Human Reproduction Vol.21, No.6 pp. 1591–1598, 2006
doi:10.1093/humrep/del032
Advance Access publication March 20, 2006.
Mycoplasma hominis attaches to and locates intracellularly
in human spermatozoa
Francisco Javier Díaz-García1,2,4,5, Alma Patricia Herrera-Mendoza3, Silvia Giono-Cerezo1
and Fernando Martín Guerra-Infante1,2
1
Departamento de Microbiología, Escuela Nacional de Ciencias Biológicas-IPN, 2Departamento de Infectología, Instituto Nacional de
Perinatología-SSa and 3Hospital de Cardiología, Centro Medico Nacional Siglo XXI-IMSS, México City, México
4
Present address: Laboratorio de Biología Molecular. 3er. Piso, Edificio de Investigación, UNAM, Circuito Escolar s/n Ciudad
Universitaria, México City D.F., C.P. 04510, México
5
To whom correspondence should be addressed at: Departamento de Infectología, INPer-SSa, Montes Urales 800, Colonia Lomas
Virreyes, México City D.F., C.P. 11000, México. E-mail: [email protected]
BACKGROUND: The study of sperm–mycoplasma interaction has been focused on the effects of infection on sperm
quality, but few studies have reported the direct interaction of this bacterium with spermatozoa. METHODS:
Selected populations of viable, motile and infection-free human spermatozoa from three healthy men were incubated
with 15–480 multiplicity of infection (MOI) units of DiIC18-labelled Mycoplasma hominis. Cells were analyzed by
means of confocal microscopy and by the eosin-Y dye exclusion test between 10 min and 24 h post-infection.
RESULTS: As early as 10 min post-infection, clusters of M. hominis were seen attached to the sperm head, midpiece
or tail. Mycoplasma showed an approximately 2.5–4.5-fold higher interaction with sperm head or tail than with midpiece. Sequential sectioning of infected spermatozoa revealed the intracellular location of M. hominis within cytosolic
spaces of head and midpiece regions. A minor proportion of infected spermatozoa showed bent or coiled tails, and/or
midpiece thickening. Sperm viability was not altered by M. hominis infection. CONCLUSIONS: These results provide specific and conclusive evidence of M. hominis attachment and invasiveness towards human sperm cells, which
seems not to affect their viability, suggesting that a short-term M. hominis interaction with spermatozoa results in
non-apparent or subtle damage, but might have implications for long-term male or couple’s fertility.
Key words: Mycoplasma hominis/spermatozoa/confocal microscopy
Introduction
The genital mycoplasmas, Mycoplasma hominis and Ureaplasma
spp., have long been suspected to cause male infertility, but
their exact role in development of this pathology is still
debated (Styler and Shapiro, 1985).
The study of in vitro sperm–mycoplasma interaction has
been focused on the effects of infection on sperm viability
(Rose and Scott, 1994; Reichart et al., 2000), morphology and
motility (Rose and Scott, 1994; Nuñez-Calonge et al., 1998;
Reichart et al., 2001), ability to undergo acrosome reaction
(Rose and Scott, 1994), capability of penetrating zona-free
hamster oocytes (Busolo and Zancheta, 1985) and sperm’s nuclear integrity (Reichart et al., 2000). Some authors have tried to
show the presence of mycoplasmas on naturally or experimentally infected spermatozoa, by means of standard light microscopy (Toth et al., 1978; Busolo et al., 1984), scanning electron
microscopy (Busolo et al., 1984; Nuñez-Calonge et al., 1998;
Reichart et al., 2000), transmission electron microscopy
(Busolo et al., 1984; Reichart et al., 2000) and transmission X-ray
microscopy (Svenstrup et al., 2003); however, despite the use
of these powerful imaging techniques, the mycoplasma-like
structures described in those reports lack the mycoplasma identity confirmation by species-specific immunogold localization.
Busolo et al. (1984) described an immunofluorescence
detection of M. hominis in spermatozoa from infertile men;
nevertheless, this technique was not sensitive enough to
show the presence of M. hominis in all the morphologically
altered spermatozoa. Svenstrup et al. (2003) used immunofluorescence to detect Mycoplasma genitalium infection on
washed spermatozoa, but the number of attached bacteria
dramatically diminished as compared with previous observations of unwashed spermatozoa made by X-ray transmission
microscopy.
