A chymotrypsin-like protease involves in motility of sperm in salmonid fish
A chymotrypsin-like protease involves in motility of sperm in salmonid fish
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胰凝乳蛋白酶样蛋白酶参与鲑鱼精子的运动
DOI：
10.2220/biomedres.12.435
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发表时间：
1991
影响因子：
1.2
通讯作者：
M. Morisawa
中科院分区：
文献类型：
--
作者：
K. Inaba;M. Morisawa
研究了十种蛋白酶抑制剂对鲑鱼脱膜精子活力的影响。其中，抑肽酶、N-甲苯磺酰苯丙氨酰氯甲烷（TPCK）和糜抑素对精子活力有较强的抑制作用。仅当用相对高浓度的 ATP (>0.5 mM) 重新激活脱膜精子时，才能观察到这三种蛋白酶抑制剂的抑制作用。结果表明，胰凝乳蛋白酶样蛋白酶以 ATP 依赖性方式参与精子运动。迄今为止，已经从两个方面对精子蛋白酶进行了研究。首先，一些蛋白酶抑制剂会抑制精子的受精和顶体反应，这表明蛋白酶参与受精时的顶体反应 (7, 8)。实际上，已经从海胆、被囊类动物或哺乳动物的精子中分离出了一些蛋白酶，如顶体蛋白 (14)、精子酶 (16) 和蛋白酶体（多催化蛋白酶）(9, 15)。其次，Gagnon 和他的同事证明，一些胰蛋白酶抑制剂和底物会抑制哺乳动物、鲤鱼和海胆的精子活力 (3, 4)。然而，负责控制精子活力的蛋白酶尚未分离出来。硬骨鱼精子没有明显的顶体结构 (1, 12)。因此，使用硬骨鱼精子来研究蛋白酶在控制精子活力中的作用似乎是有利的。我们在这里使用大马哈鱼的精子证明，胰凝乳蛋白酶样蛋白酶可能参与精子的运动。此外，蛋白酶的作用似乎取决于 ATP 的浓度。 Abbrev1'a：'i0ns。' TPCK，N-甲苯磺酰基-苯丙氨酰-氯甲烷； PMSF，苯甲磺酰氟； TLCK，N-顺式-甲苯磺酰基-赖氨酰基-氯甲烷； ST1，大豆胰蛋白酶抑制剂通过将移液管插入输精管收集大马哈鱼（Oncorhync/ms kera）的精液。用 0.04% Triton X-100 溶液对精子进行脱膜，然后在几种蛋白酶抑制剂存在的情况下用 0.5 mM ATP 重新激活脱膜的精子（表 1）。在10种蛋白酶抑制剂中，高分子量丝氨酸蛋白酶抑制剂抑肽酶(20μM)对脱膜精子的运动抑制作用最强。即使在 1 μM 的浓度下，精子活力也被抑肽酶完全阻断。 N-甲苯磺酰基苯丙氨酰氯甲烷 (TPCK) 和糜抑素（胰凝乳蛋白酶样丝氨酸蛋白酶抑制剂）也显着降低了活动精子的百分比。使用约0.2μM抑肽酶、40nM TPCK或80μM糜抑素观察到精子活力降低50％。不可逆丝氨酸蛋白酶抑制剂苯甲磺酰氟（PMSF）也在一定程度上降低了活动精子的百分比。其他类胰蛋白酶丝氨酸蛋白酶抑制剂[亮肽素、N-0:-甲苯磺酰基-赖氨酰-氯甲烷(TLCK)、止痛剂和大豆胰蛋白酶抑制剂(ST1)]、半胱氨酸蛋白酶抑制剂(E-64)和天冬氨酸蛋白酶抑制剂(胃酶抑素)最多显示50%的抑制。然后，我们检查了蛋白酶抑制剂在改变再激活培养基中 ATP 浓度时的抑制效果（图 1）。虽然精子鞭毛的跳动频率随着i的增加而增加。
Ten kinds of protease inhibitors were examined for their effects on motility of the demembranated sperm of chum salmon. Among them, aprotinin, N-tosyl-phenylalanyl-chl0r0methane (TPCK) and chymostatin showed strong inhibitory effects on the sperm motility. The inhibitory effects of these three protease inhibitors were observed only when the demembranated sperm were reactivated with relatively high concentration of ATP (>0.5 mM). The results suggest that a chymotrypsin-like protease involves in sperm motility in an ATP-dependent manner. Sperm proteases have so far been investigated in two aspects. First, fertilization and the acrosome reaction of sperm have been shown to be inhibited by some protease inhibitors, indicating that proteases involve in acrosome reaction at fertilization (7, 8). Actually, some kinds of protease, such as acrosin (14), spermosin (16) and proteasome (multicatalytic proteinase) (9, 15), have been isolated from the sperm of sea urchins, tunicates or mammals. Second, Gagnon and his co-workers have demonstrated that some trypsin inhibitors and substrates inhibit the sperm motility in mammals, carp and sea urchin (3, 4). However, the protease responsible for the control of sperm motility have not yet been isolated. Teleost fish sperm are devoid of apparent acrosomal structure (1, 12). Therefore, it seems advantageous to use teleost sperm for investigating the role of proteases in the control of sperm motility. We show here using sperm from chum salmon that a chymotrypsin-like protease is likely to involve in sperm motility. Furthermore, the action of the protease appears to depend on the concentration of ATP. Abbrev1'a:‘i0ns.' TPCK, N-tosyl-phenylalanyl-chloromethane; PMSF, phenylmethanesulfonyl fluoride; TLCK, N-cz-tosyl-lysyl-chloromethane; ST1, soybean trypsin inhibitor The semen of chum salmon (Oncorhync/ms kera) was collected by inserting a pipette into sperm duct. Sperm were demembranated with 0.04% Triton X-100 solution-and the demembranated sperm were reactivated with 0.5 mM ATP in the presence of several protease inhibitors (Table 1). Among ten kinds of protease inhibitors, aprotinin (20 ,uM), a high molecular weight serine protease inhibitor, exerted the most potent inhibitory effect on the motility of demembranated sperm. Sperm motility was completely blocked by aprotinin even at 1 ,uM. N-tosyl-phenylalanyl-chloromethane (TPCK) and chymostatin, chymotrypsin-like serine protease inhibitors, also significantly reduced the percentage of motile sperm. The 50% reduction in sperm motility was observed with approximately 0.2 /.tM aprotinin, 40 nM TPCK or 80 ,uM chymostatin. An irreversible serine protease inhibitor, phenylmethanesulfonyl fluoride (PMSF), also reduced the percentage of motile sperm to a certain extent. Other trypsin-like serine protease inhibitors [leupeptin, N-0:-tosyl-lysyl-chloromethane (TLCK), antipain and soybean trypsin inhibitor (ST1)], a cysteine protease inhibitor (E-64) and an aspartic protease inhibitor (pepstatin) showed 50% inhibition at the most. We then examined the inhibitory effect of protease inhibitors upon changing the ATP concentration in reactivation medium (Fig. 1). Although the beat frequency of sperm flagella increased with the i