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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Key words: adhesion/inhibition/Mycoplasma genitalium/spermatozoa/X-ray microscopy
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Introduction |
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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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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 spermmycoplasma 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 spermmycoplasma 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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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 1AD, 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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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 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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Submitted on May 30, 2003; accepted on June 5, 2003.