expressing cells and oocytes with micromanipulation force measurement assays coupled to confocal imaging. We observed a strong adhesion of cells expressing Izumo1 with membrane oocytes and quantified the interaction kinetics. In addition, we generated a recombinant Izumo1 extracellular domain, to investigate whether Izumo1 alone is sufficient to bind to the oocyte. We have probed the interaction of Izumo1 and the oocyte at the single molecule level using the biomembrane force probe technique. These experiments confirmed that Izumo1, and not one of its partners, is responsible for the strong adhesion between Izumo1-expressing cells and oocytes. Finally, we observed that oocyte can interact with multiple Izumo1-GFP-expressing cells at the same time and that Izumo1-GFP accumulated in the contact area. Both observations suggest that the molecular partner of Izumo1 is widely expressed on the oocyte membrane.
