Spermatozoa lacking Fertilization Influencing Membrane Protein (FIMP) fail to fuse with oocytes in mice

Yoshitaka Fujihara,Yonggang Lu,Taichi Noda,Asami Oji,Tamara Larasati,Kanako Kojima-Kita,Zhifeng Yu,Ryan M Matzuk,Martin M Matzuk,Masahito Ikawa

doi:10.1073/pnas.1917060117

Abstract

Sperm-oocyte fusion is a critical event in mammalian fertilization, categorized by three indispensable proteins. Sperm membrane protein IZUMO1 and its counterpart oocyte membrane protein JUNO make a protein complex allowing sperm to interact with the oocyte, and subsequent sperm-oocyte fusion. Oocyte tetraspanin protein CD9 also contributes to sperm-oocyte fusion. However, the fusion process cannot be explained solely by these three essential factors. In this study, we focused on analyzing a testis-specific gene 4930451I11Rik and generated mutant mice using the CRISPR/Cas9 system. Although IZUMO1 remained in 4930451I11Rik knockout (KO) spermatozoa, the KO spermatozoa were unable to fuse with oocytes and the KO males were severely subfertile. 4930451I11Rik encodes two isoforms: a transmembrane (TM) form and a secreted form. Both CRISPR/Cas9-mediated TM deletion and transgenic (Tg) rescue with the TM form revealed that only the TM form plays a critical role in sperm-oocyte fusion. Thus, we renamed this TM form Fertilization Influencing Membrane Protein (FIMP). The mCherry-tagged FIMP TM form was localized to the sperm equatorial segment where the sperm-oocyte fusion event occurs. Thus, FIMP is a sperm-specific transmembrane protein that is necessary for the sperm-oocyte fusion process.

Highlights

Sperm–oocyte fusion is a critical event in mammalian fertilization, categorized by three indispensable proteins
We found that mouse 4930451I11Rik has two isoforms, TM (+) and secreted (TM [−]; Fig. 1E)
Protein structure analysis predicted that the TM form of 4930451I11Rik is a type I single-pass transmembrane protein containing a cleavable N-terminal signal peptide and a transmembrane-encoding sequence (SI Appendix, Fig. S1 B and C)

Summary

Introduction

Sperm–oocyte fusion is a critical event in mammalian fertilization, categorized by three indispensable proteins. 4930451I11Rik encodes two isoforms: a transmembrane (TM) form and a secreted form Both CRISPR/Cas9-mediated TM deletion and transgenic (Tg) rescue with the TM form revealed that only the TM form plays a critical role in sperm–oocyte fusion. We renamed this TM form Fertilization Influencing Membrane Protein (FIMP). | | | | CRISPR/Cas fertilization infertility IZUMO1 transgenic (officially named IZUMO1R) was identified as the oocyte receptor of IZUMO1, and Juno KO female mice were infertile due to impaired sperm–oocyte fusion [15]. Few sperm–oocyte fusion factors have been unveiled to date, and only IZUMO1 has been identified as a sperm-specific fusion-mediating protein. We discovered a factor responsible for sperm–oocyte fusion in mammals, and this knowledge could be used to develop in vitro and in vivo infertility treatments as well as male contraceptives

Methods

Results

Conclusion