Human Reproduction vol.13 no.5 pp.1240–1247, 1998
The effects of antioxidant supplementation during
Percoll preparation on human sperm DNA integrity
Ciara M.Hughes1,3, Sheena E.M.Lewis1,
Valerie J.McKelvey-Martin2and W.Thompson1
1Department of Obstetrics and Gynaecology, The Queens
University of Belfast, Institute of Clinical Science, Grosvenor
Road, Belfast BT12 6BJ and2Cancer and Ageing Research Group,
School of Biomedical Sciences, The University of Ulster,
3To whom correspondence should be addressed
The integrity of sperm DNA is crucial for the maintenance
of genetic health. A major source of damage is reactive
oxygen species (ROS) generation; therefore, antioxidants
may afford protection to sperm DNA. The objectives of
the study were, first, to measure the effects of antioxidant
supplementation in vitro on endogenous DNA damage in
spermatozoausing thesinglecell gelelectrophoresis(comet)
assay and, second, to assess the effect of antioxidant
supplementation given prior to X-ray irradiation on
induced DNA damage. Spermatozoa from 150 patients
were prepared by Percoll centrifugation in the presence of
ascorbic acid (300, 600 µM), alpha tocopherol (30, 60 µM),
urate (200, 400 µM), or acetyl cysteine (5, 10 µM). DNA
damage was induced by 30 Gy X-irradiation. DNA strand
breakage was measured using the comet assay. Sperm DNA
was protected from DNA damage by ascorbic acid (600
µM), alpha tocopherol (30 and 60 µM) and urate (400 µM).
These antioxidants provided protection from subsequent
DNA damage by X-ray irradiation. In contrast, acetyl
further DNA damage. Supplementation in vitro with the
antioxidants ascorbate, urate and alpha tocopherol separ-
ately has beneficial effects for sperm DNA integrity.
Key words: alpha tocopherol/ascorbic acid/comet assay/DNA
Male infertility accounts for 40% of infertility problems
(Fleming et al., 1995). Using classical light microscopy, only
a limited diagnosis of subfertility may be made by assessing
low sperm concentration or poor motility. As a result, tests
have been introduced to determine other aspects of sperm
function: quantitative sperm motion (Zhu et al., 1994), sperm–
zona interaction (Liu et al.,1988), the acrosome reaction (Liu
and Baker, 1988) and fusion with the oocyte by the zona-
free hamster oocyte penetration assay (Kruger et al., 1988).
However with the advent of intracytoplasmic sperm injection
(ICSI) (Palmero et al., 1992; Van Steirteghem et al., 1994),
© European Society for Human Reproduction and Embryology
many of these sperm parameters have become irrelevant, as it
has been possible to fertilize oocytes using spermatozoa with
intact acrosomes, no motility, sperm heads without tails and
immature spermatozoa (Baker, 1993), and so new tests are
needed to assess sperm health. Ideally, such tests should predict
the health of the offspring and not just the success of
fertilization and/or implantation. Whatever the assisted concep-
tion technique, the quality of the sperm DNA is still of
paramount importance for accurate transmission of genetic
material to the next generation. Damaged DNA may not
prevent fertilization from occurring but may lead to fetal
abnormalities which will only be apparent later. Infertility may
be linked to DNA damage, as the sperm DNA of infertile
patients has been shown to be more susceptible to damage
in vitro than DNA from fertile men (Hughes et al., 1996). The
DNA status of individual cells may be determined using the
et al., 1993) which has been modified to measure DNA damage
in human spermatozoa (Hughes et al., 1996, 1997).
A major source of damage to DNA is by reactive oxygen
species (ROS) (Steenken, 1989). It has been shown that
spermatozoa are capable of generating ROS such as the
superoxide anion (O2
the influence of intracellular superoxide dismutase (Aitken and
Clarkson, 1987; Alvarez et al., 1987). In human semen,
defective spermatozoa and contaminating neutrophils are also
potential sources of the oxidant hydrogen peroxide (Aitken
and West, 1990; Aitken et al., 1992; Kessopoulou et al., 1992).
