Human Reproduction Vol.21, No.3 pp. 745–752, 2006
doi:10.1093/humrep/dei364
Advance Access publication October 27, 2005.
Hyper-osmotic condition enhances protein tyrosine
phosphorylation and zona pellucida binding capacity of
human sperm
D.Y.Liu1,5, G.N.Clarke3 and H.W.G.Baker1,2,4
1
Department of Obstetrics and Gynecology, University of Melbourne, 2Reproductive Services and 3Andrology Laboratory, Royal
Women’s Hospital and 4Melbourne IVF, Melbourne, Australia
5
To whom correspondence should be addressed. E-mail: [email protected]
Key words: capacitation/osmolality/sperm–zona pellucida interaction/tyrosine phosphorylation
Introduction
Human seminal plasma has high osmolality (300 to ∼400
mOsm/kg) compared with human serum or female reproductive
tract fluid (280–300 mOsm/kg; Makler et al., 1981; Polak and
Daunter, 1984; Rossato et al., 2002). A recent report by Cooper
et al. (2005) showed that osmolality increases during 30 min
incubation after ejaculation. Earlier studies had shown that seminal osmolality continues to increase for up to 6 h post-ejaculation
due to enzymatic breakdown of proteins, peptides and choline
compounds (Hofmann and Karlas, 1973). Another study
reported a negative relationship between seminal osmolality and
sperm progressive motility (Rossato et al., 2002). Therefore,
during the process of human fertilization either in vivo or in
vitro, sperm will encounter significant variations in osmotic conditions. Such marked changes in osmolality may have some
impact on plasma membranes which may affect sperm fertilizing
ability (Bielfeld et al., 1993; Rossato et al., 1996).
Previous research has clearly shown that osmolality has a
significant impact on sperm motility, capacitation, calcium
influx and the acrosome reaction (AR) in vitro (Mahi and Yanagimachi, 1973; Aitken et al., 1983; Rossato et al., 1996).
Treatment of sperm with medium of relatively low osmolality
induces plasma membrane swelling which leads to stimulation
of calcium influx and the AR of human sperm in vitro (Rossato
et al., 1996). On the other hand, hyper-osmotic conditions significantly inhibit both spontaneous and calcium ionophoreinduced AR (Bielfeld et al., 1993). It is also reported that
hyper-osmolality inhibits cortical granule exocytosis in seaurchin oocytes (Merkle and Chandler, 1989).
In standard IVF procedures, it was reported that hyperosmotic medium (345 mOsm/kg) significantly reduced human
sperm fertilizing ability compared with medium with osmolality of either 285 mOsm/kg or 315 mOsm/kg (Dumoulin et al.,
1990). However, with ICSI procedures, a recent study showed
that mouse sperm preserved in a medium with very high osmolality (800 mOsm/kg) at 4°C were able to produce normal fertilization and embryonic development, and live birth of healthy
offspring after ICSI (Van Thuan, 2005).
© The Author 2005. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved.
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BACKGROUND: The aim of this study was to determine the effect of culture medium osmolality, in the range known
to occur in the male and female reproductive tracts, on human sperm tyrosine phosphorylation and sperm–zona pellucida (ZP) interaction in vitro. METHODS: Motile sperm (2⫻106), selected by swim-up from semen of normozoospermic men with normal sperm–ZP binding, were incubated with or without four oocytes in 1 ml human tubal
fluid (HTF) medium with different osmolalities (150, 200, 280, 350, 400 mOsm/kg) adjusted by variation of the NaCl
concentration. After 2 h incubation, the number of sperm bound to the four ZP was examined, sperm motility and
velocities were assessed by Hamilton–Thorn Motility Analyzer (IVOS 10) and sperm tyrosine phosphorylation was
assessed by both western immunoblotting and immunofluorescence with an anti-phosphotyrosine monoclonal antibody (PY20). The effect of hyper-osmolality (400 mOsm/kg) on the ZP-induced acrosome reaction (AR) was also
determined. RESULTS: Incubation of human sperm in hyper-osmotic medium significantly increased tyrosine phosphorylation and the number of sperm bound to the ZP. In contrast, hypo-osmotic medium significantly decreased
both tyrosine phosphorylation and sperm–ZP binding. Medium with high osmolality (400 mOsm/kg) significantly
reduced the ZP-induced AR. Both hypo- and hyper-osmotic media significantly decreased average sperm percentage
progressive motility and velocities. CONCLUSION: Incubation of human sperm in hyper-osmotic media was associated with significantly increased tyrosine phosphorylation and ZP-binding ability but severely reduced the
ZP-induced AR.
