Localization of the CatSper1 protein and induction of hyperactivated-like motility in equine spermatozoa

In 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.