Regulation of the phosphotyrosine content of human sperm proteins by intracellular Ca2+: role of Ca2+-adenosine triphosphatases.
An increase in the concentration of intracellular free Ca2+ and in the phosphotyrosine content of specific proteins characterizes human sperm capacitation. Whether tyrosine phosphorylation regulates the intracellular free Ca2+ concentration through modulation of Ca2+-ATPase activity or the phosphotyrosine content is under Ca2+ regulation was investigated using Ca2+-ATPase modulators and tyrosine kinase inhibitors. The presence of the Ca2+-ATPase-inhibitor thapsigargin during human sperm capacitation caused an increase in the cytoplasmic free Ca2+ concentration and was associated with an increase in the phosphotyrosine content of specific sperm proteins. Conversely, a decrease in protein tyrosine phosphorylation was observed when gingerol, a Ca2+-ATPase activator, was present during the incubation period. On the other hand, thapsigargin had no effect on the phosphotyrosine content or the cytoplasmic Ca2+ concentration when spermatozoa were incubated in the presence of the phosphodiesterase-inhibitor 3-isobutyl-1-methylxanthine (IBMX). However, the effect of IBMX on phosphotyrosine-containing proteins appears to be a Ca2+-dependent phenomenon, because it was partly inhibited in spermatozoa pretreated with 1,2-bis-(o-aminophenoxy)-ethane-N,N,N,N-tetraacetic acid tetra-(acetoxymethyl)-ester (BAPTA-AM) even though, by itself, BAPTA-AM caused an increase in sperm protein phosphotyrosine content. Tyrosine kinase inhibitors prevented the increase in the phosphotyrosine content without affecting the cytoplasmic free Ca2+ concentration. Based on these findings, the present study suggests that Ca2+-ATPases are involved in the filling of internal Ca2+ stores, such as the acrosome, and are inhibited later during capacitation. Their inhibition allows an increase in cytoplasmic free Ca2+, which is involved in the subsequent increase in the phosphotyrosine content of specific sperm proteins.
Dorval
Véronique
V
Département d'Obstétrique/Gynécologie and Centre de recherche en Biologie de la Reproduction, Université Laval, and Centre de recherche du CHUQ, Québec, Québec, Canada G1L 3L5.
Dufour
Maurice
M
Leclerc
Pierre
P
D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000242:Cyclic AMP / Q000031:analogs & derivatives* / Q000494:pharmacology; D003593:Cytoplasm / Q000187:drug effects / Q000378:metabolism; D004533:Egtazic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D008297:Male; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism*; D047428:Protein Kinase Inhibitors; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D015398:Signal Transduction; D013075:Sperm Capacitation / Q000187:drug effects; D013094:Spermatozoa / Q000737:chemistry / Q000378:metabolism*; D019284:Thapsigargin / Q000494:pharmacology; D013873:Thionucleotides / Q000494:pharmacology
D004791:Enzyme Inhibitors; D047428:Protein Kinase Inhibitors; D011506:Proteins; D013873:Thionucleotides; C070379:1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester; D019000:Phosphotyrosine; C016957:adenosine-3',5'-cyclic phosphorothioate; D004533:Egtazic Acid; D019284:Thapsigargin; D000242:Cyclic AMP; D000252:Calcium-Transporting ATPases; D002118:Calcium; D015056:1-Methyl-3-isobutylxanthine