D.Y.Liu, G.N.Clarke and H.W.G.Baker
Overall evidence suggests that osmotic conditions play an
important role in sperm fertilizing ability in vitro and simple
changes of osmolality of medium may significantly impact on
sperm function. One of the most important sperm functions is the
ability to interact with the zona pellucida (ZP): sperm must be able
to bind to the ZP, undergo the AR on the surface of ZP and penetrate the ZP before fertilization takes place (Yanagimachi, 1994).
A significant correlation between phosphorylation and binding to
the ZP in vitro has been found with human sperm (Sakkas et al.,
2003). The aim of this study was to determine the effect of culture
medium osmolality on human sperm protein tyrosine phosphorylation, sperm–ZP binding and the ZP-induced AR in vitro.
Materials and methods
Human oocytes
Oocytes which showed no evidence of two or three pronuclei or cleavage at 48–60 h after insemination or immature oocytes (with germinal
vesicles) in a clinical IVF programme were used for the ZP-induced
AR test. Oocytes with any remaining cumulus and corona cells or
sperm bound to the ZP from the IVF insemination were removed by
repeated aspiration of the oocyte using a fine glass pipette with an
inner diameter (120 μm) slightly smaller than the oocyte diameter
(Liu and Baker, 1994, 1996). Degenerate, activated or morphologically abnormal oocytes as well as oocytes with >10 sperm penetrating
the ZP were not used for the test. All the oocytes were obtained on day
3 of insemination and the oocytes were pooled from several patients
and used for the test on the same day or kept in the 5% CO2 in air
incubator and used within next 3 days.
Sperm samples and preparation
Semen samples were obtained by masturbation after 3–5 days abstinence from normozoospermic men who had sperm counts >20×106/ml,
progressive motility >30% and variable normal sperm morphology.
746
Sperm–ZP interaction test
Motile sperm (2×106 in 1 ml medium) selected by swim-up were incubated with four oocytes in media with different osmolalities in 4-well
culture plates (Nunc, Rosilde, Denmark) for 2 h at 37°C in 5% CO2 in
air. After incubation, each group of four oocytes was transferred to
phosphate-buffered saline (PBS) containing 2 mg/ml bovine serum
albumin (BSA; Commonwealth Serum Laboratory, Parkville, Victoria, Australia). After incubation the oocytes were flushed several
times to dislodge loosely adherent sperm using a pipette approximately twice the diameter of the oocyte (250 μm) in three separate
wells containing 0.5 ml PBS with 0.2% BSA. The sperm which were
tightly bound to all the four ZP/test were removed by repeated aspiration using a narrow-bore pipette with diameter slightly smaller than
the oocyte (120 μm) in a small volume (<5 μl) of PBS on a slide, then
smeared in a limited area (∼4×4 mm) which was marked with a diamond pen to help find the sperm under the microscope for counting
and the AR assessment (Liu and Baker, 1996; Liu et al., 2001).
Because all semen samples were from normozoospermic men with
normal sperm–ZP binding, there were >100 sperm bound/ZP. Therefore, removing all tightly bound sperm on a slide would allow accurate counting of the number of sperm bound to the ZP as described
previously (Liu et al., 2003). The total number of sperm bound to all
the four ZP per test was used for statistical analysis.
Assessment of acrosome status of sperm bound to the ZP
The sperm bound to the ZP were removed and smeared on a glass slide
as described above. The AR of ZP-bound sperm was assessed using
PSA–FITC as described previously (Cross et al., 1986; Liu and Baker,
1996). Sperm smears were fixed in 95% ethanol for 30 min after airdrying and then stained using 25 μg/ml PSA–FITC in PBS for 2 h at
4°C. The slides were washed and mounted with distilled water and 200
sperm per sample were counted with a fluorescence microscope using
excitation wavelengths of 450–490 nm and a magnification of ×400.
