Direct measurement of the passive stiffness of rat sperm and implications to the mechanism of the calcium response

Abstract

Glass microprobes were used to measure the stiffness of the flagella of Triton X-100-extracted rat sperm models. The sperm models were treated with 50 microM sodium vanadate and 0.1 mM Mg-ATP to evaluate the stiffness of the passive flagellar structure without the influence of the dynein motor proteins. The passive stiffness was determined to be 4.6 (+/- 1.1) x 10(-19) N x m(2). Rat sperm models exposed to greater than 10(-5) M calcium ions exhibit a strong bend in the basal 40 microm of the flagellum, resulting in a fishhook-like appearance. The torque required to bend a passive rat sperm flagellum into the fishhook-like configuration was determined. The result was compared to the previously published measurement of the torque required to straighten the flagella of rat sperm in the Ca(2+)-induced fishhook configuration [Moritz et al., 2001: Cell Motil. Cytoskeleton 49:33-40]. The torque required to induce a fishhook in a passive flagellum was 2.7 (+/- 0.7) x 10(-14) N x m and the torque to straighten an active Ca(2+)-induced fishhook was 2.6 (+/- 1.4) x 10(-14) N x m. These values are identical within the limit of error of the measurement technique. This finding suggests that the fishhook configuration observed in the Ca(2+) response of rat sperm is the result of a Newtonian equilibrium, where active torque produced by dynein is counterbalanced by an equal and opposite passive torque that results from bending the flagellum. Consistent with this mechanism, the Ca(2+)-induced fishhook configuration is progressively relaxed by incremental increases in sodium vanadate concentration. This supports an active role of the dynein motors in producing the torque for the response. When rat sperm respond to Ca(2+), the bend in the flagellum always forms in the direction opposite the curvature of the asymmetric sperm head. Based on this polarity, the bending torque for the Ca(2+) response must result from the action of the dyneins on outer doublets 1 through 4.

Functional anatomy of the mammalian sperm flagellum

“…When we measured the stiffness of bull sperm and rat sperm we found great variability from cell to cell (Schmitz- Lesich and Lindemann, 2004;Lindemann et al, 2005;Lesich et al, 2008). The variability was always most pronounced in the midpiece, the stiffest portion of the flagellum.…”

Mechanical properties of the passive sea urchin sperm flagellum

“…Stiffness was measured as previously described by Schmitz-Lesich and Lindemann [2004] for rat sperm flagella. Briefly, the point of the probe tip when inducing a bend was measured and recorded.…”

Motility Measurement of a Mouse Sperm by Atomic Force Microscopy

“…To the best of our knowledge, a direct measurement of the lashing force and the torque generated from a trapped mammalian sperm using integrating the AFM and optical microscope system is presented here for the first time. Previous studies measuring sperm propulsive force provided statistical values for the force, for example, the stall force induced by a micro-glass probe [3][4][5] and the escape force from an optical tweezers. 6,7 The rapid movement of a sperm cell causes difficulty for force measurements.…”

“…Schmitz et al [3][4][5] reported a technique using glass needles to measure the force and the torque from the tail of a bull sperm and a rat sperm. The manual handling operation might cause the result to be less sensitive and precise.…”

“…In these studies, the authors measured the torques of bull and rat sperm with a force-calibrated glass microprobe, as described in Schmitz et al's work. [3][4][5] The torque was determined by the average lever-arm length of the sperm multiplied by the measured force. It was obtained by positioning a calibrated microprobe in the beat path of sperm cells where the head of sperm was trapped to a glass microscope slide.…”