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Mol. Hum. Reprod. Advance Access originally published online on February 27, 2007
Molecular Human Reproduction 2007 13(4):203-211; doi:10.1093/molehr/gal119
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© The Author 2007. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Analysis of lipid peroxidation in human spermatozoa using BODIPY C11

R.John Aitken1,2, Jordana K. Wingate1, Geoffry N. De Iuliis1 and Eileen A. McLaughlin1

1 ARC Centre of Excellence in Biotechnology and Development, Discipline of Biological Sciences, University of Newcastle, NSW, Australia

2 To whom correspondence should be addressed at: FRSE, Discipline of Biological Sciences, The University of Newcastle, Callaghan NSW 2308, Australia. E-mail: jaitken{at}mail.newcastle.edu.au


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Lipid peroxidation is known to be a major factor in the aetiology of defective sperm function. Although biochemical assays for this process exist, they are relatively insensitive and require large numbers of spermatozoa; a condition that cannot be met with many infertility specimens. Recently, a new approach for monitoring peroxidative damage has been introduced, involving the probe BODIPY (581/591) C11, which readily incorporates into cells and undergoes a spectral emission shift when attacked by reactive oxygen metabolites. We have examined the applicability of this probe as an indicator of oxidative stress in human sperm populations using flow cytometry as an end point. The measurement of peroxidation with BODIPY C11 demonstrated significant dependence on the presence of a ferrous ion promoter (P < 0.001), which was significantly enhanced in sperm recovered from low-density Percoll fractions (P < 0.05) and was particularly damaging to the sperm midpiece. Iron–induced radical formation was suppressed by ascorbate in a dose-dependent manner (P < 0.001) and could only be promoted by Fe(II) and Cu(II); nickel, zinc and Fe(III) were ineffective. The Fe(II)-promoted BODIPY C11 signal was significantly correlated with the measurement of reactive oxygen species generation with dihydroethidium. We conclude that BODIPY C11 is an extremely useful probe for indexing peroxidative damage in human spermatozoa.

Key words: BODIPY C11/human spermatozoa/lipid peroxidation/oxidative stress


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Defective sperm function is a common cause of human infertility (Hull et al., 1985), and although the aetiology of this condition is still largely unresolved, one of the few recognized contributors is oxidative stress (Aitken and Clarkson, 1987; Alvarez et al., 1987; Aitken et al., 1989; Aitken, 2004). A landmark paper by Thaddeus Mann and colleagues in 1979 (Jones et al., 1979) highlighted the particular vulnerability of human spermatozoa to peroxidative damage because of their high cellular content of polyunsaturated fatty acids. A free radical attack on such fatty acids generates peroxyl (ROO•) and alkoxyl (RO•) radicals that, in order to stabilize, abstract a hydrogen atom from an adjacent carbon, generating the corresponding acid (ROOH) or alcohol (ROH). The abstraction of a hydrogen atom from an adjacent lipid to achieve this stabilization creates a carbon-centred radical that combines with dioxygen to create another lipid peroxide and so perpetuate the propagation of peroxidative damage throughout the cell (Bielski et al., 1983). Polyunsaturated fatty acids are particularly vulnerable to this form of stress because the carbon and hydrogen dissociation energies are the lowest at the bis-allylic methylene position (Figure 1).


Figure 1
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  		Figure 1. Vulnerability of polyunsaturated fatty acids to the hydrogen abstraction step in the lipid peroxidation cascade. Approximate bond dissociation energies for carbon atoms at different locations; note the lowest dissociation energy is associated with the carbon in the bis-allylic position, which is particularly vulnerable to free radical attack (after Wagner et al., 1994).

