Recombinant human ZP3 (rhuZP3) generated by Chinese hamster ovary cells transfected with a plasmid containing human ZP3 cDNA was used to study the acrosome reaction (AR) and intracellular calcium fluxes in capacitated human spermatozoa. Conditioned medium containing rhuZP3 significantly induced the AR [P =£0.005) in 59.4 ± 4.7% of spermatozoa (control = 8.5 ± 3.1%) and caused complete acrosomal loss in a further 17.2 ± 3.8% of cells (control = 3.7 ± 0.7%; mean ± SEM, n = 5). Sperm motility was

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... ot affected and acrosomal exocytosis in response to rhuZP3 was also shown to be time-dependent. Basal concentrations of sperm intracellular calcium were measured (82 ± 7 nM; mean ± SEM, n = 9). A transient increase in intracellular calcium (typically up to 400-450 nM) occurred within 1 min of rhuZP3 addition and was followed by sustained lower values of calcium (200-400 nM). These responses were dependent on the amount of rhuZP3. This is the first report of zona protein-induced changes in intracellular calcium levels in human spermatozoa. The results support the premise that ZP3 is an agonist of the human sperm AR and that rhuZP3 generated in a eukaryotic cell is effective in this respect.