Animal Reproduction Science 120 (2010) 166–172

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Animal Reproduction Science

journal homepage: www.elsevier.com/locate/anireprosci

Cholesterol-loaded cyclodextrin enhances osmotic tolerance and

inhibits the acrosome reaction in rabbit spermatozoa
Melih Aksoy a,∗ , Orhan Akman b , Necdet Cankat Lehimcioğlu c , Hüseyin Erdem d
a
Department of Reproduction and Artificial Insemination, Faculty of Veterinary Medicine, University of Adnan Menderes, 09016 Aydın, Turkey
b
Department of Reproduction and Artificial Insemination, Faculty of Veterinary Medicine, University of Ataturk, 25240 Erzurum, Turkey
c
Department of Reproduction and Artificial Insemination, Faculty of Veterinary Medicine, University of Kafkas, 36300 Kars, Turkey
d
Department of Obstetrics and Gynaecology, Faculty of Veterinary Medicine, University of Selçuk, 42070 Konya, Turkey

a r t i c l e i n f o a b s t r a c t

Article history: The effects of cholesterol-loaded cyclodextrin (CLC) treatment on the osmotic tolerance
Received 6 October 2009 and ability to undergo the acrosome reaction of rabbit spermatozoa, with an unusually
Received in revised form 8 February 2010
high cholesterol/phospholipid ratio in plasma membranes, were examined in two suc-
Accepted 16 February 2010
cessive experiments. In the first experiment, CLC-pretreated and untreated sperm cells
Available online 23 February 2010
were exposed for 15 min to one of five fructose solutions, adjusted to 20, 80, 290, 500 or
1500 mOsm/L. After the anisoosmotic challenge, the integrity of sperm membranes in the
Keywords:
CLC-supplemented (at a dose level of 3 mg/120 × 106 spermatozoa) and control groups was
Cholesterol
Cyclodextrin estimated by a modified hypoosmotic swelling test (HOST) associated with a supravital
Spermatozoa eosin staining test (HE-test). In the second part of the study, the influence of cholesterol
Rabbit supplementation on the acrosome reaction of sperm cells stimulated by either calcium
Osmotic tolerance ionophore A23187 (CI) or lysophosphatidylcholine (LPC) was evaluated.
Acrosome reaction CLC pretreatment increased viable and live-HOST-responsive sperm rates (P < 0.01) after
incubation in anisoosmotic solutions varying from 80 to 1500 mOsm/L. However, CLC sup-
plementation did not influence the percentage of HOST-responsive sperm cells (P > 0.05). A
significant interaction was determined between CLC pretreatment and the level of osmotic
pressure in maintaining the functional and physical integrities of sperm membranes under-
going osmotic challenges. Both CI and LPC successfully induced the acrosome reaction in
rabbit spermatozoa (P < 0.001). Compared with CI, LPC was more effective (P < 0.0001). CLC
pretreatment resulted in a significant reduction (P < 0.01) in the percentage of acrosome
reacted sperm cells irrespective of the inducing agent, either CI or LPC.
In conclusion, CLC treatment enhanced the anisoosmotic tolerance of rabbit spermatozoa
and reduced their ability to undergo the acrosome reaction after stimulation by CI or LPC.
© 2010 Elsevier B.V. All rights reserved.

1. Introduction able proportion of the sperm cells are still damaged or

Cryopreservation of spermatozoa is used extensively
in artificial insemination programmes. Despite steady
progress in the cryopreservation techniques a consider-
∗ Corresponding author. Tel.: +90 256 247 07 00/109;
fax: +90 256 247 07 20.
E-mail address: aksoym@tnn.net (M. Aksoy).
die during the freezing process. The mechanism of sperm
injury during cryopreservation is not fully known. How-
ever, it is generally believed that the damage is related
to the changes in temperature, ice formation, oxidative
damage, alterations in sperm membrane, toxicity of cry-
oprotectants and osmotic stress (Watson, 1995, 2000).
The cells undergo osmotic stress during both the cooling
and warming steps. When the cell suspension is cooled
extracellular solutes and cryoprotective agents gradually

