1317
Development 130, 1317-1326
© 2003 The Company of Biologists Ltd
doi:10.1242/dev.00353
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,*
1Department
2Department
3Department
of Physiology and Biophysics, Box 357290, University of Washington, Seattle, WA 98195-7290, USA
of Anatomy and Cell Biology, Philipps University Marburg, 35037 Marburg, Germany
of Obstetrics and Gynecology, Box 356460, University of Washington, Seattle, WA 98195-6460, USA
*Author for correspondence (e-mail: [email protected])
Accepted 16 December 2002
SUMMARY
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.
INTRODUCTION
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
The bicarbonate anion is a key player in activating sperm when
they leave the mammalian epididymis. Early studies (Hamner
and Williams, 1964) noted that the bicarbonate anion
stimulates respiratory activity of sperm. Later work (Boatman
and Robbins, 1991; Garbers et al., 1982; Okamura et al., 1985;
Tajima et al., 1987) linked the HCO3– content of reproductive
fluids to the control of sperm motility and metabolism, and
strongly suggested that HCO3– increases production of cAMP
as a messenger that regulates these functions. Recent cloning
of the atypical adenylate cyclase of sperm (Buck et al., 1999)
allowed demonstration that HCO3– indeed activates the
expressed recombinant enzyme (Chen et al., 2000; Jaiswal and
Conti, 2001).
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
Key words: Capacitation, sAC, cAMP-AM, PKA, Motility, Shear
angle, Tan angle, Asymmetry
1318 G. Wennemuth and others
if they are initiated by events that share an involvement of
cAMP-mediated protein phosphorylation.
MATERIALS AND METHODS
Materials
Indo-1 AM and Pluronic 147 were from Molecular Probes (Eugene,
OR), H89 from BIOMOL Labs (Plymouth Meeting, PA), and the 8(4-chlorophenylthio) derivative of cAMP (CPT-cAMP) from
Calbiochem (San Diego, CA). The Rp- and Sp-diastereomers of
adenosine 3′,5′-cyclic monophosphothiorate (Rp- and Sp-cAMPS),
the acetoxymethyl ester of adenosine 3′,5′-cyclic monophosphate
(cAMP-AM) and all other chemicals were from Sigma (St Louis,
MO). Rotiblock™ was from Carl Roth (Karlsruhe, Germany), antiphosphotyrosine antibody clone 4G10 from Upstate Biotechnology
(Lake Placid, NY), horseradish peroxidase-labeled secondary
antibody from Santa Cruz Biotechnology (Santa Cruz, CA) and
enhanced-chemiluminescence detection reagents from Amersham
(Piscataway, NJ).
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 × 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 2× 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% Rotiblock™ 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 × 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 × 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 40× 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
(AND190H9P; 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 × 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.
Sperm activation by HCO3– 1319
RESULTS
Bicarbonate enhances evoked Ca2+ entry
Fig. 1 shows typical measurements of Ca2+ entry in sperm
using indo-1 photometry. The signals monitor spatially
averaged [Ca2+]i from small cell clusters comprising 3-7 motile
sperm loosely tethered to a coverslip. In Fig. 1A, the cells were
perfused continuously with the Ringer-like medium Na7.4,
except during brief 10-second depolarizing stimuli with
medium K8.6. [Ca2+]i rose abruptly during each stimulus, then
returned slowly towards the initial resting level. Recovery from
the first stimulus often was incomplete or absent. In a similar
experiment (Fig. 1B), the Na7.4 medium was fortified with 15
mM HCO3– for the 2 minute period before the third stimulus.
Basal [Ca2+]i remained unchanged, but the evoked response
was increased dramatically. On an expanded time scale (Fig.
1C), [Ca2+]i rose linearly during responses evoked before
exposure to HCO3– (lower two traces) and more quickly and
farther for those evoked afterwards (upper trace). This is the
enhancing effect of HCO3–. The rate of rise of [Ca2+]i is a direct
measure of the number of open Ca2+ channels. For similar
experiments to that shown in Fig. 1A, the rate of rise of the
averaged responses to the three control stimuli was 17 nM
second–1. For the averaged responses to the two control stimuli
of Fig. 1B, the rate was similar (24 nM second–1). However,
for the test stimulus applied after treatment with HCO3– the
rate increased more than fourfold (to 98 nM second–1).
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 dyeloading, 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
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 (䊏,䊉) and after (䉲) 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.
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
1320 G. Wennemuth and others
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.
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
Fig. 3. Sperm flagellar waveform analysis. (A) The initial six video
frames in a time series of images (48 × 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.
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
Sperm activation by HCO3– 1321
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 2π 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 2π 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.
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 concentrationdependent 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.
