Molecular Human Reproduction vol.2 no.12 pp. 903-909, 1996
Involvement of osmo-sensitive calcium influx in human sperm
activation
M.Rossato1, F.Di Virgilio2 and C.Foresta13
1
Patologia Medica III, University of Padova, Via Ospedale Civile 105, 35128 Padova, institute of General Pathology,
University of Ferrara, Via Borsari 46, 44100 Ferrara, Italy
^ o whom correspondence should be addressed
Mammalian spermatozoa must undergo capacrtation and acrosome reaction before fertilization. To date, the
precise mechanisms regulating these complex processes are not well understood but it is generally agreed
that they involve an influx of calcium from the extracellular space through, as yet, poorly characterized
plasma membrane pathways. Here we present evidence for a novel mechanism to increase intracellular
calcium concentration via a calcium influx pathway activated by sperm cell swelling. Activation of this influx
pathway by a mild hypo-osmotic shock and the ensuing calcium rise are a potent stimulus for sperm acrosome
reaction. Furthermore, hypo-osmolartty-activated spermatozoa are fully competent for oocyte fertilization.
During transit along male and, after ejaculation, female genital tracts spermatozoa are known to be exposed
to extracellular fluids of widely different osmolarity; thus osmo-sensitive calcium influx could have a crucial
regulatory role in the cellular events preceding fertilization.
Key words: acrosome reaction/calcium/human/spermatozoa/stretch-activated channels
Introduction
Mammalian spermatozoa, during their transit along the male
genital tract, are exposed to an extracellular environment of
high osmolarity (350-400 mOsm/1) (Polak and Daunter, 1984;
Setchell and Brooks, 1988). On the contrary, in the female
genital tract, spermatozoa come in contact with fluids and
secretions whose osmolarity is close to that of plasma (280300 mOsm/1) (Fisch et ai, 1990).
Ample experimental evidence has shown that mechanical
or osmotic stimulation is a potent trigger for numerous cell
responses including mRNA and protein synthesis (Haneda
et ai, 1989; Komuro et ai, 1990), or peptide and steroid
hormone secretion (Page et ai, 1990; Sato et ai, 1990). The
transduction process is not fully understood but it is generally
thought that stimulation is mediated by plasma membrane ion
channels sensitive to stretching after cell swelling. Such
channels are highly conserved and ubiquitous in plant and
animal cells, where they have been implicated in cell volume
and osmotic pressure regulation, morphogenesis, neuronal
growth and other cellular functions (Morris, 1990).
In the present report we show that human spermatozoa
possess Ca 2+ influx pathways activated by plasma membrane
stretching that stimulate the acrosome reaction and might be
involved in fertilization.
Materials and methods
Materials
Fura-2/AM and ix's-oxonol were purchased from Molecular Probes
(Eugene, OR, USA). Gadolinium, nifedipine, gramicidin D,
[ethylenebis (oxyethylenenitrite)] tetracetic acid (EGTA) choline
© European Society for Human Reproduction and Embryology
chloride, methylglucamine and sucrose were from Sigma (St.
Louis, MO, USA). Omega-conotoxin and ionomycin were from
Calbiochem (La Jolla, CA, USA). Verapamil was from Knoll AG
(Liestal, Switzerland). Deionized distilled water was purchased from
Monico (Venezia, Italy). Fura-2/AM and fe-oxonol were suspended
in dimethylsulphoxide (DMSO) in 1 mM and 200 |iM stock solutions
respectively. Gadolinium, omega-conotoxin, nifedipine and verapamil
were suspended in Biggers-Whitten-Whittingham (BWW) medium.
