Inhibition of the acrosome reaction by trypsin inhibitors and prevention of penetration of spermatozoa through the human zona pellucida

In this study we evaluated the effect of several trypsin inhibitors (p-aminobenzamidine: pAB; N-α-p-tosyl-L-lysine-chloromethyl-ketone: TLCK and p-nitrophenyl-p'-guanidino-benzoate NPGB) on sperm binding and penetration of the human zona pellucida. Motile spermatozoa, selected by a two-step Percoll gradient, were incubated at 1 x 107 cells ml-1 at 37°C and in 5% CO2 for 4.5 h. This was followed by the addition of 1 mmol pAB l-1 or phosphate-buffered saline (control) for 30 min. Three to four non-viable human oocytes were then added to each sperm suspension and incubated for 3 h. The numbers of spermatozoa bound to the human zona pellucida and in the perivitelline space were determined by phase contrast microscopy. The results showed that pAB significantly inhibited zona penetration by spermatozoa (56 ± 8% oocytes penetrated, control versus 0 ± 0% oocytes penetrated, pAB, mean ± SEM), without modifying spermatozoa-zona pellucida binding. The inhibition of zona penetration was due to a