0378-4320/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.anireprosci.2010.02.014
 M. Aksoy et al. / Animal Reproduction Science 120 (2010) 166–172
become more concentrated. This phenomenon results in
the occurrence of a hyperosmotic environment for the
cells and may cause changes in pH (Fishbein and Winkert,
1978), serious dehydration leading to damage of intracel-
lular organelles (Levitt, 1962), weakening cell membranes
or loss of phospholipids (Lovelock, 1957). All of these
are potential mechanisms that might have harmful con-
sequences on the viability of the cells. Therefore, it is
expected that reducing the osmotic stress or extending the
osmotic tolerance of cells would improve their cryotoler-
ance.
Cholesterol has multiple effects on plasma mem-
brane properties. It is involved in many roles including
the stabilisation of membranes, reduction of membrane
permeability, facilitation of morphological characteris-
tics and interactions between cells that influence phase
transitions and provide a suitable chemical and/or phys-
ical microenvironment for membrane proteins (Crockett,
1998).
Cyclodextrins are cyclical oligomers of six to eight glu-
cose molecules that can solubilise hydrophobic molecules,
such as cholesterol, because of their hydrophobic interior.
Recently, the use of cholesterol preloaded methyl-␤-
cyclodextrin was proposed to facilitate cholesterol transfer
into the plasma membranes of bull spermatozoa (Purdy
and Graham, 2004b). The effect of cholesterol-loaded
cyclodextrin (CLC) treatment on sperm cells has been
documented in a set of studies that used various
freezing techniques and animal species, mostly with a
low cholesterol/phospholipid ratio in sperm membranes
(Barrera-Compean et al., 2005; De Graaf et al., 2007; Moore
et al., 2005; Purdy and Graham, 2004a,b). However, to
the authors’ knowledge there has been no report evalu-
ating the influence of cholesterol treatment on sperm cells
based on an experimental model without freezing. Like-
wise, the impact of cholesterol supplementation on the
integrity of sperm membranes both during hypoosmotic
and hyperosmotic conditions was not screened in the pre-
vious studies.
The objective of the present study was to evaluate the
influence of CLC treatment, as a cholesterol provider to
sperm membranes, on the osmotic tolerance of rabbit sper-
matozoa, with an unusually high cholesterol/phospholipid
ratio in plasma membranes, incubated both in hypoosmotic
and hyperosmotic conditions without freezing. Moreover,
the impact of cholesterol supplementation on the abil-
ity of sperm cells to undergo the acrosomal reaction in
the presence of two well-known agents, which induce the
acrosomal reaction, calcium ionophore A23187 (CI) and
lysophosphatidylcholine (LPC) was also examined in the
second part of the study.
2. Materials and methods
2.1. Chemicals
All chemicals were purchased from Merck (Darmstadt,
Germany). Bovine Serum Albumin (BSA), cholesterol, chlo-
roform, methyl-␤-cyclodextrin, CI and LPC were obtained
from Sigma Chemical Company (St. Louis, MO, USA).
167
2.2. Cholesterol-loaded cyclodextrin
The CLC was prepared as described previously by
Purdy and Graham (2004a). However, the solution was
sonicated in addition to being vortexed. Briefly, 1 g methyl-
␤-cyclodextrin was dissolved in 2 ml methanol. The
cholesterol solution was prepared by dissolving 200 mg
cholesterol in 1 ml chloroform in a separate tube. A 0.45 ml
aliquot of the cholesterol solution was added to the
cyclodextrin solution and mixed. The solution was then
placed under a stream of nitrogen gas to evaporate the
solvents obtain a white CLC powder. The CLC was stored
in a glass container at room temperature. A stock solution
was prepared by dissolving 50 mg CLC powder in 1 ml tris-
buffer including 276.5 mM tris base, 90.9 mM citric acid
monohydrate and 76.8 mM fructose. The tris-buffer used
for CLC preparation contained 3 mg/ml BSA. The solution
was held in a water bath at 39 ◦ C for 5 min, then vortexed
and sonicated for 45 s to obtain a working CLC solution.
2.3. Animals and semen collection
Before the study was started approval for the animal
experiments was obtained from the Local Ethical Commit-
tee. Four New Zealand white rabbit bucks between the
ages of two and four years were used as sperm donors.
The bucks were housed in individual cages, with water
and food provided ad libitum. No special lighting regimen
was employed throughout the study. Semen samples were
collected by artificial vagina and gel plugs in semen were
removed immediately after collection.
2.4. Semen examination
Using a phase-contrast microscope at 400× magnifi-
cation equipped with a heated stage adjusted to 37 ◦ C,
individual sperm motility was assessed after semen
samples were diluted by 10–12 fold (v/v) in tris-citric
acid-glucose extender (TCG) containing 313.8 mM tris,
103.1 mM citric acid and 33.3 mM glucose (Roca et al.,
2000). Motility estimations of each sample were performed
in three different fields by the same person throughout
the study. The mean value averaged from three succes-
sive estimations was used as the final motility score. The
proportion of morphologically abnormal sperm cells were
examined on wet mount slides using two to three drops
of semen diluted in Hancock’s solution (Hancock, 1952)
with a phase-contrast microscope. Sperm concentration
per milliliter of semen was determined haemocytometri-
cally as described by Evans and Maxwell (1987) using a
Thoma counting chamber after dilution in tap water 1:200
(v/v).
The percentage of live spermatozoa and sperm mem-
brane response were evaluated by cumulative analysis
of hypoosmotic eosin staining test (HE-test), using the
eosine exclusion test and the Hypoosmotic Swelling Test
(HOST), according to Ducci et al. (2002). Briefly, semen sam-
ples were diluted 1:10 (v/v) in 80 mOsm fructose solution
including 1% (w/v) eosin Y and were incubated in a water
bath at 35 ◦ C for 15 min. Sperm smears were prepared with
10 ␮L of mixed samples, and 100 sperm cells were observed
168 M. Aksoy et al. / Animal Reproduction Science 120 (2010) 166–172
in each slide under a phase-contrast microscope at 400×
