Interaction of different Chlamydiae species with bovine spermatozoa

Background Interaction of spermatozoa and Chlamydiae spp. might contribute to reduced fertility in cattle. To proof this hypothesis, bovine semen was incubated with viable or heat inactivated Chlamydia (C.) abortus or psittaci (Multiplicity of infection = 1) and sperm motility was monitored with a computer-assisted sperm analyzer over 24 h. Additionally, the interaction with the spermatozoa was further investigated by means of light and transmission electron microscopy (TEM). Results Only viable Chlamydiae of both species decreased sperm motility and this only after about 9 h. Taking binding rates into account, the loss of sperm motility after about 9 h could likely be a consequence of Chlamydiae attachment to the spermatozoa. About two thirds of the Chlamydiae elementary bodies were bound to the front third of the sperm, the acrosomal region. No inclusions of Chlamydiae in spermatozoa were observed in TEM after 2 h co-incubation. Conclusions As initial motility was not affected following co-incubation of viable Chlamydiae and bovine sperm, it seems likely that sperm could serve as a carrier/vehicle for Chlamydiae facilitating cervical passage of Chlamydiae spp. in cattle. Additionally, our results suggest that spermatozoa carrying Chlamydiae may have no initial disadvantage in reaching the oviduct, but are immotile at the time of ovulation what might have an impact on fertilization capacities of the individual sperm. Consequently, high concentrations of the investigated Chlamydiae in the seminal plasma or female genital tract might play a role in reduced fertility in cattle.

Venereal infection is the classical route for the transmission of C. trachomatis in humans [30][31][32][33]. There is limited evidence that venereal transmission of C. abortus is possible in sheep [15]. Recently, Schautteet et al. [16] reported severe reproductive failure in sows probably related to insemination of C. suis contaminated semen. Hamonic et al. [34] confirmed that C. suis remains viable and infectious during chilled storage and is more or less unaffected by antibiotics in extenders. The authors consequently hypothesized that extended semen may act as a viable transmission mechanism for C. suis in swine [33,35]. Although it seems obvious that sperm can serve as a vector (vehicle) for Chlamydiae to infect the female genital tract, information on interactions between sperm cells and these bacteria is still patchy. There are two studies indicating that, after natural infection of man, C. trachomatis penetrates the sperm, preferentially their heads, and can also proliferate within the spermatozoa as indicated by the presence of reticulate bodies [33,35]; however, the interaction for other host-Chlamydiae spp. has not been investigated yet.
To gain further insights into the role of Chlamydiae spp. in the bovine, we studied the interaction of C. abortus and psittaci, Chlamydiae spp. previously identified in semen samples of breeding bulls [13,17], with bovine spermatozoa by means of light and transmission electron microscopy. Furthermore, we monitored the motility of spermatozoa in inoculated semen samples as a parameter directly correlated to fertility.

Animal and semen samples
Semen samples were obtained from an adult, sexually mature black Holstein Friesian bull housed in the Clinic for Obstetrics, Gynecology and Andrology of Large and Small Animals with Veterinary Ambulance in Giessen, Germany (50°35'N 8°40'O). The bull was kept in a 22 m 2 freestall barn under natural light conditions and temperatures ranging between 15 and 25°C. He had ad libitum access to water, hay and straw and was additionally fed a commercial diet once a day according to the manufacturer's instructions. Semen was collected using an artificial vagina (Minitube, Tiefenbach, Germany), and a cow or a bull as dummy. Immediately after collection, the semen samples were examined for sperm concentration as well as for total and progressive motility. For further experiments, samples were diluted to a concentration of 100 × 10 6 sperm/ml using 35°C pre-warmed Biladyl® extender without antibiotics (Minitube).

