CatSper and its CaM‐like Ca ²⁺ sensor EFCAB9 are necessary for the path chirality of sperm

Abstract

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.

3D structure and in situ arrangements of CatSper channel in the sperm flagellum

“…This is likely caused by the lack of xy -displacement response during capacitation, and highlights the importance of the intact higher-order assembly of CatSper complex for orchestrating CatSper channel activity and thus effective sperm navigation during capacitation. In addition, we have previously shown that wild type sperm exhibit a strict clockwise swimming path 33 , whereas this chirality is randomized in Efcab9 −/− sperm 37 . Based on our structural findings and the motility defects in Efcab9 −/− sperm, we propose that in wild type sperm the connected higher-order zigzag arrangement within a longitudinal nanodomain could allow for coordinated opening of the entire array of CatSper channels along the flagellar axis, ensuring a large and synchronized Ca 2+ influx to generate strong bending force (Supplementary Fig.…”

cAMP and the Fibrous Sheath Protein CABYR (Ca2+-Binding Tyrosine-Phosphorylation-Regulated Protein) Is Required for 4D Sperm Movement

“…The 4D movement analysis in the current study confirms these abnormalities in motility and extend them by identifying a decrease in the flagellar excursion in the Z-plane. In our recent work, we show a possible connection between the intracellular Ca 2+ concentration and the swimming path chirality, by demonstrating that an alteration of the Ca 2+ homeostasis after contact to ZP2 or by mutation of the CATSPER channel, the main Ca 2+ channel in sperm flagellum [15,18,21], lead to a substantial change in path chirality [43]. CABYR is a calcium-binding protein localized in the principal piece, is phosphorylated during sperm capacitation and may play a role for capacitation-dependent phosphorylation in calcium signaling [45,67].…”

“…In earlier studies, we showed that non-capacitated as well as capacitated murine sperm have a conserved CW path chirality and proposed, that this swimming pattern is necessary for reaching the oocyte [21,39,43]. We further postulate that substantial changes in the intracellular Ca 2+ concentration result in a loss of chirality and speculate that this is an important requirement to finalize fertilization [43]. In this study, we find that also a change in intracellular cAMP and ADP concentrations leads to a loss of path chirality in non-capacitated mouse sperm.…”

“…Besides rolling along their long axis, we analyzed path chirality of intact sperm and demembranated sperm under the different cAMP and ADP conditions (Figure 3E). To determine path chirality Procrustes analysis [39,43] was performed in Igor Pro TM on sequences of roll-counter-roll cycles. For this purpose, the trajectory was divided into two rolling cycles, where possible.…”

“…A role of the CatSper channel and its regulatory function in intracellular Ca 2+ homeostasis for rolling along the long axis could be disproven [42]. In contrast, by the use of digital holographic microscopy (DHM) we have shown, that trajectories of non-capacitated as well as capacitated murine wildtype (wt) sperm have a conserved CW chirality [39,43], which is lost in sperm lacking either the entire CatSper channel or its Ca 2+ sensor EF-hand calcium-binding domain-containing protein 9 (EFCAB9) [43]. Additionally, contact of capacitated sperm to the zona pellucida glycoprotein 2 (ZP2) led to an elimination of the CW path chirality indicating an important role of Ca 2+ -homeostasis for orchestrating CW swimming path chirality [43].…”