Structurally distinct Ca(2+) signaling domains of sperm flagella orchestrate tyrosine phosphorylation and motility

Abstract

Spermatozoa must leave one organism, navigate long distances, and deliver their paternal DNA into a mature egg. For successful navigation and delivery, a sperm-specific calcium channel is activated in the mammalian flagellum. The genes encoding this channel (CatSpers) appear first in ancient uniflagellates, suggesting that sperm use adaptive strategies developed long ago for single-cell navigation. Here, using genetics, super-resolution fluorescence microscopy, and phosphoproteomics, we investigate the CatSper-dependent mechanisms underlying this flagellar switch. We find that the CatSper channel is required for four linear calcium domains that organize signaling proteins along the flagella. This unique structure focuses tyrosine phosphorylation in time and space as sperm acquire the capacity to fertilize. In heterogeneous sperm populations, we find unique molecular phenotypes, but only sperm with intact CatSper domains that organize time-dependent and spatially specific protein tyrosine phosphorylation successfully migrate. These findings illuminate flagellar adaptation, signal transduction cascade organization, and fertility.

Figure 1. 3D STORM reproduces known structures in the sperm flagella

(A) Reference transmission electron micrographs. Left; longitudinal section (10,000×) near the annulus (arrowhead). Right: cross-section of principal piece (12,000×). FS, fibrous sheath; ODF, outer dense fibers; PM, plasma membrane; M, mitochondria. (B–E) 3D STORM images of (B) Glucose transporter 3 (GLUT3), (C) A-kinase anchoring protein 4 (AKAP4), (D) Outer dense fiber 2 (ODF2; V-shaped large structure is from other sperm tails), and (E) α/β-tubulin. Left, x-y projections. Right, y-z cross-sections near the annulus (white arrowheads). The color in all x-y projections encodes the relative distance from the focal plane along the z-axis (color scale bar in B). (F–G) Radial profiles of (F) TEM cross-section in A; and (G) STORM cross-sections in B–E. CP, central pair of microtubules; 9, 9 outer doublets of microtubules. See also Figure S1.

Figure 2. CatSper proteins form four linear domains

Each domain runs along each side of the longitudinal columns of the sperm flagella. (A) Confocal fluorescence (upper) and the corresponding phase-contrast (lower) images of immunostained CatSper1 in mouse sperm. (B–E) 3D STORM images of CatSper1 in x-y projection (B), in y-z cross-section (C), CatSperδ in x-y projection (D), and in y-z cross-section (E) at the initiation of the principal piece (white arrowhead). Colors in B and D indicate the z-positions (see color scale bar in B). See also Movie S1. (F) Co-localization of CatSper1 (green) and CatSperδ (magenta). (G) Silver-intensified immunogold electron microscope images of CatSper1 (3,000×). (H) CatSper gold clusters/cross-section from 3,000× images; n =56. Note that <4 clusters are only seen in smaller cross-sections of <500-nm diameter, reflecting attenuation of CatSper1 fluorescence in the distal region (A). (I) A magnified cross-section. LC, longitudinal column; CR, circumferential rib. Numbers indicate ODFs corresponding to the microtubule pairs of the axoneme. (J) Off-center longitudinal section. Arrows indicate the Ag-intensified immunogold-labeled CatSper1. (K) Cartoon of the sperm flagellum showing the orientation of the EM section shown in I and J. The off-center cut is drawn to show the location of the linear arrangement of CatSper (red) captured in J relative to the underlying cytoskeletal structures. See also Figure S2.

Figure 3. CatSper is essential to Ca²⁺ domain organization

(A–C) 3D STORM images of flagellar proteins: (A) CaMKII (P-T286), (B) Protein phosphatase 2B, catalytic subunit Aγ (PP2B-Aγ), and (C) caveolin-1. Left, x-y projections. Colors indicate z-positions (see color scale bar in A). Middle, y-z cross-sections near the annulus (white arrowheads). Right, 2-color STORM images of CatSper1 (green) and P-CaMKII/PP2B-Aγ/caveolin-1 (magenta). (A’–C’) Delocalization of P-CaMKII (T286) (A’), PP2B-Aγ (B’), and caveolin-1 (C’) in CatSper1-null spermatozoa. (D) Cartoon of the cylindrical coordinate system for defining the radius and angles of molecular coordinates in STORM images. The longitudinal axis (x) is placed at the flagellar center and the origin at the annulus. (E) Angular distributions (left) and profiles (right) of the surface-localized P-CaMKII (T286) and caveolin-1 in WT and CatSper1-null sperm shown in A, A’ and C, C’. (F) Radial profiles of P-CaMKII, caveolin-1, and PP2B-Aγ of WT (blue) and CatSper1-null (red) flagella shown in A, A’–C, C’. See also Figures S3 and S4.