An innovative approach to overcome the use of antibodybased techniques was used by Baseman et al. (1995). They
labelled Mycoplasma pneumoniae, M. genitalium and Mycoplasma penetrans cells with a lipophilic membrane fluorescent
probe, DiIC18, which allowed the authors to analyze the longterm interaction of viable mycoplasmas with several cell lines
in a relatively undisturbed microenvironment.
© The Author 2006. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved.
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F.J.Díaz-García et al.
Furthermore, the study of Baseman et al. (1995), together
with the discovery of M. penetrans, a human mycoplasma
capable of actively penetrating a variety of human cells (Lo et al.,
1992) and the findings of internalized Mycoplasma fermentans
strain incognitus within several non-phagocytic cells of AIDS
patients (Lo et al., 1989) have radically altered the pervasive
notion of mycoplasmas as exclusive cell-surface parasites.
In this work, we described the use of fluorescent labelling of
mycoplasmas, coupled to laser scanning confocal microscopy
(LSCM) examination, for analysis of M. hominis interaction
with experimentally infected human spermatozoa. The aim of
this study was to ascertain whether the bacterium interplays
with specific spermatozoon regions and to describe its cellular
distribution and their relationship with sperm viability and
morphology.
Materials and methods
Sperm specimens
Semen samples from three healthy men from the laboratory staff were
collected in the laboratory by masturbation, after 3–5 days of sexual
abstinence. Samples were kept at 37°C for 30 min after production.
Subclinical seminal infection by Chlamydia trachomatis or mycoplasmas, including M. hominis and Ureaplasma urealyticum, was determined by means of PCR, using the protocols and primer sets
described previously (Dutilh et al., 1989; Van Kuppeveld et al., 1992;
Blanchard et al., 1993; Grau et al., 1994).
Selection of viable and motile spermatozoa (Erel et al., 2000)
One millilitre aliquot of semen was diluted 1:1 with human tubal fluid
(HTF 1×; Irvine Scientific, Irvine, CA, USA); sperm suspension was
layered onto 1 ml fraction of 75% Percoll (Sigma, St. Louis, MO,
USA) prepared v/v in HTF 10× (Irvine Scientific) and centrifuged at
350 × g. The sperm pellet, rich in viable and motile spermatozoa, was
carefully drawn off from both layers of supernatant, washed twice with
HTF 1× and suspended in 2 ml HTF 1×. Viability of spermatozoa was
assessed by the 0.5% eosin-Y dye exclusion test in a Neubauer Chamber. Cell suspension was adjusted to 2 × 106 per ml.
Fluorescent labelling of M. hominis
Mycoplasma hominis ECV99030142 strain from the collection of the
Department of Infectology, Instituto Nacional de Perinatología of
Mexico, was used in this work. The strain ECV99030142 was originally isolated from the cervix of an acute bacterial vaginosis patient.
A stock culture of M. hominis at a density of ∼1 × 108 colony forming
units (CFU)/ml was prepared on modified SP-4 (mSP-4) broth and
aliquots of 1 ml were stored at –70°C.
For each experimental run, 1 ml of the M. hominis stock was
thawed, diluted with 9 ml of mSP-4 broth and incubated overnight at
37°C/5% CO2. The labelling procedure of mycoplasmas was done as
described by Baseman et al. (1995). In brief, bacterial cells were harvested by centrifugation at 12 000 × g for 10 min at 4°C and suspended
in 10 ml of fresh mSP-4 broth. Ten microlitres of 10 mM solution of
DiIC18 (1,11 dioctadecyl 3,3,3′,3′-tetramethyl indocarbocyanine perchlorate; Aldrich, Milwaukee, WI, USA) was added to the M. hominis
culture and further incubated for 1 h at 37°C/5% CO2. DiIC18-labelled
mycoplasmas were pelleted by centrifugation at 12 000 × g/10 min/
4°C, washed twice in cold phosphate-buffered saline (PBS) at pH 7.4,
resuspended in 0.5 ml PBS and immediately separated from unincorporated dye by Sephadex G-50 column chromatography (Pharmacia,
Piscataway, NJ, USA). The Sephadex column was equilibrated with
1592
PBS, the labelled bacteria was placed on top of the column and the
first three 4 ml fractions eluted with PBS were collected (based on
previous determination of mycoplasma-containing fractions). The
fractions were combined, bacteria was pelleted by centrifugation at
12 000 × g for 10 min/4°C, resuspended in 1.5 ml of HTF and filtered
through 0.45 μm syringe filter (Acrodisc filter, Gelman, Ann Arbor,
MI, USA). Viability of labelled mycoplasmas was verified by 10-fold
serial dilution in mSP-4 broth.
