help button home button Endocrine Society JCEM
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Journal of Clinical Endocrinology & Metabolism , doi:10.1210/jc.2005-2711
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a related Letter to the Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by De Iuliis, G. N.
Right arrow Articles by Aitken, R. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by De Iuliis, G. N.
Right arrow Articles by Aitken, R. J.
Related Collections
Right arrow Male Endocrinology
The Journal of Clinical Endocrinology & Metabolism Vol. 91, No. 5 1968-1975
Copyright © 2006 by The Endocrine Society

Definitive Evidence for the Nonmitochondrial Production of Superoxide Anion by Human Spermatozoa

Geoffry N. De Iuliis, Jordana K. Wingate, Adam J. Koppers, Eileen A. McLaughlin and R. John Aitken

Centre of Excellence in Biotechnology and Development and Discipline of Biological Sciences, University of Newcastle, Callaghan, New South Wales 2308, Australia

Address all correspondence and requests for reprints to: R. John Aitken, Discipline of Biological Sciences, School of Environmental and Life Sciences, University of Newcastle, Callaghan, New South Wales 2308, Australia. E-mail: jaitken{at}mail.newcastle.edu.au.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Context: Oxidative stress in the male germ line has been associated with poor fertility, impaired embryonic development, miscarriage, and childhood disease. Such stress is known to be associated with the peroxidation of unsaturated fatty acids in the sperm plasma membrane and oxidative DNA damage to both the nuclear and mitochondrial genomes. However, the source of the free radicals responsible for such damage is still unresolved.

Objective: The objective of this study was to chemically validate the use of dihydroethidium (DHE) as a probe for detecting the generation of superoxide anion by human spermatozoa and to examine the relationship between this activity and defective sperm function.

Method: DHE and SYTOX green were used in conjunction with flow cytometry and HPLC to investigate superoxide generation by human spermatozoa. Cause and effect relationships were established using menadione to artificially drive superoxide production by these cells.

Results: HPLC, mass spectrometry, nuclear magnetic resonance (NMR) spectroscopy, and spectrofluorometry were used to demonstrate that human spermatozoa generate the superoxide-specific product, 2-hydroxyethidium, from DHE. Spontaneous superoxide production by human spermatozoa was found to originate from a nonmitochondrial source and was inversely correlated with sperm motility. A causative relationship between superoxide generation and sperm function was demonstrated when the pharmacological stimulation of this activity with menadione was shown to result in both severe motility loss and DNA damage.

Conclusions: These studies validate a methodology for investigating the origins of oxidative stress in the male germ line and demonstrate, for the first time, the significance of superoxide generation by human spermatozoa in the etiology of this condition.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
OXIDATIVE STRESS IN the male germ line has been associated with a diverse array of pathological consequences including reduced rates of fertilization in vivo and in vitro, impaired preimplantation development of the embryo, increased rates of early pregnancy loss, and high rates of morbidity in the offspring (1, 2, 3, 4, 5). Such stress involves free radical attacks on human spermatozoa that induce peroxidative damage in the sperm plasma membrane (6, 7, 8, 9) and compromise the integrity of both the nuclear and mitochondrial genomes (10, 11, 12). Despite the vulnerability of these cells to oxidative assault (12, 13, 14), the source of the free radicals responsible for such damage remains unresolved.

Although studies employing chemiluminescence or cytochrome C reduction have suggested free radical generation by spermatozoa, none of this evidence is definitive (15, 16, 17). A new opportunity to address this question was recently presented by the discovery of a unique reaction product between dihydroethidium (DHE) and superoxide anion (O–&2dot;), 2-hydroxyethidium (2OHEt+). The latter differs from ethidium (Et+) by the presence of an additional oxygen atom (18) and, critically, this product is specific to the oxidation of DHE by O–&2dot;; it cannot be replicated using other commonly encountered oxidizing species such as H2O2, hypochlorous acid, or peroxynitrite (19). In this study, we have employed DHE in conjunction with analytical nuclear magnetic resonance spectroscopy, mass spectrometry (MS), and spectrofluorometry to demonstrate that human spermatozoa are capable of generating O–&2dot; and that this activity is associated with defective sperm function.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials

DHE and SYTOX Green were purchased from Molecular Probes, Invitrogen (Mount Waverley Australia). Dynabeads coated with a monoclonal antibody directed against the common leukocyte antigen, CD45, were from Dynal (Oslo, Norway). Xanthine oxidase and the superoxide dismutase (SOD) mimetic, Mn (III)tetrakis(1-methyl-4-pyridyl)porphyrin pentachloride, were obtained from Calbiochem (San Diego, CA). Percoll was sourced from Amersham Biosciences (Piscataway, NJ), and DNAse was from Promega (Madison, WI). All other chemicals were purchased from Sigma-Aldrich (St. Louis, MO).

