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(166) THE IMPORTANCE OF EXTRACELLULAR CALCIUM TO THE BICARBONATE-MEDIATED ELEVATION OF FLAGELLAR BEAT FREQUENCY IN MOUSE SPERMATOZOA. Carlson, Anne1, Hille, Bertil1, Babcock, Donner1, 1 University of Washington, Seattle, WA ABSTRACT- Past studies implicate Ca2+ and HCO3- as potential regulators of adenylyl cyclases present within intact mammalian spermatozoa. Exposure of sperm to HCO3- at ejaculation generally is thought to be the physiologically relevant signal to increase motility, an important early step in the preparation of sperm for fertilization. The atypical adenylyl cyclase of sperm responds directly to HCO3- and is hypothesized to mediate elevation of flagellar beat frequency by HCO3-. The role of Ca2+ in the HCO3- -mediated enhancement of motility has not been previously investigated. Here we use flagellar waveform analysis to compare the response of mouse epididymal sperm to 15 mM NaHCO3 in media with or without 2 mM Ca2+. Pulsed LED illumination synchronized to a cooled CCD camera generates a time series of stop-motion images. A semi-automated analysis system fits a sine wave to each flagellar image. Flagellar beat frequency is then determined from the time derivative of the phase lag of this sine wave. We find that 30 s of local perfusion with 15 mM NaHCO3 and 2 mM Ca2+ increased flagellar beat frequency from 2.63 ± 0.08 (mean ± sem; n = 19) to 6.86 ± 0.51 Hz. A Ca2+ -free 15 mM NaHCO3 solution was much less effective (2.75 ± 0.11 Hz). Similar results were obtained using bath perfusion of longer duration (1-5 min). Average flagellar beat frequency was 2.80 ± 0.19 Hz in medium without NaHCO3, 6.83 ± 0.68 Hz with NaHCO3 and 2 mM Ca2+, and only 3.90 ± 0.44 Hz with NaHCO3 and 0 mM Ca2+ (n = 9-10). These results indicate that extracellular Ca2+ is a cofactor for the HCO3- -mediated acceleration of flagellar beat frequency. We may need to include a role for Ca2+ ions in the explanation of how HCO3- elevates sperm motility via a cAMP-PKA pathway. Support by the NICHD through agreement U54-HD12629 of the Specialized Cooperative Centers Program in Reproductive Research. KEY WORDS: adenylyl cyclase, motility, cAMP, semen |
