The fusion of egg and sperm requires an egg surface protein, JUNO in mammals or Bouncer in fish, and several conserved sperm surface proteins, including IZUMO1, SPACA6, DCST1 and DCST2. The binding of JUNO and IZUMO1 is known but is not sufficient for fusion, and the specific roles of SPACA6 and DCST1/DCST2 remain unclear. Deneke, Blaha et al. have now identified a conserved sperm complex that includes IZUMO1 and interacts with JUNO/Bouncer to bridge the sperm and egg for fusion.

To identify other sperm surface proteins that may bind to IZUMO1, SPACA6 and DCST1/DCST2, they performed an AlphaFold-Multimer screen of pairwise interactions between approximately 1,400 proteins known to be expressed in zebrafish testes. The top-scoring predicted interactions were between Izumo1 and Spaca6 and between Izumo1 and the protein Tmem81, with an unknown function in fertilization. AlphaFold-Multimer predicted a trimer between IZUMO1, SPACA6 and TMEM81, with JUNO able to access the binding site of IZUMO1. However, Bouncer was predicted to bind to a distinct site at an interface between Izumo1 and Spaca6, which was validated experimentally. Co-immunoprecipitation of IZUMO1, SPACA6 and TMEM81 confirmed the AlphaFold-Multimer prediction that these proteins interact, and mutations in various interfacial residues within the trimer led to sterility in zebrafish. To test the role of the newly identified protein TMEM81 in fertilization, the team generated Tmem81-knockout lines in zebrafish and mice; these animals turned out to be sterile with their sperm unable to fertilize eggs.