Human spermatozoa

Institutional and state government ethical approval was secured for the use of human semen samples for the purposes of this research. After initial inspection of the samples for liquefaction, consistency, debris, and volume, assessments of cell count and motility were conducted, and cell viability was measured using the eosin exclusion test (20). Motility assessments were conducted using a phase contrast microscope and an eyepiece graticule to subdivide the visual field. At least 100 cells were counted, and any cell exhibiting flagellar movement was classified as motile. After allowing at least 30 min for liquefaction to occur, the spermatozoa were fractionated on a discontinuous two-step Percoll gradient, as described (21). Spermatozoa were ultimately recovered from the base of the high-density portion of the gradient as well as the low- to high-density Percoll interface (denoted 100% spermatozoa and 50% spermatozoa, respectively), washed with 10 ml Biggers-Whitten-Whittingham medium (BWW) (22), centrifuged at 600 x g for 15 min, and finally resuspended in HEPES-buffered BWW medium supplemented with 1 mg/ml polyvinyl alcohol at a concentration of 20 x 106/ml. All samples were tested for leukocyte contamination using a luminol-peroxidase based zymosan provocation assay (23) and cleared of any contaminating white cells using magnetic Dynabeads coated with a monoclonal antibody directed against the common leukocyte antigen, CD45 (21).

DHE assay

DHE is a poorly fluorescent molecule that is produced by the two-electron reduction of Et+. When DHE is oxidized within a cell, it produces DNA-sensitive fluorochromes, Et+ and 2OHEt+, that generate a red nuclear fluorescence in cells excited at 510 nm. Because the fluorochromes generated from DHE interact with DNA to generate a signal, the probe cannot provide information on the subcellular source of reactive oxygen species (ROS); it is always the nucleus that fluoresces. However, if carefully validated, this molecule is a potentially sensitive indicator of intracellular ROS generation. The fundamental chemistry underpinning the use of DHE to measure O–&2dot;, formation is outlined in Fig. 1AGo.


Figure 1
View larger version (14K):
[in this window]
[in a new window]
  		FIG. 1. DHE as a probe for ROS generation by human spermatozoa. A, Diagrammatic representation of the chemistry underlying the use of DHE as a probe for ROS generation. DHE may either undergo a nonspecific two-electron oxidation to generate Et+ or react with O–&2dot; to generate the specific reaction product, 2OHEt+. Both of these fluorochromes will interact with DNA in the sperm nucleus to generate a red fluorescence. To be certain that spermatozoa were producing O–&2dot;, it was essential to isolate the putative 2OHEt+ by HPLC and verify the chemical identity of the product using a variety of analytical tools including NMR spectroscopy, MS, and spectrofluorometry. In the course of these studies, o-chloranil was used to drive the nonspecific oxidation of DHE to Et+, whereas the closely related compound, menadione, was used to stimulate the O–&2dot;-induced formation of 2OHEt+. B, Confocal image of human spermatozoa stained with DHE and SYTOX Green; overlay image of red and green fluorescence. Nonviable cells are green or orange, whereas viable cells that are generating reactive oxygen species are red. Scale bar, 10 µm.

 
Our preliminary studies on the application of this probe to human sperm samples indicated a need to account for nonviable cells (see Results). This problem was addressed by incorporating into the reaction mixture a fluorescent vitality stain, SYTOX Green, with emission characteristics that were quite distinct from the products of DHE oxidation. For the assay, DHE and SYTOX Green were diluted in BWW and added to 2 x 106 spermatozoa at 2 and 0.5 µM, respectively, in a final volume of 200 µl. The cells were then incubated in the dark at 37 C for 15 min, this treatment having no significant influence on sperm motility or viability. The DHE (red) and SYTOX Green (green) fluorescence was then measured on a FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA). Argon laser excitation at 488 nm was coupled with emission measurements using 530/30 band pass (green) and 585/42 band pass (red) filters for FL1 and FL2, respectively. Nonsperm-specific events were gated out, and 10,000 cells were examined. Images of the DHE and SYTOX green fluorescence were collected on a Zeiss LSM510 confocal microscope (Carl Zeiss GmbH, Jena, Germany) employing argon laser excitation (488 nm) with emission collection at 500–530 nm (green) and helium neon laser excitation (543 nm) with emission collection at more than 560 nm (red).

Sample preparation for HPLC analysis

Purified spermatozoa (4 x 106) were treated with DHE (2 µM for 15 min). The cells were then washed with one volume of TE buffer (10 mM Tris and 1 mM EDTA, pH 8.0) and lysed by three freeze-thaw cycles (–80 C to +22 C). The lysed cell preparation was then subjected to a DNAse digestion. Initially, 4 U of the RQ1 DNAse (RNAse free preparation of deoxyribonuclease I) was added to the cells and incubated at 37 C for 40 min. The temperature was then elevated to 65 C for 10 min to stop the DNAse reaction. The fluorescent dyes were extracted by the addition of 1 volume of 1-butanol. This mixture was then vortexed for 1 min and placed in a microfuge. The butanol phase was removed and evaporated to dryness, then resuspended in 100 µl of Milli Q water before HPLC injection.

