Biochimica et Biophysica Acta 1674 (2004) 215 – 221

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l-Arginine promotes capacitation and acrosome reaction

in cryopreserved bovine spermatozoa
Cristian O’Flahertya,*, Pablo Rodrigueza, Sudha Srivastavab
a
Biochemistry Area, School of Veterinary Sciences, University of Buenos Aires, Av. Chorroarı́n 280 (C1427CWO) Buenos Aires, Argentina
b
National Facility for High Field NMR, Tata Institute of Fundamental Research, Homi Bhabha Road, Mumbai-400005, India

Received 20 October 2003; received in revised form 22 June 2004; accepted 24 June 2004
Available online 23 July 2004

Abstract

Sperm capacitation and acrosome reaction are essential for fertilization and they are considered as part of an oxidative process involving
superoxide and hydrogen peroxide. In human spermatozoa, the amino acid l-arginine is a substrate for the nitric oxide synthase (NOS)
producing nitric oxide (NO!), a reactive molecule that participates in capacitation as well as in acrosome reaction. l-Arginine plays an
important role in the physiology of spermatozoa and has been shown to enhance their metabolism and maintain their motility. Moreover, l-
arginine has a protective effect on spermatozoa against the sperm plasma membrane lipid peroxidation. In this paper, we have presented, for
the first time, the effect of l-arginine on cryopreserved bovine sperm capacitation and acrosome reaction and the possible participation of
NOS in both processes. Frozen-thawed bovine spermatozoa have been incubated in TALP medium with different concentrations of l-
arginine and the percentages of capacitated and acrosome reacted spermatozoa have been determined. l-Arginine induced both capacitation
and acrosome reaction. NO! produced by l-arginine has been inhibited or inactivated using NOS inhibitors or NO! scavengers in the
incubation medium, respectively. Thus, the effect of NOS inhibitors and NO! scavengers in capacitated and non-capacitated spermatozoa
treated with l-arginine has also been monitored. The data presented suggest the participation of NO!, produced by a sperm NOS, in
cryopreseved bovine sperm capacitation and acrosome reaction.
D 2004 Elsevier B.V. All rights reserved.

Keywords: l-Arginine; Nitric oxide; Nitric oxide synthase; Sperm capacitation; Acrosome reaction

1. Introduction reaction, an essential event for oocyte fertilization [4].

Mammalian spermatozoa must undergo a series of
membranous and metabolic changes before they can
fertilize the egg. These physiological changes represent a
complex process called capacitation [1–3]. Various in vitro
studies have indicated that the process of capacitation is
biochemical in nature. Capacitation regulates transient
changes in the sperm motility pattern, termed hyper-
activation, and enables the exocytotic event of acrosome
* Corresponding author. Present address: Urology Research Labora-
tory, Room H6.44, Royal Victoria Hospital, McGill University, 687 Ave des
Pins Ouest, Montreal, Quebec, Canada H3A 1A1. Tel.: +1 514 842
1231x35429.
E-mail address: coflaher@yahoo.com (C. O’Flaherty).
Though the capacitation is achieved synergistically and
efficiently in the female reproductive tract, it can also be
accomplished in vitro in various well-defined media for
several species of mammals.
Nitric oxide (NO!), a highly reactive oxygen species
(ROS), has been found in several physiological systems
and plays a decisive role in regulating multiple functions
within the male as well as female reproductive systems. It
is derived from l-arginine by the enzyme nitric oxide
synthase (NOS) [5], using oxygen and different cofactors
such as nicotinamide adenine dinucleotide phosphate,
flavin mononucleotide, flavin adenine dinucleotide, and
tetrahydrobiopterin, as well as calmodulin and calcium [6].
These enzymes are found on the acrosome and tail region
of non-capacitated spermatozoa [7,8]. At low concentra-

