Intracellular calcium store depletion and acrosome reaction in human spermatozoa: role of calcium and plasma membrane potential.
We evaluated the presence and role of internal calcium stores in human uncapacitated spermatozoa by determining the effects of two inhibitors of Ca2+ ATPase of the sarco-endoplasmic reticulum (SERCA-ATPase), thapsigargin and cyclopiazonic acid (CPA) on intracellular calcium concentrations, [Ca2+](i), plasma membrane potential and acrosome reaction. Using a fluorescent conjugate of thapsigargin, we localized internal Ca2+ stores on the acrosome, post-acrosomal region and sperm midpiece. SERCA-ATPase inhibitors induced a rise in [Ca2+](i) both in Ca2+ and Ca2+-free media but under these latter conditions it was reduced with a progressive decline to baseline values; the re-addition of Ca2+-stimulated a rise in [Ca2+](i). This demonstrated that internal Ca2+ store depletion can evoke the opening of Ca2+-channels on sperm plasma membrane, thus showing the existence of "capacitative" Ca2+ entry into these specialized cells. The addition of thapsigargin to human spematozoa induced a dose-dependent increase in acrosome reaction percentages, but only when Ca2+ was present in the external medium. Plasma membrane potential monitoring showed that these inhibitors induced a depolarization dependent on Ca2+ influx from external medium and that this was preceded by a transient hyperpolarization caused by activation of Ca2+-dependent K+ channels. When K+-dependent plasma membrane hyperpolarization was inhibited, the thapsigargin- and CPA-stimulated rise in [Ca2+](i) plasma membrane depolarization and acrosome reaction were abolished. In conclusion, the present study demonstrates that human spermatozoa possess internal Ca2+ stores and that the capacitative Ca2+ entry pathway present in these cells regulates important biological processes that are fundamental for the acrosome reaction.
Rossato
M
M
University of Padova, Department of Medical and Surgical Sciences, Clinica Medica 3, Via Ospedale 105, 35128 Padova, Italy.
Di Virgilio
F
F
Rizzuto
R
R
Galeazzi
C
C
Foresta
C
C
D020101:Acrosome Reaction / Q000187:drug effects / Q000502:physiology*; D000328:Adult; D001896:Boron Compounds; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005456:Fluorescent Dyes; D006801:Humans; D066298:In Vitro Techniques; D007211:Indoles / Q000494:pharmacology; D007424:Intracellular Fluid / Q000378:metabolism; D008297:Male; D008564:Membrane Potentials / Q000187:drug effects; D013094:Spermatozoa / Q000187:drug effects / Q000378:metabolism*; D019284:Thapsigargin / Q000494:pharmacology
C095489:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; D001896:Boron Compounds; D015220:Calcium Channels; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D007211:Indoles; D019284:Thapsigargin; D000252:Calcium-Transporting ATPases; D002118:Calcium; C000543:cyclopiazonic acid