HPLC

To characterize the products of DHE metabolism, a mixture of 2OHEt+ and Et+ was prepared by exposing DHE (6 mM) to the mixture of ROS generated by xanthine (1 mM) and xanthine oxidase (0.025 U). The products of this reaction were extracted with ethyl alcohol, lyophilized, and made up in MilliQ water before separation by an HPLC system (Waters Breeze System, Rydalmere, NSW, Australia) equipped with a UV detector (Waters 486) set at 280 nm and a fluorescence detector (Waters 2475) set at an excitation wavelength of 480 nm and emission wavelength of 586 nm. Solution A was MilliQ water with 1% acetic acid, whereas solution B was 90% acetonitrile with 1% acetic acid. A gradient from 10–25% B over 18 min at 1 ml/min was passed through a reverse-phase column (Waters Symmetry, C18 5.0 µm, 4.6 x 150 mm), after sample injection (25 µl). Two clearly resolved peaks were collected separately and further analyzed. For analysis of the products of DHE metabolism by spermatozoa, 25 µl containing approximately 1 µM reaction product was typically injected into the HPLC.

Electrospray ionization MS (ESIMS)

Mass spectra were produced using a LTQ ion-trap instrument (Thermo-Finnigan, GE Healthcare, Little Chalfont, UK). A cone voltage of 1.6 kV with a capillary temperature of 200 C was employed for electrospray. The two fractions isolated by HPLC were directly analyzed by MS. MS and subsequent tandem MS fragmentation were performed on the primary MS ion for further characterization.

Nuclear magnetic resonance (NMR) spectroscopy

NMR spectra were recorded on a Bruker Avance DPX-300 spectrometer (Bruker BioSpin AG, Fällanden, Switzerland), with deuterated dimethyl sulfoxide as the solvent in 5-mm NMR tubes. Dimethyl sulfoxide was also used as the reference (2.5 ppm for 1H and 39.5 ppm for 13C). 1H NMR spectra were typically recorded with 64 scans, whereas 13C spectra were recorded typically with 80,000 scans.

Spectrofluorometric analysis after quinone treatment

Fluorescence spectra were obtained on a Shimadzu RF-5301PC spectrofluorometer [Shimadzu Scientific Instruments (Oceania) Pty., Rydalmere, NSW]. The excitation wavelength was 510 nm (in the presence of DNA) or 480 nm (in the absence of DNA), whereas the emission wavelengths ranged from 540–800 nm with an emission slit width of 10 µm. To study the impact of SOD (300 IU) on the free radical responses of human spermatozoa to menadione and o-chloranil (50 µM), it was necessary to permeabilize the cells (32 x 106/ml in PBS) to the enzyme by freeze-thawing (–80 C to +22 C) three times. The permeabilized cells were then exposed to quinone at 37 C in the presence of DHE (10 µM) and a mixture of NADH (1 mM) and NADPH (1 mM). The latter were included to provide a potential source of electrons for the one-electron reduction of quinone to the unstable semiquinone radical. The latter would then be expected to revert back to the parent quinone releasing an electron to oxygen, with the consequent generation of O–&2dot;. To prove that quinone-induced DHE signals really represented the production of O–&2dot;, SOD was added to the mixture for 15 min before the addition of NAD(P)H and quinone. Spectrofluorometric analysis of the reaction products was then performed 15 min later. Attempts to use a membrane permeant SOD mimetic [Mn (III) tetrakis (1-methyl-4-pyridyl)porphyrin pentachloride] to confirm O–&2dot; production by intact cells were thwarted by the powerful ability of this compounds to quench DHE fluorescence. For certain experiments, menadione (50 µM) was added to suspensions of intact human spermatozoa maintained at 37 C, and their motility was assessed for the following hour.

Carbonyl cyanide m-chlorophenylhydrazone (CCCP) and rotenone treatment

To determine whether the DHE signals generated by defective spermatozoa involved electron leakage from the sperm mitochondria, the mitochondrial inhibitors rotenone (0.1 and 1.0 mM) and CCCP (10 µM) were employed. For these studies, defective sperm populations from the low-density/high-density Percoll interface were treated with anti-CD-45-coated Dynabeads to remove any leukocyte contaminants. The effectiveness of this treatment was subsequently verified by luminol-peroxidase-dependent chemiluminescence after provocation with opsonized zymosan (23). These purified sperm suspensions were then adjusted to a concentration of 2 x 106/ml and preincubated with inhibitor for 15 min at 37 C before the addition of DHE (2 µM) and SYTOX green (0.5 µM) and a further 15-min incubation at the same temperature. The sample was then subjected to analysis by flow cytometry as described above. The medium employed for these studies, BWW, contained energy substrates in the form of glucose, lactate, and pyruvate.

Terminal deoxynucleotidyl transferase dUTP nick-end labeling assay

DNA fragmentation was detected by labeling free 3'-OH termini with fluorescein isothiocyanate-labeled dUTP using an assay kit was obtained from Roche (Mannheim, Germany) as previously described (23).

Statistics

All experiments were replicated at least three times on independent samples, and the results were analyzed using the SuperANOVA program (Abacus Concepts Inc., Berkeley, CA) on a MacIntosh G5 computer; post hoc comparison of group means was by Fisher’s protected least significant difference test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Development of flow cytometry methodology

DHE analysis of O–&2dot; generation by human spermatozoa isolated from the high-density region of Percoll gradients revealed a cohort of positively staining cells comprising 15.2 ± 4.5% of the sperm population (n = 14). Although this result was suggestive of ROS generation, subsequent analysis revealed a significant correlation (P < 0.001) between the percentage of DHE-positive cells and cell viability as assessed by eosin exclusion (r = 0.514; n = 42). Moreover, if sperm vitality was completely destroyed by a single round of freeze-thawing (–80 C to +22 C), 100% of cells became DHE positive. These results indicated that DHE assays are liable to interference, due to the presence of nonviable cells. This occurs because DHE preparations invariably contain trace amounts of Et+, which can label the nuclei of cells with disrupted plasma membranes. Such contamination is a potential confounder of any analysis conducted with DHE because the positively stained population would represent a mixture of nonviable cells and spermatozoa that were actively engaged in the process of free radical generation.

