1 Division of Genomic Medicine, Floor F, University of Sheffield Medical School, Beech Hill Road, Sheffield S10 2RX and 2 Academic Unit of Reproductive and Developmental Medicine, The Jessop Wing, Central Sheffield University Hospitals Trust, Sheffield S10 2SF, UK
3 To whom correspondence should be addressed. Email: a.r.eley{at}sheffield.ac.uk
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Abstract |
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Key words: apoptosis/Chlamydia trachomatis/lipopolysaccharide/serovars E and LGV/sperm function
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Introduction |
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Chlamydial infection is a major cause of subfertility in both males and females (Westrom, 1996). The mechanism by which this occurs is thought to be via the acute inflammatory reaction associated with infection, leading to permanent scarring and functional impairment of the infected mucous membranes (Schachter, 1990
). In women this is typically associated with salpingitis, which can lead to the blockage of Fallopian tubes (Kossein and Brunham, 1986
). In men, chlamydial infection is associated with epididymitis and/or prostatitis that can lead to stenosis of the duct system, orchitis, or an impairment of accessory gland function (Purvis and Christiansen, 1995
). Few studies, however, have considered whether exposure to C. trachomatis itself may compromise gamete function directly and thereby lead to subfertility by a route which is independent of any damage to the reproductive epithelium.
The possibility that gametes may be directly affected by C. trachomatis arises as a result of the unique development cycle of the chlamydia. Briefly, the bacterium exists in two forms: alternating between an extracellular but metabolically inactive infectious form called the elementary body (EB) and an intracellular metabolically active reproducing form called the reticulate body (RB) which uses the intracellular machinery of a host cell to reproduce. Consequently, the epithelium of an infected individual of either sex will periodically release large numbers of EBs which themselves will further release lipopolysaccharide either the EBs or the lipopoly saccharide may then be encountered by any gametes within the reproductive tract.
We have shown that co-incubation of human sperm with C. trachomatis serovar E, and to a lesser extent serovar LGV, causes a significant decline in the percentage of motile sperm and results in premature sperm death (Hosseinzadeh et al., 2001). It is known that serovar E is the one most commonly seen in the UK as a cause of genital infection (Eley et al., 1993
). In addition, we showed (Hosseinzadeh et al., 2003
) that C. trachomatis-induced death of human sperm is primarily caused by lipopolysaccharide (LPS).
Bacterial components such as LPS are known to induce apoptosis in a variety of cell types (Zhang et al., 1993; Lakics and Vogel, 1998
). Indeed, Gorga et al. (2001)
have reported that both Salmonella and Pasteurella LPS can induce apoptosis in sperm. However, only one method for determining apoptosis [terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL)] was used and semen samples were tested rather than purified sperm cells.
In this study we wanted to investigate further the mode of sperm death upon exposure to C. trachomatis LPS. We did this by comparing three different methods for demonstrating apoptosis in sperm and by looking at the role of caspases.
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Materials and methods |
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LPS extraction and quantification
LPS was extracted from C. trachomatis serovars E and LGV as previously described (Hosseinzadeh et al., 2003). A Limulus Amebocyte Lysate (Cambrex Biosciences, Wokingham, UK) assay was used to quantify chlamydial LPS. The method was performed as recommended by the manufacturer.
Apoptosis and necrosis controls
To find the optimal induction of apoptosis in sperm, actinomycin (2.510 µg/ml), cycloheximide (0.502.0 µg/ml) and staurosporine (1 µmol/l1 mmol/l) were added to 5x106/ml sperm, incubated for 6 h at 37°C/5% CO2 and samples were investigated by flow cytometry. Necrotic sperm were produced by heat treatment at 56°C for 1 h. In all experiments, untreated Percoll-prepared sperm (Moohan and Lindsay, 1995) were used as a control.
Exposure of sperm to LPS
A total of 5x106 sperm were incubated at 37°C/5% CO2 for 6 h with 0.1 µg/ml LPS from both serovars of C. trachomatis. In caspase inhibition experiments, prior to adding LPS, 200 µmol/l of pan-caspase inhibitor (Z-VAD-FMK) (Calbiochem, Darmstadt, Germany) or caspase 3 inhibitor (C3I) (Calbiochem) were added to the cells and incubated for 1 h at room temperature.
To prepare the samples (including controls) for flow cytometry, sperm were washed twice with annexin binding buffer (BD Biosciences Pharmingen, San Diego, USA) and diluted to give a final concentration of 1x106/ml sperm.
Detection of apoptosis using flow cytometry
The technique used for the annexin V assay and its characteristics are described elsewhere (Oosterhuis et al., 2000). To remove in vivo bound annexin V, 200 µl Percoll-prepared sperm was washed twice with annexin V binding buffer.
