A conserved fertilization complex of Izumo1, Spaca6, and Tmem81 mediates sperm-egg interaction in vertebrates

Abstract

Fertilization, the fusion of sperm and egg, is essential for sexual reproduction. While several proteins have been demonstrated to be essential for the binding and fusion of gametes in vertebrates, the molecular mechanisms driving this key process are poorly understood. Here, we performed a protein interaction screen using AlphaFold-Multimer to uncover protein-protein interactions in fertilization. This screen resulted in the prediction of a trimeric complex composed of the essential fertilization factors Izumo1 and Spaca6, and Tmem81, a protein previously not implicated in fertilization. We show that Tmem81 is a conserved, testis-expressed transmembrane protein that is evolutionarily related to Izumo1 and Spaca6 and is essential for male fertility in fish and mice. Consistent with trimer formation in vivo, zebrafish izumo1-/-, spaca6-/-, and tmem81-/- mutants exhibit the same sperm-egg binding defect and show co-depletion of all three proteins in sperm. Moreover, we provide experimental evidence that Izumo1, Spaca6, and Tmem81 interact in zebrafish sperm. Strikingly, the Izumo1-Spaca6 interaction is predicted to form a cleft that serves as a binding site for Bouncer, the only identified egg protein essential for fertilization in zebrafish. Together, these results provide compelling evidence for a conserved sperm factor complex in vertebrates that forms a specific interface for the sperm-egg interaction required for successful fertilization.

Deep learning insights into the architecture of the mammalian egg-sperm fusion synapse

“…Of direct relevance to these questions is an independent study, submitted back to back with the original preprint of the present manuscript, which provides experimental data for the existence of a trimeric IZUMO1/SPACA6/TMEM81 complex in zebrafish and suggests that this interacts with egg Bouncer (Deneke et al, 2023). Considering that the mammalian orthologue of Bouncer is expressed on sperm instead of the egg (Fujihara et al, 2021) and that role of CD9 in egg-sperm fusion is much more important in mammals than in fish (Greaves et al, 2022), the combination of our studies raises the intriguing possibility that, during the course of evolution, CD9 may have substituted Bouncer as a binding partner of the IZUMO1/SPACA6/TMEM81 complex.…”

No evidence for a direct extracellular interaction between human Fc receptor–like 3 (MAIA) and the sperm ligand IZUMO1

“…1D ); however, no direct interactions between FCRL3/MAIA and either IZUMO1 or JUNO could be detected using this approach. IZUMO1 has recently been shown to form a complex with two structurally related sperm cell surface proteins: SPACA6 and TMEM81 ( 22 , 26 ). Both mice and zebrafish with targeted deletions in any one of these three genes results in male sterility ( 4 , 15 , 17 , 18 , 22 ).…”

“…It does not appear that the IZUMO1-JUNO interaction has a direct role in membrane fusion because neighboring cells expressing these proteins do not fuse ( 6 ), nor do IZUMO1-expressing cells fuse with oocytes ( 13 – 15 ), suggesting that this interaction has an adhesive rather than fusogenic role. Targeted gene deletions in mice have more recently identified a range of other cell surface and secreted proteins that are essential for mammalian fertilization, including the sperm proteins TMEM95 ( 15 , 16 ), SPACA6 ( 17 , 18 ), SOF1 ( 15 ), FIMP ( 19 ), DCST1, DCST2 ( 20 , 21 ), TMEM81 ( 22 ), and egg CD9 ( 23 , 24 ) [reviewed in ( 25 )]. Biochemical and modeling studies have suggested that some of these proteins may function as part of a larger multiprotein complex ( 22 , 26 ); however, whether this complex is directly involved in membrane fusion is not yet clear.…”

“…Targeted gene deletions in mice have more recently identified a range of other cell surface and secreted proteins that are essential for mammalian fertilization, including the sperm proteins TMEM95 ( 15 , 16 ), SPACA6 ( 17 , 18 ), SOF1 ( 15 ), FIMP ( 19 ), DCST1, DCST2 ( 20 , 21 ), TMEM81 ( 22 ), and egg CD9 ( 23 , 24 ) [reviewed in ( 25 )]. Biochemical and modeling studies have suggested that some of these proteins may function as part of a larger multiprotein complex ( 22 , 26 ); however, whether this complex is directly involved in membrane fusion is not yet clear. Recent research has suggested that IZUMO1 could itself directly act as a membrane fusogen ( 27 ), although some questions remain ( 28 ).…”

Sperm induction of somatic cell-cell fusion as a novel functional test

“…Considering our previous work showing that IZUMO1 can mediate cell-cell fusion (Brukman et al 2023) and that significantly lower levels of this protein were detected in the sperm cells from patients with total fertilization failure (Enoiu et al 2022), we expect that the SPICER assay will be able to resolve some unexplained cases of male infertility that result from loss of function of components of the fusion machinery. In this context, other proteins that are essential for gamete fusion, such as SPACA6, TMEM95, TMEM81, FIMP, SOF1 and DCST1/2 (Barbaux et al 2020; Noda et al 2020; Lorenzetti et al 2014; Lamas-Toranzo et al 2020; Fujihara et al 2020; Inoue, Hagihara, and Wada 2021; Noda et al 2022; Elofsson et al 2023; Deneke et al 2023), might be transferred to the somatic cells together with IZUMO1 during sperm-induced cell-cell fusion. Finally, considering that antibodies against IZUMO1 were detected on the sera of infertile women (Clark and Naz 2013) and here we observed that a monoclonal antibody against this protein was able to block sperm-induced syncytia, SPICER could serve as tool to readily analyze cases of immuno-infertility.…”