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From the * ARC Centre of Excellence in
Biotechnology and Development, Discipline of Biological Sciences, Faculty of
Science and IT, University of Newcastle, Callaghan, Australia; and the
Monash Institute of Reproduction and
Development, Monash University, Melbourne, Australia.
| Correspondence to: Dr R. John Aitken, Discipline of Biological Sciences, The University of Newcastle, Callaghan, New South Wales 2308, Australia (e-mail: jaitken{at}mail.newcastle.edu.au). |
| Received for publication May 4, 2004; accepted for publication May 4, 2004. |
Two major discoveries in this field have recently been made: 1) the Y-chromosome deletions that are found in a significant proportion of severely oligo- and azoospermic patients (Tiepolo and Zuffardi, 1976; Ma et al, 1992; Vogt et al, 1996; Aitken and Krausz, 2001), and 2) the oxidative stress that appears to characterize the infertility observed in many male patients (Aitken and Clarkson, 1987; Alvarez et al, 1987; Aitken, 1994; Aitken and Fisher, 1994; Sharma and Agarwal, 1996; Sikka, 2001, 2004; Zorn et al, 2003). It is even possible that these 2 phenomena are linked through the oxidative DNA fragmentation of the father's spermatozoa, which leads to Y-chromosome deletions as a consequence of aberrant DNA repair in the oocyte (Aitken and Krausz, 2001; Aitken and Marshall Graves, 2002).
The purpose of this study was to concentrate on oxidative stress, with special emphasis on the chemiluminescent techniques that are being widely used in andrology laboratories to measure redox activity in populations of human spermatozoa (Sikka, 2004). These techniques are certainly sensitive and powerful. However, if used indiscriminately without any understanding of the underlying chemistry, false conclusions will be, and indeed have been, drawn.
Oxidative Stress and Male Infertility
The notion that oxidative stress can be a significant cause of male
infertility goes back to the pioneering observations of John MacLeod
(1943) and Thaddeus Mann
(Jones et al, 1979). These
authors provided data indicating that reactive oxygen species, particularly
H2O2, were potentially harmful to mammalian spermatozoa
by virtue of the high sensitivity of these cells to peroxidative damage. This
vulnerability stems from the fact that human spermatozoa are richly endowed
with unsaturated fatty acids to give the plasma membrane the fluidity it will
need to perform the membrane fusion events associated with fertilization
(Jones et al, 1979;
Aitken and Clarkson, 1987;
Alvarez et al, 1987;
Ollero et al, 2001). In
addition, because these cells are largely bereft of cytoplasm, the cytosolic
antioxidant systems that protect most cells from oxidative stress (eg,
catalase, glutathione peroxidase, superoxide dismutase [SOD], indoleamine
oxygenase, aldehyde dehydrogenase) are not readily available, particularly in
vulnerable regions of the cell such as the flagellum. As a result, spermatozoa
are largely dependent on antioxidants located in the extracellular fluids
(epididymal and seminal plasma) to protect them during maturation, storage,
and ejaculation (Jones et al,
1979; Aitken and Fisher,
1994).
The possibility that excessive reactive oxygen species generation by human spermatozoa contributes to the etiology of male infertility was indicated by Aitken and Clarkson (1987) and Alvarez et al (1987). These authors provided evidence suggesting that human spermatozoa can generate reactive oxygen species and that, in cases of male infertility, this activity was developed to the point that it overwhelmed the limited antioxidant defenses offered by these cells. The result of such oxidative stress is the induction of lipid peroxidation in the sperm plasma membrane, the suppression of sperm function, and the precipitation of DNA damage to both the nuclear and mitochondrial genomes (Aitken et al, 1998; Aitken, 1999; Sawyer et al, 2003). These results have now been independently confirmed in many different laboratories (Sharma and Agarwal, 1996; Gil-Guzman et al, 2001; Sikka, 2004); as a result, there is now intense interest in the development and evaluation of simple, convenient techniques for monitoring the generation of reactive oxygen species by human spermatozoa.