 
Such lipid peroxidation chain reactions can be promoted by the presence of transition metals that can vary their valency state by gaining or losing electrons. Monitoring the generation of lipid-peroxide-breakdown products such as malondialdehyde and/or 4-hydroxyalkenals in the presence of ferrous ion promoters has been found to generate a significant amount of information about human sperm populations (Gomez et al., 1998). However, a major problem with such assays is the amount of material that is required to make an accurate determination of the lipoperoxidative potential of a given sample. Recently, a novel fluorescence assay has been developed for detecting lipid peroxide formation, which has been successfully applied to bull, boar and stallion spermatozoa (Naguib, 1998; Ball and Vo, 2002; Brouwers and Gadella, 2003; Christova et al., 2004; Brouwers et al., 2005). This assay is dependent upon the sensitivity of the fluorophore BODIPY (581/591) C11 to oxidation by radicals (peroxyl and alkoxyl) formed from lipid hydroperoxides (Drummen et al., 2002). This probe readily incorporates into biological membranes and responds to free radical attack with a spectral emission shift from red to green, which can be readily monitored and quantified by flow cytometry (Neild et al., 2005). In this study, we have evaluated the use of this probe for assessing peroxidative damage in human spermatozoa.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Sperm preparation
The study population comprised a population of unselected donors who had been counselled to exclude individuals exhibiting a high risk for sexually transmitted diseases such as HIV, which might have influenced the quality or cellular composition of their semen. Institutional and State Government ethical approval was secured for the use of human semen samples in this research. The semen samples were produced by masturbation and collected into sterile containers for immediate transportation to the laboratory. Macroscopic parameters were assessed, including liquefaction, consistency, debris and volume, after which both the motility and vitality (nigrosin–eosin stain) of the spermatozoa were assessed. After allowing at least 30 min for liquefaction to occur, the spermatozoa were processed on a discontinuous two-step Percoll gradient, as previously described (Aitken et al., 2003), to generate low- (50% Percoll) and high- (100% Percoll, isotonic) density Percoll fractions. The medium used for these studies was the HEPES-buffered Biggers–Whitten–Whittingham medium (BWW; Biggers et al., 1971) supplemented with 1 mg ml–1 polyvinyl alcohol (BWW/PVA). Sperm movement characteristics were assessed using an IVOS computer-aided sperm analysis (CASA) system supplied by Hamilton Thorne Biosciences (Beverly, MA, USA) and a standard set-up (30 frames acquired at a frame rate of 60 Hz and a temperature of 37°C in 20 µm deep chambers). The movement characteristics assessed included curvilinear velocity (VCL), straight line velocity (VSL) and average path velocity (VAP).

BODIPY C11 loading
BODIPY® 581/591 C11 (D3861, Molecular Probes) was added to 2 x 106 spermatozoa at a final concentration of 5 µM and allowed to incubate for 30 min at 37°C. The cells were washed twice at 650g for 5 min and then treated with either vehicle control or promoter (80 µM ferrous sulphate unless specified otherwise) in BWW/PVA for 15 or 60 min. At each time point, an aliquot was removed and assessed using an FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA, USA). For these assessments, excitation was achieved with an argon laser (488 nm), and 10 000 sperm-specific events were calculated. Green fluorescence was detected with an FL1 band pass (BP) filter 530/30, and red fluorescence was measured using an FL3 long pass filter (>670 nm). Initial experiments employing propidium iodide at 10 µg ml–1 and fluorescence detection by FL3 (620 nm long pass) confirmed the findings of Brouwers and Gadella (2003) that BODIPY C11 did not compromise cell viability over the time course of these experiments (data not shown). Images of the labelled cells were captured using a Zeiss LSM 510 scanning microscope incorporating a Zeiss Axiovert 100 stand; excitation was effected with an argon laser (488 nm), and emission of red wavelength photons was monitored using the 560 LP filter, and emission of green wavelength photons was assessed using a BP 500–530 nm filter.

Impact of ascorbic acid
Spermatozoa loaded with 5 µM BODIPY C11 were washed twice (650g for 5 min) and treated with a fixed dose of ferrous sulphate (80 µM) in the presence of ascorbic acid at doses ranging from 0.312 to 20 mM in BWW/PVA. At time points of 15, 30 and 60 min, aliquots were taken out and analysed immediately by flow cytometry.

Comparison of different metals
Spermatozoa were loaded with 5 µm BODIPY C11, washed twice (650g for 5 min) and treated with 10, 80 and 640 µM of the following metals: nickel nitrate [Ni(NO3)2], zinc sulphate (ZnSO4), copper nitrate [Cu(NO3)2], ferric chloride (FeCl3) and ferrous sulphate (FeSO4) in BWW/PVA. At time points of 15, 30 and 60 min, aliquots were removed and analysed immediately by flow cytometry.