When more than half the head of a sperm was brightly and uniformly
fluorescing, the acrosome was considered intact. Sperm with a fluorescing band at the equatorial segment or without fluorescence (a rare
pattern) were considered acrosome-reacted.
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Chemicals and culture medium
Human tubal fluid (HTF) with osmolality adjusted by variation of
NaCl concentration was prepared with the following final values: 150,
200, 280, 350 and 400 mOsm/kg determined by freezing point depression using Advanced DigiMatic Osmometer Model 3D II (Advanced
Instrument Inc., Needham Heights, MA, USA). The final concentrations of NaCl in these different osmotic media (150, 200, 280, 350 and
400 mOsm/kg) were 12, 42, 101, 132 and 165 mmol/l respectively. In
some experiments, sucrose (115 mmol/l) was used instead of NaCl to
increase the osmolality of standard HTF medium up to 400 mOsm/kg.
Repeated measurements of osmolality for the same media by osmometer was within ± 1%. The pH of the medium was 7.4–7.6 and was
supplemented with 5% heat-inactivated human serum. Pisum sativum
agglutinin (PSA) conjugated with fluorescein isothiocyanate (FITC),
anti-mouse IgG-FITC and phosphotyrosine antibody inhibitor, a solution of o-phospho-L-tyrosine conjugated to BSA (specific inhibitor of
the reactivity of anti-phosphotyrosine antibody) were purchased from
Sigma Chemical Company (St Louis, MO, USA). Human serum and
Anti-phosphotyrosine monoclonal antibody (PY20, mouse IgG)
labelled with FITC were obtained from ICN Pharmaceuticals Inc
(Costa Mesa, CA, USA). Polyvinylidene difluoride (PVDF) membrane was purchased from Pall Life Sciences (Australia). Horseradish
peroxidase (HRP)-conjugated goat anti-mouse IgG was obtained from
Zymed Laboratories (San Francisco, CA, USA). A chemiluminescence ECL kit was obtained from Amersham Biosciences UK Ltd,
(Little Chalfont, Buckinghamshire, UK).
Sperm count and motility were performed after liquefaction and
within 1 h of collection of semen, according to World Health Organization (1999) procedures. Sperm morphology was assessed on Shorrstained smears under oil immersion with magnification ×1000 and
bright-field illumination. Percentage normal sperm morphology was
assessed according to strict criteria (Kruger et al., 1988; World Health
Organization, 1999).
Motile sperm were selected by swim-up technique as follows: 0.3
ml of semen was carefully added to the bottom of a test tube (12×75
mm) containing 0.7 ml standard HTF (280 mOsm/kg) supplemented
with 5% heat-inactivated human serum. Care was taken to avoid bubbles and not disturb the interface between semen and the medium.
After incubation for 1 h, 0.5 ml of the top layer of the medium containing motile sperm was aspirated. The motile sperm suspension was
then centrifuged at 1000 g for 5 min, the supernatant removed and the
sperm pellet washed again with 1 ml fresh HTF by centrifugation at
1000 g for 5 min. The washed sperm pellet was resuspended with
serum-supplemented standard HTF to a motile sperm concentration of
40×106/ml for subsequent experiments. Only sperm samples with
>90% progressive motility after swim-up preparation were used for
the study. Viability of sperm was assessed by counting 200 sperm
using Eosin Y exclusion (World Health Organization, 1999).
All patients signed consent forms permitting use of their gametes (unfertilized oocytes and sperm samples) for research. The Royal Women’s
Hospital Research and Ethics Committees approved the project.
Osmolality and sperm–ZP binding
Assessment of sperm motility, velocity and movement
characteristics
After 2 h incubation of sperm in medium with various osmolality values, sperm motility, velocity (VSL, straight line velocity; VCL, curvilinear velocity) and other movement characteristics (LIN, linearity
and STR, straightness) were measured by Hamilton–Thorn Motility
Analyzer (IVOS 10, 60 Hz; Hamilton–Thorn Research, Danvers, MA,
USA). For this study, total motility or progressive motility was defined as
the percentage of sperm with VAP >7.5 or >25 μm/s respectively.
Because the sperm concentration was only 2×106/ml when the sperm
were incubated in culture medium, the sperm concentration was
adjusted to ∼20×106/ml by centrifugation at 800 g for 5 min and resuspended in 100 μl of the same medium. A sample of 5 μl was placed in
a Microcell (20 μm depth) for assessment of motility and velocities.