Chemistry of Chemiluminescence
The 2 major probes that have been used to assess reactive oxygen species
generation by human spermatozoa are luminol
(5-amino-2,3-dihydro-1,4-phthalazinedione; also, 3-aminophthalic hydrazide)
and lucigenin (N,N'-dimethyl-9,9'-biacridinium
dinitrate). Because lucigenin carries a positive ionic charge, it is generally
thought to be relatively membrane impermeant and to respond to reactive oxygen
species, particularly superoxide anion (O2•-), in
the extracellular space. In contrast, the uncharged luminol molecule is
membrane permeant and can react (in the form of luminol or as a univalently
oxidized luminol radical) with a variety of reactive oxygen species, including
O2•-, H2O2, and
OH•. The particular sensitivity of luminol toward
H2O2 can be greatly accentuated by the addition of
horseradish peroxidase (HRP) (Cormier and
Pritchard, 1968; Aitken et al,
1992b). Although these probes are extremely sensitive and
convenient for diagnostic purposes, the data they generate have to be
interpreted with care. Confounding factors such as incubation time, leukocyte
contamination, medium composition and pH, presence of seminal plasma, and type
of albumin supplementation can have a profound impact on the signals obtained.
In the following sections, we will examine the factors contributing to the
activity of these probes and review their utility in the diagnosis of male
infertility.
Luminol— It has been presumed that both H2O2 and O2•- are involved in luminol-dependent chemiluminescence because both catalase and SOD can disrupt the signal with great efficiency (Figure 1A through C). The luminol signal generated by human spermatozoa is initiated by a one-electron oxidative event mediated by H2O2 and either endogenous peroxidase (Aitken et al, 1992b; Faulkner and Fridovich, 1993) or, to sensitize the assay for extracellular H2O2, the addition of HRP (Aitken et al, 1992a; Gomez et al, 1998). The one-electron oxidation of luminol leads to the creation of a radical species (L•). The latter interacts with ground state oxygen to produce O2•-, which then participates in the oxygenation of L• to create an unstable endoperoxide, which in turn breaks down with the release of light (Figure 1A). According to this scheme, O2•- is an essential intermediate in the creation of luminol-dependent chemiluminescence, and for this reason, SOD is a very effective inhibitor of this reaction cascade. However, the activity of this scavenger should never be taken to indicate the primary production of O2•- by human spermatozoa; O2•- is simply an artificially created intermediate that is essential for luminol-dependent chemiluminescence (Aitken et al, 1992a). Indeed, any univalent oxidant has the potential to generate O2•-, and hence chemiluminescence, in the presence of luminol, including ferricyanide, persulfate, hypochlorite, ONOO-, and xanthine oxidase (Figure 1A).
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Hydrogen peroxide lies upstream of O2•- in the reaction scheme depicted in Figure 1A, and its involvement in the initial oxidation of luminol partly accounts for the inhibitory effects of catalase. In addition, H2O2 will react directly with the azaquinone (L+) and thereby contribute to the formation of excited aminophthalic acid, the chemiluminescent species (Nakamura and Nakamura, 1998). In some species (rats and mice, but not humans), secondary radical species are created by the spermatozoa (eg, NO, ONOO), possibly as a consequence of H2O2–mediated attacks on arginine (Aitken et al, in press).
One of the most important points to emphasize about Figure 1 is the opportunity that this chemiluminescent signaling system presents for redox cycling. All that is needed is a source of H2O2 and peroxidase (or alternative oxidizing agent) to initiate the one-electron oxidation of luminol, and an azaquinone reductase, such as diaphorase (Gavella and Lipovac, 1992), to reduce L+ back to the parent luminol (L). The remaining elements of the chemiluminescent cascade can be generated by the detection system itself (O2•- by the interaction of L• with ground state oxygen, H2O2 by the SOD-induced dismutation of O2•-, L+ by the dismutation of L•, etc).
Although Figure 1 is a major simplification of the chemistry involved in luminol-dependent chemiluminescence, from a diagnostic andrology point of view, there are 4 points worth emphasizing: 1) the potential redox cycling activity associated with this probe will lead to a significant amplification of the signal and may explain why alternative methods for measuring H2O2 have failed to detect this oxidant in purified suspensions of human spermatozoa (Richer and Ford, 2001); 2) the complexity of this redox chemistry is such that we cannot state with certainty what is being measured with the luminol assay, and yet it is certainly not just H2O2 (Aitken et al, in press); 3) fundamentally, such assays measure redox activity that is characterized by the cellular generation of oxidizing species capable of creating L•; and 4) notwithstanding the reservations that might be expressed concerning the specificity of this probe, the luminol assay is robust (Kobayashi et al, 2001) and generates results that are highly correlated with sperm function (see below).