Spermatozoa are uniquely susceptible to oxidative damage
because of their differentiation (Jones et al., 1973). As sperma-
tozoa discard the majority of their cytoplasm during the final
stages of spermatogenesis, they lose most of the cytoplasmic
defence enzymes which protect somatic cells from peroxidative
damage. The capacity for DNA repair is also lost as mature
spermatozoa do not have any repair enzymes (Chandley and
Kofman-Alfaro, 1971; Van Loon et al., 1991) making them
more vulnerable to damage than any other cell type. The
antioxidants that are present in the seminal plasma are therefore
an important source of protection.
it is necessary to remove the spermatozoa from the seminal
plasma and therefore from their antioxidant protection. This
leaves the spermatozoa vulnerable to oxidative attack. The
present study determines the effects of two sperm preparation
techniques on DNA integrity and the role of antioxidant
supplements in vitro in protecting sperm DNA during prepara-
tion and from induced damage. In a previous study (Lewis
et al., 1997), we found that ascorbic acid and urate made up
most of the chain-breaking antioxidant capacity of seminal
–) which subsequently forms H2O2under
Antioxidants benefit sperm DNA integrity
plasma in fertile men; therefore, these antioxidants were used
in the present study at similar concentrations. Alpha tocopherol
and acetyl cysteine have been found in previous studies (Aitken
and Clarkson, 1989; Kessopoulou et al., 1995; Baker et al.,
1996) to be of benefit to sperm parameters; therefore these
antioxidants were also studied here to assess any beneficial
effect on sperm DNA. To ascertain the extent of any protection
Materials and methods
Semen samples were obtained from 150 patients after recommended
abstinence from sexual activity for 3 days, so that spermatozoa from
30 subjects could be studied following treatment with each of the
five selected antioxidants. Routine semen analysis was carried out by
light microscopy and the percentage normal morphology (Kruger
et al., 1987) for each sample was determined. Ten additional samples
were used to examine the DNA quality of the spermatozoa following
two different preparation techniques; Percoll centrifugation and direct
swim-up. Twenty samples were also used to determine at which stage
of the protocol the antioxidants exerted their effect.
In order to determine whether sperm preparation technique has any
effect on sperm DNA quality, 10 samples were each divided into
three aliquots and a fraction of each sample was prepared by Percoll,
direct swim-up or direct swim-up in the presence of endotoxins (10
ng/ml). This chosen concentration of endotoxins added to the swim-
up media is the same as found in Percoll (Sigma Chemical Co.,
Poole, Dorset, UK). The swim-up process involved layering semen
(1 ml) beneath 1 ml of Biggers–Whitten–Whittingham (BWW)
medium and incubating the test tube at a 45% angle at 37°C for 30
min, to allow the spermatozoa to swim out of the seminal plasma
into the BWW media.
Percoll preparation of freshly liquefied semen by two-step discon-
tinuous Percoll gradient centrifugation was carried out as follows: (i)
two-layer Percoll (95.0–47.5%) centrifugation at 500 g for 20 min,
(ii) concentration step by centrifugation at 250 g for 10 min, and (iii)
the sperm cells were diluted with BWW medium (Biggers et al.
1971) to a concentration of 1?105/50 µl.
Semen from each of the 150 subjects was divided into three aliquots.