magnification. Four types of sperm cells were identified
according to staining status of the sperm head and curl-
ing of the sperm tail in smears and they were each counted
separately.
2.5. Experiment 1
The first experiment in the present study was designed
to evaluate the effect of CLC pretreatment on the osmotic
tolerance limits of sperm membranes. The resistance of
sperm membranes in anisoosmotic conditions in CLC-
supplemented and control groups were compared using
the HE-test (eosin exclusion and swelling response of the
tail).
Semen samples were pooled by mixing two or three
individual ejaculates. Pooled samples with a progressive
motility lower than 50% and abnormal sperm rate higher
than 10% were discarded. The samples that fulfilled the
requirements were diluted to 80 × 109 spermatozoa per ml
in TCG extender and were divided into two equal aliquots.
The first aliquot was supplemented with CLC at the dose
level of 3 mg per 120 × 106 spermatozoa. An equal volume
of tris-buffer including 3 mg/ml BSA with no CLC was added
to the second aliquot of the extended semen to equalize
total volume of the both aliquots. After 15 min of incuba-
tion at 35 ◦ C for cholesterol to bind to sperm membranes,
sperm cells in both aliquots were diluted in one of the fruc-
tose solutions adjusted to 20, 80, 290, 500 or 1500 mOsm/L.
They were then incubated for 15 min at 35 ◦ C. After incu-
bation, the HE-test was performed as described above. In
addition, in the hyperosmotic incubation groups (500 and
1500 mOsm/L), the samples were centrifuged at 300 × g
for 5 min to remove the supernatant. The sperm pellet in
the bottom of the tube was re-suspended in 80 mOsm/L
fructose solution. The HE-test was performed following
incubation of sperm cells at 35 ◦ C for 15 min. The percent-
age of sperm cells with stained or unstained heads and
curled or straight tails were counted separately.
2.6. Experiment 2
The second experiment in the present study was con-
ducted to examine the impact of CLC supplementation on
the ability of sperm cells to undergo the acrosome reaction
after treatment by capacitating agents. CI and LPC were uti-
lized as capacitor agents to induce the acrosome reaction in
sperm cells. Each experimental replication was conducted
on sperm samples consisting of two to three successive
ejaculates from a single buck. The ejaculates with a pro-
gressive motility higher than 50% and abnormal sperm rate
lower than 10% were allocated for the experiments.
Sperm samples were placed into 15-ml conical tube and
washed twice each in 10 ml of TCG extender to remove
seminal fluids. During each washing step, sperm were cen-
trifuged at 800 × g for 5 min. After washing, the final sperm
pellet was re-suspended in 3 ml TCG. Sperm concentration
was determined using a haemocytometer. Immediately
after the washing steps, 100 ␮L of the sample was fixed to
estimate the number of sperm with an intact acrosome in
the ejaculate. The remaining part of the sample was divided
into two equal aliquots, which were incubated in a water
bath at 35 ◦ C for 60 min. Fifteen minutes before the end of
the incubation period, the first aliquot was supplemented
with CLC at a dose level of 3 mg per 120 × 106 spermatozoa.
An equal volume of tris-buffer including 3 mg/ml BSA with
no CLC was added to the second aliquot to equalize total
volume of the both aliquots. The CLC and control groups
were both further divided into three equal portions and
were supplemented with one of the capacitating agents,
CI or LPC, or remained as a control. CI from a 2 mM stock
in dimethylsulfoxide was added to the extended semen
to make a final concentration of 20 ␮M during an addi-
tional incubation period of 1 h in a 37 ◦ C water bath.
Likewise, LPC from a 10 mg/ml stock in TCG solution was
mixed into the sample to make a final concentration of
100 ␮g/ml.
To identify acrosome reacted cells, spermatozoa were
stained using a Coomassie Blue G-250 staining procedure
described by Larson and Miller (1999). Briefly, sperm cells,
incubated with or without CI or LPC, were fixed with
4% paraformaldehyde solution (110 mM Na2 HPO4 , 2.5 mM
NaH2 PO4 , 4% paraformaldehyde, pH 7.4) for 10 min at
24 ◦ C. Sperm were centrifuged and washed twice using
1.5 ml of 100 mM ammonium acetate (pH 9.0). The final
sperm pellet was re-suspended in 1 ml of 100 mM ammo-
nium acetate, and 50 ␮l of the sperm suspension was
smeared on glass microscope slides using another glass
slide and air-dried. Sperm on the slides were incubated
in freshly made Coomassie stain (0.22% Coomassie Blue
G-250, 50% methanol, 10% glacial acetic acid, 40% water)
for 2 min at room temperature. Slides were washed thor-
oughly using distilled water to remove any excess stain.
Slides were air-dried and coverslips were placed on the
slides and sealed. Stained spermatozoa were examined
under bright field microscopy at 1000× magnification.
A minimum of 10 fields per slide and a total of two
slides (a total of 200 spermatozoa) per experiment were
observed.
2.7. Statistical analyses
Experiments were replicated at least five times. The
mean values were calculated simply by averaging the
results obtained in replications and were presented
together with standard errors throughout the study. In
the first experiment, the data were initially analysed by
two-way analysis of variance (ANOVA) to reveal poten-
tial interaction between the variables (osmotic pressure
and treatment) and the difference between the mean val-
ues of the main effects. Sperm parameters including live,
HOST-responsive and live-HOST-responsive sperm rates
were then compared between the treatment and control
groups by using Student’s t-test. In the second experi-
ment, CLC and control groups were also compared by
Student’s t-test. This followed the initial analyses of the
data by two-way ANOVA to reveal potential interaction
between the variables (inducing agents used for the acro-
some reaction and treatment) and the difference between
the means of the main effects. The statistical differences
were regarded as significant where P values were smaller
than 0.05.
 M. Aksoy et al. / Animal Reproduction Science 120 (2010) 166–172 169

