Figure 1.
Dose-response effect of E. coli on sperm motility and viability.
Motile spermatozoa were exposed to E. coli for 3 h in a Transwell system, avoiding direct contact between sperm suspension and microorganisms. Mean ± SD of 3 experiments. Overall significance for treatment variations: p<0.001 for both motility and viability; *p<0.05 vs. all the others.
Figure 2.
Sperm motility inhibition induced by E. coli soluble products and its prevention by lactobacilli.
Spermatozoa were exposed for 3 h to E. coli (1.5×106 CFU/mL) in a Transwell system with and without a 30 min-preincubation with a mix of L. brevis CD2, L. salivarius FV2, and L. plantarum FV9 (1×108 CFU/mL), before evaluating sperm motility by Computer-Aided Semen Analysis (CASA). No preventive effects were exerted by gamma ray-inactivated lactobacilli. Top, effects on the percentage of spermatozoa with average pathway velocity (VAP) >5 μm/sec; Bottom, effects on sperm motility quality; VCL, curvilinear velocity; VSL, straight line velocity. Mean ± SD of 6 experiments. Overall significance for treatment variation: p<0.0001; *p<0.05 vs. “untreated”, “Lactobacilli + E. coli” and “Lactobacilli”.
Figure 3.
E. coli and lactobacilli effects on sperm mitochondrial function.
Effect of 3 h exposure in the Transwell system to E. coli (1.5×106 CFU/mL) on (A) sperm mitochondrial membrane potential (ΔΨm), evaluated with JC-1 and (B) mitochondrial generation of reactive oxygen species (ROS), evaluated with MitoSOX red (MSR). Mitochondrial effects of E. coli were not prevented by the mix of lactobacilli (1×108 CFU/mL). Top, typical flow cytometric dot plot (A) and histogram (B) of fluorescence; Bottom, percentages of spermatozoa with JC-1 and MSR fluorescence. Mean ± SD of 6 experiments. Overall significance for treatment variation: p<0.00001; *p<0.05 vs. untreated.
Figure 4.
Correlations between E. coli effects on spermatozoa.
After 3 h exposure to E. coli (1.5×106 CFU/mL) in the Transwell system, the increase (%) in sperm mitochondrial generation of reactive oxygen species (ROS) and membrane lipid peroxidation as well as the decrease (%) in mitochondrial membrane potential (ΔΨm) and sperm motility were evaluated with respect to the untreated samples. The percentage of increment/decrement and not crude values were analyzed in order to overcome masking effects due to the inter-donors variability.
Figure 5.
Sperm lipid peroxidation induced by E. coli soluble products and its prevention by lactobacilli.
Spermatozoa were exposed for 3 h to E. coli (1.5×106 CFU/mL) in a Transwell system with and without a 30 min-preincubation with a mix of L. brevis CD2, L. salivarius FV2, and L. plantarum FV9 (1×108 CFU/mL), before evaluating sperm membrane lipid peroxidation with BODIPY C11 at flow cytometer. No preventive effects were exerted by gamma ray-inactivated lactobacilli. Top, typical double fluorescence dot plots of flow cytometry BODIPY C11 analysis. Bottom, percentages of spermatozoa with green BODIPY C11 fluorescence (indicating membrane lipid peroxidation). Mean ± SD of 6 experiments. Overall significance for treatment variation: p=0.00004; *p<0.05 vs. “untreated” and “Lactobacilli + E. coli”.