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Characterization of an L-Amino Acid Oxidase in Equine Spermatozoa

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DOI: 10.1095/biolreprod.114.126052 · Source: PubMed

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BIOLOGY OF REPRODUCTION (2015) 92(5):125, 1–13
Published online before print 4 March 2015.
DOI 10.1095/biolreprod.114.126052
Characterization of an L-Amino Acid Oxidase in Equine Spermatozoa1
Joanna B. Aitken,3 Nenad Naumovski,4 Ben Curry,4 Christopher G. Grupen,3 Zamira Gibb,4 and R. John
Aitken2,4
3
Faculty of Veterinary Science, University of Sydney, Camperdown, New South Wales, Australia
4
Priority Research Centre for Reproductive Science, Discipline of Biological Sciences, Faculty of Science and IT,
University of Newcastle, Callaghan, New South Wales, Australia
ABSTRACT
This study demonstrates for the first time the presence of an L-
amino acid oxidase (LAAO) enzyme in equine spermatozoa that
is able to generate significant amounts of reactive oxygen
species (ROS) and create a state of oxidative stress. RT-PCR
analysis revealed that the mRNA for this enzyme was present in
the equine testis and spermatozoa, while immunocytochemical
studies demonstrated that the mature LAAO protein was located
in the sperm head, particularly in the acrosomal and post-
acrosomal domains. Experimental studies demonstrated that the
aromatic amino acids (L-phenylalanine . L-tryptophan . L-
tyrosine) were substrates for this enzyme, eliciting the dose- and
time-dependent generation of ROS via mechanisms that were
enhanced by cell death. This unexpected result was confirmed
by analyses of ROS generation in subcellular sperm fractions,
which again located a majority of LAAO activity to the sperm
head. Equine cryopreservation medium was shown to contain
sufficient quantities of aromatic amino acids to activate the
LAAO system and generate ROS. The biological significance of
this activity was established in an experiment in which
physiological concentrations of aromatic amino acids were
found to suppress sperm motility but only if dead spermatozoa
were present in the same suspension. The combination of
aromatic amino acids and nonviable cells was also found to
enhance the levels of lipid peroxidation in live spermatozoa.
These results suggest the potential significance of LAAO activity
in generating the oxidative stress associated with the cryopres-
ervation of equine spermatozoa. It is possible that inhibitors of
this enzyme system may facilitate the development of modified
cryostorage regimes for clinical validation in vivo.
cryopreservation, gamete biology, oxidative stress, sperm, sperm
motility and transport
INTRODUCTION
Research on the molecular mechanisms regulating sperm
function have emphasized the importance of redox chemistry in
both promoting the acquisition of functional competence
during capacitation and disrupting the fertilizing potential of
mammalian spermatozoa in cases of infertility [1–4]. Reactive
oxygen species (ROS) such as the superoxide anion and
1
Supported by the ARC Linkage grant program, ARC Linkage grant
LP120100219.
2
Correspondence: R. John Aitken, Discipline of Biological Sciences,
Faculty of Science and IT, University of Newcastle, Callaghan, NSW
2308, Australia. E-mail: john.aitken@newcastle.edu.au
Received: 28 October 2014.
First decision: 24 November 2014.
Accepted: 19 February 2015.
Ó 2015 by the Society for the Study of Reproduction, Inc.
eISSN: 1529-7268 http://www.biolreprod.org
ISSN: 0006-3363
1 Article 125
hydrogen peroxide (H2O2) promote capacitation via several
mechanisms, including the activation of adenylyl cyclase [5,
6], the suppression of tyrosine phosphatase [6, 7], and the
oxidation of sterols [8]. Conversely, ROS impair sperm
function through the rapid depletion of ATP, the induction of
peroxidative damage to the sperm plasma membrane, and the
promotion of DNA fragmentation in the sperm nucleus [9–13].
It has been proposed that these events commonly represent a
continuum, such that the ROS production that drives
capacitation will, when sustained, overwhelm the limited
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antioxidant defenses of these cells and create a state of
oxidative stress culminating in senescence and initiation of the
intrinsic apoptotic cascade [14]. Oxidative stress can also be
created by a variety of additional factors, including antioxidant
depletion [15], cryostorage [16], exposure to redox active
metals [17, 18], electromagnetic radiation [19], and a wide
variety of xenobiotics [20].
The induction of oxidative stress in spermatozoa is
commonly associated with the enhanced production of ROS.
Thus, increased levels of ROS generation have been observed
in association with sperm damage or abnormality in several
species, including man [21], bull [22, 23], horse [24], and
mouse [25]. However, the source of ROS production under
such circumstances has not been fully resolved. The situation is
clearly complex in that spermatozoa are known to possess a
variety of redox systems that are potentially capable of
generating reactive oxygen metabolites [26]. For example,
mitochondria are known to be powerful generators of ROS and
actively produce oxygen free radicals when these cells engage
the intrinsic apoptotic cascade [27]. Equine and human
spermatozoa are also known to possess NADPH oxidases
such as Nox 5 that are potentially capable of generating ROS,
although definitive evidence that this enzyme is actually
responsible for ROS generation in spermatozoa is currently
lacking [28, 29]. In this paper, we have investigated yet another
ROS-generating system in mammalian spermatozoa that was
originally discovered by Tosic and Walton more than half a
century ago in bovine spermatozoa [30] but has since received
very little attention. The enzyme responsible for this activity
was an L-amino acid oxidase (LAAO) capable of deaminating
aromatic amino acids with the generation of ammonia and
H2O2. In this study, we have investigated the possibility that
equine spermatozoa might also possess a similar oxidase
system with the potential to generate ROS and thereby
influence the functionality of these cells.
MATERIALS AND METHODS
Materials
Unless otherwise stated, all the chemicals were purchased from Sigma-
Aldrich. Biggers, Whitten, and Whittingham (BWW) medium containing
containing 95 mM NaCl, 4.7 mM KCl, 1.7 mM CaCl2.2H2O, 1.2 mM KH2PO4,
1.2 mM MgSO4.7H2O, 25 mM NaHCO3, 5.6 mM D-glucose, 275 lM sodium
 AITKEN ET AL.

