Chakravarty S, Kadunganattil S, Bansal P, Sharma R K, Gupta S K
Gamete Antigen Laboratory, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi, India.
Mol Reprod Dev. 2008 Jan;75(1):75-88. doi: 10.1002/mrd.20726.
To delineate the functional aspects of zona pellucida (ZP) glycoproteins during fertilization in human, in the present study, fluorochrome-conjugated Escherichia coli (E. coli)- and baculovirus-expressed recombinant human ZP glycoprotein-2 (ZP2), -3 (ZP3), and -4 (ZP4) were employed. In an immunofluorescence assay, capacitated human sperm exhibited binding of the baculovirus-expressed recombinant ZP3 as well as ZP4 to either acrosomal cap or equatorial region whereas acrosome-reacted sperm failed to show any binding to the acrosomal cap. Using double labeling experiments, simultaneous binding of ZP3 and ZP4 to the acrosomal cap was observed suggesting the possibility of different binding sites of these proteins on the sperm surface. No binding of ZP2 was observed to the capacitated sperm. However, acrosome-reacted sperm (20.00 +/- 1.93%) showed binding of ZP2 that was restricted to only equatorial region. Interestingly, E. coli-expressed recombinant human zona proteins also showed very similar binding profiles. Competitive inhibition studies with unlabeled recombinant human zona proteins revealed the specificity of the above binding characteristics. Binding characteristics have been further validated by an indirect immunofluorescence assay using native human heat solubilized isolated zona pellucida. Employing baculovirus-expressed recombinant ZP3 and ZP4 with reduced N-linked glycosylation and respective E. coli-expressed recombinant proteins, it was observed that glycosylation is required for induction of acrosomal exocytosis but its absence may not compromise on their binding ability. These studies have revealed the binding profile of individual human zona protein to spermatozoa and further strengthened the importance of glycosylation of zona proteins for acrosomal exocytosis in spermatozoa.
为了阐明人受精过程中透明带（ZP）糖蛋白的功能方面，在本研究中，使用了荧光染料偶联的大肠杆菌（E. coli）和杆状病毒表达的重组人ZP糖蛋白-2（ZP2）、-3（ZP3）和-4（ZP4）。在免疫荧光测定中，获能的人精子表现出杆状病毒表达的重组ZP3以及ZP4与顶体帽或赤道区的结合，而顶体反应的精子未能显示与顶体帽有任何结合。通过双重标记实验，观察到ZP3和ZP4同时与顶体帽结合，提示这些蛋白质在精子表面可能有不同的结合位点。未观察到ZP2与获能精子结合。然而，顶体反应的精子（20.00±1.93%）显示ZP2的结合仅限于赤道区。有趣的是，大肠杆菌表达的重组人透明带蛋白也显示出非常相似的结合模式。用未标记的重组人透明带蛋白进行的竞争性抑制研究揭示了上述结合特性的特异性。通过使用天然人热溶解分离的透明带进行间接免疫荧光测定，进一步验证了结合特性。使用N-连接糖基化减少的杆状病毒表达的重组ZP3和ZP4以及各自大肠杆菌表达的重组蛋白，观察到糖基化是诱导顶体胞吐所必需的，但其缺失可能不会损害它们的结合能力。这些研究揭示了个体人透明带蛋白与精子的结合模式，并进一步强化了透明带蛋白糖基化对精子顶体胞吐的重要性。
