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

Seminal plasma proteins are essential for preserving sperm survival and functional integrity after ejaculation. Semenogelin-1 (SEMG1) is the predominant seminal plasma protein and the major structural component of the semen coagulum and copulatory plug in humans and rodents, respectively. On the sperm surface, SEMG1 binds to EPPIN (epididymal protease inhibitor), transiently suppressing the acquisition of sperm motility after ejaculation and preventing sperm hyperactivation. However, the molecular mechanisms by which SEMG1 inhibits sperm hyperactivation remain unclear. We hypothesize that SEMG1 exerts its effects through inhibition of CatSper, a sperm-specific calcium channel crucial for hyperactivation. Supporting this, we observed that recombinant mouse SEMG1 (mSEMG1) inhibited both progressive motility and hyperactivation in a concentration-dependent manner. Notably, the latter was not recovered with NH4Cl-induced alkalinization, suggesting that the effects of mSEMG1 are downstream of capacitation-associated intracellular alkalinization. Electrophysiological recordings revealed that mSEMG1 impaired CatSper currents at physiologically relevant concentrations. Recombinant mSEMG1 truncations mSEMG1Q32-V118 and mSEMG1R98-G375, but not mSEMG1Y221-G375, independently inhibited sperm hyperactivation and CatSper currents similar to full-length mSEMG1. However, only mSEMG1R98-G375 displayed similar EPPIN-binding capacity comparable to full-length mSEMG1, whereas mSEMG1Q32-V118 and mSEMG1Y221-G375 showed only minor binding. Our findings reveal that two distinct functional domains of mouse SEMG1, spanning the Q32-V118 and R98-S220regions, inhibit sperm hyperactivation by targeting the CatSper channel, both independently and dependent on EPPIN binding. These functional domains represent promising prototypes for the design of spermiostatic molecules, offering additional avenues for non-hormonal male contraception.