PARENT SESSION
BIOLOGY OF MALE AND FEMALE GAMETES
Harborside C
7:30 AM-10:00 AM

(175) PHARMACOLOGIC INVESTIGATION OF SIGNALING PATHWAYS IN BICARBONATE-MEDIATED ACCELERATION OF SPERMATOZOAN FLAGELLAR BEAT FREQUENCY.

Harper, Andrew^1,2, Carlson, Anne¹, Hille, Bertil¹, Babcock, Donner¹, ¹ Dept. of Physiology & Biophysics, Seattle, WA² Div. of Reproductive Endocrinology & Infertility, Seattle, WA

ABSTRACT- The spermatozoa stored in the cauda epididymis and vas deferens are relatively immotile. Motility increases at ejaculation when sperm are transported from this low-HCO₃^- environment to the HCO₃^--rich semen and fluids of the female reproductive tract. Exposure to HCO₃^- activates sperm adenylyl cyclase and increases cellular cAMP content. We hypothesize that the enhancement of sperm motility by HCO₃^- occurs primarily through cAMP-mediated activation of protein kinase A (PKA). We have used a pharmacological approach to test this proposal. Washed cauda epididymal mouse sperm were placed in an incubation chamber and bathed in various media. Flagellar motility was assessed using a pulsed LED illumination system synchronized to a cooled CCD camera. A flexible software program permits semi-automated analysis of the time series of stop-motion images. Flagellar beat frequency is determined from the phase lag of a simple sine function fitted to the flagellar waveform. We found that 15 mM NaHCO₃ increased beat frequency from 2.88 ± 0.11 to 7.02 ± 0.22 hertz (Hz). In the absence of HCO₃^-, incubation with 1 mM of the permeant cAMP analogs 8-4-(chlorophenylthio)-cAMP and 8-bromo-cAMP increased beat frequency relative to saline controls (2.48 ± 0.11 and 2.86 ± 0.16 vs. 2.17 ± 0.07 Hz, respectively; p < 0.05), a finding consistent with the cAMP-mediated action of HCO₃^-. In contrast to these stimulatory effects, preliminary incubation with the PKA inhibitors H-89 (30 mM) or Rp-cAMPS (1 mM) blunted the acceleration of beat frequency by HCO₃^- (4.14 ± 0.19 vs. 7.02 ± 0.22 Hz; p < 0.01) and (4.65 ± 0.32 vs. 6.22 ± 0.28; p < 0.01). We conclude that the pharmacological responses to both cAMP analogs and to PKA inhibitors are consistent with the proposal that HCO₃^- accelerates flagellar beat frequency primarily by the cAMP-PKA pathway. Further elucidation of this pathway may yield pharmacologic opportunities for the treatment of motility-related male subfertility. Support by the NICHD through agreement U54-HD12629 of the Specialized Cooperative Centers Program in Reproduction Research

KEY WORDS: sperm, motility, bicarbonate, PKA