For each sperm sample, the average of six (five to seven) fields with a
total of >400 sperm was assessed.
Western immunoblotting
Western immuoblotting for tyrosine phosphorylation was performed
using protein extracts from 2×106 sperm incubated in media with various
Figure 1. Effect of iso-osmotic (280 mOsm/kg A and B) and hyper-osmotic (400 mOsm/kg C and D) conditions on tyrosine phosphorylation of
sperm from one subject determined with PY20-FITC.(A and C) Light microscopy showing all sperm. (B and D) Fluorescence microscopy showing bright fluorescence on two (B) and four (D) sperm tails.
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Immunofluorescent detection of tyrosine phosphorylation
in human sperm
Motile sperm (2×106/ml) selected by swim-up were incubated for 2 h
in HTF medium with various osmolalities in standard culture conditions. After incubation, sperm were washed with 10 ml normal saline
and the sperm pellet resuspended in ∼20 μl saline and 5 μl smeared
on a glass slide. After drying in air, the smear was fixed in 90%
ethanol for 30 min to permeabilize plasma membranes. Tyrosine
phosphorylation of sperm tails was assessed by direct immunofluorescence using the monoclonal antibody PY-20 (ICN Pharmaceuticals Inc., Costa Mesa, CA, USA) labelled with FITC. The antibody is
diluted 1:300 in PBS containing 10 mg/ml BSA. For each smear, 50
μl of diluted PY-20 is added to cover the smear (∼10×10 mm) which
was then incubated for 4 h in a humidified box at 37°C. After incubation, the slide was washed and mounted with distilled water and covered with a coverslip (22×22 mm). Sperm tails with positive binding
of PY-20 antibody had bright fluorescence and the percentage of
sperm with positive stain (tyrosine phosphorylated sperm) was determined by scoring 400 sperm for each sample using a combination of
light and fluorescent microscopy (Figure 1). Antibody specificity was
confirmed by control tests demonstrating that phosphotyrosine
anti-body inhibitor (a solution of o-phospho-L-tyrosine conjugated to
BSA, 10 μg/ml) could block the binding of PY20-FITC on the sperm tails.
In addition, mouse IgG-FITC used as an extra control for non-specific
binding did not show positive fluorescence on any of the sperm.
D.Y.Liu, G.N.Clarke and H.W.G.Baker
A
TM
Statistical analysis
The significance of the differences for the number of sperm bound to
the ZP, sperm motility and velocities, sperm tyrosine phosphorylation
between medium with various osmolalities were examined by nonparametric two-way analysis of variance (Friedman test) for all the
data and paired comparisons between any two osmolality results.
Results
As compared to sperm in iso-osmotic (280 mOsm/kg) medium,
sperm in both hypo- (150–200 mOsm/kg) and hyper- (350–400
mOsm/kg) osmotic media had significantly reduced motility,
velocities, movement characteristics: straightness and linearity,
and viability (Figure 2). Sperm viability, progressive motility
and velocities reduced more severely in media with lower osmolality (150 mOsm/kg) than media with higher osmolality (400
mOsm/kg). There were highly significant relationships between
osmolality of medium and both tyrosine phosphorylation of
100
PM
80
80
60
60
%
%
100
Effect of hyper-osmotic conditions on tyrosine phosphorylation and
ZP binding capacity of sperm in men with defective sperm–ZP
binding
To determine if hyper-osmotic conditions could enhance tyrosine
phosphorylation and ZP binding capacity of sperm from men with
defective sperm–ZP binding, tyrosine phosphorylation and sperm–ZP
binding tests were performed using both iso-osmotic (280 mOsm/k)
and hyper-osmotic (400 mOsm/k) media on sperm samples from six
men with defective sperm–ZP binding (normal: ≥40 sperm bound/
ZP). All these men had sperm counts >20×106/ml, progressive motility >30% and variable sperm morphology.