Fig. 1. Effect of cholesterol-loaded cyclodextrin (CLC) supplementation of the extender on the viability of sperm cells after exposure to different osmotic
pressures. Asterisks indicate significant differences. *P < 0.05; **P < 0.01.

3. Results
3.1. Experiment 1
CLC supplementation enhanced the viability of sperm
cells (P < 0.001) incubated in anisoosmotic conditions vary-
ing between 80 and 1500 mOsm/L (Fig. 1). The level of the
osmotic pressure that sperm cells were exposed to also sig-
nificantly affected sperm viability (P < 0.001). However, no
interaction was determined between CLC treatment and
the osmotic pressure level in maintaining sperm viability
(P > 0.05).
CLC addition did not influence the percentage of
HOST-responsive sperm cells incubated in anisoosmotic

Fig. 2. Effect of cholesterol-loaded cyclodextrin (CLC) supplementation of the extender on (A) hypoosmotic swelling test (HOST)-responsive and (B)
live-HOST-responsive sperm rates during hypoosmotic (20 and 80 mOsm/L) and isoosmotic (290 mOsm/L) incubation. *P < 0.05.
170 M. Aksoy et al. / Animal Reproduction Science 120 (2010) 166–172

Fig. 3. Effect of cholesterol-loaded cyclodextrin (CLC) supplementation of the extender on live, hypoosmotic swelling test (HOST)-responsive and live-
HOST-responsive sperm rates during hyperosmotic (500 and 1500 mOsm/L) incubation. *P < 0.05.