To circumvent this problem, it was essential that the ability of spermatozoa to activate DHE was assessed in parallel with their viability. For this purpose, we chose the viability stain, SYTOX Green. To validate the use of SYTOX Green in the assessment of sperm vitality by flow cytometry, the results obtained with this technique were compared with the outcome of a conventional eosin exclusion test, and the results were found to correlate with an r value of 0.89 (P < 0.001; n = 42).

Figure 1BGo shows a merged confocal image of a sperm population stained with these two probes in which viable cells generating a positive DHE signal (red) can be readily distinguished from nonviable cells (green and orange). Flow cytometry with these probes permitted the ready identification of a viable sperm population in semen that was capable of generating a putative O–&2dot; signal with DHE and migrated to the low-density region of Percoll gradients on centrifugation (Fig. 2Go).


Figure 2
View larger version (33K):
[in this window]
[in a new window]
  		FIG. 2. Flow cytometry of human spermatozoa stained with DHE and SYTOX Green. In semen, a population of viable, O–&2dot; -generating cells is evident (circled). These cells are excluded from the high-density Percoll fractions but represented in the population of defective spermatozoa isolated at the high-density/low-density Percoll interface.

 
DHE processing by spermatozoa

In the presence of O–&2dot;, DHE has been shown to yield a unique product, 2OHEt+ (18, 19). However, in the presence of other oxidizing species, DHE generates a second, two-electron oxidation product, Et+.

The ability of human spermatozoa to generate both Et+ and 2OHEt+ from DHE was demonstrated by treating these cells with two redox active quinones, o-chloranil and menadione (Fig. 3AGo). o-Chloranil treatment (50 µM) of permeabilized spermatozoa resulted in the nonspecific oxidation of DHE, to generate a product with a fluorescence maximum of approximately 590 nm, characteristic of Et+ intercalated into DNA. The inability of SOD (300 IU) to suppress this fluorescence signal further emphasized that the generation of Et+ by o-chloranil was independent of O–&2dot; (Fig. 3AGo). In contrast, treatment of spermatozoa with menadione (50 µM) produced a fluorescence spectrum with an emission maximum (564 nm) characteristic of 2OHEt+ intercalated into DNA (19). The O–&2dot;-dependent conversion of DHE to 2OHEt+ induced by menadione was confirmed by the complete suppression of this signal with 300 IU SOD (Fig. 3AGo). Under these conditions, menadione is being reduced by sperm oxidoreductases to a semiquinone radical using the NAD(P)H present in the reaction mixture as a source of electrons. The unstable semiquinone is then reverting back to the parent quinone releasing an electron to oxygen to generate the O–&2dot; detected by DHE.


Figure 3
View larger version (23K):
[in this window]
[in a new window]
  		FIG. 3. Emission spectra of human spermatozoa treated with DHE. A, Fluorescence spectra of spermatozoa (10 x 106 permeabilized cells in 200 µl BWW) treated with menadione (50 µM) or o-chloranil (50 µM) and incubated with DHE (10 µM) with and without SOD (300 U) in the presence of an electron source (NADH/NADPH, 1/1 mM). Only the menadione signal exhibited a fluorescence spectrum characteristic of O–&2dot;-induced 2OHEt+ formation, and only this signal could be inhibited with SOD. B, Shift in fluorescence wavelength was observed when different ratios of Et+ and 2OHEt+ were mixed with DNA (50 µg). 1, Pure 2OHEt+ (2.91 µM) + DNA; 2, 2OHEt+:Et+ (3:1) + DNA; 3, 2OHEt+:Et+ (1:1) + DNA; 4, 2OHEt+:Et+ (1:3) + DNA; 5, pure Et+ (2 91 µM) + DNA.

 
Although Et+ and 2OHEt+ exhibit distinct emission spectra, the spectral properties of these compounds cannot be used to monitor their presence in cells if a mixture of the two compounds is present, as is usually the case (19, 20). Although the purified products (2OHEt+ and Et+) are spectrally resolvable, mixtures produce spectra with intermediate emission maxima; the more Et+ in the mixture, the more the peak shifts to a higher wavelength (Fig. 3BGo). Apart from the closeness of the emission spectra, the lack of resolution inherent in mixtures of 2OHEt+ and Et+ may be further exacerbated by the aromatic and planar nature of both compounds, which will allow them to closely interact ({pi}-{pi} stack) with each other in solution. Given these difficulties in the spectral resolution of 2OHEt+ and Et+ in mixtures of these compounds, we used HPLC to achieve their separation and both MS and NMR spectroscopy to confirm their chemical identity.

