From the Center for Research in Contraception and
Reproductive Health, Department of Cell Biology, and the
¶ Department of Biochemistry and Molecular Genetics, University of
Virginia, Charlottesville, Virginia 22908, § Universidad Autonoma de Mexico, Cuebnavaca, Mexico,
and
Center for Research in Reproduction and Women's Health,
University of Pennsylvania, Philadelphia, Pennsylvania 19104
Received for publication, June 24, 2002, and in revised form, December 16, 2002
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ABSTRACT |
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Mammalian sperm are incapable of
fertilizing eggs immediately after ejaculation; they acquire
fertilization capacity after residing in the female tract for a finite
period of time. The physiological changes sperm undergo in the female
reproductive tract that render sperm able to fertilize constitute the
phenomenon of "sperm capacitation." We have demonstrated that
capacitation is associated with an increase in the tyrosine
phosphorylation of a subset of proteins and that these events are
regulated by an HCO Upon ejaculation, mammalian sperm are not able to fertilize; they
become fertilization-competent during transit through the female
reproductive tract (1). The molecular, biochemical, and physiological
changes that occur in sperm while in the female tract are collectively
referred to as capacitation. During capacitation, changes in membrane
dynamics and properties, enzyme activities, and motility render
spermatozoa responsive to stimuli that induce the acrosome reaction and
prepare these cells for penetration of the egg vestments prior to
fertilization. These changes are facilitated by the activation of sperm
cell signaling cascades during residence of these cells in the female
reproductive tract in vivo or in defined media in
vitro. Molecular events implicated in the initiation of
capacitation have been partially defined and include the removal of
cholesterol by cholesterol acceptors such as bovine serum albumin
(BSA)1 from the sperm plasma
membrane, modifications in plasma membrane phospholipids, fluxes of
HCO Mammalian sperm capacitation is also accompanied by the
hyperpolarization of the sperm plasma membrane (3). Hyperpolarization is observed as an increase in the intracellular negative charges when
compared with the extracellular environment. Although it is not clear
how sperm plasma membrane potential is regulated during capacitation,
it appears that membrane hyperpolarization may be partially because of
an enhanced K+ permeability as a result of a decrease in
inhibitory modulation of K+ channels (3). Recently,
Muñoz-Garay et al. (4) demonstrated with patch clamp
techniques that inward rectifying K+ channels are expressed
in mouse spermatogenic cells and proposed that these channels may be
responsible for the capacitation-associated membrane hyperpolarization.
Interestingly, Ba2+ blocks these K+ channels
with an IC50 similar to that shown to inhibit the
capacitation-associated hyperpolarization and the zona
pellucida-induced acrosome reaction (4). The functional role of sperm
plasma membrane hyperpolarization during capacitation is at present not
well understood. However, one may speculate that because capacitation
prepares the sperm for the acrosome reaction, capacitation-associated
membrane hyperpolarization may regulate the ability of sperm to
generate transient Ca2+ elevations during acrosome reaction
by physiological agonists (e.g. zona pellucida of the egg or
progesterone) (5). This hypothesis is consistent with the presence of
low voltage-activated (LVA) Ca2+ T channels in
spermatogenic cells (6, 7) that may also be present in mature sperm.
Numerous studies have demonstrated that capacitation is an
HCO In the present work, we have investigated the role of bovine serum
albumin (BSA) and HCO Materials--
Chemicals were obtained from the following
sources: dibutyryl cyclic AMP (Bt2-cAMP),
(Rp)-cAMPS, 3-isobutyl-1-methylxanthine (IBMX),
cholesterol 3-sulfate (cholesterol SO
Procedures for preparing stock solutions varied according to solubility
and the desired final concentrations. The following were prepared in
dimethyl sulfoxide (Me2SO) at the stock
concentrations noted in parentheses and stored at Preparation of Sperm--
In most experiments, cauda epididymal
mouse sperm were collected from CD1 retired male breeders by placing
minced cauda epididymis in a modified Krebs-Ringer medium
(Whitten's/HEPES-buffered) (25). This medium, which does not support
capacitation, was first prepared in the absence of bovine serum albumin
(BSA) and NaHCO3 and contains 1 mM
polyvinylpyrrolidone (average Mr,
40,000). After 5 min, sperm in suspension were washed in 10 ml of the
same medium by centrifugation at 800 × g for 10 min at
room temperature (24 °C). Sperm were then resuspended to a final
concentration of 2 × 107 cells/ml and diluted 10 times in the appropriate medium depending on the experiment performed.
