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

Mariani, N. A. P.; Santos, N. C. M.; Andrade, J. J.; Andrade, A. D.; Rezende, B.; Steeman, T. J.; Novero, A. G.; De Lima-Jr, J. C.; Calderaro, G.; Themer, A. C. F.; Kushima, H.; Buffone, M. G.; Krapf, D.; Lishko, P. V.; Silva, E. J. R.

doi:10.1101/2025.09.05.674523

bioRxiv · 10.1101/2025.09.05.674523

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

Mariani, N. A. P.·Santos, N. C. M.·Andrade, J. J.·Andrade, A. D.·Rezende, B.·Steeman, T. J.·Novero, A. G.·De Lima-Jr, J. C.·Calderaro, G.·Themer, A. C. F.·Kushima, H.·Buffone, M. G.·Krapf, D.·Lishko, P. V.·Silva, E. J. R.

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Abstract

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.

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Mariani, N. A. P., Santos, N. C. M., Andrade, J. J., Andrade, A. D., Rezende, B., Steeman, T. J., Novero, A. G., De Lima-Jr, J. C., Calderaro, G., Themer, A. C. F., Kushima, H., Buffone, M. G., Krapf, D., Lishko, P. V., Silva, E. J. R.. 2025-09-10. Semenogelin-1 Inhibition of Mouse Sperm Hyperactivation Reveals Two Functional Domains Modulating CatSper Channel. https://doi.org/10.1101/2025.09.05.674523

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