Separation of 2OHEt+ and Et+ by HPLC and characterization

To establish the methodology, a chemical mixture of Et+ and 2OHEt+ was generated using ROS produced by xanthine/xanthine oxidase. HPLC analysis of the reaction products revealed the presence of two discrete peaks, putatively Et+ and 2OHEt+, respectively (Fig. 4AGo). These identities were subsequently confirmed by fluorescence spectroscopy, ESIMS, and 1H and 13C nuclear magnetic resonance spectroscopy.


Figure 4
View larger version (15K):
[in this window]
[in a new window]
  		FIG. 4. Resolution of the reaction products formed from DHE. A, Fluorescent chromatogram of the two products yielded by HPLC after incubation of DHE with a xanthine/xanthine oxidase free radical-generating system. B, Spectrofluorometry of the isolated products yielded the predicted emission spectrum of 2OHEt+ (fraction 2; emission maximum 564 nm) and Et+ (fraction 1; emission maximum approximately 590 nm); excitation wavelength of 480 nm, excitation slit width of 3 nm, emission slit width of 10 nm.

 
Figure 4BGo shows that fluorescence spectrum of fraction 1 exhibited an emission maximum of around 600 nm, consistent with pure Et+ in the absence of DNA (19), whereas the emission maximum of fraction 2 (575 nm) was consistent with that of pure 2OHEt+ (19).

Both fractions were also characterized by ion-trap ESIMS (Fig. 5Go). Fraction 1 yielded a dominant peak at 314 m/z, consistent with the mass of the Et+ cation. The MS2 spectrum of this parent ion yielded the predicted fragmentation pattern, a major peak at 286 m/z corresponding to the loss of an ethyl group (28 m/z) by the breakage of the N+-ethyl bond. Fraction 2 yielded a dominant peak at 330 m/z, which is consistent with the mass of the 2OHEt+ cation (Fig. 5Go), the difference of 16 Da corresponding to the presence of an oxygen atom in the O–&2dot;-induced product. Again, the MS2 fragmentation pattern of the parent ion was as predicted, a major fragment at 302 m/z representing the loss of an ethyl group (302 m/z). NMR spectra, both 1H and 13C, were also secured on fractions 1 and 2 and were found to correspond to the patterns previously reported (18) for Et+ and 2OHEt+ (data not shown). These results validated our HPLC methodology for separating the products of DHE oxidation and confirmed that the two fractions so isolated were Et+ and 2OHEt+, respectively.


Figure 5
View larger version (22K):
[in this window]
[in a new window]
  		FIG. 5. MS and tandem MS of Et+ isolated from fraction 1 and 2OHEt+ isolated from fraction 2.

 
The validity of the method was further established by engineering human sperm populations that were actively generating O–&2dot; as a result of treatment with menadione (Fig. 6AGo). Thus, when intact spermatozoa were exposed to menadione in the presence of DHE, a significant increase in the size of the 2OHEt+ peak was observed (P < 0.05; Fig. 6AGo). Surprisingly, this increase was accompanied by a highly significant decrease in the size of the Et+ peak recorded after treatment with menadione (P < 0.001; Fig. 6AGo), possibly reflecting competition between Et+ and 2OHEt+ for a limited number of accessible binding sites in the highly condensed chromatin typical of spermatozoa. As a consequence of this competition, the ratio of 2OHEt+ to Et+ gave a very sensitive confirmation of O–&2dot; generation by populations of human spermatozoa (P < 0.01; Fig. 6AGo).


Figure 6
View larger version (33K):
[in this window]
[in a new window]
  		FIG. 6. Application of the DHE/SYTOX Green assay to populations of human spermatozoa. A, HPLC analysis of 2OHEt+ and Et+ demonstrating that the stimulation of O–&2dot; production in intact human spermatozoa with menadione (50 µM) resulted in a significant increase in the generation of 2OHEt+. The yield of Et+ significantly declined under these circumstances such that the ratio of 2OHEt+ to Et+ gave a very sensitive indication of O–&2dot; generation in the presence of menadione. *, P < 0.05; **, P < 0.01; ***, P < 0.001. B, Highly significant (P < 0.001) negative correlation was observed between the DHE signal generated by viable human spermatozoa and sperm motility; P50, samples recovered from the high-density/low-density Percoll interface; P100, samples recovered from the high-density Percoll pellet. C, Stimulation of O–&2dot; production with menadione (50 µM) induced a highly significant decline in sperm motility after 60 min (***, P < 0.001); solid bars, menadione treated; white bars, controls. D, Induction of DNA damage in populations of human spermatozoa incubated with menadione for 15 h. ***, P < 0.001 compared with untreated control incubated for the same period of time. E, Mitochondrial inhibitor CCCP (10 µM) had no impact on either the vitality of human spermatozoa or their ability to generate O–&2dot;.

