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.

Axonemal dynein ATPases direct ciliary and flagellar beating via adenosine triphosphate (ATP) hydrolysis. The modulatory effect of adenosine monophosphate (AMP) and adenosine diphosphate (ADP) on flagellar beating is not fully understood. Here, we describe a deficiency of cilia and flagella associated protein 45 (CFAP45) in humans and mice that presents a motile ciliopathy featuring situs inversus totalis and asthenospermia. CFAP45-deficient cilia and flagella show normal morphology and axonemal ultrastructure. Proteomic profiling links CFAP45 to an axonemal module including dynein ATPases and adenylate kinase as well as CFAP52, whose mutations cause a similar ciliopathy. CFAP45 binds AMP in vitro, consistent with structural modelling that identifies an AMP-binding interface between CFAP45 and AK8. Microtubule sliding of dyskinetic sperm from Cfap45−/− mice is rescued with the addition of either AMP or ADP with ATP, compared to ATP alone. We propose that CFAP45 supports mammalian ciliary and flagellar beating via an adenine nucleotide homeostasis module.

The sperm-specific CatSper channel controls the influx of Ca2+ into the flagellum and, thereby, the swimming behavior of sperm. A hallmark of human CatSper is its polymodal activation by membrane voltage, intracellular pH, and oviductal hormones. Whether CatSper is also activated by signaling pathways involving an increase of cAMP and ensuing activation of protein kinase A (PKA) is, however, a matter of controversy. To shed light on this question, we used kinetic ion-sensitive fluorimetry, patch-clamp recordings, and optochemistry to study transmembrane Ca2+ flux and membrane currents in human sperm from healthy donors and from patients that lack functional CatSper channels. We find that human CatSper is neither activated by intracellular cAMP directly nor indirectly by the cAMP/PKA-signaling pathway. Instead, we show that non-physiological concentrations of cAMP and membrane-permeable cAMP analogs used to mimic the action of intracellular cAMP activate human CatSper from the outside via a hitherto unknown extracellular binding site. Finally, we demonstrate that the effects of common PKA inhibitors on human CatSper rest predominantly, if not exclusively, on off-target drug actions on CatSper itself rather than on inhibition of PKA. We conclude that the concept of an intracellular cAMP/PKA-activation of CatSper is primarily based on unspecific effects of chemical probes used to interfere with cAMP signaling. Altogether, our findings solve several controversial issues and reveal a novel ligand-binding site controlling the activity of CatSper, which has important bearings on future studies of cAMP and Ca2+ signaling in sperm.