40
40
20
20
C
0
0
150
200
280
350
150
400
B
100
VCL
VSL
Live sperm ( %)
μm/second
100
80
60
40
20
200
280
350
400
Osmolality (mOsm/kg)
Osmolality (mOsm/kg)
120
STR
LIN
D
80
60
40
20
0
0
150
200
280
350
Osmolality (mOsm/kg)
400
150
200
280
350
400
Osmolality (mOsm/kg)
Figure 2. Effect of osmolality of culture medium on sperm motility: (A) total motility (TM) and progressive motility (PM), (B) straight line
velocity (VSL) and curvilinear velocity (VCL), (C) movement characteristics: linearity (LIN) and straightness (STR), and (D) viability. A bar
graph of means with error bars of SEM was obtained from sperm samples from 10 different men. Hyper- and hypo-osmotic media significantly
reduced sperm motility (A, all comparisons P < 0.05 except 350 and 400 mOsm/kg: not significant), velocities (B, all comparisons P < 0.005
except 350 and 400 mOsm/kg for VCL: not significant), movement characteristics (C, all comparisons P < 0.05 except 150 and 200 mOsm/kg,
and 350 and 400 mOsm/kg for STR: not significant) and viability (D, P < 0.01 only for 150 and 400 versus 280 mOsm/kg). Medium with 150
mOsm/kg had a more negative effect on sperm motility, velocity and viability than did medium with 400 mOsm/kg.
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osmolalities as described above using the method of Buffone et al.
(2005). After 2 h incubation, sperm were pelleted and washed twice
with PBS and then solubilized in 2% sodium dodecyl sulphate (SDS
in 0.375 mol/l Tris, pH 6.8, 10% sucrose) for 5 min with vigorous
mixing by vortex but without boiling and then centrifuged at 6000 g at
4°C for 5 min. The supernatants were recovered and stored at –20°C
until required. Solubilized sperm proteins were diluted (1:1) with Laemmli (1970) sample buffer and then heated at 100°C for 5 min in a
water bath before loading onto a 10% polyacrylamide gel. The proteins were separated by sodium dodecyl sulphate polyacrylamide gel
electrophoresis (SDS–PAGE) under denaturing conditions. For each
lane of the gel, the protein extract from the equivalent of 2×106 sperm
was applied.
After SDS–PAGE, the proteins were transferred onto the PVDF
membrane at a constant current of 50 mA for 90 min. To block nonspecific binding sites, the membrane was first incubated with 5%
skimmed milk powder dissolved in Tris-buffered saline (TBS, pH
7.6). After that, it was incubated for 2 h with the monoclonal antiphosphotyrosine antibody PY20 diluted 1:5000 in blocking solution.
After three 10 min washes with TBS, HRP-conjugated goat antimouse IgG diluted 1:5000 in blocking solution was added. Following
a further 2 h incubation, the membrane was washed for 1 h in 0.05%
Tween-20 in TBS, followed by a further three 10 min washes in TBS
alone. Reactive bands were detected by enhanced chemiluminescence
using the ECL kit.
To quantify changes in protein tyrosine phosphorylation of
sperm from four subjects incubated in media with different osmolalities, rectangular boxes were drawn around the major bands on
scanned digital images of ECL contact photographs of western
immunoblots and adjusted optical densities for each lane were
obtained.
Osmolality and sperm–ZP binding
Arbitrary densitometry units
5000
4000
3000
2000
1000
0
No sperm bound/4ZP
TP sperm (%)
60
A
50
40
30
20
3000
A
2400
1800
1200
600
150
0
200
280
350
Osmolality (mOsm/kg)
400
2400
B
40
30
20
10
200
280
350
400
Osmolality (mOsm/kg)
No sperm bound/4ZP
150
TP sperm (%)
400
0
10
50
200
280
350
Osmolality (mOsm/kg)
Figure 4. Relationship between osmolality of culture medium and
sperm tyrosine phosphorylation after 2 h incubation detected by western immunoblotting of extracts from 2×106 sperm per lane. There are
significant increases in tyrosine phosphorylation (assessed on the
major bands of ∼95–170 kDa) with increasing osmolality of medium.
Bar graphs represent means with error bars of SEM for sperm samples from four different men (all comparisons P < 0.05).
3600
70
150
B
1800
1200
600
0
150
0
150
200
280
350
Osmolality (mOsm/kg)
200
280
350
Osmolality (mOsm/kg)
400
400
Figure 3. Relationship between osmolality of culture medium and
sperm tyrosine phosphorylation (TP) after 2 h incubation detected by
immunofluorescence with PY20-FITC. (A) Line graph showing individual results, and (B) bar graph of means with error bars of SEM, for
11 sperm samples from different men (all comparisons P < 0.01).
