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BIOLOGY OF REPRODUCFION 40, 183-197 (1989)
183
Generation of Reactive Oxygen Species, Lipid Peroxidation,
and Human Sperm Function
R. JOHN AITKEN,1’2 JANE S. CLARKSON,2 and SIMON FISHEL3
MRC Reproductive Biology Unit2
Centre for Reproductive Biology
Edinburgh EH3 9EW
Scotland
and
The Park Hospital3
Arnold Nottingham NG5 8RY
England
ABSTRACT
Recent studies have demonstrated that hwnan spermatozoa are capable of generating reactive oxygen
species and that this activity is significantly accelerated in cases of defective sperm function. In view of the
pivotal role played by lipid peroxidation in mediating free radical dwnage to cells, we have examined the
relationships between reactive oxygen species production, lipid peroxidazion, and the functional competence
of human spermatozoa.
Using malondialdehyde production in the presence of ferrous ion promoter as an index of lipid
peroxidation, we have shown that lipid peroxidation is significantly accelerated in populations of defective
spennafozoa exhibiting high levels of reactive oxygen species production or in normal cells stimulated to
produce oxygen radicals by the ionophore, A23187. The functional consequences of lipid peroxidation
included a dose-dependent reduction in the ability of human spermatozoa to exhibit sperm oocyte-fusion,
which could be reversed by the inclusion of a chain-breaking antioxidant, cs-tocopherol. Low levels of lipid
peroxidation also had a slight enhancing effect on the generation of reactive oxygen species in response to
ionophore, without iifluencing the steady-state activity. At higher levels of lipid peroxidation, both the basal
level of reactive oxygen species production and the response to A23187 were significantly diminished. In
contrast, lipid peroxidation had a highly significant, enhancing effect on the ability of human spermatozoa to
bind to both homologous and heterologous zonae pellucidae via mechanisms that could again be reversed by
ct-tocopherol.
These results are consistent with a causative role for lipid peroxidation in the etiology of defective sperm
function and also suggest a possible physiological role for the reactive oxygen species generated by hwnan
spermatozoa in mediating sperm-zona interaction.
INTRODUCTION
Defective sperm function has been identified as the
largest defined cause of human infertility, accounting
for about 27% of all couples attending infertility clinics
(Hull, 1986). Despite its high incidence, little is known
of the aetiology of male infertility and there are few, if
any, rational effective therapies to treat this condition.
This relative paucity of therapeutic options is largely a
Accepted March 14, 1989.
Received December 12,1988.
1Reprint requests: R. John Aitken, MRC Reproductive Biology Unit,
Centre for Reproductive Biology, 37 Chalmers Street. Edinburgh EH3 9EW,
Scotland.
consequence of our lack of knowledge concerning the
precise biochemical nature of the defects responsible
for the spermatozoa’s loss of fertilizing potential.
To address this problem we have focused our re-
seareh on the molecular mechanisms responsible for
defective sperm function, with emphasis on the mem-
brane fusion events associated with fertilization (Aitken
et al., 1984, 1987, 1988; Aitken and Clarkson, 1987).
These events, comprising the acrosome reaction and
sperm-oocyte fusion, can be monitored in vitro with the
zona-free hamster oocyte penetration assay (Yanagi-
machi et al., 1976; Aitken, 1986). Studies using this test
in conjunction with the divalent cation ionophore,
A23187, indicate that in a high proportion of cases,
184 AITKEN El AL.
defective sperm function is associated with an inability
of the sperm plasma membrane to respond to an intra-
cellular calcium signal with spenn-oocyte fusion (Alt-
ken et al., 1984, 1987).
Our understanding of the chemical nature of the
damage to the sperm plasma membrane responsible for
this refractory state has been advanced by numerous,
independent studies suggesting a key role for lipid
peroxidation in the etiology of male infertility. (Jones
and Mann, 1973; Jones et al., 1978, 1979; Aitken and
Clarkson, 1987; Alvarez et al., 1987). Human spermato-
zoa are particularly susceptible to peroxidative damage
because they contain an extremely high concentration
of polyunsaturated fatty acids (predominantly 22:6),
exhibit no capacity for membrane repair, and possess a
significant ability to generate reactive oxygen species,
chiefly superoxide anion and hydrogen peroxide (Jones
et al., 1979, Aitken and Clarkson, 1987; Alvarez et al.,
1987). The possibility that peroxidative damage to the
sperm plasma membrane might be involved in those
cases of infertility characterized by a failure to exhibit
sperm-oocyte fusion was suggested by studies indicat-
ing an association between the appearance of such
defects and the hyperactive production of reactive oxy-
gen species by the spermatozoa (Aitken and Clarkson,
1987; Aitken et al., 1988). We postulate that under such
conditions, the main defense mechanism of human
spermatozoa, superoxide dismutase (Mennella and
Jones, 1980; Alvarez et al., 1987), would be over-
whelmed, and the resulting combination of hydrogen
peroxide and excess superoxide anion would favor the
production of hydroxyl radicals, via the Haber-Weiss
reaction
H2O2 +O2#{149}=OH#{149}+OH- +02
The rate constant for this reaction is considerably en-
hanced by the presence of transition elements such as
iron, the availability of which has recently been demon-
strated in human seminal plasma (Kwenang et al.,
1987). The hydroxyl radicals formed as a result of the
Haber-Weiss reaction are powerful initiators of lipid
peroxidation and would be expected to impair human
sperm function through peroxidation-induced changes
in membrane fluidity and integrity (Jones et al., 1979;
Slater, 1984).
To obtain data pertinent to the above hypothesis, we
have investigated the relationships between reactive
oxygen species production, lipid peroxidation, and the
functional competence of human spermatozoa. Previous
studies have demonstrated that the addition of exoge-
nous lipid peroxides to human spermatozoa (Jones et
al., 1978) or the stimulation of endogenous lipid peroxi-
dation by ferrous ion and ascorbate (Jones et al., 1979)
lead to a loss of cell viability, as reflected by a dramatic
loss of motility. The results of the present study indi-
cate that less extensive peroxidation recreates the
pathological situation characterized by an inability to
respond to A23187-induced, intracellular calcium sig-
nals with sperm-oocyte fusion. Paradoxically, such
treatment was also found to result in a considerable
enhancement of sperm-zona interaction, possibly re-
flecting the underlying biological function of the super-
oxide-generating system in the human sperm plasma
membrane.
