ORIGINAL ARTICLE

Nitric oxide induces acrosome reaction in cryopreserved

bovine spermatozoa
P. C. Rodriguez, C. M. O’Flaherty, M. T. Beconi & N. B. Beorlegui
Area of Biochemistry, School of Veterinary Sciences, University of Buenos Aires, Buenos Aires, Argentina

Keywords
Acrosome reaction—bovine sperm—nitric
oxide—protein kinases—reactive oxygen
species
Correspondence
Pablo C. Rodriguez, Area of Biochemistry,
School of Veterinary Sciences, University of
Buenos Aires, Chorroarı́n 280, C1427CWO
Buenos Aires, Argentina.
Tel.: +54-11-4524-8452;
Fax: +54-11-4524-8452;
E-mail: prodriguez@fvet.uba.ar
Accepted: September 14, 2005
Summary
The aim of this work was to study the effect of nitric oxide on acrosome reac-
tion (AR) and the participation of protein kinases and reactive oxygen species
in the AR of cryopreserved bovine spermatozoa. Spermatozoa were capacitated
in Tyrode’s albumin lactate pyruvate medium with heparin (10 IU ml)1) and
then incubated with different concentrations of sodium nitroprusside (SNP)
(1–200 lmol l)1). Methylene blue and haemoglobin were used to confirm the
role of nitric oxide as an inducer of the AR. The participation of protein kinase
A (PKA) , protein kinase C (PKC) and protein tyrosine kinase was evaluated
using specific inhibitors of these enzymes (H-89, 50 lmol l)1; bisindolylmalei-
mide I, 0.1 lmol l)1 and genistein, 3 lmol l)1). The role of hydrogen peroxide
or superoxide anion was evaluated by incubation with catalase or superoxide
dismutase respectively. AR percentages were determined by the fluorescence
technique with chlortetracycline. The highest levels of AR were obtained in
capacitated spermatozoa treated with 5–200 lmol l)1 SNP (24.8 ± 1.8%). The
presence of PKA, PKC and protein tyrosine kinase inhibitors likewise decreased
AR percentages. The addition of superoxide dismutase had no effect on the AR
level but catalase completely blocked it. These results indicate that nitric oxide
induces AR in capacitated spermatozoa involving hydrogen peroxide and the
participation of PKA, PKC and protein tyrosine kinase as part of the sig-
nal transduction mechanism which lead to the AR in cryopreserved bovine
spermatozoa.

A23187 (Griveau et al., 1995), lysophosphatidylcholine

Introduction
The acrosome reaction (AR) of mammalian spermatozoa is
an exocytotic process necessary for fertilization. In order
for the AR occur in vivo and under in vitro conditions the
spermatozoa must first undergo capacitation. AR involves
fusion between the outer acrosomal membrane and the
overlying sperm plasma membrane and exposure of acro-
somal enzymatic contents and the inner acrosomal mem-
brane. The AR enables the sperm cell to penetrate the zona
pellucida and fuse with the egg plasma membrane overlying
the postacrosomal region (Yanagimachi, 1994).
Several extracellular ligands were reported to induce
the AR of sperm cells in vitro. These include steroids
(Osman et al., 1989) prostaglandins (Joyce et al., 1987),
(LPC; Fleming & Yanagimachi, 1981) and zona pellucida
(Yanagimachi, 1994). There are evidences that capacita-
tion and AR are part of an oxidative process. Superoxide
anion (O

2 ) is required to induce AR in human sperma-
tozoa (Griveau et al., 1995; de Lamirande et al., 1998)
while hydrogen peroxide (H2O2) is involved in AR of
human (Aitken et al., 1995) and bovine spermatozoa
(O’Flaherty et al., 1999). In human sperm the AR was
prevented by catalase but not by superoxide dismutase
(SOD) (Aitken et al., 1995). However, LPC-induced AR
was inhibited by the presence of SOD and catalase in
bovine (O’Flaherty et al., 2001) and in human capacitated
spermatozoa, indicating that both O
 2 and H2O2 would
be involved in the AR (de Lamirande et al., 1998).

166 ª 2005 The Authors Journal Compilation ª 2005 Blackwell Publishing Ltd Andrologia 37 (2005) 166–172
P. C. Rodriguez et al.
Nitric oxide (NO•) is a free radical synthesized in vivo
during the conversion of l-arginine to l-citrulline by
nitric oxide synthase (NOS) and is implicated in a variety
of physiological cellular signalling mechanisms in many
tissues by activating guanylyl cyclase. It has been demon-
strated that exogenous NO• stimulates the capacitation in
bovine spermatozoa (Rodriguez et al., 2005) and the AR
in bovine (Zamir et al., 1995) and human spermatozoa
(Revelli et al., 1999). In experimental systems, haemoglo-
bin acts as NO• scavenger when binding to the haeme
group (Herrero et al., 1994) and methylene blue blocks
the effects of nitric oxide by acting on guanylyl cyclase.
Methylene blue rapidly reduces the endogenous NO• con-
centrations available to spermatozoa thereby reducing
motility, rather than by a more gradual toxic effect
(Donnelly et al., 1997).
The sequence of events that culminates in the AR has
been shown to involve the cAMP-dependent protein kin-
ase A (PKA) and protein kinase C (PKC) pathways in
human sperm (Thomas & Meizel, 1989; De Jonge et al.,
1991a; Rotem et al., 1992) and nonhuman mammalian
sperm (Garbers & Kopf, 1980; Roldan & Harrison, 1989;
Thomas & Meizel, 1989). The first signal transduction
pathway demonstrated to have a role in the human sperm
AR involves the generation of the second messenger
cAMP by the amplifying enzyme adenylate cyclase, ulti-
mately leading to the activation of PKA (De Jonge et al.,
1991a). The second signalling pathway demonstrated to
play a role in the AR involves the amplifying enzyme
phospholipase C that converts phosphatidylinositol-4–5
bisphosphate in InsP3 and diacylglycerol (DAG). DAG, in
a Ca2+ and phospholipid-dependent process, activates
PKC (De Jonge et al., 1991b).
Another possible mechanism would be through an
increase in tyrosine phosphorylation of specific proteins,
a post-translational modification often regulated by redox
reactions in many types of cells and systems (Fialkow
et al., 1993). Redox reactions are important for the regu-
lation of tyrosine phosphorylation of these proteins as
ROS scavengers, such as SOD and catalase, prevented this
post-translational modification, whereas exogenously
added reactive oxygen species (H2O2 or the combination
of xanthine and xanthine oxidase) promoted it (Aitken
et al., 1995; Leclerc et al., 1997).
A better understanding of the effects of nitric oxide on
AR process will contribute to clarify intracellular mecha-
nisms in order to improve the conditions required to
optimize bovine fertilization in vitro. The aim of this
work was to determine the effect of NO• in the AR of
cryopreserved bovine spermatozoa, and to determine
whether the presence of other reactive oxygen species and
the participation of protein kinases are necessary in this
process.
ª 2005 The Authors Journal Compilation ª 2005 Blackwell Publishing Ltd Andrologia 37 (2005) 166–172
Nitric oxide in acrosome reaction
Materials and methods
Semen freezing
Semen was collected by artificial vagina from four Hol-
stein bulls (4–5 years old) of proven fertility. The bulls
belonged to a controlled programme of artificial insemin-
ation and were maintained under uniform nutritional
conditions and management during the period of
research. For all ejaculates, progressive motility was >70%
and the abnormal spermatozoa was <20%. Two ejaculates
from each bull were obtained once a week for 12 weeks.
Ejaculates were pooled and diluted to obtain a final con-
centration of 3.0–4.5 · 107 sperm ml)1, in a buffer con-
taining Tris (0.20 mmol l)1), citric acid (0.06 mmol l)1),
glycine (0.12 mmol l)1), fructose (0.06 mmol l)1), 20%
egg yolk and 7% glycerol. A slow cooling curve to 5
C
(1
C min)1) was performed, and the semen was then
equilibrated at 5
C for a further 90 min. It was frozen at
)76
C on dry ice, and pellets were preserved at )196
C
in liquid nitrogen (O’Flaherty et al., 1999, 2001).
Evaluation of sperm motility and viability
Progressive motility was evaluated by light microscopy
(400· magnification) with a thermal stage (37
C) three
times by the same observer, in the following cases:
10 min after thawing, after 60 min of incubation and
after each treatment (45 min + 15 min; Beorlegui et al.,
1997). The percentage of live spermatozoa was deter-
mined by the supravital technique with trypan blue
0.25% (Griffiths, 1995). At least 200 spermatozoa were
counted in each sample.
Determination of sperm concentration
Sperm concentration in semen or in sperm suspensions
was carried out in a Neubauer chamber.
Preparation of capacitated spermatozoa
Pooled frozen semen samples were thawed for 10 min in
Tyrode’s albumin lactate pyruvate (TALP) medium, pH
7.4, at 36
C, without calcium and without bovine serum
albumin (BSA; Parrish et al., 1988) in a 1 : 3 ratio. Sam-
ples were washed twice by centrifugation at 600 · g for
5 min to separate the seminal plasma and the freezing
buffer. The pellet was resuspended to a final concentra-
tion of 1.5 · 107 spermatozoa ml)1, in TALP with the
addition of CaCl2 (2 mmol l)1) and BSA (6 mg ml)1),
used as incubation medium (completed TALP; Rodriguez
et al., 2005) for all the experiments. In order to obtain
capacitated spermatozoa, cells were incubated with
10 IU ml)1 heparin for 45 min at 38
C (Fukui et al.,
167
Nitric oxide in acrosome reaction
1990; Rodriguez et al., 2005) under 5% CO2 in humid-
ified air.
Acrosome reaction in capacitated spermatozoa
Spermatozoa previously capacitated with heparin, were
incubated for 15 min at 38
C in the presence/absence of
sodium nitroprusside (SNP; 1, 5, 10, 100 and
200 lmol l)1, as an AR inducer; Revelli et al., 2001). It
was used 0.1 mg ml)1 LPC as positive control of AR
(Fleming & Yanagimachi, 1981). For each experiment the
sperm concentration and the percentage of progressive
motility and viability were evaluated.
Determination of acrosome reaction
The chlortetracycline (CTC) fluorescent technique was
used to detect changes in the plasma membrane of the
bovine spermatozoon (Fraser et al., 1995 modified by
Beorlegui et al., 1997). Three patterns were observed: F
(fluorescent), intact noncapacitated sperm displaying
fluorescence throughout their surface; C (capacitated),
intact capacitated spermatozoa that lost fluorescence in
the post-acrosomal region; and AR (acrosome reacted),
spermatozoa with a reacted acrosome that lost fluores-
cence in the post-acrosomal and acrosomal regions,
expressing fluorescence only in the midpiece and the tail.
To an equal volume of the medium containing the sper-
matozoa 500 ll CTC was added. Glutaraldehyde was then
added to the mixture reaching a final concentration of
0.1%. Slides were examined at 400· magnification under
epifluorescence excitation at 410 nm using a Carl Zeiss
Jenamed 2 epifluorescence microscope (Zeiss, Jena,
Germany). The percentage of acrosome-reacted spermato-
zoa (pattern AR) at zero time was subtracted to the con-
trol and treated samples incubated during 45 + 15 min to
rule out cells damaged during freezing–thawing. At least
200 spermatozoa were counted in each sample.
Experiment 1: effect of NO• on acrosome reaction
Sperm previously incubated in capacitation medium, were
incubated for 15 min at 38
C, in the presence of differ-
ent concentrations of SNP (1, 5, 10, 100 and
200 lmol l)1), a generator of NO• in vitro (Herrero et al.,
1994).
Experiment 2: effect of NO• scavengers on acrosome
reaction
Spermatozoa, previously capacitated with heparin
(10 IU ml)1) were incubated for 15 min at 38
C, with
the addition of SNP (100 lmol l)1) as AR inducer and
168 ª 2005 The Authors Journal Compilation ª 2005 Blackwell Publishing Ltd Andrologia 37 (2005) 166–172
P. C. Rodriguez et al.
haemoglobin (20 and 40 lg ml)1; Herrero et al., 1994) or
methylene blue (10, 100 and 200 lmol l)1; Donnelly
et al., 1997) as NO• scavengers.
Experiment 3: effect of protein kinases on NO• induced
acrosome reaction
Sperm previously incubated in capacitation medium, were
incubated for 15 min at 38
C, with SNP (100 lmol l)1)
in the presence or absence of 50 lmol l)1 H-89, a specific
PKA inhibitor (Galantino-Homer et al., 1997),
0.1 lmol l)1 bisindolylmaleimide I (BM), a specific PKC
inhibitor (O’Flaherty et al., 2001) or 3 lmol l)1 genistein
(GEN), a specific PTK inhibitor (Rodriguez et al., 2005).
Experiment 4: effect of ROS on NO• induced acrosome
reaction
Spermatozoa previously incubated in capacitation med-
ium, were incubated for 15 min at 38
C, with SNP
(100 lmol l)1) in the presence or absence of SOD (0.05,
0.1 and 0.5 mg ml)1) and catalase (20, 50 and
100 lg ml)1).
Statistical analysis
Percentages of acrosome reacted spermatozoa are given as
mean ± SD. Treatments in the different experiments were
analysed by anova and the Bonferroni test was used for
means comparison. P < 0.05 value was considered as sta-
tistically significant.
Results
The effect of NO• as AR inducer was determined in sper-
matozoa previously capacitated with heparin
(10 IU ml)1) using SNP concentrations ranging between
1 and 200 lmol l)1. A significant increase in AR
(P < 0.05) was observed from 1 lmol l)1 SNP, reaching a
plateau with concentrations between 5 and 200 lmol l)1
(Fig. 1).
Progressive motility (60% ± 5) was not affected by
any of the SNP concentrations used. The level reached
by adding LPC (a membrane disturbing agent) to sper-
matozoa capacitated with heparin was used as positive
control of AR. NO• scavengers were employed to con-
firm the participation of NO• (100 lmol l)1 SNP was
selected to be used as AR inducer) in this experimental
model. Both haemoglobin and methylene blue lowered
AR levels significantly in spermatozoa capacitated with
heparin (Fig. 2). With 40 lg ml)1 of haemoglobin, a
percentage of AR similar to the control group was
obtained, without modifying sperm motility (60 ± 5%)
P. C. Rodriguez et al. Nitric oxide in acrosome reaction

35 c 30
Acrosomal reaction (%)

Acrosomal reaction (%)

30 d d b
d d 25
25
b 20 d
20
15
15 a a c c
10 10 a
5 5
0 0
C H HLPC 1 5 10 100 200
C SNP H89 BM GEN
SNP µmol l–1
Fig. 3 Effect of specific inhibitors of protein kinase A (H-89), protein
Fig. 1 Effect of sodium nitroprusside (SNP) on the acrosome reaction kinase C (BM) and protein tyrosine kinase (genistein) on SNP
in bovine spermatozoa. C, control; H, heparin; HLPC, lysophosphati- acrosome reaction in bovine spermatozoa. C, control; SNP, sodium
dilcoline acrosome reaction on spermatozoa previously capacitated nitroprusside 100 lmol l)1; H-89, SNP + 50 lmol l)1 H-89; BM,
with heparin; SNP, sodium nitroprusside (lmol l)1). Samples were SNP + 0.1 lmol l)1 bisindolylmaleimide I; GEN, SNP + 3 lmol l)1
incubated for 15 min at 38
C in completed TALP medium. Different genistein. Samples were incubated for 15 min at 38
C in completed
letters indicate significant differences (P < 0.05; n ¼ 4). TALP medium. Different letters indicate significant differences
(P < 0.05; n ¼ 4).

30
Acrosomal reaction (%)

a
Acrosomal reaction (%)
25 a 35
b
b 30 b b
20 b
c,d c 25
15 d d c,d
20 a
15 a a a
10
10
5
5
0 0
C H SNP MB10 MB100 MB200 Hg20 Hg40 C SNP S0,05 S0,1 S0,5 C20 C50 C100
µmol l–1 µg/ml mg/ml µg/ml

Fig. 2 Effect of nitric oxide scavengers on the acrosome reaction of
spermatozoa previously capacitated with heparin. C, control; H, hep-
arin; SNP, 100 lmol l)1 SNP; MB, SNP + methylene blue (lmol l)1);
Hg, SNP + haemoglobin (lg ml)1). Samples were incubated for
15 min at 38
C in completed TALP medium. Different letters indicate
significant differences (P < 0.05; n ¼ 4).
Fig. 4 Effect of catalase and superoxide dismutase (SOD) on the ac-
rosome reaction induced by SNP in bovine spermatozoa. C, control;
SNP, sodium nitroprusside 100 lmol l)1; SOD, SNP + SOD (mg ml)1);
CAT, SNP + catalase (lg ml)1). Samples were incubated for 15 min at
38
C in completed TALP medium. Same letters indicate that there
are no significant differences (P > 0.05; n ¼ 4).

and viability (59 ± 2%). Methylene blue not only inhib-

ited AR to a lesser degree than haemoglobin but it also
affected negatively sperm motility at all concentrations;
sperm viability percentages (59 ± 2%) did not differ
from the control group.
A significant decrease was observed in AR induced with
100 lmol l)1 SNP in response to the addition of specific
inhibitors of protein kinases: H-89 (PKA), BM (PKC)
and genistein (PTK) to capacitated spermatozoa
(P < 0,05; Fig. 3).
The relevance of other reactive oxygen species (O
 2
and H2O2) was studied in our experimental model. When
capacitated spermatozoa were incubated with SNP
(100 lmol l)1) in the presence of different superoxide
dismutase concentrations, significant changes were not
observed in AR percentage; however, when the samples
were incubated in the presence of catalase the AR was
blocked and the percentages of AR obtained were similar
to the control group (Fig. 4).
Discussion
Once capacitation has occurred, spermatozoa developed
AR in response to a variety of substances found around
the oocyte, like follicular fluid, cumulus oophorus, prog-
esterone and the zona pellucida (Yanagimachi, 1994). The
wide localization of NOS-positive cells in the male and
female genitourinary systems (Roselli, 1997) suggests that
spermatozoa could represent one of the targets of NO•,
and the role for NO• in controlling the functions of male
gametes (Revelli et al., 1999), such as motility, viability
and metabolism. NO• could even participate in mecha-
nisms leading to fertilization as in bovine and human
sperm it increases capacitation (Zini et al., 1995; Rodri-
guez et al., 2005) and AR (Zamir et al., 1995; Revelli
et al., 1999) respectively. The results of this study show
that exogenous NO•, released from SNP, induces AR in
cryopreserved bovine spermatozoa, previously capacitated.
The presence of haemoglobin and methylene blue, NO•

ª 2005 The Authors Journal Compilation ª 2005 Blackwell Publishing Ltd Andrologia 37 (2005) 166–172 169
Nitric oxide in acrosome reaction
scavengers, inhibited SNP AR, confirm the participation
of NO• as inducer of this process.
Possible NO• targets have been studied but the intra-
cellular mechanism, through which NO• induces AR, has
not been completely clarified. In human spermatozoa,
intracellular levels of cAMP are elevated during AR
(Ward & Kopf, 1993), indicating the activation of the
adenylyl cyclase. In mammalian cells, the main cAMP
action is mediated through the activation of PKA. The
AR induced with A23187 was associated with an increase
in intracellular cAMP levels and PKA activity (Lefièvre
et al., 2002). According to our data the prevention of the
SNP-induced AR by H-89 (a specific PKA inhibitor;
Fig. 3) demonstrate that the activation of this enzyme is
involved in mechanisms trigged by NO•, regulated by the
concentration of cAMP. These data are in agreement with
De Jonge et al. (1991a), who showed that the presence of
PKA inhibitors in the incubation medium prevented the
AR induced by dbcAMP (an analogue of the second-
messenger cAMP), in human-capacitated spermatozoa.
Protein tyrosine phosphorylation is an important bio-
chemistry mechanism, which regulates the sperm function
in response to extracellular signals. In human spermato-
zoa, tyrosine phosphorylation of sperm proteins is modi-
fied by and appeared regulated by redox reactions during
the AR (Aitken, 1995; de Lamirande et al., 1998), indica-
ting that AR influences the level of phosphorylation of
sperm proteins. In all mammalian species studied, tyro-
sine phosphorylation in spermatozoa is regulated by the
second messenger, cAMP (Furuya et al., 1992; Duncan &
Fraser, 1993; Visconti et al., 1995; Leclerc et al., 1996;
Galantino-Homer, 1997; Visconti et al., 1997; Aitken
et al., 1998a,b; Leclerc et al., 1998) and is mediated
through a PKA-dependent mechanism. As PKA is a ser-
ine/threonine kinase, it cannot play a direct role in tyro-
sine phosphorylation and therefore must activate tyrosine
kinases indirectly (Leclerc et al., 1996). The fact that geni-
stein (a specific PTK inhibitor) blocks the AR induced by
SNP, suggests the participation of this enzyme in intracel-
lular mechanisms, which lead to the exocytotic event
induced by NO• in spermatozoa previously capacitated.
Revelli et al. (2001) demonstrated that the NO•
induced AR does not occur in a Ca2+-free medium or in
the presence of a PKC inhibitor. Besides, PKC activators
such as 12-o-tetradecanoylphorbol-13-acetate and 1-oleo-
yl-2-acetylglycerol are powerful inducers of the AR in
human-capacitated spermatozoa (Rotem et al., 1992).
Our results show that the SNP-induced AR may require
the activation of PKC because this process is prevented
by a specific inhibitor of the enzyme in bovine spermato-
zoa previously capacitated.
In bovine spermatozoa, the AR induced by LPC was
inhibited by SOD and catalase (O’Flaherty, 2001)
170 ª 2005 The Authors Journal Compilation ª 2005 Blackwell Publishing Ltd Andrologia 37 (2005) 166–172
P. C. Rodriguez et al.
suggesting the importance of both reactive species in
this process; while in human spermatozoa, the AR,
induced by A23187, was prevented by catalase but not
by SOD (Aitken et al., 1995). The AR is considering as
part of an oxidative process, which involves O
 2 (de
Lamirande et al., 1998) and H2O2 (de Lamirande et al.,
1998; O’Flaherty et al., 1999). In diverse cell types, the
intracellular redox state is important in the regulation of
enzymes presumably involved in signal transduction
mechanisms (Aitken et al., 1995; Hancok et al., 2001).
Indeed, the alteration of protein functions by oxidants
may be in many ways analogous to phosphorylation
except that protein modification occurs in redox-sensi-
tive amino acids (such as cystein and histidine; Finkel,
1998). There are evidences of the fact that mammalian
spermatozoa are intensively redox active cells and that
this activity controls many different aspects of sperm
function (Baker & Aitken, 2004). Several possible path-
ways have been described by which NO• interacts with
O

2 and H2O2. The addition of SOD and catalase failed
to modify capacitation induced by SNP in bovine sper-
matozoa indicating that neither O
 2 nor H2O2 is
required for the action of NO• or that NO• stimulates
capacitation independently of O
 2 generation (Rodriguez
et al., 2005). In the present study, it is shown that the
addition of catalase diminished the AR level but the
addition of SOD does not, indicating that the removal
of H2O2 prevents AR, because H2O2 is being generated
by the bovine spermatozoa during incubation with SNP.
This is in agreement with previous work in bovine sper-
matozoa, which showed an increased in the production
of H2O2 during the AR induced by LPC (O’Flaherty
et al., 2001). Therefore, the NO• could interact with
H2O2, suggesting that the stimulatory effect of NO• on
AR involves H2O2 as part of an oxidative process that
requires both reactive species.
In summary, our results suggests that NO• as inducer
of AR requires the generation of H2O2 and stimulates a
cascade of intracellular events that involves the participa-
tion and activation of PKA, PKC and PTK in cryopre-
served bovine spermatozoa.
Acknowledgements
We are deeply grateful to Genética Los Nogales for semen
sample supplies. This research was supported by the Sec-
retarı́a de Ciencia y Técnica de la Universidad de Buenos
Aires (V016, UBACYT).
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