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
* Author for correspondence (e-mail: donner{at}u.washington.edu)
Accepted 16 December 2002
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SUMMARY |
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Key words: Capacitation, sAC, cAMP-AM, PKA, Motility, Shear angle, Tan angle, Asymmetry
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INTRODUCTION |
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An obligatory maturational sequence known as `capacitation' prepares sperm
to fertilize the egg (Yanagimachi,
1994). Some events in this multistep process require
HCO3-, are mediated by cAMP, and follow activation of
protein kinase A (PKA). These include the transbilayer movement of plasma
membrane phospholipids (Gadella and
Harrison, 2000
; Harrison and
Miller, 2000
), lateral redistribution of membrane cholesterol
(Flesch et al., 2001
) and a
delayed tyrosine phosphorylation of several sperm proteins
(Visconti et al., 1995a
;
Visconti et al., 1995b
).
Several types of voltage-gated Ca2+ channel proteins have been
detected in mouse sperm by immunomethods
(Quill et al., 2001;
Ren et al., 2001
;
Serrano et al., 1999
;
Wennemuth et al., 2000
;
Westenbroek and Babcock,
1999
). Past work shows that depolarizing stimuli open some of
these channels to allow Ca2+ entry, and that this evoked channel
activity increases after conditioning of sperm with
HCO3- (Wennemuth et
al., 2000
). We characterize this additional action of
HCO3- more fully and examine its mechanistic basis.
We now show that HCO3- also accelerates the flagellar beat, as reported and quantified here for the first time by flagellar waveform analysis. We find that the actions of HCO3- on channel and flagellar function are similar in many respects, as if they are initiated by events that share an involvement of cAMP-mediated protein phosphorylation.
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MATERIALS AND METHODS |
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Sperm preparation and incubation conditions
As in prior work (Wennemuth et al.,
2000; Westenbroek and Babcock,
1999
), cauda epididymal sperm were prepared from male mice (Swiss
Webster, retired breeders) that were euthanized by CO2
asphyxiation. Briefly, the cleaned, excised epididymides were rinsed with
medium Na7.4: 135 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM
MgCl2, 20 mM HEPES, 5 mM glucose, 10 mM lactic acid, 1 mM pyruvic
acid, adjusted to pH 7.4 with NaOH. The tissue was transferred to 1 ml of a
`swimout/capacitation medium' that comprised medium Na7.4 with bovine serum
albumin (BSA) (5 mg/ml) and NaHCO3 (15 mM). Semen was allowed to
exude (15 minutes at 37°C, 5% CO2) from three to five small
incisions. All subsequent operations were at room temperature (22-25°C) in
medium Na7.4, unless otherwise noted. Sperm were washed twice, then dispersed
and stored at 1-2 x 107 cells ml-1. For
capacitation in vitro, sperm were transferred to `swimout/capacitation medium'
and incubated 90 minutes at 37°C in a 95% air/5% CO2
atmosphere. For subsequent waveform analysis, sperm were washed twice in
medium Na7.4 and examined at room temperature.
Potassium-evoked responses were produced with medium K8.6: 135 mM KCl, 5 mM
NaCl, 2 mM CaCl2, 1 mM MgCl2, 30 mM TAPS, 5 mM glucose,
10 mM lactic acid, 1 mM pyruvic acid, adjusted to pH 8.6 with NaOH, as in past
work (Wennemuth et al.,
2000).
