BIOLOGY OF REPRODUCTION 66, 1775–1780 (2002)
Progesterone Induces Hyperpolarization after a Transient Depolarization Phase
in Human Spermatozoa1
C. Patrat,2 C. Serres, and P. Jouannet
GREFH-Laboratoire d’Histologie-Embryologie et Biologie de la Reproduction, Hôpital Cochin,
Faculté de Médecine Cochin—Port-Royal, Université Paris V, 75014 Paris, France
ABSTRACT
Progesterone (P4) induces a membrane depolarization and
various ion fluxes (chloride efflux, sodium and calcium influxes),
which are required for the human sperm acrosome reaction
(AR). By use of the potentiometric fluorescent dye DiSC3(5) and
two different technical approaches, the present study aimed to
quantify and further analyze P4-induced modifications in membrane potential in capacitated human spermatozoa. Spectrofluorimetric analysis revealed that the mean resting membrane
potential of sperm was 258 6 2 mV (n 5 12). When 10 mM P4
was added, the sperm membrane depolarized by ;115 mV,
partly driven by a Cl2 efflux. It subsequently repolarized to
reach a significant lower potential than the initial resting potential in two thirds of the tested samples. The flow cytometry analysis showed a heterogeneous resting membrane potential and
revealed that the depolarization-hyperpolarization events concerned only subpopulations, between 3% and 40% of the sperm
cells according to the samples (n 5 7). We hypothesize that P4
has a beneficial effect on the ability of zona pellucida to promote the AR in a sperm subpopulation by increasing the number
of hyperpolarized cells presenting a membrane potential that is
compatible with the opening of T-type calcium channels by subsequent zona pellucida-induced depolarization.
acrosome reaction, fertilization, mechanisms of hormone action,
progesterone, sperm
INTRODUCTION
The acrosome reaction (AR) is a key step in fertilization.
It enables the spermatozoa to pass through the zona pellucida (ZP) and to fuse with oocytes. The physiological AR
is induced by ZP3 glycoprotein [1]. In vitro, the sperm ARtriggering function of ZP is optimized by prior exposure of
the spermatozoa to progesterone (P4) for a few minutes [2,
3]. In vivo, before their interaction with the ZP, the spermatozoa swim between the P4-secreting cumulus cells that
surround the oocyte. In this microenvironment, spermatozoa can suddenly be exposed to high local levels of P4 (.1
mg/ml, ;3.14 mM) [4]. Therefore, prior exposure to P4 may
be an advantage for a subpopulation of spermatozoa by
making them responsive to ZP. The mechanism by which
progesterone sensitizes spermatozoa to the subsequent action of ZP is still unknown.
This work was supported by grant 1752 from the Direction de la Recherche et des Etudes Doctorales.
2
Correspondence: C. Patrat, GREFH-Laboratoire d’Histologie-Embryologie
et Biologie de la Reproduction, Hôpital Cochin, Faculté de Médecine
Cochin-Port-Royal, Université Paris V, 24 rue du Fg St Jacques, 75014
Paris, France. FAX: 33 1 58 41 15 75;
e-mail: [email protected]
1
Received: 4 October 2001.
First decision: 29 October 2001.
Accepted: 9 January 2002.
Q 2002 by the Society for the Study of Reproduction, Inc.
ISSN: 0006-3363. http://www.biolreprod.org
In spermatozoa, the effects of P4 seem to depend on its
binding to the plasma membrane [5]. Within sperm, the
transduction of signals triggered by P4 involves changes in
the intracellular concentrations of ions, including a biphasic
increase in the calcium concentration [5–7], a sustained increase in the sodium concentration [8], and a rapid transient
decrease in the chloride concentration due to Cl2 efflux [9].
The intraspermatic increase in calcium and sodium concentrations is due to the entry of these ions from the extracellular medium [6, 8, 10]. In addition to inducing these ion
fluxes, P4 depolarizes the plasma membrane of human spermatozoa, as shown by the accumulation of a lipophilic cation, triphenylmethylphosphonium [11], or by the use of
fluorescent probes [12, 13]. On the basis of qualitative data,
it has been reported that P4-induced depolarization was not
carried by calcium influx but mainly by sodium influx [12].
