Calcium-induced quiescence in reactivated sea urchin sperm.

Abstract

Sperm flagella of the sea urchin Tripneustes gratilla beat with asymmetrical bending waves after demembranation with Triton X-100 in the presence of EGTA and reactivation at pH 8.1 with 1 mM ATP in the presence of 2 mM MgSO4. Addition of 0.1--0.2 mM free Ca2+ to these reactivated sperm induces 70--95% of them to become quiescent. This quiescence can be reversed by reduction of the free Ca2% concentration with EGTA, or by dilution to reduce the MgATP2- concentration below 0.3 mM. The quiescent waveform is characterized by a sharp principal bend of approximately 5.6 rad in the proximal region of the flagellum, a slight reverse bend in the midregion that averages approximately 0.3 rad, and a principal bend of approximately 1.1 rad in the tip. The quiescent sperm are highly fragile mechanically, and disruption, including microtubule sliding, occurs spontaneously at a slow rate upon standing or immediately upon gentle agitation. Mild digestion by trypsin causes a gradual appearance of normal, symmetrical flagellar beating. Addition of increasing concentrations of vanadate to quiescent sperm causes a graded decrease in the proximal bend angle, with 50 micrometers vanadate reducing it to approximately 2.6 rad. In the presence of 0.1 mM free Ca2% and 10 micrometers vanadate, a characteristic, crescented stationary bend is induced in the demembranated sperm, without intermediate oscillatory beating, by the addition of either 0.1 or 1 mM ATP. In the absence of vanadate, these two concentrations of ATP produce asymmetric beating and quiescence, respectively. The results support the hypothesis that quiescence in live sperm is induced by an elevated concentration of intracellular Ca2%. In addition, they demonstrate that bending can occur in flagella in which oscillatory beating is inhibited and emphasize the close relationship between asymmetric beating and quiescence.