Immunoblots
Total proteins from mouse sperm were extracted by adding an equal volume of
a 2x Laemmli buffer (Laemmli et al.,
1970). After heating for 5 minutes at 100°C, the samples were
clarified by centrifugation at 5000 g for 5 minutes and
adjusted to 5% mercaptoethanol. Proteins were separated on a 4-12% gradient
SDS-PAGE gel and transferred onto nitrocellulose membranes. Prior to the
incubation with antibodies, non-specific binding was blocked with PBS
containing 10% RotiblockTM for 1 hour. Blots were incubated with a 1:1000
dilution of the anti-phosphotyrosine primary antibody for 1 hour then washed
with 0.1% Tween in PBS (4 x 5 minutes). Under the same conditions, blots
were then incubated with horseradish peroxidase-labeled secondary antibody
diluted 1:2000 in PBS. Finally, blots were washed with 0.1% Tween in PBS (4
x 5 minutes) and treated with enhanced-chemiluminescence detection
reagents for 5 minutes.
Dye loading and photometry
Indo-1 AM was dispensed from 2 mM stocks in DMSO, dispersed in 10% Pluronic
147, diluted to 20 µM in 0.25 ml medium Na7.4, then immediately mixed with
an equal volume of the sperm suspension. After 15-20 minutes, medium Na7.4 (1
ml) was added and the cells sedimented. After resuspension in 0.25 ml fresh
medium, incubation continued for a minimum of 40 minutes and no more than 5
hours before use.
Incubation chambers were constructed from 35 mm tissue culture dishes and
#0 glass coverslips. An uncoated, 5 mm square #00 coverslip was placed in
the dish and 10 µl of cell suspension added. After
5 minutes, the
chamber was gently flooded with medium and transferred to the microscope
stage. Test solutions were applied by a solenoid-controlled, gravity-fed,
multibarreled, local perfusion device with an estimated exchange time of
<0.5 second. Photometric measurements were as described
(Herrington et al., 1996
;
Wennemuth et al., 2000
). The
photometric signals were corrected for background fluorescence and calibrated
with the constants Rmin (1.067), Rmax (6.488) and K*
(1470 nM) obtained from cells equilibrated in solutions fortified with
ionomycin (10 µM) and containing 50 mM EGTA, 15 mM CaCl2, or 20
mM EGTA with 15 mM CaCl2 (calculated free Ca2+
concentration of 251 nM). Automated correction, calibration and kinetic
analysis of digital photometric records were performed in Igor (Wavemetrics,
Lake Oswego, OR). Statistical analyses were performed in Excel (Microsoft,
Redmond, WA). All results are presented as mean±s.e.m., except where
noted.
Ester loading of cAMP
The cAMP-AM was dispensed from a 20 mM stock in DMSO, dispersed in 10%
Pluronic 147, diluted to 120 µM in 0.25 ml medium Na7.4, then immediately
mixed with an equal volume of a sperm suspension that had or had not received
preliminary loading with indo-1 AM. After 30 minutes, an aliquot (5-10 µl)
was added to the sample chamber for imaging or photometry. For evoked
[Ca2+]i (intracellular Ca2+ concentration)
responses, cells were examined with protocols that minimized the duration of
perfusion to thereby reduce washout of the membrane-permeant ester
(Schultz et al., 1994).
Waveform analysis
Images for waveform analysis were collected from cells examined in the
sample chambers described above, with a 40x 0.65 N.A. objective on an
inverted microscope (Nikon Diaphot) in which the stage lamp was replaced by a
10 mm ultrabright (15 candella at 20 mA) red LED (AND 190H9P; Newark
Electronics, Chicago, IL). Brief flashes of illuminating light were produced
by a custom-built stroboscopic power supply that provided 3-5 V pulses of 1-2
ms duration. For visual observation, the flash frequency was 150 Hz. For data
collection, the pulse was triggered once-per-frame by a synchronization signal
from the controller module of the frame-transfer CCCD camera (TCP512; Roper
Scientific, Trenton, NJ). Images were collected at 30 Hz from a 128 x
128 pixel region of the camera chip, under the direction of Metamorph
(Universal Imaging; West Chester, PA). After initial adjustments of intensity
and contrast, images were stored in TIFF format for subsequent analysis with
software routines written in Igor (Wavemetrics; Lake Oswego, OR). These
routines provide for semiautomated tracing of the flagellum in each image and
subsequent fitting of the digitized waveform with a simple sine function.
