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Male Fertility Laboratory, The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 52900, Israel
Correspondence
Tidhar Zan Bar
tidharz{at}gmail.com
Received February 6, 2008
Accepted May 20, 2008
Campylobacter fetus subsp. fetus infection can occur in female sheep, causing infertility or abortion. Despite extensive research on the effect of these bacteria on female fertility, little research has been done on the influence of C. fetus subsp. fetus on the male factor. Our objective was to examine the influence of C. fetus subsp. fetus on ram sperm. Motility index, percentage of live spermatozoa, mean 
t value (an indication of the chromatin stability of the sperm cell) and percentage of sperm cells expressing the FAS receptor were measured in sperm incubated in the presence or absence of C. fetus subsp. fetus. The motility index and viability of sperm incubated with the bacteria were lower than those of untreated sperm samples after 5 h. In bacteria-incubated sperm cells, the percentage expressing FAS receptor was already significantly elevated at 15 min. Bacteria-incubated sperm showed a greater prevalence of morphological damage. The bacteria were attached to tail and acrosome regions, and the sperm damage was concentrated in both the motility and chromatin regions. Bacteria-infected sperm cells showed a decrease in motility, increase in early acrosome reaction and chromatin damage. Similar effects were induced by incubation of the sperm with supernatants from C. fetus subsp. fetus cultures. Thus this study demonstrates that C. fetus subsp. fetus has a detrimental effect on the quality of ram sperm.
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INTRODUCTION |
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 TOP INTRODUCTION METHODSRESULTS AND DISCUSSIONReferences |
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In humans, C. fetus subsp. fetus has been reported to be an aetiologic agent in human abortions in general (Hood & Todd, 1960), and of septic abortion with intact fetal membranes in particular (Steinkraus & Wright, 1994). To the best of our knowledge, no association between infection by C. fetus subsp. fetus and male fertility potential has been reported. Therefore, the aim of this study was to determine the possible effect of infection by C. fetus subsp. fetus on sperm quality and fertility potential in rams.
We show here that incubation of ram sperm with C. fetus subsp. fetus or with bacterial supernatant from C. fetus subsp. fetus cultures causes defects in the sperm that were previously associated with decreased sperm quality and viability.
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METHODS |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONReferences |
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Sperm and bacteria preparation. Using a sterile artificial vagina, ram semen was collected from Assaf breed rams after male stimulation by a rutted female. The sample was then washed twice and diluted in Ringer's phosphate medium with glucose (pH 5).
Bacterial cultures of C. fetus subsp. fetus were a kind gift from Dr M. Bernstein of the Department of Bacteriology, Israeli Veterinary Institute in Beit Dagan, Israel. Three strains of isolated bacteria from aborted sheep fetus were obtained. Bacteria were maintained on blood agar plates under anaerobic conditions (Lancaster & Simon, 2002).
Before each experiment, the bacteria were transferred to grow on trypticase soy agar with 5% DSB plates (Novamed) for 2 days at 37 °C. After that, the bacteria were harvested and transferred to the medium (suspension).
Other bacteria, including Escherichia coli and Erwinia amylovora, were cultured in the same media.
Using spectrophotometry, we found that a 0.1 OD660 bacterial suspension equalled a bacterial concentration of 27x108 ml–1. To examine the influence of the bacteria on sperm quality, we incubated the 0.1 OD bacteria with 50x106 ml–1 sperm cells, which created a bacteria : sperm ratio of 54 : 1. This ratio was determined to be the lowest concentration of the bacteria that caused a sperm motility decrease after 5 h of incubation. Moreover, it is a similar infection ratio to that in Reichart et al. (2001). A similar concentration of the bacterium Ureaplasma urealyticum was used in a study by Reichart et al. (2000). After 2 days of bacterial growth, bacterial supernatant was produced by centrifugation and the bacterial suspension was passed through a 0.2 µm filter.
Sperm morphology analysis was performed using scanning electron microscopy as described previously (Evenson & Wixon, 2005).
Parameters of sperm viability. The parameters examined included motility, viability, morphology, sperm chromatin structure and FAS receptor expression, as these have been shown to predict sperm cell damage (Reichart et al., 2000). The motility parameter indicates a tail defect, while all the other parameters are indicative of a head defect.
The mean t value (an indication of the chromatin stability of the sperm cell) and %COMPt (percentage of cells outside the main peak) were assessed by testing 10 aliquots of fixed sperm nuclei by FACS analysis (Reichart et al., 2000).
The sperm were incubated in Ringer's glucose phosphate with 0.5 % glucose at room temperature (25 °C) for the duration of the experiment after dilution to 50x106 cells ml–1.