Chlamydia stock solution
Chlamydiae were propagated on Buffalo-Green-Monkey-Kidney cells (ZBV Friedrich-Löffler-Institute, 17,493 Greifswald, Insel Riems, Germany), cultured in sterile filtrated medium [440 ml Eagle's Minimum Essential Medium supplemented with 0.425 g NaHCO 3 , 5 ml 200 mM L-glutamine (Biochrom GmbH, Berlin, Germany), 5 ml Vitamin 100x (Biochrom GmbH) and 50 ml heat-inactivated fetal bovine serum (FBS, all Biochrom GmbH)] at 37°C for about 4-7 days. After adding C. abortus or C. psittaci to the cells, vials were centrifuged for 1 h at 1935 rcf (centrifuge J2-21, rotor JS 7.5, Beckman Coulter GmbH, München, Germany) to promote infection of cells. Chlamydiae were allowed to grow for about 4-7 days at 37°C before being harvested and separated from cell debris as described elsewhere [36]. Analysis with a transmission electron microscope (Zeiss EM 109, Oberkochen, Germany) revealed that the harvested pellet was mainly composed of elementary bodies. Chlamydiae concentrations were assessed by counting particles in Gimenez stained smears using an Ortholux II microscope with a counting tube (Leitz Wetzlar, Germany). Briefly, suspensions were diluted 1:50, 1:100 and 1:200 with sterile saline. 10 μl of each dilution were air-dried on a 1 cm 2 area on a slide and fixated with 100% methanol (Sigma Aldrich, Seelze, Germany) for 1 h. Slides were incubated for 6 min in 0.5 ml carbol fuchsine solution (1.5 mg/ml Neofuchsine, Merck, Darmstadt, Germany, 3 mg/ml phenol, Merck, in phosphate buffer). Afterwards they were rinsed twice with water and counterstained with 0.5 ml malachite green solution (8 g malachite green, Merck, in 1000 ml distilled water) for a minute. Concentration of chlamydial particles was calculated based on the number of particles counted in 100 fields of a counting ocular considering optical magnification (787.5 fold).
Influence of Chlamydiae spp. on total and progressive motility Ten [10] μl of either a suspension of viable Chlamydiae spp. (C. abortus or C. psittaci, 100 × 10 8 particles/ml; viability was confirmed by infection of Buffalo-Green--Monkey-Kidney cells), a corresponding suspension of heat-inactivated (95°C, 1 h) Chlamydiae spp., or sterile saline were added to 100 × 10 6 spermatozoa diluted in 990 μl Biladyl®. The Chlamydia: sperm ratio corresponds to a multiplicity of infection (MOI) of 1. Samples were incubated at 35°C for 24 h. Every three hours, 3 μl were then transferred into a 20-μl sample chamber (Leja® Standard Count 4 Chamber Slide, 20 μm, Leja Products B. V., Nieuw Vennep, Netherlands) for motility analysis at 37°C. Percentages of motile and progressively motile spermatozoa were assessed as six repeated measurements with the computer-assisted sperm analyzer (CASA; SpermVisionTM Software Version 3.5.6.2; Minitube) using the settings as given in Table 1. All experiments were performed five times with semen from different preparations.

Chlamydia-sperm interactions
To investigate the interactions of Chlamydiae spp. and bovine spermatozoa by light microscopy, 1 ml Biladyl® diluted spermatozoa-Chlamydiae suspension (100 × 10 6 sperm and respective number of Chlamydiae spp./ml, C. abortus or C. psittaci) were incubated at 35°C for 24 h (long-term co-incubation). In 3-h intervals, 25 μl of the initial suspension were diluted with sterile saline up to 1 ml and centrifuged (tabletop centrifuge, 3 min, 4025 rcf). The supernatant containing unbound Chlamydiae was discarded. The pellet was re-suspended with sterile saline and centrifuged again. This centrifugation-washing procedure was performed five times in total. After the last centrifugation step (1935 rcf, 10 min, centrifuge JS-21, Rotor JS-7.5, Beckman Coulter GmbH), spermatozoa were transferred on a cover slide in a 6 ml polystyrene vial (Greiner Bio-One). Afterwards, slides were fixed with 1 ml 100% methanol (Sigma Aldrich) and Gimenez stained (see above). Using a Leica DMRIIC microscope (Leica, Wetzlar, Germany), 50 spermatozoa were examined at a magnification of × 1000. Presence (yes/no) and localization (acrosomal region/remaining other parts of the sperm) of Chlamydiae were recorded. The same experiment was performed with samples taken every 30 min over a total of 3 h (short-term co-incubation). All experiments were repeated four times.
For both Chlamydiae spp., the presence, localization, and size of chlamydial particles as well as signs of invasion into spermatozoa were further analyzed by electron microscopy. One ml of the above described Chlamydiae-sperm suspension (100 × 10 6 Biladyl® diluted spermatozoa and C. abortus or C. psittaci/ml) was incubated at 35°C for 2 h. Removal of unbound Chlamydiae was performed as described above. For electron microscopic investigations, the resulting pellets (n = 5) were fixed for 12 h with 0.1 mol/l sodium cacodylate buffer (Merck) containing 6% glutaraldehyde (Plano, Wetzlar, Germany). Afterwards, the samples were centrifuged for 10 min at 447 rcf (tabletop centrifuge). The pellets were washed three times with 0.1 mol/l sodium cacodylate buffer. Briefly, they were fixed for 1 h with 1% osmium tetroxide (Plano), dehydrated with ethanol (Merck), embedded in epoxy resin (Plano), sectioned, stained with 0.2% lead citrate and 0.5% uranyl acetate using an Ultrastainer (Leica Reichert, Bensheim, Germany) and evaluated with a transmission electron microscope (Zeiss EM 109, Zeiss, Oberkochen, Germany) at 80 kV. Pure Chlamydia particles in Buffalo-Green-Monkey-Kidney cell culture treated in the same way as indicated above served as positive control.

