Leyton L, Saling P
Department of Obstetrics and Gynecology, Duke University Medical Center, Durham, North Carolina 27710.
Cell. 1989 Jun 30;57(7):1123-30. doi: 10.1016/0092-8674(89)90049-4.
In the mouse, the zona pellucida (ZP) glycoprotein ZP3 both binds intact sperm and induces acrosomal exocytosis. The subsequent signaling pathway(s) is still uncertain, but Gi-like proteins have been implicated. By analogy with other signal transduction mechanisms, we examined anti-phosphotyrosine antibody reactivity in mouse sperm. Antibodies reacted with three proteins of 52, 75, and 95 kd. Indirect immunofluorescence localized reactivity to the acrosomal region of the sperm head. The 52 kd and 75 kd phosphoproteins are detected only in capacitated sperm, whereas the 95 kd protein is detected in both fresh and capacitated sperm. For the 95 kd protein, the level of immunoreactivity is not related to sperm motility but is enhanced by both capacitation and sperm interaction with solubilized ZP proteins. In addition, binding of radiolabeled whole ZP or purified ZP3 to blots of separated sperm proteins identified two ZP binding proteins of 95 kd and 42 kd. 95 kd sperm proteins that bind to ZP3 also react with anti-phosphotyrosine antibodies (in a ZP concentration-dependent manner), supporting the idea that the same 95 kd sperm protein serves as a ZP3 receptor and as a tyrosine kinase substrate. These findings and our evidence on acrosome reaction triggering via sperm receptor aggregation suggest that a 95 kd protein in the sperm plasma membrane is aggregated by ZP3, which stimulates tyrosine kinase activity leading to acrosomal exocytosis.
在小鼠中，透明带（ZP）糖蛋白ZP3既能结合完整的精子，又能诱导顶体胞吐作用。随后的信号通路仍不确定，但已涉及类似Gi的蛋白质。通过与其他信号转导机制类比，我们检测了小鼠精子中抗磷酸酪氨酸抗体的反应性。抗体与三种分子量分别为52、75和95kd的蛋白质发生反应。间接免疫荧光将反应定位在精子头部的顶体区域。52kd和75kd的磷蛋白仅在获能精子中检测到，而95kd的蛋白在新鲜精子和获能精子中均能检测到。对于95kd的蛋白，免疫反应水平与精子活力无关，但获能以及精子与可溶性ZP蛋白相互作用均能增强这种反应。此外，放射性标记的完整ZP或纯化的ZP3与分离的精子蛋白印迹结合，鉴定出两种分子量分别为95kd和42kd的ZP结合蛋白。与ZP3结合的95kd精子蛋白也能与抗磷酸酪氨酸抗体发生反应（呈ZP浓度依赖性），这支持了同一种95kd精子蛋白既是ZP3受体又是酪氨酸激酶底物的观点。这些发现以及我们关于通过精子受体聚集触发顶体反应的证据表明，精子质膜中的一种95kd蛋白被ZP3聚集，从而刺激酪氨酸激酶活性，导致顶体胞吐作用。
