Protease-Induced Hyperactivation of Canine Spermatozoa Associated with Disappearance of Lectin-Binding Glycoproteins on their Surface

Abstract

The relationship between the disappearance of glycoproteins from the surface of canine sperm and sperm capacitation was investigated in vitro. The protease (PR) concentration in flush fluids of the uterine horns and oviducts removed from 6 estrous, 5 diestrous, and 5 anestrous bitches was measured with a protease assay kit. Ejaculated sperm collected from 10 dogs were incubated for 4 hr in Eagle's MEM supplemented with 1 or 5 μg/ml PR, or to which no PR had been added (control). The glycoproteins on the surface of the sperm were stained with 4 different FITC-lectins (Con A, PHA-E, PNA, and WGA), and the percentages of hyperactivated (HA-) sperm and acrosome-reacted (AR-) sperm were evaluated. The mean PR concentration (5.95 μg/ml) in the flush fluid from the oviducts of the estrous bitches was significantly higher than in the fluid from their uterine horns (1.00 μg/ml; P<0.01). The PR concentrations of the flush fluids from the uterine horns and oviducts of both the diestrous and anestrous bitches were less than 0.05 μg/ml. Before incubation the acrosomal regions or entire heads of all sperm clearly stained with each FITC-lectin, but the percentages of sperm binding the 4 FITC-lectins decreased after incubation. The percentages of lectin-binding sperm in the MEM containing 5 μg/ml PR were significantly lower than in the control MEM (P<0.05 and 0.01). The mean percentages of motile sperm and HA-sperm after incubation in the MEM with PR were higher than in the control MEM, but there were no differences in the percentages of AR-sperm. The results indicate that HA-movement of sperm is induced by the disappearance of glycoproteins from the surface of canine sperm as a result of the action of PR in the oviductal fluid of estrous bitches.