Oocyte ERM and EWI Proteins Are Involved in Mouse Fertilization.

J Cohen,A Ziyyat,S Marques,B Lefèvre,C Gourier,L Wang,C Ialy-Radio,S Barbaux

doi:10.3389/fcell.2022.863729

Abstract

In mammalian fertilization, the link between the oocyte plasma membrane and underneath cytoskeleton has often been associated to key elements of successful gamete fusion, like microvilli shaping or CD9 function, but its effective role has poorly been studied. EWI-2 and EWI-F as cis partners of CD9, and ERM proteins (Ezrin, Radixin and Moesin) that both attach to the actin cytoskeleton and to the EWI are part of the molecules that make the link between the oocyte membrane and its cytoskeleton. This study aims to assay through siRNA inhibition, the involvement of these ERM and EWI molecules in mouse fertilization, their role in the microvilli morphology of the egg but also their possible contribution to the cortical tension, a parameter that reflects the mechanical behavior of the oocyte cortex. Whereas inhibiting separately the expression of each protein had no effect on fertilization, the combined inhibition of either EWI-2/EWI-F or the three ERM triggered a significant decrease of the fertilization index. This inhibition seems to correlate with an increase in the radius of curvature of the oocyte microvilli. It also causes a decrease of the oocyte cortical tension. These results show the importance of EWI-2 and EWI–F and ERM proteins in the smooth running of a fertilization event and support their involvement in the microvilli architecture of the oocyte and in its mechanical properties.

Highlights

Fertilization is the process of union of two gametes
Effect of the EWI-2 and EWI-F small interfering RNA (siRNA) Injection on the Corresponding Protein Membrane Expression When observed by epifluorescence after immunostaining 24 h after microinjection of combined EWI-2 and EWI-F siRNA or CTRL siRNA, EWI-2 was localized on the egg membrane with no difference detectable to the naked eye between the two groups
A clear reduction of expression of both EWI-2 and EWI-F was observed on the oocytes injected with EWI-2 and EWI-F siRNA compared to the CTRL group (Figure 2A d vs. e and g vs. h)

Summary

Introduction

Fertilization is the process of union of two gametes. It occurs in several steps and culminates by the encounter of highly differentiated gametes. One article suggested that CD9 may function in a trans manner during gamete fusion by binding to an immunoglobulin (Ig) super family member, namely PSG17 (Ellerman et al, 2003). This protein has never been found on sperm. Crystal and cryoelectron microscopic structure of human CD9 and CD9-EWI-2 complex showed the interaction between CD9 and EWI-2, mediated by small residues in the transmembrane region and protein/lipid interaction suggesting the possible role of EWI-2 as a bridge between tetraspanins and other proteins, required for remodeling of membrane resulting in the formation of complex protein network. Cryo-electron microscopic structure of the CD9-EWI-2 complex and the mutation analysis revealed the importance of the small residues in the transmembrane region for the complex formation between CD9 and EWI-2 and the partial involvement of the CD9 large extracellular loop in the complex formation with EWI-2 (Umeda et al, 2020)

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