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

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.

Sperm proteins SOF1, TMEM95, and SPACA6 are required for sperm−oocyte fusion in mice

“…Hence, the transmembrane domain may be important for the normal functioning of TMEM95 in underpinning sperm−oocyte fusion. The necessity of the transmembrane domain in fusion-related sperm factors has also been demonstrated in FIMP (31). The impaired fusion ability of Fimp KO spermatozoa is restored by Tg expression of the membrane isoform of FIMP.…”

“…While a combination of IVF and fusion-inhibiting antibodies had nominated critical proteins, IZUMO1 was, until now, the only sperm protein proven to be essential by KO mouse studies (2,4). Recently, by making KO mice using the CRISPR-Cas9 system, we identified sperm protein FIMP as a sperm−oocyte fusion-related protein (31). Specifically, FIMP localizes to the equatorial segment of acrosome-intact spermatozoa, and a faint signal remains after the acrosome reaction in some spermatozoa.…”

“…In fact, we revealed that more than 90 genes are dispensable (22)(23)(24)(25)(26), while 10 genes and two clusters are required (21,(27)(28)(29)(30) for male fertility. Recently, we reported that a testis-specific gene, 4930451I11Rik (renamed as Fertilization Influencing Membrane Protein [Fimp]), is another sperm factor critical for sperm−oocyte fusion (31). While the amount and localization of IZUMO1 in Fimp KO spermatozoa are normal, Fimp KO males are severely subfertile due to a sperm−oocyte fusion defect.…”

Find and fuse: Unsolved mysteries in sperm–egg recognition

“…Evidence suggesting that ZP3 and/or glycans are the ZP sperm receptor has also been obtained [35]; however, targeted gene deletion experiments in animal models did not settle the question because ZP2 and ZP3 are necessary for the formation of the ZP [29,36]. Notably, none of the new essential sperm proteins seem to be involved in ZP binding, as removal of the ZP does not overcome the infertility defect of gene-deleted sperm [25][26][27][28]. The crystal structure of ZP proteins might finally provide the long-awaited answer, and a recent structure of the ZP domain, a protein polymerisation module of approximately 260 amino acids found in many secreted proteins including all ZPs, suggests that the sperm-binding region might lie at the interface between the ZP2 and ZP3 subunits [37].…”

“…The introduction of gene editing technologies in the 1980s represented an invaluable tool to investigate gene functions in model organisms [21], and the remarkable advances made over the last decade [22] have made the creation of gene-deficient mice much easier and have been systematically applied to investigate the role of a large number of potential sperm candidates, many of which were found to have no role in fertility [23,24]. Remarkably, in just a few months, 4 new genes that encode sperm cell surface or secreted proteins have been reported that are essential for male fertility: SPerm ACrosome membrane-Associated protein 6 (Spaca6), Fertilisation Influencing Membrane Protein (Fimp), Sperm-Oocyte Fusion required 1 (Sof1), and TransMEMbrane protein 95 (Tmem95) [25][26][27][28]. In this article, we will discuss 2 fundamental and yet enduring mysteries concerning sperm-egg recognition and place these recent discoveries into our understanding of fertilisation.…”