Figure 3.

Assessment of the effect of direct PFAS exposure on reactive oxygen species generation in spermatozoa. Cauda epididymal spermatozoa were incubated in a PFAS mixture (low or high dose) or control solution for up to 3 h prior to being assessed for (A) mitochondrial ROS generation via labeling with MitoSOX Red (MSR), (B) cytosolic ROS via labeling with dihydroethidium (DHE) and (C) membrane fluidity using merocyanine 540 (M540), a lipophilic dye whose intercalation with membranes provides an indication of their relative fluidity. In all cases, the percentage of positively labeled live cells was determined using flow cytometry. The potential implications of PFAS-directed elevation in ROS generation were also monitored via assessment of 4HNE by immunoblotting (D) and subsequent densiometric assessment (E). Beyond lipid peroxidation, downstream impacts of any potential PFAS mediated elevation in ROS were assessed by determination of sperm DNA integrity utilizing sperm chromatin structure assays (SCSA) (F), and the alkaline comet assay (G) to assess DNA strand breaks. In all cases, graphical data represent the mean ± s.e.m. for each treatment group, calculated on n = 3 biological replicates. Data were subjected to two-way ANOVA with Tukey’s multiple comparison test. *P < 0.05.