Publications

In order to fertilize the egg, spermatozoa must go through the capacitation process where they experience Ca2+ uptake, increases in cyclic 3',5' adenosine monophosphate (cAMP) concentrations, superoxide anion production, and protein tyrosine phosphorylation. Although the importance of these processes has been described, the interactions between them, as well as the temporal sequence of these events, remain to be demonstrated. Previous studies from our laboratory have demonstrated that tyrosine phosphorylation of p105 and p81 (p105/81), the two major human sperm phosphotyrosine-containing proteins, was under cAMP and oxygen derivatives regulation. In the present study, we investigated the importance of intra- and extracellular Ca2+, as well as the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine and the phosphatase inhibitors calyculin A and okadaic acid, in the production of superoxide anion and p105/81 tyrosine phosphorylation. An increase in p105/81 phosphotyrosine content was observed when spermatozoa were incubated in the absence of extracellular Ca2+ or with the calmodulin antagonist N-(6-aminohexyl)-1-naphthalenesulfonamide. However, the human sperm capacitation inducer FCSu (ultrafiltrate of fetal cord serum) requires the presence of the extracellular Ca2+ to induce capacitation, superoxide anion production, and tyrosine phosphorylation of p105/ 81, whereas free intracellular Ca2+ had no effect on these last two processes. The production of superoxide anion by spermatozoa was stimulated by inhibitors of phosphodiesterases and serine/threonine phosphoprotein phosphatases. The tyrosine phosphatase inhibitor vanadate decreased by 40% the FCSu-stimulated superoxide anion production, although it had no effect when used alone. These results suggest that, during sperm capacitation, Ca2+ induces an elevation in cAMP levels; this cAMP, through undefined serine/threonine protein phosphorylation, stimulates the generation of superoxide anion, which, in turn, causes the increase in p105/81 phosphotyrosine contents.