Human Reproduction, Vol. 18, No. 10, 2103-2109,
October 2003
© 2003 European Society of Human Reproduction and Embryology
Mycoplasma genitalium attaches to human spermatozoa
1 Department of Medical Microbiology and Immunology, The Bartholin Building, 2 Institute for Storage Ring Facilities Aarhus University, DK-8000 Aarhus C and 3 The Fertility Clinic, Braedstrup Hospital, DK-8740 Braedstrup, Denmark
4 To whom correspondence should be addressed. e-mail: hellef{at}biobase.dk
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Abstract |
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BACKGROUND: Mycoplasma genitalium causes urogenital diseases in men and women and is presumed to be sexually transmitted. We wanted to investigate whether spermatozoa could serve as vectors for M.genitalium in order to cause upper genital diseases in women. METHODS: By use of Nomarski light microscopy and transmission X-ray microscopy, the attachment of M.genitalium to spermatozoa was studied. Semen was incubated in vitro with M.genitalium. Purified, motile spermatozoa were examined for attachment of M.genitalium by immunofluorescence microscopy. RESULTS: Mycoplasma genitalium was shown to adhere to the head, midpiece and tail of the spermatozoa. The spermatozoa became immotile when many M.genitalium were attached. However, the motile spermatozoa were demonstrated to carry M.genitalium and in this case the mycoplasmas were seen to attach mostly to the midpiece or neck region. Occasionally, M.genitalium was seen at the head but not at the tail. By X-ray microscopy, it was possible to observe the diffentiated structure of M.genitalium, and the attachment seemed to be mediated by the tip. CONCLUSIONS: Mycoplasma genitalium can bind to human spermatozoa and thus could be carried by motile sperm. This ability may be important in the process of causing female genital diseases and infertility.
Key words: adhesion/inhibition/Mycoplasma genitalium/spermatozoa/X-ray microscopy
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Introduction |
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Mycoplasma genitalium is a human pathogen causing urogenital diseases. Originally, it was isolated from male patients with urethritis (Tully et al., 1981
). Since then, only a few laboratories have isolated this fragile organism successfully from human samples. PCR studies have shown that M.genitalium is an important cause of non-gonococcal, non-chlamydial urethritis in men (Horner et al., 1993; Jensen et al., 1993; Bjornelius et al., 2000; Totten et al., 2001; Yoshida et al., 2002). PCR and serological studies of women have associated M.genitalium with pelvic inflammatory disease (Moller et al., 1984), cervicitis (Uno et al., 1997), endometritis (Cohen et al., 2002) and infertility (Clausen et al., 2001). Findings of M.genitalium in the lower genital tract of women support the hypothesis that the organism is sexually transmitted (Palmer et al., 1991; Taylor-Robinson et al., 1993).
Sexually transmitted microorganisms are known to spread directly by invasion through the female genital tract or via the mesosalpingeal lymphatics to the Fallopian tubes (Eschenbach, 1984). Alternatively, spermatozoa have been suggested as vectors for the bacterial spread. In-vitro experiments have shown that Chlamydia trachomatis, Escherichia coli, Neisseria gonorrhoeae, Ureaplasma urealyticum and Mycoplasma hominis attach to human spermatozoa (James-Holmquest et al., 1974; Friberg and Fullan, 1983; Keith et al., 1984). Furthermore, C.trachomatis has been detected by monoclonal antibodies on spermatozoa retrieved from the peritoneal fluid from patients with acute salpingitis (Friberg et al., 1985, 1987).
Mycoplasma genitalium is the smallest bacterium isolated, having a genome size of only 580 kbp and a cell diameter of 300 nm. Mycoplasmas differ from other bacteria by the complete lack of a cell wall (International Committee on Systematic Bacteriology, 1995). The small size of M.genitalium lies at the threshold for light microscopy, but it is possible to see coccoid bodies with immunofluorescence, phase-contrast and dark-field optics. Electron microscopy has shown that M.genitalium is not spherical but flask-shaped, with a specialized terminal structure with an electron-dense centre termed the tip, similar to the closely related Mycoplasma pneumoniae (Tully et al., 1983). Protrusions at both ends have also been observed (Kirchhoff et al., 1984). The tip is used for adhesion to host cells and it may be involved in gliding motility, which is exhibited by the organism (Taylor-Robinson and Bredt, 1983). A major adhesin of M.genitalium is the 150 kDa protein denoted MgPa (Inamine et al., 1989).
In this study, we examined the attachment of M.genitalium to human spermatozoa and determined whether motile sperm could carry M.genitalium. We have used different microscopy methods: Nomarski and immunofluorescence light microscopy, and X-ray microscopy to investigate the interaction between M.genitalium and the spermatozoa.