The possible involvement of P4-induced Cl2 efflux in its
depolarizing effect has also been suggested but not investigated [9]. So these previously published studies have provided incomplete data because they lack information about
either the extent of P4-stimulated depolarization or the ionic
nature of its depolarizing effect.
In a previous study, we postulated that the ;10 mM
change in [Na1]i induced by P4 would correspond to about
110 mV of the depolarizing effect [8]. To confirm this
assumption, we aimed to quantify P4-induced membrane
potential changes in capacitated human spermatozoa, using
the fluorescent potentiometric probe DiSC3(5) in spectrofluorimetry. Moreover, we further analyzed in flow cytometry these membrane effects of P4 to detect possible differential responses of subpopulations. To complete our knowledge of the ionic fluxes responsible for the depolarizing
effect of P4, we also wanted to determine whether a chloride efflux is involved in the P4-induced sperm membrane
modifications.
MATERIALS AND METHODS
Media
Biggers Whitten Whittingham (BWW) 1 BSA medium consisted of
95 mM NaCl, 4.8 mM KCl, 1.7 mM CaCl2, 1.2 mM KH2PO4, 1.2 mM
MgSO4, 5.6 mM glucose, 0.25 mM sodium pyruvate, 20 mM acid-free
HEPES, 25 mM NaHCO3, 20 mM lactic acid, and 5 mg/ml BSA (pH 7.5)
adjusted with NaOH; osmolarity was 300–310 mOsm/L.
In some experiments we used modified BWW medium: Ca21-free,
Na1-free, and high K1 medium (high K1 was BWW medium in which
NaCl was replaced by KCl, NaHCO3 by KHCO3, and CaCl2 and sodium
pyruvate were omitted) pH 7.5; adjusted with KOH. Osmolarity was 270–
300 mOsm/L.
Products
Progesterone, bicuculline, valinomycin, nigericin, gramicidin D, and
fluorocarbonyl cyanide m-chlorophenyl hydrazone (FCCP) dissolved in
absolute ethanol, biscarboxyethylcarboxyfluorescein acetoxy methyl ester
(BCECF-AM) dissolved in dimethyl sulfoxide (DMSO), sodium pyruvate,
and HEPES were purchased from Sigma-Aldrich (Saint-Quentin Fallavier,
France). 3,39-Dipropylthiodicarbocyanin iodide (DiSC3(5)) diluted in
1775
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PATRAT ET AL.
FIG. 1. Effects of P4 on the membrane potential of human spermatozoa
assessed by DiSC3(5) fluorescence variations in spectrofluorimetry. Ten
micromolar P4 was added (arrow) to sperm samples incubated in BWW
medium in the presence of 250 nM DiSC3(5). Traces represent 2 typical
sperm membrane potential modifications induced by P4.
DMSO was purchased from Molecular Probes (Interchim, Asnières,
France). All other chemicals (salts for buffers) were purchased from Merck
(Merck-Clévenot, Nogent-sur-Marne, France).
Sperm Preparation
Human semen with normal sperm characteristics according to World
Health Organization criteria (concentration $20 3 106 cells/ml, motility
$50%, normal morphology $30%) was collected by masturbation from
14 different donors. After liquefaction at 378C, motile spermatozoa were
selected by centrifugation (20 min, 300 3 g) through a two-layer Percoll
density gradient (95% and 47.5%) [14] This technique is considered as
the most appropriate to select normal mature spermatozoa for clinical application [15, 16]. The pellets were washed (10 min, 600 3 g), suspended
at a concentration of 1 3 107 spermatozoa/ml in BWW1BSA, and incubated for 18 h at room temperature to allow capacitation to occur. These
conditions of capacitation of human spermatozoa have been proved to be
efficient for the AR to be induced by the ZP [17]. Sperm motility was
checked in every sperm sample and was always greater than 70% after
incubation.
