Effect of Protease Inhibitors on the Acrosome Reaction and Sperm‐Zona Pellucida Binding in Bovine Sperm

Acrosomal proteases participate in several events during fertilization process and are necessary during the acrosome reaction (AR) and sperm‐zona pellucida (ZP) binding process. In this study, the participation of sperm trypsin‐like, chymotrypsin‐like, and metalloprotease enzymes in the AR and ZP binding in cattle was investigated using protease inhibitors. Motile bovine sperm were obtained by a swim‐up method (4 × 10⁶ cells / ml) in Brackett and Oliphant medium. The sperm were capacitated and then incubated with Antithrombin III (trypsin and chymotrypsin inhibitor); N‐α‐p‐tosyl‐l ‐lysine‐chloromethyl‐ketone (trypsin inhibitor); Trypsin inhibitor (I‐S Type from soybean); N‐tosyl‐l ‐phenylalanine‐chloromethyl‐ketone (chymotrypsin inhibitor); or disodium salt from the hydrated ethylenediaminetetraacetic acid (metalloprotease inhibitor). Then, the AR was induced with lysophosphatidylcholine and evaluated with the double stain technique. Sperm‐zona binding capacity was evaluated using cumulus cell‐free oocytes. A significant decrease (p < 0.05) in the percent of true acrosome‐reacted sperm was observed only in cells incubated with trypsin (10.2 ± 1%) and chymotrypsin inhibitors (18.5 ± 1%) in relation to the control (52.2 ± 1%). Treatment with the metalloprotease inhibitor did not affect the AR percentage (51.8 ± 1%). On the contrary, there was no significant change in the number of sperm bound to the ZP with any of the inhibitors used. The results suggest a role for trypsin and chymotrypsin proteases, but not metalloproteases, in the AR in bovine sperm. In addition, these proteases do not seem to be involved in the binding of bovine sperm to the ZP.