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Materials and methods |
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Purification of spermatozoa
This study was approved by the relevant local Scientific Ethical Committees (2001/0161). Semen samples were retrieved from 30 healthy donors with a normal sperm count (>20 x 106 spermatozoa/ml; World Health Organization, 1992). The semen samples were kept at room temperature, in darkness and for no longer than 3 h before use.
The spermatozoa were purified by a ‘swim-up’ procedure. Aliquots of the semen sample (0.5 ml) was placed beneath 1 ml of IVF medium (Medi-Cult, Jyllinge, Denmark). During a 1 h incubation at 37°C, the spermatozoa were allowed to ‘swim-up’ into the IVF medium. The spermatozoa were harvested by centrifugating the supernatant at 1000 g for 10 min. The pellet was resuspended carefully in fresh IVF medium and the centrifugation was repeated. The final concentration of spermatozoa was adjusted to 20 x 106 spermatozoa/ml using a Bürker Türk counting chamber (Blaubrand®, Germany).
Culture and harvest of M.genitalium
Mycoplasma genitalium G37 (ATCC, MD) was cultured in 10 ml of SP-4 medium (Tully et al., 1979) in TTP tissue culture flasks (Medi-Cult, Copenhagen, Denmark) and incubated at 37°C. After 48 h growth, the medium changed colour from red to orange, which indicated an exponential growth phase, and cells were harvested. The SP-4 medium was poured off and the mycoplasma cells attached to the bottom were washed once in IVF medium. Then, the mycoplasmas from an area of 25 cm2 were scraped off in 2 ml of IVF medium and diluted 1:10 ready for infection of the purified spermatozoa.
Light microscopy and Nomarski differential interference contrast microscopy (NM)
The sperm and mycoplasmas were mixed 1:10 (v/v), and examined by light microscopy using a x10 objective immediately and after overnight incubation at room temperature. For studies using NM, the sperm–mycoplasma suspension was incubated at 37°C and studied after 5 min, 30 min, 1 h, 2 h and overnight incubation. At each time point, an aliquot of 3 µl was mounted on slides, and after a few minutes the sperm stuck to the glass coverslips. A total of 3 x 100 spermatozoa were counted and the number of sperm with M.genitalium attached was determined. NM was performed with a Leitz microscope (Leica Mikroskopie and Systeme GmbH, Wetzlar, Germany) using a x100 objective.
X-ray microscopy (XRM)
Spermatozoa incubated with M.genitalium were examined with a transmission X-ray microscope (Medenwaldt and Uggerhoj, 1998) located at the ASTRID storage Ring, Aarhus, Denmark. An aliquot of 3 µl of sperm–mycoplasma mixture was placed in a chamber between two thin silicon foils. To ensure a high X-ray transmission and to assist sperm motility, the space between the silicon foils was maintained by addition of 5 µl of washed Dynospheres (Plano, Marburg-Cappel, Germany) and the liquid adjusted with the syringes connected to the chamber. Beam exposure time ranged from 3 to 15 s. Structures down to 30 nm, which is 10 times less than the size of M.genitalium, could be seen clearly in the image. More information about the X-ray microscope can be found at the ISA web site (http://www.isa.au.dk/SR/XRM/xrm.htmlMicroscope).
Production of rabbit polyclonal antibodies
Rabbit polyclonal antibody was raised against whole cells of M.genitalium and denoted Pab(G37) (Clausen et al. 2001).
Indirect immunofluorescence microscopy (IFM)
Drops (20 µl) of infected sperm cells were allowed to air-dry on glass coverslips placed in 24-well Multi dish plates (NUNC, Roskilde, Denmark). The samples were fixed in 0.5 ml of 100% methanol (4°C) for 1 min and washed in phosphate-buffered saline (PBS). To detect the M.genitalium, Pab(G37) (diluted 1:2000 in PBS) was added for 30 min at 37°C. A solution of 300 µl of secondary fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG (H+L) (Jackson Immuno Research Laboratories Inc., Pennsylvania, PA) (1:100) and Evans blue (1:10) diluted in PBS were added per well for 30 min at 37°C. The cells were washed twice in PBS before and after addition of antibodies. The samples were investigated by IFM. A drop of anti-fade solution (1 µg/ml p-phenyldiamine dihydrochloride in 10% PBS and 90% glycerol pH 9.0) was placed between the glass coverslips and microslides. Microscopy was performed with a Leitz DMR fluorescence microscope (Leica Mikroskopie and Systeme GmbH, Wetzlar, Germany) using a x100 objective.
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Results |
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Interference with sperm motility by M.genitalium
Purified spermatozoa were incubated with M.genitalium, and sperm motility was studied using ordinary light microscopy. The spermatozoa started to clump immediately after mixing. After 5 min of incubation, small sperm agglutinations could be detected and, with time, the agglutinations increased in size. Repeating the experiments showed that the sizes and number of the agglutinates varied with 10-fold dilutions of M.genitalium and sperm obtained from different donors. Nevertheless, it was common to all the experiments that after overnight incubation the majority of the spermatozoa were still free and motile.