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FIG. 1. Presence of LAAO in equine testes and spermatozoa. A) Control RT-PCR for beta actin. B) RT-PCR for LAAO demonstrating a clear reaction
product in the testes and spermatozoa. C–F) Immunohistochemical analysis revealing the subcellular localization of LAAO; C and E are phase contrast
images of equine spermatozoa while D and F are the corresponding immunocytochemical images illustrating the presence of LAAO in the sperm head.
Bar ¼ 10 lm. Arrows indicate cells that lack staining over the anterior acrosome.

pyruvate, 3.7 ll/ml 60% sodium lactate syrup, 50 units (U)/ml penicillin, 50 lg/
ml streptomycin, and 0.1% (w/v) polyvinyl alcohol, with an osmolarity of 290–
310 mOsm/kg [26, 31] was used throughout these experiments. The medium
was also supplemented with 20 mM HEPES to maintain pH stability. Amino
acids, including phenylalanine, were dissolved in H2O and vortexed for 30 sec
to make stock solutions (100 mM). Working solutions (10 mM) were then
made using BWW. L-Tryptophan required heating in a water bath (1008C) for 2
min in order to facilitate solubilization. L-Tyrosine was dissolved in 1 M HCl
and was buffered to pH 7.4 using 1 M NaOH; D-tryptophan and D-tyrosine
were also treated in this manner.
Sperm Preparation
Institutional and New South Wales State Government ethical approval was
secured for the use of equine spermatozoa in this study. The guidelines set out
in the journal’s statement on the Care and Use of Experimental animals were
adhered to throughout. Equine spermatozoa were collected from pony stallions
held on institutionally approved premises using either a pony-sized Missouri
artificial vagina or a modified Hannover artificial vagina, shortened by
approximately 10 cm (Minitube Australia). The ejaculate (approximately 15
ml) was immediately diluted with two parts Kenney’s extender (272 mM
glucose, 24 mg/ml skim milk powder, 1500 U/ml penicillin, and 1.5 mg/ml
streptomycin) in 50 ml Falcon tubes [32]. The extended semen was then kept at
ambient temperature (208C–238C) in a polystyrene box for 1.5 h during
transportation back to the laboratory. The extended semen was subsequently
fractionated on a Percoll gradient using 40% and 80% Percoll (GE Healthcare)
fractions as described for human spermatozoa [26]. Highly motile equine
spermatozoa were recovered from the base of the high density region of the
gradient, washed with BWW medium, and finally resuspended at a
concentration of 2 3 107/ml. All the sperm incubations were conducted in an
incubator at 378C in capped conical bottomed, 15 ml Falcon tubes. Each
suspension was routinely monitored for viability and motility. Viability was
assessed using 5 ll of 0.5% eosin (1:1 dilution) and counting 100 cells. Any
sample that exhibited ,65% viability was excluded from the study. Motility
involved counting 100 cells under phase contrast microscopy at a magnification
of 4003. The fresh samples used in this study had an average motility of 88.8%
6 0.87% (mean 6 SEM) and average viability of 89.5% 6 2.07%. All the
subsequent motility assessments were conducted using a computer-aided sperm
analysis (CASA) system (see below).
Cryopreservation
For cryopreservation, semen was collected and diluted with Kenney’s
extender as described above; the following procedures were then performed at
room temperature (218C). Samples were first centrifuged (500 3 g for 15 min),
the supernatant was removed, and the pellet was resuspended in lactose-
ethylenediaminetetraacetic acid-egg yolk (L-EDTA-EY) cryodiluent [33] to
obtain a concentration of 400 3 106 spermatozoa/ml. An equal volume of 6%
(v/v) dimethylformamide (DMF) in L-EDTA-EY was added to the samples in a
dropwise manner to obtain a concentration of 200 3 106 spermatozoa/ml in L-
EDTA-EY containing 3% DMF). The sperm suspension was then loaded into
0.5 ml straws using a pipette and closed with sealing powder (Minitube
Australia). The straws were refrigerated for 90 min and then suspended 3 cm
above liquid nitrogen for 10 min in a Styrofoam box, before being plunged into
and stored under liquid nitrogen. These straws were subsequently thawed by
immersing in a 378C water bath for 30 sec after which the sperm suspension
was diluted to a concentration of 20 3 106 spermatozoa/ml (1:10 dilution) by
adding 2.7 ml of BWW to 300 ll of sperm preparation. Viability and motility
were assessed as described above. The responses of these sperm preparations to
phenylalanine were compared with unfractionated spermatozoa prepared from

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FIG. 2. Analyses of the ability of individual amino acids to elicit a chemiluminescent response from populations of equine spermatozoa. A–F) Each panel
presents results for cell-free control incubations and populations of fresh and snap frozen-thawed spermatozoa. For each group of amino acids tested, L-
phenylalanine (F) was consistently incorporated as a positive control. **P , 0.01, ***P , 0.001 for differences compared with untreated control sample, Cn. Above
each panel the results of an ANOVA analysis are presented for the overall comparison of viable and snap frozen-thawed populations. Abscissa presents single letter
amino acid code while the ordinate axis represents the integration of chemiluminescent counts over 180 min (n ¼ 4 independent samples for each panel).

3 Article 125
 AITKEN ET AL.
FIG. 3. The chemiluminescent signal generated in response to arginine could be completely reversed by the presence of peroxynitrite scavengers, Mn
(III) tetrakis (4-benzoic acid) porphyrin and tetra(parasulphonatephenyl)porphyrinato ferrate (III). Snap frozen-thawed cells (A) and fresh cells (B). Single
letter amino acid code for arginine (R) is presented (n ¼ 3 independent samples).
fresh ejaculates following centrifugation (15 min at 500 3 g) and resuspension
to the same concentration of 20 3 106 spermatozoa/ml. For these frozen-thawed
samples, the average viability was 81.74% 6 1.55% and motility was 77.4% 6
1.15%. For certain experiments, spermatozoa were flash-frozen in liquid
nitrogen and thawed at 378C to obtain populations of dead, snap frozen-thawed
spermatozoa.
CASA
CASA (IVOS; Hamilton Thorne Research) settings used for equine
spermatozoa were negative phase-contrast optics, recording rate of 60 frames/
sec, minimum contrast 70, minimum cell size four pixels, low size gate 0.17,
high size gate 2.9, low intensity gate 0.6, high intensity gate 1.74, nonmotile
head size 10, nonmotile head intensity 135, progressive velocity average path
(VAP) threshold value, 50 lm/sec, slow cells VAP cutoff 20 lm/sec, slow cells
velocity straight line (VSL) cutoff 0 lm/sec, and threshold straightness (VSL/
VAP 3 100) .75%. Progressive cells were those exhibiting a VAP of .50 lm/
sec and a velocity-and-path straightness of .75%. For each sperm sample, an
average of five fields were assessed to generate data on at least 200 cells.
Following the initial motility assessment on the ejaculate, all the subsequent
motility determinations were conducted using the CASA system.
Chemiluminescence
Chemiluminescence was used in this study to quantify the amount of ROS
produced with each sample. The luminol-peroxidase assay was performed on 4
3 106 spermatozoa in 400 ll BWW supplemented with 4 ll luminol (25 mM)
and 8 ll horseradish peroxidase (11.52 U/ml). The inclusion of horse radish
peroxidase focuses the assay on H2O2 generation; however, any metabolite
capable of effecting the one electron oxidation of luminol, such as
peroxynitrite, is potentially capable of activating this probe. Activation of the
luminol probe generated photons of light, which were then read on a Berthold
AutoLumat luminometer (LB-953) at 378C for 2 h. Media blanks (cell negative
and untreated controls) were run for every treatment in order to ensure that the
signals recorded were not due to the spontaneous activation of the probe. The
values obtained in these media-only control incubations were subtracted from
those obtained in the presence of spermatozoa. For certain experiments, the
spermatozoa were separated into head and tail fractions using the technique
described by Baker et al. [34] involving sonication of the cells on ice followed
by separation of heads from tails by centrifugation through 80% Percoll.
4 Article 125
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Immunocytochemistry
In order to determine the subcellular localization of LAAO, immunocy-
tochemistry was employed. Fresh cells were isolated using a Percoll gradient
and the cell pellet diluted to a concentration of 20 3 106 spermatozoa/ml. After
fixing in 2% paraformaldehyde for 15 min at 48C, cells were washed once in
phosphate-buffered saline (PBS) and then resuspended in 0.1 M glycine-PBS at
48C for storage. The primary antibody selected for this analysis was an LAAO
antibody (IL4I1, NBP1-94050; Novus Biologicals, Inc. used at a titer of 1:50).
This polyclonal antibody was raised in a rabbit against a recombinant protein
representing the human sequence (LWQTEKDDWTVPYGRIYFAGEH-
TAYPHGWVETAVKSALRAAIKINSRKGPASDTASPEGHASD-
MEGQGHVHGVASSPSHDLAKEEGSHPPVQGQLSLQNTTHT) and its
specificity verified by the supplier on a protein array containing target protein
plus 383 other nonspecific proteins. Overall, the predicted equine sequence
exhibits a 76% homology with human LAAO while the homology over the
region covered by the recombinant peptide used for antibody generation is
50%.
The secondary antibody was Alexa Fluor 488 goat anti-rabbit immuno-
globulin G (heavy and light chains) (Invitrogen used at a titer of 1:250). To
stain the cells, 1–2 3 106 fixed spermatozoa were plated onto a poly-L-lysine
coated coverslip for approximately 3 h at 48C. Cells were then permeabilized
with 0.2% Triton X-100 for 15 min at room temperature and subsequently
blocked with 3% bovine serum albumin and 10% goat serum in PBS for 1 h at
room temperature. Primary antibody was incubated overnight at 48C at a 1:50
dilution in 1% bovine serum albumin-PBS. The coverslips were then washed
three times and secondary antibody added at 1:250 dilution for 1 h. The
coverslips were finally mounted with 10% Mowiol (Calbiochem) with 30%
glycerol in 0.2 M Tris (pH 8.5) incorporating 2.5% 1,4-diazobicyclo-(2.2.2)-
octane, and the stained cells were examined by fluorescence microscopy. In
order to control for nonspecific antibody binding, negative control incubations
were conducted involving secondary antibody alone.
Preparation of RNA from Equine Testis
Equine testes tissue was homogenized in 500 ll of Trizol reagent (Life
Technologies); 100 ll of chloroform was then added to the solution and
agitated for 1 min, after which the solution centrifuged at 13 000 rpm for 20
min at 48C. The aqueous phase was then transferred to a fresh Eppendorf tube,
and an equal volume of isopropanol added and incubated for 1 h at room
temperature. Following centrifugation at 13 000 rpm for 20 min at 48C, the
 L-AMINO ACID OXIDASE