0304-4165/$ - see front matter D 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.bbagen.2004.06.020
216 C. O’Flaherty et al. / Biochimica et Biophysica Acta 1674 (2004) 215–221
tions, NO! improves sperm motility. Besides promoting
sperm motility, NO! is also known to enhance capacitation
and acrosome reaction in mouse and human spermatozoa
[9–13]. However, higher concentrations of l-arginine can
have adverse effect on motility and fertility of human [14]
and rat [15] spermatozoa. Bovine sperm capacitation and
acrosome reaction are both essential for fertilization and
are considered parts of an oxidative process that involves
the superoxide anion (O2 S) and hydrogen peroxide (H2O2)
[16–18].
l-Arginine plays an important role in the physiology of
goat spermatozoa and has been shown to enhance the cell
metabolism severalfold [19,20]. It also has a protective
effect against lipid peroxidation on these cells [21].
Although elevated doses of l-arginine reversibly inhibit
fertility in rats [15], it has been suggested that l-arginine
deficiency decreases sperm motility [22]; the administra-
tion of l-arginine to oligospermic patients improves both
the sperm count and motility without any side effects
[23,24]. Since l-arginine increases the production of ROS
[25], collectively these reports suggest the involvement of
NO! in capacitation and acrosome reaction.
The aim of this report was to investigate the effect of l-
arginine on capacitation and acrosome reaction on cryopre-
served bovine spermatozoa and to establish the possible
participation of NO! in these processes.
2. Materials and methods
2.1. Materials
All chemicals employed were of the highest commer-
cially available purity. l-Arginine, chlortetracycline (CTC),
fructose, glycine, glycerol, trypan blue, sodium chloride,
potassium chloride, sodium phosphate monobasic mono-
hydrate, magnesium chloride hexahydrate, sodium bicar-
bonate, calcium chloride, bovine serum albumin (BSA,
fraction V), sodium lactate, sodium pyruvate, HEPES, N N-
nitro-l-arginine (l-NA) and N G-nitro-l-arginine methyl
ester (l-NAME), methylene blue, hemoglobin (from
bovine blood) and lysophosphatidylcholine (LPC, purified
from egg yolk) were supplied by Sigma Co. (St. Louis,
MO, USA). Heparin (purified from porcine intestinal
mucosa) was purchased from Calbiochem Co. (La Jolla,
CA, USA).
2.2. Semen freezing
Semen was collected from four pedigree Holstein bulls
(4 to 5 years old) of proven fertility using an artificial
vagina. The bulls belonged to a controlled program of
artificial insemination and were maintained under uniform
nutritional conditions and management during the period
of research. For all ejaculates the progressive motility was
greater than 70% and the percentage of abnormal
spermatozoa was less than 20%. Semen freezing was
performed as described previously [16]. Briefly, two
ejaculates from each bull were obtained once a week for
12 weeks; they were pooled and diluted in a buffer
containing 0.20 mM Tris, 0.06 mM citrate, 0.12 mM
glycine and 0.06 mM fructose. Twenty percent egg yolk
and 7% glycerol were used at a 2:1 ratio. Final cell
concentration was maintained within 3.0–4.5107 sperm/
ml. A slow cooling to 5 8C (at the rate 1 8C/min) was
performed, and the semen was then equilibrated at 5 8C for
a further 90 min. Pellets were then obtained on dry ice at
76 8C and stored in liquid nitrogen at 196 8C.
2.3. Sample preparation
Frozen semen samples from four bulls were thawed for
1 min in Tyrode’s albumin lactate pyruvate (TALP)
medium containing 99 mM NaCl, 3.1 mM KCl, 0.35
mM NaH 2 PO 4 d H 2 O, 10 mM HEPES, 1.1 mM
MgCld 6H2O, 25 mM NaHCO3, 1 mM sodium pyruvate,
21.6 mM sodium lactate, at pH 7.4 and 36 8C [26]. It was
then equilibrated for 5 min followed by centrifugation at
600g for 5 min to separate the seminal plasma and
buffer. The pellet thus obtained was washed in the same
medium and centrifuged as stated above. The final pellet
was resuspended in TALP medium containing 2 mM
CaCl2 and 6 mg/ml BSA, to a final concentration of
1.5107 spermatozoa/ml. Aliquots for different treatments
were incubated at 38 8C (bovine body temperature) under
5% CO2 in humidified air for 45 min.
2.4. Determination of sperm concentration, viability and
progressive motility
Sperm concentration was determined using a Neubauer
chamber (Reichert Buffalo, NY, USA). Progressive motility
and viability were determined in spermatozoa incubated
with different concentrations of l-arginine (10 to 50 mM) in
TALP medium at pH 7.4. A drop of each sample was placed
and covered over a thermostatized slide at 37 8C and the
percentage of progressively motile spermatozoa was eval-
uated by direct light microscopy at 400 magnification. The
sperm viability was determined at 400 magnification using
0.25% trypan blue [16]. Two hundred cells were counted for
each treatment.
2.5. Determination of capacitation and acrosome reaction
Percentage of capacitated and acrosome reacted sper-
matozoa was determined by the chlortetracycline (CTC)
epifluorescence assay [27,28]. Samples were evaluated
using an epifluorescence microscope (Carl Zeiss Jenamed
2, Jena, Germany) at 400 magnification (filter block
Carlz Zeiss, excitation at 410 nm). The percentage of
capacitated (pattern B) and acrosome reacted (pattern AR)
spermatozoa were calculated by subtracting the values
 C. O’Flaherty et al. / Biochimica et Biophysica Acta 1674 (2004) 215–221 217

