Insights into the Mechanism of ADP Action on Flagellar Motility Derived from Studies on Bull Sperm

Abstract

Adenosine diphosphate (ADP) is known to have interesting effects on flagellar motility. Permeabilized and reactivated bull sperm exhibit a marked reduction in beating frequency and a greatly increased beat amplitude in the presence of 1-4 mM ADP. In this study we examined the force production of sperm reactivated with 0.1 mM ATP with and without 1 mM ADP and found that there is little or no resulting change in the stalling force produced by a bull sperm flagella in response to ADP. Because bull sperm bend to a higher curvature after ADP treatment we explored the possibility that ADP-treated sperm flagella are more flexible. We measured the stiffness of 50 muM sodium vanadate treated bull sperm in the presence of 4 mM ADP, but found no change in the passive flagellar stiffness. When we analyzed the torque that develops in ADP-treated sperm at the point of beat reversal we found that the torque developed by the flagellum is significantly increased. Our torque estimates also allow us to calculate the transverse force (t-force) acting on the flagellum at the point of beat direction reversal. We find that the t-force at the switch-point of the beat is increased significantly in the ADP treated condition, averaging 0.7 +/- 0.29 nN/microm in 0.1 mM ATP and increasing to 2.9 +/- 1.2 nN/microm in 0.1 mM ATP plus 4 mM ADP. This suggests that ADP is exerting its effect on the beat by increasing the tenacity of dynein attachment at the B-subtubule. This could be a direct result of a regulatory effect of ADP on the binding affinity of dynein for the B-subtubule of the outer doublets. This result could also help to explain a number of previous experimental observations, as discussed.

Flagellar and ciliary beating: the proven and the possible

“…We have demonstrated that reactivated, de-membranated bull sperm show a significant increase in the t-force at the switch-point of the beat in the presence of ADP (Lesich et al, 2008). This is in agreement with experimental studies on the nucleotide regulation of dynein, which assert that the binding affinity of dynein for tubulin is directly regulated through a long-residence ADP-binding site on the dynein head (Inoue and Shingyoji, 2007;Cho et al, 2008).…”

“…The dynein illustration is based on the proposal put forth by Inoue and Shingyoji (Inoue and Shingyoji, 2007). Figure adapted from Lesich et al (Lesich et al, 2008).…”

Functional anatomy of the mammalian sperm flagellum

“…Its stiffness ranges from 15 3 10 221 N • m 2 in bull sperm (Lesich et al, 2008) to 300 3 10 221 N • m 2 in rat sperm (Lindemann et al, 2005) with mouse sperm being intermediary (data not published). This compares to a value of 1 3 10 221 N • m 2 in sea urchin (Pelle et al, 2009) The torque required to bend a flagellum is the product of the curvature multiplied by the stiffness.…”

“…This induced infertility could potentially be of value as a male contraceptive. It has the advantages that it would be reversible, and because the calcineurin involved is sperm specific, it could conceivably be targeted by inhibitors selective for the sperm calcineurin.When we measured the stiffness of bull sperm and rat sperm we found great variability from cell to cell (SchmitzLesich 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.…”

“…When we measured the stiffness of bull sperm and rat sperm we found great variability from cell to cell (SchmitzLesich 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.…”