Lucigenin— This probe is thought to be sensitive to the cellular generation of O2•-, largely because of the ability of SOD to suppress lucigenin-dependent cellular signals (Faulkner and Fridovich, 1993). However, the same reservations that apply to the use of luminol to detect specific reactive oxygen species also apply to lucigenin. In the case of lucigenin, activation of the probe requires a one-electron reduction, rather than the one-electron oxidation associated with luminol-dependent chemiluminescence (Faulkner and Fridovich, 1993). This one-electron reduction creates a radical (LH•+) from lucigenin (L2+) that rapidly gives up its electron to ground state oxygen to create O2•-, thus returning the lucigenin to its parent state (Figure 2A). The LH•+ that is generated from the one-electron reduction of lucigenin then combines with O2•- to produce the dioxetane (Figure 2A), which in turn decomposes with the generation of light (chemiluminescence). The O2•- involved in the last reaction could come from an independent cellular source, such as an NADPH oxidase (Figure 2A and C), in which case the chemiluminescence recorded would reflect the generation of O2•-, as originally proposed for both leukocytes (Gyllenhammar, 1987) and spermatozoa (Aitken et al, 1992b; McKinney et al, 1996). However, an unknown proportion of the O2•- involved in this reaction is an artifact created by the reaction between LH•+ and ground state oxygen. Chemiluminescence created by the cellular generation of O2•- or the redox cycling of lucigenin cannot be readily distinguished, since both sources of reactive oxygen species are suppressible by SOD. When an agonist such as PMA (12-myristate, 13-acetate phorbol ester) is used to stimulate chemiluminescence through the activation of protein kinase C, then it is probable that the cellular production of O2•- is being measured (Figure 2C). By contrast, when chemiluminescence is generated by the addition of NAD(P)H (Aitken et al, 1997; Richer and Ford, 2001), then the system may also be detecting the presence of any oxidoreductase capable of effecting the one-electron reduction of lucigenin and activating its redox cycling activity (Figure 2B). For example, we have recently reported that the lucigenin-dependent chemiluminescence associated with caput epididymal sperm suspensions can be largely accounted for by the activity of the cytochrome P-450 reductase originating from the epididymal epithelia (Baker et al, in press).
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Notwithstanding the deficiencies of lucigenin as a probe for evaluating for O2•- production, it does have value as a nonspecific redox marker for the enhanced electron transfer activity associated with defective sperm function (Aitken et al, in press). In many ways, the sensitivity and diagnostic value of the probe are enhanced, rather than diminished, by its redox cycling activity. If quantification of O2•- production is required, then there are alternative chemiluminescent probes that do not create a redox cycle, including the Cypridina luciferin analog MCLA (de Lamirande and Gagnon, 1995) and coelenterazine (Tarpey et al, 1999).
Laboratory Application of Chemiluminescent Assays
From the above descriptions, it will be evident that, although
chemiluminescent probes such as luminol and lucigenin cannot yield specific
data on reactive oxygen species generation by human spermatozoa, they are
nevertheless sensitive, quantifiable indicators of redox activity in these
cells. The sensitivity of these probes is extremely valuable, but it makes
them very susceptible to interference in ways that may distort their
diagnostic information content. Some of these confounding factors are
described in the paragraphs that follow.
Leukocyte Contamination— When chemiluminescence is used to assess human semen samples, the first point of caution to be considered is the confounding effect of leukocyte contamination. Leukocytes (particularly neutrophils) are inherent generators of reactive oxygen species and, on a cell-by-cell basis, are log orders of magnitude more active than spermatozoa in stimulating chemiluminescence (Aitken and West, 1990). If luminol is added to unfractionated semen samples, then chemiluminescent signals are generated that are highly correlated with the levels of leukocyte contamination (Aitken et al, 1995) (Figure 3A). Unless the leukocytic infiltration is very severe, such leukocyte-generated free radicals have little impact on sperm function as long as the spermatozoa are protected by seminal plasma (Aitken et al, 1995). However, as soon as seminal plasma is removed during sperm preparation, then these cells become very susceptible to the reactive oxygen species generated by contaminating leukocytes. As a consequence, the degree to which unwashed sperm suspensions are contaminated with leukocytes is negatively correlated with sperm function in vitro (Aitken et al, 1995) as well as the outcome of in vitro fertilization therapy (Krausz et al, 1994; Sukcharoen et al, 1995; Vicino et al, 1999) (Figure 3B).