Fig. 4. Effect of cholesterol-loaded cyclodextrin (CLC) supplementation of the extender on the ability of sperm cells to undergo acrosome reaction induced
by calcium ionophore (CI) or lysophosphatidylcholine (LPC). *P < 0.05; **P < 0.01.

conditions (P > 0.05; Fig. 2). In fact, CLC pretreatment
resulted in a significant increase in the percentage of
live-HOST-responsive sperm rate (P < 0.001; Fig. 3). Like-
wise, the osmotic pressure level significantly affected the
live-HOST-responsive sperm rate (P < 0.001). An impor-
tant interaction was determined between CLC treatment
and the level of osmotic pressure (P < 0.001) in maintain-
ing functional and physical integrity of sperm membranes
incubated in anisoosmotic conditions.
3.2. Experiment 2
CLC pretreatment significantly reduced the percent-
age of sperm cells that underwent the acrosome reaction
(P < 0.05; Fig. 4). Both CI and LPC successfully induced
the acrosome reaction in rabbit spermatozoa (P < 0.001).
LPC was more effective compared with CI in inducing the
acrosome reaction (P < 0.0001). No interaction was deter-
mined between the inducing agents and CLC pretreatment
(P > 0.05).
4. Discussion
Cholesterol is one of the most important compo-
nents of plasma membranes in cells. It has multi-
ple effects on membrane properties and is involved
in a variety of roles such as stabilising the mem-
brane, reducing membrane permeability, facilitating
morphological characteristics and regulating interactions
between cells that influence phase transitions. Moreover,
cholesterol influences membrane functions by modulat-
ing the activity of various integral proteins (Crockett,
1998).
Several authors have reported the incorporation of
cholesterol in the membranes of different types of somatic
cells (Klein et al., 1995; Veronique et al., 1997), and sperma-
tozoa (Purdy and Graham, 2004a,b; Moore et al., 2005). In
bulls (Purdy and Graham, 2004b; Moce and Graham, 2006;
Purdy et al., 2005), stallions (Moore et al., 2005; Zahn et
al., 2002; Combes et al., 1998), goats (Barrera-Compean et
al., 2005) and mice (Movassaghi et al., 2009) the beneficial
effect of CLC treatment on cryosurvival of sperm cells has
been reported. This effect has been mostly attributed to the
increased fluidity of sperm membranes and, thus, broad-
ened phase transition temperature (Moce and Graham,
2006; Purdy et al., 2005; Moore et al., 2005). The results
of the present study demonstrated that cholesterol sup-
plementation not only enhances membrane fluidity but
also increases the osmotic tolerance of sperm membranes
within a range varying between 80 and 1500 mOsm/L.
These data indicate that CLC treatment might also have
potential consequences in the freezing of somatic cell lines.
The ordering effect of cholesterol on biological mem-
branes results in a considerable increase in membrane
thickness (Stockton and Smith, 1976); however, the nature
of this effect depends largely on the phase of the mem-
brane and the number of carbon atoms in the phospholipid
chains (McIntosh, 1978). Nezil and Bloom (1992) reported
that the addition of 30 mole percent of cholesterol to
phosphatidylcholine vesicles, including one saturated and
 M. Aksoy et al. / Animal Reproduction Science 120 (2010) 166–172
one unsaturated acyl chain, increased the thickness of
the hydrophobic region from 25.8 to 29.9 Å. This obser-
vation may, at least, partly explain the beneficial effect of
cholesterol leading to enhanced osmotic tolerance of sperm
membranes in the present study.
The sperm cells from different animal species differ
widely in their cholesterol/phospholipid ratios as well
as their tail lengths and the degree of unsaturation of
the membrane phospholipids (Giraud et al., 2000; White,
1993). Human and rabbit spermatozoa contain consider-
ably high molar ratio of cholesterol to phospholipid (0.99
and 0.88, respectively; Watson, 1981) and, thus, it was con-
sidered that the protective influence of CLC might be less
prominent when compared with the other animal species
possessing a lower cholesterol to phospholipid ratio i.e.,
bull, stallion and ram (Moce and Graham, 2006).
Although we did not evaluate the effect of cholesterol
pretreatment on frozen sperm cells, the beneficial impact
