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doi: 10.1242/10.1242/dev.00353

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

Gunther Wennemuth1,2, Anne E. Carlson1, Andrew J. Harper3 and Donner F. Babcock1,*

1 Department of Physiology and Biophysics, Box 357290, University of Washington, Seattle, WA 98195-7290, USA
2 Department of Anatomy and Cell Biology, Philipps University Marburg, 35037 Marburg, Germany
3 Department of Obstetrics and Gynecology, Box 356460, University of Washington, Seattle, WA 98195-6460, USA



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  		Fig. 1. Bicarbonate enhances the rate of depolarization-evoked Ca2+ entry. (A,B) Sperm were perfused locally with medium Na7.4 that contained 0 or 15 mM bicarbonate except during brief (10 second) stimuli with the depolarizing medium K8.6 as shown. [Ca2+]i was monitored by indo-1 emission ratio photometry. (C) The initial portions of the averaged responses evoked in these experiments before ({blacksquare},•) and after ({blacktriangledown}) exposure to 15 mM HCO3-. The slope of the best-fit regression line with the indicated rate of rise is a measure of the number of Ca2+ channels open during the KCl depolarization.

 


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  		Fig. 2. The rapid, dose-dependent stimulatory action of bicarbonate increases with duration of exposure and with the age of the sperm preparation. (A) Depolarization-evoked responses were monitored in sperm stored in medium Na7.4 at ambient conditions for the indicated times (t=0 marks the start of indo-1 loading). Stimulus with medium K8.6 was applied (to a total of 175 cells from 11 animals) after 2-minute perfusions with medium Na7.4 containing 15 mM HCO3-. Mean rates of rise were determined for age bins of 10 minutes duration. Regression analysis indicates that the rate of rise increases linearly with age (r=0.9917). (B) Anti-phosphotyrosine immunoblot of extracts from sperm prepared as follows: lane 1, after swim-out (15 minutes at 37°C with 5% CO, 95% air) and washing in medium Na7.4; lane 2, after 15 minutes further incubation at ambient conditions in medium Na7.4 containing 15 mM HCO3-; lane 3, after 90 minutes incubation of the washed cells in capacitation medium at 37°C with 5% CO2/95% air; lane 4, after 180 minutes incubation in storage medium Na7.4 at ambient conditions. The arrow indicates migration of a 120 kDa marker protein. Results are representative of three independent experiments. (C) Ca2+ channel activity was determined as in Fig. 1B, except that the third stimulus followed perfusions of varied durations using medium Na7.4 with 15 mM NaHCO3. The depolarization-evoked rate of rise was normalized to the mean rate for the two preceding stimuli applied before exposure to HCO3- (each time point is averaged from 12-15 trials examined in three independent experiments). (D) In a similar protocol, the third stimulus followed a 2 minute perfusion using medium Na7.4 with 0, 1 or 15 mM NaHCO3. Values shown are mean±s.e.m. (n=11, in three experiments): 1.30±0.17; 2.45±0.20; 6.41±0.87.

 


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  		Fig. 3. Sperm flagellar waveform analysis. (A) The initial six video frames in a time series of images (48 x 48 µm) of a sperm collected at 30 Hz. (B) The flagellar beat envelope was visualized from a composite of all 30 images collected in the complete 1 second series. The flagellar beat amplitude is indicated by the orthogonal arrow placed 25 µm along the midline of the flagellar beat axis. (C) The flagellar waveform in each image in A was fitted with a sine function. The original, superimposed grayscale images were overlaid with the (colored) best-fit curves. (D) Regression analysis of the phase lags of the fitted sine functions in a longer segment of this series yields the flagellar beat frequency. The slope of the regression line (39.8 rad second-1) corresponds to a beat frequency of 6.34 Hz.

 


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  		Fig. 4. Acceleration of flagellar beat frequency by bicarbonate. (A,B) In a large-scale experiment, 90 cells from a single animal were selected randomly for examination 1-10 minutes after addition to medium Na7.4 containing 0 ({circ}) or 15 ({triangleup}) mM NaHCO3. Beat frequency (A) and amplitude (B) were determined by waveform analysis as described in Fig. 3. Horizontal bars mark the mean, enclosed by boxes that extend ±1 s.d. (C-E) Beat frequencies extracted from sperm images collected during perfusion by medium Na7.4 with 0 ({circ}, {square}), or with Na7.4 containing 1 ({blacksquare}), or 15 (•) mM NaHCO3 as indicated. Data were averaged from triplicate experiments each involving three cells from one animal (n=9). Error bars that were smaller than the symbol were deleted for clarity. In C, half times were 8.8±0.2 and 17.5±0.4 seconds for the indicated bestfit sigmoidal curves.