Semen Samples
MATERIALS AND METHODS
All donors were normal healthy males who had been
subjected to a complete physical examination and
shown to be free of sexually transmitted disease, in-
cluding human immunodeficiency virus (HIV) infec-
tion. The 64 specimens employed in this study were
obtained from 48 donors characterized by a normal
semen profile (>20 x 10o spermatozoa/mi, >40% motil-
ity>40% normal motility), normal liquefaction, and no
evidence of leucocytic infiltration (<1 x 106 which
cells/mi) (World Health Organization, 1987). The sam-
ples were produced by masturbation and were analyzed
within 1.5 li of production.
Hamster Oocyte Fusion
The ability of human spermatozoa to exhibit sperm-
oocyte fusion in response to the calcium signal gener-
ated by A23187 was assessed according to the protocol
described by Aitken et al. (1984). For this procedure,
the spermatozoa were first separated from seminal
plasma by 3 cycles of centrifugation (500 x gfor 5
mm) and resuspension in medium BWW (Biggers et
al., 1971). The spermatozoa were then adjusted to a
concentration of 10 x 106/ml, and lipid peroxidation
was induced by exposing the spermatozoa to various
doses of ferrous sulphate and sodium ascorbate (Jones
et al., 1979; Alvarez et al., 1987) for 45 mm.
At the end of this period, the cells were centrifuged
at 500 x gfor 5mm and resuspended in fresh medium
BWW containing an aqueous suspension of the Ca2,
UPID PEROXIDATION AND SPERM FUNCFION 185
Mg2 salt of A23187 (0.05 mg/nil). After a further
incubation of 2.25 h at 37’C, in an atmosphere of 5%
CO2 in air, the cells were again centrifuged at 500 x g
for 5mm, after which they were resuspended in normal
BWW, at a concentration of 10 x 106 spermatozoa/mi,
and distributed as 50-jtl droplets under liquid paraffin.
At this point, portions of the sample were removed for
malondialdehyde determination (see below) and the as-
sessment of sperm motility, at least 100 cells being
assessed with the aid of a grid on an eye piece graticule
(World Health Organization, 1987).
Zona-free hamster oocytes, prepared as described by
Yanagimachi et al. (1976), were then introduced into
the droplets, at least 20 oocytes being used for each
treatment. The oocytes were removed after a 3-h incu-
bation period, compressed to a depth of about 30 J.Lm
under a 22 x 22-mm coverslip, and examined by phase-
contrast microscopy for the presence of decondensing
sperm heads. The results were assessed in terms of the
percentage of oocytes penetrated at a motile sperm
concentration of S x 106 cells/mi and the mean number
of spermatozoa penetrating each oocyte (Aitken and
Elton, 1984).
Movement Characteristics
For experiments investigating the influence of lipid
peroxidation on sperm-zona interaction, the detailed
movement characteristics of human spermatozoa were
assessed objectively with time-exposure photomicrogra-
phy (Aitken et al., 1982, 1985). For this procedure,
spermatozoa were photographed at 250x, using a 1-s
exposure under dark-field illumination, with a heated
stage set to 3TC.
Sperm-Zona Interaction
The influence of lipid peroxidation on sperm-zona
interaction was evaluated using salt-stored human zonae
pellucidae that had been preserved in 1.5 M magnesium
chloride and 1% dextran (Yanagimachi et aL, 1979).
For these studies, washed suspensions of human sper-
matozoa at 10 x 106 cells/mi were preincubated for 2.5
h and then exposed to various doses of ferrous sulfate
and sodium ascorbate for 1 h to induce lipid peroxida-
tion. At the end of this period, the spermatozoa were
centrifuged at 500 x g for 5mm, resuspended at 10 x
106 spermatozoa/mi in fresh Medium BWW, and dis-
tributed at 50-ILl droplets under liquid paraffin. Washed,
salt-stored human zonae pellucidae were introduced
into the droplets and incubated for 3 h. They were
subsequently removed, washed to remove loosely ad-
herent spermatozoa, and photographed at 250x so that
the number of spermatozoa bound to the zonae could be
determined from the photographic prints (Henderson et
al., 1987). Identical procedures were also used to evalu-
ate the binding of human spermatozoa to porcine zonae
pellucidae, isolated as described by Henderson et al.
(1987).
Where indicated, spermatozoa were incubated with
10 mM a-tocopherol prior to the introduction of ferrous
ion and ascorbate. In such instances, the a-tocopherol
was dissolved in ethanol, dried-down under a stream of
nitrogen, and finally taken up in BWW (Aitken and
Clarkson, 1988).
Reactive Oxygen Species
The generation of reactive oxygen species by human
spermatozoa was determined using the chemilumines-
cent probe, luminol (5-amino-2,3-dihydro-, 1,4-
phthalazinedione), as described previously (Aitken and
Clarkson, 1987). Five-hundred microliters of washed
human spermatozoa, at a concentration of 20 x 106
cells/mi, were treated with 1 p.1 of luminol stored as a
100-mM stock solution in dimethyl sulfoxide. These
sperm suspensions were then diluted with 500 p.1 Med-
ium BWW alone (control) or Medium BWW containing
the Ca2, Mg2 salt of A23 187 (0.05 mg/mI). After 3
mm, the chemiluminescent signal was measured on a
Berthold, Biolumat Ll39500Tluminometer, the counts
being integrated over a 10-s period. The consistency of
the luminometer’s photomultiplier response was as-
sessed by making a number of independent assessments
of the chemiluminescent signal generated when luminol
was chemically oxidized with potassium permanganate.
This procedure gives an interassay coefficient of varia-
tion of 16.8%.