obtained from the control and treated samples at zero time deviation, were analyzed by ANOVA and Bonferroni
in order to rule out the basal percentage of pattern B and tests. A Pb0.05 value was regarded as statistically
AR immediately after the freezing-thawing process. Two significant.
hundred cells were counted for each treatment.

2.6. Effect of l -arginine on bovine spermatozoa 3. Results

Spermatozoa were incubated in the presence of different
concentrations of l-arginine (1.25 to 30 mM). Heparin (10
IU/ml), a well-known capacitation inducer of bovine
spermatozoa [26], was used as a control for capacitation.
Samples without heparin were run at the same time. All the
samples were incubated at 388C for 45 min under
humidified atmosphere [29] and the percentage of capaci-
tation was determined by the CTC assay.
2.7. Effect of l -arginine on acrosome reaction in heparin-
capacitated spermatozoa
Spermatozoa previously capacitated with heparin (10 IU/
ml) were incubated with different concentrations of l-
arginine (1.25 to 30 mM) for 15 min and the percentage of
acrosome reaction was determined by CTC assay. An
aliquot of capacitated spermatozoa was incubated with
LPC (0.2 mM) for additional 15 min and used as a control.
2.8. Effect of NOS specific inhibitors on l -arginine-induced
sperm capacitation and acrosome reaction
3.1. Effect of l -arginine on sperm viability and progressive
motility
Nitric oxide can increase or decrease sperm motility
depending on its concentration. Therefore, we first studied
the effect of l-arginine on sperm viability and progressive
motility. The sperm viability was measured at zero time,
after 45 min of incubation alone (control), after incuba-
tion with heparin for 45 min, and on incubation with
different concentrations of l-arginine for 45 min. It was
observed that viability of the control sample (incubated
for 45 min) was maintained with time compared to that
observed at zero time. Secondly, the viability remains
unaffected at all concentrations of l-arginine compared to
control and heparin-treated cells.
Sperm progressive motility as a function of l-arginine
concentration is presented in Fig. 1B. It was observed that
at lower concentrations (10 mM or lower), l-arginine
maintained the sperm motility compared to the control.
However, at higher concentrations motility was signifi-
cantly decreased.

Spermatozoa were incubated in the presence of 10 mM

l-arginine with or without two specific inhibitors, l-NA and
l-NAME, both at 1 mM concentrations [12,30]. To assess
the effect of the inhibitors on l-arginine-induced acrosome
reaction, spermatozoa were capacitated with 10 IU/ml
heparin for 45 min at 38 8C, prior to the incubation with
l-arginine (10 mM) with or without l-NA and l-NAME
inhibitors for 15 min.