In experiments where capacitation was investigated, 5 mg/ml BSA and 24 mM NaHCO3 were added; to compensate for the
addition of NaHCO3, 24 mM NaCl was added to the
control medium. In all cases pH was maintained at 7.2. To study the
role of Na+ in capacitation and in the regulation of the
membrane potential, NaCl was replaced by
choline+Cl SDS-PAGE and Immunoblotting--
After incubation under
different experimental conditions, the sperm were concentrated by
centrifugation, washed in 1 ml of phosphate-buffered saline containing
1 mM sodium orthovanadate, resuspended in sample buffer
(27) without 2-mercaptoethanol, and boiled for 5 min. After
centrifuging, the supernatants were saved, and 2-mercaptoethanol was
added to a final concentration of 5%. The samples were boiled for 5 min and subjected to 10% SDS-PAGE (27). Electrophoretic transfer of
proteins to Immobilon P (Bio-Rad) and immunodetection of
tyrosine-phosphorylated proteins and hexokinase type 1 were carried out
using anti-phosphotyrosine ( Membrane Potential Assay in Cell Populations--
A number of
fluorescent indicator dyes have been commonly used by several
laboratories to evaluate membrane potential. DiSBAC2-(3) and DiSC3-(5) have been successfully used in mammalian
sperm (3, 29). These fluorescent dyes are charged and distribute across cellular membranes in response to electrochemical gradients. As a
result of binding to intracellular proteins, the dyes undergo quenching
of the fluorescent emission and exhibit a slight shift in their
spectrum (30). Because DiSBAC2-(3) is an anionic oxonol and
DiSC3-(5) is a cationic carbocyanine dye, they distribute in opposite directions across the cell membrane at equivalent membrane
potentials. Neither dye has toxic effects on sperm function at the
concentrations used (<5 µM) (3). When constant
concentrations of sperm and probe are used, these dyes provide
reproducible estimates of plasma membrane potential.
Both fluorescent dyes possess advantages and disadvantages in sperm.
For the most part, we have used DiSC3-(5) due to its greater signal and the convenience of calibration using valinomycin. One problem with the use of DiSC3-(5) is that this cationic
dye binds to mitochondria in their normal energized state; this can contribute up to 20-30% of the fluorescence signal (3). To alleviate
this problem, recordings were initiated after dissipating the
mitochondrial membrane potential with 1 µM CCCP. Under
these conditions, the plasma membrane potential of valinomycin-treated mouse sperm responds to the extracellular K+ concentration
following the Nernst equation. In selected experiments, a set of
parallel membrane potential determinations were carried out using
DiSBAC2-(3), which is negatively charged, distributes across the membrane in the opposite direction, and does not bind to
mitochondria; these experiments were performed as controls to the
determinations performed with the cationic DiSC3-(5).
DiSBAC2-(3) has the disadvantage that it forms an insoluble
complex with valinomycin, thus complicating calibration of plasma
membrane potential.
Sperm were collected as described above and, after dilution in the
appropriate medium, capacitated for different times. Eight min before
the measurement, 1 µM DiSC3-(5) (final
concentration) was added to the sperm suspension and further incubated
for 5 min at 37 °C, and when used, 1 µM CCCP (final
concentration) was added, and the sperm were incubated for 2 additional
min. After this period, 1.5 ml of the suspension was transferred to a
gently stirred cuvette at 37 °C, and the fluorescence (620/670 nm
excitation/emission) was recorded continuously. After reaching a steady
fluorescence (usually after 1-2 min), calibration was performed by
adding 1 µM valinomycin and sequential additions of KCl
(29). The initial suspension contains 5.9 mM KCl;
additional KCl was added to the final concentrations of 8, 12, 20, and
36 mM KCl, corresponding to plasma membrane potentials of
Intracellular pH Measurements--
For this purpose, the
pH-sensitive dye BCECF was used (32). Sperm (1 × 106)
in Whitten's medium were incubated for 5 min in 4 µM
BCECF-AM, the permeant non-fluorescent precursor of BCECF. Once within
the cell, non-specific esterases hydrolyze the acetoxymethyl ester (AM), yielding the non-permeant fluorescent indicator. The low leakage
rate of BCECF and the small intracellular volume result in the final
intracellular concentration being much higher than the external
incubation concentration. After incubation, cells were washed in fresh
medium once (400 × g for 5 min), and 1.5 ml of this
suspension was placed in a gentle-stirring cuvette for fluorescence
measurements. The pH-dependent spectral shift exhibited by
BCECF allows calibration of the pH response in terms of the ratio of
fluorescence intensities measured at two different excitation
wavelengths (510/450 nm, fixed emission at 540 nm). Calibration was
performed by the null point determination as described by Babcock (31).
