| dc.description.abstract | In vitro fertilization does not occur readily in the horse. Recent evidence suggests that this is due to failure to initiate hyperactivated motility in vitro; however, little is known about the induction of hyperactivated motility in equine sperm. In mice, hyperactivated motility requires the CatSper channel, a pH-gated calcium channel, therefore we investigated this channel and its related intracellular changes, alkalinization and calcium influx, in equine sperm. Motility was assessed by computer-assisted sperm motility analysis, andchanges in intracellular pH and calcium were determined via the calcium and pH-specific fluorescent probes, BCECF-AM, Fluo3-AMand Fluo4-AM. Additionally, a demembranated sperm model was developed to investigate the direct effect of major regulators of sperm motility on axonemal function.
Increasing intracellular pH induced a rise in intracellular calcium, which was inhibited by the known CatSper blocker mibefradil, supporting the presence of a pH-gated calcium channel, presumably CatSper, in equine sperm. Hyperactivation was induced by treatment with high-pH medium, procaine and 4-aminopyridine. Hyperactivation was associated with moderately increased intracellular pH, but appeared inversely related to increases in intracellular calcium. Sperm treated with procaine in calcium-deficient media both maintained motility and underwent hyperactivation, suggesting that extracellular calcium was not required for hyperactivation. CATSPER1 protein was localized to the principal piece of equine sperm on immunocytochemistry. Analysis of the predicted equine CATSPER1 protein revealed species-specific differences in structure in the pH-sensor region.
Demembranated equine sperm required ATP for reactivated motility, but did not require cAMP. Motility of demembranated equine sperm was not inhibited by elimination of calcium (chelation to below 20 pM). Excess calcium inhibited motility at concentrations lower than those reported in other species. Calcium-inhibited sperm arrested with a straight tail rather than in a curve, as seen with calcium arrest in other species. Hyperactivated-like motility was not induced at any pH or calcium concentration. Equine sperm were not inhibited by cadmium at concentrations that profoundly inhibit motility in demembranated sperm in other species. These findings indicate species-specific differences in calcium regulation of sperm motility which may relate directly to the inefficiency of functional capacitation of equine sperm under standard in vitro conditions. | en |
