Bielfeld P, Faridi A, Zaneveld L J, De Jonge C J
Heinrich Heine Universitat, Dusseldorf, Germany.
Fertil Steril. 1994 Mar;61(3):536-41.
To determine if the solubilized human zona pellucida (ZP)-induced acrosome reaction is mediated by protein kinases.
Capacitated spermatozoa were incubated with inhibitors of cyclic adenosine 3':5'-monophosphate (cAMP)-dependent kinase (KT5720), Ca(2+)- and phospholipid-dependent kinase (Calphostin C), and cyclic guanosine 3':5'-monophosphate (cGMP)-dependent kinase (KT5823) and then treated with a corresponding kinase stimulator (dibutyryl cAMP, phorbol 12-myristate 13-acetate and dibutyryl cGMP, respectively) to determine the effect on the acrosome reaction. Appropriate controls were performed. Zonae obtained from the unfertilized oocytes of women attending an IVF program were solubilized using acidic NaH2PO4, and the effect of solubilized ZP on the acrosome reaction was tested in dose-response fashion. Comparative studies with solubilized, zona-free oocyte-treated spermatozoa were performed. The effect of the kinase inhibitors on the solubilized ZP-induced acrosome reaction was then determined.
No significant stimulation of the acrosome reaction by kinase stimulators occurred when spermatozoa were pretreated with inhibitors of the kinases, in contrast to the controls. Capacitated spermatozoa incubated with 2, 4, and 6 solubilized ZP showed a dose-dependent increase in the acrosome reaction. Solubilized oocytes had no effect on the acrosome reaction. Pretreatment of spermatozoa with kinase inhibitors significantly lowered the acrosome reaction induced by solubilized ZP but not completely. When a "cocktail" of the three inhibitors was used, a significant reduction in the acrosome reaction occurred in comparison with single inhibitor treatment.
The present data indicate a role for human ZP-induced activation of multiple second messenger pathways, involving kinases A, C, and G in the human sperm acrosome reaction.
确定溶解的人透明带（ZP）诱导的顶体反应是否由蛋白激酶介导。
将获能精子与环磷酸腺苷（cAMP）依赖性激酶抑制剂（KT5720）、钙和磷脂依赖性激酶抑制剂（Calphostin C）以及环磷酸鸟苷（cGMP）依赖性激酶抑制剂（KT5823）一起孵育，然后用相应的激酶刺激剂（分别为二丁酰cAMP、佛波醇12 - 肉豆蔻酸酯13 - 乙酸酯和二丁酰cGMP）处理，以确定对顶体反应的影响。进行了适当的对照实验。从接受体外受精（IVF）程序的女性未受精卵母细胞中获取的透明带，用酸性磷酸二氢钠溶解，并以剂量反应方式测试溶解的ZP对顶体反应的影响。对用溶解的、无透明带的卵母细胞处理过的精子进行了比较研究。然后确定激酶抑制剂对溶解的ZP诱导的顶体反应的影响。
与对照组相比，当精子用激酶抑制剂预处理时，激酶刺激剂未对顶体反应产生显著刺激。与2、4和6个溶解的ZP一起孵育的获能精子显示顶体反应呈剂量依赖性增加。溶解的卵母细胞对顶体反应无影响。用激酶抑制剂预处理精子可显著降低溶解的ZP诱导的顶体反应，但未完全消除。当使用三种抑制剂的“鸡尾酒”时，与单一抑制剂处理相比，顶体反应显著降低。
目前的数据表明，人ZP诱导的多种第二信使途径的激活在人精子顶体反应中涉及激酶A、C和G。
