Yudin A I, Vandevoort C A, Li M W, Overstreet J W
Department of Obstetrics and Gynecology, University of California, Davis 95616, USA.
Mol Reprod Dev. 1999 Jul;53(3):350-62. doi: 10.1002/(SICI)1098-2795(199907)53:3<350::AID-MRD11>3.0.CO;2-9.
In this study, we investigated the functions of PH-20 and acrosin during the interaction of macaque sperm with the zona pellucida. Both of these sperm enzymes have been reported to be present on the inner acrosomal membrane of acrosome reacted sperm, and have been suggested to play a role during secondary sperm-zona binding in other species. Anti-macaque PH-20 IgG, anti-pig acrosin IgG and soybean trypsin inhibitor (SBTI) were used as probes for immunolocalization of the two proteins at the ultrastructural level, and as reagents for blocking sperm penetration of the macaque zona pellucida in vitro. As a control, we performed similar studies with antibodies to CD-46, which is also located on the inner acrosomal membrane, but has no known function in sperm-zona pellucida interaction. After labeling with anti-acrosin IgG, gold label was not present on the sperm surface before the acrosome reaction, but was detected over the entire head of sperm that were induced to acrosome react with calcium ionophore A23187. In contrast, when sperm were induced to acrosome react by binding to intact zona pellucida, acrosin was present in the acrosomal shroud but not on the inner acrosomal membrane. Similar results were obtained when SBTI was used as a probe for enzyme localization. PH-20 and CD-46 were demonstrated on the inner acrosomal membrane of sperm induced to acrosome react by ionophore treatment and by zona binding. Neither anti-acrosin IgG nor anti-CD-46 IgG affected sperm penetration of the zona at concentrations up to 300 microg/ml, but zona penetration was blocked completely when anti-PH-20 IgG (100 microg/ml) was present during sperm-oocyte interaction. Ultrastructural observations of oocytes incubated with anti-PH-20 IgG showed that acrosomal shrouds were present on the zona surface but no sperm had begun to penetrate into the zona substance. We conclude that anti-PH-20 IgG prevented sperm penetration of the macaque zona pellucida by interference with secondary sperm-zona binding, rather than primary sperm-zona binding or the zona-induced acrosome reaction. Acrosin was not detected on the inner acrosomal membrane of sperm that are induced to acrosome react after zona binding, and acrosin does not appear to be critical for sperm penetration of the macaque zona pellucida.
在本研究中，我们调查了猕猴精子与透明带相互作用过程中PH - 20和顶体蛋白酶的功能。据报道，这两种精子酶存在于顶体反应精子的顶体内膜上，并且有人提出它们在其他物种的精子与透明带的二次结合过程中发挥作用。抗猕猴PH - 20 IgG、抗猪顶体蛋白酶IgG和大豆胰蛋白酶抑制剂（SBTI）被用作在超微结构水平上对这两种蛋白质进行免疫定位的探针，以及在体外阻断猕猴精子穿透透明带的试剂。作为对照，我们用抗CD - 46抗体进行了类似的研究，CD - 46也位于顶体内膜上，但在精子与透明带的相互作用中没有已知功能。用抗顶体蛋白酶IgG标记后，在顶体反应前精子表面未检测到金标记，但在用钙离子载体A23187诱导顶体反应的精子头部整个区域检测到了金标记。相反，当精子通过与完整透明带结合诱导顶体反应时，顶体蛋白酶存在于顶体被膜中，而不在顶体内膜上。当使用SBTI作为酶定位探针时，也获得了类似的结果。在用离子载体处理和与透明带结合诱导顶体反应的精子的顶体内膜上证实了PH - 20和CD - 46的存在。在浓度高达300微克/毫升时，抗顶体蛋白酶IgG和抗CD - 46 IgG均不影响精子穿透透明带，但在精子与卵母细胞相互作用期间存在抗PH - 20 IgG（100微克/毫升）时，透明带穿透被完全阻断。用抗PH - 20 IgG孵育卵母细胞的超微结构观察表明，透明带表面存在顶体被膜，但没有精子开始穿透进入透明带物质。我们得出结论，抗PH - 20 IgG通过干扰精子与透明带的二次结合而非初次结合或透明带诱导的顶体反应来阻止猕猴精子穿透透明带。在与透明带结合后诱导顶体反应的精子的顶体内膜上未检测到顶体蛋白酶，并且顶体蛋白酶似乎对猕猴精子穿透透明带并不关键。
