Fertilization is a decisive process in the life of higher organisms. For mammals, the process is almost doomed to fail without the tetraspanin Cd9 (2000) and Juno (2014) on the oocyte and the Ig-Super Family protein Izumo1 (2005) on the sperm. Nevertheless, basic questions about their roles and action modes are still not answered. The researchers cannot explain how sperm cells fuse with the egg? One of the reasons is the lack of experimental methods available.

2018

• Ravaux B. et al. – Egg CD9 protein tides correlated with sperm oscillations tune the gamete fusion ability in mammal, Journal of Molecular Cell Biology.

Mammalian fertilization involves membrane events—adhesion, fusion, sperm engulfment, membrane block to polyspermy whose causes remain largely unknown. Recently, specific oscillations of the sperm in contact with the egg were shown to benecessary for fusion. Using a microfluidic chip to impose the venue for the encounter of two gametes allowed real-time observation of the membrane remodelling occurring at the sperm/egg interface. The spatiotemporal mapping of egg CD9 revealed that this protein concentrates at the egg/sperm interface as a result of sperm oscillations, until a CD9-rich platform is nucleated on which fusion immediately takes place. Within 2–5 min after fusion, most of the CD9 leaves the egg for the external aqueous medium. Then an egg membrane wave engulfs the sperm head in ∼25 min. These results show that sperm oscillations initiate the CD9 recruitment that causes gamete fusion after which CD9 and associated proteins leave the membrane in a process likely to contribute to block polyspermy. They highlight that the gamete fusion story in mammals is an unexpected interplay between mechanical constraints and proteins.

2016

• Ravaux B. – Effect of the flagellum beating and of the spermatozoon lipids composition on the fusion step during mammalian gametes interaction, Thesis.

Fertilization is the encounter of two gametes. Although this process is crucial for sexual organisms, the timeline of the molecular events is not yet established. The researchers cannot explain: how the spermatozoon fuses with the oocyte? One of the reasons is the lack of experimental methods available. Indeed, the gametes need a specific environment to fertilize. Nevertheless, the scientific community identified three essential proteins: Izumo1 on the spermatozoon, Juno (its receptor) and CD9 on the oocyte membrane. For our part, we tried to determine if the none-proteins environment of Izumo1 and CD9 could influence the gametic interaction. To do so, we were focused on the role of the lipids composition of the sperm membranes and on the influence of the forces developed by the flagellum beating on the oocyte. We designed two original experimental methods to offer a better understanding of the mechanisms inside the gamete contact area. With the first one, we tried to identify the minimal machinery to induce fusion. We started to reconstitute step by step the membrane of the spermatozoon head. We tested the identified lipids alone, and then we coupled these molecules with Izumo1. With the second one, we developed a microfluidic tool to observe the gametic encounter with the “best” viewpoint in the most physiological in-vitro conditions. We observed that the flagellum beating is not only involved in the crossing of the female genital tract but also in the initiation of the fusion step. Indeed, the mechanical constraints induce membrane reorganization with CD9 recruitment. So we succeed to establish the kinetic of the events with an unequaled resolution.

• Ravaux B. et al. – A specific flagellum beating mode for inducing fusion in mammalian fertilization and kinetics of sperm internalization, Scientific Report.

The salient phases of fertilization are gamete adhesion, membrane fusion and internalization of the spermatozoon into the oocyte but the precise timeline and the molecular, membrane and cell mechanisms underlying these highly dynamical events are far from being established. The high motility of the spermatozoa and the unpredictable location of sperm/egg fusion dramatically hinder the use of real time imaging optical techniques that should directly provide the dynamics of cell events. Using an approach based on microfluidics technology, the sperm/egg interaction zone was imaged with the best front view and the timeline of the fertilization events was established with an unparalleled temporal accuracy from the onset of gamete contact to full sperm DNA decondensation. It reveals that a key element of the adhesion phase to initiate fusion is the oscillatory motion of the sperm head on the oocyte plasma membrane generated by a specific flagellum-beating mode. It also shows that the incorporation of the spermatozoon head is a two steps process that includes simultaneous diving, tilt and plasma membrane degradation of the sperm head into the oocyte and subsequent DNA decondensation.

2014

• Chalbi M., Barraud-Lange V., Ravaux B. et al. – Sperm protein Izumo binds an egg partner of CD9 conserved between mouse and human to drive fast and robust sperm-egg adhesion prior to fusion, Development.

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.

Talks and Poster Presentation

2017

• Invited speaker at the Institute for Advanced Biosciences: Role of the flagellum beating and of the spermatozoon lipids composition on the fusion step during mammalian gametes interaction, Grenoble, France.

2016

• Selected speaker at the EMBO Workshop on Membrane Fusion in health and disease: Kinetics of mammalian fertilization with microfluidics, Paris, France.
• Selected speaker at the Biophysical Meeting on Membrane Dynamics session: Remodeling of Gamete Membrane during Mammalian Fertilization, Los Angeles, USA.

2014

• Written communication at the Molecular Basis for Membrane Remodeling and Organization conference: Molecular mechanisms involve in gamete interaction during mammalian fertilization, Roscoff, France.
• Written communication at the Membrane, morphology and function conference of the Biochemical Society: Molecular mechanisms involve in gamete interaction during mammalian fertilization, Fara San Martino, Italy.
• Written communication at the Curie Institute workshop on Cellular membranes: Dynamics of membrane reorganizations triggered by the spermatozoon and oocyte encounter, Paris, France.