Mycoplasma hominis experimental infection of spermatozoa
Sperm suspension for each experiment was distributed on 30 wells of a
96-well microplate, at a density of 4 × 105 spermatozoa/100 μl HTF 1×
per well. One hundred microlitres of the DiIC18-labelled M. hominis
suspension was then added to each of 15 test wells, and 100 μl of HTF
1× were added to each of 15 control wells. The microplate was incubated at 37°C/5% CO2 for 24 h. The infective dose multiplicity of
infection (MOI) was determined by 10-fold serial dilution cultures in
mSP-4 broth and agar plates, directly from test wells. Aliquots of test
and control wells were taken at different time-intervals: 10, 30, 60, 120
min and 24 h, for confocal microscopy analysis and sperm viability
assessment. Since sperm from donor 1 served to standardize the interaction assay, their analysis was carried out as far as 2 h, so we decided
to extend the incubation period up to 24 h for subsequent assays.
LSCM examination of mycoplasma-infected spermatozoa
Fifty microlitres of DiIC18-labelled M. hominis, control uninfected or
DiIC18-labelled M. hominis-infected spermatozoa was smeared on 25 ×
75 mm glass slides (Corning, Nagog Park Acton, MA, USA). The
smears were fixed in 4% paraformaldehyde for 7 min at room temperature and immediately rinsed with PBS, air-dried and mounted with a 24 ×
50 mm coverslip and Vectashield mounting fluid (Vector Laboratories,
Burlingame, CA, USA); the coverslip edges were sealed with nail polish.
Slides were examined under a high-magnification Plan Neofluar
100×/1.3 oil Ph3 objective in a Carl Zeiss’ Axiovert 100 M microscope equipped with LSM 510 confocal system, using Helium/Neon
laser illumination with the 543 nm wavelength along with the LP 560
optical filter set. Fluorescent and Nomarski’s differential interference
contrast (DIC) optics were used simultaneously to visualize DiIC18labelled bacteria and non-fluorescent unstained sperm structures, as
well as a through-focal series of optical slices 1.0–1.1 μm apart taken
along the z-axis and moving from the slide-to-coverslip boundaries of
individual cells. Optical sectioning allowed imaging of sequential
focal planes with each plane containing exclusively the fluorescence
in focus, therefore, the relative distribution and localization of DiIC18labelled M. hominis in infected spermatozoa could be determined.
Assessment of sperm viability
In parallel to confocal microscopy analysis, the viability of sperm suspension was quantified by the eosin-Y dye exclusion method. A 10 μl
aliquot of the sperm suspension was mixed with an equal volume of
0.5% eosin-Y dye solution in PBS, and the sperm suspension was enumerated for dye uptake by light microscopy under high-power Achroplan 40×/0.65 objective and bright field optics in a Carl Zeiss’ Axiolab
E-er microscope. For each determination, 200 sperm were evaluated.
Results
Screening for sperm subclinical infection
Semen samples obtained from apparently healthy donors were
analyzed by PCR to rule out subclinical infections caused by
C. trachomatis or mycoplasmas. None of the samples gave
positive PCR results, whereas the specific amplicons of 129,
M. hominis interaction with human sperm
715, 170 and 429 base pairs of C. trachomatis, Mycoplasma
spp., M. hominis and Ureaplasma spp., respectively, were
present in the corresponding positive controls.
Analysis of DiIC18-labelled Mycoplasma hominis
After the fluorochrome labelling and purification, M. hominis
grew adequately in mSP-4 liquid and semisolid media, without
any apparent difference with unlabelled bacteria. The DiIC18labelled bacterial count was decreased in 2log10 of CFU/ml
with regard to unlabelled stock inoculum (data not shown).