 
Application of the DHE assay to human spermatozoa

Flow cytometry assays of human sperm populations labeled with DHE and SYTOX Green were repeatable, replicate assays of a single highly active, free radical-generating sample, giving a coefficient of variation of 15% (34.6 ± 1.7% of cells positive; n = 10). In addition, the percentage of DHE-positive cells appeared to remain constant over a 24-h time period, giving a mean of 2.4 ± 0.8% positive cells after 15 min that was unchanged (P > 0.05) after 24 h (3.0 ± 0.2%; n = 3). Overall, the percentage of viable cells generating O–&2dot; was significantly lower in the functional sperm populations recovered from the high-density Percoll pellet (9.9 ± 3.8%) compared with the original ejaculate (29.3 ± 4.5%; P < 0.001), and the poor quality spermatozoa recovered from the high-density/low-density Percoll interface (23.5 ± 3.9%; P < 0.05; n = 14). Moreover, there were highly significant negative correlations between the percentage of free radical-generating viable cells (DHE-positive, SYTOX green-negative) and percentage motility within sperm populations recovered from unprocessed semen (r = –0.74; P < 0.01; n = 14), the high-density Percoll pellet (r = –0.90; P < 0.001; n = 14), and the low-density/high-density Percoll interface (r = –0.72; P < 0.01; n = 14), giving an overall correlation across of entire data set of r = 0.81 (P < 0.001; n = 42) (Fig. 6BGo).

The causative nature of these associations between sperm motility and O–&2dot; production was demonstrated using menadione to stimulate the generation of this radical in otherwise normal cells. The results of this analysis revealed that the induction of O–&2dot; generation through the redox cycling of this quinone resulted in a dramatic loss of sperm motility over a 60-min period (P < 0.001; Fig. 6CGo) and the subsequent induction of significant DNA damage (P < 0.001; Fig. 6DGo) and loss of sperm viability (data not shown). Significantly, the excessive generation of O–&2dot; by viable human spermatozoa did not appear to involve electron leakage from the mitochondria because neither rotenone (0.1 and 1.0 mM) nor CCCP (10 µM, Fig. 6EGo) had any significant impact on the vitality of these cells or the DHE signals they generated.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Oxidative stress is a major factor in the etiology of defective sperm function (10, 24, 25, 26, 27). Although the potential significance of ROS generation by human spermatozoa in the etiology of his condition was first indicated in 1987 (16, 28), definitive evidence for this assertion has not been forthcoming due to inadequacies in the purity of the sperm suspensions analyzed and the techniques used for ROS detection (29, 30, 31, 32, 33, 34).

The current study reports the development and validation of a method for detecting the generation of O–&2dot; by populations of human spermatozoa. The results demonstrate beyond reasonable doubt that human spermatozoa are capable of generating O–&2dot; and that the production of this oxygen radical is inversely correlated with defective sperm function. Because the oxidation of DHE generates DNA-sensitive fluorochromes that then stain the sperm nucleus, the subcellular origins of the ROS cannot be determined with this probe. Nevertheless, the present results indicate that spontaneous O–&2dot; production by populations of human spermatozoa does not involve electron leakage from the mitochondrial electron transport chain. Neither rotenone nor CCCP inhibited the DHE signal recorded in suspensions of defective human spermatozoa; moreover, we could not induce O–&2dot; production by supplying the mitochondria with additional energy substrates in the form of succinate or glutamate (data not shown). Nevertheless, this validated methodology now opens the way for definitive studies on the biochemical origins of O–&2dot; generation, the impact of environmental toxicants on this activity, and the relationship among O–&2dot; production, male infertility, and DNA damage in the male germ line.


   Acknowledgments
 
We gratefully acknowledge the financial support provided by the Centre of Excellence in Biotechnology and Development.


   Footnotes
 
G.N.D.I., J.K.W., A.J.K., E.A.M., and R.J.A. have nothing to declare.

First Published Online February 28, 2006

Abbreviations: BWW, Biggers-Whitten-Whittingham medium; CCCP, carbonyl cyanide m-chlorophenylhydrazone; DHE, dihydroethidium; ESIMS, electrospray ionization mass spectrometry; Et+, ethidium; MS, mass spectrometry; m/z, mass to charge ratio; NMR, nuclear magnetic resonance; O–&2dot;, superoxide anion; 2OHEt+, 2-hydroxyethidium; ROS, reactive oxygen species; SOD, superoxide dismutase.

Received December 15, 2005.

