Experimental Chlamydia trachomatis infection causes apoptosis in human sperm

Abstract

BACKGROUND: Chlamydia trachomatis is responsible for a widespread sexually transmitted infection. In men, it is associated with a wide clinical spectrum causing infertility. Furthermore, C. trachomatis serovar E infection decreases motility and increases the number of non-viable sperm. No other effects of C. trachomatis have been reported on sperm despite the crucial role of DNA integrity for sperm function. The aim of this study was to investigate the effects of C. trachomatis on sperm apoptosis. METHODS: Sperm from eight normozoospermic men were incubated with increasing concentrations of C. trachomatis serovar E elementary bodies (EB) for 6 and 24 h. Sperm were then collected to evaluate phosphatidylserine (PS) membrane translocation and DNA fragmentation by Annexin V–propidium iodide staining, TUNEL assay and flow cytometry. RESULTS: After 6 h of incubation, C. trachomatis had no effect on the percentage of sperm showing PS externalization. However, a significant effect on this parameter was observed after 24 h. C. trachomatis also significantly increased the number of sperm with DNA fragmentation both after 6 and 24 h of incubation. CONCLUSIONS: C. trachomatis causes sperm PS externalization and DNA fragmentation. These effects may explain the negative direct impact of C. trachomatis infection on sperm fertilizing ability.

Introduction

Chlamydia trachomatis, an obligate intracellular parasite, has a biphasic life cycle characterized by an elementary body (EB) with infective capacity and a reticular body that is able to replicate within eukaryotic cells. C. trachomatis serotypes D–K are a common aetiological factor of cervicitis. It has been estimated that ∼20% of women with lower genital tract chlamydial infection will develop pelvic inflammatory disease (PID), ∼4% develop chronic pelvic pain, 3% infertility, and 2% adverse pregnancy outcome. Research on the effects of C. trachomatis infection on male infertility are much more limited (Paavonen and Eggert-Kruse, 1999). C. trachomatis is the recognized aetiological factor of ∼50% of non-gonococcal urethritis and of the vast majority of post-gonococcal urethritis (Oriel, 1992). 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 male sexual accessory gland function. Recent evidence suggest that it may cause male infertility by acting directly on sperm. Indeed, incubation with C. trachomatis has been reported to decrease sperm motility and to increase the number of non-viable sperm (Hosseinzadeh et al., 2001). In addition, the presence of IgA against C. trachomatis correlates positively with lipid peroxidation of the sperm membrane (Segnini et al., 2003). No other effects of C. trachomatis exposure have been reported on sperm, despite the recently highlighted relevance of sperm apoptosis on the spermatozoon fertilizing competence. Indeed, sperm DNA integrity has been associated with male infertility potential in vivo and in vitro (Zini et al., 2001). Moreover, many authors reported that couples in whom the husband’s semen exhibit a high percentage of sperm with DNA damage have a very low potential for natural fertility (Evenson et al., 1999; Spanò et al., 2000). Therefore, the aim of this study was to investigate the effects of C. trachomatis on sperm apoptosis. To accomplish this, sperm of normozoospermic men were incubated with EB of C. trachomatis serovars E; and membrane phosphatidylserine (PS) externalization, an early molecular event of apoptosis, and DNA fragmentation, a late sign of apoptosis, were evaluated by cytofluorimetric analysis.

Materials and methods

C. trachomatis EB production

C. trachomatis serovar E (ATCC VR-348B) EB were prepared by infecting McCoy cells, a human and murine synovia continuous cell line [American type culture collection (ATCC), CRL-1696], as previously reported (Black, 1997). Briefly, cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) containing glucose (0.056 mol/l), 10% fetal calf serum (FCS), gentamycin (50 µg/ml), streptomycin (100 µg/ml) and amphotericin B (25 µg/ml), to prevent bacterial and/or mycotic infections, up to their semi-confluence. Cells were then infected with serovar E EB in 2‐sucrose phosphate buffer (2SP) by centrifugation at 750 g at 32°C for 1 h, according to the instructions of ATCC, and afterwards incubated in DMEM containing 2% FCS and cycloheximide (1 µg/ml) to inhibit McCoy cell protein synthesis.