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 (䊊) or 15 (䉭) 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 (䊊, 䊐), or with Na7.4 containing 1 (䊏), or 15 (䊉) 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.
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
1322 G. Wennemuth and others
increased the averaged tangent angle and the symmetry of the
flagellar waveform (Fig. 5C).
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 (䊊)
or with 15 mM NaHCO3 (䊉). The length of the averaged segments
were limited by other traces that were shorter than those shown in A
and B.
from cells examined in the absence and presence of 15 mM
NaHCO3. For a perfectly symmetrical waveform, the timeaverage 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–
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±0.2fold. 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 H89, 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
Sperm activation by HCO3– 1323
Table 1. Modest actions of cAMP analogs on evoked Ca2+
entry and flagellar activity
Addition
None
Dibutyryl-cAMP
8Br-cAMP
Sp-cAMPS
Sp-cAMPS + IBMX
Rp-cAMPS
CPT-cAMP
CPT-cAMP + IBMX
NaHCO3
Rp-cAMPS + NaHCO3
Normalized rate of rise
Beat frequency (Hz)
1.20±0.08 (n=20)
n.d.
n.d.
0.47±0.08 (n=10)
0.36±0.03 (n=11)
1.65±0.22 (n=9)
0.41±0.07 (n=10)
0.45±0.08 (n=8)
4.92±1.24 (n=11)
n.d.
2.72±0.01 (n=147)
2.86±0.03 (n=22)
2.67±0.03 (n=36)
3.04±0.03 (n=36)
n.d.
n.d.
2.66±0.03 (n=24)
n.d.
6.37±0.01 (n=128)
5.08±0.05 (n=36)
For evoked Ca2+ entry, cells perfused with Na7.4 received two 10-second
stimuli with K8.6. After 1 minute of recovery in Na7.4, the perfusate was
changed to Na7.4 alone or with 15 mM NaHCO3, or to Na7.4 with the
indicated additions of cAMP analogs (1 mM) alone or with 0.2 mM IBMX. A
third stimulus was applied after 2 minutes. Rates of rise during the third
stimulus were normalized to that of the mean for the first two. Beat frequency
was assessed from waveform analysis of cells selected at random from
samples bathed for 5-20 minutes in Na7.4 with the indicated additions of
analogs (1 mM), alone or with NaHCO3. The Rp-cAMPS analog was added
25 minutes before NaHCO3. Shown are the mean of the normalized rates of
rise, beat frequencies and the number of cells examined in three independent
experiments for each of the two assays
n.d., not determined.
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.
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 (520 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 4-6 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
1324 G. Wennemuth and others
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 cAMPAM (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 timeaveraged 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.
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.
DISCUSSION
The alterations to mammalian sperm that occur between
mating and fertilization (collectively called capacitation) (see
Yanagimachi, 1994) are extremely difficult to study. Even
the component of capacitation that is most accessible
experimentally – the activation of sperm upon release from
storage in the male reproductive tract – is not entirely defined.
However, several recent insights provide an improved view
through this narrow early window of observation. Numerous
past reports proposed roles for cAMP as a regulatory
messenger in sperm (Kopf and Garbers, 1980), without
identifying the primary signal that increases cAMP content.
We now know that sperm possess an atypical adenylyl cyclase
that is stimulated directly by HCO3– (Buck et al., 1999; Chen
et al., 2000; Jaiswal and Conti, 2001). This advance has
sparked renewed interest in the role of the HCO3– anion as a
regulatory signal for other early events in capacitation. These
events include activation of sperm motility (Holt and Harrison,
2002; Okamura et al., 1985), the transbilayer movement of
aminophospholipids from the inner to the outer leaflet of the
plasma membrane (Gadella and Harrison, 2000; Harrison
and Miller, 2000) and subsequent lateral redistribution of
cholesterol in membranes of the sperm head (Flesch et al.,
2001; Gadella and Harrison, 2002). The half-time for
externalization of phosphatidylethanolamine is ~5 minutes
(Gadella and Harrison, 2002). We now report that HCO3–
facilitates voltage-gated Ca2+ channels and increases flagellar
beat frequency even more quickly, with half times of ~60 and
~10 seconds.
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).
Sperm activation by HCO3– 1325
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 Cα subunit of
PKA. Male Cα-null mice were runted and few survived to
puberty. The spermatogenic cells of the testes of the survivors
lacked Cα 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 Cα2 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.
We thank David Miller for the design and construction of the
custom-built LED power supply, Lea Miller and Anke Friedetzky for
technical help, and Drs Joe Beavo, Charles Brokaw and Linda
Wordeman for critical reading of earlier versions of this manuscript.