 


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  		Fig. 5. Waveform analysis of sperm in the absence and presence of bicarbonate. (A,B) The angular deviation (tan angle) and distance along the flagellum (arc length) were extracted from time series of waveform images. (A) Data encompassing two beat cycles (23 frames) for a representative sperm bathed in medium Na7.4. (B) Data encompassing approximately three beat cycles (11 frames) for another sperm in Na7.4 with 15 mM NaHCO3. In A,B, the heavier black lines show the time-averaged tangent angle compiled from two and six beat cycles, respectively. (C) Similar analysis was applied to 15-29 frames (two to six beat cycles) of waveform images of 20 other cells for each treatment group. Mean values (±s.e.m.) for all cells (n=21 cells in three experiments) bathed with Na7.4 alone ({circ}) or with 15 mM NaHCO3 (•). The length of the averaged segments were limited by other traces that were shorter than those shown in A and B.

 


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  		Fig. 6. Enhancement of the actions of bicarbonate by the phosphodiesterase inhibitor IBMX and blockade by the PKA inhibitor H89. (A) Enhancement by IBMX. Ca2+ channel activity was determined as in Fig. 1B, except that the third stimulus followed 1 minute perfusion by medium Na7.4 alone (none), with 15 mM NaHCO3, or with 0.2 mM IBMX and NaHCO3, as indicated. The depolarization-evoked rate of rise was normalized to the average of rates for the preceding two control stimuli. Mean values (n=13) were 0.95±0.10, 1.78±0.20 and 2.57±0.45. (B) Inhibition by H-89. In similar experiments, sperm in Na7.4 received a control stimulus, 1 minute of recovery in Na7.4 with 0, 1, 10 or 30 µM H89 then 2 minute exposures to Na7.4 with 15 mM NaHCO3 and the same concentration of H89. The test stimulus used K8.6 containing the same concentration of H89. Normalized mean values (n=11) were 6.64±0.74, 4.39±0.37, 3.85±0.82 and 2.96±0.39. (C,D) Beat frequencies for cells randomly selected after 5-15 minutes in Na7.4 with: (C) 0, 50, 100 or 200 µM IBMX; (D) no additions, 30 µM H89, 15 mM NaHCO3 (BC), or 30 µM H89 (5 minutes preincubation) and 15 mM NaHCO3 as indicated. For each averaged point in C, n=15 cells from three independent experiments. For the data groups in D, n=41-43 cells from three independent experiments. Boxes indicate ±1 s.d. from the mean.

 


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  		Fig. 7. Ester loading of cAMP mimics the actions of bicarbonate. Sperm were incubated 30 minutes with 60 µM of cAMP-AM. Aliquots of these loaded sperm (cAMP-AM) were transferred to a bath containing medium Na7.4 that also contained 60 µM of cAMP-AM (A) or Na7.4 alone (B-D) and examined immediately. Other samples of untreated sperm (stored in medium Na7.4) were transferred to a bath containing medium Na7.4 alone (none) or with 15 mM NaHCO3 (15 BC). (A) Depolarization-evoked [Ca2+]i responses were monitored in three independent experiments as in Fig. 1, except that cells were perfused continuously only during application of medium K8.6. Mean values were 17±4 (none; n=17), 41±6 (cAMP-AM; n=16), and 55±8 (15 BC; n=19) nM second-1. Waveform analysis was performed as in Fig. 3 (but without perfusion) to provide flagellar beat frequency (B) and beat amplitude (C), and as in Fig. 5 (but without perfusion) to provide the time-averaged tangent angle (D). Mean values (n=24 for each treatment group; eight cells per group in three independent experiments) were 2.6±01, 6.7±0.3 and 8.3±0.4 Hz; 24.8±1.0, 20.5±1.2 and 20.1±0.7 µm.

 

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© The Company of Biologists Ltd 2003