A total of 106 sperm/ml were incubated with 0.1 mg ml1 fluorescein isothiocyanate (FITC)labelled annexin V, incubated at 37°C/5% CO2 for 30 min in 5% CO2 in the dark followed by adding 50 mg ml1 propidium iodide (PI) (Sigma, UK). The sperm were then analysed in a FACScalibur flow cytometer (Becton Dickinson, Oxford, UK). A minimum of 10 000 sperm were examined for each experimental group. The sperm population was gated by using forward-angle light scatter. However, side-angle light scatter was used to exclude electronic noise and debris. The FITC-labelled annexin V-positive sperm cells were measured in the FL1 channel and the PI-labelled cells were measured in the FL2 channel of the flow cytometer.
Detection of apoptosis by measurement of ADP:ATP ratios
ATP was measured using a commercial kit (Bradbury et al., 2000), following the manufacturer's instructions. The ApoGlowTM kit (Cambrex Biosciences, UK) is based upon the bioluminescent measurement of ATP. The method uses luciferase enzyme which catalyses the formation of light from ATP and luciferin according to the following reaction: ATP+luciferin+O2
(luciferase/Mg2+)
oxyluciferin+AMP+Ppi+CO2+light. Measurement of ADP:ATP ratios is used to differentiate cells undergoing apoptosis or necrosis.
The emitted light intensity is linearly related to the ATP concentration and is measured using a luminometer. The non-adherent cell assay was used in our experiments due to the nature of Percoll-prepared sperm. A volume of 100 µl of the cell suspension was transferred into a fresh 96-well opaque white microtitre plate using a multichannel pipette. The microtitre plate was then loaded into the luminometer and the protocol initiated. Measurement was then recorded.
Detection of apoptosis by measurement of mono- and oligonucleosomal DNA
Apoptosis was determined using the Cell Death Detection ELISA (enzyme-linked immunosorbent assay) kit from Roche (Diagnostics Ltd, Lewes, UK) following the manufacturer's instructions on a final concentration of sperm adjusted to 5x106 per ml.
The assay is based on a quantitative sandwich enzyme immunoassay principle using mouse monoclonal antibodies directed against DNA and histones respectively. This allows the specific determination of mono- and oligonucleosomes in the cytoplasmatic fraction of cell lysates.
Quantification of caspase activity
Sperm lysates were prepared as described previously (Parvathenani et al., 1998). An aliquot of sperm lysate was diluted with a solution containing interleukin 1B converting enzyme (ICE) buffer and the fluorogenic substrate N-acetyl-aspartate-glutamate-valine-aspartate, 7-amino-4-trifluorome-thyl coumarin (Ac-DEVD-afc). Fluorescent emission (excitation 400 nm and emission 505 nm) was measured after incubation for 45 min at 37°C. Blanks without sperm were evaluated to determine background fluorescence. Standards containing 0500 pmol/
AFC were diluted to determine the amount of fluorochrome released. Fluorescence was measured at
max=505 nm using a fluorimeter (PerkinElmer, Beaconsfield, UK). Caspase activity was expressed as pmol/min/mg protein. Apoptotic human neutrophils treated with 1 mmol cycloheximide were used as positive controls.
Statistical analysis
The results of all experiments in this study were analysed using GraphPad InStat software version 3.0 (GraphPad Software Inc, USA). The statistical significance of differences between each experimental pair was evaluated by one-way analysis of variance (ANOVA) (Figures 2, 3 and 4), or by Student's t-test (Figures 5 and 6). P<0.05 was considered significant.
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Results |
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Co-incubation of sperm with C. trachomatis LPS leads to apoptosis as demonstrated by three methods
In the first experiment, apoptosis was quantified by measuring phosphatidylserine (PS) externalization using annexin V binding and flow cytometry. Untreated sperm typically showed a small number of necrotic and apoptotic cells (Figure 1a). When sperm were heat-shocked, the majority of cells became necrotic with a slight increase in apoptotic cells (Figure 1b). Staurosporine at 1 mmol/l showed a marked apoptotic effect although there was also a substantial increase in necrotic cells (Figure 1c). When sperm were exposed to C. trachomatis LPS from serovar E they showed an increased number of both apoptotic and necrotic cells (Figure 1d).
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Similar to what has been previously reported (Bradbury et al., 2000) we found that heat-shocked (i.e. necrotic) rather than staurosporine (i.e. apoptotic)-treated sperm had higher ADP:ATP ratios (Figure 3). As the ADP:ATP ratios can serve to differentiate cells which are either necrotic or apoptotic, these ratios allowed us to determine that sperm exposed to C. trachomatis LPS from serovar E were predominantly apoptotic but that a larger population of necrotic cells was seen in sperm exposed to C. trachomatis LPS from serovar LGV1.