Sperm motility was determined using the sperm quality analyser (SQA; Gameta). The SQA has a photoelectric cell that detects fluctuations in the optical density of the suspension caused by the motility of sperm and converts them to a sperm motility index value (as described by Reichart et al., 2000).
FAS receptor expression. FAS receptor expression was determined by immunofluorescence. Rabbit polyclonal anti-FAS IgG [2 µl (ml sperm suspension)–1 (MACS antibody; Miltent)] was added to each sample, which was then incubated at room temperature for 1.5 h. The samples were then washed and secondary antibody [bovine anti-rabbit IgG FITC, 1 µl (ml sample)–1; Santa Cruz Biotechnology] was added for 30 min at 37 °C. The labelled cells represented damaged sperm. The cells were analysed by FACS at an excitation wavelength of 488 nm and emission wavelength of 535 nm (Xu et al., 2001).
Statistical analysis.
All statistical analyses were performed using SPSS for Windows, version 10.0. The sperm samples analysed in this study included spermatozoa incubated with bacteria and untreated sperm cells. Therefore, the type of treatment (bacteria/control) was defined as a between-sample factor. The sperm parameters motility index, percentage of live spermatozoa, mean t value and percentage of sperm cells with FAS receptor were measured at two time points: 15 min and 5 h after the start of the experiment. Fifteen minutes is the minimum time to determine whether the bacterial influence is immediate, and 5 h reflected a time between capacitation and acrosome reaction of the sperm, as in mammals it is known that the time before fertilization with the oocyte is 4–5 h (Takahashi et al., 1992). Therefore, the time of evaluation was defined as a within-sample factor. Time changes in the sperm parameters of the treated versus untreated samples were analysed using ANOVA with repeated measures. The between-sample changes in these parameters were analysed by ANOVA and the within-sample changes were analysed using a paired t-test. With regard to sperm morphology, one-way ANOVA was used since these variables were assessed only once, after 5 h of incubation.
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RESULTS AND DISCUSSION |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONReferences |
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t value, were examined in the control group of untreated sperm (C) and in samples incubated with C. fetus subsp. fetus bacteria (B) at 15 min and 5 h. After 5 h of incubation, these parameters in both the treated and control sperm were significantly lower compared to their mean values at 15 min (for example, sperm motility index was 524±21.4 vs 463±24.4, t=7.2, and 519±16.4 vs 333±48.1, t=13.9, respectively; P 
0.01, Table 1
). Comparison of control sperm and those incubated with the bacteria revealed that the two groups were statistically similar in their 15 min motility-index values; however, after 5 h of incubation, the bacteria-treated samples exhibited significantly lower values of this variable compared to controls (F=46.9, P 0.01; Table 1).
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0.01; Table 1
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Both samples exhibited similar sperm viability after 15 min; however, after 5 h of incubation, the sperm co-incubated with bacteria exhibited significantly lower viability compared to the control (F=8.0, P 0.02; Table 1).
No change over time was observed in the percentage of control sperm cells expressing the FAS receptor; however, in sperm incubated with the bacteria, the degree of FAS expression after 5 h of incubation was significantly higher compared with expression after only 15 min incubation (61.6±11.9 % vs 36.2±7.1 %, t=–8.1, P 0.01; Table 1).
It is notable that the percentage of bacterially treated sperm cells expressing FAS receptor was already significantly higher than in the controls after 15 min (36.2±7.1 % vs 16.8±4.8 %, F=30.7, P 0.01). A similar increase in FAS expression was observed after 5 h of incubation [61.6±11.9 % (+ bacteria) vs 21.0±5.3 % (control), F= 58.1, P 0.01; Table 1]. No significant changes within or between samples were observed with regard to the t values (Table 1).
Of the five sperm morphological defects examined after 5 h of incubation with or without bacteria, i.e. percentages of abnormal acrosome, disconnected heads, agglutinated heads, broken tails and twisted tails, four of these occurred at a significantly higher prevalence in the samples incubated with bacteria (abnormal acrosome: 23.4±8.8 % vs 14.3±5.8 %, t=–6.4; disconnected heads: 28.1±9.8 % vs 16.7±4.1 %, t=–4.2; agglutinated heads: 40.6±10.7 % vs 22.5±7.0 %, t=–7.0, P 0.01; and broken tails: 25.6±10.2 % vs 14.6±7.8, t=–3.0, P 0.03; Table 2). The percentage of twisted tails was not statistically different between groups (Table 2; Fig. 1).