In the remaining experiments, we only investigated the free spermatozoa.
Attachment of M.genitalium to spermatozoa
To study the interaction between the sperm and M.genitalium, NM was performed. Small sphere-shaped bulges of M.genitalium cells were attached to the head, midpiece and tail of the spermatozoa (see Figure 1). In Figure 1A and B, a single cell or microcolony of M.genitalium was bound to the midpiece, and in Figure 1C and D, numerous cells of M.genitalium were attached to the midpiece and head of the spermatozoa. The attachment of M.genitalium to spermatozoa seemed to be random in terms of both number of cells and site of attachment. Adherence of mycoplasma to spermatozoa as shown in Figure 1A–D, where relatively few cells were bound, could be seen after 5 min incubation, whereas the multiple attachments all over the spermatozoon seen in Figure 1E could be observed after 30 min incubation. These spherical structures were not present in the negative control where sperm were incubated without mycoplasmas (Figure 1F).
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Attachment of M.genitalium to sperm was studied over time. A record of the number of spermatozoa with mycoplasma attached as a function of time is seen in Figure 2. The curve reached a plateau after 2 h, where only
30% of the sperm had at least one cell or a microcolony of M.genitalium bound.
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X-ray microscopy
The resolution of NM was not high enough to distinguish the differentiated structure of M.genitalium, and therefore the closer interaction between the sperm and the mycoplasmas could not be studied with light microscopy. Instead, XRM with higher resolution (30 nm) was applied. Even though it is not yet possible to label the mycoplasma in XRM, the characteristic flask-shape of M.genitalium known from electron microscopy (Mernaugh et al., 1993
; Jensen et al., 1994) was recognized (Figure 3A). The structure of M.genitalium appeared as a dark cell body with a fainter tip. Most of the cells had more than one tip. Some of the mycoplasmas were attached via the tip but this was not always possible to determine. The M.genitalium were seen on the tail, midpiece and head. In Figure 3B, a single cell of M.genitalium is bound to the midpiece vesicle. Small colonies or clumps of M.genitalium were also seen on sperm cells (not shown). It is impossible to distinguish a single cell of M.genitalium in a small colony, as shown in Figure 3C. A spermatozoon without M.genitalium attached is seen in Figure 3D.
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IFM
Mycoplaslama genitalium was attached to purified spermatozoa and methanol-fixed for immunoflurescence microscopy with polyclonal antibodies (PabG37) as primary antibodies. Single cells or microcolonies of M.genitalium appeared as green spots on the red coloured sperm cells (black and white representation in Figure 4). As observed with NM and XRM, M.genitalium adhered as single cells, microcolonies or clumps to all parts of the sperm without any apparent preference for a specific locus. Figure 4A and B shows M.genitalium attached to different parts of the tail, and in Figure 4C–E they are located on the midpiece and head of the sperm.
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To study whether motile sperm were carrying M.genitalium, the mycoplasma was added to the semen before purification. The motile spermatozoa were purified and treated with methanol for immunofluorescence microscopy as described above. These spermatozoa had only a single green spot attached (see Figure 4G–L). Interestingly, M.genitalium was mainly attached to the midpiece or neck region (Figure 4G–I), but it was also seen on the head (Figure 4J and K). Occasionally, M.genitalium was attached to the tail but then it was close to the midpiece (Figure 4L) and not located at the distal end, as shown in Figure 4B. Sperm completely covered by the mycoplasmas as shown with the other microscopy techniques was never seen when motile spermatozoa were selected for analysis.
The M.genitalium did not seem to cause changes of the morphological appearance of the sperm cells. A few times, the tail of a spermatozoon was seen to bend where many mycoplasmas were attached (not shown), but it was not certain if the spermatozoa were damaged before or after the attachment of M.genitalium.
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Discussion |
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Mycoplasma genitalium has been shown to attach to a number of different cell types including erythrocytes (Morrison-Plummer et al., 1987
), cells of the Fallopian tube (Collier et al., 1990), Vero cells (Jensen et al., 1994), airway cells (Baseman et al., 1996), Hep-2 cells (Svenstrup et al., 2002) and even plastic, but no previous reports have shown that M.genitalium could bind to spermatozoa, as demonstrated here. It is important to note that non-genital mycoplasmas, e.g. M.pneumoniae, have been shown to bind human spermatozoa (Taylor-Robinson and Manchee, 1967), and as M.genitalium is able to bind to different cell types, the attachment to spermatozoa is not a proof of a route of infection, but certainly it is an important first piece of evidence.
