Comparison of two techniques to evaluate the acrosomal status of zona pellucida bound sperm in humans

In this work, we have compared two procedures that evaluate the acrosomal status of human sperm bound to the human zona pellucida. Motile sperm, selected by a Percoll gradient, were capacitated by incubation at 37°C, 5% CO ₂, for 4.5 h, at 20 × 10⁶ cells ml^-1. Then, the sperm were incubated with nonviable human oocytes for 10 min at 37°C, 5% CO₂. The oocytes with bound sperm were transferred to 500 μl phosphate-buffered saline (PBS) and washed to remove loosely bound sperm. The oocytes were then processed according to the procedures of Cross et al. (1986) or Liu & Baker (1996). In the Cross's procedure, the sperm were labelled while they were bound to the zona. In the Liu's procedure, the sperm were first dislodged from the zona into a droplet of PBS and labelled in there. Both procedures gave equivalent percentages of acrosome-reacted sperm. However, the total number of zona-bound sperm available for assessment with the procedure of Liu & Baker was greater than that of Cross et al. We suggest to use the former procedure to evaluate the acrosomal status of zona-bound sperm in humans. Moreover, this procedure also provided information about sperm ability to bind to the zona pellucida.