Forty-eight to 72 h after the infection, cells underwent three freezing–thawing cycles, were scraped from the flasks, and sonicated three times at an intensity of 60 (Ultrasonic 2000 ARTEK with Titanium standard ¾ inch/19 mm Tip) for 30 s at 1 min interval. The suspension was then centrifuged at 500 g for 10 min at 4°C and the supernatant centrifuged at 30 000 g for 20 min at 4°C. The pellet made up of semi-purified EB was resuspended in 2SP and stored at –80°C (Wang et al., 1979; Yong et al., 1979). Serial dilutions of the suspension obtained were used for EB quantification using shell-vials of McCoy cells which were fixed and stained with monoclonal antibody–fluorescein isothiocyanate anti-major outer membrane proteins of Micro Trak C. trachomatis direct specimen test (Trinity Biotech plc, IDA Business Park, Co. Wicklow, Ireland), according to the manufacturer’s instructions. The mean number of inclusion-forming units (IFU) of the single dilutions, counted at the epifluorescence microscope (Leica DMLB), was used to calculate the suspension titre expressed in IFU/ml (Kuipers et al., 1995; Raulston, 1997; Hosseinzadeh et al., 2003).

Sperm preparation

Human semen samples were collected by masturbation after 3–5 days of abstinence from healthy subjects with negative sperm and urethral swab cultures (including for C. trachomatis). Sperm parameters were evaluated according to World Health Organization (1999) criteria. Only samples with sperm concentration ≥20×10⁶/ml, total motility ≥50%, progressive motility ≥40%, normal forms ≥30%, viability ≥80% and leukocyte concentration <1×10⁶/ml were used. The sample from each individual subject was tested separately.

After 30 min liquefaction at 37°C, motile sperm were isolated using a discontinuous Percoll gradient (45/90%). The final pellet was resuspended in Biggers–Whitten–Wittingham (BWW) medium containing 0.3% bovine serum albumin at a concentration of 17.5×10⁶ sperm/ml. Aliquots containing 5.25×10⁶sperm in 300 µl were infected with 0, 1, 10 or 100 µl of EB suspension (in 2SP buffer) containing 3×10⁵IFU/ml. The final volume was brought to 500 µl by adding 100, 99, 90 or 0 µl of 2SP buffer respectively, and 100 µl of BWW. Sperm were left to incubate with EB serovar E at 37°C for 6 and 24 h. At the end of the incubation, membrane PS translocation and sperm DNA fragmentation were evaluated by Annexin V–propidium iodide (PI) assay and terminal deoxynucleotidyl transferase-mediated fluorescein-dUTP nick end labelling (TUNEL) assay and flow cytometry.

Flow cytometry

Flow cytometry analysis was performed using the flow cytometer EPICS XL (Coulter Electronics, IL, Italy) equipped with a 488 nm argon laser as light source and three fluorescent detectors corresponding to the green colour (FL-1 detector 525 nm wavelength band), orange colour (FL-2 detector 620 nm wavelength band) and red colour (FL-3 detector 620 nm wavelength band). Twenty thousand events were measured for each sample at a flow rate of 50–100 events/s and analysed using the SISTEM II™ Software, Version 3.0. The debris was gated out, by drawing a region on the forward versus side-scatter dot-plot enclosing the population of cells of interest. The compensations and the settings were adapted according to the assay utilized.

Annexin V–PI assay

Staining with Annexin V–PI was performed using a commercial kit (Annexin V–FITC Apoptosis detection kit, Sigma Chemical). An aliquot of cells containing ∼0.5×10⁶/ml was resuspended in 500 µl of binding buffer, labelled with 10 µl of Annexin V–FITC plus 20 µl of PI, incubated for 10 min in the dark, according to the manufacturer’s instructions, and immediately analysed by flow cytometry. Samples were evaluated by FL-1 (Annexin V–FITC) and FL-3 (PI) detectors. The cell population of interest was gated on the basis of the forward and side-scatter properties. The different labelling patterns in the bivariate PI–Annexin V analysis identified the different cell populations where FITC negative and PI negative were designated as viable cells; FITC positive and PI negative as apoptotic cells; and FITC positive and PI positive as late apoptotic or necrotic cells.

