CatSper and its CaM-like Ca²⁺ sensor EFCAB9 are necessary for the path chirality of sperm

Successful fertilization depends on sperm motility adaptation. Ejaculated and activated sperm beat symmetrically in high frequency, move linearly, and swim with clockwise chirality. After capacitation, sperm beat asymmetrically with lower amplitude and a high lateral head excursion. This motility change called hyperactivation requires CatSper activation and an increase in intracellular Ca²⁺ . However, whether CatSper-mediated Ca²⁺ influx participates in controlling the swim path chirality is unknown. In this study, we show that the clockwise path chirality is preserved in mouse sperm regardless of capacitation state but is lost in the sperm either lacking the entire CatSper channel or its Ca²⁺ sensor EFCAB9. Pharmacological inhibition of CatSper with either mibefradil or NNC 55-0396 leads to the same loss in swim path chirality. Exposure of sperm to the recombinant N-terminal part of the zona pellucida protein 2 randomizes chirality in capacitated cells, but not in non-capacitated ones. We conclude that Ca²⁺ sensitive regulation of CatSper activity orchestrates clockwise swim path chirality of sperm and any substantial change, such as the physiological stimulus of zona pellucida glycoproteins, results in a loss of chirality.