Measurement of Membrane Potential
Capacitated spermatozoa were washed by dilution (v/v) in normal or
modified BWW media (without BSA) and centrifugation (5 min, 600 3
g). They were then suspended in normal or modified BWW medium for
spectrofluorimetry or flow cytometry measurements.
Spectrofluorimetric measurement. Sperm suspension (5–10 3 106
cells/ml), gently stirred with a magnetic bar, was exposed to 250 nM final
concentration of cationic dye DiSC3(5) in a glass cuvette maintained at
378C for fluorescence measurement in a PTI M2001 spectrofluorimeter
(BIO-TEK Kontron Instruments, Saint-Quentin en Yvelines, France). Progesterone or bicuculline was added to a final dilution of 1:1000. After
each treatment, a 30-ml sample was removed from the cuvette for estimating sperm motility by optical microscopy.
The membrane potential was determined according to the experimental
procedure described for bull and mouse sperm [18]. Fluorescence was
evaluated at an excitation wave length of 620 nm and an emission wave
length of 670 nm (5 nm bandpass). The extent to which it accumulates in
cells depends on their membrane potential. After adding the dye to sperm
suspension the intensity of fluorescence decreased, reaching equilibrium
within 5 to 10 min. This quenching of the fluorescent signal is probably
due to the accumulation of the dye in cells in an aggregated and nonfluorescent form. To facilitate the unambiguous interpretation of the data, valinomycin and KCl were added at the end of each experiment in BWW
medium (6 mM KCl) to obtain the fluorescence changes linked to hyperpolarization and depolarization, respectively. Membrane depolarization, inducing the release of the positively charged dye into the extracellular
space, was expected to increase the fluorescence level by decreasing the
formation of nonfluorescent aggregates in spermatozoa. The fluorescence
signal obtained with DiSC3(5) reflected the resting plasma membrane potential and the membrane potential of other compartments, such as mitochondria, which have a highly negative potential. The mitochondrial potential could be eliminated by adding 1 mM FCCP, a proton exchanger, to
the sperm samples [18].
The fluorescence signal was converted to apparent membrane potential
by the calibration of fluorescence emission, as described by Linares-Hernandez et al. [19] for human sperm. Change in fluorescence (DF/Fval) was
plotted against electrochemical potassium potential (Ek), assuming that K1
was the major component responsible for the plasma membrane potential
of mammalian sperm [20]. Fval was the fluorescence value obtained after
adding 2 mM valinomycin, a potassium-specific ionophore. DF was defined as the change in fluorescence observed after adding a known amount
of KCl to the medium in the presence of valinomycin (external potassium
concentration, [K1]e, varied from 6 mM [value in BWW medium] to 46
mM). Ek, calculated with the Nernst equation, was 261.5 Log ([K1]i/
[K1]e) at 378C.
The internal potassium concentration ([K1]i) of the capacitated human
spermatozoa in our experimental conditions was determined by the null
point method of Babcock [21] by use of the K1/H1 exchanger, nigericin.
Briefly, spermatozoa were incubated with 0.5 mM BCECF-AM (a fluorescent probe for pH measurement) for 30 min at room temperature, and
were then washed by centrifugation through a 95% Percoll layer. BCECFloaded cells were resuspended in medium containing various proportions
of KCl 1 LiCl (total 150 mM), 0.5 mM MgCl2, 2.5 mM CaCl2, 2 mM
glucose, and 10 mM Hepes-Li at pH 6.9. Fluorescence emission was recorded at 530 nm using a dual (430 and 500 nm) excitation wave length.
The change in the 500:430 fluorescence ratio that followed the addition
of 2 mM nigericin was determined in various media containing 30 to 150
mM KCl. If the addition of nigericin resulted in no change in fluorescence
(e.g., no change in internal pH), [K1]e was considered to be equal to [K1]i.
