CD9 tetraspanin is the only egg membrane protein known to be essential for fertilization. To investigate its role, we have measured, on a unique acrosome reacted sperm brought in contact with an egg, the adhesion probability and strength with a sensitivity of a single molecule attachment. Probing the binding events at different locations of wild-type egg we described different modes of interaction. Here, we show that more gamete adhesion events occur on Cd9 null eggs but that the strongest interaction mode disappears. We propose that sperm-egg fusion is a direct consequence of CD9 controlled sperm-egg adhesion properties. CD9 generates adhesion sites responsible for the strongest of the observed gamete interaction. These strong adhesion sites impose, during the whole interaction lifetime, a tight proximity of the gamete membranes, which is a requirement for fusion to take place. The CD9-induced adhesion sites would be the actual location where fusion occurs.force measurement | cell-cell adhesion | membrane organization | Biomembrane Force Probe | contact lifetime M embrane fusion occurs in many contexts: virus-cell fusion, intracellular fusion, cell-cell fusion. It takes place in two steps: attachment of two membranes and physical merging of the membranes and inner contents. Numerous questions on this complex process are still open. This is particularly true for gamete fusion during fertilization. So far, among the molecular actors of sperm-egg interaction identified at the gamete membranes (1-5), CD9 tetraspanin is the only egg protein that has been proven to be essential for fertilization of mice (6-10) and other mammal species (11-13). Indeed, deletion of Cd9 gene results in a dramatic reduction of female fertility due to a lack of fusion of sperm with Cd9 null eggs (7,8,10). The mechanism by which CD9 takes part in gamete fusion still needs to be elucidated. However, the main function attributed to tetraspanins is to organize networks of cis-partner proteins within the plasma membrane (14-19) Deletion of Cd9 tetraspanin gene has also been reported to alter the morphology of egg microvilli (20). The relation between CD9-dependent membrane organization and fusion ability needs to be clarified. If decisive for fusion, CD9-driven membrane morphology and molecular distribution could have crucial effects on gamete adhesion first, because adhesion is the first and necessary step in any fusion process. So far, combination of zona-free eggs in vitro fertilization and binding assays has shown that the lack of fusion observed for Cd9 null eggs was not accompanied by a loss of sperm-egg adhesion (7,8,10). However, this method, that only allows counting the number of sperm attached to the egg membrane after an extended period of coincubation, cannot reveal any adhesion detail due to CD9-controlled membrane organization. Revealing such specificity requires the characterization of the sperm-egg attachments at the single molecule level. We have recently developed a biophysical approach allowing such an accurate characteriza...

Little is known about the molecular mechanisms that induce gamete fusion during mammalian fertilization. After initial contact, adhesion between gametes only leads to fusion in the presence of three membrane proteins that are necessary, but insufficient, for fusion: Izumo1 on sperm, its receptor Juno on egg and Cd9 on egg. What happens during this adhesion phase is a crucial issue. Here, we demonstrate that the intercellular adhesion that Izumo1 creates with Juno is conserved in mouse and human eggs. We show that, along with Izumo1, egg Cd9 concomitantly accumulates in the adhesion area. Without egg Cd9, the recruitment kinetics of Izumo1 are accelerated. Our results suggest that this process is conserved across species, as the adhesion partners, Izumo1 and its receptor, are interchangeable between mouse and human. Our findings suggest that Cd9 is a partner of Juno, and these discoveries allow us to propose a new model of the molecular mechanisms leading to gamete fusion, in which the adhesion-induced membrane organization assembles all key players of the fusion machinery.

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