Yuan Y Y, Chen W Y, Shi Q X, Mao L Z, Yu S Q, Fang X, Roldan E R S
Zhejiang Academy of Medical Sciences, Hangzhou, Zhejiang 310013, People's Republic of China.
Biol Reprod. 2003 Mar;68(3):904-13. doi: 10.1095/biolreprod.102.005777.
Phospholipase A(2) (PLA(2)) is activated in spermatozoa in response to progesterone and Ca(2+) ionophores, but to our knowledge, no study has yet reported zona pellucida (ZP)-induced activation of PLA(2). We investigated whether PLA(2) is involved in ZP-stimulated acrosomal exocytosis, if Ca(2+) is required for activation of PLA(2), and signal transduction pathways modulating PLA(2) using guinea pig sperm as a model. Spermatozoa were capacitated and labeled in low-Ca(2+) medium with [(14)C]choline chloride or [(14)C]arachidonic acid and were then exposed to millimolar Ca(2+) and various reagents and stimulated with ZP. Precapacitated spermatozoa exposed to millimolar Ca(2+) and stimulated with ZP experienced increases in arachidonic acid (AA) and lysophosphatidylcholine (lysoPC) levels and a parallel decrease in phosphatidylcholine level; these changes are indicative of PLA(2) 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 PLA(2) 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 G(i) protein inhibitor, before stimulation with ZP blocked the release of AA and lysoPC as well as acrosomal exocytosis. Exposure of spermatozoa to the diacylglycerol (DAG) kinase inhibitor R59022 before ZP stimulation led to a significant increase in generation of lysoPC and exocytosis. Taken together, these results indicate very strongly that PLA(2) plays an essential role in ZP-induced exocytosis in spermatozoa, that PLA(2) activation requires Ca(2+) internalization, and that PLA(2) activation is regulated by signal transduction pathways involving G proteins and DAG.
磷脂酶A2（PLA2）在精子中会因孕酮和钙离子载体而被激活，但据我们所知，尚无研究报道透明带（ZP）可诱导PLA2激活。我们以豚鼠精子为模型，研究了PLA2是否参与ZP刺激的顶体胞吐作用、激活PLA2是否需要钙离子，以及调节PLA2的信号转导途径。精子在低钙培养基中用[14C]氯化胆碱或[14C]花生四烯酸进行获能和标记，然后暴露于毫摩尔浓度的钙离子和各种试剂中，并用ZP刺激。预先获能的精子暴露于毫摩尔浓度的钙离子并经ZP刺激后，花生四烯酸（AA）和溶血磷脂酰胆碱（lysoPC）水平升高，同时磷脂酰胆碱水平相应降低；这些变化表明PLA2被激活。用ZP刺激也导致高比例的精子发生顶体胞吐作用。如果精子在经ZP处理前暴露于马兜铃酸（一种PLA2抑制剂），则脂质变化和胞吐作用会受到抑制。在不添加钙离子的培养基中或在含有毫摩尔浓度钙离子和乙二醇双四乙酸（EGTA）或镧离子（La3+）的培养基中用ZP刺激，不会导致脂质变化或胞吐作用。在用ZP刺激前用百日咳毒素（一种G蛋白抑制剂）预处理，可阻断AA和lysoPC的释放以及顶体胞吐作用。在ZP刺激前将精子暴露于二酰基甘油（DAG）激酶抑制剂R59022，会导致lysoPC生成和胞吐作用显著增加。综上所述，这些结果有力地表明，PLA2在ZP诱导的精子胞吐作用中起重要作用，PLA2的激活需要钙离子内流，并且PLA2的激活受涉及G蛋白和DAG的信号转导途径调控。
