EVALUATION OF THE ACROSOMAL MEMBRANE IN BOVINE SPERMATOZOA - EFFECTS OF PROTEINASE-INHIBITORS

Thawed bovine spermatozoa are characterized by a lack of homogeneity i n the acrosomal membrane. Therefore, it is difficult to visualize the acrosome to assess morphology. Synthetic proteinase inhibitors were te sted on thawed bovine semen for their effect on the integrity of acros omal membranes. The proteinase inhibitors 4-nitrophenyl-4-guanidinoben zoate (NPGB) and N-L-p-tosyl-L-lysine-chloromethylketone (TLCK) were a dded to a medium containing spermatozoa separated on a percoll gradien t. After incubation for 30 min at 38 degrees C in 5% CO2, 95% air (fin al concentration 1 mM), the action of these inhibitors was controlled by measuring the activity of acrosome proteinases. The acrosomal membr ane was evaluated by means of a dual stain procedure (trypan blue, Gie msa). In contrast to spermatozoa that had been incubated with proteina se inhibitor-free solution, samples that had been incubated with TLCK showed homogeneity in 90% of the acrosomal membranes and excellent vis ualization of the acrosome itself; in the NPGB-treated samples, homoge neous staining was observed in 83% of spermatozoa (P < 0.0005). It is concluded that alteration of the acrosomal membrane in thawed semen is not directly caused by freezing-thawing, but may be due to activation of acrosomal proteinases, which is increased during staining procedur es. The addition of proteinase inhibitors before staining offers a new possibility for improved assessment of the acrosome in bovine spermat ozoa.