During interaction with the oocyte, sperrnatozoa are stimulated to undergo exocytosis of the aerosome, a process essential for fertihzation. Two main agonists of exoeytosis have been identified in the oocyte vestments...During interaction with the oocyte, sperrnatozoa are stimulated to undergo exocytosis of the aerosome, a process essential for fertihzation. Two main agonists of exoeytosis have been identified in the oocyte vestments : progesterone (P4), which is trapped in the matrix of the cumulus展开更多
Objective To investigate whether PLA2 is involved in ZP-stimulated acrosomal exocytosis,if Ca^2+ is required for activation of PLA2,and sigal transduction pathways modulating PLA2. Methods Guinea-pig spermatozoa were ...Objective To investigate whether PLA2 is involved in ZP-stimulated acrosomal exocytosis,if Ca^2+ is required for activation of PLA2,and sigal transduction pathways modulating PLA2. Methods Guinea-pig spermatozoa were capacitated and labeled in low Ca^2+ medium with [^14C]choline chloride or [^14C] arachidonic acid,and were then exposed to millimolar Ca^2+ and various reagents and stimulated with ZP. Results Precapacitated spermatozoa exposed to millimolar Ca^2+ and stimulated with ZP experienced increases in arachidonic acid(AA)and lysophosphatidylcholine (lysoPC) and a parallel decrease in phosphatidylcholine (PC);these chages are indicative of PLA2 activation.Simulation with ZP also led to acrosomal exocytosis in a high proportion of spermatozoa. Lipid changes and exocytosis were prevented if spermatozoa were exposed to aristolochic acid,a PLA2 inhibitor, before treatment with ZP.Stimulation with ZP in medium without added Ca^2+,or in medium with millimolar Ca^2+ and EGTA or La^3+ resulted in no lipid changes or exocytosis. Pretreatment with pertussis toxin,a G1 protein inhibitor, before stimulation with ZP,blocked the release of AA and lysoPC and acrosomal exocytosis.Exposure of spermatozoa to the DAG kinase inhibitor R59022 before ZP stimulation led to a significant increase in the generation of lysoPC and exocytosis.Conclusion These results indicate very strongly that PLA2 plays an essential role in ZP-induced exocytosis in spermatozoa, that PLA2 activation requires Ca^2+ internalization,and that it is regulated by signal transduction pathways involving G proteins and DAG.展开更多
To investigate whether GABA/progesterone (P4) stimulates PPI breakdown and its role in the acrosome reaction (AR), spermatozoa of guinea pig were preincubated in MCM-LCa2+ for 5.5 h and then labeled with [32P]pi for 1...To investigate whether GABA/progesterone (P4) stimulates PPI breakdown and its role in the acrosome reaction (AR), spermatozoa of guinea pig were preincubated in MCM-LCa2+ for 5.5 h and then labeled with [32P]pi for 1 h. Samples were washed through a three-step gradient Percoll, adjusted to 5×107 cells/mL and exposed to 2 mmol/L Ca2+, 5 mmol/L GABA, 10 mmol/L P4 and other agents. Lipids were separated by t.l.c. and radioactivity in spots determined by scintillation counting. The AR was assessed by phase-contrast microscopy. The results showed that (i) when spermato-zoa were treated with GABA, 32P-label diminished rapidly in phosphatidylinositol 4, 5-bisphosphate (PIP2), phosphatidylinositol 4-phosphate (PIP), and increased in phosphatidic 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 reached 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≥GABA; (iii) 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 increase in PA labeling abolished when spermatozoa were exposed to EGTA or Ca2+ channel blocker. These re-sults indicate that GABA or P4-induced PPI breakdown is an important and essential event in the series of changes to membrane fusion during the AR of guinea pig spermatozoa and this effect is mediated via calcium by activation of phosphatidylinositol-specific phospholipase C.展开更多
文摘During interaction with the oocyte, sperrnatozoa are stimulated to undergo exocytosis of the aerosome, a process essential for fertihzation. Two main agonists of exoeytosis have been identified in the oocyte vestments : progesterone (P4), which is trapped in the matrix of the cumulus
文摘Objective To investigate whether PLA2 is involved in ZP-stimulated acrosomal exocytosis,if Ca^2+ is required for activation of PLA2,and sigal transduction pathways modulating PLA2. Methods Guinea-pig spermatozoa were capacitated and labeled in low Ca^2+ medium with [^14C]choline chloride or [^14C] arachidonic acid,and were then exposed to millimolar Ca^2+ and various reagents and stimulated with ZP. Results Precapacitated spermatozoa exposed to millimolar Ca^2+ and stimulated with ZP experienced increases in arachidonic acid(AA)and lysophosphatidylcholine (lysoPC) and a parallel decrease in phosphatidylcholine (PC);these chages are indicative of PLA2 activation.Simulation with ZP also led to acrosomal exocytosis in a high proportion of spermatozoa. Lipid changes and exocytosis were prevented if spermatozoa were exposed to aristolochic acid,a PLA2 inhibitor, before treatment with ZP.Stimulation with ZP in medium without added Ca^2+,or in medium with millimolar Ca^2+ and EGTA or La^3+ resulted in no lipid changes or exocytosis. Pretreatment with pertussis toxin,a G1 protein inhibitor, before stimulation with ZP,blocked the release of AA and lysoPC and acrosomal exocytosis.Exposure of spermatozoa to the DAG kinase inhibitor R59022 before ZP stimulation led to a significant increase in the generation of lysoPC and exocytosis.Conclusion These results indicate very strongly that PLA2 plays an essential role in ZP-induced exocytosis in spermatozoa, that PLA2 activation requires Ca^2+ internalization,and that it is regulated by signal transduction pathways involving G proteins and DAG.
基金973" Project (Grant No. G199905992), National Natural Science Foundation of China (Grant No. 39870364) and Natural Science Foundation of Zhejiang Province (Grant No. 398028).
文摘To investigate whether GABA/progesterone (P4) stimulates PPI breakdown and its role in the acrosome reaction (AR), spermatozoa of guinea pig were preincubated in MCM-LCa2+ for 5.5 h and then labeled with [32P]pi for 1 h. Samples were washed through a three-step gradient Percoll, adjusted to 5×107 cells/mL and exposed to 2 mmol/L Ca2+, 5 mmol/L GABA, 10 mmol/L P4 and other agents. Lipids were separated by t.l.c. and radioactivity in spots determined by scintillation counting. The AR was assessed by phase-contrast microscopy. The results showed that (i) when spermato-zoa were treated with GABA, 32P-label diminished rapidly in phosphatidylinositol 4, 5-bisphosphate (PIP2), phosphatidylinositol 4-phosphate (PIP), and increased in phosphatidic 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 reached 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≥GABA; (iii) 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 increase in PA labeling abolished when spermatozoa were exposed to EGTA or Ca2+ channel blocker. These re-sults indicate that GABA or P4-induced PPI breakdown is an important and essential event in the series of changes to membrane fusion during the AR of guinea pig spermatozoa and this effect is mediated via calcium by activation of phosphatidylinositol-specific phospholipase C.