Dihydroethidium analysis
Dihydroethidium (DHE) is a poorly fluorescent 2-electron reduction product of ethidium (Et+), which when attacked by reactive oxygen species (ROS) produces DNA-sensitive fluorochromes (ethidium and 2-hydroxyethidium) that generate a red nuclear fluorescence on excitation at ~510 nm. For the assay, DHE (2 µM) and the vitality stain SYTOX® Green (0.5 µM) (Molecular Probes, Invitrogen, Mount Waverley, Australia) were diluted in BWW/PVA and added to 2 x 106 spermatozoa in a final volume of 200 µl. The cells were then incubated in the dark at 37°C for 15 min, washed once (650g for 5 min) and the resultant red and green fluorescence measured on an FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA, USA) as described (De Iuliis et al., 2006).

Statistics
All experiments were repeated at least three times on independent samples and the results analysed by ANOVA using the SuperANOVA program (Abacus Concepts Inc., CA, USA) on a MacIntosh G5 computer; post hoc comparison of group mean values was by Fisher's PLSD test. Paired comparisons were conducted using a paired t-test. Differences with a P-value of <0.05% were regarded as significant.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Impact of time, promoter and Percoll fractionation on lipid peroxidation
The stimulation of lipid peroxidation in BODIPY C11-loaded human spermatozoa with a ferrous ion promoter resulted in the anticipated spectral emission shift from red to green (Figure 2A–D). The population of peroxidized cells identified by flow cytometry was heterologous with respect to the intensity of green fluorescence, indicating that clear differences existed in the extent to which individual cells experience peroxidative damage when exposed to promoter (Figure 2B–D). For quantification purposes, the entire population of cells displaying an increase in the green fluorescence (circled in Figure 2C and D) was counted as positive. ANOVA analysis of the impact of time, Percoll fractionation and the presence of a ferrous ion promoter (80 µM Fe2+) on lipid peroxidation levels in 12 independent sperm samples demonstrated that both the presence of iron (P < 0.001) and incubation time (P < 0.001) had highly significant impacts on probe activation (Figure 2A). Moreover, the results revealed a significant interaction between time and the presence of a promoter (P < 0.001), reflecting the fact that only in the presence of Fe(II) did the duration of incubation have a dramatic effect on lipid peroxidation levels (Figure 2A). Lipid peroxidation was also significantly influenced by the Percoll fractionation. The low-density Percoll fractions contained significantly more peroxidized spermatozoa than the high-density Percoll pellets (69.9 ± 6.2 versus 52.8 ± 7.6%, after 60 min incubation in the presence of 80 µM iron; P < 0.001). These low-density Percoll sperm populations would therefore appear to be particularly susceptible to peroxidative damage, as previously suggested by Ollero et al. (2001).


Figure 2
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  		Figure 2. Analysis of the impact of time and ferrous ion promotion on lipid peroxidation in human spermatozoa as recorded by BODIPY C11. (A) ANOVA analysis revealed highly significant impacts due to the presence of promoter and the duration of incubation on spermatozoa recovered from the high-density region of Percoll gradients. White bars indicate control incubations and black bars indicate the presence of promoter. *P < 0.01; **P < 0.001. (B) Flow cytometry analysis of control cells; (C) and (D) flow cytometry analysis demonstrating the progressive increase in BODIPY C11 green fluorescence in human sperm samples treated with 80 µM Fe(II) for 15 (C) or 60 min (D).

 
Confocal imaging of spermatozoa labelled with BODIPY C11 revealed that the dye had become readily incorporated into all regions of the cell, as indicated by the uniform red fluorescence recorded using the 560LP filter (Figure 3A and E). However, following incubation with a ferrous ion promoter, the green fluorescence appeared. This was most commonly observed in the midpiece of the cell incorporating the mitochondria and any excess residual cytoplasm retained following spermiation (Figure 3B, C, F and G). It was also notable that individual spermatozoa that had undergone extensive lipid peroxidation revealed green fluorescence in the head as well as the midpiece of the cell (Figure 3B and C).