Additional routines fully automated were: (1) regression analysis of the
timecourse of the phase lags of the fitted sine functions to provide the
flagellar beat frequency; (2) tabulation of the distance along the flagellum
(arc length), the angular deviation (tangent angle) evaluated at 0.5 µm
intervals along the traced flagellum, and the time-averaged tangent angle
data; (3) presentation of the time-averaged tangent angle versus arc length
data (shear curves) as a measure of flagellar beat asymmetry (see
Brokaw, 1979) [for a general
discussion of polymer mechanics and their analysis see Howard
(Howard, 2001
)]; and (4)
tabulation of the maximal excursion of the traced flagellum to define the
flagellar beat envelope and the beat amplitude at regular intervals along the
beat axis.
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RESULTS |
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Characterization of bicarbonate action on Ca2+ channel
activity
In early trials, the rates of evoked Ca2+ rise varied
considerably between experiments. Fig.
2A identifies the age of the preparation (t=0 marks the
beginning of dye loading) as the source of much of this variation. After the
60 minutes of dye-loading, sperm were stored for various periods of at room
temperature in medium Na7.4, then exposed to 15 mM HCO3-
for 2 minutes prior to stimulus. In records collected immediately, indo-1
signals were weak (probably indicating that hydrolysis of the indo-1 AM ester
was still incomplete) and the rates of rise were 20 nM
second-1, similar to those seen for the control stimuli in
Fig. 1. Sperm examined 1-6
hours later had stronger indo-1 signals. During this interval, the mean evoked
rates of rise increased more than threefold. Regression analysis shows that
the age of the sperm preparation correlates strongly with this increase
(r=0.992). Usually, we limit the experimental window to preparations
of 2-5 hours in age and compare the rates of rise observed during control and
test stimuli applied in protocols like those of
Fig. 1.
|
Our conditions for storage of sperm are intended to prevent capacitation in
vitro. As protein tyrosine phosphorylation is considered a marker of
progression towards capacitation, we used phosphotyrosine immunoblots to
assess the effectiveness of our strategy.
Fig. 2B compares extracts of
sperm prepared in various ways. Lane 1 is after `swimout' from the epididymal
exudate, conditions that include 15 minutes incubation with 15 mM
NaHCO3 at 37°C. Lane 2 is after a further 15 minute incubation
in Na7.4 with 15 mM NaHCO3 at 22-24°C, a longer exposure to
HCO3- than we use in actual experiments. Lane 3 is after
90 minutes in the swimout/capacitation medium at 37°C (conditions reported
to induce capacitation in vitro) (Visconti
et al., 1995a). Lane 4 is after 180 minutes at 22-24°C in
Na7.4 alone, our usual storage conditions. All extracts show a prominent band
migrating at
116 kDa, a less-prominent band migrating at
200 kDa and
minor bands of lower apparent Mr. The 116 kDa band
presumably is the constitutively phosphorylated hexokinase reported by Kalab
et al. (Kalab et al., 1994
);
the identity of the higher and lower Mr bands is not
known. The extracts of sperm incubated under capacitating conditions (lane 3)
show large increases in a band migrating at
90 kDa, and in three bands
migrating between 40 and 60 kDa, as reported in prior work
(Visconti et al., 1995a
). The
90 kDa band also is present but substantially weaker in extracts prepared from
cells after 3 hours storage in Na7.4. Thus, the gradual increases in evoked
Ca2+ channel responses that occur during prolonged storage at room
temperature (Fig. 2A) are
accompanied by some of the increases in protein tyrosine phosphorylation that
are produced by incubation under capacitating conditions. However, no
increases in phosphorylation were observed for cells incubated with
HCO3- for 15 minutes, several times longer than the 1-2
minutes exposures that acutely enhance Ca2+ channel responses
(Fig. 1B).