Accepted February 22, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Aitken RJ 2004 Founders’ lecture. Human spermatozoa: fruits of creation, seeds of doubt. Reprod Fertil Dev 16:655
  2. Loft S, Kold-Jensen T, Hjollund NH, Giwercman A, Gyllemborg J, Ernst E, Olsen J, Scheike T, Poulsen HE, Bonde JP 2003 Oxidative DNA damage in human sperm influences time to pregnancy. Hum Reprod 18:1265–1272[Abstract/Free Full Text]
  3. Saleh RA, Agarwal A, Nada EA, El-Tonsy MH, Sharma RK, Meyer A, Nelson DR, Thomas AJ 2003 Negative effects of increased sperm DNA damage in relation to seminal oxidative stress in men with idiopathic and male factor infertility. Fertil Steril 79(Suppl 3):1597–1605
  4. Agarwal A, Saleh RA, Bedaiwy MA 2003 Role of reactive oxygen species in the pathophysiology of human reproduction. Fertil Steril 79:829–843[CrossRef][Medline]
  5. Aitken RJ, Baker MA, Sawyer D 2003 Oxidative stress in the male germ line and its role in the aetiology of male infertility and genetic disease. Reprod Biomed Online 7:65–70[Medline]
  6. Jones R, Mann T, Sherins RJ 1979 Peroxidative breakdown of phospholipids in human spermatozoa: spermicidal effects of fatty acid peroxides and protective action of seminal plasma. Fertil Steril 31:531–537[Medline]
  7. Aitken RJ, Harkiss D, Buckingham DW 1993 Analysis of lipid peroxidation mechanisms in human spermatozoa. Mol Reprod Dev 35:302–315[CrossRef][Medline]
  8. Ollero M, Powers RD, Alvarez JG 2000 Variation of docosahexaenoic acid content in subsets of human spermatozoa at different stages of maturation: implications for sperm lipoperoxidative damage. Mol Reprod Dev 55:326–334[CrossRef][Medline]
  9. Ollero M, Gil-Guzman E, Lopez MC, Sharma RK, Agarwal A, Larson K, Evenson D, Thomas Jr AJ, Alvarez JG 2001 Characterization of subsets of human spermatozoa at different stages of maturation: implications in the diagnosis and treatment of male infertility. Hum Reprod 16:1912–1921[Abstract/Free Full Text]
  10. Kodama H, Yamaguchi R, Fukuda J, Kasi H, Tanak T 1997 Increased deoxyribonucleic acid damage in the spermatozoa of infertile male patients. Fertil Steril 65:519–524
  11. Barroso G, Morshedi M, Oehninger S 2000 Analysis of DNA fragmentation, plasma membrane translocation of phosphatidylserine and oxidative stress in human spermatozoa. Hum Reprod 15:1338–1344[Abstract/Free Full Text]
  12. Lewis SE, Aitken RJ 2005 DNA damage to spermatozoa has impacts on fertilization and pregnancy. Cell Tissue Res 322:33–41[CrossRef][Medline]
  13. Aitken RJ, Koopman P, Lewis SE 2004 Seeds of concern. Nature 432:48–52[CrossRef][Medline]
  14. Aitken RJ, Buckingham DW, Brindle J, Gomez E, Baker HW, Irvine DS 1995 Analysis of sperm movement in relation to the oxidative stress created by leukocytes in washed sperm preparations and seminal plasma. Hum Reprod 10:2061–2071[Abstract/Free Full Text]
  15. Aitken RJ, Baker MA, O’Bryan MK 2004 Shedding light on chemiluminescence: the application of chemiluminescent assays in diagnostic andrology. J Androl 25:455–465[Free Full Text]
  16. Alvarez JG, Touchstone JC, Blasco L, Storey BT 1987 Spontaneous lipid peroxidation and production of hydrogen peroxide and superoxide in human spermatozoa. J Androl 8:338–348[Abstract/Free Full Text]
  17. Richer SC, Ford WC 2001 A critical investigation of NADPH oxidase activity in human spermatozoa. Mol Human Reprod 7:237–244[Abstract/Free Full Text]
  18. Zhao H, Kalivendi S, Zhang H, Joseph J, Nithipatikom K, Vasquez-Vivar J, Kalyanaraman B 2003 Superoxide reacts with hydroethidine but forms a fluorescent product that is distinctly different from ethidium: potential implications in intracellular fluorescence detection of superoxide. Free Radic Biol Med 34:1359–1368[CrossRef][Medline]
  19. Fink B, Laude K, McCann L, Doughan A, Harrison DG, Dikalov S 2004 Detection of intracellular superoxide formation in endothelial cells and intact tissues using dihydroethidium and an HPLC-based assay. Am J Physiol Cell Physiol 287:C895–902
  20. World Health Organization 1992 WHO laboratory manual for the examination of human semen and semen-cervical mucus interaction. 3rd ed. Cambridge, United Kingdom: Cambridge University Press
  21. Aitken RJ, Ryan AL, Curry BJ, Baker, MA 2003 Multiple forms of redox activity in human spermatozoa. Mol Human Reprod 9:645–661[Abstract/Free Full Text]
  22. Biggers JD, Whitten WK, Whittingham DG 1971 The culture of mouse embryos in vitro. In: Daniel JC, ed. Methods in mammalian embryology. San Francisco: Freeman; 86–116
  23. Ainsworth C, Nixon B, Aitken RJ 2005 Development of a novel electrophoretic system for the isolation of human spermatozoa. Hum Reprod 20:2261–2270[Abstract/Free Full Text]
  24. Irvine DS 2000 Male reproductive health: cause for concern? Andrologia 32:195–208[Medline]
  25. Aitken RJ 2005 Sperm function tests and fertility. Int J Androl 29:69–75
  26. Aitken RJ 1999 The human spermatozoon-a cell in crisis? The Amoroso Lecture. J Reprod Fertil 115:1–7[Abstract/Free Full Text]
  27. Aitken RJ 2004 Founders’ lecture. Human spermatozoa: fruits of creation, seeds of doubt. Reprod Fertil Dev 16:655–664[Medline]
  28. Aitken RJ, Clarkson JS 1987 Cellular basis of defective sperm function and its association with the genesis of reactive oxygen species by human spermatozoa. The Walpole lecture. J Reprod Fertil 83:459–469
  29. Said TM, Agarwal A, Sharma RK, Mascha E, Sikka SC, Thomas Jr AJ 2004 Human sperm superoxide anion generation and correlation with semen quality in patients with male infertility. Fertil Steril 82:871–877.[CrossRef][Medline]
  30. Aitken RJ, Buckingham D, West K, Wu, FC, Zikopoulos K, Richardson DW 1992 Differential contribution of leucocytes and spermatozoa to the high levels of reactive oxygen species recorded in the ejaculates of oligozoospermic patients. J Reprod Fertil 94:451–462[Abstract/Free Full Text]
  31. Whittington K, Ford WC 1999 Relative contribution of leukocytes and of spermatozoa to reactive oxygen species production in human sperm suspensions. Int J Androl 22:229–235[CrossRef][Medline]
  32. Baker MA, Krutskikh A, Curry BJ, Hetherington L, Aitken RJ 2005 Identification of cytochrome-b5 reductase as the enzyme responsible for NADH-dependent lucigenin chemiluminescence in human spermatozoa. Biol Reprod 73:334–342[Abstract/Free Full Text]
  33. Baker MA, Krutskikh A, Curry BJ, McLaughlin EA, Aitken RJ 2004 Identification of cytochrome P450-reductase as the enzyme responsible for NADPH-dependent lucigenin and tetrazolium salt reduction in rat epididymal sperm preparations. Biol Reprod 71:307–318[Abstract/Free Full Text]
  34. Ford WC 2002 Male infertility: tales of progress and frustration. Hum Fertil (Camb) 5 (Suppl 1):S53–S60