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In light of these results, the combination of luminol and HRP has been used in conjunction with leukocyte-specific agonists such as formyl methionyl leucyl phenylalanine (FMLP) (Krausz et al, 1994) or opsonized zymosan (Figure 4A) to generate highly sensitive assays with which to screen human sperm suspensions for leukocyte contamination. These procedures can also be used to monitor the effectiveness of techniques, based on the use of CD45-coated magnetic beads (Aitken et al, 1996b), to selectively remove leukocytes from human sperm suspensions. The application of such leukocyte-removal strategies, in conjunction with FMLP/zymosan provocation assays, is essential if chemiluminescent techniques are to be used to assess the redox activity of human sperm suspensions. If such techniques are not applied, there is a strong possibility that the responses recorded are more reflective of the level of leukocyte contamination than of the abnormal redox activity on the part of the spermatozoa (Aitken and West, 1990; Whittington and Ford, 1999). For example, the results that led to the assertion that nerve growth factor will stimulate reactive oxygen species generation by human spermatozoa (Weese et al, 1993) may simply reflect the levels of leukocyte contamination in the sperm suspensions (Figure 4B).
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Apart from leukocyte contamination, other practical factors that may confound the outcome of laboratory chemiluminescent assays using human spermatozoa include the following:
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Diagnostic Application
With the above practical considerations in mind, chemiluminescent assays
have been found to give clinically significant results that correlate with the
quality of the ejaculate and the fertilizing potential of the spermatozoa in
vitro and in vivo. Thus, Aitken and Clarkson
(1987), using
A23187-stimulated, luminol-dependent chemiluminescence for assessment
purposes, demonstrated a significant increase in the redox activities
associated with defective human spermatozoa. This negative association between
A23187-induced, luminol-dependent chemiluminescence has been found in patients
with overt disruptions in their semen profile, such as oligozoospermia (Aitken
et al, 1989b,
1992a), and in the low-density,
defective sperm populations recovered from Percoll gradients
(Aitken et al, 1989a). The
clinical significance of this assay was also emphasized in a long-term
prospective study of 139 couples that was characterized by a lack of any
detectable pathology in the female partners; in this study, a negative
association was observed between the luminol-dependent chemiluminescence of
spermatozoa and the incidence of spontaneous pregnancy
(Aitken et al, 1991). Furthermore, within this group of patients, the conventional criteria of semen
quality were of no diagnostic value whatsoever
(Aitken et al, 1991).
The incorporation of A23187 in these protocols is probably not necessary for these luminol-based assays to yield diagnostic information, because the basal and A23187-induced signals are highly correlated (r = 0.805) (Aitken et al, 1991). Indeed, recent studies have demonstrated that spontaneous, unstimulated luminol-dependent signals are, just like the A23187-stimulated version of the assay, perfectly capable of identifying the high levels of redox activity associated with defective sperm populations (Gil-Guzman et al, 2001; Ollero et al, 2001).
One of the problems that beset these early (and some current) studies is that they failed to discriminate between redox activities that were due to extrinsic factors (leukocyte contamination) and those that were due to intrinsic factors (defective spermatozoa). Both sources of redox activity should be monitored in order to interpret the outcome of diagnostic chemiluminescent assays.
Leukocyte Contamination— As indicated, the presence of these cells can be readily monitored using luminol/HRP in conjunction with a leukocyte-specific agonist such as FMLP or opsonized zymosan (Krausz et al, 1994; Sukcharoen et al, 1995). The presence of significant leukocyte contamination is undoubtedly damaging to spermatozoa, particularly in the absence of seminal plasma protection, and can have a negative impact on fertilization rates (Sukcharoen et al, 1995; Whittington and Ford, 1998). The reactive oxygen species generated by contaminating leukocytes can also affect all of the spermatozoa in a sperm suspension, whereas the increased redox activity associated with subpopulations of defective spermatozoa is not readily transmissible to normal spermatozoa within the same suspension (Plante et al, 1994).
Intrinsic Factors— The importance of the redox activity emanating from the spermatozoa is that it reflects the developmental normality of these cells. Thus, when defective sperm populations are isolated from the low-density region of Percoll gradients and freed of detectable leukocyte contamination, the chemiluminescence elicited by PMA in the presence of luminol/HRP shows a very tight correlation with key attributes of the original semen profile, including sperm morphology, motility, and count (Gomez et al, 1998) (Figure 6). In other words, the presence of defective spermatozoa exhibiting high levels of redox activity signifies the underlying quality of the spermatogenic process. This conclusion is supported by Gil-Guzman et al (2001), who also recorded a correlation between the luminol signals generated by defective spermatozoa recovered from the low-density region of Percoll gradients and the quality of sperm morphology in the original ejaculate, implying "that dysregulation of normal spermatozoa may lead to the release of high reactive oxygen species–producing immature spermatozoa into the seminiferous tubules."
