Browsing by Subject "CatSper"
Now showing 1 - 2 of 2
Results Per Page
Sort Options
Item Hyperactivated Motility of Stallion Spermatozoa(2013-12-02) Loux, Shavahn CIn 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.Item Localization of the CatSper1 protein and induction of hyperactivated-like motility in equine spermatozoa(2009-05-15) Rolke, Kristin RoseIn vitro fertilization is not efficient in the horse, and this may be due to a failure of induction of hyperactivated motility in stallion sperm in vitro. Hyperactivated motility is characterized by high curvilinear velocity and amplitude of lateral head movement, and is required for the sperm to penetrate the zona pellucida of the oocyte in order to achieve fertilization. In mice, hyperactivated motility is induced by calcium influx into the flagellum of the sperm through the CatSper channel, a tetrameric cation channel made up of CatSper proteins 1 to 4. The CatSper channel is located specifically in the principal piece of the sperm tail, and opens in response to an increase in intracellular pH. Factors associated with the induction of hyperactivated motility of sperm have been reported in most domestic and laboratory species, with the notable exception of the horse. In addition, presence of CatSper proteins has not been demonstrated in the horse. Our objective was to determine the presence and location of the CatSper1 protein on stallion sperm, and to determine whether alkalinization of the intracellular pH induces hyperactivated motility in stallion sperm. Presence of the CatSper1 protein on the principal piece of the flagellum of stallion sperm was confirmed by immunocytochemistry with an anti-human CatSper1 C-terminus antibody. Incubation of stallion sperm with the cell-permeant weak base NH4Cl increased curvilinear velocity and amplitude of lateral head movement values in stallion sperm as measured by computer-assisted sperm analysis. These measures are indicative of hyperactivated motility in other species. Hyperactivated-like motility in response to NH4Cl was dependent upon the CO2 atmosphere in which the sperm were incubated and was enhanced by presence of glucose in the medium. Maximum motility values were reached with 25 mM NH4Cl at 60 minutes in a 5% CO2 atmosphere. These results indicate that the CatSper1 protein, and thus presumably the CatSper channel, is present on the principal piece of stallion sperm and that treatments inducing a rise in intracellular pH increase hyperactivated-like motility. These findings represent a basis for establishment of in vitro fertilization protocols that include induced hyperactivated motility to ensure zona penetration.