One control aliquot was then prepared by Percoll as described, while
the other two aliquots were prepared by Percoll in the presence of
an antioxidant at one of two different concentrations that was added
to the Percoll layers and the BWW. Thirty subjects were therefore
studied in this manner for each of the following antioxidants:
ascorbic acid (300 and 600 µM) (L-ascorbic acid product A4034;
(iii) ascorbic acid (300 µM) ? alpha tocopherol (30 µM) (TROLOX)
and ascorbic acid (600 µM) ? alpha tocopherol (60 µM)
(iv) urate (200 and 400 µM),
(v) acetyl cysteine (5 and 10 µM) (N-acetyl cysteine product A8199;
To determine at which stage of the protocol the antioxidants might
be protecting the DNA, an additional 20 samples were each divided
into three aliquots. One part was untreated; in the second part ascorbic
acid (600 µM) was present in the Percoll preparation but not
throughout the comet assay protocol; and the third part was prepared
by Percoll without ascorbic acid, followed by the comet assay protocol
in which ascorbic acid was present throughout (600 µM).
Each of the three aliquots of spermatozoa from the 150 subjects (the
control and two treated with antioxidants) was divided into two
further aliquots, so that spermatozoa from each of the three treatment
groups could be additionally treated with a damaging agent. To induce
oxidative DNA damage, spermatozoa from each sample were X-ray
irradiated with a dose of 30 Gy at room temperature (Hughes et al.,
1996). X-ray irradiations were performed using a 300 kV Siemens
Stabilipan X-ray source at a dose rate of 2.6 Gy min–1. A set of six
aliquots of spermatozoa from each subject was therefore obtained;
(i) control spermatozoa, (ii) irradiated spermatozoa, (iii) spermatozoa
treated with a low concentration of an antioxidant, (iv) spermatozoa
treated with a low concentration of an antioxidant plus irradiation,
(v) spermatozoa treated with a higher concentration of an antioxidant
and (vi) spermatozoa treated with a higher concentration of an
antioxidant plus irradiation.
Single cell gel electrophoresis assay
The modified alkaline comet assay for spermatozoa (Hughes et al.,
1996, 1997) was carried out on the prepared samples. For each
sample, six slides were prepared, one for each of the aliquots treated
as described above.
Fully frosted slides (Richards Supply Company Limited, London,
UK) were covered with 100 µl of 0.5% normal melting point agarose
(Sigma), a coverslip was added and the agarose was allowed to
solidify. The coverslips were removed and 1?105cells in 50 µl
BWW were mixed with 50 µl of 1.2% low melting point agarose
(Sigma) and used to form the second layer. The slides with coverslips
removed were then placed in lysis buffer for 1 h [2.5 M NaCl, 100
mM NaEDTA, 10 mM Tris, 1% Triton X (Sigma) at a pH of 10].
The slides were then incubated at 37°C in 100 µl/ml of proteinase K
(Sigma) in lysis buffer overnight. Antioxidants were present in the
lysis buffer throughout the incubation.
After draining the proteinase K solution from the slides, they were
placed in a horizontal electrophoresis unit filled with freshly prepared
alkaline electrophoresis solution containing 300 mM NaOH and 1
mM EDTA (Sigma) for 20 min to allow the DNA to denature.
Electrophoresis was performed at room temperature, at 25 V (0.714
V/cm) and 300 mA, obtained by adjusting the buffer level, for 10
min. The slides were then washed with a neutralizing solution of 0.4
M Tris (Sigma) at pH 7 to remove alkali and detergents. After
neutralization, the slides were each stained with 50 µl of 20 µg/ml
ethidium bromide (Sigma) and mounted with a coverslip. All steps
were carried out under yellow light to prevent further DNA damage.
Analysis of cells and statistics
Fifty cells from each slide were selected randomly and analysed by
image analysis using Hewlett and Packard Super VGA and Fenestra
Komet Software (version 3). Observations were made at magnification
?400 using an epifluorescent microscope (Olympus BH2). Following
preparation by the SCGE assay, each cell has the appearance of a
‘comet’ with a brightly fluorescent head and a ‘tail’ to one side,
formed by the DNA which contains strand breaks being drawn away
from the comet head into a tail during the electrophoresis (Figure 1).
The DNA which remains in the ‘head’ of the comet, after specified
electrophoresis times, gives a value for the amount of intact DNA
C.M.Hughes et al.