Sperm collection
Sperm samples were obtained from 20 healthy fertile sperm donors
(aged 22-30 years) after 3 days of sexual abstinence. Semen samples
were allowed to liquefy at room temperature for 30 min; then
they were analysed for semen volume, pH, sperm concentration,
morphology, motility and viability (as measured by the Eosin Red
exclusion test). Samples with motility >60% and viability >90%
were utilized. All experiments were performed using spermatozoa
isolated by the swim-up technique as previously described (Foresta
et ai, 1992). Spermatozoa isolated by this method were collected
and centrifuged for 10 min at 800 g. The final pellets were resuspended
in BWW medium containing (in mM): 95 NaCl, 4.8 KC1, 1.2 MgSO4,
1.2 KH2PO4, 5.6 fructose, 25 NaHCO3, 1.7 CaCl2 0.25 sodium
pyruvate, 20 HEPES (pH 7.4, 37°C), 3.7 ml sodium lactate syrup
(60%), 104 IU/ml penicillin, 10 mg/ml streptomycin. Sperm concentration was adjusted to 15X106/ml. Osmolarity of BWW medium was
300 ± 2 mOsm/1 in all cases. In parallel experiments, spermatozoa
were isolated and incubated with modified BWW medium containing
135 mM NaCl to reach a final osmolarity of 380 ± 2 mOsm/1.
In some experiments extracellular sodium (Na + ) was isotonically
replaced with choline chloride, methylglucamine or sucrose. Since
results of experiments with these Na+-replaced media were similar,
only those obtained with sucrose-medium are shown in Figure 5.
Measurement of intracellular calcium concentration
[Ca2+]j was measured using the fluorescent probe fura-2/AM (Foresta
et ai, 1992): spermatozoa isolated as above were incubated in BWW
903
M.Rossato, F.Di Virgilo and C.Foresta
10
Table I. Effects of hypo-osroolar shock on sperm motility
T
8"
Osmolarity
(mOsm/1)
Motility (%)
+Ca
4"
300
270
240
210
180
150
2"
60
0
2
87.4 ±
86.5 it
87.7 it
86.8 it
85.0 ii
86.1 1t
-Ca
2.9
2.8
1.9
2.8
2.0
3.1
+Ca 2 +
2+
87.8 :t
86.1 ;t
86.3 it
84.2 2t
84.8 it
84.4 it
2.5
2.6
2.5
3.1
2.9
3 1
85.8 ±
80 1 ±
83.7 ±
74.5 ±
37.1 ±
19.3 ±
-Ca 2 +
2.5
2.5
3.2
3.6
3.9
4.3
68.9
67.5
69.1
70.1
40.0
14.1
±
±
±
±
±
±
3.4
2.2
2.8
4.1
3.9
3.1
10 20 30 40 50
Osmolarity reduction (%)
Table II. Effects of hypo-osmolar shock on sperm viability
Figure 1. Effects of hypotonic shock on fura-2 leakage from
human spermatozoa. Osmolarity reduction was obtained by adding
different volumes of distilled water, controls being reacted with an
equivalent volume of isotonic sucrose solution to preserve isoosmolarity. Sperm samples were then centrifuged and the
supernatant fluorescence measured. Left-hand bars of each pair =
controls; right-hand bars = distilled water. Fluorescence is
expressed as arbitrary units (AU). Data represent the results
obtained in a typical experiment out of three similar experiments.
for 30 min at 37°C in the presence of fura-2/AM (2 u.M). After
loading, spermatozoa were washed free of extracellular fura-2/AM
by centrifugation at 800 g for 10 min, resuspended in BWW and
maintained at room temperature until used. [Ca 2+ ]j was measured in
an LS50B Perkin Elmer fluorometer equipped with a thermostatically
maintained and magnetically-stirred cuvette holder and using 1.0 ml
sperm aliquots. Addition of distilled water caused dilution of the
sperm suspension and consequently a small drop in fluorescence.
This possible source of artefact was accounted for by measuring the
fluorescence decrease caused by addition of equal volumes of isotonic
sucrose solution. The excitation wavelength was alternated between
340 and 380 nm and emission fluorescence was continuously
monitored at 505 nm.
In experiments evaluating the involvement of voltage-operatedCa 2 + channels in hypo-osmolarity-induced Ca 2 + influx, fura-2-loaded
sperm samples were incubated for 15 min in the presence of nifedipine
(1.0 (iM), verapamil (10 (iM) or omega-conotoxin (100 nM) before
osmolarity reduction. In control experiments aliquots of spermatozoa
isolated as described above, were loaded with fura-2/AM (2 (iM) at
37"C for 30 min. After loading, spermatozoa were washed and
resuspended in BWW. Separate samples of this suspension were
treated by adding different volumes of distilled water to decrease the
final osmolarity by 10, 20, 30, 40 and 50%. In parallel experiments,
sperm samples were treated with equal volumes of isotonic (300
mOsm/1) sucrose solution to maintain an unchanged final osmolarity
(controls). After incubation for 15 min sperm samples were centrifuged
and supematants were analysed for fluorescence that was compared
with that of the respective controls. No differences were observed
between fluorescence of supematants from the treated samples and
controls demonstrating that hypotonic shock did not induce fura-2
leakage from spermatozoa (Figure 1).
