Characterization of the Biological Functions of Human Recombinant Zona Pellucida Protein 3

Recombinant human zona pellucida protein 3 (rhZP3), expressed. isolated and purified from PA-1 cells, is characterized for its biological activity and the role in the signal transduction pathway. Characterization of the biological activity of rhZP3 was detected by hemizona assay and immunofluorescence staining of acrosome reaction The results indicated that rhZP3 exhibited an inhibition in the binding assay (HZI 43.6 +/-3.3; n = 9; 30 ng/mL rhZP3) and induction of acrosome reaction (198.6% +/- 77.2% increase from baseline; n = 29; 30 ng/mL rhZP3). It was further confirmed by the transmission electron microscopy that there was no difference in morphology of rhZP3-induced, acrosome-reacted spermatozoa and the calcium ionophore A23187-induced, acrosome-reacted spermatozoa. Recombinant human zona pellucida protein 3 also exhibited a dose-dependency in both inhibition of the binding assay and immunofluorescence staining for acrosome reaction. Furthermore, the antagonistic action of pertussis toxin on the G$\rm\sb{i}$-protein resulted in a decrease in the stimulation of acrosome reaction by the rhZP3. Also, there were no detectable changes in [Ca2+]i in the rhZP3-treated spermatozoa as well as in the potentiation study of progesterone and rhZP3 by FURA-2 spectrofluorometry. Hence the present study concludes that rhZP3 is both a binding ligand for the sperm-zona pellucida interaction and acrosome reaction inducer. It is deduced that the optimal experimental conditions of the rhZP3 in the immunofluorescence staining of acrosome reaction are a concentration of rhZP3 protein of at least 30 ng/mL, capacitation time of 4 hours, and 0.5-4 million spermatozoa per mL. In addition, the acrosome reaction induced by the rhZP3 utilizes Gi-protein dependent pathway. Finally, there are no changes in [Ca2+]i detected in populations of sperm with use of FURA-2 spectrofluorometry