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FIG. 4. Time-dependent analyses of the ability of the aromatic amino acids to trigger a chemiluminescent response in populations of fresh (A) and snap
frozen-thawed (B) equine spermatozoa. C, D) Time- and dose-dependent response to phenylalanine in viable (C) and snap frozen-thawed (D)
spermatozoa. Single letter amino acid code is presented (n ¼ 4 independent samples).

RNA pellet was washed with 75% ethanol (made up in diethylpyrocarbonate-
treated water) and dissolved in diethylpyrocarbonate -treated water.
Preparation of RNA from Equine Spermatozoa
Spermatozoa recovered from pooled ejaculates were purified on Percoll
gradients and adjusted to 1 3 108 cells, washed twice in PBS, and pelleted.
Total RNA was prepared from the final cell pellets using Trizol reagent (Life
Technologies), as described above, except that prior to isopropanol
precipitation, 5 ll of 2 mg/ml glycogen (Ambion) was added to facilitate
RNA precipitation.
RT-PCR
To determine the presence of horse LAAO mRNA, 10 lg of total RNA was
reverse transcribed with oligo (dT)15 primer (Promega Corporation) and M-
MLV Reverse Transcriptase (Promega). RT-PCR for equine LAAO was then
performed to detect known sequences of the gene. PCR primers were designed
to detect the predicted Equus caballus LAAO sequence in the GenBank
database (GenBank accession number XM_005596742). For the first round of
PCR, the forward primer sequence was 5 0 -AGGACAAAGCCATCACCGTT-
3 0 ; the reverse primer sequence was 5 0 -CTTGCAGAGCTTGTGGAGGA-3 0 .
These primers are predicted to generate bands of 489 bp. The PCR reaction
conditions were as follows: one cycle of 948C for 5 min; 35 cycles of 958C for
45 sec, 678C for 45 sec, 728C for 2 min; and one cycle of 728C for 10 min. For
nested PCR, a 1:100 dilution of the first-round product was used as the
template, and the conditions were the same as above. For nested PCR, the
forward primer sequence was 5 0 -ACCCGACGATGACTTCTGTG-3 0 ; the
reverse primer sequence was 5 0 -GGGCTTCAAGGTCCGATTGA-3 0 . These
primers are predicted to generate bands of 241 bp. The PCR products were run
on 1.5% agarose gels, and the DNA was purified from the gel using the Wizard
Gel Clean-Up Kit (Promega). The DNA was sequenced at the Australian
Genomic Research Facility (Brisbane, Australia) to confirm that the correct
target had been amplified.
Flow Cytometry Measurements
In order to assess levels of oxidative stress in equine spermatozoa, a product
of lipid peroxidation, 4-hydroxynonenal (4HNE), was monitored by flow
cytometry. For this purpose, spermatozoa were suspended in 49 ll of BWW,
and 1 ll of anti-4HNE polyclonal rabbit antiserum (Jomar Diagnostics) was
added (final titer 1:50) for 30 min at 378C. Cells were centrifuged at 650 3 g for

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FIG. 5. Analyses of the chemiluminescent responses generated by head and tail fractions to L-phenylalanine (F). Time-dependent analysis (A) and
integrated responses (B) over 120 min. Head fractions were significantly more active than the tail fractions (**P , 0.01; n ¼ 3 independent samples). C, D)
Time-dependent analyses of the impact of DPI, a flavoprotein inhibitor, on the chemiluminescent response to F in fresh (C) and snap frozen-thawed (D)
spermatozoa (n ¼ 4 independent samples).

5 min and washed twice with BWW. They were then resuspended in 99 ll of
BWW, and 1 ll of secondary antibody (Alexa Fluor 488 goat anti-rabbit IgG)
was added (final titer 1:100) for 10 min at 378C. Cells were centrifuged again,
washed twice with BWW, and resuspended in 400 ll of BWW for analysis by
flow cytometry using a FACSCalibur flow cytometer (Becton Dickinson) with
a 488 nm argon-ion laser. Simultaneous measurements of cell viability were
achieved using the LIVE/DEAD Fixable Far Red Dead Cell stain (Invitrogen).
This probe was used at a final dilution of 1:10 000 and was added to the sperm
suspensions at the same time as the anti-4HNE antibody prior to a 30 min
incubation period and subsequent analysis by flow cytometry. Emission
measurements were made using 530/30 band pass (green/FL-1), 585/42 band
pass (red/FL-2), 661/16 band pass (red/FL-4), and .670 long pass (far red/FL-
3) filters. Debris was gated out using a forward scatter/side scatter dot plot, and
10 000 cells were analyzed per sample. All the data were analyzed using
CellQuest Pro software (Becton Dickinson).
Statistical Analysis
All the experiments were replicated at least three times on independent
samples and the results analyzed by one- and two-way ANOVA using the
Super-ANOVA program (Abacus Concepts Inc.) on a MacIntosh G4 Power-
book computer; post hoc comparison of group means was by Fisher protected
6 Article 125
least significant difference. Paired comparisons were conducted using a paired
t-test. Differences with a P value of , 0.05% were regarded as significant.
RESULTS
Confirmation of the Presence of LAAO in Stallion
Spermatozoa
A single intense reaction product of the predicted size (241
bp) was detected following nested PCR with equine testicular
RNA, which on sequencing was found to align perfectly with
the predicted equine LAAO sequence. An identical PCR
product was detected when RNA extracted from equine
spermatozoa was used as a target (Fig. 1, A and B). In order
to demonstrate that the translated protein product was also
present in these cells, we used immunocytochemistry to
confirm the presence of LAAO in mature equine spermatozoa.
This analysis revealed a clear cross reactivity with equine
spermatozoa localized to the sperm head (Fig. 1C–F). A
distinct granular labeling pattern was observed in the
acrosomal region of these cells along with a signal of varying
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FIG. 6. Comparison of L- and D-isomers of aromatic amino acids to elicit a chemiluminescent response in populations of equine spermatozoa. A)
Response kinetics of fresh cells. B) Response kinetics of snap frozen-thawed cells. C) Histogram depicting the mean chemiluminescent response integrated
over 180 min. Cn, control; L-F, L-phenylalanine; D-F, D-phenylalanine, D-W, D-tryptophan; D-Y, D-tyrosine. Combination of D-F and L-F was
significantly different compared to L-F alone (*P , 0.05, ***P , 0.001; n ¼ 4 independent samples). Single letter amino acid code is presented.

intensity in the postacrosomal domain. Spermatozoa that had
lost their acrosomal staining were labeled exclusively in the
postacrosomal region or were unlabeled (Fig. 1, C and D,
arrows). Only very faint labeling of the sperm tail was
observed in these experiments.
Generation of H2O2 in Response to Aromatic Amino Acids
The substrate specificity of equine sperm LAAO was
assessed using a sensitive chemiluminescence method to
determine the production of H2O2. Each chemiluminescence
run included cell-free controls and Percoll-purified populations
of spermatozoa that were either viable (viability .90%;
motility .80%) or had been rendered nonviable by a single
freeze-thaw cycle. An initial screen was conducted at a single
dose (1 mM) employing all natural amino acids with the
exception of cysteine, which is redox active in the luminol
peroxidase system, generating intense chemiluminescent sig-
nals in the absence of spermatozoa. Of the remaining amino
7 Article 125
acids, phenylalanine was associated with significantly greater
chemiluminescence (P , 0.001) than any other amino acid
tested and was incorporated into every run as a positive control
(Fig. 2A). The other amino acids to exhibit activity were
tryptophan (W) and, to a lesser extent, tyrosine (Y) (Fig. 2F), in
keeping with predicted substrate specificity of this aromatic
amino acid oxidase and previous observations on bovine
spermatozoa [35, 36]. The only other active substrate was the
nonaromatic amino acid, arginine (R), which also exhibited a
signal in the chemiluminescent system (Fig. 2, A and B),
possibly because this compound is a substrate for nitric oxide
synthase and, in the presence of superoxide anion, NO is
known to generate a positive signal in the presence of luminol
as a consequence of peroxynitrite formation [37]. Consistent
with this observation, we found that the chemiluminescent
signal generated in response to arginine could be reversed by
the presence of peroxynitrite scavengers, Mn (III) tetrakis (4-
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FIG. 7. Comparison of the chemiluminescent responses generated by populations of freshly prepared and cryostored spermatozoa. A) Dose-dependent
comparison between these two populations of cells revealed a significant difference in overall activity (**P , 0.010 with chemiluminescence integrated
over 120 min. B) Time-dependent analyses of fresh cells. C) Time dependent analyses of cryostored spermatozoa (n ¼ 4 independent samples).

benzoic acid) porphyrin and tetra(parasulphonatephenyl)por-
phyrinato ferrate (III) (Fig. 3, A and B).
This single-dose comparison of live versus dead spermato-
zoa revealed that the generation of ROS by equine spermatozoa
in response to arginine, tryptophan, and phenylalanine were
significantly enhanced in snap frozen-thawed spermatozoa
(Fig. 2A–F). In order to evaluate whether the increased
chemiluminescence seen in response to aromatic amino acids
with snap frozen-thawed spermatozoa was compatible with
improved access to intracellular enzymes, dose- and time-
dependent studies were conducted using tyrosine (Y) trypto-
phan (W), and phenylalanine (F) to stimulate oxidase activity,
with valine (V) as a negative control. In the time-dependent
analysis, the chemiluminescent response given by freshly
prepared cells was shown to evolve slowly over the first hour
of incubation and, in the case of phenylalanine, then plateaued
after 60 min incubation, whereas with tryptophan the response
was still rising after 120 min (Fig. 4A). Tyrosine was different
in that the response increased over the first 60 min and then
gradually declined (Fig. 4A). With snap frozen-thawed cells,
the response to amino acid exposure was extremely rapid,
8 Article 125
consistent with the notion that a loss of viability simply
increases the substrates’ ability to access LAAO; the response
was also significantly more intense than that elicited in intact
cells. In terms of kinetics, the ROS response of permeabilized
spermatozoa to phenylalanine and tryptophan increased rapidly
and then plateaued, whereas the tyrosine response increased
rapidly and then fell, reflecting the bell-shaped response curve
seen in intact cells (Fig. 4, A and B). Dose-dependent studies
with phenylalanine confirmed the increased intensity and
accelerated onset of the ROS response to this amino acid
following a loss of cell viability (Fig. 4, C and D).
Because the immunocytochemical analyses revealing the
presence of LAAO in the acrosome and postacrosomal regions
was at variance with previously published data on bovine
spermatozoa localizing LAAO to the sperm tail [35], we sought
to confirm our observations biochemically by physically
separating sperm heads from tails and testing the isolated
fractions for amino acid oxidase activity. Using methods that
we have previously published to separate the head and tail
domains of mammalian spermatozoa [34], we were able to
confirm that a majority of the activity remained associated with
 L-AMINO ACID OXIDASE

TABLE 1. Amino acid composition of equine cryopreservation media before and after freezing.a

Prefreeze (20% egg yolk þ DMF) Postthaw (20% egg yolk þ DMF)