Statistical analysis
For all parameters assessed, the different Chlamydiae spp., C. abortus and C. psittaci, were evaluated individually. In general, data was presented as mean ± standard deviation (SD). For statistical analysis, samples were evaluated as repeated measures over the different time points.
To test for a significant effect of Chlamydiae spp. on semen motility and progressive motility, respectively, a mixed-effect model for a four-factorial analysis of variance with repeated measures and equal cell sizes was used (fixed factors: treatment and time as repeated measures; random effects: ejaculate and replication; program BMDP8V). A two-way analysis of variance (factors: time and localization) with repeated measures (program BMDP2V) was performed to analyze binding sites of Chlamydiae on the sperm, i.e. at the acrosomal region of the sperm head versus any other region of the sperm cell, such as midpiece and tail. Calculations for shortand long-term co-incubation, 3 and 24 h, respectively, were performed separately, excluding the time point 0 (The time when the first sample was takentime 0). For all tests, the statistical software program package BMDP Release 8.1 was used [37]. Values were considered to be statistically significant at P ≤ 0.05.

Influence of chlamydia on total and progressive motility
Sperm motility constantly decreased during the 24 h of observation in all experiments. The effect varied strongly between the different ejaculates. Saline controls showed a final reduction of total and progressive motility of 26-90% and 19-81% respectively (data not shown). Nevertheless, test sets from single ejaculates showed a high repeatability. The relative standard deviation within 36 duplicates (saline control, 4 different ejaculates, and 9 different time points) was 0.83%, only. For evaluation of the chlamydial influence on sperm motility, the results for total and progressive motility of the Chlamydiae-inoculated samples were related to the results of the respective saline controls (normalized) and presented as the % difference (Fig. 1). Heat inactivated Chlamydiae of both species did not significantly reduce sperm motility during the course of the experiment. Spermatozoa co-incubated with viable Chlamydiae spp. showed motility results comparable to saline controls in the first nine hours. Interestingly, at all later examination times the immotile fraction was increasingly higher than in the samples inoculated with heat inactivated Chlamydiae spp. This effect was statistically significant for both Chlamydiae spp. with P < 0.0001 after 24 h. The average reduction of the motile fraction was more distinct in samples inoculated with C. abortus (19%) than with C. psittaci (15%). This effect was even stronger in terms of the mean reduction of progressive motility (C. abortus 34%, and C. psittaci 18%).

Chlamydia sperm interaction
Light microscopy revealed that viable C. abortus and psittaci were both able to attach to bovine spermatozoa ( Fig. 2) with no significant differences between the two investigated Chlamydiae spp.. Detailed results on the time course and localization of attachment of the two different Chlamydiae spp. studied are given in Fig. 3. Binding rates increased in a time-dependent manner. About two thirds of the Chlamydiae (in mean 60.3% of C. abortus and 62.9% of C. psittaci) were found to be attached to the acrosomal region of the sperm head comprising only one third of the sperm surface.
In the course of the experiment, this ratio did not change significantly.
Using transmission electron microscopy, Chlamydiae were visualized as small round particles of 0.3 to 0.4 μm attached to the spermatozoon's surface (Fig. 4B). Double membranes surrounded the dark and electron dense particles allowing for easy differentiation from larger cytoplasmic droplets. Chlamydia particles in Buffalo-Green-Monkey-Kidney cell culture pellet were shown after harvesting as positive control (Fig. 4A). Similar to what has been observed with light microscopy (see Fig.  2), most of the Chlamydiae seemed to be located at the apical part of the sperm in the acrosomal area. No particles or structures strongly ressembling Chlamydiae could be identified inside the sperm head or its nucleus after 2 h of co-incubation by using TEM, nor were any changes in the acrosomal membrane of spermatozoon obvious at the attachment site following 2 h co-incubation.