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This association may be due, in part, to cytoplasmic retention by defective spermatozoa. The retention of excess residual cytoplasm by human spermatozoa has repeatedly been associated with high levels of redox activity in these cells, as indicated in Figure 7 (Gomez et al, 1996; Gil-Guzman et al, 2001). The association between excess residual cytoplasm and defective sperm function explains the positive correlations that have repeatedly been observed between male infertility and cytosolic sperm enzymes, including creatine kinase (Huszar et al, 1988), lactic acid dehydrogenase (Casano et al, 1991), SOD (Aitken et al, 1996a), and glucose-6-phosphate dehydrogenase (Gomez et al, 1996). The cellular content of such enzymes is highly correlated with the amount of residual cytoplasm retained in the midpiece of human spermatozoa (Gomez et al, 1996). Cytoplasmic retention is also associated with infertility in cases of varicocele (Zini et al, 2000). Furthermore, studies of in vitro fertilization patients have demonstrated a strong negative correlation between fertilization rate and the presence of residual cytoplasm in the sperm midpiece (Keating et al, 1997).
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The signals generated by NAD(P)H in the presence of lucigenin are also elevated in populations of defective human spermatozoa isolated from the low-density Percoll fractions, regardless of whether NADH or NADPH is used as the stimulus (Aitken et al, in press). Even though these signals are probably more reflective of the presence of oxidoreductases capable of effecting the one-electron reduction of lucigenin than are reactive oxygen species, they are highly correlated with the luminol/HRP signals induced by A23187 (Figure 8). The intercorrelations between the results obtained with various chemiluminescent assays in the absence of leukocyte contamination probably reflect each assay's dependence on some feature of cytoplasmic retention. Whether it is the cytoplasmic NAD(P)H oxidoreductases responsible for NAD(P)H-induced lucigenin chemiluminescence (Vernet et al, 2001) or the putative plasma membrane NAD(P)H oxidases responsible for generating the oxidizing species detected with luminol/HRP (Aitken et al, 1997), the chemiluminescence will be enhanced in cases of failed cytoplasmic extrusion, because both the cytoplasmic volume and the plasma membrane area of such cells will be elevated. This common dependence on some aspect of cytoplasmic retention would also explain the relationship between semen quality and the excessive redox activity observed in the subpopulations of spermatozoa migrating to the low-density region of Percoll gradients (Gomez et al, 1998; Gil-Guzman et al, 2001). The task that now lies ahead is to understand which elements within the residual cytoplasm account for the excessive redox activity and the role of such activity in the suppression of sperm function and the induction of DNA damage (Aitken, 1994, 1999). It will also be of fundamental importance to resolve the environmental and genetic factors responsible for the disruption of spermiogenesis and the resultant increases in cytoplasmic retention and cellular redox activity. Errors pertaining to Sertoli cell function seem to present a reasonable proposition worthy of investigation.
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Conclusions
Chemiluminescent assays are currently being used in andrology laboratories
to assess the generation of reactive oxygen species by human sperm
suspensions. In this review, we have outlined the chemistry that underpins the
activation of these probes and emphasized that their activity does not
necessarily equate with the generation of reactive oxygen species. If used
with care, however, reagents such as luminol and lucigenin can be used to
monitor the redox activity associated with human sperm suspensions and to
differentiate the source of this redox activity between spermatozoa and
contaminating leukocytes. The leukocytic contribution is important to resolve
because, in washed sperm preparations, these cellular contaminants have the
capacity to create oxidative stress and disrupt both fertilization and DNA
integrity in the spermatozoa. The chemiluminescent signals generated by the
spermatozoa are also important to assess because they are associated with
lipid peroxidation and DNA damage. Moreover, these signals are linked to the
retention of excess residual cytoplasm and thereby reflect the overall quality
of the spermatogenic process. Given that excess redox activity is the only
known biochemical signature of defective human spermatozoa, such
chemiluminescent assays will be instrumental in researching our understanding
of the etiology of male infertility and thereby improving our capacity to both
manage and prevent this condition.
Acknowledgments
We gratefully acknowledge the support of the ARC Centre of Excellence in Biotechnology and Development.
Footnotes
* Andrology Lab Corner welcomes the submission of unsolicited
manuscripts, requested reviews, and articles in a debate format. Manuscripts
will be reviewed and edited by the Section Editor. All submissions should be
sent to the Journal of Andrology Editorial Office. Letters to
the editor in response to articles as well as suggested topics for future
issues are encouraged. 
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