Figure 3
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  		Figure 3. Imaging of cells loaded with BODIPY C11 and exposed to a ferrous ion promoter. (A and E) Red fluorescence associated with the widespread incorporation of the non-oxidized probe into the spermatozoa; (B and F) green fluorescence associated with the oxidized probe; (C and G) overlaid images in which areas of lipid peroxidation appear yellow; (D and H) phase contrast images. Note the particular susceptibility of the sperm midpiece (MP) to peroxidative damage; peroxidized mitochondria (M) and excess residual cytoplasm (RC) are clearly discernable in certain cells. In addition, some cells show a strong BODIPY C11 signal over the sperm head (H).

 
Impact of ascorbate
Biochemical assays of lipid peroxidation based on malondiadehyde and 4-hydroxyalkenals normally employ ascorbate in conjunction with ferrous sulphate in order to maintain the latter in a reduced state (Jones et al., 1979; Gomez et al., 1998). In order to determine the relative benefit of incorporating this reducing agent into the promoter system used with BODIPY C11, a dose- and time-dependent study was undertaken employing sodium ascorbate in conjunction with a ferrous ion promoter. ANOVA analysis of the data revealed highly significant effects of time (P < 0.001) and ascorbate supplementation (P < 0.001) on the levels of lipid peroxidation recorded with BODIPY C11. Ascorbate did not promote the lipid peroxidation process, as observed with malondialdehyde assays (Gomez et al., 1998), but instead induced a highly significant suppression of the signal generated in the presence of iron (P < 0.001); doses above 1 mM reducing the response by more than 50% (Figure 4). It should be noted, however, that even at the highest dose of ascorbate assessed (20 mM), Fe(II) supplementation still managed to induce a significant response when compared with the non-promoted control incubation (Figure 4).


Figure 4
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  		Figure 4. Impact of ascorbate on the ability of a ferrous ion promoter to stimulate lipid peroxidation in human spermatozoa. Spermatozoa were loaded with BODIPY C11 and then exposed to 80 µM ferrous sulphate in the presence of various doses of ascorbate. The sperm populations were then assessed at 15, 30 and 60 min. ANOVA revealed highly significant effects due to treatment (P < 0.001) and time of incubation (P < 0.001). Ascorbate induced a profound dose-dependent suppression of lipid peroxidation under these conditions. Significance values refer to the difference between the control samples lacking iron and the ascorbate-supplemented incubations. *P < 0.001; **P < 0.01; ***P < 0.05.

 
Impact of inorganic ions
A variety of different inorganic ions were assessed for their ability to stimulate lipid peroxidation in human spermatozoa in the absence of ascorbate supplementation (Figure 5). The results of this analysis revealed that nickel, zinc and ferric ions were ineffective promoters of lipid peroxidation in human spermatozoa. However, the reduced transition metals Cu(II) and Fe(II) were both able to stimulate significant lipid peroxidation, particularly the latter, which induced a highly significant time- and dose-dependent increase in BODIPY C11 oxidation (*P < 0.001; Figure 5). Cu(II) was also effective in this respect, although at the highest dose tested (640 µM), the lipid peroxidation levels fell because of a concomitant decrease in sperm viability (data not shown).


Figure 5
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  		Figure 5. Impact of different transition metals on lipid peroxidation in human spermatozoa using BODIPY C11 as the detection reagent. Time- and dose-dependent analysis demonstrating the relative importance of Cu(II) and Fe(II) ions in the propagation of peroxidative damage in these cells. Significance values refer to the differences between the control incubations and those supplemented with a given metal. *P < 0.05; **P < 0.001.

 
Relationship between reactive oxygen species generation of lipid peroxidation
Since Fe(II) was clearly the most active metal in stimulating lipid peroxidation, this metal was selected as the promoter in studies designed to determine whether a relationship existed between the levels of ROS being generated by human spermatozoa and the lipid peroxidation potential. For this analysis, spermatozoa were recovered from the Percoll gradients and incubated, either untreated or promoted with two doses of iron (80 and 640 µM) for two periods of time (15 and 60 min). The percentage of cells exhibiting peroxidative damage were then correlated with spontaneous ROS generation using DHE as the probe and an incubation time of 15 min; previous studies having demonstrated that the DHE signal generated by human spermatozoa is stable for several hours (De Iuliis et al., 2006). The results of this analysis revealed that in the absence of iron supplementation, no correlation was observed between the ROS and the very low levels of lipid peroxidation detected at either the 15 or 60 min time points (n = 25). However, in the presence of iron, significant correlations were observed between ROS generation and lipid peroxidation at the 80 (r = 0.541; P < 0.01) and 640 µM (r = 0.644; P < 0.001; Figure 6) doses of Fe(II) at 15 min and, similarly, the 80 (r = 0.536; P < 0.01) and 640 µM (r = 0.588; P < 0.01) doses of Fe(II) at 60 min.


