Figure 3.

CT does not attach to spermatozoa in vitro. Fractions of highly motile human sperm samples containing 1 × 10⁶ sperm/ml were in vitro incubated with 3% BSA supplemented BWW medium alone or with 1 × 10⁵, 1 × 10⁶ and 1 × 10⁷ EBs/mL of GFP-CT (green fluorescent) during 6 h at 37 °C. Sperm suspensions were then subjected to five consecutive washes and centrifuged at 300 × g for 5 min to remove free bacteria (Post-washes). Sperm suspensions were smeared onto glass slides, stained with DAPI (stain for DNA) and counterstained with 0.01% Evans blue and analyzed by confocal microscopy. Negative control corresponds to fractions of sperm samples incubated with supplemented BWW medium alone [Negative control (Vehicle), Pre-washes]. Positive control corresponds to fractions of sperm samples incubated with 1 × 10⁷ EBs of GFP-CT without subsequent washings [Positive control (1 × 10⁷ EBs), Pre-washes]. Green fluorescent EBs of GFP-CT are observed as green/blue co-localizing dots (marked with arrows). Results shown are from one representative experiment out of three performed (using fractions of n = 10 different sperm samples) with essentially the same results. Images were captured using an Olympus FV1200 laser scanning confocal microscope with an objective PLAPON 60X (1.42 NA). Fluorophore signals were acquired in sequential mode.