Figure 1. The typical appearance of a sperm ‘comet’ following
preparation by the comet assay, showing the brightly fluorescent
circular ‘head’ of the comet and the diffusely stained ‘tail’ of
damaged DNA to one side of the head.
and is measured as ‘percentage head DNA’ by the software. Statistical
analysis was carried out using non-parametric statistics with the
Statistica (Statsoft Inc.) package on the values obtained for the percent
head DNA of each cell.
The effect of sperm preparation technique on DNA integrity was
assessed by the Mann–Whitney U-test. Analysis was carried out on
the effect of the antioxidants on the sperm DNA using the Wilcoxon
signed rank test by comparing the percentage head DNA values
obtained for the control spermatozoa and those treated with an
antioxidant. Further analysis using the Wilcoxon signed rank test was
carried out to determine if the damage induced by the X-ray irradiation
was significantly different from the control values within each sample
and if the antioxidants had any protective effect from the induced
Comparison of sperm preparation techniques; Percoll and
The comparison of the two techniques showed that Percoll
preparation isolated spermatozoa with significantly better DNA
integrity (P ? 0.05) than the direct swim-up technique. This
was visualized by plotting the median value and interquartile
range for the percentage head DNA readings obtained for the
spermatozoa following each of the preparation techniques
(Figure 2). There was no significant difference (P ? 0.05) in
DNA from spermatozoa isolated by swim-up with and without
Comparison of morphology and percent head DNA
Following Percoll preparation of the 150 samples used in the
main study, there was no significant difference in percentage
head DNA values between those samples for which the
percentage normal morphology prior to Percoll was greater or
less than 14% (Figure 3).
Stage of protocol influenced by antioxidants
Spermatozoa prepared by Percoll in the presence of ascorbic
acid showed significantly higher percent head DNA values
than both control spermatozoa (P ? 0.01) and spermatozoa
treated with ascorbic acid during the comet protocol only
(P ? 0.05). This can be observed by the distribution of percent
Figure 2. The median values and interquartile ranges for the %
head DNA for spermatozoa obtained following preparation by
either Percoll, direct swim-up or swim-up in which the BWW
contained endotoxins. *Significantly different from Percoll
Figure 3. The median value and interquartile ranges for percent
head DNA in spermatozoa from two groups divided according to
percentage normal morphology before Percoll, i.e. those with
normal morphology of greater or less than 14%.
head DNA (Figure 4), as a greater percentage of spermatozoa
appear to fall into the higher percent head DNA ranges
following Percoll preparation in the presence of the antioxidant
in comparison to the control.
Ascorbic acid supplementation
Sperm percentage head DNA (percent intact DNA) was signi-
ficantly higher when ascorbic acid (600 µM) had been present
duringsperm preparation(P ?0.001), indicatedbythe increase
in values determined for the median of each group (Table I).
The values for the 1500 spermatozoa analysed (50 for each of
the 30 subjects) were divided up into ranges of percentage
head DNA, (0–10, 11–20, ... 91–100%), so that the distribution
of DNA damage could be visualized. When this was carried
Antioxidants benefit sperm DNA integrity
Figure 4. The DNA integrity of spermatozoa after Percoll
preparation with no antioxidants (control) (w), plus ascorbic acid
(600 µM) present in the Percoll preparation (m) or ascorbic acid
(600 µM) added during the comet assay (q). The graph shows the
1000 values obtained for each of the three treatments following
division into ranges of percent head DNA, so that the percentage of
spermatozoa within each range was plotted to give a distribution of
Table I. The effect of antioxidant present in vitro during Percoll
preparation, X-ray irradiation and the comet assay protocol on sperm DNA.
Values are percentage head DNA medians (interquartile range). Baseline
values are those obtained from spermatozoa which were not treated by
AntioxidantConcentration Baseline values Irradiated (30 Gy)
Ascorbic acid Control 70.7 (80.6/54.8)
77.2 (87.8/57.5)** 73.0 (85.7/57.4)**,††
71.5 (80.7/53.2)61.5 (73.3/41.5)††
75.7 (87.0/56.1)** 64.8 (80.5/37.4)*,††
76.7 (86.9/52.3)** 65.4 (82.2/42.9)*,††
70.8 (83.2/60.5)61.2 (71.0/43.0)††
71.8 (85.7/62.6)64.1 (74.4/45.6)*,††
75.9 (88.3/65.3)** 66.8 (74.8/47.4)*,††
68.6 (88.5/48.7)55.5 (80.2/39.5)††
30 ? 300 µM 57.2 (81.0/39.2)** 53.5 (802./35.8)
60 ? 600 µM 52.6 (77.5/34.5)** 50.5 (70.1/35.5)*,†
Control 71.1 (80.2/55.0)
5 µM 60.1 (70.1/46.6)** 49.6 (64.3/34.0)*,††
10 µM47.1 (63.3/35.5)** 42.5 (52.3/33.8)*,††
Ascorbic acid Control
*Significantly different from control without antioxidant (P ? 0.01).
**Significantly different from control without antioxidant (P ? 0.001).
†Significantly different from spermatozoa without irradiation (P ? 0.01).
††Significantly different from spermatozoa without irradiation
(P ? 0.001).
out for the control spermatozoa and those treated with the
ascorbic acid, it was clear from these graphs that there was a
shift in the distribution for percent head DNA towards a
greater number of spermatozoa having more intact DNA
following preparation in the presence of antioxidants (Figure
Following irradiation (30 Gy), there was a significant
decrease in percent head DNA of the sperm population (P ?
0.001), also indicated by the decrease in the median value for
the population (Table I). In the presence of ascorbic acid (300
µM) and subsequent irradiation, the median percent head DNA
value did not decrease to the same extent, and there was a
significant increase in percent head DNA values for the
population from the irradiated control values (P ? 0.01). In
fact, when spermatozoa were irradiated in the presence of
ascorbic acid (600 µM), the resulting median percent head
DNA value was similar to the median baseline control value
(P ? 0.05) (Table I).
In the presence of alpha tocopherol (30 or 60 µM), percent
head DNA was significantly higher than the control values
(P ? 0.001), shown also as an increase in the median value
for percent head DNA from that obtained for the control
(Table I). This protective effect was also evident from plotting
the distribution graph which shows that a greater number of
spermatozoa with more intact DNA are obtained following
Percoll in the presence of the antioxidant (Figure 5B).
Irradiation produced a significant decrease in percent head
DNA which was lessened by the presence of alpha tocopherol
(30 or 60 µM) (Table I). In the presence of the antioxidants,
there was a significant increase in percent head DNA from the
irradiated control (P ? 0.01), as demonstrated by an increase
in the median value for the population of spermatozoa in the
presence of alpha tocopherol (Table I).
In the presence of urate (400 µM), there was a significant
increase in the percent head DNA over the control values
(P ? 0.001), along with an increase in the median value
(Table I). There was also a shift to the right in the distribution
graph (Figure 5C), again showing a greater number of sperma-
tozoa with more intact DNA in the presence of urate.
The antioxidant also provided protection from induced DNA
damage as the percent head DNA in the presence of urate
(200 or 400 µM) was significantly improved from the irradiated
control level (P ? 0.01). Again, this can be observed as an
increase in the median value for the population from the
irradiated control in the presence of urate (Table I).
Ascorbic acid ? alpha tocopherol
The presence of ascorbic acid and tocopherol together through-
out the protocol (Table I, Figure 5D) caused a decrease in
percent head DNA (P ? 0.001), indicating that DNA damage
had been induced. These antioxidant supplementations also
resulted in significant DNA damage additional to that induced
by the irradiation shown in Table I by the decrease in
the median value in the presence of antioxidants from the
The presence of acetyl cysteine (Figure 5E) also induced
significant DNA damage with a decrease in percent head DNA
for the sperm population (P ? 0.001) and a decrease in the
median value (Table I). Following irradiation further DNA
The most important sperm parameter for assisted conception
is intact DNA, especially for the techniques which select
C.M.Hughes et al.