Amino acid (lM)b S1 S2 S3 S1 S2 S3

Alanine 321.9 320.8 362.8 335.6 355.5 367.6

Glycine 273.2 272.4 310.2 285.0 278.9 316.2
Valine 410.2 392.2 416.2 416.4 423.8 422.2
Leucine 527.5 528.8 580.5 522.6 567.0 597.9
Isoleucine 284.9 285.4 303.2 279.2 295.7 329.9
Threonine 513.7 528.3 578.2 529.6 558.3 586.7
Serine 729.7 755.1 815.4 751.7 820.8 810.8
Proline 389.6 379.4 414.3 411.7 413.0 430.3
Asparagine 248.8 269.6 288.7 262.5 278.4 291.4
Aspartic acid 476.5 462.8 466.4 464.6 530.7 495.5
Methionine 142.1 132.1 140.0 132.4 140.7 138.6
Glutamic acid 1050.3 962.6 1068.5 1083.9 1242.0 1124.7
Phenylalanine 264.0 241.7 274.6 249.5 267.0 279.5
Glutamine 424.8 445.9 464.7 464.0 462.3 515.1
Ornithine 12.3 10.3 13.4 12.3 14.5 13.8
Lysine 495.9 463.9 540.6 506.9 582.4 544.4
Histidine 98.2 98.6 110.0 98.1 113.2 116.6
Hydroxylysine 32.4 39.9 32.3 28.7 37.5 31.6
Tyrosine 320.0 292.4 324.0 277.5 292.2 330.8
Tryptophan 63.3 61.8 68.2 68.1 69.5 74.1
Cystathionine 724.7 327.9 548.2 849.5 583.6 581.1

Downloaded from www.biolreprod.org.

a
S1, S2, and S3 indicate individual semen samples.
b
Boldface type indicates the aromatic amino acids.

the head fraction, in keeping with the immunocytochemistry
results (Fig. 5, A and B).
Because LAAO is a flavoprotein we reasoned that a
flavoprotein inhibitor such as 10 lM diphenyleneiodonium
(DPI) should be able to inhibit this enzyme. This was shown to
be the case (Fig. 5, C and D); however, the fact that this
enzyme is also a known inhibitor of flavoproteins in the
mitochondrial electron transport chain and equine spermatozoa
are heavily dependent on oxidative phosphorylation for their
ATP meant that this compound induced a concomitant loss of
sperm motility (data not shown).
D-Amino Acids Are Ineffective
The equine amino acid oxidase was specific for L-amino
acids because D-phenylalanine, D-tryptophan, and D-tyrosine
were all incapable of stimulating ROS generation before and
after permeabilization of the plasma membrane (Fig. 6, A and
B). Furthermore, the concomitant presence of a 10-fold excess
concentration of D-phenylalanine significantly inhibited the
signal generated by L-phenylalanine under these conditions
(Fig. 6A–C).
Cryostorage and LAAO Activity
In light of the above results, we next sought to determine
whether the loss of viability exhibited by equine spermatozoa
following cryostorage would result in an increase in LAAO
activity. A comparison was therefore conducted of fresh and
frozen-thawed equine spermatozoa across a range of phenyl-
alanine doses (0.625–1.0 mM) following preparation using a
simple repeated washing procedure. The results of this analysis
revealed a clear dose-dependent response to phenylalanine that
was significantly (P , 0.01) enhanced following cryostorage
(Fig. 7A–C).
In order to determine whether cryostored equine spermato-
zoa would be exposed to sufficient concentrations of free
amino acid to trigger a ROS response, an analysis of the free
amino acid content of equine semen before and after
cryostorage in a conventional egg yolk-based cryopreservation
9 Article 125
medium was undertaken. The results of this analysis are
presented in Table 1 and reveal significant concentrations of
phenylalanine (;250 lM), tyrosine (;300 lM), and trypto-
phan (;70 lM) before and after cryostorage in such media.
Spermatozoa incubated in the presence of this combination of
amino acid concentrations exhibited a significant increase in
ROS generation in both intact and permeabilized cells (Fig.
8A). Importantly, the concentrations of aromatic amino acids in
the Kenney’s extended semen were low prior to the addition of
egg-yolk based cryopreservation medium with respect to
phenylalanine (7.30 6 0.7 lM), tryptophan (0.00 6 0.00
lM), and tyrosine (7.13 6 0.77 lM).
To determine whether the ROS generated by snap frozen-
thawed cells under such circumstances might influence the
motility of viable cells in the immediate vicinity, an experiment
was conducted involving the coincubation of live and dead
(frozen-thawed) cells in vitro in the presence and absence of
the above concentrations of phenylalanine, tyrosine, and
tryptophan, typical of cryostorage media. The results of this
analysis demonstrated that coincubation of live and dead
spermatozoa (1:1) together for 24 h generated sperm
populations exhibiting, overall, 28.0% 6 6.5% motility and
13.7% 6 2.6% progressive motility as determined by CASA.
However, the concomitant presence of phenylalanine reduced
both forms of motility to 0% (Fig. 8B). This dramatic reduction
of motility was associated with a significant increase in the
levels of lipid peroxidation (i.e., 4HNE) observed in live cells
that had been coincubated with amino acids in the presence (P
, 0.001) but not the absence of snap frozen-thawed
spermatozoa (Fig. 9A–C).
DISCUSSION
This is the first study to demonstrate the presence of an
LAAO in equine spermatozoa. Consistent with previous
studies on the LAAO in bovine and ram spermatozoa, this
enzyme appears to have a particular preference for L-aromatic
amino acids [35, 36, 38]. The most active substrate was L-
phenylalanine, which elicited a clear dose-dependent response
 AITKEN ET AL.
FIG. 8. Analyses of physiological concentrations of amino acids on ROS
generation and motility in populations of equine spermatozoa. A)
Chemiluminescence responses of cell-free controls, fresh, and snap
frozen-thawed populations, respectively (**P , 0.01, ***P , 0.001 in
relation to the corresponding control, Cn). Above this panel, the results of
an ANOVA analysis are presented representing an overall comparison of
fresh and snap frozen-thawed sperm populations (***P , 0.001, n ¼ 3). B)
Total motility (TM; closed bars) and progressive motility (PM; open bars) of
control and amino acid-treated samples. The live/dead control was
significantly different to live/dead treated with aromatic amino acids (AA)
for both total motility and progressive motility, arrowed (*P , 0.05, **P ,
0.01; n ¼ 3 independent samples). Single letter amino acid code is
presented. FC, phenylalanine (1 mM) positive control. F, phenylalanine;
W, tryptophan, Y, tyrosine.
in terms of H2O2 generation, increasing gradually over the 3 h
observation period such that the higher the dose, the more rapid
the evolution of the response. The kinetics of the ROS response
elicited by L-phenylalanine was largely determined by the
integrity of the sperm plasma membrane. Exposing spermato-
zoa to a flash-freezing protocol creates osmotic stress and
irreversibly damages the plasma membrane, rendering the cells
10
nonviable [39]. When the plasma membrane was damaged in
this way, the cellular response to L-phenylalanine exposure
was rapid and sustained over the ensuing 3 h observation
period. Furthermore, the absolute level of response observed in
these flash-frozen, nonviable cells was significantly higher (P
, 0.001) than in viable cells exposed to the same doses of L-
phenylalanine. The rapid response to amino acid exposure in
snap frozen-thawed cells is in keeping with the notion that a
loss of viability increases the substrates ability to access the
enzyme. The activity observed in the presence of snap frozen-
thawed cells therefore emphasizes the fundamental biochem-
istry of this enzyme, namely, that it requires only the amino
acid substrate, oxygen, and water to generate H2O2, ammonia,
and the corresponding keto acid, as described in the
deamination reaction:
L  amino acid þ H2 O þ O2 ¼ NH3 þ H2 O2 þ 2  oxo acid
ð1Þ
No other intracellular factors are required. The only amino
acids to exhibit LAAO activity with equine spermatozoa were
the aromatic amino acids and arginine. The signals generated
by tryptophan and tyrosine were less than those triggered by
Downloaded from www.biolreprod.org.
phenylalanine. Indeed, tryptophan only generated a response
that was significantly greater than control levels once cell
viability had been compromised. In the case of tyrosine, the
response kinetics associated with this amino acid were different
from any of the other stimulatory amino acids examined in that
the response was transient, declining rapidly after reaching a
peak at 17 min. This sudden decline could suggest the rapid
dissociation of this aromatic amino acid from the enzyme;
however, further studies will be needed to substantiate this
point.
These results are in keeping with the predicted substrate
specificity of this aromatic amino acid oxidase and previous
observations in bovine spermatozoa [35, 36]. MacMillan et al.
[40] demonstrated a decrease in the viability of bovine
spermatozoa as the concentrations of L-phenylalanine, L-
tryptophan, and L-tyrosine increased. This paper also analyzed
the D-forms of these aromatic amino acids and found no impact
on sperm viability when they were added individually or in
addition to L-forms. In the present study, the D-forms of these
aromatic amino acids were again shown to be incapable of
stimulating the production of H2O2, confirming the specificity
of this enzyme for L-aromatic amino acids in equine
spermatozoa. However, we did generate some evidence for
competitive inhibition when the D-form was present in 10-fold
excess over the L-form (Fig. 6).
The only nonaromatic amino acid to generate a ROS
response in the presence of these cells was arginine, possibly
because this compound can act as a substrate for nonenzymatic
nitric oxide generation, and, in the presence of superoxide, NO
is known to generate a positive signal in the presence of
luminol and peroxidase as a consequence of peroxynitrite
formation [37]. Previous studies have demonstrated that
mammalian spermatozoa can be stimulated to generate NO
by the addition of both D- and L-arginine via mechanisms that
can be inhibited by scavengers of peroxynitrite, including uric
acid and ascorbate [41].
Since the pioneering work of Shannon and Curson [35, 36]
on bovine spermatozoa, it has been assumed that LAAO
activity is localized in the sperm tail. However, this is the first
study to demonstrate the presence of LAAO predominantly in
the equine sperm head. Using an anti-LAAO antibody to detect
the enzyme, intense signals were seen in the sperm acrosome
and postacrosomal region separated by an unreactive equatorial
Article 125
 L-AMINO ACID OXIDASE

