ABSTRACT. The effects of hyaluronic acid, which comprises the cumulus intercellular matrix, on Ca 2+ influx, lactate dehydrogenase (LDH) activity, and cyclic AMP synthesis in canine sperm during capacitation was investigated. Ejaculated sperm were collected from 10 Beagle dogs and the sperm were incubated for 4 hr in Eagle's MEM containing 10 µg/ml of hyaluronic acid. The percentages of actively motile sperm, hyperactivated sperm (HA-sperm), acrosome-reacted sperm (AR-sperm), and sperm labeled with fluoresceinated Ca 2+ indicator (Ca 2+ -labeled sperm) were evaluated to assess Ca 2+ influx into the sperm. LDH activity and cAMP concentration were measured in homogenized sperm. The mean percentages of motile sperm, HA-sperm, and Ca 2+ -labeled sperm in the MEM containing hyaluronic acid were higher than in the control medium (P<0.05, 0.05, and 0.01, respectively), but there was no difference between the percentages of AR-sperm. Mean LDH activity and mean cAMP concentration were also significantly higher than the control values (P<0.05). Sperm capacitation consists of changes in movement in the form of hyperactivation (HA) and in morphology in the form of acrosome reaction (AR), and HA and AR are necessary for canine sperm to be able to penetrate oocytes [28,29]. The oviductal fluids of the estrous cow [2,13,25,39] and bitch [19] have been reported to contain a large amount of glycosaminoglycans (GAGs). GAGs are mucopolysaccharides, which includes hyaluronic acid and heparin, that induce sperm capacitation in the oviducts [27,31]. The cumulus intercellular matrix of oocytes is comprised of hyaluronic acid in many species [3,33,43] It is well known that sperm capacitation is induced by Ca 2+ influx into the cytoplasm of sperm [11,41]. Energy metabolism of sperm is closely related to the activity of lactate dehydrogenase (LDH) enzyme in the sperm [6,7,17,24,35,41], and high LDH activity is maintained during sperm capacitation [35]. The flagellar movement of the canine sperm tail has been reported to be induced by an increase in cyclic adenosine monophosphate (cAMP) concentration in the sperm [47]. In other species [1,15,16], energy for sperm movement is produced by an increase in the cAMP concentration in the sperm as well as an increase in LDH activity. It is thought that cAMP production is stimulated by an increase of Ca 2+ concentration in the sperm cytoplasm [14,41] and that sperm capacitation is subsequently induced [40].In the present study, canine ejaculated sperm were incubated in culture medium supplemented with hyaluronic acid to assess the effect of hyaluronic acid on Ca 2+ influx, LDH activity, and cAMP concentration in the sperm. The binding site of GAGs containing hyaluronic acid on the ejaculated sperm surface was investigated using fluorescein isothiocyanate (FITC)-labeled heparin.