Figure 5. Relationship between osmolality of culture medium and
the number of sperm bound/four zona pellucida (ZP) after 2 h incubation (all comparisons P = 0.005). (A) Line graph showing individual
results for eight sperm samples from different men. (B) Bar graph of
means with error bars of SEM. There were zero sperm bound/four ZP
with 150 mOsm/kg and an average of 37 sperm bound/four ZP with
200 Osm/kg.
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sperm detected by immunofluorescence (Figure 3) and western immunoblotting (Figure 4) and the number of sperm bound
to the ZP of human oocytes (Figure 5). There were three major
bands ∼100 kDa in the immunoblots that increased with
osmola-lity (Figure 4). Other faint bands were not measured.
When sperm and oocytes were incubated in hyper-osmotic
medium of 350 and 400 mOsm/kg, the number of sperm bound
to the ZP was 2–3-fold higher than with sperm in iso-osmotic
medium (Figure 5). Also, hyper-osmotic medium enhanced tyrosine phosphorylation of sperm 2-fold over the level observed on
sperm incubated in iso-osmotic medium (Figures 3 and 4).
Hypo-osmotic medium (150 and 200 mOsm/kg) severely
impaired sperm–ZP binding and no sperm bound to the ZP when
sperm and oocytes were incubated in medium with osmolality of
150 mOsm/kg. On the other hand, the ZP-induced AR was significantly lower in hyper-osmotic medium (400 mOsm/kg) than
in iso-osmotic medium (280 mOsm/kg, Figure 6).
To confirm that the enhancement of sperm–ZP binding and
tyrosine phosphorylation was due to high osmolality rather
than to high concentrations of NaCl, we also used sucrose to
adjust osmolality of standard HTF medium (280 mOsm/kg) up
to 400 mOsm/kg. Hyper-osmotic medium (400 mOsm/kg)
adjusted with either NaCl or sucrose gave similar enhancement
of both sperm–ZP binding and tyrosine phosphorylation when
compared with standard medium (280 mOsm/kg, Table I).
D.Y.Liu, G.N.Clarke and H.W.G.Baker
100
Table II. Comparison of sperm–zona pellucida (ZP) binding and the
percentage of tyrosine-phosphorylated sperm determined with PY20-FITC
after sperm were incubated with iso-osmotic (280 mOsm/kg) or hyperosmotic (400 mOsm/kg) media
ZP-induced AR (%)
80
60
Subjects
40
20
0
280
400
Osmolality (mOsm/kg)
Table I. Comparison of sperm–zona pellucida (ZP) binding and the
percentage of tyrosine phosphorylated sperm determined with PY20-FITC
after sperm from four men were incubated in iso-osmotic medium (standard
human tubal fluid with osmolality of 280 mOsm/kg) or hyper-osmotic (400
mOsm/kg) medium achieved by the addition of NaCl (Na) or sucrose (Su)
1
2
3
4
Mean
No sperm bound/four ZPa
Phosphorylated sperm (%)b
280
400-Na
400-Su
280
400-Na
400-Su
390
326
498
309
380
1521
1011
2154
1214
1475
1475
1076
2242
1159
1488
9
11
12
8
10
48
31
40
33
38
50
34
41
31
39
a
P < 0.05 only for 280 versus either 400-Na or 400-Su.
P < 0.05 only for 280 versus either 400-Na or 400-Su.
b
Interestingly, for sperm samples with defective sperm–ZP
binding, hyper-osmotic medium also significantly increased
tyrosine phosphorylation but did not enhance the number of
sperm bound to the ZP (Table II). In other words, hyperosmotic conditions promoted tyrosine phosphorylation of any
sperm samples but only enhanced sperm–ZP binding capacity
in those samples with normal sperm–ZP binding.