2.9. Effect of NO! scavengers on l -arginine-induced sperm

capacitation and acrosome reaction

Spermatozoa were incubated with 10 mM l-arginine in

the presence or absence of NO! scavengers, namely, 29 mg/
ml hemoglobin [31] or 100 mM methylene blue [32].
Further, to understand if NO! is also involved in the l-
arginine-induced acrosome reaction, spermatozoa were
capacitated with 10 IU/ml heparin for 45 min prior to the
incubation with 10 mM l-arginine for 15 min, with or
without 29 mg/ml hemoglobin or 100 mM methylene blue,
and the percentage of acrosome reaction was determined by
the CTC assay.

2.10. Statistical analysis

Fig. 1. Percentage of viability (A) and progressive motility (B) in sperm
sample at time zero, incubated for 45 min alone (control), with heparin (10
Percentages of capacitation and acrosome reaction IU/ml) or with different concentrations of l-arginine (10 to 50 mM).
obtained by CTC assay, presented as meansFstandard *Values higher than all others ( Pb0.05); n=4.
218 C. O’Flaherty et al. / Biochimica et Biophysica Acta 1674 (2004) 215–221

was comparable to that of LPC (0.2 mM) (Fig. 2B).

However, higher concentrations of l-arginine (beyond 10
mM) did not show any significant effect on acrosome
reaction.