Mycoplasma genitalium was demonstrated to adhere to all parts of the spermatozoa. Numerous M.genitalium cells bound to the spermatozoa were shown to cause sperm agglutination and immotility. Inhibition of sperm motility has also been documented for other human and animal mycoplasmas (Panangala et al., 1981; Rose and Scott, 1994). When single cells of M.genitalium were bound to motile spermatozoa, the midpiece/neck was the preferred site of attachment. This location is natural in the sense of minimizing the effect on sperm motility. From tests for determination of the presence of antisperm antibodies, it is well known that spermatozoa are able to show progressive motility even with latex particles bound (Bohring and Krause 1999). Such latex particles (2.4 µm) are 8 times the size of M.genitalium, suggesting that a possible motility-inhibiting effect caused by the microorganism should be of a biological rather than ‘mechanical’ nature. Binding to the tail, midpiece and head of the spermatozoa has also been shown for U.urealyticum (Nunez-Calonge et al., 1998), N.gonorrhoeae (James-Holmquest et al., 1974) and E.coli (Friberg and Fullan, 1983). Interestingly, C.trachomatis was demonstrated to attach to the midpiece of sperm cells obtained from peritoneal fluids (Friberg et al., 1985, 1987). This means that the spermatozoa were able to transport the midpiece-bound chlamydia.
It is characteristic for E.coli to readily bind and agglutinate spermatozoa, as shown in the study by Wolff et al. (1993). Almost 100% of the motile sperm cells were agglutinated after 60 min. In contrast to this, we could not show any significant difference in the sperm count on free spermatozoa between the sample incubated with M.genitalium and the negative control without mycoplasmas. Although we observed sperm agglutination caused by M.genitalium, the number of spermatozoa was too small to be measured by this method. However, the ability to bind readily to spermatozoa also observed for M.genitalium is probably required because of the fast moving sperm.
The adhesion of M.genitalium to the spermatozoa seemed to be mediated by the tip, as shown by X-ray microscopy, but, in order to conclude whether the attachment is specific, further experiments are needed. Nevertheless, a specific binding is likely. The host cell receptor for M.genitalium is probably a sialoglycoconjugate as suggested by Jensen et al. (1994) because neuraminidase treatment of human erythrocytes inhibits adsorption of M.genitalium (Tully et al., 1983). Sialoglycoconjugates are also the host cell receptor for M.pneumoniae (Krivan et al., 1989). Spermatozoa possess many different kinds of receptors and they have been proven to expose sialoglycoproteins at their surface (Czuppon, 1984), which could function as receptors for M.genitalium.
Only one clinical study has investigated semen for the presence of M.genitalium (Kjaergaard et al., 1997). Semen from 115 men attending fertility clinics was studied, and M.genitalium was only found in one sample (0.9%) and could therefore not be associated with infertility. Previously we published a serological study of infertile women where we could not correlate M.genitalium with male factor infertility but could with tubal factor infertility (Clausen et al., 2001). Even though these two studies do not indicate an association of M.genitalium with male infertility, we showed that M.genitalium inhibited sperm motility and consequently showed the potential to cause male infertility.
The ability to cause physiological damage to sperm has been suggested for many animal and human genital mycoplasmas, but there have been conflicting results. It may be that XRM is the method of choice to study the damage to spermatozoa caused by mycoplasmas. The benefits of XRM are that live cells can be studied with high resolution and minimum preparation of the samples. Long exposure times, however, can cause damage to the sperm cells (Abraham-Peskir et al., 1998). XRM has been used to investigate the ultrastructure of spermatozoa, revealing new information on sperm vesicles (Abraham-Peskir et al., 2002) and mitochondria (Vorup-Jensen et al., 1999).
The purification of spermatozoa by the swim-up procedure has been shown to remove microbes from the sperm cells (Wong et al. 1986). Despite this, we succeeded in purifying motile sperm with M.genitalium attached, but we do not know whether these single attached cells are able to establish an epithelial infection. However, based on this result, it is tempting to speculate that M.genitalium could be transported to the uterus and Fallopian tubes to colonize and destroy the ciliated epithelia, and thereby cause infertility in women.
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Acknowledgements |
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We are grateful to Karin Skovgaard Sørensen and Inger Andersen for skilled laboratory practice, and to Lisbet Wellejus Pedersen for excellent linguistic assistance with this paper. This work was supported financially by ‘Vestdansk Sundhedsvidenskabeligt Forskningsforum’ (grant no. 1999-043-33), Aarhus University Research Foundation and ‘Fonden til Lægevidenskabens Fremme’.
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Submitted on May 30, 2003; accepted on June 5, 2003.
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J D C Ross and J S Jensen Mycoplasma genitalium as a sexually transmitted infection: implications for screening, testing, and treatment. Sex Transm Inf, August 1, 2006; 82(4): 269 - 271. [Abstract] [Full Text] [PDF]
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