Characterization of two second messenger pathways and their interactions in eliciting the human sperm acrosome reaction.

Abstract

The human sperm acrosome reaction (AR) occurs via the activation of at least two signal transduction pathways. The purpose of this investigation was to characterize two of the pathways, the protein kinase A (PKA) and C (PKC) pathways, and determine whether pathway "crosstalk" occurs between them in eliciting the AR in capacitated spermatozoa. Stimulators of each pathway were tested in a dose-dependent manner. ARmax, ED50, and delta ARmax (%ARmax-%ARcontrol) values were calculated. The PKA pathway stimulators forskolin and dibutyryl cyclic AMP (dbcAMP) induced an ARmax at 1.0 microM and 1.0 mM, respectively. The ED50 and delta ARmax values were: 0.01 microM and 17% for forskolin and 0.069 mM and 13% for dbcAMP. Two stimulator types of the PKC pathway were tested: synthetic diacylglycerols (DG) and a phorbol diester. 1,2-dioleoyl-sn-glycerol and 1,2-dioctanoyl-sn-glycerol, analogues of the PKC-activating second messenger DG, each induced an ARmax at 50 microM. The ED50 and delta AR max values were: 33 microM and 24% for 1,2-dioleoyl and 34.8 microM and 34% for 1,2-dioctanoyl. 4 beta-Phorbol-12,13-didecanoate, a PKC stimulator, induced an ARmax at 0.1 microM. The ED50 and delta ARmax were 0.021 microM and 26%. An inhibitor of each kinase was added at the end of the capacitation period and prior to stimulation by inducers at their ARmax dose. KT5720, a PKA inhibitor, caused a dose-dependent reduction of the forskolin and dbcAMP-induced AR. Calphostin C, a PKC inhibitor, prevented stimulation of the AR by 1,2-dioleoyl and 4 beta-phorbol-12,13-didecanoate. To investigate pathway "crosstalk," the following experiments were conducted: (1) stimulators of each pathway were combined and tested at the ARmax and ED50 concentrations for each; (2) spermatozoa were pretreated with a kinase inhibitor and then stimulated using an alternative pathway stimulator; and (3) a PKA or PKC inhibitor and a combination of PKA and PKC stimulators, at ED50 concentrations, were tested. The results for (1) indicate an additive AR response of ED50 concentrations but not for ARmax doses. The results for (2) demonstrate that a kinase inhibitor for one pathway prevents induction of the AR by a stimulator of the alternative pathway. Finally, the results for (3) show that a kinase inhibitor for one pathway prevents induction of the AR by the combined use of separate pathway stimulators. When taken collectively, the present results suggest a convergent mechanism of crosstalk between the PKA and PKC pathways leading to the human sperm AR.