A method for preparation, storage, and activation of large populations of immotile sea urchin sperm.
Reversible protein phosphorylation is associated with initiation and modulation of sperm flagellar motility. Many studies aimed at examining the signal transduction mechanisms underlying the expression of motility have relied on detergent-permeabilized sperm reactivated with exogenous 32P-ATP. However, the reactivation conditions allow variable levels of motility to be expressed and phosphorylation of many proteins that appear to be unrelated to sperm motility. Thus, identification of the few relevant proteins is difficult. We have developed a method to collect and keep sperm immotile until reactivated for analysis to normal motility levels. Artificial sea water (ASW) buffered with 5 mM 2-[N-morpholino]ethanesulfonic acid at pH 6.0 and containing 50 mM KCl allows collection and storage of immotile sea urchin sperm for up to 96 h at 4-5 degrees C. Motility under these conditions is essentially zero, but sperm is rapidly reactivated to normal motility by diluting with ASW to standard pH (8.0) and KCl concentration (10 mM).
Bracho
G E
GE
Department of Molecular & Integrative Physiology, University of Kansas Medical Center, Kansas City 66160-7401, USA.
Fritch
J J
JJ
Tash
J S
JS
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002021:Buffers; D002463:Cell Membrane Permeability; D003902:Detergents; D006863:Hydrogen-Ion Concentration; D008297:Male; D010766:Phosphorylation; D012617:Sea Urchins; D012623:Seawater; D012662:Semen Preservation / Q000379:methods*; D015398:Signal Transduction; D013081:Sperm Motility; D013094:Spermatozoa / Q000166:cytology / Q000502:physiology*; D013997:Time Factors
D002021:Buffers; D003902:Detergents; D000255:Adenosine Triphosphate