Fig. 2: Stress recovery alters miRNA and protein content of epididymal epithelial cell-secreted extracellular vesicles (EVs).

a Time course of corticosterone (Cort) treatment (3 days) and recovery (1, 4, or 8 days) in DC2 mouse caput epididymal epithelial cells, using physiologically-relevant concentrations for baseline (50 ng/mL) and stress (500 ng/mL) levels of corticosterone. Gray triangles indicate time points for EV collection for transcriptomic, proteomic, and size assessment. b Rank–rank hypergeometric overlap analysis was used on total sequenced miRNA. The differential expression profiles of sperm 12 weeks post-stress were plotted against the differential expression profiles of EVs collected 1, 4, or 8 days post-corticosterone treatment (left, middle, and right, respectively). Overlap data are plotted as sperm miRNA ratios increasing down the y-axis and EV miRNA ratios increasing left along the x-axis, with each pixel representing the −log₁₀(nominal p-value) of overlapping miRNA via the hypergeometric distribution and color coding according to degree of significance (as shown). Each RRHO heatmap is divided into four quadrants, where the bottom-left and upper-right squares represent concordant miRNA changes in both models, quantified below each heatmap (N = 3–4 EVs/treatment/time, max −log₁₀(p-value) = 5). c RNA-sequencing examination of all detectable miRNA from secreted DC2 EVs 8 days following stress levels of corticosterone treatment as indicators of changes in EV programming compared with vehicle treatment (N = 3–4 EVs/treatment). d Proteomic mass spectrometry comparison of secreted DC2 EV protein content analyzed by orthogonal partial least squares analysis show the impact of corticosterone programming, accounting for 26.2% of EV protein variation at 1 day after (left) and progressed to 51.1% at 8 days after treatment (right, N = 5–6 EVs/treatment/time). e Heatmap of total identified EV proteins by mass spectrometry 8 days post-corticosterone treatment compared with vehicle with hierarchical clustering of samples (N = 6 EVs/treatment). f Nanosight particle tracking identified a significant reduction in size distribution of the total population from secreted EVs 8 days post-corticosterone treatment compared with vehicle, plotted as average values across replicates (N = 4 EVs/treatment). g Quantification of Nanosight plots showing the mean size of EVs secreted by DC2 cells was significantly reduced 8 days following corticosterone treatment compared with vehicle (two-tailed unpaired Student’s t-test, t(6) = 8.68, ***p = 0.0001, N = 4 EVs/treatment). Error bars are SEM.