In those experiments in which the effect of the Na+
concentration was assayed, the sperm were collected and washed in
Na+-free medium (Whitten's/choline+).
Assay for Acrosome Reaction--
On the premise that only
capacitated sperm will undergo exocytosis in response to zonae
pellucidae, the zona pellucida-induced acrosome reaction in sperm was
analyzed as end point of capacitation. Zona pellucidae were prepared
from homogenized ovaries of virgin female 22-day-old outbred CD1 mice
(Charles River Laboratories) as described (33) and solubilized for all
experiments by the procedures outlined previously (11). The percentage
of acrosome reaction was measured using Coomassie Blue G-250 as
described (34). Briefly, sperm were incubated at 37 °C for 45 min
followed by the addition of 5 zona pellucida equivalents/µl. After an
additional 30 min of incubation at 37 °C, an equal volume of 2×
fixative solution (10% formaldehyde in phosphate-buffered saline) was
added to each tube. Following fixation, 10-µl aliquots of suspension were spread onto glass slides and air-dried. The slides were then stained with 0.22% Coomassie Blue G-250 in 50% methanol and 10% glacial acetic acid for 3-5 min, gently rinsed with deionized H2O, air-dried, and mounted with 50% (v/v) glycerol in
phosphate-buffered saline. To calculate the percentage of acrosome
reaction, at least 100 sperm were assayed per experimental condition
for the presence or absence of the characteristic dark blue acrosomal crescent.
Cholesterol Acceptors and HCO
To evaluate the role of HCO HCO
Several hypotheses that can account for this HCO Na+/HCO
Stilbenes in general and DIDS in particular have been shown to inhibit
a variety of anionic transporters; accordingly, DIDS inhibition is one
of the criteria used to describe Na+/HCO Mammalian sperm do not acquire full fertilizing capacity
immediately after ejaculation; the ability to fertilize is achieved in
the female tract in a process known as capacitation. Capacitation is
associated with a cAMP/protein kinase A-dependent pathway
that is upstream of an increase in protein tyrosine phosphorylation (2). Capacitation is also associated with the hyperpolarization of the
sperm plasma membrane. The functional role of sperm plasma membrane
hyperpolarization during capacitation is at present not well
understood. Because the presence of LVA Ca2+ T channels has
been demonstrated in mouse spermatogenic cells (6, 7), Florman and
co-workers (40) have hypothesized that hyperpolarization of the sperm
plasma membrane is required for the activation of these LVA
Ca2+ T channels by physiological agonists (e.g.
zona pellucida or progesterone). A signature property of LVA
Ca2+ channels is a low threshold for
voltage-dependent inactivation. This means that these
Ca2+ channels are inactive when the plasma membrane is
depolarized, such as is observed before capacitation, thus suppressing
premature exocytosis until the completion of capacitation. After
capacitation, hyperpolarization of the sperm plasma membrane will allow
the agonist-dependent opening of LVA Ca2+ T
channels during the acrosome reaction (5, 40).
In the present work we have analyzed whether components of the
capacitation media that regulate the cAMP pathway and the increase in
protein tyrosine phosphorylation have a role in the regulation of the
sperm plasma membrane potential. The central observations of this study
are as follows: 1) HCO Previously, an essential role of HCO BSA is also required for capacitation; its role is linked to its
ability to bind cholesterol and facilitate its efflux from the sperm
plasma membrane. Supporting this hypothesis, BSA can be replaced in the
capacitation media by other protein and non-protein cholesterol
acceptors such as high density lipoprotein and the high affinity
cholesterol-binding cyclic sugars, Sperm plasma membrane potential was measured using fluorescent dyes;
this method gives reliable measurements of the plasma membrane
potential and has already been used to measure membrane potential in
mammalian sperm (3, 29). The addition of CCCP 2 min before the
fluorimetric measurements precludes the contribution of mitochondrial
membrane potential to the final calibration. The calibration procedure
followed in this study compensates for variation in sperm concentration
and viability and ensures an accurate determination of the average
sperm population plasma membrane potential. By using this methodology,
we have demonstrated that addition of HCO The functional family of HCO How the Na+/HCO Intracellular pH has been implicated in the control of several
mammalian sperm functions such as the development of progressive motility, capacitation, and the acrosome reaction. However, the transport mechanisms that regulate pHi in mammalian sperm
are not well understood. A Na+-dependent
Cl induces a hyperpolarizing current in
mouse sperm plasma membranes. This
HCO
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-cyclodextrin (2-OH-p-
-CD), carbonyl
cyanide m-chlorophenylhydrazone (CCCP), valinomycin,
nigericin, ouabain, H89, DIDS,
4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS),
choline chloride (choline+Cl
), choline
bicarbonate (choline+HCO
20 °C except
when otherwise stated: cholesterol SO
-CD was added directly to Whitten's medium for a
final concentration of 1 mM. Solutions of 800 mM trimethylamine hydrochloride,
choline+Cl
,
choline+HCO
up to the concentration indicated
in the respective experiment. Other Na+ salts present in
Whitten's/HEPES were replaced with their respective K+
salts, and the total K+ concentration was maintained at 5.9 mM. Because
-cyclodextrins are able to replace BSA as a
cholesterol sink and capacitate mouse sperm (26), in some experiments
these compounds were used to analyze the role of plasma membrane
cholesterol release in different signaling events.