Evaluation of sperm plasma membrane potential changes
Sperm plasma membrane changes were monitored using the potential
sensitive fluorescent dye fcis-oxonol as previously described (Foresta
et aL, 1992). Briefly, 1.5X106 spermatozoa were placed in a cuvette
thermostatically maintained at 37°C containing the bis-oxono\ solution
(200 nM) in BWW. After stabilizing the fluorescent signal, distilled
904
Osmolarity
Viability (%)
(musm/lj
60
0
+Ca +
2
300
270
240
210
180
150
93.3
92.4
93.9
93.7
94.4
93.3
:t
:t
:t
:t
:t
±
-Ca
2.9
1.8
2.9
2.8
3.0
2.1
+Ca 2 +
2+
95.9
96.5
96.5
94.3
94.9
94.7
±
±
±
±
±
±
3.5
2.9
3.0
2.9
2.6
2.3
93.8
94.8
95.5
94 8
90.8
90.9
±
±
±
±
±
±
-Ca 2 +
3.9
2.5
3.4
3.2
3.9
3.2
95.1
92.4
91.3
90.2
90 1
90.9
±
±
±
±
±
±
3.0
3.2
3.8
3.1
3.6
3.1
water or isotonic sucrose solution were added. Excitation and emission
wavelengths were 540 and 580 nm respectively. As in the case of
[Ca 2+ ], measurement (see above), addition of distilled water caused
dilution of the sperm suspension and consequently a reduction in
fluorescence. This dilution artefact can be corrected by measuring
the dilution response to addition of equal volumes of isotonic
sucrose solution.
Evaluation of sperm motility and viability
Sperm motility of each sample was assessed by light microscopy at
the beginning and end of each experiment. Sperm motility was
unaffected by medium osmolarity reduction of up to 30% as evaluated
by light microscopy. A greater degree of hypo-osmolarity produced
a significant loss of motility (Table I). Thus, a 20-30% dilution was
chosen as the optimal treatment in most experiments. Sperm viability
was determined by Red Eosin exclusion test and was greater than
90% at the end of all experiments (Table II).
Evaluation of acrosome reaction
Spermatozoa, isolated as above, were resuspended at a final concentration of lSxlOfyml. After medium dilution, sperm samples were
incubated for 60 min in a humidified atmosphere containing 95% O2
and 5% CO2, at 37°C. After 0, 30 and 60 min of incubation, aliquots
of sperm samples were evaluated for motility, viability and acrosome
reaction. Acrosome reaction was determined by the triple-stain
technique as previously described (Foresta et ai, 1992).
Evaluation of fertilization potential
Zona-free hamster oocytes, isolated as previously described (Foresta
et al., 1992), were incubated in a humidified atmosphere (95% O2
and 5% CO2) in 35 mm culture dishes for 3 h with sperm suspensions
pre-incubated for 30 min in hypo-osmolar medium (260 or
210 mOsm). Spermatozoa pre-incubated for 30 min in BWW diluted
with equal volumes of 300 mOsm/1 sucrose solution were used as
controls. At the end of sperm-oocyte incubation, penetration rates
Osmosensitive calcium channels in human spermatozoa
nM
250 1
200
160
200
120
150 •
SO
40
100
50
0J
0
1 min
10
20
30
40
SO
60
% OSMOLARITY REDUCTION
Figure 2. (A) Kinetics of sperm intracellular concentration [Ca 2+ ]; increase triggered by reduction of medium osmolarity. Arrow indicates
point of sperm medium (1.0 ml) dilution with either distilled water (0.11 ml to reduce osmolarity by 10% , trace b; 0.25 ml to reduce
osmolarity of 20%, trace c; 0.428 ml to reduce osmolarity of 30%, trace d; 1.0 ml to reduce osmolarity of 50%, trace e) or an isotonic
(300 mOsm/1) sucrose solution (to preserve iso-osmolarity, trace a). (B) Dose-response relationship between peak [Ca 2+ ], rise and extent of
medium osmolarity reduction (0-50%). Peak [Ca 2+ ], increases above basal level were plotted against percentage of osmolarity reduction.
