The acrosome reaction in digitonin-permeabilized sea urchin sperm in the absence of the natural inducer.
In many species, the acrosome reaction of sperm is an obligatory step in fertilization. Increases in [Ca2+]i and pHi, activation of adenylyl cyclase and inositol trisphosphate generation accompany the egg jelly-induced acrosome reaction of sea urchin sperm. The signaling mechanisms involved are unknown. We used digitonin, a cholesterol-complexing compound, to selectively permeabilize the plasma membrane of sea urchin sperm suspended in a medium that mimics the cytosolic ion composition. Within 6 to 8 min, 30 to 50 microM digitonin allowed incorporation of the membrane-impermeant dye Hoechst 33258 into the sperm, staining exclusively the nucleus. No alterations in sperm morphology were caused by digitonin at the concentrations used, however, it irreversibly permeabilized the plasma membrane. Permeabilized sperm retained lactate dehydrogenase and actin. When incubated in Ca(2+)-containing permeabilization buffer (pH 7.8), sperm were capable of undergoing spontaneously the acrosome reaction; this reaction was pH dependent and displayed an absolute Ca2+ requirement. Electron microscopy indicates that the acrosome reaction undergone by permeabilized sperm resembled that induced by egg jelly. Additionally, rhodaminyl-phalloidin staining of sperm reacted under permeabilizing conditions revealed a fluorescent filament in the acrosomal tubule region, demonstrating the occurrence of actin polymerization. Thus, in permeabilized sperm the machinery necessary to perform a [Ca2+]i- and pHi-sensitive acrosome reaction is functionally preserved. Permeabilized sperm offer new avenues to study the molecular bases of the sea urchin sperm acrosome reaction.
Castellano
L E
LE
Instituto de Investigación en Biología Experimental, Facultad de Química, Universidad de Guanajuato, Mexico.
López-Godínez
J
J
Aldana
G
G
Barrios-Rodiles
M
M
Obregón
A
A
García de De la Torre
L
L
Darszon
A
A
García-Soto
J
J
D000177:Acrosome / Q000187:drug effects*; D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D002463:Cell Membrane Permeability / Q000187:drug effects*; D004072:Digitonin / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration; D008297:Male; D008854:Microscopy, Electron; D010590:Phalloidine; D012617:Sea Urchins; D013094:Spermatozoa / Q000166:cytology / Q000187:drug effects*; D013194:Staining and Labeling
D010590:Phalloidine; D004072:Digitonin; D002118:Calcium