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Objective: This study was undertaken to clone and express a recombinant human zona pellucida protein 3 and to characterize its biologic activities as a sperm ligand and an inducer of the acrosome reaction., Study Design: Human ovarian teratocarcinoma (PA-1) cells were transfected with an expression vector containing human zona pellucida protein 3 complementary deoxyribonucleic acid with a sequence coding for a 6-histidine tail introduced into its 3' end. Purification of the secreted glycoprotein was performed by sequential affinity (lectin and nickel--nitrilotriacetic acid) and ion-exchange chromatography., Results: Western blot analysis confirmed a molecular weight of approximately 65 kd for the purified product. A cell-free translation system revealed a correctly sized protein backbone of 47 kd. The recombinant human zona pellucida protein 3 demonstrated specific, potent, and dose-dependent competitive inhibition of sperm-zona pellucida binding in vitro under hemizona assay conditions. Recombinant human zona pellucida protein 3 also stimulated the acrosome reaction of live sperm. This effect was fast, dose dependent, and capacitation time dependent. Furthermore, advance incubation with pertussis toxin, an inactivator of heterotrimeric G proteins, blocked recombinant human zona pellucida protein 3--induced acrosomal exocytosis., Conclusion: The recombinant human zona pellucida protein 3 expressed in PA-1 cells manifested the full spectrum of expected biologic activities. It therefore represents a valuable tool for examination of human fertilization and the design of new strategies in diagnosis of male factor infertility and in contraception.