The microscopic appearance of DiIC18-labelled M. hominis
are shown in Figure 1A–D. Bright red signals bound to slide surface were observed against a blue–green background in the composite fluorescence-Nomarski channel. The smallest fluorescent
signals were compatible in size (0.35–0.45 μm diameter) and
morphology (sphere-shaped particles) with Mycoplasma single
cells. Greater fluorescent signals were likely to be cell aggregates rather than leakage of fluorochrome.
Analysis of uninfected spermatozoa
Microscopic analysis of uninfected spermatozoa was done by
observation of cells in the fluorescence channel. The paraformaldehyde fixation of sperm cells and the use of Vectashield
mounting fluid produced no detectable fluorescent signals, neither in the background nor in the sperm cells, thus ruling out
autofluorescence of uninfected spermatozoa. At Nomarski
channel, the entire single sperm cells were readily visible and
needed no further contrast staining (Figure 1E–G).
shape of the structure involved. Free round-shaped mycoplasmas bound to slide surface and non-infected spermatozoa were
also observed. As soon as 10 min post-infection, tight clusters
or microcolonies of M. hominis were seen attached to sperm
head, midpiece or tail (Figure 2), but no specific patterns of
interaction were observed. Differences in the sperm–mycoplasma
interaction among the three sperm samples were observed
throughout the experiment (Table I); the lowest interaction was
obtained for donor 1 during the 30 min period after infection
(16%) while the highest interaction reached up to 73% for
donor 2 at 60 min post-infection; however, despite the numerical differences between donors 1 and 2, the overall distribution
of spermatozoa with positive fluorescent signals increased to
maximum at 60 min and thereafter started to decline. Conversely, distribution of mycoplasma-infected spermatozoa from
donor 3 was opposed to that of the other donors, since the highest and lowest interaction frequencies were observed at 10 and
60 min post-infection, respectively. The calculated infective
doses of DiIC18-labelled M. hominis for each experiment were
36, 15 and 480 MOI for donors 1, 2 and 3, respectively. It is
worthy to note that despite the higher infective dose used with
donor 3, examination of slides revealed moderate spermatozoa
with attached bacteria, but the rest of the slide surface appeared
crowded with large M. hominis clusters.
The frequencies of interaction of mycoplasma with specific
sperm structures are shown in Table I. Roughly, there was a
2.5–4-fold higher proportion of interaction with sperm’s head
and 3–4.5–fold higher interactions with tail than for midpiece.
Intracellular location of Mycoplasma hominis
Mycoplasma hominis interaction with spermatozoa
At LSCM examination, mycoplasmas were seen attached to
spermatozoa, and its distribution on the cell frequently took the
Evidence that M. hominis is capable of entering the cytoplasmic spaces of the spermatozoon is presented in Figure 3. Here,
sequential sectioning of a series of focal planes from infected
Figure 1. Microscopic appearance of DiIC18-labelled Mycoplasma hominis and of uninfected spermatozoa. Bright round-shaped red signals of
mycoplasma cells are readily visible in the fluorescence channel (A and D) but not in the Nomarski channel (B); a composite image of the fluorescence and Nomarski channels is seen in (C). Fluorescent signals were compatible in size and morphology with single mycoplasma cells or small
bacterial aggregates (D). Non-specific fluorescent signals were absent in uninfected spermatozoa (E), instead unstained spermatozoa was
observed appropriately in the Nomarski (F) and the composite channels (G). All images are from interaction assay with 15 multiplicity of infection (MOI) units.
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F.J.Díaz-García et al.
all cases the presence of attached or internalized mycoplasmas
was demonstrated.
Effect on sperm viability
The initial percentages of viability of purified spermatozoa
were 86%, 84% and 69% for donors 1, 2 and 3, respectively.
These values remained constant during the first 120 min of
incubation, both in uninfected controls and in M. hominisinfected sperm suspensions (Figure 5). However, at 24 h of
incubation, the viability of sperm decreased by approximately
20%–30%, but no significant differences were observed
between control and infected cells.
Discussion
The LSCM examination of experimentally infected human
spermatozoa allowed us to show both adherence and intracellular location of DiIC18-labelled M. hominis on sperm cells,
without affecting their viability within the 24 h period of this
study.