Figure 6
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  		Figure 6. Regression analysis of the relationship between the spontaneous ROS generation in human spermatozoa (recorded using DHE as the probe after 15 min incubation in the control medium) and the level of lipid peroxidation recorded in the presence of a ferrous ion (640 µM) promoter after 15 min. r = 0.644; P < 0.001; 50% Percoll fraction depicted as P50 and symbolized as circles; 100% Percoll fraction depicted as P100 and symbolized as squares.

 
Relationship between BODIPY and sperm function
In order to determine the functional significance of BODIPY C11 fluorescence, samples from 14 individuals were fractionated on Percoll gradients and each fraction analysed for motility, vitality and BODIPY C11 fluorescence after 60 min incubation in the presence of 80 µM Fe(II). As anticipated, the BODIPY C11 signal was significantly (P < 0.01) higher in the poor quality spermatozoa isolated from the low-density Percoll fraction (65.8 ± 7.3%) when compared with their mature, high-density counterparts (53.7 ± 7.3%). The high levels of peroxidative damage recorded in these low-density Percoll fractions were associated with significantly reduced levels of motility (46.2 ± 6.4 versus 88.1 ± 1.7%; P < 0.001) and vitality (86.8 ± 1.8 versus 96.3 ± 0.6%; P < 0.001; Figure 7B and C).


Figure 7
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  		Figure 7. Relationship between BODIPY C11 fluorescence and sperm function. Poor quality spermatozoa isolated from the low-density region of Percoll gradients exhibited: (A) significantly elevated levels of BODIPY C11 fluorescence in concert with significantly diminished motility (B) and significantly impaired vitality (C). *P < 0.01; **P < 0.001. (D) An analysis of the cause and effect relationship between lipid peroxidation and disrupted sperm motility was initiated by demonstrating that high levels of BODIPY C11 fluorescence could be generated by 60 min exposure to a combination of 80 µM Fe(II) and 1 mM mercaptosuccinate (MS), a glutathione peroxidase inhibitor (A); **P < 0.001 for the difference between the Fe(II)- and Fe(II)/MS-treated groups, relative to the dimethylsulphoxide (DMSO) control. The Fe(II)/MS treatment also induced significantly more BODIPY C11 positive cells than Fe(II) alone (P < 0.001). It was subsequently shown that the creation of peroxidative damage in otherwise normal cells using this combination of reagents induced a highly significant decline in the quality of sperm movement as exemplified by (E) the average path velocity and (F) the straight line velocity of the spermatozoa.

 
In order to ensure that this inverse association between sperm function and BODIPY was causative and not simply correlative, an experiment was performed in which functional spermatozoa from the high-density region of Percoll gradients were induced to undergo peroxidation and the impact on their movement characteristics assessed by CASA. In the initial stages of this analysis, a 1 h incubation in the presence of 80 µM Fe(II) and the glutathione peroxidase (GPx) inhibitor, mercaptosuccinate (1 mM), was shown to significantly enhance the BODIPY C11 signal (P < 0.001; Figure 7D) beyond the level achieved with Fe(II) alone (Figure 7D). Having optimized a system for inducing lipid peroxidation in human spermatozoa, the impact of this damage on sperm movement was determined. This analysis revealed that after a 1 h incubation in the presence of mercaptosuccinate and Fe(II), the quality of sperm movement was seriously impaired, as indicated by significant changes in VAP (P < 0.01; Figure 7E), VSL (P < 0.01; Figure 7F) and VCL (P < 0.01).


