Andrology Lab Corner*: Shedding Light on Chemiluminescence: The Application of Chemiluminescence in Diagnostic Andrology

Traditional diagnostic andrology places great store on a descriptive assessment of semen in terms of sperm number, morphology, and motility. However, the reality is that these criteria cannot assist in developing a diagnosis but can only provide a description of the seminal phenotype. If we are to improve the management of infertile patients, we must understand how the superficial defects we are cataloging at the level of conventional seminology reflect the underlying pathophysiology of sperm production and maturation. It is only with a knowledge of these mechanisms that we can address the key issues of etiology, prevention, and treatment. 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. 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. 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. 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). . Luminol-dependent chemiluminescence. (A) Schematic representation of the underlying chemistry; L indicates luminol; L•, a luminol radical created by the one-electron oxidation of L. L+ is an azaquinone formed by the further one-electron oxidation of L• by oxygen, generating O2•− as a by-product. The reaction of L• with O2•− or the reaction of L+ with H2O2 generates an unstable endoperoxide whose decomposition leads to the production of the chemiluminescent species, an electronically excited aminophthalate. Redox cycling of the probe could result if human spermatozoa possessed an appropriate reductase to convert L+ back to the parent L. Any reactant that can achieve the univalent oxidation of luminol will generate chemiluminescence in this assay, including H2O2 and ONOO−. (B) PMA (12-myristate, 13-acetate phorbol ester)-induced chemiluminescence quenched by catalase. (C) PMA-induced chemiluminescence quenched by superoxide dismutase (SOD). 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). 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. . Lucigenin-dependent chemiluminescence. (A) Schematic representation of the underlying chemistry; Luc2+ indicates lucigenin; LH•+, a lucigenin radical created by the one-electron reduction of Luc2+. The reaction of LH•+ with oxygen generates O2•−. The latter then participates in an oxygenation reaction with LH•+, generating a dioxetane that decomposes with the generation of chemiluminescence. Any entity that can effect the one-electron reduction of lucigenin will, in the presence of oxygen, create a redox cycle that produces high levels of O2•− and chemiluminescence. It is impossible to distinguish between the relative contribution of such probe-dependent and cell-dependent chemiluminescent signals. (B) The chemiluminescence generated by the addition of exogenous NADPH is likely to involve a significant contribution because of the secondary O2•− production following the univalent reduction of the probe by NAD(P)H-dependent oxidoreductases (Vernet et al, 2001). (C) In contrast, the PMA (12-myristate, 13-acetate phorbol ester)-induced signal is more likely to involve the primary cellular production of O2•−. 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 is used to chemiluminescence through the activation of then it is that the cellular production of O2•− is being measured (Figure contrast, chemiluminescence is generated by the addition of (Aitken et al, and Ford, 2001), then the system may also be the presence of any capable of the one-electron reduction of lucigenin and its redox cycling activity (Figure we have recently that the lucigenin-dependent chemiluminescence associated with sperm suspensions can be largely for by the activity of the reductase from the et al, in press). the of lucigenin as a probe for for O2•− it have as a redox for the electron activity associated with sperm function (Aitken et al, in press). In many the sensitivity and diagnostic of the probe are rather than by its redox cycling If of O2•− production is then there are alternative chemiluminescent probes that not create a redox including the and and et al, the it will be that, chemiluminescent probes such as luminol and lucigenin cannot specific data on reactive oxygen species generation by human spermatozoa, they are of redox activity in these cells. The sensitivity of these probes is extremely but it them very to in that may their diagnostic of these factors are in the that When chemiluminescence is used to assess human semen the point of to be is the effect of leukocyte are of reactive oxygen species on a are of more than spermatozoa in chemiluminescence (Aitken and If luminol is to semen then chemiluminescent signals are generated that are highly correlated with the levels of leukocyte (Aitken et al, (Figure the is very such have impact on sperm function as as the spermatozoa are by seminal plasma (Aitken et al, However, as as seminal plasma is during sperm then these cells very to the reactive oxygen species generated by As a the to which sperm suspensions are with leukocytes is correlated with sperm function in (Aitken et al, as as the of in fertilization et al, et al, et al, (Figure . and oxidative (A) between the luminol signals generated in human semen and leukocyte indicates that luminol-dependent chemiluminescence can by a of in the of leukocyte contamination, the underlying contribution of spermatozoa to the luminol signals obtained. (B) observed between the in fertilization observed in an in fertilization and the fertilization on the of a criteria of semen The most important in this were the level of leukocyte in the sperm and sperm et al, In light of these the of luminol and HRP has been used in with such as et al, or (Figure to generate highly sensitive assays with which to human sperm suspensions for leukocyte These can also be used to the of on the use of (Aitken et al, to leukocytes from human sperm The of such in with is essential if chemiluminescent techniques are to be used to assess the redox activity of human sperm If such techniques are not there is a possibility that the recorded are more of the level of leukocyte than of the redox activity on the of the spermatozoa (Aitken and and Ford, the results that to the that will reactive oxygen species generation by human spermatozoa et al, 1993) may simply reflect the levels of leukocyte in the sperm suspensions (Figure . of leukocyte (A) or can be used to a chemiluminescent from leukocytes in the presence of luminol and horseradish peroxidase (B) These signals are highly correlated with the generated with such as from leukocyte contamination, factors that may the of chemiluminescent assays human spermatozoa the to activity to with following the of spermatozoa from seminal plasma (Aitken and Clarkson, As a assays are 1 of sperm (Kobayashi et al, 2001). that not give very chemiluminescent because the spermatozoa are to from the in seminal plasma, such as et al, and et al, 2001), that with the underlying radical The generated by artificially the production of chemiluminescent signals by human spermatozoa (Aitken and Clarkson, et al, of the used in spermatozoa for chemiluminescent is The supplementation