Selection of viable, motile and infection-free sperm cells
was done to guarantee a population of ‘high quality’ spermatozoa and to avoid the possible biased effect of underlying genital infection and defective sperm cells. Despite the fact that the
DiIC18 fluorochrome is partially inserted into the lipid bilayer
of plasma membrane and diffuses laterally (Haughland, 1996),
it does not appreciably affect mycoplasma viability, metabolic
activity or other physiological properties. Thus, DiIC18labelled mycoplasmas retained the properties of unlabelled
bacteria, and consequently, the infectious process of our model
was reliable.
Unlike other pathogenic mycoplasmas, M. hominis cells do
not exhibit the typical elongated flask-shaped morphology
(Razin et al., 1998) but a sphere-shaped morphology instead
(Krause and Taylor-Robinson, 1992). Thus in this study, the
round-shaped appearance of DiIC18-labelled M. hominis
either attached to spermatozoa or bound to the slide was consistent with single cells or large tight bacterial aggregates.
Girón et al. (1996) described that M. penetrans initially
attaches to HEp-2 cells as small clusters that increased in size
as the infection proceeds, and the attached bacteria underwent
morphological changes from the flask shape to the spherical
forms.
Figure 2. Interaction of DiIC18-labelled Mycoplasma hominis with
human spermatozoa in vitro. (A) Sperm cells incubated with mycoplamas but without any interaction. Arrows point to clusters of mycoplasmas attached to different spermatozoon regions, such as head (B
and C), tail (D and E) or midpiece (F). Observations were made in the
composite fluorescence-Nomarski channel. Images are from interaction assays with 15 multiplicity of infection (MOI) (C and E), 30
MOI (B) and 480 MOI (A, D and F).
spermatozoa indicates that mycoplasmas parasitize the entire
head and midpiece regions. Three-dimensional reconstruction
and projection of the sequential optical slices allowed discrimination between attached and internalized bacteria, because
intracellular bacteria were seen clearly surrounded by sperm’s
plasma membrane.
Morphological alterations attributable to infection with M.
hominis were observed in a low proportion of spermatozoa.
Such alterations include several grades of coiling and bending
of tail (Figure 4A–C) or midpiece thickening (Figure 4D); in
Table I. Interaction kinetics between DiIC18-labelled Mycoplasma hominis and viable spermatozoa
Time (minutes)
Percentage of interactiona
Donor 1
10
30
60
120
1440
a
Donor 2
Total
(H/M/T)
Total
(H/M/T)
Total
(H/M/T)
16
16
36
28
ND
(50/12.5/37.5)
(37.5/12.5/50)
(44.4/11.2/44.4)
(42.9/21.4/35.7)
ND
43
47
73
51
55
(23.2/–/76.8)
(19.2/29.8/51)
(48/15/37)
(33.3/5.9/60.8)
(80/1.8/18.2)
42
36
20
26
20
(38.1/4.8/57.1)
(33.3/16.7/50)
(40/30/30)
(23.1/15.4/61.5)
(40/–/60)
Data in parenthesis indicate percentages of interaction with specific sperm’s structures.
H, sperm head; M, sperm midpiece; T, sperm tail; ND, not determined.
1594
Donor 3
M. hominis interaction with human sperm
Figure 3. Intracellular location of Mycoplasma hominis within human spermatozoa infected in vitro. A series of optical slices 0.9–1.0 μm apart
were taken along the z-axis, and the resulting sequential images displayed only the fluorescence in focus. M. hominis is clearly located within the
cytoplasmic spaces of the head (A) and midpiece (B) as indicated by the strong fluorescent signal shown in the inner slices. In C and D, the series
of optical slices shown in A and B were stacked (blue-outlined panels) and subjected to orthogonal sectioning along the specified x-axis (green
horizontal line) and y-axis (red vertical line), confirming the internal location of fluorescent signals in the corresponding lateral views of the
x- and y-sections, shown as green- and red-outlined panels, respectively. The blue line across the lateral views indicates the current optical slice
displayed in the stack. All observations were made in the composite fluorescence-Nomarski channel and are from interactions assays with 15
multiplicity of infection (MOI).
Nuñez-Calonge et al. (1998) reported the presence of mycoplasma-like structures attached to in vitro-infected human
sperm, a feature confirmed in this study, since the DiIC18labelled M. hominis was demonstrated in close interplay with
sperm’s membrane surface. Despite attachment of M. hominis
to the entire spermatozoon regions, interaction kinetics
revealed a marked affinity towards tail and head of spermatozoa. The predilection of attachment to sperm structures other
than midpiece was also observed by Reichart et al. (2000),
which showed a 4-fold higher adherence of U. urealyticum to
sperm head than to the tail; in contrast, M. genitalium appears
to attach more efficiently to midpiece (Svenstrup et al., 2003).