Means of replicated determinations (at least 10) from different donors are shown.
were assessed by counting oocytes showing a swollen sperm head
within the cytoplasm. At least 50 oocytes in each sample were
counted. Results are expressed as percentage average of penetrated
eggs from triplicate determinations.
Measurement of osmolarity
Medium osmolarity was measured with an automatic Osmometer
(Advanced DigiMate Osmometer, Advanced Instruments, Needham
Heighs, MA, USA) with a range of 0-2000 mOsm and a sensitivity
of 1 mOsm.
Data analysis
Statistical analysis was performed using the StatView II (Abacus
Concepts, Berkeley, CA, USA) statistical package. Statistical analysis
was carried out using analysis of variance and Student's r-test.
P <0.05 was determined as the limit for statistical significance.
nM
200150
EGTA
100
50
H2O
1 mill
Results
Figure 3. Effect of extracellular Ca2+ chelation on intracellular
concentration [Ca2+]; changes triggered by hypo-osmotic shock.
Spermatozoa were suspended in Ca2+-free medium. EGTA 0.1 mM
was added shortly before medium dilution. Where indicated,
distilled water (H2O) was added to dilute osmolarity by 30%.
Finally 1.8 mM Ca2+ was added.
Figure 2A shows that hypo-osmolarity caused a long-lasting
rise in [Ca2+]j in fura-2-loaded spermatozoa. Both the extent
and the kinetics were dependent on the degree of medium
osmolarity reduction: a 10% dilution with distilled water
caused a rather small and slow [Ca2+]j increase, while a
more drastic decrease in osmolarity (20-30%) triggered a
much larger and faster rise. A similar, albeit smaller, Ca 2+
rise was also triggered by shifting spermatozoa from a
hyper- (3?0 mOsm) to an iso-osmotic (300 mOsm/1) solution
(not shown). In parallel experiments the incubation medium
was diluted with a volume of iso-osmotic sucrose solution
equal to the volume of distilled water added to achieve a
30% decrease in osmolarity. As shown in Figure 2A (trace
a), addition of iso-osmotic sucrose solution did not trigger
a rise in [Ca2+]i. Figure 2B shows the relationship between
the increase in [Ca2+]j and decrease in medium osmolarity.
Figure 3 shows the effects of hypo-osmolar shock on [Ca2+]j
in spermatozoa suspended in Ca2+-free medium (to which
0.1 mM EGTA was added prior to medium dilution). Hypoosmolar shock performed in these experimental conditions
did not induce any modification in [Ca2+]j. However, when
Ca 2+ was added back to the medium, a rapid rise in [Ca 2+ ],
occurred, resembling that observed when Ca 2+ was present
in the medium from the beginning of the experiment.
Similar results were obtained when Ca 2+ was added up to
15 min after medium dilution (not shown). Pre-incubation
of spermatozoa in the presence of voltage-operated Ca 2+
channel (VOC) blockers, nifedipine, verapamil or omegaconotoxin, did not influence the rise in [Ca2+]j induced by
reduction of osmolarity (Figure 4). These results were
confirmed by experiments performed in Na+-free media (in
which Na+-dependent plasma membrane depolarization and
VOC activation does not occur). As shown in Figure 5,
omission of Na + from the extracellular medium did not
inhibit the hypo-osmolarity-induced rise in [Ca 2+ ] L Figure 6
905
M.Rossato, F.Di Virgilo and C.Forests
120n
Table i n . Effect of hypo-osmolar shock on hamster egg fertilization
Fertilization rate (%)
Control (300 mOsm)
20% dilution (240 mOsm)
30% dilution (210 mOsm)
Control
Nif
Ver
Omega
Figure 4. Effects of sperm pre-incubation with voltage-operated
Ca2"1" channel blockers on hypo-osmolarity induced [Ca2+], rise.