Flow cytometry measurement. Sperm suspension (2 3 106 cells/ml)
was exposed to 250 nM DiSC3(5) for 10 min before recording fluorescence
by flow cytometry (Facs Calibur, Becton Dickinson, Pont-de-Claix,
France) at an excitation wave length of 633 nm and an emission wave
length of 680 nm. Debris and cell aggregates were excluded from analysis
by gating side versus forward scatter cytogram. P4 and gramicidin D were
added to a final dilution of 1:1000. Valinomycin was added to a final
dilution of 1:500. The timing of loading and incubation of the samples
was precisely determined and was the same for each of the aliquots. Results were expressed as the fluorescence intensity (reflecting membrane
potential) of sperm cells plotted against the number of cells displaying that
fluorescence. Markers were positioned to quantify the observed sperm subpopulations.
To interpret the fluorescence modifications in terms of hyperpolarizing
or depolarizing effects, capacitated human spermatozoa were incubated in
BWW medium (6 mM KCl) in the presence of 2 mM valinomycin or 1%
gramicidin D, respectively. These experimental conditions resulted in a
shift in sperm cell repartition toward higher and lower fluorescence, respectively (data not shown).
Statistical Analysis
Data are expressed as means 6 SEM. Means were compared using the
Student t-test for paired or unpaired samples, and P , 0.05 was considered
to be statistically significant.
RESULTS
Effect of P4 on Human Sperm Membrane Potential
The spectrofluorimetric recordings showed that the addition of 10 mM P4 caused a transient depolarization of the
membrane of human spermatozoa incubated in capacitating
conditions. This depolarizing effect was followed by a repolarization phase, during which the membrane potential
either became more negative than it was initially in 8 out
of 12 experiments, or it returned to its initial level in the 4
remaining sperm samples. These membrane events occurred rapidly within 3 to 5 min (Fig. 1, upper curve) or
more slowly within 5 to 10 min (Fig. 1, lower curve).
Using flow cytometry, two or three sperm subpopulations according to the samples could be distinguished, reflecting a heterogeneous resting membrane potential of human spermatozoa incubated in capacitating conditions,
P4-INDUCED SPERM HYPERPOLARIZATION
FIG. 2. Effects of P4 on the membrane potential of human spermatozoa
assessed by DiSC3(5) fluorescence variations and flow cytometry. The
sperm sample was incubated in BWW medium in the presence of 250
nM DiSC3(5) and fluorescence variations reflecting the relative sperm
membrane potential were registered by flow cytometry before (T0) and
after the addition of 10 mM P4 (T 5 30 to 300 sec). A) Histograms showing
distribution of the relative membrane potential in the sperm population
before and after addition of P4. The sperm membrane potential expressed
in arbitrary DiSC3(5) fluorescence units (x-axis) is plotted against the relative number of cells displaying that fluorescence (y-axis). The whole population has been divided into three subpopulations (m 5 I, m 5 II, and
□ 5 III) and markers have been positioned to quantify these subpopulations. B) Quantification of sperm cell subpopulations (I to III) (y-axis) at
different times (x-axis) following P4 treatment. Two typical experiments
from 7 similarly performed in duplicate or triplicate are shown.
ranging from depolarized cells with low fluorescence intensity to hyperpolarized cells with high fluorescence intensity
(Fig. 2A, T0). This heterogeneous profile of resting membrane potential was reproducible for the sperm samples
tested in duplicate or triplicate. The addition of 10 mM P4
to seven different sperm samples modified the sperm membrane potential in a similar manner as observed in spectrofluorimetry. Three percent to 30% of sperm cells (12.7%
6 3.2%, n 5 7) shifted toward lower fluorescence, reflecting an increase in the number of depolarized cells (Fig. 2,
marker I). In 4 out of these 7 experiments, the depolarizing
effect was associated with a hyperpolarizing effect, represented by an increase of 4% to 40% in the number of hy-
1777
FIG. 3. [K1]i determination and calibration of DiSC3(5) fluorescent signal. Upper panel shows determination of [K1]i in human spermatozoa by
null point measurement. Capacitated and BCECF-loaded spermatozoa
were incubated in media containing various concentrations of KCl (30 to
150 mM). The change in 500 nm/430 nm fluorescence ratio (DF) following
the addition of 2 mM nigericin was measured in these media. [K1]i was
equal to [K1]e when no change in fluorescence (DF 5 0) was observed
after nigericin addition (null point). At the null point, Log [K1]e 5 1.87
(arrow) indicating that [K1]i equals 75 mM. Each point is the mean (6
SD) of 3 to 5 independent experiments and the equation, DF 5 0.9865
Log [K1]e 2 1.85, fits the data. Lower panel shows the calibration curve
for converting fluorescence intensity to membrane potential. The sperm
membrane potential was monitored with the fluorescent probe, DiSC3(5).