TUNEL assay

DNA fragmentation was evaluated by attaching to the breaks dUTP, conjugated to a fluocrome, in a reaction catalysed by exogenous terminal deoxynucleotidyl transferase (TdT), as previously reported (Vicari et al., 2002). Briefly, sperm were labelled with the Apoptosis Mebstain kit (Beckman Coulter, IL, Milan, Italy). To obtain a negative control, TdT was omitted from the reaction mixture. The positive control was obtained by pre-treating the sperm with 1 µg/ml deoxyribonuclease I, RNAse-free (Sigma Chemical) at 37°C for 60 min before labelling. Light-scattering and fluorescence data were obtained at a fixed gain setting in logarithmic mode. The percentage of FITC-labelled sperm was determined with the FL-1 detector of the flow cytometer.

Statistical analysis

Results are reported as mean ± SEM throughout the study. Results were analysed by one-way analysis of variance (ANOVA) followed by the Duncan’s multiple range test. The software SPSS 9.0 for Windows was used for statistical evaluation (SPSS Inc., Chicago, IL, USA). P < 0.05 was accepted as significantly different.

Results

Infection with C. trachomatis EB at the concentration of 300, 3000 or 30 000 IFU had no effect on the percentage of sperm with PS translocation after 6 h of incubation (Figure 1). A significant effect on this parameter was instead observed after 24 h of incubation (P < 0.001, ANOVA). The lowest effective concentration was 3000 EB which increased significantly the percentage of sperm with PS externalization compared to sperm incubated without C. trachomatis EB (P < 0.05, Duncan test). A further statistically significant increase was observed at the concentration of 30 000 EB (P< 0.05 versus 0, 300, 3000 EB, Duncan’s test).

C. trachomatis also caused a statistically significant increase in the percentage of sperm with DNA fragmentation both after 6 and 24 h of incubation (Figure 2). The effect reached statistical significance only at the highest concentration (30 000 EB) of C. trachomatis EB (P < 0.05 versus 0 EB, Duncan’s test) after 6 h of incubation, whereas it was effective at 3000 EB (P < 0.05 versus 0 and 300 EB, Duncan’s test) after 24 h of incubation.

Discussion

In vitro infection with C. trachomatis EB induced apoptosis in sperm isolated, by a discontinuous Percoll gradient, from normozoospermic healthy men. Although EB did not have any effect on PS externalization—an early molecular sign of apoptosis—after 6 h of incubation, a significant effect was observed on DNA fragmentation with the highest concentration of EB used. After 24 h of incubation, a significantly higher number of sperm had membrane PS externalization, suggesting that a great proportion of cells had undertaken the pathway towards apoptosis. Accordingly, an elevated number of sperm showed signs of DNA fragmentation, a late apoptotic sign. Interestingly, however, a much lower percentage of sperm had DNA fragmentation compared to that externalizing PS after 24 h of incubation. This suggests that DNA fragmentation may require a longer time of exposure to become fully evident.

It is noteworthy that the pro-apoptotic effects were observed with a low number of C. trachomatis EB which were added at concentrations two to four orders of magnitude lower than sperm. The ability of C. trachomatis to cause sperm apoptosis at a low concentration and in a short span of time suggests that sperm may also be damaged during their transit in an infected female genital tract. Therefore, infection of the female partner may contribute to infertility by this additional mechanism. Chlamydia is not the only microrganism which has been shown to cause sperm apoptosis. Recently, it has been shown that experimentally induced E. coli or S. aureus infection causes sperm apoptosis with a mechanism independent from reactive oxygen species production (Villegas et al., 2005). In addition, we showed that a lack of oocyte fertilization due to an unrecognized Candida albicans infection was associated with an increased percentage of motile sperm with chromatin packaging abnormalities, PS externalization and DNA fragmentation (Burrello et al., 2004).

The reported sperm pro-apoptotic effects of the infection with C. trachomatis should be added to the direct detrimental effects on other sperm parameters. Hosseinzadeh and colleagues reported that incubation with EB of serovar E (0.54×10⁶/ml) resulted in a significant decline in the percentage of motile sperm and an increase in the proportion of dead sperm after 1, 3 and 6 h of incubation. Incubation with serovar LGV caused only an increase in the number of dead sperm after 6 h. The amount of acrosome-reacted sperm, the other sperm function examined, was not affected by the infection with serovar E or LGV. Dose–response experiments indicated that increasing the concentration of EB to 2.5×10⁶/ml did not significantly alter the results. These effects were the result of the live organism and not of soluble components or membrane elements since they were not present after co-incubation of sperm with EB killed by heat (Hosseinzadeh et al., 2001).

