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

Abstract

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 x 10(6) cells / ml) in Brackett and Oliphant medium. The sperm were capacitated and then incubated with Antithrombin III (trypsin and chymotrypsin inhibitor); N-alpha-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.

Participation of the sperm proteasome during in vitro fertilisation and the acrosome reaction in cattle

“…, 2004). With regard to cattle, a previous report indicates that sperm‐zona binding was not affected when the spermatozoa had been treated with trypsin and chymotrypsin inhibitors, enzymatic activities characteristic of the proteasome (Deppe et al. , 2008).…”

Changes in the Cellular Distribution of Tyrosine Phosphorylation and Its Relationship with the Acrosomal Exocytosis and Plasma Membrane Integrity during In Vitro Capacitation of Frozen/Thawed Bull Spermatozoa

“…The release of these hydrolytic enzymes is thought to facilitate the sperm penetration of the cumulus oocyte complex [29]. However, recent publications have shown that mouse spermatozoa that reach the fertilization site have already undergone AE [30] and the same seems to occur in cattle [31][32][33]. The AE is a calcium-dependent exocytotic event [28] that can be artificially induced using calcium ionophores, such as ionomycin and A23187, and also with physiological molecules, such as progesterone or glycoproteins from the zona pellucida of some species [34,35].…”

Venom of the ChileanLatrodectus mactansAlters Bovine Spermatozoa Calcium and Function by Blocking the TEA-sensitive K+Current

“…After depolarization of the membrane or exposure to the venom for 15 min, the spermatozoa were then incubated during 30 min at 381C, 5% CO 2 with protease inhibitors as previously described [Deppe et al 2008]. The spermatozoa were subsequently incubated with Tripan blue 2% for 15 min at 371C in a water bath and were centrifuged at 480 Â g for 3 min.…”

“…The tube was inclined to 45 degrees and incubated for 40 min at 38.51C, 5% CO 2 . After incubation the upper 800 mL of the medium (containing motile spermatozoa) was removed with a sterile transfer pipette [Deppe et al 2008]. The concentration of spermatozoa was measured in a Neubauer counting chamber and diluted in an appropriate volume of external solution to give a final concentration of 3 Â 10 6 spermatozoa ml À 1 .…”