Aliquots of spermatozoa were incubated for 15 min with nifedipine
(Nif, 1.0 U.M), verapamil (Ver, 10 uM) or omega-conotoxin
(omega, 100 nM) before addition of distilled water to reduce
medium osmolarity by 30%. In control experiments sperm samples
were treated with equal amounts of an isotonic (300 mOsm/1)
sucrose solution. Results are mean ± SD of three different
experiments.
shows the effects of Gd 3+ , a trivalent lanthanide shown to
block mechano-sensitive channels (Yang and Sachs, 1989)
on the rise in [Ca 2+ ]| induced by hypo-osmolar shock:
medium dilution in the presence of Gd 3+ blocked the [Ca 2+ ],
increase in a dose-dependent manner.
Glycerol is known to induce cell swelling as it is taken
up and concentrated intracellularly (Wong and Chase, 1986).
Figure 7, trace a, shows that glycerol addition to the sperm
suspension triggered a slow increase in [Ca 2+ ],. The effects
of glycerol were dependent on Ca 2+ influx from extracellular
medium since, in the absence of extracellular Ca 2+ , glycerol
was without effect on sperm [Ca 2+ ],. When Ca 2+ was added
back a few minutes after the addition of glycerol addition
in the absence of extracellular Ca 2+ , a fast rise in [Ca 2+ ]|
was observed, similar to that seen following medium dilution.
Figure 8 shows the results of an experiment in which
0
69 ± 4
76 ± 5
the sperm plasma membrane was depolarized by substitution
of Na + by K + (95 mm) in the external medium. After
medium dilution, the influx of Ca 2+ was significantly
slowed down.
Figure 9 shows the percentages of acrosome reacted
spermatozoa before and after medium osmolarity reduction.
Hypo-osmolarity was able to trigger the sperm acrosome
reaction; this effect was completely inhibited by the presence
of Gd 3+ . Finally, the fertilization potential of hypotonicallyshocked spermatozoa was assessed using zona-free hamster
oocytes. As reported in Table III, spermatozoa exposed to
a 20 and 30% decrease in osmolarity had a fertilization
rate of 69 ± 4 and 76 ± 5% respectively.
Discussion
Stretch-activated ion fluxes have been investigated by measuring membrane conductance changes in cells exposed to treatments that induce the plasma membrane to stretch. These
treatments include exposing cells to a hypo-osmotic shock
(Falke and Misler, 1989); prodding the plasma membrane with
a glass pipette (Morris and Horn, 1991); and applying suction
through a patch pipette (Gustin et ai, 1988).
In human spermatozoa, transmembrane ion fluxes cannot be
easily studied using electrophysiological techniques because it
is very difficult to patch cells as small and motile as living
spermatozoa. Therefore we used the fluorescent indicator fura2 to monitor Ca 2+ fluxes caused by hypo-osmotic shock. The
results of the present study demonstrate that a reduction in
[Ca 2+ ], nM
250 n
200
150
100
50
1 min
Figure 5. Kinetics of [Ca 2+ ]j rise induced by hypo-osmolarity in sperm suspended in Na+-containing (trace a) and Na+-free medium (trace
b), in which Na + was isotonically replaced by sucrose. Where indicated, distilled water to reduce osmolarity by 30% was added.
Replacement of Na + by choline chloride or methylglucamine gave similar results.
906
Osmosensrtive calcium channels in human spermatozoa
|Ca2*]i nM
nM
250
250
200 -
200
150
150
100 -
100
50
50
1mln
1 mln
Figure 6. Effect of Gd 3+ on [Ca2+]i changes induced by hypoosmolarity. Where indicated, distilled water (H2O) to reduce
osmolarity by 30% was added in the absence (trace a) or presence
of 0.1 mM (trace b), 0.25 mM (trace c), 0.5 mM (trace d) and 1.0
mM (trace e) Gd 3+ (added at least 5 min before medium dilution).
Figure 8. Kinetics of [Ca2+]j rise induced by reduction of medium
osmolarity (30%) in spermatozoa suspended in BWW medium
(trace a) and in high-K+ medium, where the NaCl in the BWW
medium was replaced by KC1 (95 mM, trace b). The arrow
indicates the point of medium dilution with distilled water.