Lipid Peroxidation
The thiobarbituric acid (TBA) assay has been widely
used to provide a convenient index of lipid peroxida-
tion, the outcome of which correlates well with alterna-
tive techniques for assessing peroxidation, including
chemiluminescence, pentane or ethane formation, and
colorimetric reactions based on the reduction of phos-
phoilpid hydroperoxides with potassium iodide (Jones
et aL, 1979; Smith et al., 1982). Despite a certain lack
of selectivity (Bird and Draper, 1984) arguments in
E
C
C’)
a)L()
QLC)
C
1)
ow
(I)..
U)
-‘C
-Q
LLi- L()
><
2000
1000 -
00 10 20 40
186 AffKEN ET AL.
#{149}Control
oFe Promoter
Sperm Conc [x 106 /mI]
FIG. 1. Differences in the rates of malondialdehyde production in the pres-
ence (0) and absence (#{149})of ferrous stilfate:ascorbate promoter.
favor of using the TBA-assay to give a general indica-
tion of the extent of lipid peroxidation in mammalian
spermatozoa have been carefully presented (Alvarez
and Storey, 1982; Alvarez et al., 1987). The major
drawback of the assay is that only a small fraction of
membrane lipids, chiefly prostaglandin-like cyclic en-
doperoxides (Pryor et al., 1976 Shimizu et al., 1981),
yield TBA-reactive products on peroxidation, the con-
version of lipid hydroperoxides to malondialdehyde in
the TBA assay being less than 5% (Smith and Ander-
son, 1987; van Kuijk and Dratz, 1987).
As a consequence, spontaneous malondialdehyde
production by human spermatozoa is slow and long
incubation periods have to be used to monitor the
peroxidation process (Alvarez et al., 1987). However,
the rate of reaction can be considerably enhanced if a
suitable promoter system is incorporated into the proto-
col, such as ferrous ion and ascorbate (Jones and Mann,
1977; Jones et al., 1978, 1979). The high rate of malon-
dialdehyde production that can be achieved within 1 11
by adding 0.03 mM ferrous sulphate and 0.17 mM
sodium ascorbate to the medium is graphically illus-
trated in Figure 1.
In view of these findings, we have elected to use a
promoted TBA assay to provide an index of lipid
peroxidation in human spermatozoa. Unless otherwise
stated, this assay was performed by incubating 1 ml of
spermatozoa at 20 x 106 cells/mi with 0.25 ml ferrous
sulphate (0.2 mM) and 0.25 ml sodium ascorbate (1
mM) at 37#{176}Cfor 1 h. One milliliter of this reaction
mixture was then supplemented with 0.5 ml 40% th-
chloroacetic acid and 0.5 ml of medium BWW and
centrifuged for 10 mm at 2500 x g. One milliliter of the
supernatant was then added to 0.25 ml 2% TBA and
incubated at 90#{176}Cfor 10 mm. After cooling them to
room temperature, the samples were read on a Perkin
Elmer spectrofluorimeter using excitation and emission
wavelengths of 510 nm and 553 nm, respectively
(McMillan et aL, 1977). The standards were generated
by incubating serial dilutions of 1,1,3,3-
tetraethoxypropane with equal volumes of 0.2 N HC1
overnight at room temperature (Okuma et al., 1970).
Percoll Preparation
Experiments on the relationship between lipid perox-
idation and reactive oxygen species production also
involved the fractionation of human semen samples on
discontinuous Percoll gradients as described by Aitken
and Clarkson (1988). Isotonic Percoll was prepared
with 10 ml of 10-strength Medium 199 (Flow Laborato-
ries, hvine, Scotland) supplemented with 300 mg bo-
vine serum albumin (BSA; Fraction V. Sigma, St.
Louis, MO), 3 mg sodium pyruvate, and 0.37 ml of a
60% sodium lactate syrup (Sigma) and diluted with 90
ml of Percoll (Pharmacia, Uppsala, Sweden): this prep-
aration was designated 100% stock isotonic Percoll
(Lessley and Gamer, 1983). Unprocessed human semen
was then layered on top of a 6-step discontinuous
gradient containing 100%, 90%, 80%, 70%, 55%, and
40% Percoll in sequence (Berger et al., 1985). After
centrifugation for 20 mm at 500 x g, the spermatozoa
were collected into 3 fractions comprising the cells
located at the base of the 40% and 55% steps (Fraction
1), the 70% and 80% steps (Fraction 2), and the 90%
and 100% steps (Fraction 3) (Aitken and Clarkson,
1988). The spermatozoa in each fraction were collected,
diluted with 8 ml of Medium BWW, and centrifuged at
500 x g for 5 mm before being resuspended at a
concentration of 20 x 106 cells/ml.
Statistics
Nonparametric statistics (the Mann-Whitney U-test
for unpaired data, and the Wilcoxon matched-pairs
signed-ranks test for paired data) have been used
throughout this study.
oCONTROL
#{149} A23187
0
a) 5oooo
C.)
a)
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30000
C.)
a)
20000
C)
C.)
U) 10000
E
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b)
I.-
.3
E
0.
<1
0
c) 100
80
60
>
40
20
0
FiG. 2. Relationship between motility, reactive oxygen species production,
and lipid peroxidation for subpopula*ioas of human spermatozoa fractionated
on Percoll gradients: a) Luminol-dependent chemiluminescence measured be-
fore and 3mm after the addition of A23187; b) Lipid peroxidation as reflected
by malondialdehyde production, and C) motility. **P =0.01. n =10.
12
Percofi Fraction
UPID PEROXIDATION AND SPERM FUNCI’ION 187
RESULTS
Relationship between Reactive
Oxygen Species Production
and Lipid Peroxidation
To evaluate the relationship between reactive oxygen
species production and lipid peroxidation, spermatozoa
from 10 separate ejaculates were fractionated on dis-
continuous Percoll gradients and examined for their
motility, rate of reactive oxygen species generation, and
degree of lipid peroxidation. The results, presented in
Figure 2, indicate that relative to Fraction 3, the frac-
tions (1 and 2) from the upper layers of the Percoll
gradient exhibited a significantly (p<O.Ol) reduced level
of sperm motility in association w9i marked (p<0.Ol)
increases in the generation of reactive oxygen species
in terms of both the steady-state activity and the re-
sponse to A23187. The particularly high levels of reac-
tive oxygen species production recorded in Fraction 1
were associated with a significant increase (p<O.Ol) in
the levels of malondialdehyde production relative to
Fractions 2and 3 (Fig. 2).