Fig. 2C examines the
timecourse of this enhancing action of HCO3- on channel
responses. Using a protocol like that of
Fig. 1B we varied the duration
of exposure to HCO3-. In this and most subsequent
experiments, the rates of Ca2+ rise for the test stimulus was
normalized to the rate of rise in the preceding control stimulus. The
half-time for the action of 15 mM HCO3- is 60
seconds. Fig. 2D shows that
HCO3- acts even at 1 mM, although this concentration
appears to be below the EC50 with respect to the rate of rise.
Sperm flagellar waveform analysis
Simultaneously with indo-1 photometry, we observe real-time, red-light
video images of cells within the 25 µm diameter recording window to
ascertain that the recorded sperm remain motile. Although these low-quality
images show only the sperm head and midpiece, they clearly reveal a rapid
increase in movement when cells are perfused with HCO3-.
To study this action of HCO3- more quantitatively, we
assembled an apparatus that captures higher-quality, stop-motion images of the
sperm flagellum, and developed software to facilitate extraction of parameters
that describe the frequency, amplitude and symmetry of the flagellar
waveform.
Fig. 3A shows the initial segment of a typical time series of images of a single sperm collected with this waveform analysis system. As in our photometry work, the cell has attached spontaneously to the cover slip at the posterior head. These six successive images show the cell pivoting about this point of attachment as the flagellum goes through one beat cycle. Fig. 3B is a composite image formed from a longer segment of this series. The extremes of flagellar excursion outline the flagellar beat envelope, and the midline of the envelope approximates the flagellar beat axis. Beat amplitude can be estimated from the perpendicular distance between the extremes of the envelope (the double-headed arrow), here measured arbitrarily at 25 µm from the point of attachment.
|
The image series of Fig. 3A contains much additional information related to the spatial coordinates of the flagellum. Extraction of these coordinates is not a trivial task. The simplest approach begins with manual tracing of projected images to provide a binary bit-map. This is easy to apply, but tedious and time consuming. Image recognition algorithms hold the promise of fully automated determination of flagellar coordinates. However, we examined several and rejected them on the basis of unacceptably high error rates. Instead, we have implemented semi-automated tracing, with a various fully automated calculations.
For the range of conditions explored here, the flagellar waveform is
relatively symmetrical and sinusoidal. Analysis therefore includes fitting of
a simple sine function to each image. The fit usually is quite good, as shown
in Fig. 3C, which contains a
grayscale composite of the images of Fig.
3A, each overlaid with a colored, best-fit sine wave. The phase
lag of the fitted sine function must change by 2 radians (360°) during
each beat cycle. For the cell examined here, regression analysis of the phase
lag for 15 successive images indicates a slope of 39.8 rad
second-1. Dividing this slope by 2
gives a beat frequency of
6.34 Hz (Fig. 3D).
Bicarbonate accelerates flagellar beat frequency
Simple visual observation indicated that HCO3-
stimulates sperm motility. Fig.
4A examines the distribution of flagellar beat frequencies for
cells selected at random from samples of the sperm from a single animal that
were bathed in media lacking or containing 15 mM NaHCO3. Without
HCO3- the beat frequencies ranged from 1.5-6.0 with a
mean of 3.1±0.2 Hz. With HCO3- the range was
3.8-10.3 with a mean of 7.9±0.3 Hz. The distributions within both
groups were well fitted by single Gaussian functions that showed little
overlap. Beat amplitude (Fig.
4B) for the two treatment groups showed larger variability and
more overlap. Here, and consistently in subsequent experiments, the trend was
toward decreased beat amplitude for sperm exposed to
HCO3-. A statistically significant correlation between
beat frequency and amplitude was not found here.