50
[Caw], nM
O
250
40 •
30
200 -
i
O
150 -
u
20
10
100 0
30
60
Minutes of incubation
50
Glycerol
1mln
Figure 7. Effect of glycerol (addition indicated by an arrow) on
[Ca2+], in spermatozoa suspended in Ca2+-containing medium
(trace a) or Ca2+-free medium (to which 0.1 mM EGTA had been
added, trace b). The addition of Ca2+ (1.8 mM) to trace b is shown
by an arrow.
medium osmolarity caused a rapid rise in sperm [Ca2+]j. This
was shown to be dependent on an influx from the extracellular
space since, in the absence of Ca 2+ in the extracellular medium,
hypo-osmolar shock had no effect on sperm [Ca2+]j. The
subsequent rapid rise in [Ca2+]j following restoration of Ca 2+
to the extracellular medium up to 15 min after its removal,
demonstrates that the channels do not inactivate over several
min. The curve describing the relationship between the increase
in [Ca2+]i and the decrease in medium osmolarity had a double
sigmoidal shape (Figure 2B) suggesting that two different
mechanisms may be responsible for the rise in [Ca2+]j operating
at low-intermediate (10-40%) and high (>40%) osmolarity
reduction. It is likely that a large reduction in osmolarity also
caused some plasma membrane damage and resultant nonspecific leakage to Ca 2+ , as suggested by the decrease in sperm
motility which followed reductions in osmolarity of >30%.
Hypo-osmolarity-induced Ca 2+ influx may be due to the
Figure 9. Dose-response and time-course of hypotonicity-induced
acrosome reaction. Medium osmolarity was decreased by 10%
(A), 20% (O), 30% ( • ) , and 50% (A) by adding at time 0
prewarmed (37°C) distilled water. In control experiments an
amount of an isotonic sucrose solution equivalent to that needed to
reduce osmolarity by 30% was added (•)• Parallel samples were
exposed to hypoosmotic shock (30% dilution) in the presence
of 1.0 mM Gd 3+ ( • ) . Data are means of triplicate determinations
from different sperm donors, a = P <0.01 and b = P <0.001 in
comparison with both the control and Gd 3+ treated samples.
opening of voltage-operated Ca 2+ channels (VOCs) as a
consequence of stretch-induced plasma membrane depolarization due to Na + influx. We think that this hypothesis is unlikely
for the following reasons: (i) iso-osmotic replacement of
extracellular Na + with choline, methylglucamine or sucrose
did not abolish, and in fact increased, the rise in [Ca 2+ ]|; (ii)
specific blockers of L- and N-VOCs (nifedipine, 1 |iM;
verapamil, 10 |iM; omega-conotoxin, 0.1 |iM) did not block
the [Ca2+]j rise; and (iii) Gd 3+ , a trivalent lanthanide shown
to block mechano-sensitive channels (Yang and Sachs, 1989),
also blocked the rise in [Ca2+]j in a dose-dependent manner.
Since osmolarity was decreased by diluting the incubation
medium with distilled water, it cannot be excluded that sperm
activation was due to dilution of medium components rather
than decrease of osmolarity. To rule out this possibility, a
907
M.Rossato, F.Di Virgilo and C.Foresta
series of parallel experiments were performed in which the
incubation medium was diluted by adding a volume of isoosmotic sucrose solution equivalent to the volume of distilled
water added to achieve the correspondent reduction in
osmolarity. Under these experimental conditions, the addition
of iso-osmotic sucrose solution did not trigger a rise in [Ca2+]j,
ruling out the involvement of medium components dilution in
the effects induced by hypo-osmotic shock.
It is known that glycerol is able to permeate cell plasma
membrane causing cell swelling (Wong and Chase, 1986). Our
results demonstrate that this agent is able to induce an increase
in [Ca 2+ ], at a slow kinetic rate that is probably a reflection
of the slow kinetics of glycerol uptake, since the rise in [Ca2+]j
is much faster when glycerol is administered in the absence
of extracellular Ca 2+ and this ion is added a few minutes later.
In both cases the increase in [Ca2+]j reached the same plateau.