A relationship between the level of reactive oxygen
species production and lipid peroxidation was also indi-
cated in experiments involving the use of the divalent
cation ionophore, A23 187. Addition of this compound
to suspensions of human spermatozoa (n =20) resulted
in a significant (p =0.006) increase in reactive oxygen
species production that peaked after 3 miii, at which
point the chemiluminescent counts, integrated over 10
s, were 670.7 ± 160.3 in the controls and
3530.1 ± 849.8 in the presence of A23 187. This in-
crease was associated with a significant rise (p =0.045)
in the rate of malondialdehyde production from
2.45 ± 1.l0/10 spermatozoa/h to 4.24 ± 1.50 pmoll
108 spermatozoa/h.
The nature of the reactive oxygen species generated
under these conditions was investigated by examining
the ability of exogenous superoxide dismutase and cata-
lase to quench the chenuluminescent signal. In the
absence of A23 187, the baseline signal generated by
human spermatozoa was unaffected by superoxide dis-
mutase, although a dose-dependent suppression was
achieved by catalase (Fig. 3). In contrast, the addition
of A23 187 resulted in a burst of chemilumunescence
that could be suppressed to basal levels by superoxide
dismutase (Fig. 3). Catalase also exerted a statistically
significant (p<O.O5) suppressive effect on this A23 187-
a)
6000
--5000
4000
o 3000
2000
-1000 IiCATALASE C) A23 187 CATALASE
3000
-92000
h00
b) SUPEROXIDE DISMUTASE d) A23187 SUPEROXIDE DISMUTASE
4000-
3000
0
2000
0
1000
S
- 0Ii
0 9 18 36 72
Dose IU
C 0 9 18 36 72
DoseU 103j
188 AITKEN ET AL.
FIG. 3. Influence of exogenous superoxide disinutase and catalase on the
luininol-dependent chemilwninescent signal generated by human spermatozoa
in the absence, (a and b) and presence (C and d) of A23 187. p<O.O5. Separate
groups of 6 donors were used for analyses in the absence (a and b) and the
presence (C and d) of A23187.
induced response although, even at the highest dose
tested, the chemiluntinescent activity was still
69 ± 4.8% of the levels observed with A23187 alone
(Fig. 3). An analysis of possible synergism indicated
that a combination of superoxide dismutase (3.5 x iO
U) and catalase (60 x 10 U) was no more efficient at
suppressing the response to A23187 than superoxide
dismutase alone.
In the absence of promoter, the rate of malondial-
dehyde production was unifonnly low (0.28 ± 0.06
pmol/108 spermatozoa/h; n =15) and did not signifi-
cantly increase after incubation in the presence of
A23187 for as long as 3 h (0.31 ± 0.05 pmol/108
spermatozoa/h). Within this same data set, the addition
of A23 187 significantly enhanced the generation of
reactive oxygen species from a control level of
1990 ± 723 counts/lO s to 5126 ± 1438 counts/lO s in
the presence of ionophore. Furthermore, within this
series of samples, both the steady-state level of reactive
oxygen species production and the response to A23187
were negatively correlated with hamster oocyte penetra-
tion rate (r =-0693 and -0.650, respectively), as
observed in previous studies (Aitken and Clarkson,
1987).
Influence of Lipid Peroxidation
on Reactive Oxygen Species Production
Having established that factors influencing the level
of reactive oxygen species production also influenced
the rate of lipid peroxidation, it was of interest to
determine whether the converse relationship also exist-
ed, i.e. whether lipid peroxidation could influence the
ability of human spermatozoa to generate reactive oxy-
gen species. To investigate this possibility; human sper-
matozoa were incubated for 1 h, at 10 x 106 cells/mi, in
the presence of 3 different levels of promoter (n =7).
At the end of this period, the cells were centrifuged
(500 x g for 5 mm) and resuspended in normal Medium
BWW, at which point the motility, rate of malondial-
dehyde production, and capacity for generating reactive
oxygen species were assessed (n =7). The results,
presented in Figure 4, revealed the anticipated dose-
dependent induction of lipid peroxidation, without any
concomitant change in motility. At the two lowest
doses of ferrous sulfate:ascorbate used (12.5 pM; 62.5
pM and 50 pM:250 pM), the rate of malondialdehyde
production increased from the control value of
0.18 ± 0.04 pmol/108 spermatozoa/h to 1.83 ± 0.48
and 3.45 ± 0.26 pmol/i08 spermatozoa/h, respectively.
This increase in lipid peroxidation was associated with
a significant (p’<O.OS) enhancement in the ability of the
spermatozoa to generate reactive oxygen species in
response to A23187, while the basal, steady-state activ-
ity remained unchanged.
This enhanced response to A23 187 in the presence of
promoter could be significantly suppressed by the anti
oxidant a-tocopherol. For this analysis, human spermat-
ozoa were preincubated at 20 x 106 cells/nd, with or
without a-tocopherol, at a dose (10 mM) that we have
previously shown to be optimal for inhibiting lipid
peroxidation in human spermatozoa (Aitken and Clark-
son, 1988). The spermatozoa were then diluted to 10 x
106/ml cells with Medium BWW containing promoter
(12.5 pM ferrous sulphate: 62.5 pM ascorbate) and
incubated for 1 h before being assessed for their basal
level of reactive oxygen species generation and their
responsiveness to A23 187. In the absence of A23l87,
a-tocopherol did not exert a significant effect on reac-
tive oxygen species productionn (738.6 ± 304.9 vs.
964.3 ± 435.3); however, in the presence of A23187,
this antioxidant significantly suppressed the response
recorded 3 mm after the addition of ionophore
(2717.6 ± 1354.7 vs. 5474.6 ± 2959.9; ii =; p<0.05).
At the highest dose of promoter used (200 pM
ferrous sulphate: 1000 pM ascorbate), the rate of
malondialdehyde production increased to 5.24 ± 0.04
pmol/108 spermatozoa/h (n =7), and although the mo-
tility of the cells remained unchanged, both the basal
oCONTROL
#{149}A23187
50-
- 40-
. 30-
0
20-
10-
0- CONTROL 12.5/62 50/250 200/1000
LIPID PEROXIDATION AND SPERM FUNCFION 189
a)
b)
c)
8000-
U)
0
6000
4000-
2000
C
E
-J 0
7.
a,
0.
U)
0
0
E
0.
5-
4-
3-
2-
1-
0-
60-
Dose of Ferrous Ion Promoter (jiM)
FIG. 4. Influence of lipid peroxidation on the generation of reactive oxygen
species. a) Luminol-dependentchemiluminescence measured before and 3mm
after the addition of A23 187; b) lipid peroxidation as reflected by malondial-
dehyde production and c) motility. p<Q.05. **p =0.02. n =7. Paired numbers
at the foot of each column = dose of ferrous sulfate/dose of ascorbate in pro-
moter system.
and the A23187-induced rates of reactive oxygen spe-
cies production significantly (p =0.02) declined relative
to the control values (Fig. 4).
Jtfluence of Lipid Peroxidation
on Hamster Oocyte Fusion
To examine the influence of lipid peroxidation on the
ability of human spermatozoa to exhibit sperm-oocyte
fusion in response to the calcium signal generated by
A23187, a dose-response analysis was performed with
ferrous sulfate: ascorbate used at concentrations ranging
from 200 pM: 1000 pM to 6.25 pM: 31.25 pM (n =7).
Exposure of human spermatozoa to this promotion sys-
tem for 45 mm resulted in a dose-dependent increase in
malondialdehyde formation that ranged from a maxi-
mum of 7.34 ± 1.91 pmol/108 spermatozoa/mi with
the highest concentration of ferrous sulfate:ascorbate
(200 pM: 1000 pM) to 0.81 ± 0.09 pmol/108 spermat-
ozoa with the lowest dose employed (6.25 pM: 31.25
pM). At all doses of promoter, the rate of malondial-
dehyde production was significantly elevated (p =0.02;
n=7) over the control value of 0.18 ± 0.05 pmol/108
spermatozoa/h (Fig. 5). These values for malondial-
dehyde production are well below the lipoperoxidative
lethal end point for human spermatozoa of 100 pmol/
108 spermatozoa (Alvarez et al., 1987), and, as a conse-
quence, sperm motility was not significantly influenced
by the promoter system, even at the highest dose em-
ployed (Fig. 5). In contrast the capacity of these cells
for sperm-oocyte fusion declined as a reciprocal of the
rate of lipid peroxidation. In the absence of promoter,
the addition of A23187 was responsible for elevating
the levels of sperm oocyte fusion from a control value
of 13.01 ± 10.9% (0.14 ± 0.12 spermatozoa/egg) to
86.2 ± 8.7% (3.65 ± 1.14 spermatozoa/egg). In the
presence of the ferrous sulphate:ascorbate promoter, the
levels of sperm-oocyte fusion observed in response to
A23 187 were significantly (p =0.02) inhibited at all
doses tested, reaching a nadir of 19.6 ± 9.1%
(0.32 ± 0.21 spermatozoa/egg) at 200 pM ferrous sul-
fate: 1000 pM ascorbate. Across a data set comprising
70 independent specimens (56 from the above experi-
ment), the rate of malondialdehyde production was
negatively correlated with hamster oocyte penetration
rate (r =-0.598) but did not show any relationship with
motility.
If the influence of ferrous ion and ascorbate on
human sperm function was mediated by the induction
of lipid peroxidation, we reasoned that the presence of
antioxidants should have an ameliorating effect on the
fertilizing ability of these cells. To investigate this
possibility, we used the chain-breaking anti-oxidant, a-
tocopherol, at the optimal dose described previously
b)
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601i-I-
0- -
Dose of Fetrous Ion Pronloteq (MM) 23
8
6
b) 12
10
0)
0.
(1)4
2
0
C) 100
80
60
>‘
40
20
0
9
(Aitken and Clarkson, 1988). For these studies, each
sperm sample was prepared at a concentration of 20 x
106 cells/mi and divided into 4 portions, 2 of which
were preincubated with and 2 without 10 pM a-tocoph-
erol. After a 1-h preincubation period, the samples were
exposed to A23 187; where necessary, cx-tocopherol was
added to maintain the concentration at 10 pM. At this
point, -promoter was added to one of the portions that
had been preincubaled with a-tocopherol and also to
one of the portions preincubated in BWW alone. An
equal volume of Medium BWW was added to the
remaining two portions to give 4 treatment groups:
untreated control, ct-tocopherol alone, promoter alone,
and a-tocopherol plus promoter. After these additions,
the spermatozoa, now at a concentration of 10 x 106
cells/nil, were incubated for a further 3 h prior to being
centrifuged (500 x g for 5mm) and resuspended in
normal Medium BWW for the hamster oocyte penetra-
tion test. Separate experiments were performed with 3
doses of ferrous sulfate:ascorbate (12.5 pM: 62.5 pM [n
=8], 25 pM:125 pM En =6], and 50 p.M:250 pM En =
9]), and the results are presented in Figure 6.
0/0 pM 12 5/625 pM 25/125 pM 50/250 pM
190 A1TKEN ET AL.
Ii
I
Do.. ol F.qrous Ian PromoIa. (MM)
FIG. 5. Influence of lipid peroxidation on the capacity of human spermato-
zoa for spurn oocyte fusion, a) Perceptage of hamster oocytcs penetrated at a
motile sperm concentration of 5 x 10” cdilshnl; b) mean number of spermato-
zoa penetrating each oocyie, C) rates of malondialdchyde production, d) motili-
ty. 4p = 0.02. n =7. All spurn preparations were exposed to promoter for 45
main and then cenuifloged (500 x g for 5miii) before being resuspended in fresh
medium containing A23 187 for 2.25 h. At the end of this period, the cells were
again centrifuged (500 x gfor5 miii) and resuspended in mmaupplcmemed. fresh
Medium BW’W. At this point. samples were removed for assessments of asian-
dialdehyde production, motility, and sperm-oocyte fusion. See Materials and
Method$ for further details. Paired nambers at the foot of each colw,,na dose of
ferrous sulfate/dose of ascorbate in promoter system.
a) 100
80
c60
0
(0
- 40
a,
0
20
Dose of Ferrous Ion Promoter
FIG. 6. Influence of a-tocopherol on the inhibition of sperm-oocy*e fusion
by lipid peroxidation. Results preseqjed as a) percentage oocytc fusion at a
motile spenn concentration of 5x 10” ccli Wml, b) mean number of spermato-
zoa penetrating each oocy*e, and C) motility. Values at the head of each column
=number of independent experiments performed for each dose of prosnotcr
data from control incubations have been pooled. *p<05 **p<.02. Paired
numbers at the foot of each column =dose of ferrous sulfate/dose of ascorbate
in promoter system.
LIPID PEROXIDATION AND SPERM FUNCTION 191
TABLE 1. Influence of a-tocopherol on the sustained motility (%) of human spermatozoa in presence and absence of ferrous ion promoter.a
Duration of
incubation Control ct-Tocopherol Fe Promoter Fe Promoter +
a-tocopherol
0 hb
24 h66.0
39.0 ±3.9
± 8.0 62.5 ± 5.6
62.5 ± 5.6*5* 61.2
33.5 ±534
± 7.7t 70.3 ±4.3
57.9 ± 7#{149}9**
48h 26.7 ±5.1 40.9 ± 5.45* 15.5 ± 33.0 ± 5.8
5Values expressed as means ± SEM for n =12.
bsamples exposed to pnsnoter± a-tocopherol in presence of A23 187 before being centrifuged at 500 xgand resuspended in BWW alt =Oh.
5p4J.5, p<fJ.O1, **p<O.OOl, relative to control incubations.
p<f).O5, pO.Ol. mP.dlOOl, relative to Fe promoter +cr-tocopherol.
This analysis revealed the anticipated dose-depend-
ent suppression of sperm-oocyte fusion in the presence
of promoter, without any concomitant effect on motili-
ty. Even in the presence of a-tocopherol, a dose-de-
pendent suppression of hamster oocyte fusion was ob-
served, reflecting the fact that this reagent does not
inhibit the initiation of lipid peroxidation, merely its
propagation (Mak et al., 1983). However, relative to the
results achieved with promoter alone, the concomitant
presence of a-tocopherol resulted in a highly significant
enhancement in the level of sperm-oocyte fusion ob-
served at all doses of ferrous sulfate and ascorbate used
(Fig. 6). There was even a statistically significant
enhancement of sperm motility when a-tocopherol was
used in conjunction with the two lowest doses of pro-
moter (p =0.04; Fig. 6). Furthermore this enhancement
of motility was sustained over the ensuing 48 h, even
though the a-tocopherol had been removed after 4 h in
preparation for the sperm-oocyte fusion assay (Table 1).
In the absence of promoter, the presence of z-to-
copherol did not significantly influence sperm motility
but did exert a stimulatory effect on sperm-oocyte
fusion as judged by a significant increase in the degree
of polyspermy observed (p =0.034; Fig. 6).
Influence of Lipid Peroxidation
on Sperm-Zona Interaction
In the first experiment in this series, the spermatozoa
was preincubated for 2.5 h and then exposed to 2 doses
of promoter (ferrous sulphate:ascorbate concentrations
of 12.5 pM: 62.0 pM and 200 pM: 1000 pM, respec-
tively) or control Medium BWW for 1 h before being
washed and incubated with human zonae (n =7). With
this particular protocol, only the higher dose of ferrous
ion promoter induced a significant increase in the rate
of malondialdehyde production and only this dose of
promoter influenced sperm-zona interaction, signifi-
cantly increasing the number of spermatozoa tightly
bound to the zona pellucida from a control value of
47.4 ± 13.4 spermatozoa/b4 pm2 to 107.2 ± 18.6
spermatozoa/b4 J.tm2 (Figs. 7 and 8). This increase in
sperm-zona interaction was achieved without any sig-
nificant changes in percentage motility or the detailed
movement characteristics of these cells, including their
velocity, amplitude of lateral sperm head displacement,
frequency of rotation, and percentage rolling/yawing
(data not shown).
A second independent experiment was performed
that was of similar design except only the higher dose
of promoter was used and the preincubation of the
spermatozoa was carried out in the presence or absence
of 10 mM cz-tocopherol. This experiment yielded very
similar results, in that the induction of lipid peroxida-
tion with promoter was associated with a highly signifi-
cant (p<O.O2) increase in the ability of human spermat-
ozoa to bind to the zona pellucida (n =9; Fig. 9). Even
in the presence of cx-tocopherol, the addition of pro-
moter significantly (p<O.O2) enhanced sperm-zona
binding, although the magnitude of this increase was
significantly suppressed compared with the samples
incubated with promoter alone (p<O.02).
To evaluate the specificity of this enhanced binding
of human spermatozoa to the zona pellucida, limited
lipid peroxidation was again induced by a 1-h exposure
to 200 pM ferrous sulfate: 1000 pM ascorbate, only
this time the target consisted of isolated porcine zonae
pellucidae. Under normal condition, human spermato-
zoa exhibit no demonstrable affinity for the porcine
zona pellucida (Fig. 10). However, after the induction
of lipid peroxidation, the numbers of spermatozoa asso-
ciating with the zona surface increased from a control
value of 0.6 ± 0.5/104 pm2 to 109.3 ± 7.6/104 pm2
(p<O.OO1) in a manner reflecting the enhanced binding
of lipo-peroxidized spermatozoa to the human zona
pellucida.
140
a120
100’
80
U)
60
N
2 40
0)
C
20
0J
b) 120
0
. 100
8
80
.60
40
0
c)
FIG. 7. Influence of lipid peroxidation on sperm-zona interaction: a) spam
binding to human zonac pellucidac. b) lipid peroxidazifm as indicated by malon-
dialdehyde production, and c) motility. **p= 0.02, n =7. Paired nuners at the
foot of each column =dose of ferrous sulfateldose of ascorbate in promoter
syem.
This increase in sperm-zona interaction did not ap-
pear to be associated with a nonspecific increase in the
adhesiveness of the spermatozoa; hence, a b-h exposure
to the higher dose of promoter (200 pM ferrous sul-
phate: 1000 pM ascorbate) was not associated with
0
detectable autoagglutination of the spermatozoa in any
of the specimens examined (5100 x microscopic fields
examined for each specimen). Furthermore, in the zona-
free hamster oocyte experiments, the reduction in
sperm-oocyte fusion observed after the induction of
lipid peroxidation was not associated with an increase
in the number of spermatozoa binding to the oolemma.
DISCUSSION
Anumber of independent lines of evidence suggest a
role for lipid peroxidation in the etiology of defective
sperm function (Jones et al., 1978, 1979, Aitken and
Clarkson, 1987; Aitken et al., 1987, 1988; Alvarez et
a!., 1987). Analysis of the fatty acid composition of
human spermatozoa has revealed a high degree of un-
saturation (Jones et al., 1979), and this factor together
with the capacity of these cells to generate reactive
oxygen species, render them particularly susceptible to
oxidative stress. In previous studies (Aitken and Clark-
son, 1987; Aitken et al., 1988), we have recorded an
inverse relationship between the generation of reactive
oxygen species by samples of human spermatozoa and
their capacity for sperm-oocyte fusion. On the basis of
such results, we have postulated that lipid peroxidation
is initiated when the generation of superoxide anion by
human spermatozoa overwhelms the superoxide dismu-
tase system and conditions are created for the genera-
tion of hydroxyl radicals via the Haber-Weiss reaction,
as discussed in the Introduction.
The results obtained in the present study lend support
to this hypothesis in that samples exhibiting high rates
of reactive oxygen species production, either spontane-
ously or following activation with A23187, demon-
strated a correspondingly high rate of lipid peroxidation
in the presence of promoter. Furthermore, the studies
with exogenous catalase and superoxide dismutase indi-
cate that the two reactive oxygen species required to
generate hydroxyl radicals in a Haber-Weiss reaction
(superoxide anion and hydrogen peroxide) are both
produced by human spermatozoa. Under steady-state
conditions, the suppressive effect of catalase suggests
that the major product released to the exterior of the
cell is hydrogen peroxide, arising through the action of
endogenous superoxide dismutase (Alvarez et al.,
1987). However, when the spermatozoa are stimulated
with A23187, the predominant form of reactive oxygen
emanating from the spermatozoa is superoxide anion
(Fig. 3).
CONTROL 12.5/62 200/1000
Dose of Ferrous Ion Promoter (jiM)
192 AITKEN ET AL.
60
50
30
20
10
0
4’
...l;.
s:e1i’-. #{149}.
**
**
Control Fe/Asc
UPID PEROXIDATION AND SPERM FUNCtiON 193
FIG. 8. Phetomicrograph of the enhanced binding of human spermatozoa to the human zona peliucida following the induction of limited lipid peroxidation with
ferrous ion promoter. a) Control. b) Pcroxidized. x375.
.- 120
c’JE
100
0
80
60
a-
N
040
0)
D20
C
0
Treatment
FIG. 9. Influence of a-tocopherol on the lipoperoxidation.induced increase
in sperm-zona interaction in the human. 55p<0.02. a =9.
In the absence of promoter, high levels of reactive
oxygen species generation were not associated with
significantly enhanced rates of malondialdehyde pro-
duction even though, within the same data set, an
inverse relationship between reactive oxygen species
production and sperm-oocyte fusion was still observed.
These results emphasize the limitations of malondial-
dehyde production as an index of lipid peroxidation,
since only a small proportion of lipids, chiefly cyclic
endoperoxides, break down to yield this product
(Asakawa and Matsoshita, 1980). As a consequence,
the levels of lipid peroxidation associated with defec-
tive sperm function may not be detectable unless pro-
longed incubation times (Alvarez et al., 1987) or pro-
moters (Jones et a!., 1979) are used to accelerate the
peroxidative process and generate detectable levels of
malondialdehyde. The current intense interest in the
role of lipid peroxidation in pathological processes has
resulted in the development of new techniques for
FIG. 10. Photomicrograph of the enhanced binding of human spermatozoa to porcine zoom pdllucidac following the induction of limited lipid peroxidation with
ferrous ion promoter. a) Control. b) Peroxidized. x375.
194 AITKEN ET AL.
directly, and specifically, measuring the lipid peroxide
content of cells (Smith and Anderson, 1987). The appli-
cation of these alternative techniques to the study of
defective sperm function will be of high priority in the
future.
A frequent characteristic of patients exhibiting defec-
tive sperm function is that their spermatozoa develop a
refractoriness to calcium signals, such that sperm-oo-
cyte fusion will not occur in the presence of the iono-
phore A23187 (Aitken et a!., 1984, 1987). The present
study supports the concept that this refractoriness is due
to changes in the properties of the plasma membrane
induced by lipid peroxidation (Aitken and Clarkson,
1987). Induction of limited lipid peroxidation in other-
wise normal cells resulted in the production of spermat-
ozoa that exhibited the same refractoriness to calcium
activation as observed in the patient population, without
any loss of cell viability. The mechanism responsible
for the impairment of sperm-oocyte fusion may involve
a reduction in the fluidity of the plasma membrane
(Ohyashiki et al., 1988) as well as changes in the
activities of key membrane-bound enzymes, including
ion channels (Slater, 1984).
To determine whether the enhanced generation of
reactive oxygen species observed in cases of defective
sperm function is a cause or consequence of lipid
peroxidation, we examined the free-radical-generating
activity of spermatozoa exposed to ferrous ion promot-
er. At low levels of lipid peroxidation, the basal rate of
reactive oxygen species generation was unchanged, a!-
though there was a slight, but significant, stimulation of
the response to A23 187. This stimulation was observed
at a rate of malondialdehyde production (3.4 ± 0.2
pmol/108 spermatozoa/h for 50 pM ferrous sulphate:
250 p.M ascorbate), which was similar to the rate
produced spontaneously by defective sperm cells iso-
lated from Fraction 1 of the Percoll gradients
(3.7 ± 0.5 pmol/108 spermatozoa/h), which were also
LIPID PEROXIDATION AND SPERM FUNCtiON 195
characterized by elevated levels of reactive oxygen
species production. In this case, however, the basal
activity, as well as the response to A23 187 was elevat-
ed. Hence, although limited peroxidation of human
spermatozoa may enhance the capacity of these cells to
generate reactive oxygen species, presumably by inter-
fering with the cellular mechanisms that normally regu-
late this activity, such damage is not the primary insti-
gator of enhanced oxygen radical production but, more
probably, its consequence.
A logical corollary of this postulated role for lipid
peroxidation in the etiology of defective sperm function
is that antioxidants may be of value in treating this
condition. In the present study, we have demonstrated
that the damage inflicted on the sperm plasma mem-
brane by iron-catalyzed peroxidation can be limited by
the presence of a-tocopherol, a chain-reacting antioxi-
dant that inhibits the propagation of lipid peroxidation
within biological membranes without influencing the
initiation of this process. As a consequence, a-tocoph-
erol is competent to prevent the lateral diffusion of
damage in the sperm plasma membrane but cannot
completely reverse the disruption induced by a ferrous
ion promoter. An ever growing catalogue of antioxi-
dants, attacking different stages of the peroxidative
process, are becoming available, it will be of interest to
compare a-tocopherol with these alternative com-
pounds for their therapeutic potential in correcting de-
fective human sperm function in vitro and in vivo.
The superoxide anion generated by human spermato-
zoa appears to derive from an NADPH oxidase system
located in the sperm plasma membrane that is similar,
in principle, to the mechanisms responsible for the
oxidative burst of phagocytic macrophages and neutro-
phils (R. J. Aitken, unpublished observations). If this
highly specialized superoxide-generating system is po-
tentially so detrimental to the spermatozoa, it can only
have evolved if it conferred a significant advantage
upon these cells in terms of their capacity for fertiliza-
tion. The unexpected enhancement of sperm-zona inter-
action following the induction of lipid peroxidation
may provide a clue to the normal biological role of
superoxide in the regulation of sperm function. Lipid
peroxidation appears to lead to a sudden increase in the
capacity of human spermatozoa to bind to the zona
pellucida in a manner that appears to lack species-
specificity but may be zona-specific, since limited per-
oxidation of spermatozoa does not induce autoagglutin-
ation or enhanced binding to zona-free hamster oocytes.
Intriguingly, in other cell types whose functions are
characterized by phases of increased adhesiveness, such
as neutrophils or platelets, new evidence has indicated a
mediating role for superoxide anion and lipid peroxida-
tion (Lafuze et a!., 1983; Bearpark et at., 1988) in the
mechanism of adhesion. Similarly, peroxidation of
liposomes containing unsaturated fatty acids has been
shown to induce increased vesicle aggregation and,
significantly, fusion (Sevanian et al., 1988). In addition
to the effects of peroxidation on membrane adhesion
and fusion, this process also activates phospholipase A2
(Ungemach, 1985; Koshio et a!., 1988, Spom et al.,
1988) as a consequence of which membrane instability
should be further enhanced through the generation of
lysophospholipids. Previous studies in a variety of
mammalian species have suggested that the sudden
induction of the acrosome reaction on the surface of the
zona pellucida may involve the activation of phospholi-
pase A2 and the subsequent generation of lysophospho-
lipids and arachidonic acid (Fleming and Yanagimachi,
1981; Llanos et al., 1982; Ohzu and Yanagimachi,
1982; Bennet et a!., 1987). Furthermore exogenous lipid
peroxides have been shown to result in the selective
destabilization and loss of the acrosomal cap in ram
spermatozoa (Jones and Mann, 1977). In light of such
data, it is possible to postulate a role for reactive
oxygen species in sperm-zona interaction that could be
readily tested experimentally.
The initial recognition event uniting the spermato-
zoon with the zona pellucida appears to involve multi-
ple entities (Benau and Storey, 1988) including sperm
glycosyl transferases, which bind to the oligosaccharide
side chains of glycoproteins concentrated on the outer
zona surface (Macek and Shur, 1988). This recognition
event then triggers an intracellular calcium signal, a
consequence of which would be to stimulate the pro-
duction of reactive oxygen species (Aitken and Clark-
son, 1987). The resultant induction of local lipid perox-
idation should then result in the creation of a
multiphasic phospholipid arrangement within the
plasma membrane, as a result of which the binding of
phospholipase A2 to its substrate will be enhanced
(Ungemach, 1985). In addition, the presence of hydro-
gen peroxide may activate phospholipase A2 by means
of a tyrosine kinase-induced inactivation of lipocortin
(Hirata et a!., 1984; Koshio et a!., 1988). The resulting
stimulation of phospholipase A2 should then enhance
the fusogemcity of the sperm plasma membrane
through the destabilizing action of lysophospholipids
196 AITKEN ET AL
and, possibly, the release of substrates for prostaglandin
or leukotriene synthesis (Fleming and Yanagimachi,
1981; Ohzu and Yanagimachi, 1982; Aitken and Kelly,
1985). As a result of these changes in membrane orga-
nization and order, the peroxidation-dependent activa-
tion of phospholipase A2 would be expected to induce
an increase in sperm-zona adhesion, the acrosome reac-
tion (Llanos et at., 1982; Bennet et at., 1987), and the
generation of a fusogenic equatorial segment capable of
initiating sperin-oocyte fusion.
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