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

Abstract

<jats:p>Sperm−oocyte membrane fusion is one of the most important events for fertilization. So far, IZUMO1 and Fertilization Influencing Membrane Protein (FIMP) on the sperm membrane and CD9 and JUNO (IZUMO1R/FOLR4) on the oocyte membrane have been identified as fusion-required proteins. However, the molecular mechanisms for sperm−oocyte fusion are still unclear. Here, we show that testis-enriched genes, sperm−oocyte fusion required 1 (<jats:italic>Sof1</jats:italic>/<jats:italic>Llcfc1</jats:italic>/<jats:italic>1700034O15Rik</jats:italic>), transmembrane protein 95 (<jats:italic>Tmem95</jats:italic>), and sperm acrosome associated 6 (<jats:italic>Spaca6</jats:italic>), encode sperm proteins required for sperm−oocyte fusion in mice. These knockout (KO) spermatozoa carry IZUMO1 but cannot fuse with the oocyte plasma membrane, leading to male sterility. Transgenic mice which expressed mouse<jats:italic>Sof1</jats:italic>,<jats:italic>Tmem95,</jats:italic>and<jats:italic>Spaca6</jats:italic>rescued the sterility of<jats:italic>Sof1</jats:italic>,<jats:italic>Tmem95</jats:italic>, and<jats:italic>Spaca6</jats:italic>KO males, respectively. SOF1 and SPACA6 remain in acrosome-reacted spermatozoa, and SPACA6 translocates to the equatorial segment of these spermatozoa. The coexpression of SOF1, TMEM95, and SPACA6 in IZUMO1-expressing cultured cells did not enhance their ability to adhere to the oocyte membrane or allow them to fuse with oocytes. SOF1, TMEM95, and SPACA6 may function cooperatively with IZUMO1 and/or unknown fusogens in sperm−oocyte fusion.</jats:p>

Large-scale discovery of male reproductive tract-specific genes through analysis of RNA-seq datasets

“…Since Spint4/5 null male mice are severely subfertile, without an apparent difference in epididymis histology, sperm number, sperm morphology, and sperm motility parameters in comparison to the wild-type (WT) mice, this phenocopies the reproductive phenotype of several null mice of testis-, epididymis-, or prostate-specific genes (Sof1, Tmem95, and Spaca6; Pate8, and Pate10), which reveal a requirement in regulating sperm migration through the oviduct and sperm-oocyte fusion in mice [92,93]. A severe fertility defect associated with normal sperm number, morphology, and motility is also shared among mice lacking the sperm membrane protein ADAM3, thought to be crucial in sperm-ZP binding and sperm migration through the uterotubular junction (UTJ) [94,95].…”

Find and fuse: Unsolved mysteries in sperm–egg recognition

“…There is also a tacit assumption that the candidate fusogen maintains its activity when isolated from its native biological environment and expressed in a heterologous system. Like IZUMO1, SPACA6, FIMP, TMEM95, and SOF1 are not able to induce cell fusion either alone or together when assessed in a cell fusion assay [25][26][27][28]. These novel sperm proteins, therefore, seem unlikely to be the long-sought-after molecule that induces fusion in mammalian gametes.…”

“…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 use of CRISPR technologies has led to the recent and remarkable identification of 4 new sperm proteins that are essential for mammalian fertilisation (Fig 2). They are 3 membrane-anchored proteins, FIMP [28], SPACA6 [25,27], and TMEM95 [25,26], and a predicted secreted protein, SOF1 [25]. Similarly to IZUMO1, SPACA6 is a type I transmembrane protein with a short cytoplasmic C-terminus and an immunoglobulin (Ig)-like domain in the midst of its extracellular region.…”

“…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.…”