EXTRACELLULAR CALCIUM NEGATIVELY MODULATES TYROSINE PHOSPHORYLATION AND TYROSINE KINASE-ACTIVITY DURING CAPACITATION OF HUMAN SPERMATOZOA

Capacitation of spermatozoa, a complex process occurring after sperm e jaculation, is required to obtain fertilization of the oocyte in vivo and in vitro. Although most of the biochemical/biophysical events that occur during capacitation in vitro have been characterized, the molec ular mechanisms underlying these complex events are still obscure. Inc reases of intracellular free Ca2+ concentrations ([Ca2+](i)) and prote in tyrosine phosphorylation have previously been demonstrated during i n vitro capacitation of human spermatozoa, In the present study we inv estigated the relationship between extracellular/intracellular Ca2+, p rotein tyrosine phosphorylation, and tyrosine kinase and phosphatase a ctivities during sperm capacitation. We report that the increase in ty rosine phosphorylation of several protein bands that occurs during spe rm capacitation is independent of the presence of Ca2+ in the external medium and, at least partially, of the increase in [Ca2+](i) occurrin g during the process. Indeed, the spontaneous increase in phosphorylat ion was still present in Ca2+-free/EGTA-containing-medium and in the p resence of the intracellular Ca2+ chelator BAPTA/AM. Moreover, phospho rylation of proteins and protein tyrosine kinase (PTK) activity was en hanced if spermatozoa were incubated in Ca2+-free medium, suggesting t he presence of Ca2+-inhibited tyrosine kinase(s) in human sperm. This hypothesis is further substantiated by the lower tyrosine phosphorylat ion observed after incubation with the ionophore A23187 and the endopl asmic Ca2+-ATPase inhibitor thapsigargin, which promote Ca2+ influx in human sperm. The ability of the cells to undergo acrosome reaction in response to progesterone, which can be considered a functional endpoi nt of capacitation, was highly compromised when spermatozoa were incub ated in Ca2+-free medium or in the presence of EGTA, confirming that C a2+ is required for sperm capacitation. Conversely, in the presence of erbstatin, a inhibitor of tyrosine kinase activity, which blunts tyro sine phosphorylation during capacitation, response to progesterone was maintained, suggesting that tyrosine phosphorylation must be kept at a low level (physiologically by the presence of Ca2+ in the external m edium, or pharmacologically by the presence of erbstatin) in order to obtain response to progesterone. This mechanism may be important in vi vo during sperm transit in the female genital tract to ensure appropri ate timing of full capacitation in the proximity of the oocyte.