The equilibrium potential for K1(Ek) was changed by increasing [K1]e from
6 mM in normal BWW medium to 46 mM in the presence of 2 mM
valinomycin. Ek was calculated using the Nernst equation, with [K1]i 5
75 mM, and was plotted against the corresponding percentage of change
in fluorescence (DF) with respect to basal fluorescence in BWW medium
(Fval). Each point is the mean 6 SD of 6 to 9 independent experiments.
The regression line was Y 5 1.33 X 1 89.02, and the correlation coefficient was 0.98.
perpolarized cells (19.1% 6 7.8%, n 5 4; Fig. 2, marker
III). The relative changes in the number of cells included
in the various subpopulations occurred within the initial
range of the resting membrane potential so that no more
hyperpolarized or depolarized subpopulations were observed when P4 was added. These effects of P4 were reproducible when tested in duplicate or triplicate for the
same sperm samples.
Addition of ethanol (1%), the P4 solvent, did not induce
any noticeable modifications in sperm fluorescence over a
5-min period (data not shown).
Quantification of Membrane Potential Changes Induced
by P4 in Human Spermatozoa
For the quantification of membrane potential in spectrofluorimetry, we calculated the [K1]i of human spermatozoa
incubated in capacitating conditions. It was found to be 75
mM (Fig. 3). This value of [K1]i was inserted into the
Nernst equation to calculate Ek during calibration experiments (see Materials and Methods). The change in fluorescence, DF/F, was linearly related to Ek (r 5 0.98, P , 0.01;
Fig. 3). Therefore, the fluorescence measurement could be
used as an estimation of membrane potential.
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PATRAT ET AL.
TABLE 1. Individual values of human sperm membrane potential before and after exposure to 10 mM P4 and measured in spectrofluorimetry.a
After P4 exposure
Resting
membrane
potential (Vm1)
Depolarization
(Vm2)
Repolarization
(Vm3)
1
2
3
4
5
6
7
8
Mean
SEM
255
244
256
255
256
253
258
262
254.9bc
1.8
225
226
230
240
253
248
250
251
240.4
4.2
262
247
259
260
259
257
264
270
259.9b
2.3
130
118
126
115
13
15
18
111
14.5
3.4
9
10
11
12
Mean
SEM
253
274
265
262
263.8c
4.3
241
265
252
235
248.4
6.6
253
274
265
262
263.8
4.3
112
19
113
127
15.2
4.1
Sperm
samples
Depolarizing
effect
(Vm2–Vm 1)
Hyperpolarizing
effect
(Vm3–Vm 1)
27
23
23
25
23
24
26
28
24.9
0.7
0
0
0
0
0
0
0
a
The values are expressed in mV; Vm2 was measured at maximal depolarization; Vm3 was measured after the repolarization phase, 5 to 10 min after
10 mM P4 exposure.
b P , 0.01, with paired t-test.
c P , 0.05, with unpaired t-test.