of the cholesterol treatment of sperm membranes of rab-
bit spermatozoa by increasing the osmotic tolerance in
this study was surprising. This indicates that the benefi-
cial effect of cholesterol supplementation on sperm cells
to undergo cryopreservation would not be limited to the
animal species possessing a low cholesterol/phospholipid
ratio in the plasma membranes. Although the cholesterol to
phospholipid molar ratio is not as high as in rabbit sperma-
tozoa, enhanced osmotic tolerance was also reported after
cholesterol supplementation in unfrozen boar spermato-
zoa (Walters et al., 2008).
In the present study cholesterol pretreatment increased
the viability of sperm cells without having a prominent
effect on the functional integrity of sperm membranes
undergoing a set of osmotic challenges varying from 80
to 1500 mOsm/L. A significant interaction was also deter-
mined between CLC and the level of the osmotic pressure.
Likewise, Barrera-Compean et al. (2005) demonstrated that
treating buck spermatozoa with cholesterol before cry-
opreservation improved the post-thaw motility without
affecting the integrity of plasma membranes. In contrast,
cholesterol pretreatment was reported to enhance the
membrane integrity of both chilled (Torres et al., 2006) and
deep-frozen (Zahn et al., 2002) stallion and boar (Walters
et al., 2008) spermatozoa.
The functional and morphological changes that occur
during capacitation in sperm cells include loss of sterols,
altered distribution of phospholipids in the plasma mem-
brane, loss of molecules from the cells surface, membrane
hyperpolarisation, production of reactive oxygen species,
elevated concentrations of calcium and cyclic AMP, pro-
tein phosphorylation and increased intracellular pH (Cross,
2003). Loss of sperm sterols begins just after removal of
the sperm cells from seminal plasma and is obligatory for
capacitation of human sperm (Cross, 1996). Incubation of
sperm cells in media including high level of sterols to main-
tain high sterol levels in spermatozoa inhibit progesterone
and CI-induced acrosome reactions in human (Cross, 1996).
The sperm cholesterol content gradually decreases when
sperm cells are incubated in a capacitating medium in vitro
(Go and Wolf, 1985). The loss of cholesterol is initially lin-
ear in human spermatozoa, but acrosomal responsiveness
appears several hours later, suggesting that cholesterol loss
171
precedes the development of responsiveness (Zarintash
and Cross, 1996).
In agreement with the previous reports noted CLC
treatment in the present study significantly reduced the
percentage of acrosome reacted sperm cells although the
mechanism is unclear. The inhibitory effect of CLC treat-
ment on the ability of sperm cells to undergo the acrosome
reaction was reported previously in human (Khorasani et
al., 2000), equine (Zahn et al., 2002) and bovine (Purdy and
Graham, 2004b).
The chemical substances that are used to induce the
acrosome reaction in sperm cells act by different mecha-
nisms. The CI induces the acrosome reaction by altering
the composition of the plasma membrane, which leads
to transportation of calcium through the membrane and
successively results in the elevated intracellular calcium
concentrations required for capacitation and the acro-
some reaction (Talbot et al., 1976; Roldan and Harrison,
1989). However, LPC uses several different kinase path-
ways together (Liguori et al., 2005). Thus, some of the
inhibitory substances, such as PD98059 (mitogen-activated
protein kinase inhibitor), prevent the acrosome reaction
when induced by LPC (De Lamirande and Gagnon, 2002) but
not when induced by CI (Du Plessis et al., 2002). The signif-
icantly higher rates of acrosome reactions obtained by LPC,
compared with CI, in the present study might indicate that
the ability of rabbit spermatozoa to undergo capacitation
is dependent on the inducing agent and its mechanism of
action both before and after CLC treatment.
In conclusion, CLC treatment enhanced the tolerance of
rabbit spermatozoa against both hypoosmotic and hyper-
osmotic challenges, but reduced their ability to undergo the
acrosome reaction induced by CI or LPC.
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