Discussion
The present study is apparently the first report that hyperosmotic conditions (350 and 400 mOsm/kg) are associated
with increased sperm tyrosine phosphorylation and binding to
the ZP. There was a 2-fold increase in the percentage of tyrosine-phosphorylated sperm with hyper-osmotic media over that
in standard (iso-osmotic) medium. Enhancement of sperm
tyrosine phosphorylation by hyper-osmotic conditions was
detected by both immunofluorescence and western immunoblotting. The number of sperm bound to the ZP tripled in hyperosmotic media. The promotion of both ZP binding capacity
and tyrosine phosphorylation of sperm by hyper-osmotic
conditions further supports previous findings that tyrosine
750
1
2
3
4
5
6
Mean
15
16
17
5
2
1
9.3
No sperm
bound/four ZPa
Phosphorylated
sperm (%)b
280
400
280
400
5
4
7
10
1
0
4.5
6
3
8
10
2
0
4.8
7
3
2
5
3
4
4.0
44
51
38
30
34
37
39.0
The sperm samples were from six men with defective sperm–ZP binding
(all men had sperm count >20×106/ml, progressive motility >30% and variable
sperm morphology). For comparison, under these experimental conditions,
sperm from fertile men would consistently have >400 sperm bound/four ZP.
a
P > 0.05 for 280 versus 400 mOsm/kg.
b
P < 0.02 for 280 versus 400 mOsm/kg.
phosphorylation is related to sperm–ZP binding (Sakkas et al.,
2003). We have recently confirmed that the proportion of
sperm showing tyrosine phosphorylation on the tails as
detected with PY20–FITC is strongly correlated with human
sperm–ZP binding (D.Y.Liu et al., unpublished data).
In this study, we did not use medium with osmolality >400
mOsm/kg since it would severely reduce sperm motility which
would then affect the sperm–ZP interaction test. The primary
aim was to test the effect of osmolalities, in the range known to
occur in the male and female reproductive tracts, on sperm–ZP
interaction. Rossato et al. (2002) showed that sperm motility
was nearly completely lost when sperm were exposed to
medium with 600 mOsm/kg. Low sperm–ZP binding in hypoosmotic (150 mOsm/kg) medium may be explained by
severely reduced sperm motility or velocities. However, for
200 mOsm/kg osmolality medium, low sperm–ZP binding was
not due to reduced sperm motility since results of sperm motility and velocities were very similar to those in hyper-osmotic
(350 and 400 mOsm/kg) media. We confirmed that the
increased sperm–ZP binding and tyrosine phosphorylation was
due to high osmolality per se rather than to higher concentrations of NaCl, by obtaining similar results when sucrose was
used instead of NaCl to increase osmolality to 400 mOsm/kg.
Interestingly, enhancement of sperm–ZP binding by hyperosmotic conditions was limited to those sperm samples with
initially normal sperm–ZP binding capacity. For sperm with
defective ZP binding, hyper-osmotic conditions had no effect
on the sperm–ZP binding capacity (Table II). Therefore, tyrosine phosphorylation may be an indirect indicator for sperm–
ZP binding and may not be directly involved with the sperm
receptors for the ZP. Simple enhancement of tyrosine phosphorylation alone by increasing osmolality of medium would not
improve sperm–ZP binding capacity in men with defective
sperm–ZP receptors.
Hyper-osmotic medium significantly reduced the ZP-induced
AR. Previous studies showed that hyper-osmotic conditions
reduced ionophore A23187-induced AR and calcium-influx
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Figure 6. Comparison of the zona pellucida (ZP)-induced AR of
sperm cultured in iso-osmotic (280 mOsm/kg) and hyper-osmotic
(400 mOsm/kg) media. Line graph showing individual results of
experiments with sperm samples from 18 men. The ZP-induced acrosome reaction was significantly lower (P < 0.001) in hyper-osmotic
medium than in iso-osmotic medium.
Samples
Normal sperm
morphology (%)
Osmolality and sperm–ZP binding
binding, but severely inhibit the ZP-induced AR. Using hyperosmotic conditions to increase tyrosine phosphorylation and
sperm–ZP binding appears to have no therapeutic application
because of the reduced ZP-induced AR. While changing the
osmotic conditions may be not useful for clinical IVF, it will
be useful for studying aspects of sperm function.
Acknowledgements
The authors thank Mingli Liu and Franca Agresta for technical assistance, the scientists in both Royal Women’s Hospital and Melbourne
IVF Laboratories for collecting the oocytes, the scientists in the
Andrology Laboratory for collecting sperm samples.
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Submitted on April 29, 2005; resubmitted on September 27, 2005; accepted on
September 28, 2005
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