Figure 5. The graphs show the DNA integrity of spermatozoa obtained
following Percoll preparation. The 1500 values obtained for each of the
3 treatments for each graph were divided into ranges of % head DNA,
so that a percentage of sperm within each range was plotted to give a
distribution of DNA damage. The graphs show the DNA integrity of
control sperm with no antioxidants present w, compared to the DNA
integrity of sperm prepared in the presence of; (a) ascorbic acid (300 q
or 600 m µM) (b) alpha tocopherol (30 q or 60 m µM) (c) urate (200
q or 400 m µM) (d) alpha tocopherol (30 µM) ? ascorbic acid (300
µM) q, or alpha tocopherol (60 µM) ? ascorbic acid (600 µM) m (e)
N-acetylcysteine (5 q or 10 m µM).
few spermatozoa for fertilization purposes, such as subzonal
insemination (SUZI), or indeed only one spermatozoon, as is
the case with ICSI. Although DNA integrity has not been
correlated with in-vitro fertilization (IVF) rates, probably due
to the competition between the large number of spermatozoa
used, it has been directly correlated with SUZI, as defective
DNA packaging due to lack of protamines resulted in reduced
fertilization rates (Bianchi et al., 1993). Damage to this DNA
may be due to the increased generation of reactive oxygen
species that, under normal conditions, is limited to the low
steady generation of superoxide and hydrogen peroxide
there is a potential for increased generation of ROS from
defective spermatozoa, the mechanism of which is unclear but
Antioxidants benefit sperm DNA integrity
which may involve increased availability of NADPH from
excess cytoplasm, or electron leakage from damaged mitochon-
dria (Aitken, 1997).
Spermatozoa have only two defence mechanisms against
oxidative attack of their DNA; the packaging arrangement of
the DNA, and the seminal plasma. During spermatogenesis,
the chromatin becomes highly condensed within a protamine
matrix (Sidney et al., 1986). The DNA is organized into loops,
attached at their bases to the nuclear matrix, anchored to the
base of the sperm tail by the nuclear annulus and stabilized
by disulphide bonds (Ward, 1993; Barone et al., 1994). This
tight packing of the DNA reduces exposure to free radical
attack. The second line of defence is the antioxidant capacity
of its seminal plasma (Lewis et al., 1995; 1997).
Sperm preparation techniques are used routinely in assisted
conception units to isolate the spermatozoa with the best
motility and morphology from seminal plasma, damaged
spermatozoa and other debris present in the semen. Although
studies with acridine orange have shown that both Percoll and
swim-up procedures may select spermatozoa with more mature
nuclei (Golan et al., 1997), here we have shown that Percoll
gradients concentrate spermatozoa with more intact DNA than
the swim-up process (Figure 2) which is in agreement with
other studies (Le Lannou and Blanchard, 1988; Colleu et al.,
1996). As direct swim-up depends only on the swimming
properties of the spermatozoa, those with defective nuclei may
also swim-up into the top layer of BWW. In contrast, the
Percoll technique also relies on the density of the spermatozoa.
Since an intact nucleus is tightly packed (Ward, 1993; Barone
et al., 1994) and therefore more dense, it is predictable that
these should be concentrated in the bottom layer of Percoll
(Pasteur et al., 1991).
In the present study, the percentage normal morphology
before Percoll preparation was not related to the DNA integrity
after Percoll (Figure 3). This is in agreement with Bianchi et al.
(1993), who showed that morphologically normal spermatozoa
may possess loosely packed defective chromatin. On the other
hand, Hall et al. (1995) showed that Percoll centrifugation
decreases the percentage of spermatozoa with head abnormalit-
ies which may also reflect the lack of relationship between the
pre-Percoll morphology and DNA packaging, as the ratio
of normal morphology may change pre- and post-Percoll.
