A DPY19L2/FAM209-associated protein–Arp complex–CAPZA3 scaffold is essential for perinuclear theca assembly and fertilization

Fertilization is a fundamental biological process that enables the fusion of a spermatozoon with an oocyte to initiate embryonic development. However, the underlying mechanisms leading to fertilization failure remain poorly understood. Here, we demonstrated that the actin-related protein (Arp) family members ACTRT2 and ACTRT3 are essential for fertilization in both humans and mice, whereas ACTL10 is dispensable for fertility and the human-specific ACTL8 may play a role in early spermatogenesis. Ultrastructural and immunofluorescence analyses revealed that, during spermatogenesis, ACTRT2 or ACTRT3 deficiency disrupts the assembly of subacrosomal layer of the perinuclear theca (SAL-PT), leading to acrosome detachment and subsequent manchette abnormalities, which in turn impairs the formation of the postacrosomal sheath of the PT. These structural defects were accompanied by reduced abundance of the candidate sperm-borne oocyte-activating factors PAWP and PLCζ. Moreover, knockout of Actrt2 or Actrt3 severely impaired spermatozoa’s ability to penetrate the zona pellucida during in vitro fertilization. Mechanistically, ACTRT2, ACTRT3, ACTL7A, ACTL7B, and ACTL9 form a testis-enriched Arp complex in the SAL-PT and maintain the stability of one another. This complex interacts with the PT protein CAPZA3, which subsequently binds and stabilizes PLCζ. Additionally, the Arp complex interacts with DPY19L2/FAM209-associated proteins to mediate acrosome anchoring to the nuclear envelope. Therefore, these findings established a sequential DPY19L2/FAM209-associated protein–Arp complex–CAPZA3 scaffold extending from the nuclear envelope to the acrosome that is indispensable for fertilization. Collectively, we defined a previously unidentified structural and molecular framework for fertilization and provided insights into the diagnosis and treatment of fertilization failure.