Two-dimensional slither swimming of sperm within a micrometre of a surface

Abstract

Sperm motion near surfaces plays a crucial role in fertilization, but the nature of this motion has not been resolved. Using total internal reflection fluorescence microscopy, we selectively imaged motile human and bull sperm located within one micron of a surface, revealing a distinct two-dimensional (2D) 'slither' swimming mode whereby the full cell length (50-80 μm) is confined within 1 μm of a surface. This behaviour is distinct from bulk and near-wall swimming modes where the flagellar wave is helical and the head continuously rotates. The slither mode is intermittent (∼1 s, ∼70 μm), and in human sperm, is observed only for viscosities over 20 mPa·s. Bull sperm are slower in this surface-confined swimming mode, owing to a decrease in their flagellar wave amplitude. In contrast, human sperm are ∼50% faster-suggesting a strategy that is well suited to the highly viscous and confined lumen within the human fallopian tube.

Figure 1. TIRF microscopy set-up for near-field imaging of sperm.

(a) Schematic view of the TIRF microscopy set-up. A representative TIRF microscopy image showing both sperm head and tail for the sperm cell closest to the surface. Scale bar, 20 μm. The image intensity was inverted and contrast adjusted for clarity. (b) Cartesian coordinate system used to quantify sperm motion.

Figure 2. 3D bulk swimming versus 2D slither swimming.

Image sequences of bull sperm swimming in bulk fluid observed through (a) bright-field and (b) fluorescence microscopy, where both show continuous sperm head rotation. (c) Sequence of TIRF microscopy images showing a non-rotating, surface-aligned motion of the sperm head confined within a few hundred nanometres of the glass surface. The sperm tail also appears as a continuous line within the 1-μm depth of field. (d) Schematic of sperm swimming in bulk liquid, where the torque resulting from the 3D helical beating pattern of sperm's flagellum is balanced by counter-rotation of the sperm head. (e) Schematic of sperm in the observed slither mode, where both the head and the tail are confined to a 2D plane parallel to the surface with no rotation. (f) Sample of sperm surface density fluctuations for both near-wall swimmers and slither swimmers, with the full histogram and probability distribution function (PDF) shown on the right. (g) Schematic showing a typical transition from bulk swimming to slither swimming modes, with head areas shown below as would be imaged by traditional (2–3, 7–8, blue) and TIRF (4–6, red) microscopy. Scale bars, 20 μm; the image was inverted and contrast-adjusted for clarity.

Figure 3. Schematic of drag-based sperm locomotion in bulk swimming and slither swimming modes.

(a) Bulk swimmer sperm propulsion. The net drag force, f, acting on each segment of flagellar helix has a propulsive, f_prop, and rotational component, f_rot. The result is propulsion with continuous rotation in opposite direction of the flagellar wave. (b) Slither swimmer propulsion. The net drag force, f, on each segment has a surface-aligned propulsive component, f_prop, and an oscillating perpendicular component, f_oci, which also lies in the 2D plane of the surface. With all forces acting within the same plane, slither swimmers achieve forward progression with no rotation.

Figure 4. TIRF characterization of the slither swimming mode.

(a) Preferential circling direction of bull and human sperm in slither swimming mode (n≥126). An overlay of consecutives images taken at 50 frames per second showing swimming trajectories of five bull sperm shown in the inset. Red and blue arrows indicate counter-clockwise and clockwise trajectories, respectively. (b) A reconstructed trajectory of a bull sperm and its projected trajectory in the 2D plane of the surface (out-of-plane axis is magnified × 40). The colour corresponds to time, as shown in the legend, and the black and red lines show the projected swimming trajectory and average path, respectively. The corresponding overlay of TIRF microscopy images is shown in the inset. (c) Curvilinear velocity (top) and local curvature (bottom) measured as a function of distance from the surface (bull sperm). Open circles represent local data points and the solid black circles are averages binned over 200-nm intervals. P values were determined by a one-tailed z-test, *P≤0.05 and ***P≤0.001. Scale bars, 20 μm.

Figure 5. Human sperm motility parameters for slither swimming mode as compared with bulk swimming mode, as a function of viscosity.

(a) Curvilinear velocity (VCL), (b) average path velocity (VAP), (c) straight line velocity (VSL), (d) linearity (LIN), (e) wobble (WOB), (f) mean curvature (MCR), (g) amplitude of lateral head displacement (ALH) and (h) beat cross frequency (BCF) for slither swimmer human sperm compared with bulk swimmer sperm in media with viscosity ranging from 20 to 250 mPa·s (n≥126). Human sperm swimming in slither mode showed significantly higher velocities compared with sperm swimming in bulk fluid in media with the same viscosity. Increase in viscosity negatively affect sperm motility. Values are reported as mean±s.d., and P values were determined by the two-tailed t-test, *P≤0.05, **P≤0.01 and ***P≤0.001.

Figure 6. Synchronized motion of sperm in slither swimming mode.

(a) An image sequence showing a pair of synchronized slither swimming bull sperm, adopting a shared beating frequency and flagellar waveform and (b) their overlaid trajectories. Synchronized motion of (c) four bull sperm and (d) two human sperm in the slither swimming mode. The head of each individual sperm is colour coded along its swimming path. Scale bars, 20 μm. The image was inverted and contrast-adjusted for clarity.