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FIG. 9. Analyses of the impact of aromatic amino acids and snap frozen-thawed cells on lipid peroxidation. A) 4-Hydroxynonenal (4HNE) expression by
flow cytometry (***P , 0.001) for the increase in lipid peroxidation associated with the presence of aromatic amino acids (AA) and dead cells (n ¼ 3
independent samples). B, C) Scattergrams revealing the pattern of 4HNE expression in live/dead preparations incubated in the absence (B) and the
presence (C) of aromatic amino acids. Note the increased number of cells located in the lower right-hand quadrant (arrowed) representing live cells with
extensive lipid peroxidation. Vitality was determined with the LIVE/DEAD Fixable Far Red Dead Cell stain.

segment. This finding was confirmed by the existence of
substantially more oxidase activity in purified heads than tails
when these subcellular fractions were compared in the
chemiluminescence assay. These observations open up the
possibility that the biological significance of this ROS-
generating system stretches beyond the regulation of motility,
as originally proposed by Tosic and Walton [30] to include the
physiological induction of acrosomal exocytosis.
A potential role for H2O2 in the induction of acrosomal
exocytosis has been evident since Griveau et al. [42]
demonstrated that catalase could inhibit the acrosome reaction
in human spermatozoa, while exposure to H2O2 induced this
process. Stimulation of the acrosome reaction with H2O2 was
also observed by Oehninger et al. [43] in human spermatozoa
while Hsu et al. [44] found that this oxidant rapidly induced
acrosomal exocytosis in the rat. However, detailed analysis of
the impact of phenylalanine on the tyrosine phosphorylation
status of equine spermatozoa and their ability to acrosome react
in response to progesterone failed to reveal any significant
effects in the present study (data not shown). We therefore
11
suggest that the primary role for this amino acid oxidase may
be to support the energy metabolism of equine spermatozoa
through the oxidative deamination of aromatic amino acids,
generating keto acids that are then oxidized by the sperm
mitochondria.
From a pathological point of view, the results of this study
have revealed a potential contribution of LAAO activity to the
loss of sperm motility seen when equine spermatozoa are
cryopreserved. Analyses of free amino acid concentrations in
equine seminal plasma and cryostorage media revealed that
exposure to the egg yolk-based medium significantly increased
the concentrations of phenylalanine (i.e., from 7.3 lM to 260
lM), tyrosine (i.e., from 7.1 lM to 312 lM) and tryptophan
(i.e., from undetectable to 64 lM). Thus, there would appear to
be insufficient quantities of aromatic amino acids to support
significant LAAO activity in the absence of egg yolk-based
diluents; it is only when equine spermatozoa are exposed to the
latter that LAAO activity becomes pathologically relevant. The
elevated amino acid levels present in egg yolk diluents were
found to significantly increase H2O2 generation in both viable
Article 125
 AITKEN ET AL.
and snap frozen-thawed equine spermatozoa. Furthermore,
because dead spermatozoa appeared to be more responsive to
the presence of L-aromatic amino acids than viable cells, we
reasoned that any situation capable of disrupting cell viability,
such as emersion in cryopreservation medium, is likely to
enhance ROS generation by nonviable cells and ultimately
influence the viability of live cells in the immediate vicinity.
Experimental investigation of this concept revealed that a
combination of snap frozen-thawed spermatozoa and free L-
aromatic amino acids effectively suppressed the motility of live
cells in the same suspension, with a concomitant increase in
lipid peroxidation (i.e., 4HNE) levels, as predicted. Thus, even
though LAAO is located in the sperm head, the H2O2 generated
by this system is freely membrane permeable and readily
capable of traversing the extracellular space to influence the
motility of spermatozoa in the immediate vicinity.
If the generation of H2O2 by LAAO in equine spermatozoa
is so potentially detrimental to sperm function, strategies
should be put in place to scavenge this reactive oxygen
metabolite or prevent its production. In terms of scavenging
activity, equine seminal plasma contains a range of antioxidant
enzymes, including glutathione peroxidase and catalase, both
of which are active against H2O2 [45, 46]. In addition, equine
seminal plasma contains small molecular mass ROS scavengers
such as ergothionine, uric acid, vitamins E and C, and citric
acid [47–49]. In order to ensure that the spermatozoa are
protected from oxidative stress following their dilution with
cryodiluents, a number of studies have examined the possible
benefits of adding a variety of antioxidants to the cryostorage
medium. These antioxidants include melatonin, phenols,
vitamins C and E, reduced glutathione, superoxide dismutase,
and catalase [50, 51]. In keeping with the results of this study,
the addition of antioxidants to equine semen has generally been
found to have a positive effect on sperm viability and motility,
although this is not always the case [52, 53].
There have been no previous investigations into the
development of possible inhibitors of LAAO for use with
equine spermatozoa. In the present study, DPI, an inhibitor of
flavoproteins, effectively suppressed ROS generation by these
cells. However, because spermatozoa possess many flavopro-
tein-dependent enzyme systems, including NADPH oxidases
[54] and the mitochondrial electron transport chain [55], such
an inhibitor indiscriminately blocks ROS generation from
multiple sources. It also suppresses sperm motility in the horse
