|
Pilot Studies on the Role of Calcium Signaling in Equine Sperm Motility |
|
|
In spite of the importance of sperm motility for male fertility, our understanding of the mechanisms that regulate equine sperm motility is incomplete. In mammals, it is well accepted that increases in intracellular calcium and the subsequent activation of a calcium signaling pathway are important components of normal motility. However, the individual components of this calcium signaling pathway remain unclear. Calmodulin is a universal intracellular calcium sensor and previous work from our laboratory suggests that calmodulin, as well as other members of a putative calcium/calmodulin pathway, are present in the flagellum of equine sperm. Therefore, as a first step towards a better understanding of the role of calcium signaling in equine sperm motility, we tested the effect of N-(6-aminohexyl)-5-chloro-1-napthalenesulfonamide hydrochloride (W-7), a calmodulin antagonist, on motile horse sperm. The hypothesis is that inhibition of calmodulin through W-7 will cause a decline in equine sperm motility. Ejaculated sperm were collected routinely from a variety of fertile stallions presenting to our reproduction clinic. After collection, sperm were washed 1X and resuspended in TALP buffer to an appropriate concentration. Resuspended ejaculates were divided into seven 400 uL aliquots and treated with either DMSO (control sample) or with a range of concentrations (2.5 to 200 micromolar final concentration) of W-7 in DMSO. The percentages of total and progressively motile sperm were analyzed in each sample at 0, 5 and 15 minutes following treatment using a computer assisted semen analysis (CASA) system (IVOS, Hamilton-Thorne). W-7 significantly reduced both total and progressive sperm motility in a concentration-dependent fashion. To confirm that the effect of W-7 was a result of the inhibition of calmodulin, and not just a toxic effect on the cells, paired sperm samples from each control and treatment group were stained with propidium iodide and 5-carboxyfluorescein diacetate (5-CFDA) to determine the percentage of live cells that were present at each time point. The percentage of live cells for each W-7 treatment group did not differ significantly from the control sample in spite of the significant decline in sperm motility. From this we conclude that the adverse effect of W-7 on sperm motility results from a direct inhibitory effect on calmodulin, rather than from a toxic effect on the sperm. Thus, we have preliminary evidence that equine sperm motility is regulated by a calcium pathway that involves calmodulin. Future studies will attempt to systematically study other potential downstream members of this signaling pathway and to determine their roles in equine sperm motility. Once these pathways are determined, it might be possible in the future to utilize specific agonists to enhance motility in subfertile stallions with reduced sperm motility.
|
|
|