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In the absence of added HCO3-, the beat frequency of
most sperm remained steady for long periods of observation. If the perfusing
medium was changed to include 15 mM NaHCO3, the beat frequency
quickly increased by several-fold and remained at this faster rate for many
minutes so long as HCO3- was present
(Fig. 4C,D). The effects were
reversible. Thus, if HCO3- was removed after 60 seconds,
beat frequency slowly returned to the basal rate in 10 minutes
(Fig. 4E). Then if
HCO3- was reapplied, the sperm responded rapidly and
strongly again. Fig. 4C also
examines the concentration-dependent kinetics of HCO3-
action on a shorter time scale. At 15 mM, HCO3- robustly
increased beat frequency. Acceleration was detectable within 2 seconds and
proceeded linearly, perhaps with a slight overshoot. The half-time for
development of response was 8.8±0.2 seconds. At 1 mM
HCO3-, the increase in beat frequency was nearly as
large but only half as rapid with a half-time of 17.5±0.2 seconds.
Bicarbonate decreases flagellar beat asymmetry
Sperm swimming behavior is determined both by the frequency of the
flagellar beat and by its symmetry. Flagellar beat symmetry determines
linearity of swimming paths (Brokaw,
1979; Cook et al.,
1994
). Our analysis of flagellar waveform images (like those of
Fig. 3A) includes calculation
of the angular deviation (tangent angle) along the traced flagellum.
Fig. 5A,B show examples of
typical data obtained from cells examined in the absence and presence of 15 mM
NaHCO3. For a perfectly symmetrical waveform, the time-average of
such distributions of the tangent angle along the length of the flagellum (the
arc length) would be zero. The extent of divergence from zero therefore
provides a measure of flagellar beat asymmetry. Arbitrarily, we assign a
positive value to asymmetry that is biased in the same direction as the hook
of the rodent sperm head (the reverse bend of the flagellum)
(Woolley, 1977
). For the cell
examined in Fig. 5A, the
tangent angle averaged over two beat cycles showed a small negative deviation
that increased along the length of the flagellum. If the cell were not
tethered to the slide, such asymmetry would produce circular swimming that
curved opposite to the direction of the hook of the head. The cell of
Fig. 5B, examined in the
presence of 15 mM HCO3-, showed a reduced negative
asymmetry. Data averaged from populations that comprised the cells of
Figs. 5A,B and 20 other cells
confirmed that HCO3- increased the averaged tangent
angle and the symmetry of the flagellar waveform
(Fig. 5C).
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Mechanisms of bicarbonate action on Ca2+ channel activity
and flagellar function
How is HCO3- coupled to enhancement of evoked
Ca2+ entry and acceleration of flagellar beat frequency? We used a
pharmacological approach to evaluate the hypothesis that stimulation of sperm
adenylyl cyclase, increased cAMP content, and activation of PKA underlie the
actions of HCO3-.
Fig. 6A shows measurements of Ca2+ rise using protocols similar to Fig. 1. Sperm received a pair of control stimuli, then were perfused for 1 minute with medium Na7.4 alone or with 15 mM HCO3-, or with 15 mM HCO3- and 0.2 mM of the phosphodiesterase inhibitor IBMX. A third and final test stimulus then was applied. With short 1-minute exposures to HCO3-, facilitation of Ca2+ entry was only increased 2.3±0.2-fold. When cells were perfused with HCO3- together with IBMX, facilitation increased to 3.3±0.2-fold. Perfusion with IBMX alone produced only a 1.6±0.1-fold enhancement (data not shown). These results are consistent with the interpretation that IBMX enhances HCO3- action by shifting the balance between competing pathways for synthesis and degradation of the cAMP messenger.
|
Fig. 6B summarizes
experiments testing the ability of the PKA inhibitor H89 to prevent
enhancement of Ca channel responses by HCO3-. In
protocols similar to Fig. 1,
sperm received first a pair of control stimuli. After brief recovery in Na7.4,
alone or with 0-30 µM H89, sperm were perfused for 2 minutes with media
that contained HCO3- (at 15 mM) and the same
concentration of H89. The third test stimulus was then applied. As in
Fig. 2D, this longer exposure
to HCO3- alone produced a nearly sevenfold increase in
the evoked rate of [Ca2+]i rise. H-89 inhibited this
response with an IC50 of 10 µM, consistent with a required
phosphorylation by PKA in the pathway of HCO3-
action.
