Semenogelin-1 Inhibition of Mouse Sperm Hyperactivation Reveals Two Functional Domains Modulating CatSper Channel

Seminal plasma is essential for sperm survival and function after ejaculation. Semenogelin-1 (SEMG1), the predominant seminal plasma protein, transiently suppresses sperm motility and hyperactivation after ejaculation through epididymal protease inhibitor (EPPIN) binding. However, the molecular mechanism underlying SEMG1-mediated inhibition of hyperactivation remains unclear. Here, we tested the hypothesis that SEMG1 inhibits CatSper, a sperm-specific calcium channel crucial for hyperactivation. Full-length recombinant mouse SEMG1 (mSEMG1; Q32-G375) inhibited both progressive motility and hyperactivation; the latter was not recovered with NH4Cl-induced alkalinization, indicating an effect downstream of capacitation-associated intracellular alkalinization. Electrophysiological recordings revealed that mSEMG1 reduced CatSper currents at physiologically relevant concentrations. Truncated mSEMG1 fragments mSEMG1Q32-V118 and mSEMG1R98-G375, but not mSEMG1Y221-G375, inhibited sperm hyperactivation and CatSper currents to a similar extent as full- length mSEMG1. Notably, only mSEMG1R98-G375 retained full EPPIN-binding capacity. Together, our findings identify two functional domains within mSEMG1 (Q32-V118 and R98-S220) that inhibit sperm hyperactivation by suppressing CatSper activity and differ in their EPPIN-binding capacities. These functional domains represent promising prototypes for the design of spermiostatic molecules, offering additional avenues for non-hormonal male contraception. Summary statementWe report a novel mechanism by which SEMG1, a major seminal plasma protein, modulates sperm hyperactivation by inhibiting a key calcium channel, offering new insights into sperm function and non- hormonal male contraception.