GABA/progesterone-induced polyphosphoinositide (PPI) breakdown and its role in the acrosome reaction of guinea pig spermatozoa in vitro

To investigate whether GABA/progesterone (P-4) stimulates PPI breakdown and its role in the acrosome reaction (AR), spermatozoa of guinea pig were pre incubated in MCM-LCa2+ for 5.5 h and then labeled with [P-32]pi for 1 h. Sa mples were washed through a three-step gradient Percoll, adjusted to 5 X 10 (7) cells/mL and exposed to 2 mmol/L Ca2+, 5 mu mol/L GABA, 10 mu mol/L P-4 and other agents. Lipids were separated by t.l.c. and radioactivity in spo ts determined by scintillation counting. The AR was assessed by phase-contr ast microscopy. The results showed that (1) when spermatozoa were treated w ith GABA, P-32-label diminished rapidly in phosphatidylinositol 4, 5-bispho sphate (PIP2), phosphatidylinositol 4-phosphate (PIP), and increased in pho sphatidic acid (PA). The loss of label from PPI was almost completed by 10 min. The time-course of the AR was much slower than PPI when spermatozoa re ached a maximal response by 15 min; (ii) the pattern of PPI hydrolysis and stimulation of AR was similar for the three agonists tested; their potency followed the order A23187 > progesterone greater than or equal to GABA; (ii i) GABA-induced PIP2 hydrolysis and rise in PA and the AR were prevented by inclusion of 10 mmol/L neomycin; (iv) the loss of PIP2 labeling and the in crease in PA labeling abolished when spermatozoa were exposed to EGTA or Ca 2+ channel blocker. These results indicate that GABA or P-4-induced PPI bre akdown is an important and essential event in the series of changes to memb rane fusion during the AR of guinea pig spermatozoa and this effect is medi ated via calcium by activation of phosphatidylinositol-specific phospholipa se C.