Lee M A, Kopf G S, Storey B T
Biol Reprod. 1987 Apr;36(3):617-27. doi: 10.1095/biolreprod36.3.617.
Capacitated mouse sperm undergo the spontaneous acrosome reaction in suspension and the zona-induced acrosome reaction when bound to isolated, intact zonae pellucidae. The zona-induced acrosome reaction in the mouse resembles, in part, ligand-receptor-mediated exocytotic processes that occur in some somatic cells. Since such processes have been shown to be mediated in part by protein kinase C-catalyzed protein phosphorylation, the effects of phorbol esters, which are potent activators of this kinase, on both the spontaneous and the zona-induced acrosome reaction were examined. At concentrations up to 10 microM, 12-tetradecanoyl phorbol-13-acetate (TPA) had no effect on the time course of the spontaneous acrosome reaction as scored by the chlortetracycline (CTC) fluorescence assay. Capacitated, acrosome-intact sperm display Pattern B in the CTC assay with fluorescence on the anterior head; fully acrosome-reacted sperm display Pattern AR with no fluorescence on the head. The time course of the loss of Pattern B in the zona-induced acrosome reaction was markedly accelerated by 65 nM TPA as compared to controls, whereas the appearance of Pattern AR was retarded. The appearance of Pattern S, which is characterized by punctate fluorescence on the head and which marks an intermediate state between Pattern B and Pattern AR in the controls, was accelerated by 65 nM TPA to the same extent as the loss of Pattern B at early times post-binding to zonae. The disappearance of Pattern S at later times post-binding to zonae was retarded by 65 nM TPA to the same extent as the appearance of Pattern AR. The transitions between the fluorescence patterns, designated the B-to-S and the S-to-AR transitions, therefore define two stages of the zona-induced acrosome reaction, which are affected in opposite directions by TPA. The effects of 65 nM TPA are mimicked by 60 nM 4-beta-phorbol-12,13-didecanoate (4-beta-PDD) while the 4-alpha isomer is without effect. Such stereospecificity is similar to that reported for the activation of protein kinase C. The diacylglycerol, 1-oleyl-2-acetylglycerol, which is also known to activate protein kinase C, mimicked the effects of TPA and 4-beta-PDD on the time courses of the B-to-S and S-to-AR transitions. These results suggest that protein kinase C may play an intermediary role in the zona-induced mouse sperm acrosome reaction.
获能的小鼠精子在悬浮液中会自发发生顶体反应，而当与分离的完整透明带结合时会发生透明带诱导的顶体反应。小鼠的透明带诱导顶体反应部分类似于某些体细胞中发生的配体 - 受体介导的胞吐过程。由于已表明此类过程部分由蛋白激酶C催化的蛋白质磷酸化介导，因此研究了佛波酯（这种激酶的有效激活剂）对自发和透明带诱导顶体反应的影响。通过金霉素（CTC）荧光测定法评估，在浓度高达10微摩尔时，12 - 十四酰佛波醇 - 13 - 乙酸酯（TPA）对自发顶体反应的时间进程没有影响。在CTC测定中，获能的、顶体完整的精子呈现模式B，头部前部有荧光；完全顶体反应的精子呈现模式AR，头部无荧光。与对照相比，65纳摩尔TPA显著加速了透明带诱导顶体反应中模式B消失的时间进程，而模式AR的出现则延迟。模式S的出现，其特征是头部有斑点状荧光，在对照中标志着模式B和模式AR之间的中间状态，在与透明带结合后的早期，65纳摩尔TPA加速模式S出现的程度与模式B消失的程度相同。在与透明带结合后的后期，65纳摩尔TPA延迟模式S消失的程度与模式AR出现的程度相同。荧光模式之间的转变，即B到S和S到AR的转变，因此定义了透明带诱导顶体反应的两个阶段，TPA对这两个阶段的影响方向相反。65纳摩尔TPA的作用被60纳摩尔4 - β - 佛波醇 - 12,13 - 二癸酸酯（4 - β - PDD）模拟，而其4 - α异构体则无作用。这种立体特异性与报道的蛋白激酶C激活的情况相似。二酰基甘油1 - 油酰 - 2 - 乙酰甘油，也已知可激活蛋白激酶C，模拟了TPA和4 - β - PDD对B到S和S到AR转变时间进程的影响。这些结果表明蛋白激酶C可能在透明带诱导的小鼠精子顶体反应中起中介作用。