because the spermatozoa of this species are heavily dependent
on oxidative phosphorylation for ATP generation [56]. As a
consequence, DPI is of no practical value when designing
optimized cryoprotection media. Further studies are therefore
needed to identify inhibitors of this oxidase that could be used
to limit the levels of oxidative stress suffered by equine
spermatozoa during the cryopreservation procedure.
In conclusion, this study has successfully characterized an
LAAO in equine spermatozoa. The major substrates for this
enzyme are aromatic amino acids, particularly L-phenylala-
nine, and the activity is enhanced once spermatozoa have lost
their vitality. The enzyme is located in the sperm head and
plays a potential role in the pathological suppression of sperm
motility during cryopreservation. This enzyme and its meta-
bolic products might be targeted in future attempts to refine the
procedures used to cryopreserve stallion semen.
REFERENCES
1. Aitken RJ, Curry BJ. Redox regulation of human sperm function: from the
physiological control of sperm capacitation to the etiology of infertility
and DNA damage in the germ line. Antioxid Redox Signal 2011; 14:
367–381.
12
2. Alvarez JG, Touchstone JC, Blasco L, Storey BT. Spontaneous lipid
peroxidation and production of hydrogen peroxide and superoxide in
human spermatozoa. Superoxide dismutase as major enzyme protectant
against oxygen toxicity. J Androl 1987; 8:338–348.
3. Bize I, Santander G, Cabello P, Driscoll D, Sharpe C. Hydrogen peroxide
is involved in hamster sperm capacitation in vitro. Biol Reprod 1991; 44:
398–403.
4. Ecroyd HW, Jones RC, Aitken RJ. Endogenous redox activity in mouse
spermatozoa and its role in regulating the tyrosine phosphorylation events
associated with sperm capacitation. Biol Reprod 2003; 69:347–354.
5. Zhang H, Zheng RL. Promotion of human sperm capacitation by
superoxide anion. Free Radic Res 1996; 24:261–268.
6. Lewis B, Aitken RJ. A redox-regulated tyrosine phosphorylation cascade
in rat spermatozoa. J Androl 2001; 22:611–622.
7. Aitken RJ, Harkiss D, Knox W, Paterson M, Irvine DS. A novel signal
transduction cascade in capacitating human spermatozoa characterised by
a redox-regulated, cAMP-mediated induction of tyrosine phosphorylation.
J Cell Sci 1998; 111:645–656.
8. Brouwers JF, Boerke A, Silva PF, Garcia-Gil N, van Gestel RA, Helms
JB, van de Lest CH, Gadella BM. Mass spectrometric detection of
cholesterol oxidation in bovine sperm. Biol Reprod 2011; 85:128–136.
9. de Lamirande E, Gagnon C. Reactive oxygen species and human
spermatozoa. II. Depletion of adenosine triphosphate plays an important
role in the inhibition of sperm motility. J Androl 1992; 13:379–386.
10. Jones R, Mann T, Sherins R. Peroxidative breakdown of phospholipids in
human spermatozoa, spermicidal properties of fatty acid peroxides, and
protective action of seminal plasma. Fertil Steril 1979; 31:531–537.
Downloaded from www.biolreprod.org.
11. Aitken RJ, Harkiss D, Buckingham DW. Analysis of lipid peroxidation
mechanisms in human spermatozoa. Mol Reprod Dev 1993; 35:302–315.
12. De Iuliis GN, Thomson LK, Mitchell LA, Finnie JM, Koppers AJ, Hedges
A, Nixon B, Aitken RJ. DNA damage in human spermatozoa is highly
correlated with the efficiency of chromatin remodeling and the formation
of 8-hydroxy-2 0 -deoxyguanosine, a marker of oxidative stress. Biol
Reprod 2009; 81:517–524.
13. Kodama H, Yamaguchi R, Fukuda J, Kasai H, Tanaka T. Increased
oxidative deoxyribonucleic acid damage in the spermatozoa of infertile
male patients. Fertil Steril 1997; 68:519–524.
14. Aitken RJ. The capacitation-apoptosis highway: oxysterols and mamma-
lian sperm function. Biol Reprod 2011; 85:9–12.
15. Fraga CG, Motchnik PA, Wyrobek AJ, Rempel DM, Ames BN. Smoking
and low antioxidant levels increase oxidative damage to sperm DNA.
Mutat Res 1996; 351:199–203.
16. Peña FJ, Garcı́a BM, Samper JC, Aparicio IM, Tapia JA, Ferrusola CO.
Dissecting the molecular damage to stallion spermatozoa: the way to
improve current cryopreservation protocols? Theriogenology 2011; 76:
1177–1186.
17. Kiziler AR, Aydemir B, Onaran I, Alici B, Ozkara H, Gulyasar T,
Akyolcu MC. High levels of cadmium and lead in seminal fluid and blood
of smoking men are associated with high oxidative stress and damage in
infertile subjects. Biol Trace Elem Res 2007; 120:82–91.
18. Aitken RJ, Finnie JM, Muscio L, Whiting S, Connaughton HS, Kuczera L,
Rothkirch TB, De Iuliis GN. Potential importance of transition metals in
the induction of DNA damage by sperm preparation media. Hum Reprod
2014; 29:2136–2147.
19. De Iuliis GN, Newey RJ, King BV, Aitken RJ. Mobile phone radiation
induces reactive oxygen species production and DNA damage in human
spermatozoa in vitro. PLoS One 2009; 4:e6446.
20. Aitken RJ, Baker MA. Oxidative stress, sperm survival and fertility
control. Mol Cell Endocrinol 2006; 250:66–69.
21. Aitken RJ, Clarkson JS. Cellular basis of defective sperm function and its
association with the genesis of reactive oxygen species by human
spermatozoa. J Reprod Fertil 1987; 81:459–469.
22. Brouwers JF, Gadella BM. In situ detection and localization of lipid
peroxidation in individual bovine sperm cells. Free Radic Biol Med 2003;
35:1382–1391.
23. Chatterjee S, Gagnon C. Production of reactive oxygen species by
spermatozoa undergoing cooling, freezing, and thawing. Mol Reprod Dev
2001; 59:451–458.
24. Ball BA, Vo AT, Baumber J. Generation of reactive oxygen species by
equine spermatozoa. Am J Vet Res 2001; 62:508–515.
25. Alvarez JG, Storey BT. Lipid peroxidation and the reactions of superoxide
and hydrogen peroxide in mouse spermatozoa. Biol Reprod 1984; 30:
833–841.
26. Aitken RJ, Ryan AL, Curry BJ, Baker MA. Multiple forms of redox