Similar experiments examined HCO3- action on
flagellar beat frequency. Fig.
6C shows that bath application of 50-200 µM IBMX alone
increased beat frequency with an EC50 of 50 µM, suggesting
that basal phosphodiesterase activity may limit resting beat frequency.
Fig. 6D shows that when 30
µM H89 was applied for the 5 minutes before and during exposure to
HCO3-, average beat frequency increased to only
3.3±0.2 Hz. This rate is higher than the 2.3±0.2 Hz of resting
cells, but much less than the 7.3±0.2 Hz observed for cells treated
directly with HCO3-. Interestingly, 30 µM H89 alone
had little or no effect on beat frequency, suggesting that the basal beat rate
is not determined by a dynamic balance between PKA and competing phosphatases.
In other experiments to test the reversibility of H89 action (not shown)
individual sperm were perfused sequentially with Na7.4 alone, then with 30
µM H-89, then 30 µM H-89 and 15 mM NaHCO3, then finally with
Na7.4 containing only 15 mM NaHCO3. It was difficult to maintain
recording during experiments of such long duration. However, in 3 out of 18
trials the results clearly showed that a strong stimulation by
HCO3- returned within 1 minute after removal of H-89,
indicating that the inhibitory actions of H89 are readily reversible.
The results with IBMX and those with H-89 are consistent with a simple
working hypothesis: bicarbonate acts on flagellar beat frequency and on evoked
Ca2+ entry by raising cAMP and stimulating protein phosphorylation
via PKA. This view predicts that membrane-permeant analogs of cAMP should
mimic (or block) the actions of HCO3-.
Table 1 summarizes several
tests of that prediction. Rates of evoked Ca2+ rise (first column)
were assessed before and after 2 minutes perfusion with various analogs alone
or with IBMX. Medium Na7.4 alone or with 15 mM NaHCO3 served as
negative and positive controls. The effects were not those expected. Stimuli
applied after perfusion with CPT-cAMP or Sp-cAMPS gave normalized rates of
rise 50% lower than for stimuli applied before exposure to these agents.
For Rp-cAMPS the rates were
50% higher than for the stimuli applied after
Na7.4 alone. By comparison, for control experiments in which no treatment was
applied before the test stimulus, the normalized rates were elevated by 20%.
Thus, the effects, if any, of the analogs are modest compared with the robust
nearly 500% increase produced by NaHCO3.
|
Similar tests were carried out on flagellar beat frequency (Table 1, second column). The effects of some of the nucleotide analogs were in the expected direction, but still much smaller than the effect of HCO3-. Although these longer exposures (5-20 minutes by inclusion in the bath medium) to CPT-, dibutyryl- or 8-Br-cAMP produced no detectable effect, the Sp-cAMPS increased mean beat frequency slightly, and the Rp-cAMPS analog partially blocked the action of HCO3-.
We reasoned that poor permeability may have limited the efficacy of these
cAMP analogs. Tsien's laboratory has noted
(Schultz et al., 1994) that
the acetoxylmethyl ester of cAMP masks the charged phosphate of the parent
compound, making a more permeant analog that can undergo esterolytic
hydrolysis to generate intracellular cAMP.
Fig. 7 shows evoked
[Ca2+]i responses and flagellar waveform analysis for
sperm sampled after incubation with 60 µM of the cAMP ester and compares
them with untreated sperm examined in the presence or absence of 15 mM
NaHCO3. Consistent with the experiments shown in Figs
4,5,6
and Table 1, NaHCO3
increased the evoked rates of [Ca2+]i rise from
17±4 to 55±8 nM second-1
(Fig. 7A). It also increased
mean beat frequency from 2.6±0.1 to 8.3±0.4 Hz
(Fig. 7B), decreased beat
amplitude slightly from
25 to
20 µm
(Fig. 7C), and increased
flagellar symmetry (Fig. 7D).
