Rotational motion and rheotaxis of human sperm do not require functional CatSper channels and transmembrane Ca²⁺...

Navigation of sperm in fluid flow, called rheotaxis, provides long-range guidance in the mammalian oviduct. The rotation of sperm around their longitudinal axis (rolling) promotes rheotaxis. Whether sperm rolling and rheotaxis require calcium (Ca²⁺ ) influx via the sperm-specific Ca²⁺ channel CatSper, or rather represent passive biomechanical and hydrodynamic processes, has remained controversial. Here, we study the swimming behavior of sperm from healthy donors and from infertile patients that lack functional CatSper channels, using dark-field microscopy, optical tweezers, and microfluidics. We demonstrate that rolling and rheotaxis persist in CatSper-deficient human sperm. Furthermore, human sperm undergo rolling and rheotaxis even when Ca²⁺ influx is prevented. Finally, we show that rolling and rheotaxis also persist in mouse sperm deficient in both CatSper and flagellar Ca²⁺ -signaling domains. Our results strongly support the concept that passive biomechanical and hydrodynamic processes enable sperm rolling and rheotaxis, rather than calcium signaling mediated by CatSper or other mechanisms controlling transmembrane Ca²⁺ flux.