Frintrop L et al. (2022), cAMP and the Fibrous Sheath Protein CABYR (Ca2+...

ECB-ART-50878

Int J Mol Sci 2022 Sep 13;2318:. doi: 10.3390/ijms231810607.

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

Frintrop L , Wiesehöfer C , Stoskus A , Hilken G , Dubicanac M , von Ostau NE , Rode S , Elgeti J , Dankert JT , Wennemuth G .

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A new life starts with successful fertilization whereby one sperm from a pool of millions fertilizes the oocyte. Sperm motility is one key factor for this selection process, which depends on a coordinated flagellar movement. The flagellar beat cycle is regulated by Ca2+ entry via CatSper, cAMP, Mg2+, ADP and ATP. This study characterizes the effects of these parameters for 4D sperm motility, especially for flagellar movement and the conserved clockwise (CW) path chirality of murine sperm. Therefore, we use detergent-extracted mouse sperm and digital holographic microscopy (DHM) to show that a balanced ratio of ATP to Mg2+ in addition with 18 µM cAMP and 1 mM ADP is necessary for controlled flagellar movement, induction of rolling along the long axis and CW path chirality. Rolling along the sperm's long axis, a proposed mechanism for sperm selection, is absent in sea urchin sperm, lacking flagellar fibrous sheath (FS) and outer-dense fibers (ODFs). In sperm lacking CABYR, a Ca2+-binding tyrosine-phosphorylation regulated protein located in the FS, the swim path chirality is preserved. We conclude that specific concentrations of ATP, ADP, cAMP and Mg2+ as well as a functional CABYR play an important role for sperm motility especially for path chirality.