The mean resting membrane potential of human spermatozoa incubated in capacitating conditions, measured in
12 semen samples from 12 different donors, was 257.8 6
2.2 mV (range, 274 mV to 244 mV). The addition to the
sperm suspension of P4 at a final concentration of 10 mM
resulted in a mean membrane depolarization of 15.0 6 2.6
mV, the membrane potential reaching 242.9 6 3.5 mV, a
level significantly different from the resting potential level
(P , 0.001, n 5 12). The sperm membrane then repolarized
to a mean value of 261.0 6 2.3 mV (range, 274 mV to
247 mV; n 5 12). In 8 out of the 12 sperm samples, the
membrane potential reached 5 to 10 min after P4 addition
was significantly more negative than the initial resting
membrane potential (259.9 6 2.3 mV vs. 254.9 6 1.8 mV
respectively, P , 0.01, n 5 8; Table 1). This resulted in a
significant hyperpolarization of the sperm membrane in
these samples. It is interesting that the four remaining
sperm samples which did not hyperpolarize after P4 expo-
FIG. 4. Role of ion movements in P4-induced membrane potential
change in human spermatozoa. Spermatozoa were incubated in a Ca21free, Na1-free and high K1 medium (high K1) with 250 nM DiSC3(5). After
stabilization of the fluorescent signal, 2 aliquots of 10 mM P4 were successively added to the cuvette (arrows). Then the cells were pelleted by
centrifugation (5 min, 600 3 g), and resuspended in normal BWW medium. They were returned to the cuvette in the presence of 250 nM
DiSC3(5) and 10 mM P4 was added.
sure had an initial resting membrane potential more negative than that of the group of the 8 samples (263.8 6 4.3
mV vs. 254.9 6 1.8 mV, respectively; P , 0.05; Table 1).
In order to evaluate the respective contribution of mitochondrial and plasma membrane potential, the mitochondrial membrane potential was dissipated by the addition of
1 mM FCCP in 6 other experiments. The residual resting
plasma membrane potential was 20% more positive in
FCCP-treated samples compared with controls (251.8 6
3.1 mV vs. 261.2 6 1.4 mV, respectively; P , 0.02, n 5
6). In FCCP-treated spermatozoa, P4 elicited a transient depolarizing effect, with a pattern similar to that observed in
untreated spermatozoa, but with a significantly smaller amplitude (8.5 6 1.4 mV vs. 14.2 6 2.9 mV, respectively, P
, 0.05, n 5 6).
Involvement of Ion Fluxes in the Membrane
Depolarization-Repolarization Process Induced by P4
When spermatozoa were incubated with DiSC3(5) in
Ca21-free, Na1-free, high K1 medium to prevent the ion
fluxes that were believed to be primarily responsible for the
membrane potential, the addition of 10 mM P4 had no effect
on the membrane potential (Fig. 4). The high K1 medium
was not toxic for spermatozoa because they remained motile, although the flagellar beat slowed down. When these
spermatozoa, treated in such high K1 conditions, were then
washed and resuspended in normal BWW medium, P4-induced membrane depolarization occurred (n 5 2) (Fig. 4).
It has been suggested that the depolarizing effect of P4
was mainly driven by the Na1 influx and that the Ca21
influx was not involved [12]. Because P4 also stimulates a
Cl2 efflux [9], we investigated whether the Cl2 efflux could
be involved in P4-induced depolarization. The prior incubation of human spermatozoa for 3 min with 10 mM bicuculline, a GABAA receptor/Cl2 channel antagonist that
abolishes P4-induced Cl2 efflux in human spermatozoa [9],
significantly decreased the depolarizing effect induced by
P4 (9.0 6 2.5 mV vs. 23.0 6 6.5 mV for treated and control
groups, respectively; P , 0.05, n 5 4).
P4-INDUCED SPERM HYPERPOLARIZATION
DISCUSSION
The effect of progesterone on the membrane potential of
human sperm was investigated by using the carbocyanine,
DiSC 3 (5), to quantify P 4 -induced membrane potential
changes.
