Takano H, Yanagimachi R, Urch U A
Department of Anatomy and Reproductive Biology, University of Hawaii School of Medicine, Honolulu 96822.
Zygote. 1993 Feb;1(1):79-91. doi: 10.1017/s0967199400001325.
The sperm plasma membrane over the equatorial segment of the acrosome gains the ability to fuse with the oolemma some time during, or after, the acrosome reaction. Since acrosin is a major component of the acrosome matrix that dissolves during the acrosome reaction, we sought to determine the effect of acrosin inhibitors on the sperm's ability to fuse with the oolemma. Five acrosin inhibitors (soybean trypsin inhibitor (SBTI), leupeptin, benzamidine, N-p-tosyl-1-lysin-chloromethyl ketone (TLCK) and phenylmethylsulphonyl fluoride (PMSF) and one non-acrosin inhibitor (N-p-tosyl-1-phenylalanine chloromethyl ketone (TPCK) were tested at non-toxic levels (below motility-disturbing concentrations). These inhibitors were added at three different times: (1) during the acrosome reaction of spermatozoa, (2) during sperm-oocyte contact and fusion, and (3) soon after sperm-oocyte fusion was completed. TLCK prevented sperm-oocyte fusion by inhibiting the acrosome reaction. PMSF inhibited gamete fusion, without inhibiting the acrosome reaction. SBTI, leupeptin and benzamidine also inhibited gamete fusion, but they had no effect if spermatozoa were allowed to acrosome-react in inhibitor-free medium. TPCK was without any inhibitory effects, suggesting that chymotrypsin-like enzymes are not involved in gamete fusion. Although acrosin inhibitors prevented acrosome-reacted spermatozoa from becoming fusion-competent, acrosin (and trypsin) alone could not make the plasma membrane of acrosome-intact spermatozoa fusion-competent. The data suggest that (1) the plasma membrane of the acrosomal region first undergoes dramatic changes immediately before or during the acrosome reaction and (2) acrosin released from the acrosome during the acrosome reaction further alters biophysical and biochemical characteristics of the plasma membrane over the equatorial segment. Such dual changes make the plasma membrane of this specialised region of the spermatozoon competent to fuse with the oolemma. Acrosin may not be the only acrosomal enzyme to participate in these changes.
在顶体反应期间或之后的某个时间，顶体赤道段上方的精子质膜获得了与卵质膜融合的能力。由于顶体蛋白酶是顶体基质的主要成分，在顶体反应期间会溶解，因此我们试图确定顶体蛋白酶抑制剂对精子与卵质膜融合能力的影响。测试了五种顶体蛋白酶抑制剂（大豆胰蛋白酶抑制剂（SBTI）、亮抑蛋白酶肽、苯甲脒、N-对甲苯磺酰-L-赖氨酸氯甲基酮（TLCK）和苯甲基磺酰氟（PMSF））以及一种非顶体蛋白酶抑制剂（N-对甲苯磺酰-L-苯丙氨酸氯甲基酮（TPCK））在无毒水平（低于干扰运动浓度）下的作用。这些抑制剂在三个不同时间添加：（1）在精子的顶体反应期间，（2）在精子与卵母细胞接触和融合期间，（3）在精子与卵母细胞融合完成后不久。TLCK通过抑制顶体反应来阻止精子与卵母细胞融合。PMSF抑制配子融合，但不抑制顶体反应。SBTI、亮抑蛋白酶肽和苯甲脒也抑制配子融合，但如果精子在无抑制剂的培养基中发生顶体反应，则它们没有作用。TPCK没有任何抑制作用，这表明类胰凝乳蛋白酶不参与配子融合。尽管顶体蛋白酶抑制剂阻止了发生顶体反应的精子获得融合能力，但仅顶体蛋白酶（和胰蛋白酶）不能使顶体完整的精子质膜具有融合能力。数据表明：（1）顶体区域的质膜在顶体反应之前或期间立即首先发生显著变化；（2）顶体反应期间从顶体释放的顶体蛋白酶进一步改变赤道段上方质膜的生物物理和生化特性。这种双重变化使精子这个特殊区域的质膜能够与卵质膜融合。顶体蛋白酶可能不是参与这些变化的唯一顶体酶。