Hypo-osmolarity-induced Ca 2+ influx was significantly
retarded by previous plasma membrane depolarization, an
indication that the ion flux is electrogenic (Di Virgilio et al,
1987; Penner et al, 1988). Not surprisingly, steady state
[Ca2+]j concentrations were very similar in KCl-depolarized
and control spermatozoa, since addition of distilled water
also caused a slow depolarization that reached a plateau at
approximately the same concentration in both experiments
(data not shown). In human spermatozoa, Ca 2+ influx across
the plasma membrane is thought to be the first and crucial
step in the chain of events leading to the acrosome reaction
(Yanagimachi and Usui, 1974; Yanagimachi, 1988; Kopf and
Gerton, 1990). Therefore it can be anticipated that a hypoosmolar shock may also be a trigger for this event.
The data reported in the present study show that Ca 2+ influx
induced by hypo-osmolar shock is a potent stimulus for the
acrosome reaction that closely correlates with the induction of
the capacity for fertilization, as demonstrated by the zona-free
hamster oocytes penetration assay. Furthermore Gd 3+ , a well
known blocker of mechano-sensitive Ca 2+ channels (Yang and
Sachs, 1989), completely inhibited the acrosome reaction, thus
confirming the pivotal role of Ca 2+ influx in hypo-osmolarityinduced sperm activation.
Mechanical stimulation has been shown to activate ion
channels in many different cell types (Haneda et al, 1989;
Page et al, 1990; Sato et al, 1990; Filipovic and Sackin,
1991; Komuro et al, 1991). The data shown in this paper
provide the first evidence to our knowledge for the presence
of swelling-activated Ca 2+ influx and its possible physiological
role in human spermatozoa. Although we cannot formally
prove that Ca 2+ influx occurred via Ca 2+ channels, this
suggestion is supported by the fast kinetics of activation, the
sensitivity to membrane depolarization and the blockade by
Gd 3+ . Although the physiological role of swelling-activated
channels is not fully understood, they may be involved in the
regulation of important sperm functions. During their transit
along the male genital tract (seminiferous tubules, epididymis,
vas deferens), spermatozoa are exposed to environments of
different chemical composition but with continuous high osmolarity (>350 mOsm) (Setchell and Brooks, 1988). The osmolarity of human seminal plasma has been reported to be >380
mOsm/1 (Polak and Daunter, 1984; and personal unpublished
908
observations). It is believed that in these regions, spermatozoa
may remain quiescent in order to prevent activation prior to
their ejaculation into the female genital tract, the secretions of
which have an osmolarity similar to that of the plasma (Fisch
et al, 1990). These observations are in agreement with our
previous results which indicated that cervical mucus retrieved
during the ovulatory period of fertile healthy women had an
osmolarity of 286.8 ± 30.7 mOsm/1 (personal unpublished
observations). Furthermore, during their migration through
cervical mucus crystals in the female genital tract sperms are
able to direct their trajectories, swimming in linear paths in
the direction of mucus alignment (Overstreet and Davis, 1991).
Under these conditions it can be postulated that swimming
against mucus crystals may mechanically stimulate plasma
membrane Ca 2+ channels, thus regulating sperm progression
and eventually leading to activation.
Krausz et al. (1995) have recently proposed the ability of
spermatozoa to respond to progesterone by a rise in [Ca 2+ ],
as a prognostic test for couples undergoing in-vitro fertilization
(TVF). In agreement with these authors we suggest that the
ability of spermatozoa to undergo a rise in [Ca2+], and
acrosome reaction after exposure to hypo-osmolar medium
could be a simple, non-toxic tool to verify their fertilizing
ability. Equally, it has been long known that the results of the
hypo-osmotic swelling test are highly correlated with human
fertilization and pregnancy rates (Van der Ven et al, 1986;
Check et al, 1989).
The observations reported in the present paper make it
tempting to speculate that specialized 'mechanotransducers'
located on human sperm plasma membrane are able to convert
mechanical stimuli into biological signals (stretch into Ca 2+
influx) which in turn modulate crucial events in fertilization
including motility and the acrosome reaction. Finally, the
demonstration that hypo-osmolarity-activated spermatozoa
exhibit a rate of fertilization suggests a simple, rapid and nontoxic means to activate spermatozoa during different procedures
of assisted reproduction, and suggests that the osmolarity of
media used for sperm washing, isolation and fertilization
should be carefully checked.
Acknowledgements
This work was in part supported by grants from the Ministry of
Scientific Research, the National Research Council of Italy, the Italian
Association for Cancer Research (AIRC) and Telethon of Italy.
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Received on May 13, 1996; accepted on November 7, 1996
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