Discussion
The present data shows that viable Chlamydiae of both investigated species, C. abortus and C. psittaci, at an MOI of 1 are capable to affect motility of bovine sperm. The effect on motility, a reduction by 15-19%, as observed in this study, is rather impressive, as spermatozoa were inoculated with Chlamydiae spp. at an MOI of only 1 resulting in low attachment rates (Fig. 2). Data on natural chlamydial load in semen is rare and, to the best of our knowledge, is not available for bovine ejaculates. Low chlamydial loads, i.e. 1.5 × 10 4 [38] or 4.9 × 10 4 [39] particles, were detected in ejaculates of infected men by Real time-quantitative polymerase chain reaction (RT-qPCR). Due to the lack of details about sperm concentration in the aforementioned studies [38][39][40], it is impossible to compare those with this study in terms of chlamydial load necessary to elicit effects on sperm. Results are depicted as mean values ± SD of 5 replicates. Motility of spermatozoa co-incubated with viable Chlamydiae spp. was significantly reduced from 9 h onwards compared to motility of spermatozoa incubated with heat inactivated Chlamydiae (p < 0.0001) However, comparison with the WHO reported minimum and average sperm concentrations in the human ejaculate (9-73 × 10 6 /mL; [41]) reveal possible MOIs between 2.1 × 10 − 4 and 5.4 × 10 − 3 . Different to the likely low chlamydial load in semen, a higher chlamydial load might be expected in the female genital tract where up to 8.5 × 10 7 C. trachomatis per ml genital fluid had been found in women [39] indicating that similar chlamydial and sperm concentrations as used here, might be possible naturally.
Sperm motility started to decrease nine hours of co-incubation with the respective Chlamydiae spp.. In contrast binding of chlamydial particles to sperm was already microscopically observed immediately after the start of co-incubation, and was correlated with time. It seems possible that the negative effect of Chlamydiae spp. on total and progressive motility might be due to chlamydial viability, since this effect was absent when spermatozoa were co-incubated with saline only (negative control) or heat-inactivated Chlamydiae. Heat-inactivated Chlamydiae spp. showed a certain lightmicroscopical integrity and typical Gimenez-staining, however, detailed examinations on the morphology by transmission electron microscopy were not performed as well as details on binding partners/receptors were lacking. These analyses should be included in future studies to identify the binding mechanism. In contrast to this study, other investigations did not find such negative effects on semen parameters, which might be, at least in part, due to differences in the study designs. In particular, semen parameters were evaluated shortly after starting co-incubation (e.g. [27,28,35]) at a time where also in this study, no effects of Chlamydiae spp. on motility were observed.
Taken together, the results of the current study suggest that spermatozoa start to display a reduced motility around 9 h post co-incubation due to effects of Chlamydiae spp.. Moreover, C. abortus induced a slightly stronger effect than C. psittaci. The reasons for this difference remain to be investigated.
Basic knowledge of the sperms' fate in the bovine female genital tract is necessary for understanding possible clinical effects of Chlamydiae-mediated reduced sperm motility on female reproduction. Following male infection, Chlamydiae spp. are located in the accessory sex glands in man [19,20], bulls and boars [42]. During ejaculation, spermatozoa get in contact to the pathogen that is released from the accessory sex glands together with the seminal plasma. In bovines, the spermatozoa are deposited into the anterior vagina during breeding, and are then required to rapidly enter the cervix. However, the cervical passage of sperm is hampered by the cervical mucus, which acts as a mechanical barrier particularly to sperm with abnormal motility patterns or reduced motility thus being a mechanism of sperm selection [43][44][45][46]. Additionally, the cervical mucus is also considered to have a filter function for seminal plasma and free microbes [47]. It might thus be that binding to motile spermatozoa is necessary for Chlamydiae spp. to not be caught by the cervical mucus in order to be able to reach the uterus and eventually also the oviduct. After having passed the cervix, spermatozoa quickly reach the utero-tubal junction where the apical surface of the sperm head binds to the oviductal epithelium [48,49] at the site of sperm reservoir (for review see [47,50]). At this site, sperm remain viable for 18 to 24 h or even longer as earlier studies have shown that artificial insemination with frozen semen (as common in cattle) is most successful, if it is performed in oestrus 12-24 h before ovulation [51]. As motility was first significantly affected after nine hours of co-incubation and the percentages of spermatozoa with bound spermatozoa are similar, it seems, however, likely that Chlamydiaecarrying spermatozoa are immotile at the time of fertilization. It remains to be investigated why motility was affected from nine hours after co-incubation and what were the reasons of immotility of spermatozoa. It is noteworthy that heat inactivated Chlamydiae spp. had no impact on sperm motility and it deserves further investigation if addition of specific antibiotics to commercial semen extenders or semen freezing is capable to induce the same effects on Chlamydiae sp.. in semen samples. The inactivation process might have resulted in denaturation of chlamydial structures relevant e. g. for the binding to the host cell, the observed reduction of motility occurred when Chlamydiae spp. in other host cells might start intracellular replication, energy parasitism and induction of other severe metabolic changes as a consequence of invasion [4,52].
Our data did, however, not show an invasion of the studied Chlamydiae spp. into the bovine spermatozoa, only adhesion of 0.3-0.4 μm particles considered as infectious elementary bodies was visualised by means of light and transmission electron microscopy. Adherence of chlamydial particles to the sperm surface has been described before for C. trachomatis following in vitro [53] and in vivo infection [33,35], with the latter authors also describing elementary and reticulate bodies within the sperm head [33,35] and tail [33] by means of transmission and scanning electron microscopy. Interestingly, the described changes resemble previously described genetic or fixative-related membrane changes and defects, crater-like changes in the acrosome and chromatin defects in the sperm head (for review see [54]). It remains to be clarified if the lack of invasion into spermatozoa is real in this Chlamydiae spp.sperm interaction or could have been related to the use of different Chlamydiae spp. (C. trachomatis versus C. abortus and C. psittaci) and hosts (human versus bovine), the duration of Chlamydiae-sperm interaction (2 h) or due to the fact that the experiments were based on in vitro co-incubation and not on natural infection.
Considering the attachment of Chlamydiae spp. to the sperm cells, seminal plasma proteins coating the spermatozoa deserve further attention as binding mediators. As about two thirds of the Chlamydiae particles were attached to the apical part of the sperm head, PDC-109 and osteopontin can be considered as strong candidates. Whereas PDC-109 modulates binding to the oviductal epithelium [55][56][57] and is bound predominantly to the midpiece, but also to the acrosome as well as the post-equatorial and equatorial segments [57], osteopontin, predominantly identified on the acrosome, is hypothesized to be involved in sperm-oocyte interaction, thereby affecting fertilization [57].

