Roldan E R, Harrison R A
Department of Molecular Embryology, AFRC Institute of Animal Physiology and Genetics Research, Babraham, Cambridge, U.K.
Biochem J. 1989 Apr 15;259(2):397-406. doi: 10.1042/bj2590397.
An investigation was made of the modifications in phospholipids that occur during the exocytotic event known as the 'sperm acrosome reaction'. Phospholipids were prelabelled with 32P, and exocytosis was induced with Ca2+ and the ionophore A23187. When incubated with [32P]Pi in various media suitable for supporting sperm survival or fertilization in vitro, spermatozoa from all five species examined (ram, boar, guinea pig, mouse and human) incorporated 32P rapidly into the components of the phosphoinositide cycle. There were differences both between species and between media with respect to the actual rate of incorporation of label, and also between species with respect to other phospholipids labelled. Treatment of spermatozoa with Ca2+ and A23187 to induce the acrosome reaction resulted in a rapid breakdown of phosphatidylinositol 4, 5-bisphosphate and phosphatidylinositol 4-phosphate, which was complete within 3 min; there was also a great increase in labelling of phosphatidate. Occurrence of acrosome reactions in the sperm population was only observed after 5-10 min and reached a maximum response of greater than 90% after more than 30 min. The phosphoinositide breakdown was related to subsequent exocytosis: after EGTA/ionophore treatment, neither inositide breakdown nor exocytosis took place; however, later addition of Ca2+ resulted in immediate inositide breakdown, and exocytosis followed, with a delay relative to Ca2+ addition exactly similar to that following standard Ca2+/ionophore treatment. Neomycin inhibited both inositide breakdown and subsequent exocytosis provided it was added together with Ca2+ and ionophore; however, if the drug was added 3 min after Ca2+ and ionophore (by which time inositide breakdown was already complete), exocytosis was not inhibited. Ca2+ seemed to have several consecutive roles in the acrosome reaction. Low (micromolar) levels of free Ca2+ were needed both for phosphoinositide breakdown and for an event downstream of this breakdown; no other bivalent cation could substitute for Ca2+ in either event, and inositide breakdown was actually inhibited by Mg2+. In addition, millimolar levels of Ca2+ were needed for later stages of exocytosis, although this requirement could be satisfied by Sr2+. We conclude that breakdown of polyphosphoinositides is an essential early process after Ca2+ entry in the chain of events that lead to exocytosis in the mammalian sperm acrosome reaction.
对在被称为“精子顶体反应”的胞吐事件过程中发生的磷脂变化进行了研究。磷脂用³²P预先标记，并用Ca²⁺和离子载体A23187诱导胞吐作用。当在各种适合支持精子在体外存活或受精的培养基中与[³²P]Pi一起孵育时，所检测的所有五个物种（公羊、公猪、豚鼠、小鼠和人类）的精子都迅速将³²P掺入磷酸肌醇循环的成分中。在标记掺入的实际速率方面，物种之间和培养基之间存在差异，并且在其他被标记的磷脂方面物种之间也存在差异。用Ca²⁺和A23187处理精子以诱导顶体反应导致磷脂酰肌醇4,5 - 二磷酸和磷脂酰肌醇4 - 磷酸迅速分解，在3分钟内完成；磷脂酸的标记也有很大增加。在精子群体中顶体反应的发生仅在5 - 10分钟后观察到，并且在30多分钟后达到大于90%的最大反应。磷酸肌醇分解与随后的胞吐作用相关：用EGTA/离子载体处理后，既不发生肌醇磷脂分解也不发生胞吐作用；然而，随后添加Ca²⁺导致立即发生肌醇磷脂分解，随后发生胞吐作用，相对于Ca²⁺添加的延迟与标准Ca²⁺/离子载体处理后的延迟完全相似。新霉素抑制肌醇磷脂分解和随后的胞吐作用，前提是它与Ca²⁺和离子载体一起添加；然而，如果在Ca²⁺和离子载体后3分钟添加该药物（此时肌醇磷脂分解已经完成），则胞吐作用不受抑制。Ca²⁺在顶体反应中似乎有几个连续的作用。低（微摩尔）水平的游离Ca²⁺对于磷酸肌醇分解和该分解下游的一个事件都是必需的；在这两个事件中没有其他二价阳离子可以替代Ca²⁺，并且肌醇磷脂分解实际上被Mg²⁺抑制。此外，胞吐作用的后期阶段需要毫摩尔水平的Ca²⁺，尽管Sr²⁺可以满足这一需求。我们得出结论，多磷酸肌醇的分解是Ca²⁺进入导致哺乳动物精子顶体反应中胞吐作用的事件链后的一个重要早期过程。
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