Single cell analysis of tyrosine kinase dependent and independent Ca2+ fluxes in progesterone induced acrosome reaction.
In this study we developed a single cell analysis protocol with which protein tyrosine kinase (PTK)-dependent and independent Ca2+ fluxes occurring in human spermatozoa in response to progesterone were evaluated. By recording the fluorescence emitted by fluo-3-loaded spermatozoa using a confocal laser scanning microscopy system it was possible not only to monitor relative changes in the intracellular free Ca2+ concentration ([Ca2+]i) but also to determine the time at which the acrosomal exocytosis began. The addition of progesterone produced a rapid transient [Ca2+]i increase in 35% of spermatozoa. In approximately 10% of spermatozoa, this initial [Ca2+]i increase was followed by a secondary [Ca2+]i increase beginning 2-10 min after the progesterone addition and leading to the acrosomal exocytosis in most of these spermatozoa. On the other hand, a rapid triggering of exocytosis during the initial [Ca2+]i increase was a relatively infrequent observation. The inhibition of PTK with genistein or herbimycin A did not influence the initial progesterone-induced [Ca2+]i increase but inhibited the secondary [Ca2+]i increase and the ensuing acrosomal exocytosis. The initial PTK-independent Ca2+ response could be induced by progesterone in both non-capacitated and capacitated spermatozoa, whereas the ability to generate the secondary, PTK-dependent response developed during in-vitro capacitation.
Tesarik
J
J
Department of Biochemistry and Molecular Biology, University of Granada Faculty of Sciences, Spain.
Carreras
A
A
Mendoza
C
C
D000177:Acrosome / Q000502:physiology*; D002118:Calcium / Q000378:metabolism*; D006801:Humans; D008297:Male; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D015398:Signal Transduction; D013075:Sperm Capacitation; D013094:Spermatozoa / Q000502:physiology*
D011505:Protein-Tyrosine Kinases; D002118:Calcium