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
These results support previous studies suggesting that BODIPY C11 is an extremely effective means of monitoring lipid peroxidation in mammalian sperm populations (Brouwers and Gadella, 2003; Brouwers et al., 2005; Neild et al., 2005). BODIPY C11 does not detect the ROS responsible for the first chain initiation of the lipid peroxidation cascade, such as the superoxide anion or hydrogen peroxide. However, it is readily incorporated into sperm cells and responds to dynamic changes in chain-propagating species, such as alkoxyl (RO•) and peroxyl (ROO•) radicals, following exposure to a ferrous ion promoter (Drummen et al., 2002). Furthermore, the levels of lipid peroxidation detected with this probe were significantly correlated with the ROS production by the spermatozoa (Figure 6). This association is consistent with the hypothesis that defective sperm function is frequently associated with peroxidative damage that may be induced by a variety of pro-oxidant factors (xenobiotic exposure, antioxidant deficiency, leukocytospermia), but is most commonly a consequence of aberrant free radical generation by the spermatozoa themselves (Aitken and Clarkson, 1987).

The minimal spontaneous activation of BODIPY C11 observed in human sperm populations incubated in the absence of promoter suggests that if transition elements are not available, lipid radical formation is maintained at a very low level in these cells. This reflects the fact that human spermatozoa possess an active defence against lipid peroxidation in the form of the GPx/glutathione reductase system (Alvarez and Storey, 1989; Williams and Ford, 2004). The classical GPx1 works in tandem with phospholipase A2 to detoxify free lipid peroxides liberated from phospholipids according to the general equation:


Formula 119UM1

(119UM1)
In addition, spermatozoa possess a form of phospholipid hydroperoxide, GPx4, that can detoxify membrane lipid peroxides in situ, without the need for prior phopholipase A2 (PLA2) action (Tramer et al., 2004). Moreover, the activity of this form of GPx is known to be deficient in the spermatozoa of infertile men (Imai et al., 2001). In addition to the GPx system, lipid peroxides may be stabilized in the sperm plasma membrane by chain-breaking antioxidants such as vitamin E (Aitken et al., 1989; Sheweita et al., 2005; Drevet, 2006). However, in the presence of a ferrous ion promoter, these peroxides are induced to break down with the generation of alkoxyl radicals (RO•). The latter may then attack existing lipids or lipid peroxides in the membrane to generate the mixture of alkoxyl and peroxyl radicals that activate the BODIPY probe (Alvarez and Storey, 1995).

The importance of GPx in the protection of human spermatozoa against peroxidative damage was further emphasized by the ability of mercaptosuccinate to significantly enhance (P < 0.001) the BODIPY C11 signal generated in the presence of Fe(II). The high levels of peroxidation associated with this treatment were, in turn, associated with a significant impairment of sperm movement as revealed by CASA analysis (Figure 7). These results suggest that the association between high BODIPY signals and impaired motility is causative. Such results are in keeping with a wealth of published data indicating that lipid peroxidation has a negative impact on human sperm movement (Jones et al., 1979; Alvarez and Storey, 1989; Aitken et al., 1993a,b; Williams and Ford, 2005).

In keeping with previous studies in this area, ferrous ion promotion was found to be the most effective means of triggering a peroxidative response in these cells (Jones et al., 1979; Aitken et al., 1993a,b). Ferrous ion is clearly much more effective than ferric ion in the catalysis of lipid peroxidation in human spermatozoa. This is not invariably the case. In aortic segments, for example, Fe(III) is much more effective than Fe(II) in the induction of lipid peroxidation (Kostellow and Morrill, 2004). Since there is no evidence for hydroxyl radical (OH•) formation when human spermatozoa are exposed to Fe(II) (Aitken et al., 1993b), the dependence of BODIPY C11 on the presence of ferrous ions would not appear to depend upon the first chain initiation of the lipid peroxidation cascade. Rather, as indicated earlier, Fe(II) induces the break down of pre-existing lipid peroxides to create the alkoxyl and peroxyl radicals responsible for activating BODIPY C11. The fact that Fe(III) is not effective in this regard suggests that there is insufficient superoxide anion (O2–•) being generated by normal human spermatozoa to bring about the first step in the Haber–Weiss reaction, which involves the O2–•-mediated reduction of Fe(III) to Fe(II):