of with albumin has the potential to generate chemiluminescent signals in the presence of human seminal plasma (Figure and These signals are generated because most are with which will generate luminol-dependent chemiluminescence on with the and in human seminal plasma the of human spermatozoa et al, and 1994; Aitken, particularly dependent on luminol, are sensitive to in (Figure of 1 from to has a effect on the of luminol, in a significant in the of the observed to PMA (Figure assessment of the diagnostic of chemiluminescent assays at and would be extremely of will with the chemiluminescence generated by spermatozoa by either or artificially the including (Figure or such as (Figure will artificially generate chemiluminescent signals in the of particularly with the many radical such as or acid, will cellular chemiluminescence, as will (Figure a to sperm In light of this to it is to as an of chemiluminescent . that the chemiluminescent signals generated by human spermatozoa. (A) The presence of albumin in the medium for will generate a chemiluminescent signal with peroxidase (HRP) assays on with seminal (B) This signal because most sources of are with that, on with the in seminal plasma, such as generate (C) The chemiluminescence generated in luminol-dependent is highly dependent on a in 1 from to has a effect on the chemiluminescent to of PMA (12-myristate, 13-acetate phorbol in the presence of signals can be generated assays with that will form such as In this such as will also generate signals with assays because of the formation of have the potential to chemiluminescence, being a medium that can have this the in chemiluminescent assays have been found to give significant results that with the of the and the potential of the spermatozoa in and in Aitken and Clarkson luminol-dependent chemiluminescence for assessment purposes, a significant in the redox associated with human spermatozoa. This between luminol-dependent chemiluminescence has been found in patients with in their semen such as (Aitken et al, and in the sperm populations from (Aitken et al, The of this assay was also in a study of that was characterized by a of any in the in this a was observed between the luminol-dependent chemiluminescence of spermatozoa and the of (Aitken et al, this of patients, the conventional criteria of semen were of diagnostic (Aitken et al, The of in these is not for these assays to diagnostic because the and signals are highly correlated (Aitken et al, Indeed, have that luminol-dependent signals just the of the assay, capable of the high levels of redox activity associated with sperm populations et al, 2001; Ollero et al, 2001). One of the that these some is that they failed to between redox that were to factors and that were to factors sources of redox activity should be in to the of diagnostic chemiluminescent As the presence of these cells can be readily in with a agonist such as or et al, 1994; et al, The presence of significant leukocyte is to spermatozoa, particularly in the of seminal plasma and can have a impact on fertilization et al, and Ford, 1998). The reactive oxygen species generated by leukocytes can also of the spermatozoa in a sperm the redox activity associated with of spermatozoa is not readily to spermatozoa the same et al, 1994). The of the redox activity from the spermatozoa is that it the of these cells. sperm populations are from the of and of leukocyte contamination, the chemiluminescence by PMA in the presence of a very with key of the semen including sperm morphology, and et al, (Figure In the presence of spermatozoa high levels of redox activity the underlying of the This is by Gil-Guzman et al also recorded a between the luminol signals generated by spermatozoa from the of and the of sperm in the of spermatozoa may lead to the release of high reactive oxygen spermatozoa the . between the of the PMA (12-myristate, 13-acetate phorbol ester)-induced chemiluminescent signals generated in sperm suspensions from the of and the of the semen (A) of cells in the semen (B) of cells in the semen (C) in the semen et al, 1998). This may be in to by spermatozoa. The of by human spermatozoa has been associated with high levels of redox activity in these as indicated in Figure et al, 1996; Gil-Guzman et al, 2001). The between and sperm function the positive that have been observed between male infertility and cytosolic sperm including et al, et al, SOD (Aitken et al, and et al, 1996). The cellular of such is highly correlated with the of in the of human spermatozoa et al, 1996). is also associated with infertility in cases of et al, of in fertilization patients have a between fertilization and the presence of in the sperm et al, . between in the of human spermatozoa and chemiluminescence. was to these sperm suspensions to the of cellular H2O2 by glutathione peroxidase et al, 1996). The signals generated by in the presence of lucigenin are also in populations of human spermatozoa from the of or NADPH is used as the (Aitken et al, in press). these signals are more of the presence of oxidoreductases capable of the one-electron reduction of lucigenin than are reactive oxygen species, they are highly correlated with the signals by (Figure The between the results with chemiluminescent assays in the of leukocyte reflect on some of it is the oxidoreductases for lucigenin chemiluminescence (Vernet et al, 2001) or the plasma membrane for generating the oxidizing species with (Aitken et al, the chemiluminescence will be in cases of failed because both the and the plasma membrane of such cells will be This on some of would also explain the between semen and the excessive redox activity observed in the of spermatozoa to the of et al, 1998; Gil-Guzman et al, 2001). The that now lies is to understand which elements the for the excessive redox activity and the of such activity in the suppression of sperm function and the induction of DNA damage It will also be of to the and factors for the of and the in and cellular redox to cell function to a of . between lucigenin chemiluminescence by and peroxidase (HRP) chemiluminescence by These signals are highly correlated even they measure different of cellular redox populations of human spermatozoa from the populations from the of the All been with and were of leukocyte assays are being used in andrology laboratories to assess the generation of reactive oxygen species by human sperm In this we have the chemistry that the activation of these probes and that their activity not with the generation of reactive oxygen If used with such as luminol and lucigenin can be used to the redox activity associated with human sperm suspensions and to the source of this redox activity between spermatozoa and The contribution is important to in sperm these cellular have the to create oxidative stress and disrupt both fertilization and DNA 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. these signals are linked to the of and thereby reflect the of the that redox activity is the only of human spermatozoa, such chemiluminescent assays will be in understanding of the etiology of male infertility and thereby to both and this the of the of in and