3.3. NOS and NO! are involved in bovine sperm capacita-

tion and acrosome reaction

To know if the effect of l-arginine on bovine sperm

capacitation process and acrosome reaction is mediated by
NOS, the above experiments were performed after inhibit-
ing the enzyme activity using known inhibitors. Thus,
spermatozoa were incubated with l-arginine (10 mM) in the
presence of two NOS inhibitors (l-NA and l-NAME).
Results indicate a significant inhibition of capacitation in the
presence of the two inhibitors (1 mM) (Fig. 3A). A similar
inhibition has been observed for acrosome reaction induced
by l-arginine in heparin-capacitated spermatozoa (Fig. 3B).
Fig. 2. Effect of the addition of l-arginine during the incubation of bovine
The concentration of each inhibitor used did not affect the
spermatozoa in TALP medium. (A) Percentage of capacitation when cells viability and the progressive motility of spermatozoa (data
are incubated for 45 min in TALP in the presence of different not shown).
concentrations of l-arginine and (B) percentage of acrosome reaction in In order to support the above results and to determine
heparin-treated spermatozoa incubated with different concentrations of l- whether NO! is involved in the l-arginine-induced capaci-
arginine. LPC: heparin-treated spermatozoa incubated with LPC (100 mg/
ml) for 15 min. *Values higher than all others ( Pb0.05); n=4.
tation, the experiments were performed in the presence of
NO! scavengers. For this purpose, the effect of l-arginine
on capacitation and acrosome reaction in spermatozoa was
3.2. l -Arginine induced sperm capacitation and acrosome monitored in the presence of methylene blue and hemoglo-
reaction in bovine spermatozoa bin (both NO! scavengers). The results indicate inhibition of
both capacitation and acrosome reaction, which otherwise
In order to know the effect of l-arginine on capacitation,
cells were incubated for 45 min in TALP medium with
different concentrations of the amino acid. Cells incubated
in TALP medium alone and those treated for 45 min with
heparin were used for comparison. It was observed that l-
arginine promoted capacitation at all concentrations (used in
the present experiment) compared to the control sample
(Fig. 2A). The extent of capacitated spermatozoa is about
fourfold higher (40% at 30 mM l-arginine concentration)
compared to the control sample (10%). At lower concen-
trations (1.25 to 5 mM), the effect of l-arginine was
comparable to that of the known inducer heparin. At higher
concentrations (40–50 mM), l-arginine had an adverse
effect on capacitation (data not shown). This is in
accordance with our earlier report on the effect of higher
concentration of l-arginine on sperm metabolism [19].
As described above for sperm capacitation, similar
experiments were carried out to test the hypothesis that l-
arginine could induce the acrosome reaction in spermatozoa
previously capacitated with heparin. Only cells in TALP
medium were used as control and cells previously capaci-
tated with heparin and incubated with LPC, a known
inducer for acrosome reaction in bovine spermatozoa [26], Fig. 3. Effect of NOS inhibitors on l-arginine-induced sperm capacitation
were used for comparison. The results indicate that the and acrosome reaction. (A) Spermatozoa incubated with l-arginine (10
mM) alone ( ) or with the NOS inhibitors l-NA or l-NAME (1 mM each)
percentage of spermatozoa with reacted acrosomes for 45 min. (B) Heparin-treated spermatozoa incubated with l-arginine (10
increased when cells were treated with l-arginine as mM) alone ( ) or in the presence of l-NA or l-NAME, for 15 min.
compared to the control. At 10 mM of l-arginine, the effect *Values higher than all others ( Pb0.05); n=4.
 C. O’Flaherty et al. / Biochimica et Biophysica Acta 1674 (2004) 215–221
S
Fig. 4. Effect of NO scavengers on l-arginine-induced sperm capacitation
and acrosome reaction. (A) Spermatozoa incubated with l-arginine (10
mM) alone ( ) or with methylene blue (100 mM) or hemoglobin (29 mg/
ml) for 45 min. (B) Heparin-treated spermatozoa incubated with l-arginine
(10 mM) alone ( ) or with methylene blue (100 mM) or hemoglobin (29
mg/ml). *Values higher than all others ( Pb0.05); n=4.
would have been occurred if l-arginine alone was present in
the incubation medium (Fig. 4A and B).
4. Discussion
The participation of O2 S and H2O2 in bovine sperm
capacitation and acrosome reaction was previously demon-
strated [16–18]. In the present report, we suggest a positive
effect of l-arginine on bovine sperm function. This amino
acid induced sperm capacitation and acrosome reaction
altering neither progressive motility nor viability of bovine
spermatozoa.
Nitric oxide, derived from l-arginine by a reaction
catalyzed by the enzyme NOS [5], plays a decisive role in
regulating multiple functions within the male as well as
female reproductive systems. In this study, viability of the
cells remains unchanged with time (as compared to time
zero) as well as at all concentrations of l-arginine (Fig. 1A).
This finding indicates that l-arginine does not has any
adverse effect on viability of the cells throughout the time of
the experiment. Therefore, all the experiments were
performed on viable samples.
Low concentration of NO! improves sperm motility [8].
However, higher concentrations of l-arginine can have
adverse effect on motility and fertility of human [14] and rat
[15] spermatozoa. We observed that, at low concentrations
219
of l-arginine, motility was maintained. However, higher
concentration of l-arginine significantly decreased the
progressive motility of bovine spermatozoa (Fig. 1B), in
accordance with previous studies performed in other species
[8,14,15]. This result is in contrast to the effect of l-arginine
on motility of goat epididymal spermatozoa obtained within
1 h of sacrifice of the animal, where a direct correlation
between sperm motility and rate of metabolism or ATP
generation in these cells has been established [19]. This
discrepancy could be attributed to the higher susceptibility
of cryopreserved bovine spermatozoa to high concentrations
of ROS [29].
NO! is a vital molecule, produced in the male repro-
ductive tract of several species and participates in repro-
duction [9,33–35]; moreover, it is well known as a
requirement of ROS for capacitation and acrosome reaction
[9–13] and for in vitro fertilization [36,37]. l-Arginine is a
known precursor for the synthesis of nitric oxide by the
enzyme NOS in various biological systems including
spermatozoa [7,8]. This is achieved by the conversion of
l-arginine to l-citrulline and NO!; this reaction is inhibited
by l-arginine analogs such as l-NA and l-NAME [12,30].
We have shown that l-arginine induces both capacitation
and acrosome reaction in cryopreserved bovine spermatozoa
(Fig. 2). Further, we have observed that hemoglobin and
methylene blue (scavengers of NO!) and l-NA and l-
NAME (inhibitors of NOS) prevent the induction due to l-
arginine. This supports the possible role of NO!, which is
generated due to the action of NOS on l-arginine during
these processes. These results are in accordance with the
earlier observation on the effect of l-arginine on metabolism
of goat spermatozoa [19] where a mechanism of action of l-
arginine through the NO! production was proposed and the
positive effect of NO! on sperm capacitation and acrosome
reaction [12,13,30].
The fact that high concentrations of l-arginine (40–50
mM) inhibit the progressive motility without altering the
sperm viability suggests that a higher concentration of NO!
produced by the amino acid could render the spermatozoon
immotile. This finding is supported by the observation made
by Roselli et al. [14] and Weinberg et al. [38], where high
concentration of nitric oxide decreases motility and induces
toxicity in human spermatozoa. Another plausible reason for
the reduced motility may be a significantly reduced cAMP
concentration at higher concentrations of NO! [39], an
important requisite for the motility machinery.
The results presented here suggest, for the first time, that
l-arginine promotes sperm capacitation and acrosome
reaction in bovine spermatozoa. Although l-arginine might
induce a premature capacitation and/or acrosome reaction
affecting the fertilization rates in the artificial insemination
programs, this amino acid maintains the motility and
viability of spermatozoa and could be added (at low
concentration) to the semen extender.
The mechanism of l-arginine action is through the
production of NO! in the presence of NOS, and it was
220 C. O’Flaherty et al. / Biochimica et Biophysica Acta 1674 (2004) 215–221
confirmed by the inhibition of the enzyme action as well
as by scavenging the NO! free radical. A similar role of
NO! has been demonstrated in human spermatozoa where
this ROS has been shown to trigger tyrosine phosphor-
ylation [39] and double serine/threonine phosphorylation
[40], both events being closely related with capacitation.
Here, we have made an attempt to indicate, for the
first time, the role of NO! on bovine sperm function. This
free radical is involved in capacitation and acrosome
reaction, indispensable processes required for the acquis-
ition of the fertilizing ability of bovine spermatozoon.
Further studies are being conducting to better understand
the participation of NO! in the signaling pathways of
these processes.
Acknowledgements
We are grateful to Genética Los Nogales for semen
sample supplies. This research was supported by the
Secretarı́a de Ciencia y Técnica de la Universidad de
Buenos Aires (V016, UBACYT).
References
[1] C.R. Austin, The capacitation of the mammalian sperm, Nature
(Lond.) 170 (1952) 326.
[2] E. de Lamirande, P. Leclerc, C. Gagnon, Capacitation as a regulatory
event that primes spermatozoa for the acrosome reaction and
fertilization, Mol. Hum. Reprod. 3 (1997) 175 – 194.
[3] P.E. Visconti, V.A. Westbrook, O. Cherithin, I. Demarco, S. Sleight,
A.B. Diekman, Novel signalling pathways involved in sperm
acquisition of fertilizing capacity, J. Reprod. Immunol. 53 (2002)
133 – 150.
[4] R. Yanagimachi, Mammalian fertilization, in: E. Knobil, J.D. Neill
(Eds.), The Physiology of Reproduction, Raven Press, New York,
1994, pp. 189 – 318.
[5] R.M.J. Palmer, D.S. Ashton, S. Moncada, Vascular endothelial cells
synthesize nitric oxide from l-arginine, Nature 33 (1988) 664 – 668.
[6] S. Moncada, E.A. Higgs, Molecular mechanisms and therapeutic
strategies related to nitric oxide, FASEB J. 9 (1995) 1319 – 1330.
[7] M.B. Herrero, S. Perez Martinez, J.M. Viggiano, J.M. Polak,
Localization by indirect immunofluorescence of nitric oxide synthase
in mouse and human spermatozoa, Reprod. Fertil. Dev. 8 (1996)
931 – 934.
[8] S.E.M. Lewis, E.T. Donnelly, E.S.L. Sterling, M.S. Kennedy, W.
Thompson, U. Chakravarthy, Nitric oxide synthase and nitrite
production in human spermatozoa: evidence that endogenous nitric
oxide is beneficial to sperm motility, Mol. Hum. Reprod. 2 (1996)
873 – 878.
[9] A. Zini, E. de Lamirande, C. Gagnon, Low levels of nitric oxide
promote human sperm capacitation in vitro, J. Androl. 16 (1995)
424 – 431.
[10] M.B. Herrero, J.M. Viggiano, S. Perez Martinez, M.F. de Gimeno,
Evidence that nitric oxide synthase is involved in progesterone-
induced acrosomal exocytosis in mouse spermatozoa, Reprod. Fertil.
Dev. 9 (1997) 433 – 439.
[11] K. Sengoku, K. Tamate, T. Yoshida, Y. Takaoka, T. Miyamoto, M.
Ishikawa, Effects of low concentrations of nitric oxide on the zona
pellucida binding ability of human spermatozoa, Fertil. Steril. 69
(1998) 522 – 527.
[12] M.B. Herrero, E. de Lamirande, C. Gagnon, Nitric oxide regulates
human sperm capacitation and protein-tyrosine phosphorylation in
vitro, Biol. Reprod. 61 (1999) 575 – 581.
[13] A. Revelli, C. Costamagna, F. Moffa, E. Aldieri, S. Ochetti, A. Bosia,
M. Massobrio, B. Lindblom, D. Ghigo, Signaling pathway of nitric
oxide-induced acrosome reaction in human spermatozoa, Biol.
Reprod. 64 (2001) 1708 – 1712.
[14] M. Rosselli, R.K. Dubey, B. Imthurn, E. Macas, P. Keller, Effects of
nitric oxide on human spermatozoa: evidence that nitric oxide
decreases sperm motility and induces sperm toxicity, Hum. Reprod.
10 (1995) 1786 – 1790.
[15] W.D. Ratnasooriya, M.G. Dharmasiri, l-Arginine, the substrate of
nitric oxide synthase, inhibits fertility of male rats, Asian J. Androl. 3
(2001) 97 – 103.
[16] C.M. O’Flaherty, N.B. Beorlegui, M.T. Beconi, Reactive oxygen
species requirements for bovine sperm capacitation and acrosome
reaction, Theriogenology 52 (1999) 289 – 301.
[17] C. O’Flaherty, N. Beorlegui, M. Beconi, Role of superoxide anion and
hydrogen peroxide in acrosome reaction of bovine spermatozoa, in: B.
Robaire, H. Chemes, C. Morales (Eds.), Andrology in the 21st
Century, Medimond Publications, Milan, 2001, pp. 103 – 108.