Formula 119UM2

(119UM2)
The lack of activity on behalf of Fe(III) may also reflect the fact that reactions of ferric ions with lipid peroxides are slower than those recorded with Fe(II). Fe(III) may also be unable to form the appropriate complexes within human spermatozoa, since the specific nature of Fe(III) chelates makes a significant difference to the reaction rates observed in the promotion of lipid peroxidation (Halliwell and Gutteridge, 1999). In this context, it may be particularly important that spermatozoa are coated with lactoferrin (Goodman and Young, 1981; Jin et al., 1997), which binds two atoms of Fe(III) per molecule in a manner that does not support the catalysis of peroxyl radical formation.

Nickel is also known to induce oxidative stress, but with this cation, the mechanism is generally thought to involve thiol complexation and depletion of glutathione (Valko et al., 2005). In addition, one series of reports suggest that nickel complexed with protamine P2 is redox active and can promote oxidative catalysis, stimulating the formation of ROS including the superoxide anion and the hydroxyl radical (Bal et al., 1997; Liang et al., 1999). Notwithstanding the potential redox activity of nickel complexes in the sperm nucleus, no significant impact was observed on the levels of lipid peroxidation recorded with BODIPY C11. Zinc was also inactive in this regard. Although both pro- and antioxidant activities have been described for this transition metal in previous studies, it was incapable of promoting peroxyl radical formation in human spermatozoa. Given the amount of zinc in human seminal plasma (Behne et al., 1988) and the fact that this ion only exists in one valency state and is therefore redox inactive, this lack of pro-oxidant activity might be anticipated. However, it should be noted that Gavella and Lipovac (1998) have reported a promoting effect of zinc on lipid peroxide formation in a subset of semen specimens. The significance of these differences in the behaviour of individual sperm samples towards zinc supplementation is uncertain at the present time, but deserves further investigation.

Ascorbate can act as a pro-oxidant in biochemical systems by virtue of its ability to maintain transition metals such as Cu(II) and Fe(II) in a reduced state. However, this vitamin can also act as a powerful antioxidant through the direct scavenging of free radical species (Chakraborty et al., 1994; Whiteman and Halliwell, 1996; Regoli and Winston, 1999) or through its ability to regenerate chain-breaking antioxidants such as {alpha}-tocopherol (Liebler et al., 1986). Assays of lipid peroxidation in whole sperm cells using the biochemical assessment of malondialdehyde and/or 4-hydroxyalkenals as an end-point have previously shown ascorbate to promote lipid peroxide formation (Jones et al., 1979; Gomez et al., 1998; Rhemrev et al., 2001). However, using BODIPY C11 as the probe, a dose-dependent inhibitory effect of ascorbate on Fe(II)-induced lipid peroxidation was clearly observed (Figure 3). The ability of ascorbate to regenerate hydrophobic, membrane-associated antioxidants such as {alpha}-tocopherol, as well as its reactivity towards the free radical products of lipid peroxide decomposition, may explain this protective effect at the high ascorbate/Fe(II) ratios employed in this study (Liebler et al., 1986; Hu and Shih, 1997). This explanation would be in keeping with the apparent importance of {alpha}-tocopherol for the maintenance of male fertility (Suleiman et al., 1996) and the significance of ascorbic acid as a free radical trap in human seminal plasma (Rhemrev et al., 2000).

In summary, these studies indicate that BODIPY C11 is an effective probe for monitoring peroxyl radical formation in human spermatozoa. The flow cytometry end-point is rapid and convenient and is not obfuscated by the presence of contaminating cells such as leukocytes. The spontaneous rates of lipid peroxidation in these unselected specimens were low, but could be significantly enhanced by the presence of a ferrous ion promoter. In this promoted form, the analysis of lipid peroxidation with BODIPY C11 exhibited the anticipated correlation with ROS production by the spermatozoa and should serve as a valuable tool in research, designed to elucidate the role of oxidative stress in the aetiology of male infertility.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Aitken RJ. (2004) Founders' lecture. Human spermatozoa: fruits of creation, seeds of doubt. Reprod Fertil Dev 16:655–664.[Medline]

Aitken RJ and 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.

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Submitted on November 20, 2006; accepted on January 2, 2007.


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