Figure 4. Ca²⁺ domains confine P-Tyr to the axoneme and inhibit premature P-Tyr

P-Tyr proteins are potentiated and delocalized in CatSper1-null spermatozoa. (A) Confocal fluorescence of P-Tyr (green) from mouse sperm incubated for the indicated time under capacitating conditions. Mitochondrial import receptor subunit, Tom20 (red), serves as reference for normalizing the intensity of P-Tyr under 4 different conditions. Upper, WT; Lower, CatSper1-null spermatozoa. (B) Immunoblot of total sperm cell extract by α-P-Tyr. (C) Fluorescence image of a capacitated WT spermatozoon co-immunostained by α-CatSper1 (red) and α-P-Tyr (green). MP, mid-piece. PP, principal piece. White lines mark the boundary. (D) A 2-color STORM cross-section of CatSper1 (red) and P-Tyr (green) in a capacitated WT sperm cell. (E) Two-color STORM images of P-Tyr (green) and GLUT3 (red) in capacitated WT and CatSper1-null cells. The y-z cross-sections are obtained from the proximal to the distal part of the principal piece (same cells). (F) Radial profiles of pY (green) and GLUT3 (red) in the STORM cross-section in E. (G) STORM cross-sections of P-Tyr in CatSper1-null spermatozoa at different times during capacitation. (H) Time-course population variation of P-Tyr development in all spermatozoa imaged with 3D STORM. See also Figure S5.

Figure 5. Profiling P-Tyr sites from capacitated sperm

Related to Figure S6, Tables S1 and S2. (A) Annotation of 45 P-Tyr proteins from capacitated WT and CatSper1-null spermatozoa. (B) Normalized amino acid frequency plots (WebLogo) for ± 6 residues neighboring all 62 tyrosine phosphorylation (P-Tyr) sites (upper) and 41 P-Tyr sites with the 2-fold ratio cutoff between WT and CatSper1-null spermatozoa (lower). (C) Kinase motifs extracted from the CatSper-dependent P-Tyr sites with P + 1 restriction. Three motifs (y[DE], y[AG], and y[ST]) fit to known Src consensus sites. Number of peptides for each given motif in parenthesis (see Table S2 for other motif sequences with restriction on different positions). (D) Protein expression and phosphorylation of SFKs in sperm before (0-min) and after (90-min) capacitation. Recognition of P-Src (Y416), P-Lyn (Y507) and P-Lck (Y505). Lck and tubulin are loading controls. (E) STORM cross-sections of SFKs shown in D. (F–G) Validation of a pY target identified from this study. Expression (F) and sub-flagellar localization (G) of phosphorylated CaM at Y100 in capacitated (90-min) sperm.

Figure 6. Sperm with intact CatSper domains hyperactivate and migrate efficiently

(A–B) Capacitation alters the levels of P-CaMKII, PP2B-Aγ, caveolin-1 and CatSper1 in sperm. (A) Representative immunoblots from 3 – 4 experiments. (B) Relative densitometry of proteins in A. Signals normalized to tubulin and compared to uncapacitated sperm. Error bars denote s.e.m. of paired two-tailed t-tests (*P<0.05, **P<0.01). Protein declines after capacitation (P=0.013 for P-CaMKII, P=0.013 for PP2B-Aγ, and P=0.009 for caveolin-1). Caveolin-1 is upregulated in CatSper1-null spermatozoa (P=0.014). (C–D) Capacitation generates heterogeneous molecular phenotypes. (C) Variation in sub-flagellar localization in capacitated sperm; 2 representative 3D STORM cross-sections. (D) Population variation in structures of CatSper domain components in capacitated sperm. (E) Hyperactivated sperm have intact CatSper domains. Capacitated WT sperm cells on grid coverslips were videotaped for motility correlation, fixed and labeled for 3D STORM. Two representative hyperactivating sperm cells from grid 4P. Left, phase-contrast images. Right, STORM cross-sections of CatSper1. Also see Movie S2. (F) Confined P-Tyr in hyperactivated spermatozoa. 100× fluorescence images; CatSper1 (red) and P-Tyr (green). (G–I) Impaired sperm migration from the uterus into the oviduct in CatSper1-null mice. (G) Cartoon of sperm migration in the female. (H–I) Uterus and oviduct collected from WT females mated with DsRed2-tagged WT (H) and CatSper1-null (I) males 3-h after coitus. Two insets indicate areas including the end of uterus and uterotubal junction (UTJ, dotted line) (1) and the isthmus (2). See also Figure S7.