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M. Llanos, P. Vigil, A. M. Salgado, and P. Morales

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 × 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 block of the acrosome reaction normally induced by the human zona pellucida. In separate experiments, sperm suspensions pretreated with 1 mmol pAB l−1 or 10 μmol NPGB l−1 exhibited a marked decrease in the percentage of acrosome reactions on the zona surface (85 ± 4% and 76 ± 3% inhibition, respectively). In addition, the inhibitors prevented the acrosome reaction induced by human follicular fluid (percentage of acrosome-reacted spermatozoa: control 8 ± 2; follicular fluid 25 ± 3; pAB 6 ± 2; NPGB 8 ± 1; TLCK 12 ± 2). Electron microscope studies suggested a significant inhibition of the membrane fusion events of the acrosome reaction in the inhibitor-treated spermatozoa. These results are the first to show that trypsin inhibitors block sperm penetration of the human zona pellucida owing to an inhibition of the acrosome reaction. In addition, they suggest a role for a trypsin-like enzyme during the acrosome reaction of human spermatozoa.