CHARACTERIZATION OF 2 2ND-MESSENGER PATHWAYS AND THEIR INTERACTIONS IN ELICITING THE HUMAN SPERM ACROSOME REACTION

The human sperm acrosome reaction (AR) occurs via the activation of at least two signal transduction pathways. The purpose of this investiga tion was to characterize two of the pathways, the protein kinase A (PK A) and C (PKC) pathways, and determine whether pathway ''crosstalk'' o ccurs between them in eliciting the AR in capacitated spermatozoa. Sti mulators of each pathway were tested in a dose-dependent manner. AR(ma x), ED(50), and Delta AR(max) (%AR(max) - %AR(control)) values were ca lculated. The PKA pathway stimulators forskolin and dibutyryl cyclic A MP (dbcAMP) induced an AR(max) at 1.0 mu M and 1.0 mM, respectively. T he ED(50) and Delta AR(max) values were: 0.01 mu M and 17% for forskol in and 0.069 mM and 13% for dbcAMP. Two stimulator types of the PKC pa thway were tested: synthetic diacylglycerols (DG) and a phorbol dieste r. 1,2-dioleoyl-sn-glycerol and 1,2-dioctanoyl-sn-glycerol, analogues of the PKC-activating second messenger DG, each induced an AR(max) at 50 mu M. The ED(50) and Delta AR max values were: 33 mu M and 24% for 1,2-dioleoyl and 34.8 mu M and 34% for 1,2-dioctanoyl. 4 beta-Phorbol- 12,13-didecanoate, a PKC stimulator, induced an AR(max) at 0.1 mu M. T he ED(50) and Delta AR(max) were 0.021 mu M and 26%. An inhibitor of e ach kinase was added at the end of the capacitation period and prior t o stimulation by inducers at their AR(max) dose. KT5720, a PKA inhibit or, caused a dose-dependent reduction of the forskolin and dbcAMP-indu ced 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) s timulators of each pathway were combined and tested at the AR(max) and ED(50) concentrations for each; (2) spermatozoa were pretreated with a kinase inhibitor and then stimulated using an alternative pathway st imulator; and (3) a PKA or PKC inhibitor and a combination of PKA and PKC stimulators, at ED(50) concentrations, were tested. The results fo r (1) indicate an additive AR response for ED(50) concentrations but n ot for AR(max) doses. The results for (2) demonstrate that a kinase in hibitor for one pathway prevents induction of the AR by a stimulator o f the alternative pathway. Finally, the results for (3) show that a ki nase inhibitor for one pathway prevents induction of the AR by the com bined use of separate pathway stimulators. When taken collectively, th e present results suggest a convergent mechanism of crosstalk between the PKA and PKC pathways leading to the human sperm AR.