Molecular Basis of Sperm Flagellar Axonemes

] 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...

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“…Calaxin binds to the β heavy chain of outer-arm dynein (12,19) and suppresses microtubule sliding at high Ca 2+ concentrations (Fig. 4 B and C).…”

Section: Resultsmentioning

confidence: 93%

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

Mizuno

Shiba

Okai

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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“…Motile axonemes are complex molecular machineries composed of dyneins and regulatory components attached to scaffolding MTs (Inaba, 2007(Inaba, , 2011. Axonemal MTs are arranged in so-called 9+2 structures, comprising nine outer doublet MTs and two central singlet MTs.…”

Section: Introductionmentioning

confidence: 99%

Ttll9 −/− mice sperm flagella show shortening of doublet 7, reduction of doublet 5 polyglutamylation and a stall in beating

Konno

Ikegami

Konishi

et al. 2016

Journal of Cell Science

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Nine outer doublet microtubules in axonemes of flagella and cilia are heterogeneous in structure and biochemical properties. In mammalian sperm flagella, one of the factors to generate the heterogeneity is tubulin polyglutamylation, although the importance of the heterogeneous modification is unclear. Here, we show that a tubulin polyglutamylase Ttll9 deficiency (Ttll9) causes a unique set of phenotypes related to doublet heterogeneity. Ttll9 −/− sperm axonemes had frequent loss of a doublet and reduced polyglutamylation. Intriguingly, the doublet loss selectively occurred at the distal region of doublet 7, and reduced polyglutamylation was observed preferentially on doublet 5. Ttll9 −/− spermatozoa showed aberrant flagellar beating, characterized by frequent stalls after antihook bending. This abnormal motility could be attributed to the reduction of polyglutamylation on doublet 5, which probably occurred at a position involved in the switching of bending. These results indicate that mammalian Ttll9 plays essential roles in maintaining the normal structure and beating pattern of sperm flagella by establishing normal heterogeneous polyglutamylation patterns.

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“…However, difference in the usage of axonemal proteins has been observed between Chlamydomonas and animal sperm [28,37,151]. The composition of the ODA-DC is likely different between sperm and Chlamydomonas flagella [40].…”

Section: Notes On the Differences Between Sperm And Chlamydomonasmentioning

confidence: 99%

“…In Chlamydomonas flagella, the ODA-DC is composed of three subunits, DC1 (a coiled-coil protein), DC2 (coiled-coil protein), and DC3 (an EF-hand protein). However, orthologs of DC1 and DC3 are not found in animal sperm flagella [28,40].…”

Section: Structure Of the Axonemementioning

confidence: 99%

“…Outer arm dyneins (OADs) from Chlamydomonas flagella are composed of three heavy chains (HCs), two intermediate chains (ICs), and ten light chains (LCs). Work using sperm of tunicates (a subphylum of marine invertebrates) and sea urchins revealed similar molecular composition of the IADs but not the ODAs [26][27][28][29]. 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?…”

Section: Structure Of the Axonemementioning

confidence: 99%

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Sperm dysfunction and ciliopathy

Inaba

Mizuno

2015

Reprod Medicine & Biology

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Molecular Basis of Sperm Flagellar Axonemes

Cited by 83 publications

References 90 publications

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

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

Ttll9 −/− mice sperm flagella show shortening of doublet 7, reduction of doublet 5 polyglutamylation and a stall in beating

Sperm dysfunction and ciliopathy

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Molecular Basis of Sperm Flagellar Axonemes

Cited by 83 publications

References 90 publications

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

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

Ttll9 −/− mice sperm flagella show shortening of doublet 7, reduction of doublet 5 polyglutamylation and a stall in beating

Sperm dysfunction and ciliopathy

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

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