Our study established 3 major new findings: 1) P4-induced membrane depolarization is not a long-lasting but a
transient event and is followed by a membrane hyperpolarization in a majority of sperm samples; 2) P4-induced membrane potential variations concern only subpopulations,
consisting of between 3% and 40% of the sperm cells; and
3) the amplitude of P4-induced depolarization is ;115mV
from a mean resting membrane potential of about 258 mV
in human spermatozoa incubated in capacitating conditions
and is partly driven by Cl2 efflux.
The dye used, DiSC3(5), was suitable for studying
changes in membrane potential that are directly linked to
ion fluxes, because its partitioning into sperm cells did not
vary in the absence of ionic fluxes as demonstrated by experiments in high K1 medium. The fluorescence signal generated by DiSC3(5), a positively charged carbocyanine
probe, reflects the potentials of the plasma membrane and
of other compartments, such as the mitochondria. However,
DiSC3(5) has a short alkyl tail (3 carbon atoms), which
renders it poorly hydrophobic and does not favor its incorporation into mitochondria [22]. This weak contribution of
mitochondrial potential to the total membrane potential
measured with DiSC3(5), about 20% in our study, is consistent with the observations of Linares-Hernandez et al.
[19] on noncapacitated human spermatozoa and GonzalezMartinez and Darszon [23] on sea urchin spermatozoa, using carbonyl cyanide m-chlorophenyl hydrazone and KCN,
respectively, to collapse the mitochondrial potential.
As is usually reported in humans, our results are
throughout characterized by intersperm and intrasperm
sample variations, not only in the resting membrane potential values, but also in the amplitude of the response to P4.
Similar interdonor variations were detected in spectrofluorimetry for basal intracellular calcium concentration and
also in single cell imaging for the responsiveness to P4
when 43% to 94% of the sperm cells exhibited an intracellular calcium increase, according to sperm samples [24,
25]. In the present study, there is also evidence for a cellular
heterogeneity within a same sperm sample because only
some cell subpopulations display membrane potential modifications after P4 addition. This observation is in agreement
with the study by Kirkman-Brown et al. [25], who reported
an individual variability in the pattern of calcium increase
induced by P4. The reasons for such a cellular heterogeneity
remain under speculation. As suggested by others, it could
be due, at least in part, to the capacitation status [24, 25].
There is indeed more and more evidence that sperm cells
within a population do respond to capacitating conditions
to different degrees and at different rates [26]. Given the
inherent heterogeneity of spermatozoa, capacitation appears
as a specific process, which affects sperm cells differentially.
The resting plasma membrane potential of human spermatozoa incubated in capacitating conditions, measured
with DiSC3(5) after the dissipation of mitochondrial potential, was found to be 252 6 3 mV. This is close to the
values reported for capacitated mouse sperm (255 6 2
mV) and bull sperm (261 6 4 mV) [18]. These values of
membrane potential determined by spectrofluorimetry are
the means for the total sperm population and mask a cel-
1779
lular heterogeneity as revealed by the analysis in flow cytometry showing that the population of human spermatozoa
incubated in capacitating conditions is composed of 2 or 3
subpopulations with various membrane potentials. This observation does not depend on methodological bias because
the same repartition for a same sperm sample was found in
experiments performed in duplicate or triplicate. Our observations differ from the recent data that report a heterogeneous but unimodal repartition of resting membrane potential of human spermatozoa submitted to capacitating
conditions [13]. In this last study, another cyanine fluorescent dye, DiOC6(3), was used and interdonor variations
were not examined in any detail. Recently, a single-cell
analysis also reported individual membrane potential values
of 292 to 232 mV (mean, 263 6 17 mV) for mouse
spermatozoa incubated in capacitating conditions, demonstrating large differences in membrane potential between
cells [27].
In our study, the effect of capacitating conditions on
sperm membrane potential was not investigated. A mean
resting membrane potential of 240 6 16 mV, measured
with DiSC3(5), has been reported for noncapacitated human
spermatozoa (i.e., just after selection of motile spermatozoa) [19]. This value, which is more positive than the value
that we obtained with cells that are submitted to capacitating conditions, is consistent with the development of sperm
plasma membrane hyperpolarization during capacitation
[13, 18, 27]. Membrane hyperpolarization probably results
from K1 efflux during capacitation [18], which is consistent
with the [K1]i of 75 mM found in this study for human
cells incubated in capacitating conditions, which is lower
than the [K1]i of 120 mM obtained with the same method
by Linares-Hernandez et al. [19] for noncapacitated human
cells.
