Bicarbonate actions on flagellar and Ca2+-channel responses:initial events in sperm activation

Abstract

At mating, mammalian sperm are diluted in the male and female reproductive fluids, which brings contact with HCO3- and initiates several cellular responses. We have identified and studied two of the most rapid of these responses. Stop-motion imaging and flagellar waveform analysis show that for mouse epididymal sperm in vitro, the resting flagellar beat frequency is 2-3 Hz at 22-25°C. Local perfusion with HCO3- produces a robust, reversible acceleration to 7 Hz or more. At 15 mM the action of HCO3- begins within 5 seconds and is near-maximal by 30 seconds. The half-times of response are 8.8±0.2 seconds at 15 mM HCO3- and 17.5±0.4 seconds at 1 mM HCO3-. Removal of external HCO3- allows a slow return to basal beat frequency over ∼10 minutes. Increases in beat symmetry accompany the accelerating action of HCO3-. As in our past work,HCO3- also facilitates opening of voltagegated Ca2+ channels, increasing the depolarization-evoked rate of rise of intracellular Ca2+ concentration by more than fivefold. This action also is detectable at 1 mM HCO3- and occurs with an apparent halftime of ∼60 seconds at 15 mM HCO3-. The dual actions of HCO3- respond similarly to pharmacological intervention. Thus, the phosphodiesterase inhibitor IBMX promotes the actions of HCO3- on flagellar and channel function, and the protein kinase A inhibitor H89 blocks these actions. In addition, a 30 minute incubation with 60 μM cAMP acetoxylmethyl ester increases flagellar beat frequency to nearly 7 Hz and increases the evoked rates of rise of intracellular Ca2+ concentration from 17±4 to 41±6 nM second-1. However, treatment with several other analogs of cAMP produces only scant evidence of the expected mimicry or blockade of the actions of HCO3-, perhaps as a consequence of limited permeation. Our findings indicate a requirement for cAMP-mediated protein phosphorylation in the enhancement of flagellar and channel functions that HCO3- produces during sperm activation.