Conclusions
Data shows that viable C. abortus and psittaci (MOI = 1) attach to spermatozoa. Initially, spermatozoa with attached Chlamydiae are not hampered. However, taking binding rates into account, our data also suggests that Chlamydiae spp. reduce sperm motility after 9 h of co-incubation in vitro, and thus possibly lead to a reduced fecundity of bull semen. As about two thirds of the Chlamydiae particles were bound to the front third of the sperm, the acrosomal region, it is suggested that specific binding proteins, like e.g. PDC-109, are involved in Chlamydiae-spermatozoa interaction.

Funding
This study was founded by LOEWE-MIBIE (Initiative of the County Hesse for development of scientific-economic excellence, program: male infertility in case of infection and inflammation). The funding did not affect or influence the design of the study, the collection, analysis, and interpretation of data nor the writing of the manuscript.

Availability of data and materials
The data that support the findings of this study are available from the corresponding author upon reasonable request.
Authors' contributions AW, JK and SGP designed the study, TE did the experimental work, CH was responsible for the culture of Chlamydiae (partly together with TE) and critically discussed the results with SGP, MB did TEM analysis. KF performed all statistical analysis. SGP critically analyzed the data and wrote the initial draft of the manuscript. Manuscript revision was by AW, CH, MB and JK, finalized by SGP. All authors read and approved the final manuscript.

Ethics approval
This study was carried out in strict accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The housing facilities and the protocol were approved by the Regierungspräsidium Giessen, Germany (AZ No. V 54-19c2015h02 GI 18/14 Nr. A 27/2012).

Consent for publication
Not applicable.

Competing interests
The authors declare no conflict of interest. I confirm that I have read BioMed Central's guidance on competing interests and have included a statement indicating that none of the authors have any competing interests in the manuscript.

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