Therefore, it would be of interest to determine any relationship
Percoll has recently been removed from the market for use
in assisted conception due to the presence of ‘uncontrolled
substances’ (Pharmacia Biotech, formal notification, 1997).
This study has shown that, in terms of DNA quality, Percoll
appears to have no detrimental effects and that endotoxins
present at these concentrations are of no consequence
Baseline DNA integrity in spermatozoa is lower than that
of somatic cell types (Hughes et al., 1996), possibly a reflection
of its physiological role (Singh et al., 1989). Human sperm
samples were also found to exhibit a wide variation in DNA
damage as measured by the comet assay (Hughes et al., 1996),
which is in agreement with the variation found in CMA3
fluorochrome staining measuring the protamine content of the
nucleus, and the amount of endogenous DNA nicks measured
by in-situ nick translation (Bianchi et al., 1993; Sakkas et al.,
1995). Spontaneous lipid peroxidation in human spermatozoa
occurs at highly variable rates in different sperm samples, and
is largely dependent on the intracellular content of superoxide
dismutase which varies between spermatozoa (Storey et al.,
1997). This variability in antioxidant protection may explain
the variation in DNA damage found in individual spermatozoa
within one sample. The present study suggests that some of
the DNA damage found in a sperm population may be due to
the removal of the seminal plasma during preparation for IVF,
as this procedure also removes the spermatozoa from their
main source of antioxidant protection. Addition of various
chain-breaking antioxidants to the media resulted in protecting
the DNA from subsequent damage.
Ascorbic acid is the major contributor to the chain-breaking
antioxidant capacity of seminal plasma (Lewis et al., 1997)
the concentration of which is 10 times higher than that of
blood plasma and is actively secreted by the seminal vesicles
during ejaculation (Berg et al., 1941). When ascorbic acid was
added to the media at concentrations found by Lewis et al.
(1997) in seminal plasma of fertile men, it protected spermato-
zoa from DNA damage in vitro. This protection was previously
shown in vivo (Fraga et al., 1991; Jacob et al., 1991), by
measuring the oxidized nucleoside 8-hydroxy-2?-dehydroxy-
guanosine (oxo8dG), one of the major products of DNA
damage. Ascorbic acid has been shown to scavenge most ROS
(Sies et al., 1992) which undoubtedly explains its efficiency.
Alpha tocopherol has been shown to inhibit sperm lipid
peroxidation in vitro (Aitken and Clarkson, 1988), to enhance
the ability of spermatozoa to fuse with zona-free hamster
oocytes (Aitken et al., 1989) and improve zona pellucida
binding (Kessopoulou et al., 1995). In this study, we found
alpha tocopherol (30 µM) provided protection to DNA, which
is in agreement with protection afforded in vitro by this
antioxidant (Fraga et al., 1996), making it a multifunctional
antioxidant and a potentially useful supplement to sperm
Although ascorbic acid and alpha tocopherol supplemented
separately produced protective effects from DNA damage,
when added together they produced a damaging effect. It is
known that in high concentrations, ascorbic acid can be
potentially oxidative (Gutteridge, 1994). However, the ascorbic
acid concentrations used with alpha tocopherol were the same
as those used separately. The trace tocopherol levels found by
Lewis et al. (1997) may be sufficient to combat oxidative
assault when present with ascorbic acid due to its regeneration
(Kagan, 1992), so the higher concentration used in this study
may have caused an abnormal redox balance. Similar results
were found by Sweetman et al. (1997), who found a protective
effect from ascorbic acid or tocopherol individually, but not
together, on human lymphoblast cells. It was suggested that
there is a narrow physiological range in which these anti-
in our laboratory in a pilot study in vivo (results not shown).
Supplementation with ascorbic acid (350 mg) and alpha
tocopherol (250 mg) together for 1 month induced substantial
damage to the sperm DNA, whereas supplementation with the
C.M.Hughes et al.
antioxidants individually had no damaging effect and in some
cases enhanced DNA integrity.
