The effect of A23187 concentration and exposure time on the outcome of the hamster egg penetration test

Abstract

We investigated the conditions required to enhance the performance of human sperm in the hamster egg penetration test with the free acid form of A23187. The best performance was observed after stimulation with 2 microM A23187 for 1 h when the median penetration rate with sperm from fertile donors was 100% of eggs with 5.8 decondensed sperm heads/egg. Extending the stimulation period with 2 microM A23187 to 2 or 3 h, resulted in a progressive decrease in the penetration rate. In the absence of A23187, the penetration rate was lower (0.7 decondensed sperm heads/egg after 1 h) but increased with stimulation time. A similar picture was observed with sperm from patients taken for an IVF programme. For a pool of cryopreserved semen, the coefficient of variation of the penetration rate after stimulation with 2 microM A23187 for 1 h, expressed as decondensed sperm heads/egg, was 11% within and 20% between assays. There was no correlation between the outcome of the hamster egg penetration test and the percentage motility, velocity or lateral head displacement of the sperm measured after the same stimulation regime. However, in IVF patients the initial velocity and lateral head displacement of the sperm (zero time) were correlated with the best result from the hamster egg penetration test (r = 0.62 and 0.57 respectively). No motility changes characteristic of capacitation were detected. We conclude that stimulation with 2 microM A23187 (free acid) for 1 h prior to the addition of the zona free hamster eggs can produce a high penetration rate with fertile samples and provides a convenient and robust protocol for the assay. However, when carried out in this way the test does not assess the ability of the sperm to capacitate.