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t values were examined in the control sperm samples and compared to those in sperm samples incubated with the supernatant of C. fetus subsp. fetus cultures. As above, in both treated and control groups, the motility index was significantly decreased after 5 h of incubation compared to the initial state (518.7±12.8 vs 442.6±31.3, t=8.6; and 367.3±45.3 vs 131.2±45.3, t=17.0, respectively, P 0.01; Table 3). Comparison of supernatant and control groups demonstrated that at both time points, 15 min and 5 h, the mean motility index in the treated sperm was significantly lower compared to that in the control (F=123.7 and F=383.3, respectively, P 0.01; Table 3). In both groups, the percentage of live spermatozoa was significantly decreased after 5 h of incubation compared to the 15 min time point (84.2±9.0 % vs 72.6±8.6 %, t=2.9, P 0.04; and 76.2±11.3 % vs 40.2±4.5 %, t=7.6, P 0.01, respectively; Table 3).
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0.01; Table 3).
As above, no significant changes over time were observed for FAS receptor expression in the control group; however, for sperm treated with bacterial supernatant, the mean value of this parameter after 5 h of incubation was significantly higher compared to that at 15 min (94.0±3.1 % vs 83.7±2.6 %, t=–12.8, P 0.01; Table 3). At both points tested (15 min and 5 h), the percentage of sperm cells expressing FAS receptor in supernatant-treated cultures was significantly higher compared with the control (83.7±2.6 % vs 14.0±2.4 %; and 94.0±3.1 % vs 17.7±6.3 %, respectively, F=2674.5 and F=779.0, respectively, P 0.01). No significant changes were observed for the mean t values.
After 1 h of incubation in bacterial supernatant, all five morphological defects studied occurred at a significantly higher prevalence in samples treated with bacterial supernatant relative to the control (Table 4) (t=–5.8, t=–6.2, t=–7.3, t=–4.3 and t=6.9, respectively, P 0.03; Table 4).
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0.01), viability (P 0.05) and FAS expression (P 0.05), no significant effect was induced by the other bacterial species (Table 5). This shows that C. fetus subsp. fetus has a unique effect on ram sperm quality.
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Bacterial infection may cause occlusion of the male tract or the accessory glands and thus lead to a decrease in the amount of sperm cells. It may even be related to a decrease in the quality of the sperm cells secreted into the semen, as expressed by a decrease in sperm cell motility and an increase in the number of sperm cells exhibiting abnormal morphology (Diemer et al., 1996).
Vizzier-Thaxton et al. (2006) reported that horizontal and vertical transmission of Salmonella and Campylobacter can occur in broiler breeder flocks. Although the mechanism of this transmission is still unclear, scanning electron microscopy showed that Salmonella was associated with all three segments (head, midpiece and tail) of the spermatozoa and was, apparently, equally distributed. Campylobacter was mainly associated with the midpiece and tail segments; few isolates were located on the head segment (Vizzier-Thaxton et al., 2006). In this study, we found that C. fetus subsp. fetus was attached to two main regions: the tail (close to the mitochondrial area) and the acrosome, especially the post-acrosome region (Fig. 1). The damage to sperm was manifested in characteristics associated with both the motility and chromatin regions. Bacteria-infected sperm cells showed a decrease in motility and an increase in premature acrosome reaction as well as chromatin damage. C. fetus subsp. fetus caused the sperm head to separate from the tail, a significant increase in the development of apoptosis, and acrosome damage (Fig. 1).
Sperm morphology is considered disturbed in the presence of more than 20 % abnormal sperm cells (Liu et al., 2004). Incubation (up to 5 h) of sperm cells with C. fetus subsp. fetus caused up to 30 % of cut-off heads, early acrosome reaction and agglutination. Recent reports show that the presence of bacteria in semen may lead to early abortions as a result of damage to sperm cells (Reichart et al., 2000). Thus the bacteria can cause damage to sperm that will only be manifested following fertilization. This could explain why the bacteria have been recognized more as a trigger of abortion than a reason for infertility (Agerholm et al., 2006).
The nature of the agent secreted by C. fetus subsp. fetus causing toxicity to sperm is not known. LPS has been suggested as mediating the toxic effects of the bacterium (Brooks et al., 2002). Kajihara et al. (2006) found that LPS-treated mice demonstrate an increase in FAS-positive cells in many germ cell populations, especially in spermatocytes and spermatids. Their study suggests that the Fas/FasL system mediates apoptosis of germ cells in the testis of LPS-treated mice (Kajihara et al., 2006). Nevertheless, it has been shown that different species of bacteria produce LPS with different structures and toxicity (Braun et al., 2005). The synthesis of toxic LPS is regulated by environmental and nutritional factors (such as sugar). C. fetus subsp. fetus demonstrates significantly greater toxicity than other bacteria such as Erwinia amylovora and E. coli. It is possible that this difference reflects the production by C. fetus subsp. fetus of higher levels of toxic LPS than other bacteria.
This study provides an important basis for understanding the mechanism of bacterial effects on the male and female genital tracts by showing that C. fetus subsp. fetus can be toxic to the sperm cells.
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References |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONReferences |
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