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	Figure 1. 4D motility of demembranated wt sperm. (A) Representative transmission electron microscopy cross sections of flagella mid-pieces of membranated (left) and demembranated (right) sperm. MT—microtubules, FS—fiber sheath, PM—plasma membrane, ODF—outer dense fiber. Scale bar = 500 nm. (B) Analysis of beat frequency and beat amplitude of demembranated mouse sperm with 1 mM ATP and different Mg2+ concentrations (1–3 mM) (left) or with 1 mM Mg2+ and different ATP concentrations (1–3 mM) (right). An increase in ATP or Mg2+ concentration results in an increase in beat frequency and a decrease in the beat amplitude compared to membrane intact control sperm (c). mean ± SEM; N = 4, n = 12. (C) 4D waveform analysis of intact sperm (first panel) compared with demembranated sperm analyzed without ADP but with increasing cAMP concentrations (0, 3, 18 µM). Traces of a flagellum in 3D at different time points (0, 40, …, 280 ms) are color-coded and the projections onto the XY-and XZ-plane are shadowed in black. (D) Statistical analysis of XY-(first panel) and Z-excursions of flagella (second panel), 3D curvilinear velocity (VCL) (third panel) and lateral head displacement (forth panel) of intact and demembranated sperm in the presence of different cAMP and ADP concentrations (color-coded). N = 4, n = 15, * p < 0.05, p < 0.01, * p < 0.001, median: thick dashed lines; interquartile range: thin dashed lines.
	Figure 2. 4D motility of demembranated wt sperm can be recovered by adding 18 µM cAMP and 1 mM ADP. (A) 4D waveform analysis of demembranated sperm analyzed with 1 mM ADP and 0 cAMP (first panel), 1 mM ADP and 3 µM cAMP (second panel) and 1 mM ADP and 18 µM cAMP (third panel). Color-coded lines are traces of a flagellum in 3D at different time points (0, 40, …, 280 ms) and the projections onto the XY-and XZ-plane are shadowed in black. (B) Comparative statistical analysis of flagellar XY-(upper panel, left) and Z-excursions (upper panel, right), 3D curvilinear velocity (lower panel, left) and lateral head displacement (lower panel, right) of demembranated sperm measured with 1 mM ADP and 0 µM cAMP or in the presence of 3 µM or 18 µM cAMP. (C) Comparative statistical analysis of flagellar XY-(upper panel, left) and Z-excursions (upper panel, right), 3D curvilinear velocity (lower panel, left) and lateral head displacement (lower panel, right) of demembranated sperm measured in the presence of cAMP or cGMP. cGMP does not recover sperm motility in demembranated sperm. N = 4, n = 15, p < 0.01, * p < 0.001, median: thick dashed lines; interquartile range: thin dashed lines.
	Figure 3. Rolling of sperm requires cAMP and ADP. (A–C) Statistical analysis of demembranated murine sperm in the presence of defined cAMP and ADP concentrations (color-coded as in Figure 1) compared to membrane intact sperm. (A) Percentage of rolling sperm, (B) number of rolling cycles during a 2.5 s recording, where a rolling cycle is defined as RCh-LCh-RCh or vice versa, and (C) the number of rolling movements characterized by an increase in intensity of light reflected from the head analyzed. (D) Analysis of rolling behavior along the long axis of intact sperm (upper panel, left) and of demembranated sperm under different cAMP and ADP concentrations (upper and lower panels) by measuring the oscillating light intensity which scattered from the sperm head. Red and blue bars indicate periods of “RCh” and “LCh” orientation for the sperm head. The short white areas represent an indeterminate orientation and the curved arrows indicate CW or CCW rolling. (E) Path chirality analysis using averaged Procrustes alignments. Positive dθ/dt values indicate a CW and negative values a CCW chirality. Depending on the different rolling behavior of demembranated sperm due to different cAMP and ADP concentration the number of analyzed sperm varied. N = 4, n = 12, mean ± SEM; p < 0.01, * p < 0.001.
	Figure 4. 4D motility analyses of sea urchin sperm. (A) 50 ms swimming path of a sea urchin sperm overlaid on the reconstructed XY projection of the final frame in the 0.5 s holographic record (63X objective) (left) and illustrated in 4D (right). The swimming trajectory is visualized by tracing the head position. (B) 4D flagellar waveform analysis whereas the time-lapse trace of a flagellum at 3D position (laboratory-fixed frame of reference xyz) is visualized in color and its projections onto XY- and XZ-planes are shadowed in black (left). Statistical analysis of beat frequency and flagellar XY- and Z-excursion (right). (C) Statistical analysis of curvilinear velocity. N = 3, n = 15, median: thick dashed lines; interquartile range: thin dashed lines. ** p < 0.01.
	Figure 5. Rolling requires longitudinal stabilizing structures along the flagellum. (A) Rolling analysis by measuring the intensity of light scattered from the moving sperm head shows no periodic fluctuations in intensity of reflected light. Based on sea urchin sperm head morphology orientation could not be determined. (B) Simulation snapshot of a sperm with an attached bead. Arrows indicate the three main axes of rotation. The different rotation angles (Ωp, Ωb, Ωe) are illustrated. (C) Simulation results for the main rotation frequencies, as a function of bead size for various beating amplitudes (A = amplitude of the curvature wave). (D) XY-projections of a 250 ms holographic record sampled at 198 frames per second (fps) illustrate no rolling of a sea urchin sperm along its long axis by using polystyrene latex beads (0.8 µm mean particle size). Scale bar = 5 µm, N = 3, n = 15.
	Figure 6. Cabyr–a Ca2+ binding tyrosine-phosphorylation-regulated protein—is required for 4D sperm motility. (A) Computer-assisted sperm analysis (CASA) reveals decrease of motile sperm in Cabyr−/− mice (left), which correlates with a decrease in the number of sperm showing a progressive sperm movement (right). N = 4, n > 800 sperm. (B) Flagellar beat amplitude and beat frequency analyzed by CASA are not affected by Cabyr knockout. N = 4, n = 15. (C) Statistical analysis of curvilinear velocity by DHM. N = 4, n = 15. (D) Statistical analysis of lateral head displacement of Cabyr knockout in comparison to WT sperm. (E) Analysis of flagellar excursion in XY- and Z-plane in Cabyr−/− sperm shows a decrease of flagellar movement. N = 4, n = 15. (F) Representative 4D flagellar excursions of wt (left) and Cabyr−/− (right) sperm in which 3D flagellar excursions at different time points (0, 40, …, 280 ms) are color-coded and its projection onto the XY- and Z-plane are shadowed in black. * p < 0.05, ** p < 0.01, median: thick dashed lines; interquartile range: thin dashed lines.
	Figure 7. Cabyr facilitates conserved CW path chirality. (A) Analysis of rolling behavior along the long axis of wt (left panel) or Cabyr−/− (right panel) sperm during a time interval of 2.5 s. Red and blue bars indicate periods of “RCh” and “LCh” orientation for the sperm head. The short white areas represent an indeterminate orientation and the curved arrows indicate CW or CCW rolling. In the diagram, the oscillating light intensity, which is scattered from the sperm head, is shown. Cabyr−/− sperm show a comparable rolling behavior as wt sperm. (B) Averaged Procrustes alignments of five representative wt (upper, left) or five Cabyr−/− sperm (upper, right) translocated to place the initial point at the origin. The curved arrow indicates a CW path chirality analyzed by visual assessment. Determined dθ/dt (mean ± SEM) for 15 wt (lower, left) and 15 Cabyr−/− sperm (lower, right). Positive dθ/dt values indicate a CW and negative values a CCW path chirality. CABYR facilitates conserved CW path chirality.

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