PY) monoclonal antibodies (Clone 4G10,
Upstate Biotechnology, Inc., Lake Placid, NY) and a polyclonal
anti-hexokinase type 1 (
HK1) as described previously (28).
Immunoblots were developed with the appropriate secondary antibody
conjugated to horseradish peroxidase (Sigma) and an ECL kit (Amersham
Biosciences) according to the manufacturer's instructions.
80,
72,
61,
47, and
32 mV, respectively. These values were
obtained using the Nernst equation, assuming an intracellular
K+ concentration of 120 mM (31) and considering
that the membrane potential corresponds to the K+
equilibrium in the presence of CCCP and valinomycin. The sperm membrane
potential in each case was linearly interpolated using these data as
plasma membrane potential versus arbitrary units of
fluorescence (Fig. 1). The fluorescence
emission of DiSC3-(5) depends on variables such as sperm
number, dye concentration, sperm viability, fluorimeter cuvette
dimensions, and the presence of compounds that interact with
DiSC3-(5). The internal calibration of each single
determination compensates for variables that influence the absolute
fluorescence values. As mentioned before, in some cases membrane
potential was measured using DiSBAC2-(3). In these cases,
sperm were incubated as described above, except that 0.5 µM DiSBAC2-(3) was added 5 min before the
fluorescence measurements, and fluorescence (535/560 nm
excitation/emission wavelength pair) was recorded.
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Fig. 1.
Membrane potential calibration using
valinomycin and sequential addition of KCl. A,
mouse caudal epididymal sperm were incubated at 37 °C in Whitten's
media. After a 7-min incubation, 1 µM
DiSC3-(5) was added to the sperm suspension and further
incubated for 5 min, then CCCP (1 µM) was added, and the
sperm were incubated for an additional 2 min, and 1.5 ml of the
suspension was added to the fluorimeter chamber for recording the
fluorescence emission. After the signal reached stability (usually
within 1-2 min), calibration was performed adding 1 µM
valinomycin (Val.) and sequential additions of KCl. The
initial suspension contains 5.9 mM KCl, and the subsequent
KCl additions resulted in final concentrations (mM KCl) in
the sperm suspension of 8 (a), 12 (b), 20 (c), and 36 (d), which correspond to membrane
potentials of 80,
72,
65,
47, and
32 mV, respectively. These
values were obtained using the Nernst equation as explained under
"Experimental Procedures." B, plasma membrane
potential values obtained with valinomycin and sequential addition of
KCl versus the arbitrary units of fluorescence. Sperm
membrane potential is then linearly interpolated using these data. In
this case the plasma membrane potential =
36 mV.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-Cyclodextrins are cyclic heptasaccharides known for their ability
to bind cholesterol; we have demonstrated previously (26) that
in the presence of these compounds cholesterol is released from the
sperm plasma membrane. This cholesterol-efflux initiates signaling
events leading to capacitation (26). When BSA is replaced by 1 mM 2-OH-p-
-CD in complete medium, cauda
epididymal mouse sperm are hyperpolarized after 45 min of incubation,
similarly to the control using 5 mg/ml BSA (Fig.
2 and data not shown). In contrast, in
media lacking BSA or
-cyclodextrin the capacitation-associated hyperpolarization did not occur (Fig. 2). In addition, when the cholesterol-binding sites of BSA were saturated with 5 µM
cholesterol SO
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Fig. 2.