Incubation with C. trachomatis EB increased sperm protein tyrosine phosphorylation, a function which mediates important aspects of sperm capacitation and function. Indeed, it has been reported that the serovar E, but not LGV, increased tyrosine phosphorylation of proteins mainly located in the sperm tail. Immunoblotting revealed a marked increase in tyrosine phosphorylation of sperm proteins with a mol. wt of 80 and 95 kDa, whereas the serovar LGV increased phosphorylation of only the 80 kDa moiety (Hosseinzadeh et al., 2000). In the attempt to establish the C. trachomatis EB component responsible for these effects on sperm function, the same authors showed that lipopolysaccharide (LPS) from both serovars resulted in a marked reduction in sperm motility (and a concomitant increase in the proportion of dead sperm) in a manner similar to that seen in response to EB of serovar E. In addition, when sperm were incubated with various doses of EB and LPS, the greater spermicidal effects of serovar E EB (compared with serovar LGV) were not observed when sperm were incubated with LPS from the two serovars. This suggests that the more potent effect of serovar E EB cannot be explained entirely by differences in the composition of LPS. However, that chlamydial LPS was spermicidal was demonstrated by the use of polymyxin B (a polycationic antibiotic known to neutralize LPS effects), confirming that the effects observed were primarily a result of LPS activity. Interestingly, the authors reported that E. coli LPS was required in doses 500 times higher than chlamydial LPS to kill a similar proportion of sperm, suggesting that bacterial LPS may differ in their spermicidal properties (Hosseinzadeh et al., 2003).

The results on the effects of Chlamydia on sperm parameters in men are controversial. Recently, Veznik et al. (2004) evaluated sperm parameters of men without or with chlamydial infection. Patients with chlamydial infection had seminal plasma volume, sperm motility, velocity and normal morphology worse than those observed in samples without infection. On the other hand, Hosseinzadeh et al. (2004) could not confirm that chlamydial infection was associated with any abnormality of sperm parameters. Indeed, C. trachomatis detected by PCR in the semen sample of 31 men (4.9% of the cases examined) was associated with significantly higher mean concentration of leukocytes and a higher mean ejaculate volume than in those whose ejaculates were PCR negative. However, there was no difference in the mean percentage motility between the two groups. The reason for this discrepancy is not clear. It may relate to the different methods (immunofluorescence versus PCR) used to detect Chlamydia in the semen samples and/or to the various adaptative host inflammatory responses. It is well known that PCR, being more sensitive, may have detected tiny amounts of Chlamydia in some samples without any negative effect on sperm parameters. Since chlamydial polysaccharide seems to be the major factor leading to sperm damage, a new investigational line has been proposed to measure its levels in the semen and to relate them with sperm parameters and sperm function (Eley et al., 2005).

Our findings contribute to the understanding of the mechanisms through which Chlamydia may cause male infertility. Indeed, sperm from infertile men have various nuclear alterations, including abnormal chromatin structure, microdeletions, chromosomal rearrangements, aneuploidy and DNA strand breaks (Sharma et al., 2004). Apoptosis is a programmed cell death based on a genetic mechanism that induces a series of cellular morphological and biochemical alterations leading the cell to suicide. In the final stage, apoptosis accounts for a significant proportion of DNA strand breaks. The extent of DNA damage is closely related to sperm function and male infertility (Aitken, 1999). In fact, intact human sperm DNA is an essential prerequisite for successful IVF and embryo development. With advanced assisted reproductive technology (i.e. IVF, ICSI), sperm selection is largely a random process that may result in the inadvertent use of DNA-damaged sperm that can fertilize an oocyte, produce high-quality early stage embryos, and then, in relationship to the extent of DNA damage, fail to produce a successful term pregnancy (Aitken et al., 1999) or transmit pathology to the offspring, including childhood cancer (Aitken and Krausz, 2001).

In conclusion, these data suggest that C. trachomatis causes sperm PS externalization and DNA fragmentation. These effects may explain the negative impact of C. trachomatis infection on sperm fertilizing ability.

References

Author notes

¹Section of Endocrinology, Andrology and Internal Medicine and Master in Andrological and Human Reproduction Sciences and ²Deparment of Microbiological and Gynaecological Sciences, University of Catania, Catania, Italy