The obvious metabolic and pathogenic differences among genital mycoplasmas (Krause and Taylor-Robinson, 1992; Razin
et al., 1998) can explain why particular attachment patterns are
observed.
In a few cases, examination of infected cells revealed fluorescent signals on the midpiece/postacrosomal region slightly
larger than the cellular structure involved (data not shown),
suggesting the presence of cytoplasmic droplet with adhered
bacteria, although those cases were scored as ‘midpiece’ or
‘head’ interaction.
The cytadherence exhibited by M. hominis at low infective
doses (15–30 MOI) was in a time-dependant manner,
because the number of spermatozoa with attached mycoplasmas increased with the time course of infection to maximum at 1 h of incubation. Similar adherence kinetic curves
for other mycoplasmas have been described previously
(Giron et al., 1996; Svenstrup et al., 2003). However, with
an infective dose of 480 M. hominis cells per sperm, the
maximum adherence was observed at 10 min post-infection
probably as a result of rapid saturation and subsequent loss
of functionality of sperm’s receptor caused by excessive
release of toxic by-products of mycoplasmal metabolism
(Rottem and Naot, 1998).
The sulfogalactoglycerolipid (SGG) is the major sulphated
glycolipid located in the outer leaflet of the plasma membrane
of mammalian male germ cells (Vos et al., 1994), which is
asymmetrically distributed throughout spermatozoon, depending on the stage of maturation (Lingwood, 1986). By thin-layer
chromatography overlay binding assays, sulfoglicolipids were
confirmed as the only receptor molecules for M. hominis
(Olson and Gilbert, 1993), and SGG-mediated adherence to
sperm cells has been suggested for M. hominis, U. urealyticum,
Ureaplasma diversum and Mycoplasma pulmonis (Lingwood
et al., 1990b); furthermore, an arylsulphatase activity that completely degrades the SGG after binding was also identified in
M. pulmonis (Lingwood et al., 1990a). The high prevalence of
SGG in the human spermatozoa would provide M. hominis
with considerable numbers of receptor molecules through
which adherence and subsequent internalization could be
achieved. A 70 kDa heat shock protein-related molecule has
been proposed as the SGG ligand in M. hominis (Boulanger
et al., 1995).
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F.J.Díaz-García et al.
Figure 4. Morphologic alterations observed in Mycoplasma hominis infected human spermatozoa. A spermatozoon with a nearly perpendicular tail bending is shown in (A). Sperm cell with different grades
of tail coiling are shown in (B) and (C). A marked midpiece thickening
was also observed in some cells (D). Arrows points out to the morphologic alterations described above, whereas arrowheads indicate
the presence of M. hominis. Observations were made in the composite
fluorescence-Nomarski channel. Images are from interaction assays
with 15 multiplicity of infection (MOI) (C and D) and 30 MOI (A and B).
Figure 5. Viability of Mycoplasma hominis-infected spermatozoa as a
function of time. Both control uninfected (continuos line) and M. hominsinfected (dotted line) spermatozoa showed a similar distribution of viability percentages, which indicate that M. hominis had no effect on
sperm viability. Symbols: ▲ = donor 1; ■ = donor 2; ● = donor 3.
The uneven interaction of M. hominis towards the different
sperm cell structures could be the result of both, presence of
cells at different stages of maturation and incubation-related
premature capacitation of spermatozoa, since these processes
are characterized by modifications in sperm surface protein
1596
distribution, increased plasma membrane fluidity and changes
in enzymatic activities and motility (Baldi et al., 1996).