Cholesterol acceptors and NaHCO3
are necessary for the capacitation-associated membrane
hyperpolarization. Mouse caudal epididymal sperm were incubated in
Whitten's media for the time indicated in the figure at 37 °C with
only 1 mM 2-OH-p- -CD (A,
),
with only 24 mM NaHCO3 (B,
), or
with both (C,
). Sperm plasma membrane potential
determination in each case was performed as described in Fig. 1.
D, the data in this graph represent the mean ± S.E. from three independent experiments.
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Fig. 3.
Saturation of cholesterol-binding sites by
cholesterol SO
-CD or 5 mg/ml BSA, and the plasma membrane
potential was measured at different periods. In the absence of
HCO
-CD-HCO
(Fig. 4, A and B),
I
, Br
, NO
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Fig. 4.
HCO
antiporter
(model 2) is present in sperm as suggested previously (15, 16, 35).
Because HCO
exchanger is
electroneutral, HCO
/H+
antiporter (model 3) (18). Similarly to the
HCO
, this exchanger is
electroneutral. Nevertheless, hyperpolarization could occur by coupling
this antiporter with the electrogenic Na+/K+
ATPase. To analyze this possibility, cauda mouse sperm were incubated for 5 min with 10 µM ouabain or in the absence of
extracellular K+. Although both treatments are able to
inhibit the Na+/K+ pump, neither of them
inhibited the HCO
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Fig. 5.
A, different models to explain the
HCO /HCO
/H+
antiporter, and the resultant increase in intracellular Na+
could activate the electrogenic Na+/K+ ATPase,
in this way hyperpolarizing the cell. 4, The
HCO
, and the HCO
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Fig. 6.
The HCO
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Fig. 7.
Capacitation and capacitation-associated
increase in protein tyrosine phosphorylation are
Na+-dependent. A,
Na+-dependent increase in protein tyrosine
phosphorylation. Caudal epididymal sperm were recovered in
Na+-free medium (Whitten's/choline+) and then
incubated in the same media or in Whitten's media with different NaCl
concentrations prepared as described. In the last lanes,
sperm were incubated in Na+-free or in
Na+-containing medium with the addition of 1 mM
Bt2-cAMP and 100 µM IBMX as indicated in
the figure. After 1.5 h of incubation, the sperm were
extracted and proteins subjected to SDS-PAGE, transferred to Immobilon
P, and probed with anti-phosphotyrosine antibody (clone 4 G10).
B, zona pellucida-induced acrosome reaction is
inhibited in Na+-free medium. Mouse caudal
epididymal sperm were incubated in Whitten's/choline+ with
the addition of 24 mM
choline+HCO
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Fig. 8.
DIDS and SITS inhibit the
HCO PY antibodies (Ab) were
performed as described. The arrows show the position of
hexokinase type 1 (28). The Western blot on the right was
developed with
HK1. A representative experiment (out of 3) is
shown.
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Fig. 9.
The
HCO
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-cyclodextrins (26, 34). In the
present work, we have demonstrated that 2-OH-p-CD is able to
induce the capacitation-associated hyperpolarization in the absence of
BSA. Moreover, when sperm are incubated in the presence of the
cholesterol analogue, cholesterol-SO
/HCO
/HCO
/HCO
/HCO
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants HD38082 (to P. E. V.), HD06274, and HD22732 (to G. S. K.), by CONACyT, and DGAPA IN201599 (to A. D.), and by the Mellon Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
** Present address: Wyeth Ayerst P. O. Box 8299, Philadelphia, PA 19101-8299.
To whom correspondence should be addressed: Department of
Veterinary and Animal Sciences, Paige Labs, University of
Massachusetts, Amherst, MA 01003. E-mail: pevb61@yahoo.com.
Published, JBC Papers in Press, December 19, 2002, DOI 10.1074/jbc.M206284200
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ABBREVIATIONS |
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The abbreviations used are:
BSA, bovine serum
albumin;
LVA, low voltage-activated;
DIDS, 4,4'-diiodothiocyanatostilbene-2,2'-disulfonic acid;
pHi, intracellular pH;
Bt2-cAMP, dibutyryl cyclic AMP;
IBMX, 3-isobutyl-1-methylxanthine;
2-OH-p--CD, 2-hydroxypropyl-
-cyclodextrin;
CCCP, m-chlorophenylhydrazone;
SITS, 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid;
DiSC3, 3,3'-dipropylthiadicarbocyanine iodide;
BCECF-AM, 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, acetoxymethyl
ester;
sAC, soluble adenylyl cyclase;
DiSBAC2, bis-(1,3-diethylthiobarbituric acid trimethine oxonol.
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REFERENCES |
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