Oocyte-triggered dimerization of sperm IZUMO1 promotes sperm-egg fusion in mice.

Naokazu Inoue,Takahisa Suzuki,Ikuo Wada,Yoshihisa Hagihara,Danelle Wright

doi:10.1038/ncomms9858

Abstract

Sperm–egg fusion is indispensable for completing mammalian fertilization. Although the underlying molecular mechanisms are poorly understood, requirement of two spermatozoon factors, IZUMO1 and SPACA6, and two oocyte factors, CD9 and the IZUMO1 counter-receptor JUNO, has been proven by gene disruption, and the binding of cells to an oocyte can be reconstituted by ectopic expression of IZUMO1. Here we demonstrate that robust IZUMO1-dependent adhesion of sperm with an oocyte accompanies the dimerization of IZUMO1. Despite the intrinsic dimeric property of its N-terminal region, IZUMO1 is monomeric in spermatozoa. Interestingly, JUNO associates with monomeric IZUMO1, which is then quickly removed as tight adhesion of the two cells is subsequently established. We therefore propose that global structural rearrangement of IZUMO1 occurs on JUNO recognition and that this rearrangement may then initiate force generation to overcome repulsion between the juxtaposing membranes, through an unidentified receptor on the egg.

Highlights

Sperm–egg fusion is indispensable for completing mammalian fertilization
This study focuses on the mechanism of the structural changes of IZUMO1 that occur at the moment of fusion, which can only be viewed in a cultured cell zona-free oocyte-binding system, as it is incapable of proceeding to fusion
By using newly produced IZUMO1 monoclonal antibodies, bimolecular fluorescence complementation (BiFC), and a photon-counting histogram (PCH), we found that dimerization of IZUMO1 can occur at the adhesive surface between cultured cells or between spermatozoa and an oocyte, and appears to be critical for the tight binding

Summary

Introduction

Sperm–egg fusion is indispensable for completing mammalian fertilization. the underlying molecular mechanisms are poorly understood, requirement of two spermatozoon factors, IZUMO1 and SPACA6, and two oocyte factors, CD9 and the IZUMO1 counter-receptor JUNO, has been proven by gene disruption, and the binding of cells to an oocyte can be reconstituted by ectopic expression of IZUMO1. We specified the critical domain of IZUMO1 for sperm–egg fusion and clarified its function by epitope mapping fusion-inhibiting monoclonal antibodies and by a series of biophysical measurements[12]. This fragment, including the potential functional site, binds directly to zona-free eggs and has fusion-inhibitory activities in an in vitro fertilization system. By using newly produced IZUMO1 monoclonal antibodies, bimolecular fluorescence complementation (BiFC), and a photon-counting histogram (PCH), we found that dimerization of IZUMO1 can occur at the adhesive surface between cultured cells or between spermatozoa and an oocyte, and appears to be critical for the tight binding. The analyses demonstrated that JUNO no longer existed at the interface, strongly suggesting the presence of an alternative egg receptor other than JUNO

Methods

Results

Conclusion