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C. trachomatis LPS-induced apoptosis of sperm can be blocked with a pan-caspase inhibitor
To give us an indication as to whether caspases were implicated in the apoptosis of ejaculated sperm exposed to C. trachomatis LPS, we preincubated cells to the pan-caspase inhibitor (Z-VAD-FMK) and looked for apoptosis by flow cytometry and annexin V binding.
Not surprisingly, the inhibitor had a negligible (statistically insignificant) effect on both untreated and heat-treated sperm (Figure 5). In contrast, there was statistically significant inhibition of apoptosis in sperm which were treated with staurosporine or C. trachomatis LPS from either serovars E or LGV1. These findings suggest that caspases play an important role in those sperm in which apoptosis is induced.
Confirmation that C. trachomatis LPS-induced apoptosis of sperm is caspase-mediated using a fluorogenic substrate
When the fluorogenic substrate Ac-DEVD-afc was added to sperm exposed to staurosporine, increased levels of fluorescent emission were detected indicating increased caspase activity (Figure 6) in comparison to heat-shocked sperm. In addition, increased caspase activity was also seen following the exposure of sperm to C. trachomatis LPS from both serovar E and LGV1. Moreover, this was significantly reduced in the presence of the caspase-3 inhibitor C3I, confirming an important role for caspase-3 in the apoptotic pathway of ejaculated sperm.
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Discussion |
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In an attempt to understand how sperm became non-viable in the presence of C. trachomatis LPS (Hosseinzadeh et al., 2003), we performed three different types of experiments to explore the possible induction of apoptosis in sperm caused by exposure to C. trachomatis LPS. Although there were some differences in the results by the different methodologies, in general terms LPS from serovar E led to a greater amount of cell death through apoptotic mechanisms, whereas co-incubation with LPS from serovar LGV generally led to sperm death by the process of necrosis.
Further experiments demonstrated caspase activation, and inhibition by a pan-caspase inhibitor and a caspase-3 inhibitor confirm that caspases play a central role in C. trachomatis LPS-induced apoptosis of sperm. Of all downstream caspases, caspase-3 is the most important among them and works by generating DNA strand breaks (Paasch et al., 2004a).
Although the concept of apoptosis in ejaculated human sperm has been controversial, there is an increasing body of evidence to show that it is possible (Said et al., 2004). Indeed, recent work by Weng et al. (2002)
clearly demonstrates that caspases are present in ejaculated sperm undergoing apoptosis. Moreover, it has already been reported that lipopolysaccharide from both Salmonella and Pasteurella can induce apoptosis in human sperm as demonstrated by TUNEL (Gorga et al., 2001
). Similarly, Helicobacter pylori LPS has also been shown to induce caspase-mediated apoptosis in other cell types (Kawahara et al., 2001
).
Finally, an interesting question to arise from this work is why human sperm should respond differently to the LPS molecule purified from two closely related serovars of C. trachomatis. Theoretically, the structure of the LPS molecule of serovars E and LGV are very similar (Heine et al., 2003) although detailed studies of the structure of LGV1 LPS have not yet been carried out. Following the results of this study we would propose that, given the importance of LPS in compromising assisted reproduction (Snyman and Van der Merwe, 1986
; Fishel et al., 1988
), future research efforts should focus on the molecular structure of LPS in relation to its spermicidal properties.
In conclusion, our work strongly suggests that in vitro human sperm can undergo caspase-mediated apoptosis and that LPS from C. trachomatis is able to initiate this effect. These findings provide an explanation as to why C. trachomatis, when co-incubated with sperm, causes their death (Hosseinzadeh et al., 2001, 2003
). They also support the suggestion of Greene et al. (2004)
that LPS is a toxic material released by chlamydia and, as our results show, this appears to play a role in promoting apoptosis. Why sperm should undergo apoptosis in the presence of C. trachomatis is an interesting question with possible relevance to human fertility. It is plausible that the exposure of sperm to chlamydia either in the male or female genital tract, or both, could lead to sperm dysfunction. Furthermore, it is interesting to speculate that apoptosis in sperm may have evolved to prevent the transmission of genetic defects, as sperm infected by C. trachomatis may be sufficiently damaged to prevent the successful development of embryos should fertilization be successful. It is clear that further work is warranted to see if these in vitro findings can also be observed in infected individuals with a subsequent effect on semen quality.
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Submitted on March 16, 2005; resubmitted on April 12, 2005; accepted on April 19, 2005.
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