This article has been cited by other articles:


Home page
 Hum ReprodHome page
L.K. Thomson, S.D. Fleming, R.J. Aitken, G.N. De Iuliis, J.-A. Zieschang, and A.M. Clark
Cryopreservation-induced human sperm DNA damage is predominantly mediated by oxidative stress rather than apoptosis
Hum. Reprod., September 1, 2009; 24(9): 2061 - 2070.
[Abstract] [Full Text] [PDF]

Home page
 Biol. Reprod.Home page
G. N. De Iuliis, L. K. Thomson, L. A. Mitchell, J. M. Finnie, A. J. Koppers, A. Hedges, B. Nixon, and R. J. Aitken
DNA Damage in Human Spermatozoa Is Highly Correlated with the Efficiency of Chromatin Remodeling and the Formation of 8-Hydroxy-2'-Deoxyguanosine, a Marker of Oxidative Stress
Biol Reprod, September 1, 2009; 81(3): 517 - 524.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Pharmacol.Home page
L. M. Hughes, R. Griffith, A. Carey, T. Butler, S. W. Donne, K. W. Beagley, and R. J. Aitken
The Spermostatic and Microbicidal Actions of Quinones and Maleimides: Toward a Dual-Purpose Contraceptive Agent
Mol. Pharmacol., July 1, 2009; 76(1): 113 - 124.
[Abstract] [Full Text] [PDF]

Home page
 J. Clin. Endocrinol. Metab.Home page
A. J. Koppers, G. N. De Iuliis, J. M. Finnie, E. A. McLaughlin, and R. J. Aitken
Significance of Mitochondrial Reactive Oxygen Species in the Generation of Oxidative Stress in Spermatozoa
J. Clin. Endocrinol. Metab., August 1, 2008; 93(8): 3199 - 3207.
[Abstract] [Full Text] [PDF]

Home page
 Biol. Reprod.Home page
V. Kumar, V. Kota, and S. Shivaji
Hamster Sperm Capacitation: Role of Pyruvate Dehydrogenase A and Dihydrolipoamide Dehydrogenase
Biol Reprod, August 1, 2008; 79(2): 190 - 199.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
H. A. Mostefai, A. Agouni, N. Carusio, M. L. Mastronardi, C. Heymes, D. Henrion, R. Andriantsitohaina, and M. C. Martinez
Phosphatidylinositol 3-Kinase and Xanthine Oxidase Regulate Nitric Oxide and Reactive Oxygen Species Productions by Apoptotic Lymphocyte Microparticles in Endothelial Cells
J. Immunol., April 1, 2008; 180(7): 5028 - 5035.
[Abstract] [Full Text] [PDF]

Home page
 Biol. Reprod.Home page
D. C. Wathes, D. R. E. Abayasekara, and R. J. Aitken
Polyunsaturated Fatty Acids in Male and Female Reproduction
Biol Reprod, August 1, 2007; 77(2): 190 - 201.
[Abstract] [Full Text] [PDF]

Home page
 Mol Hum ReprodHome page
R.J. Aitken, J. K. Wingate, G. N. De Iuliis, and E. A. McLaughlin
Analysis of lipid peroxidation in human spermatozoa using BODIPY C11
Mol. Hum. Reprod., April 1, 2007; 13(4): 203 - 211.
[Abstract] [Full Text] [PDF]

Home page
 J. Clin. Endocrinol. Metab.Home page
R. J. Aitken, J. K. Wingate, G. N. De Iuliis, A. J. Koppers, and E. A. McLaughlin
Cis-Unsaturated Fatty Acids Stimulate Reactive Oxygen Species Generation and Lipid Peroxidation in Human Spermatozoa
J. Clin. Endocrinol. Metab., October 1, 2006; 91(10): 4154 - 4163.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a related Letter to the Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by De Iuliis, G. N.
Right arrow Articles by Aitken, R. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by De Iuliis, G. N.
Right arrow Articles by Aitken, R. J.
Related Collections
Right arrow Male Endocrinology

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals