Soluble ZP2 N-terminal fragments activate CatSper-dependent Ca2+ entry and regulate motility and acrosomal exocytosis in mammalian sperm

Sperm motility and function are central to mammalian fertilization and are tightly regulated by intracellular calcium (CaCa2+) signaling. This signaling is primarily orchestrated by the CatSper Ca2+; channel complex located in the flagella of spermatozoa. However, the natural ligands that activate CatSper remain largely unknown in many species, despite the conservation of CatSper in mammals. Here, we present a signaling role for soluble N-terminal ZP2 fragments in regulating CatSper activity and sperm physiology in mice and humans. ZP2 has been implicated in mediating sperm binding and recognition at the oocyte surface interface; however, new evidence is starting to unveil the molecular mechanisms and function of ZP2 during fertilization transition. Here, we show that the during fertilization, cleaved ZP2 N-terminal fragment triggers a rapid and robust increase in intracellular CaCa2+ levels in sperm. This increase depends strictly on CatSper function, as demonstrated through pharmacological analysis and CatSper1 knockout mice. This calcium influx is sufficient to induce acrosomal exocytosis in a subset of human and mouse sperm. AlphaFold-based structural modeling suggests a potentially conserved extracellular interaction site between the soluble N-terminal ZP2 fragments and the CatSper complex. In human sperm, ZP2 treatment significantly modulates motility parameters, including flagellar movement and velocity, while inducing a CatSper-dependent increase in intracellular CaCa2+ similar in magnitude to that evoked by progesterone. Species-matched ZP2 stimulation elicits the stronger calcium response, underscoring evolutionary adaptations in ligand-channel protein pairs. Taken together, our findings reveal a conserved signaling pathway from ZP2 to CatSper that integrates oocyte-derived signals into the regulation of sperm motility and acrosomal exocytosis. This pathway provides new mechanistic insights into fertilization and highlights potential targets.