Effect of protease inhibitors on the acrosome reaction and sperm-zona pellucida binding in bovine sperm.
Effect of protease inhibitors on the acrosome reaction and sperm-zona pellucida binding in bovine sperm.
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DOI：
10.1111/j.1439-0531.2007.00977.x
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发表时间：
2008-12
期刊：
影响因子：
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通讯作者：
M. Deppe;P. Morales;R. Sánchez
中科院分区：
文献类型：
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作者：
M. Deppe;P. Morales;R. Sánchez
顶体蛋白酶参与受精过程中的几个事件，并且在顶体反应（AR）和精子-透明质酸（ZP）结合过程中是必需的。在这项研究中，参与精子胰蛋白酶样，糜蛋白酶样，和金属蛋白酶在AR和ZP结合牛进行了研究，使用蛋白酶抑制剂。在Brackett和Oliphant培养基中通过上游法（4 × 10（6）个细胞/ ml）获得活动牛精子。使精子获能，然后与抗凝血酶III（胰蛋白酶和胰凝乳蛋白酶抑制剂）、N-α-对甲苯磺酰基-L-赖氨酸-氯甲基-酮（胰蛋白酶抑制剂）、胰蛋白酶抑制剂（来自大豆的I-S型）、N-对甲苯磺酰基-L-苯丙氨酸-氯甲基-酮（胰凝乳蛋白酶抑制剂）或来自水合乙二胺四乙酸的二钠盐（金属蛋白酶抑制剂）孵育。然后，用溶血磷脂酰胆碱诱导AR，并用双重染色技术进行评价。使用卵丘无细胞卵母细胞评价精子结合能力。与对照组（52.2 +/- 1%）相比，仅在胰蛋白酶（10.2 +/- 1%）和胰凝乳蛋白酶抑制剂（18.5 +/-1%）孵育的细胞中观察到真正顶体反应精子的百分比显著降低（p < 0.05）。用金属蛋白酶抑制剂处理不影响AR百分比（51.8 +/- 1%）。相反，使用任何抑制剂时，与ZP结合的精子数量均无显著变化。结果表明，胰蛋白酶和糜蛋白酶的作用，但不是金属蛋白酶，在AR牛精子。此外，这些蛋白酶似乎不参与牛精子与ZP的结合。
Acrosomal proteases participate in several events during fertilization process and are necessary during the acrosome reaction (AR) and sperm-zona pellucida (ZP) binding process. In this study, the participation of sperm trypsin-like, chymotrypsin-like, and metalloprotease enzymes in the AR and ZP binding in cattle was investigated using protease inhibitors. Motile bovine sperm were obtained by a swim-up method (4 x 10(6) cells / ml) in Brackett and Oliphant medium. The sperm were capacitated and then incubated with Antithrombin III (trypsin and chymotrypsin inhibitor); N-alpha-p-tosyl-l-lysine-chloromethyl-ketone (trypsin inhibitor); Trypsin inhibitor (I-S Type from soybean); N-tosyl-l-phenylalanine-chloromethyl-ketone (chymotrypsin inhibitor); or disodium salt from the hydrated ethylenediaminetetraacetic acid (metalloprotease inhibitor). Then, the AR was induced with lysophosphatidylcholine and evaluated with the double stain technique. Sperm-zona binding capacity was evaluated using cumulus cell-free oocytes. A significant decrease (p < 0.05) in the percent of true acrosome-reacted sperm was observed only in cells incubated with trypsin (10.2 +/- 1%) and chymotrypsin inhibitors (18.5 +/- 1%) in relation to the control (52.2 +/- 1%). Treatment with the metalloprotease inhibitor did not affect the AR percentage (51.8 +/- 1%). On the contrary, there was no significant change in the number of sperm bound to the ZP with any of the inhibitors used. The results suggest a role for trypsin and chymotrypsin proteases, but not metalloproteases, in the AR in bovine sperm. In addition, these proteases do not seem to be involved in the binding of bovine sperm to the ZP.