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

Abstract

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. KEY WORDS: canine, capacitation, glycoprotein, protease, sperm.J. Vet. Med. Sci. 66(9): 1027-1031, 2004 The surface of the plasma membrane of mammalian sperm is coated with various glycoproteins [13,15,25,39,41], and some of the glycoproteins on the surface of the sperm are thought to be related to sperm maturation in the epididymal duct [15,25,39] and sperm capacitation in the female reproductive tract [10,31,40]. Studies with fluorescein-isothiocyanate (FITC) -labeled lectins have shown that the glycoproteins secreted by the epithelial cells of the epididymal duct bind to the entire heads or acrosomal regions of the sperm during their transit through the epididymis [3,6,21,27,29] and that the ejaculated sperm are coated with glycoproteins produced by the accessory reproductive glands [5,9]. Some of the glycoproteins on the surface of sperm have been suspected of acting as decapacitation factors [2,10,24,31]. The secretory fluids in the lumen of the female reproductive tract contain various enzymes [9,29], and the concentrations and activities of the enzymes increase in the follicular phase of the ovarian cycle [38]. It has been found that some of the glycoproteins on the surface of the sperm of the rabbit [29] and hamster [38] are degraded and disappear as a result of the ...

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