The effect of P4 on membrane potential has been studied
to 10 mM final concentration, a dose that induces maximal
effects of this steroid on both human sperm Ca21 influx and
the acrosome reaction [2, 5]. In other studies on the action
of P4 on human sperm potential, the steroid used was between 3.14 mM and 20 mM [11–13]. In the present work,
the sperm membrane depolarization induced by P4 is immediate and transient, reaching a mean amplitude of ;115
mV, not far from the value (120 mV) found in the only
quantitative study in humans [11]. This depolarization concerns 3% to 30% of the cells, without widening the initial
range of potential. This observation is in disagreement with
a previous study [13] reporting that membrane depolarization concerned the majority of the sperm cells, and a larger
depolarized subpopulation was observed after treatment
with P4. Because previous studies have shown that P4-promoted Ca21 influx was essentially ubiquitous [28, 29]
whereas P4-induced membrane depolarization would concern, in our study, only a subpopulation, the involvement
of voltage operated-calcium channels (VOCCs) in P4-induced Ca21 entry is still a matter of debate. VOCCs might
be responsible for only a part of P4-promoted Ca21 influx,
as suggested recently by Rossato et al. [30].
It has been suggested that P4-initiated sperm membrane
depolarization is mainly due to a Na1 influx and not to a
Ca21 influx [12]. We have recently shown that P4 elicits a
sodium influx of about 10 mM in human spermatozoa, with
sodium concentration peaking in spermatozoa within 1 min.
Using the Nernst equation, we postulated that the [Na1]i
change induced by P4 would correspond to about 110 mV
of the depolarizing effect [8]. The quantitative data obtained in our study confirm that Na1 influx could actually
1780
PATRAT ET AL.
account for the majority of the depolarizing effect. However, our work provides evidence that the chloride efflux
induced by P4 could also play a minor role in membrane
depolarization. As the internal Cl2 concentration decreases
within a few seconds after P4 is added [9], Cl2 efflux could
be involved in an initial phase of depolarization. Consequently, we suggest that P4-promoted membrane depolarization is mainly driven by Na1 influx and, to a lesser extent, also by a Cl2 efflux.
After the P4-induced depolarization phase, a long repolarization phase was observed. The new resting membrane
potential reached after P4 exposure was usually more negative than that before P4 treatment. Our studies in flow cytometry show that this hyperpolarization would concern
4%–40% of sperm cells. The physiological relevance of
this hyperpolarizing effect is not demonstrated and remains
under speculation. However, already it has been shown that
a prior exposure of spermatozoa to P4 would enhance the
AR rate induced by ZP in mouse [5] and is increasing the
Ca21 influx in human sperm induced by mannose-BSA neoglycoprotein, an AR inducer used to replace the ZP [31].
Moreover, the mouse spermatozoa undergoing the AR in
response to the ZP must have a resting membrane potential
that maintains a closed but activation-competent state of Ttype calcium channels (i.e., #260 mV), allowing their activation by the ZP [27]. Therefore, we assume that P4-induced hyperpolarization could be the main mechanism by
which P4 exerts a sensitizing effect on the ability of the ZP
to induce the human AR. The physiological role of P4induced hyperpolarization would be to render a subpopulation of spermatozoa (4%–40% in our study) responsive
to ZP, by decreasing their membrane potential to below
260 mV. To confirm our hypothesis, it should be demonstrated that the sperm samples that hyperpolarize after P4
addition exhibit a higher number of acrosome-reacted spermatozoa induced by human ZP compared with the same
spermatozoa exposed to ZP alone.
ACKNOWLEDGMENT
We thank Christophe Arnoult for fruitful and critical discussions.
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