We especially thank Dr Bertil Hille for providing laboratory space and
for his continued encouragement of these studies. This work was
supported by the NICHD, National Institutes of Health through
cooperative agreement U54-HD12629 as part of the Specialized
Cooperative Centers Program in Reproduction Research, by the W.
M. Keck Foundation, and by the Fonds der Chemischen Industrie and
the Deutsche Forschungsgemeinschaft (We 2344/4-1 DFG,
Germany).
REFERENCES
Boatman, D. E. and Robbins, R. S. (1991). Bicarbonate: carbon-dioxide
regulation of sperm capacitation, hyperactivated motility, and acrosome
reactions. Biol. Reprod. 44, 806-813.
Brokaw, C. J. (1979). Calcium-induced asymmetrical beating of tritondemembranated sea urchin sperm flagella. J. Cell Biol. 82, 401-411.
Buck, J., Sinclair, M. L., Schapal, L., Cann, M. J. and Levin, L. R. (1999).
Cytosolic adenylyl cyclase defines a unique signaling molecule in mammals.
Proc. Natl. Acad. Sci. USA 96, 79-84.
Chen, Y., Cann, M. J., Litvin, T. N., Iourgenko, V., Sinclair, M. L., Levin,
L. R. and Buck, J. (2000). Soluble adenylyl cyclase as an evolutionarily
conserved bicarbonate sensor. Science 289, 625-628.
Cook, S. P., Brokaw, C. J., Muller, C. H. and Babcock, D. F. (1994). Sperm
chemotaxis: egg peptides control cytosolic calcium to regulate flagellar
responses. Dev. Biol. 165, 10-19.
Flesch, F. M., Brouwers, J. F., Nievelstein, P. F., Verkleij, A. J., van Golde,
L. M., Colenbrander, B. and Gadella, B. M. (2001). Bicarbonate
stimulated phospholipid scrambling induces cholesterol redistribution and
enables cholesterol depletion in the sperm plasma membrane. J. Cell Sci.
114, 3543-3555.
Gadella, B. M. and Harrison, R. A. (2000). The capacitating agent
bicarbonate induces protein kinase A-dependent changes in phospholipid
transbilayer behavior in the sperm plasma membrane. Development 127,
2407-2420.
Gadella, B. M. and Harrison, R. A. (2002). Capacitation induces cyclic
adenosine 3′,5′-monophosphate-dependent, but apoptosis-unrelated,
exposure of aminophospholipids at the apical head plasma membrane of
boar sperm cells. Biol. Reprod. 67, 340-350.
Garbers, D. L., Tubb, D. J. and Hyne, R. V. (1982). A requirement of
bicarbonate for Ca2+-induced elevations of cyclic AMP in guinea pig
spermatozoa. J. Biol. Chem. 257, 8980-8984.
Hamner, C. E. and Williams, W. L. (1964). Identification of sperm
stimulating factor of rabbit oviduct fluid. Proc. Soc. Exp. Biol. Med. 117,
240-243.
Harrison, R. A. and Miller, N. G. (2000). cAMP-dependent protein kinase
control of plasma membrane lipid architecture in boar sperm. Mol. Reprod.
Dev. 55, 220-228.
Herrington, J., Park, Y. B., Babcock, D. F. and Hille, B. (1996). Dominant
role of mitochondria in clearance of large Ca2+ loads from rat adrenal
chromaffin cells. Neuron 16, 219-228.
Holt, W. V. and Harrison, R. A. (2002). Bicarbonate stimulation of boar
sperm motility via a protein kinase A-dependent pathway: between-cell and
between-ejaculate differences are not due to deficiencies in protein kinase
A activation. J. Androl. 23, 557-565.
Howard, J. (2001). In Mechanics of Motor Proteins and the Cytoskeleton, pp.
99-116. Sunderland, MA: Sinauer.
Jaiswal, B. S. and Conti, M. (2001). Identification and functional analysis of
splice variants of the germ cell soluble adenylyl cyclase. J. Biol. Chem. 276,
31698-31708.
Kalab, P., Visconti, P., Leclerc, P. and Kopf, G. S. (1994). p95, the major
phosphotyrosine-containing protein in mouse spermatozoa, is a hexokinase
with unique properties. J. Biol. Chem. 269, 3810-3817.
Kopf, G. S. and Garbers, D. L. (1980). The regulation of spermatozoa by
calcium cyclic nucleotides. Adv. Cyclic Nucleotide Res. 13, 251-306.
Laemmli, U. K., Molbert, E., Showe, M. and Kellenberger, E. (1970). Formdetermining function of the genes required for the assembly of the head of
bacteriophage T4. J. Mol. Biol. 49, 99-113.