The cAMP-AM was nearly as effective as NaHCO3, increasing the mean
rate of rise to 41±6 nM second-1 and beat frequency to
6.7±0.3 Hz, and decreasing the amplitude to
21 µm.
Interestingly, the action of cAMP-AM on waveform symmetry was even stronger
than that of NaHCO3; with cAMP-AM the time-averaged asymmetry
parameter became positive over the entire proximal 40 µm of the
flagellum.
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DISCUSSION |
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Pharmacological evidence supports the hypothesis that the activation of a
phospholipid scramblase, which underlies HCO3- action on
membrane lipid remodeling, requires protein phosphorylation by the
cAMP-dependent protein kinase A (Gadella
and Harrison, 2002). We find that, like their actions on
HCO3--mediated lipid remodeling, the phosphodiesterase
inhibitor IBMX promotes and the PKA inhibitor H89 blocks the actions of
HCO3- on flagellar beat frequency and the rate of evoked
Ca2+ entry.
Moreover, we find that incubation with cAMP-AM ester strongly increases
rates of evoked Ca2+ entry and flagellar beat frequency, presumably
indicating an effective elevation of cAMP without involvement of adenylyl
cyclase. Unexpectedly, several other analogs of cAMP were less effective in
mimicking these actions of HCO3- than in their actions
on sperm reported elsewhere (Gadella and
Harrison, 2002; Harrison and
Miller, 2000
; Visconti et al.,
1995b
). The most likely explanation for limited efficacy of the
cAMP analogs examined in Table
1 is that they are much less permeant than the cAMP-AM ester in
sperm. Comparison of the kinetics of responses to cAMP-AM and other analogs in
somatic cell preparations support this interpretation
(Schultz et al., 1994
;
Zhang et al., 2001
).
If the action of HCO3- on flagellar function indeed
results from stimulation of adenylyl cyclase, we can now also estimate the
time scale for this activation. Thus, the region of the signaling pathway
between HCO3- exposure and elevation of cAMP (perhaps
involving only entry of HCO3- and direct activation of
adenylyl cyclase) must operate on a time scale at least as fast as that found
for the acceleration of flagellar beat frequency (t1/2
<10 seconds). As the kinetics of Ca2+ channel facilitation are
slower than those of flagellar responses, the signaling pathway between cAMP
and the ultimate target that facilitates Ca2+ entry channels may
contain additional steps. The ability of concentrations as low as 1 mM
HCO3- to stimulate these responses is consistent with
reported stimulation of the adenylyl cyclase of rat spermatogenic cells by 1
mM HCO3- (Jaiswal
and Conti, 2001).
In concept, targeted disruption of the catalytic (C) subunits of PKA
provides an alternate approach to study of the signaling pathways that control
sperm function. Skalhegg et al. (Skalhegg
et al., 2002) produced mice carrying a null mutation in the widely
expressed gene that encodes the C
subunit of PKA. Male C
-null
mice were runted and few survived to puberty. The spermatogenic cells of the
testes of the survivors lacked C
protein. Intact sperm were recovered
from the cauda epididymis, but they did not show progressive motility and
presumably were unable to fertilize. Subsequent work
(Nolan et al., 2002
) has
produced mice carrying the more selective disruption of the unique C
2
transcript that is expressed exclusively in spermatocytes and spermatids
(Reinton et al., 2000
;
San Agustin et al., 2000
;
San Agustin and Witman, 2001
).
These animals should provide an improved transgenic model to test the proposed
requirement for PKA-mediated phosphorylation in the actions of
HCO3- during sperm activation.
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ACKNOWLEDGMENTS |
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