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.

卵子和精子的融合需要卵子表面蛋白（哺乳动物中的 JUNO）或鱼类中的 Bouncer，以及几种保守的精子表面蛋白，包括 IZUMO1、SPACA6、DCST1 和 DCST2。JUNO 和 IZUMO1 的结合是已知的，但不足以进行融合，并且 SPACA6 和 DCST1/DCST2 的具体作用仍不清楚。Deneke、Blaha 等人现在已经确定了一种保守的精子复合物，其中包括 IZUMO1，并与 JUNO/Bouncer 相互作用，在精子和卵子之间架起桥梁进行融合。

为了鉴定可能与 IZUMO1、SPACA6 和 DCST1/DCST2 结合的其他精子表面蛋白，他们对斑马鱼睾丸中已知表达的大约 1,400 种蛋白质之间的成对相互作用进行了 AlphaFold-Multimer 筛选。得分最高的预测相互作用是 Izumo1 和 Spaca6 之间以及 Izumo1 和蛋白质 Tmem81 之间，受精功能未知。AlphaFold-Multimer 预测了 IZUMO1、SPACA6 和 TMEM81 之间的三聚体，JUNO 能够进入 IZUMO1 的结合位点。然而，预测 Bouncer 会与 Izumo1 和 Spaca6 之间界面处的不同位点结合，这在实验中得到了验证。IZUMO1、SPACA6 和 TMEM81 的免疫共沉淀证实了 AlphaFold-Multimer 预测，即这些蛋白质相互作用，三聚体内各种界面残基的突变导致斑马鱼不育。为了测试新发现的蛋白质 TMEM81 在受精中的作用，该团队在斑马鱼和小鼠中生成了 Tmem81 敲除细胞系;这些动物被证明是不育的，它们的精子无法使卵子受精。