Nolan, M. A., Wennemuth, G., Burton, K. A., McKnight, G. S. and
Babcock, D. F. (2002). Bicarbonate actions on sperm Ca2+ channels and
flagellar function require protein kinase A. Mol. Biol. Cell Suppl. 13,
385a.
Okamura, N., Tajima, Y., Soejima, A., Masuda, H. and Sugita, Y. (1985).
Sodium bicarbonate in seminal plasma stimulates the motility of mammalian
spermatozoa through direct activation of adenylate cyclase. J. Biol. Chem.
260, 9699-9705.
Quill, T. A., Ren, D., Clapham, D. E. and Garbers, D. L. (2001). A voltagegated ion channel expressed specifically in spermatozoa. Proc. Natl. Acad.
Sci. USA 98, 12527-12531.
Reinton, N., Orstavik, S., Haugen, T. B., Jahnsen, T., Tasken, K. and
Skalhegg, B. S. (2000). A novel isoform of human cyclic 3′,5′-adenosine
monophosphate-dependent protein kinase, Cα-s, localizes to sperm
midpiece. Biol. Reprod. 63, 607-611.
Ren, D., Navarro, B., Perez, G., Jackson, A. C., Hsu, S., Shi, Q., Tilly, J.
L. and Clapham, D. E. (2001). A sperm ion channel required for sperm
motility and male fertility. Nature 413, 603-609.
San Agustin, J. T., Wilkerson, C. G. and Witman, G. B. (2000). The unique
catalytic subunit of sperm cAMP-dependent protein kinase is the product of
an alternative Cα mRNA expressed specifically in spermatogenic cells. Mol.
Biol. Cell 11, 3031-3044.
San Agustin, J. T. and Witman, G. B. (2001). Differential expression of the
Cs and Cα1 isoforms of the catalytic subunit of cyclic 3′,5′-adenosine
monophosphate-dependent protein kinase testicular cells. Biol. Reprod. 65,
151-164.
Schultz, C., Vajanaphanich, M., Genieser, H. G., Jastorff, B., Barrett, K.
E. and Tsien, R. Y. (1994). Membrane-permeant derivatives of cyclic AMP
optimized for high potency, prolonged activity, or rapid reversibility. Mol.
Pharmacol. 46, 702-708.
Serrano, C. J., Trevino, C. L., Felix, R. and Darszon, A. (1999). Voltagedependent Ca2+ channel subunit expression and immunolocalization in
mouse spermatogenic cells and sperm. FEBS Lett. 462, 171-176.
1326 G. Wennemuth and others
Skalhegg, B. S., Huang, Y., Su, T., Idzerda, R. L., McKnight, G. S. and
Burton, K. A. (2002). Mutation of the Cα subunit of PKA leads to growth
retardation and sperm dysfunction. Mol. Endocrinol. 16, 630-639.
Tajima, Y., Okamura, N. and Sugita, Y. (1987). The activating effects of
bicarbonate on sperm motility and respiration at ejaculation. Biochim.
Biophys. Acta 924, 519-529.
Visconti, P. E., Bailey, J. L., Moore, G. D., Pan, D., Olds-Clarke, P. and
Kopf, G. S. (1995a). Capacitation of mouse spermatozoa. I. Correlation
between the capacitation state and protein tyrosine phosphorylation.
Development 121, 1129-1137.
Visconti, P. E., Moore, G. D., Bailey, J. L., Leclerc, P., Connors, S. A.,
Pan, D., Olds-Clarke, P. and Kopf, G. S. (1995b). Capacitation of
mouse spermatozoa. II. Protein tyrosine phosphorylation and capacitation
are regulated by a cAMP-dependent pathway. Development 121,
1139-1150.
Wennemuth, G., Westenbroek, R. E., Xu, T., Hille, B. and Babcock, D. F.
(2000). CaV2.2 and CaV2.3 (N- and R-type) Ca2+ channels in
depolarization-evoked entry of Ca2+ into mouse sperm. J. Biol. Chem. 275,
21210-21217.
Westenbroek, R. E. and Babcock, D. F. (1999). Discrete regional
distributions suggest diverse functional roles of calcium channel α1 subunits
in sperm. Dev. Biol. 207, 457-469.
Woolley, D. M. (1977). Evidence for ‘twisted plane’ undulations in golden
hamster sperm tails. J. Cell Biol. 75, 851-865.
Yanagimachi, R. (1994). Mammalian Fertilization. In The Physiology of
Reproduction (ed. Knobil, E. and Neill, J. D.), pp. 189-317. New York:
Raven Press.
Zhang, J., Ma, Y., Taylor, S. S. and Tsien, R. Y. (2001). Genetically encoded
reporters of protein kinase A activity reveal impact of substrate tethering.
Proc. Natl. Acad. Sci. USA 98, 14997-15002.