The other major chain-breaking antioxidant in seminal
plasma is urate (Lewis et al., 1997). Seminal plasma concentra-
tions of urates were found to decrease in normozoospermic
infertile men (Thiele et al., 1995), although this was not
observed by Lewis et al. (1997). Urate binds transition metals,
thus preventing Fe2?-driven free radical reactions, and also
scavenges peroxyl and hydroxyl radicals (Sevanian et al.,
1991). Here, urate at the higher concentration of 400 µM
prevented damage to the sperm DNA, suggesting that an
optimal concentration of urate is required for antioxidant
N-Acetyl cysteine (NAC) can act as an antioxidant through
its role as a precursor of glutathione (GSH) synthesis and at
an extracellular level, where it acts directly on oxidants (De
Vries and De Flora, 1993). The antioxidant has been shown
to be beneficial on sperm motility in vitro (Baker et al., 1996)
and sperm DNA (Den Boer, 1990). In contrast, in the present
study the effect of acetyl cysteine was to damage the DNA.
One explanation for the damaging effect of the NAC may be
that in the spermatids thiols act to preserve the S–H groups in
et al., 1993), whereas in ejaculated spermatozoa the DNA
becomes stabilized by S–S bonds in the presence of zinc in
the seminal fluid (Molina et al., 1995). Perhaps the presence
of acetyl cysteine in the absence of zinc in the Percoll medium
has disturbed the thiol:disulphide ratio, reducing the S–S bonds
and so destabilizing the sperm DNA allowing damage to occur.
While substantial quantities of glutathione are found in the
testis, reproductive tract fluid and epididymal spermatozoa,
much less is present in ejaculated spermatozoa (Agrawal and
Vanha-Perttula, 1988). This may be a consequence of the small
cytoplasmic content of mature spermatozoa which would in
turn reduce the amount of enzymes available for the formation
of glutathione. This suggests that the effect of NAC on
spermatozoa comes not from its role as a precursor of gluta-
explanation for the DNA damage observed in the present
study is the direct extracellular action of NAC. Under these
circumstances, NAC may initiate lipid peroxidation and so
may be acting as a pro-oxidant. This is supported by the fact
that levels of thiols in general were found to be higher in
infertile men (Lewis et al., 1997), who also demonstrated an
increase in ROS activity.
Spermatozoa produced by infertile men were shown to have
DNA which was more susceptible to damage by irradiation
than that from fertile men (Hughes et al., 1996). X-ray
irradiation, the source of damaging agent used in the present
study, acts by ionizing water to produce hydroxyl radicals
(Hall, 1994) and by producing single and double strand breaks
directly. Ascorbic acid efficiently scavenges these radicals
(Sies et al., 1992) and its presence in this study prevented
induced damage from the X-ray irradiation (Table I). As a
scavengerofhydroxyl radicals,uratefulfilsasimilarrole tothat
observed with ascorbic acid. Although tocopherol prevented
induced damage to some extent, the DNA integrity did not
return to baseline as observed with ascorbic acid. Tocopherol
is found in the cell membrane, in contrast to ascorbic acid
which, being water soluble, is found in the cell cytoplasm
where the hydroxyl radicals are formed, leaving it closer to
the oxidative attack. Previous reports have indicated that
where ascorbic acid plays a dominant role in protection from
irradiation, alpha tocopherol is more important in protection
against chemicals which must cross the cell membrane to gain
access to the cell (Sweertman et al., 1997).
We have shown in this study that DNA can be protected
during sperm Percollcentrifugation preparation by the presence
of antioxidants in the media. Ascorbic acid, alpha tocopherol
and urate separately significantly increased the percentage of
spermatozoa with good DNA integrity. The antioxidants also
protected the spermatozoa from induced in-vitro oxidative
DNA damage by irradiation. Ascorbic acid and urate, being
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Received on June 23, 1997; accepted on January 21, 1998