Calaxin drives sperm chemotaxis by Ca ²⁺ -mediated direct modulation of a dynein motor

Abstract

] i ) in sperm significantly alters swimming direction during chemotaxis (3, 4). Recent studies of human sperm suggest that progesteronemediated Ca 2+ influx through a Ca 2+ channel, CatSper, plays essential role in motility changes, such as hyperactivation and chemotaxis (5, 6), although both phenomena are argued to be separated (7). However, Ca 2+ is a primary factor regulating symmetry of flagellar waveform (8, 9). Chemotactic movements are achieved by continuous changes in waveform symmetry of sperm flagella and subsequent changes in swimming direction to access the egg (10). Ca 2+ -dependent regulation of flagellar beating is reportedly governed by Ca 2+ -binding proteins that likely regulate axonemal dynein (8, 9). These proteins have not been identified, however, and the molecular mechanism of Ca 2+ -dependent control of flagellar asymmetry in sperm chemotaxis remains uncharacterized.Marine invertebrates are excellent models to study sperm motility because their sperm show clear motility changes and are produced in quantities sufficient for biochemical analysis. In the ascidian, Ciona intestinalis, a transient flagellar [Ca 2+ ] i burst is induced by a chemoattractant called sperm activating and attracting factor (SAAF), which triggers rapid sperm-turning followed by straight swimming toward eggs (4, 11). In a search for candidates that regulate Ca 2+ -dependent flagellar movement, we recently identified an axonemal Ca 2+ -binding protein, calaxin, which binds to outer-arm dynein in sperm flagella of C. intestinalis (12). Calaxin is highly conserved in metazoa, including mouse and human. In the present study, using Ciona sperm we show that calaxin is essential for Ca 2+ -dependent modulation of sperm movement necessary for chemotaxis toward the egg. We use in vitro motility assays to demonstrate that calaxin directly suppresses microtubule sliding driven by outer-arm dynein. Results and DiscussionTo test the function of calaxin in regulation of sperm motility in chemotaxis, we used an inhibitor of neuronal calcium sensor family proteins, repaglinide, which specifically binds to calaxin in sperm flagella (Fig. S1) (13). We first asked whether calaxin plays a critical role in sperm chemotaxis. During chemotactic movements, sperm show a unique turning movement associated with a flagellar change to an asymmetric waveform, followed by a straight-ahead movement (11). We observed sperm chemotactic movement toward a glass capillary filled with SAAF in the absence and presence of repaglinide (Fig. 1A). Sperm in control artificial sea water (ASW) with 0.5% (vol/vol) solvent (DMSO) showed very strong chemotaxis toward the glass capillary. However, sperm in the ASW containing 150 μM repaglinide did not exhibit the unique turn movement and showed less-effective chemotaxis (Fig. 1A). Linear equation chemotaxis index (LECI) (11) analysis quantitatively showed significantly decreased chemotactic property promoted by repaglinide at >100 μM (Fig. 1B). Sperm-swimming velocity showed no dramatic change following repagli...

Possible ability of bovine follicular fluid to attract migrating bull spermatozoa

“…However, the basic structures of the flagellar axoneme are conserved among these animals. Interestingly, a study reported the details of the sliding of dynein motor‐driven microtubules in the flagellar axoneme during the sperm chemotaxis of marine animals 25. In order to elucidate the swimming direction‐changing mechanism of chemotactic bull sperm, information on the dynamics of the structural elements of the sperm flagellar axoneme might be useful.…”

Flagellar ion channels of sperm: similarities and differences between species

“…The maintenance and regulation of intracellular calcium concentration ([Ca 2+ ] i ), is therefore, of great importance and is carried out by proteins and co-factors that import, export and/or sequester calcium ions [50]. In spermatozoa, swimming behavior is controlled by rises in [Ca 2+ ] i that changes flagellar beat pattern through Ca 2+ sensing proteins calaxins [51,52]. Calciumbound calaxins inhibit the activity of dynein motors within the Murine potassium channel KSper comprised of the Slo3 channel and associated gamma subunit LRRC52, as well as possible beta subunits.…”

Sperm dysfunction and ciliopathy

“…The OAD from animal sperm has two HCs, two WD-repeat ICs, a unique IC with thioredoxin and nucleoside diphosphate kinase domains [30][31][32], and regulatory LCs such as Tctex-2 LC [33,34] and a Ca 2? -binding protein named calaxin [35][36][37] (Fig. 1d).…”

“…OAD is involved in the elevation of ciliary beat frequency [19], force generation for penetration of sperm through the zona pellucida [141], metachronal synchrony of motile cilia [142], and propagation of asymmetric waveforms of sperm during chemotaxis [36]. The mutations in OAD reported so far include those in HCs (DNAH5, DNAH11), ICs (DNAI1, DNAI2, NME8), a LC (DNAL1), and a component of the DC (coiled-coil domain containing protein 114, CCDC114; DC2 in Chlamydomonas) ( Table 1).…”