activity in populations of human spermatozoa. Mol Hum Reprod 2003; 9:
645–661.
27. Ortega Ferrusola C, González Fernández L, Morrell JM, Salazar Sandoval
Article 125
 L-AMINO ACID OXIDASE
C, Macı́as Garcı́a B, Rodrı́guez-Martinez H, Tapia JA, Peña FJ. Lipid
peroxidation, assessed with BODIPY-C11, increases after cryopreserva-
tion of stallion spermatozoa, is stallion-dependent and is related to
apoptotic-like changes. Reproduction 2009; 138:55–63.
28. Sabeur K, Ball BA. Characterization of NADPH oxidase 5 in equine testis
and spermatozoa. Reproduction 2007; 134:263–270.
29. Musset B, Clark RA, DeCoursey TE, Petheo GL, Geiszt M, Chen Y,
Cornell JE, Eddy CA, Brzyski RG, El Jamali A. NOX5 in human
spermatozoa: expression, function, and regulation. J Biol Chem 2012; 287:
9376–9388.
30. Tosic J, Walton A. Metabolism of spermatozoa. The formation and
elimination of hydrogen peroxide by spermatozoa and effects on motility
and survival. Biochem J 1950; 47:199–212.
31. Biggers JD, Whitten WK, Whittingham DG. The culture of mouse
embryos in vitro. In: Daniels JC (ed.), Methods in Mammalian
Embryology. San Francisco, CA: Freeman; 1971:86–116.
32. Kenney RM, Bergman RV, Cooper WL, Morse GW. Minimal
contamination techniques for breeding mares: techniques and preliminary
findings. In: 21st Annual Convention of the American Association of
Equine Practitioners; 1975:327.
33. Cochran JD, Amann RP, Froman DP, Pickett BW. Effects of
centrifugation, glycerol level, cooling to 5 degrees C, freezing rate and
thawing rate on the post-thaw motility of equine sperm. Theriogenology
1984; 22:25–38.
34. Baker MA, Naumovski N, Hetherington L, Weinberg A, Velkov T, Aitken
RJ. Head and flagella subcompartmental proteomic analysis of human
spermatozoa. Proteomics 2013; 13:61–74.
35. Shannon P, Curson B. Site of aromatic L-amino-acid oxidase in dead
bovine spermatozoa and determination of between bull differences in the
percentage of dead spermatozoa by oxidase activity. J Reprod Fertil 1982;
64:469–473.
36. Shannon P, Curson B. Toxic effect and action of dead sperm on diluted
bovine semen. J Dairy Sci 1972; 55:614–620.
37. Wang JF, Komarov P, de Groot H. Luminol chemiluminescence in rat
macrophages and granulocytes: the role of NO, O2-/H2O2, and HOCl.
Arch Biochem Biophys 1993; 304:189–196.
38. Upreti GC, Jensen K, Munday R, Duganzich DM, Vishwanath R, Smith
JF. Studies on aromatic amino acid oxidase activity in ram spermatozoa:
role of pyruvate as an antioxidant. Anim Reprod Sci 1998; 51:275–287.
39. Morris GJ, Faszer K, Green JE, Draper D, Grout BW, Fonseca F. Rapidly
cooled horse spermatozoa: loss of viability is due to osmotic imbalance
during thawing, not intracellular ice formation. Theriogenology 2007; 68:
804–812.
40. Macmillan KL, Tiku JL, Hart NL. Toxic effects of aromatic amino acids
on the livability of bull spermatozoa. Aust J Biol Sci 1972; 25:1039–1045.
41. Aitken RJ, Ryan AL, Baker MA, McLaughlin EA. Redox activity
13
View publication stats
associated with the maturation and capacitation of mammalian sperma-
tozoa. Free Radic Biol Med 2004; 36:994–1010.
42. Griveau JF, Renard P, Le Lannou D. An in vitro promoting role for
hydrogen peroxide in human sperm capacitation. Int J Androl 1994; 17:
300–307.
43. Oehninger S, Blackmore P, Mahony M, Hodgen G. Effects of hydrogen
peroxide on human spermatozoa. J Assist Reprod Genet 1995; 12:41–47.
44. Hsu PC, Hsu CC, Guo YL. Hydrogen peroxide induces premature
acrosome reaction in rat sperm and reduces their penetration of the zona
pellucida. Toxicology 1999; 139:93–101.
45. Ball BA, Gravance CG, Medina V, Baumber J, Liu IK. Catalase activity in
equine semen. Am J Vet Res 2000; 61:1026–1030.
46. Baumber J, Ball BA. Determination of glutathione peroxidase and
superoxide dismutase-like activities in equine spermatozoa, seminal
plasma, and reproductive tissues. Am J Vet Res 2005; 66:1415–1419.
47. Brummer M, Hayes S, Dawson KA, Lawrence LM. Measures of
antioxidant status of the horse in response to selenium depletion and
repletion. J Anim Sci 2013; 91:2158–2168.
48. Leone E. Ergothioneine in the equine ampullar secretion. Nature 1954;
174:404–405.
49. Mann T. Biochemistry of stallion semen. J Reprod Fertil Suppl 1975; 23:
47–52.
50. da Silva CM, Macias-Garcia B, Miro-Moran A, Gonzalez-Fernandez L,
Morillo-Rodriguez A, Ortega-Ferrusola C, Gallardo-Bolanos JM, Stilwell
G, Tapia JA, Pena FJ. Melatonin reduces lipid peroxidation and apoptotic-
like changes in stallion spermatozoa. J Pineal Res 2011; 51:172–179.
51. Gibb Z, Butler TJ, Morris LH, Maxwell WM, Grupen CG. Quercetin
Downloaded from www.biolreprod.org.
improves the post-thaw characteristics of cryopreserved sex-sorted and
nonsorted stallion sperm. Theriogenology 2013; 79:1001–1009.
52. Morillo-Rodriguez A, Macias-Garcia B, Tapia JA, Ortega-Ferrusola C,
Pena FJ. Consequences of butylated hydroxytoluene in the freezing
extender on post-thaw characteristics of stallion spermatozoa in vitro.
Andrologia 2012; 44(Suppl 1):688–695.
53. Baumber J, Ball BA, Linfor JJ. Assessment of the cryopreservation of
equine spermatozoa in the presence of enzyme scavengers and
antioxidants. Am J Vet Res 2005; 66:772–779.
54. Aitken RJ, Fisher HM, Fulton N, Gomez E, Knox W, Lewis B, Irvine S.
Reactive oxygen species generation by human spermatozoa is induced by
exogenous NADPH and inhibited by the flavoprotein inhibitors
diphenylene iodonium and quinacrine. Mol Reprod Dev 1997; 47:
468–482.
55. Li Y, Trush MA. Diphenyleneiodonium, an NAD(P)H oxidase inhibitor,
also potently inhibits mitochondrial reactive oxygen species production.
Biochem Biophys Res Commun 1998; 253:295–299.
56. Gibb Z, Lambourne SR, Aitken RJ. The paradoxical relationship between
stallion fertility and oxidative stress. Biol Reprod 2014; 91:77.
Article 125