A simple method for assessment of the human acrosome reaction of spermatozoa bound to the zona pellucida: lack of relationship with ionophore A23187-induced acrosome reaction.

Abstract

Acrosome reactions induced by the calcium ionophore A23187 and zona pellucida (ZP) were studied. Sperm samples were obtained from fertile men or men with normal semen analysis and normal sperm-ZP binding. Oocytes were obtained, with the consent of the patients, after the failure of fertilization in vitro. Motile spermatozoa selected by a swim-up technique were incubated with 10 microM A23187 for 1 h, four oocytes for 2 h or solubilized ZP (4 ZP/microliters) for 2 h. Spermatozoa bound to the ZP were dislodged and collected in a small volume of phosphate-buffered saline by aspirating the oocytes with a glass pipette with an inner diameter (120 microns) slightly smaller than the diameter of the oocyte. The acrosome status of the spermatozoa was determined using fluorescein-labelled Pisum sativum agglutinin. The proportion of spermatozoa undergoing the acrosome reaction on the ZP at 2 h varied over a wide range (5-99%), but the agreement between results for the same semen sample exposed to different groups of oocytes was good: the standard deviations of the differences being 9%. Pre-incubation of spermatozoa for 2 h did not increase the ZP-induced acrosome reaction. Re-incubation of ZP with the same sperm suspension for 2 h after removing ZP-bound spermatozoa from the first 2 h incubation produced a significantly lower ZP-induced acrosome reaction in the second incubation (22 +/- 16%) than in the first incubation (30 +/- 14%; P < 0.001, n = 20). There was no significant difference in the ZP-induced acrosome reaction with oocytes with ZP which had or had not been penetrated by spermatozoa during the in-vitro fertilization insemination. Pre-incubation of spermatozoa with solubilized ZP blocked sperm-ZP binding. However, the acrosome reaction induced by solubilized ZP (4 ZP/microliters) was significantly lower than the acrosome reaction induced by intact ZP (10 +/- 5 and 30 +/- 13% respectively, n = 11, P < 0.001), but there was a high correlation (Spearman r = 0.822, P < 0.01) between the results. On the other hand, although the average of the acrosome reaction was similar for A23187 (42%) and for ZP (43%), there was no significant correlation between the results for the two stimuli (n = 60). In conclusion, a useful method for assessing the ZP-induced acrosome reaction has been developed using oocytes which failed to fertilize in vitro. The lack of a relationship between the result of the chemical (A23187) and physiological (ZP) stimuli for the acrosome reaction in the same subjects questions the biological basis of using A23187 for tests of sperm function. Solubilized human ZP in a concentration that blocks sperm-ZP binding is a less efficient inducer of the acrosome reaction than is intact ZP. It is possible that the three-dimensional structure of the ZP is important for induction of the acrosome reaction or that spermatozoa which bind to the ZP are more likely to acrosome react. Assessment of the physiological acrosome reaction for diagnosis of sperm defects which interfere with the fertilization process should be concentrated on the spermatozoa which are capable of binding to the ZP.