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. 2017 Apr 25;7:1126. doi: 10.1038/s41598-017-01262-w

Figure 4.

Figure 4

Spermatozoa do not carry attached EBs of Chlamydia spp. (a) In vitro cultured confluent HeLa cells were inoculated with human spermatozoa in vitro pre-incubated with different concentration rates (1 × 105, 1 × 106 or 1 × 107 EBs/million sperm) of CT serovar E, immediately after incubation without removing bacteria (Pre-washes), or after five consecutive washes post-incubation (Post-washes) in order to remove free bacteria. Some cultured cells were directly inoculated with 1 × 106 EBs of CT serovar E/mL (positive control) or with supplemented BWW medium alone (negative control). After 48 h of culture, infection of cultures was assessed by the detection of chlamydial inclusion bodies by direct immunofluorescence using a FITC-labeled anti-cLPS monoclonal antibody. Representative microphotographs of 1 out of 4 independent experiments performed with essentially the same results. (b and c) C. muridarum detection (omp2 gene) by conventional (b) or real time PCR (qPCR) (c) in vaginal lavages (at 7 days post insemination) and female genital tract tissue samples (at 15 days post insemination) from female C57BL/6 mice that were intravaginally inseminated with murine sperm in vitro pre-incubated with C. muridarum in capacitating conditions and then washed 5 times to remove free bacteria. Four experimental groups were included: the sham infected group (n = 6) inseminated with 30 µl of a solution containing 1 × 106 sperm that were pre-incubated with vehicle alone (BWW buffer); the positive control group (n = 6) inseminated with 30 µl of a solution containing 1 × 106 sperm that were pre-incubated with 1 × 107 EBs of C. muridarum without subsequent washings; one group (n = 6) inseminated with 30 µl of a solution containing 1 × 106 sperm that were pre-incubated with 1 × 106 EBs of C. muridarum and then washed 5 times; and one group (n = 6) inseminated with 30 µl of a solution containing 1 × 106 sperm that were pre-incubated with 1 × 107 EBs of C. muridarum and then washed 5 times. The expression of the housekeeping gene eef2 was assayed. Representative data from 1 out of 3 independent experiments performed with essentially the same results. Data are shown as mean ± SD. Statistical analysis was performed using two-way ANOVA with Bonferroni post hoc test analysis. *p < 0.01.