Subsequent three-dimensional analysis of infected spermatozoa revealed the intracellular distribution of M. hominis
clearly located throughout the cytoplasmic spaces of the head
and midpiece. These findings demonstrate that M. hominis has
the ability to gain entry into spermatozoa, which could have
implications for in vivo evasion of host immune responses and
persistence (Rottem and Naot, 1998). The invasive properties
of several Mycoplasma species towards different non-phagocytic
cell lines have been demonstrated (Taylor-Robinson et al.,
1991; Andreev et al., 1995; Baseman et al., 1995; Giron et al.,
1996). By means of transmission electron microscopy analysis,
the intracellular location of U. urealyticum in experimentally
infected human spermatozoa has been suggested (Reichart
et al., 2000). The molecular mechanism of M. hominis internalization is yet unknown, but it is likely to speculate that the
bacterium’s membrane-bound phospholipase (Krause and
Taylor-Robinson, 1992) could participate in triggering signal
transduction cascades and cytoskeletal rearrangement that
results in its own uptake by target cells (Rottem and Naot,
1998), a mechanism described for the internalization of
M. penetrans to Hep-2 cells (Giron et al., 1996). In fact, the
invasion process by M. penetrans to Molt-3 lymphocytes
results in the release of unsaturated free fatty acids from the
membranes of target cells (Salman et al., 1998).
A minor proportion (less than 1%) of M. hominis-infected
spermatozoa showed coiled and bent tails, findings similar to
those described previously by others in sperm samples from
naturally infected men (Toth et al., 1978; Busolo et al., 1984)
or in spermatozoa infected in vitro (Rose and Scott, 1994;
Nuñez-Calonge et al., 1998), but unlike those reports, the presence of M. hominis was observed mainly in morphologically
unaltered cells and in all the morphologically altered sperm. A
moderate loss of viability in control and infected spermatozoa
after 2 h of incubation could be explained by an acidic shift in
the pH of the medium by sperm’s glycolytic activity, and since
M. hominis cannot ferment glucose (Pollock et al., 1997), its
contribution to this pH shift is negligible.
Since the infection-associated sperm viability and morphology remained practically unaltered, it can be argued that the
main virulence mechanism displayed by sperm-attached M.
hominis might be the release of reactive oxygen species (ROS)
that induce host cell membrane damage (Rottem and Naot,
1998), such as lipid peroxidation, loss of membrane fluidity
and ultimate inability to undergo hyperactivation and acrosome
reaction (Miller and Britigan, 1997; Potts et al., 2000). Exposure of sperm antigens might be a side-effect of membrane disturbance by ROS, which in turn may lead to autoimmune
responses (Turek, 1999).
Another possible mechanism by which genital mycoplasmas
impairs human fertility is through induction of DNA fragmentation (Reichart et al., 2000), because its possible impact on
fertility goes beyond the fertilization process and may represent a threat for later events, such as egg implantation, embryo
development and pregnancy outcome (Kanakas et al., 1999;
Reichart et al., 2000). From the results presented here, it is
tempting to speculate that M. hominis exerts a subtle damage
M. hominis interaction with human sperm
on spermatozoa, maybe damage alike to U. urealyticum induction of nuclear decondensation, denaturation or single DNA
strand breaks (Reichart et al., 2000), which also has no shortterm consequences on viability, motility and morphology.
Indeed, mycoplasmal endonuclease-induced internucleosomal
DNA fragmentation (Paddenberg et al., 1996) and deleterious
effects on chromosome structure (Tsai et al., 1995) were demonstrated after long-term infection of cell lines.
In summary, an adhering and internalizing capability
towards human spermatozoa was demonstrated for M. hominis,
which apparently have no effect on cell viability but instead
produces a low proportion of cauda and midpiece morphological alterations during the 24 h period of infection. These findings suggest that M. hominis attachment and subsequent
internalization to human spermatozoa are early events during
the infectious process; nevertheless, it is imperative to define
the long-term effect of infection on sperm physiology and their
relationship with reproductive failure.
Acknowledgements
We thank the following persons for their technical assistance and
helpful suggestions: Jorge Sosa Melgarejo, D.Sc., Saúl FloresMedina, M.Sc., Marcela López-Hurtado, M.Sc., and laboratory technician Verónica Montoya-Hernández. This work was supported by
research grants CGPI2004753 from IPN and 21225022501 from
INPer-SSa. F.J. Díaz-García is a recipient of Institutional and PIFI
grants from Instituto Politécnico Nacional of Mexico. This work was
presented at the 43rd Interscience Conference on Antimicrobial
Agents and Chemotherapy of the ASM, held in Chicago, IL, USA on
September 14–17, 2003 (Abstract L-1065).
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Submitted on June 7, 2005; resubmitted on January 16, 2006; accepted on
January 19, 2006