[18] C. O’Flaherty, N. Beorlegui, M. Beconi, Participation of superoxide
anion in the capacitation of cryopreserved bovine sperm, Int. J.
Androl. 26 (2003) 109 – 114.
[19] A.B. Patel, S. Srivastava, R.S. Phadke, G. Govil, Arginine activates
glycolysis of goat epididymal spermatozoa: an NMR study, Biophys.
J. 75 (1998) 1522 – 1528.
[20] A.B. Patel, S. Srivastava, R.S. Phadke, G. Govil, Arginine acts as a
protective and reversal agent against glycolytic inhibitors in sperma-
tozoa, Physiol. Chem. Phys. Med. NMR 31 (1999) 29 – 40.
[21] S. Srivastava, P. Desai, E. Couthinho, G. Govil, Protective effect of l-
arginine against lipid peroxidation in goat epididymal spermatozoa,
Physiol. Chem. Phys. Med. NMR 32 (2000) 127 – 135.
[22] D.W. Keller, K.L. Polakoski, l-Arginine stimulation of human sperm
motility in vitro, Biol. Reprod. 13 (1975) 154 – 157.
[23] M. Scibona, P. Maschini, S. Capparelli, C. Pecori, P. Rossi, G.F.M.
Fabris, l-Arginine and male infertility, Minerva Urol. Nefrol. 46
(1994) 251 – 253.
[24] S. Aydin, O. Inci, B. Alagol, The role of arginine, indomethacin and
kallikrein in the treatment of oligoasthenospermia, Int. Urol. Nephrol.
27 (1995) 199 – 202.
[25] S.S. Gross, M.S. Wolin, Nitric oxide: pathophysiological mechanisms,
Annu. Rev. Physiol. 57 (1955) 737 – 769.
[26] J.J. Parrish, J.L. Susko-Parrish, M.A. Winer, N.L. First, Capacitation
of bovine sperm by heparin, Biol. Reprod. 38 (1988) 1171 – 1180.
[27] L.R. Fraser, L.R. Abeydeera, K. Niwa, Ca(2+)-regulating mechanisms
that modulate bull sperm capacitation and acrosomal exocytosis as
determined by chlortetracycline analysis, Mol. Reprod. Dev. 40
(1995) 233 – 241.
[28] N. Beorlegui, P. Cetica, G. Trinchero, M. Cordoba, M. Beconi,
Comparative study of functional and biochemical parameters in frozen
bovine sperm, Andrologia 29 (1997) 37 – 42.
[29] C. O’Flaherty, N. Beorlegui, M. Beconi, Effect of natural antioxidants,
superoxide dismutase and hydrogen peroxide on capacitation of
frozen-thawed bull spermatozoa, Andrologia 29 (1997) 269 – 275.
[30] A. Revelli, G. Soldati, C. Costamagna, O. Pellerey, E. Aldieri, M.
Massobrio, A. Bosia, D. Ghigo, Follicular fluid proteins stimulate
nitric oxide (NO!) synthesis in human sperm: a possible role for NO!
in acrosomal reaction, J. Cell. Physiol. 178 (1999) 85 – 92.
[31] M.B. Herrero, E. Cebral, M. Boquet, J.M. Viggiano, A. Vitullo, M.A.
Gimeno, Effect of nitric oxide on mouse sperm hyperactivation, Acta
Physiol. Pharmacol. Ther. Latinoam. 44 (1994) 65 – 69.
[32] E.T. Donnelly, S.E. Lewis, W. Thompson, U. Chakravarthy, Sperm
nitric oxide and motility: the effects of nitric oxide synthase
stimulation and inhibition, Mol. Hum. Reprod. 3 (1997) 755 – 762.
[33] V.D. Dixit, N. Parvizi, Nitric oxide and the control of reproduction,
Anim. Reprod. Sci. 65 (2001) 1 – 16.
 C. O’Flaherty et al. / Biochimica et Biophysica Acta 1674 (2004) 215–221
[34] S.M. McCann, V. Rettori, The role of nitric oxide in reproduction,
Proc. Soc. Exp. Biol. Med. 211 (1996) 7 – 15.
[35] W.D. Ratnasooriya, M.G. Dharmasiri, R.M. Wadsworth, Reduction in
libido and fertility of male rats by administration of the nitric oxide
(NO) synthesis inhibitor N-nitro-l-arginine methyl ester, Int. J.
Androl. 23 (2000) 187 – 191.
[36] M.B. Herrero, J.M. Viggiano, S. Perez Martinez, M.F. de Gimeno,
Effects of nitric oxide synthase inhibitors on the outcome of in vitro
fertilization in the mouse, Reprod. Fertil. Dev. 8 (1996) 301 – 304.
[37] F. Francavilla, R. Santucci, B. Macerola, G. Ruvolo, R. Romano,
Nitric oxide synthase inhibition in human sperm affects sperm–
221
oocyte fusion but not zona pellucida binding, Biol. Reprod. 63
(2000) 425 – 429.
[38] J.B. Weinberg, E. Doty, J. Bonaventura, A.F. Haney, Nitric oxide
inhibition of human sperm motility, Fertil. Steril. 64 (1995) 408 – 413.
[39] M.B. Herrero, S. Chatterjee, L. Lefièvre, E. de Lamirande, C. Gagnon,
Nitric oxide interacts with the cAMP pathway to modulate capacitation
of human spermatozoa, Free Radic. Biol. Med. 29 (2000) 522 – 536.
[40] J. Thundathil, E. de Lamirande, C. Gagnon, Nitric oxide regulates the
phosphorylation of the threonine-glutamine-tyrosine motif in proteins
of human spermatozoa during capacitation, Biol. Reprod. 68 (2003)
1291 – 1298.