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. 2020 Mar 20;11(1):1499.
doi: 10.1038/s41467-020-15305-w.

Reproductive Tract Extracellular Vesicles Are Sufficient to Transmit Intergenerational Stress and Program Neurodevelopment

Free PMC article

Reproductive Tract Extracellular Vesicles Are Sufficient to Transmit Intergenerational Stress and Program Neurodevelopment

Jennifer C Chan et al. Nat Commun. .
Free PMC article


Extracellular vesicles (EVs) are a unique mode of intercellular communication capable of incredible specificity in transmitting signals involved in cellular function, including germ cell maturation. Spermatogenesis occurs in the testes, behind a protective barrier to ensure safeguarding of germline DNA from environmental insults. Following DNA compaction, further sperm maturation occurs in the epididymis. Here, we report reproductive tract EVs transmit information regarding stress in the paternal environment to sperm, potentially altering fetal development. Using intracytoplasmic sperm injection, we found that sperm incubated with EVs collected from stress-treated epididymal epithelial cells produced offspring with altered neurodevelopment and adult stress reactivity. Proteomic and transcriptomic assessment of these EVs showed dramatic changes in protein and miRNA content long after stress treatment had ended, supporting a lasting programmatic change in response to chronic stress. Thus, EVs as a normal process in sperm maturation, can also perform roles in intergenerational transmission of paternal environmental experience.

Conflict of interest statement

The authors declare no competing interest.


Fig. 1
Fig. 1. Stress dynamics impact intergenerational transmission and sperm miRNA content.
a F0 breeding time course with periods of stress (4 weeks) and recovery (1 or 12 weeks after stress) for sperm and F1 offspring assessment. b Transmission of a stress phenotype in the corticosterone response to an acute 15-min restraint (gray bar) did not occur for offspring conceived at 9 weeks (two-way rmANOVA, time (F(3,39) = 88.07, p = 2.0375 × 10−17), paternal treatment (F(1,13) = 0.5528, NS p = 0.4704, N = 7–8 offspring/paternal treatment). c Offspring conceived at 20 weeks had altered stress reactivity (two-way rmANOVA, interaction of paternal treatment × time (F(3,42) = 3.718, p = 0.0185), time (F(3,42) = 138.5, p = 8.3427 × 10−22), paternal treatment (F(1,14) = 1.179, p = 0.2959)), with significant reduction in the maximal rise of corticosterone at 30 min (Bonferroni’s post hoc test, t(56) = 3.29, **adjusted p = 0.0069, N = 8 offspring/paternal treatment). d Doubling F0 stress (severe stress) did not transmit a stress phenotype for offspring conceived at 9 weeks (two-way rmANOVA, time (F(3,39)=92.52, p = 8.8203 × 10−18; paternal treatment F(1,13)=0.0667, NS p = 0.8002, N = 7–9 offspring/paternal treatment). e Severe stress transmission altered stress reactivity for offspring conceived at 20 weeks (two-way rmANOVA, interaction of paternal treatment × time (F(3,30) = 2.600, p = 0.0705), time (F(3,30) = 173.5, p = 4.4839 × 10−19; paternal treatment (F(1,10) = 8.199, p = 0.0169), with significant reduction in the maximal rise of corticosterone at 30 min (Bonferroni’s post hoc test, t(40) = 3.549, **adjusted p = 0.0040, N = 6 offspring/paternal treatment). Error bars represent mean ± SEM. f Expression patterns for all sperm miRNA detected by RNA sequencing as indicators of germ cell reprogramming, correlating with timing of phenotypic transmission. miRNA values were averaged within treatment groups (N = 6–8 samples/paternal treatment/time post-stress). g Examination of sperm miRNA dynamics and perceived stress in a human cohort. Principal component (PC) analysis of sperm based on all sequenced miRNA demonstrated the influence of perceived stress dynamics on sperm expression patterns, where PC2 largely segregated samples collected from ‘stable-stress’ (blue circles) and ‘recovering-stress’ subjects (red circles). N = 5–6 sperm samples/subject/stress phenotype group (see Methods). h Quantification showing significant association between a subject’s sample PC2 score and its stress phenotype group (regression model β-coefficient = −8.38, **p = 0.0112). Data are median with first and third quartiles (box) and top and bottom quartiles (whiskers) indicated. Individual data points are colored to denote subject. i Hypothesized model of maturing sperm interacting with extracellular vesicles within the caput epididymal lumen.
Fig. 2
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.
Fig. 3
Fig. 3. Intracytoplasmic sperm injection (ICSI) of sperm incubated with extracellular vesicles (EVs) secreted following stress recovery alter offspring neurodevelopment.
a Schematic representation of ICSI paradigm using sperm incubated with vehicle (EVVeh) or corticosterone-treated (EVCort) EVs, followed by microinjection into super-ovulated oocytes obtained from the same female donors. Zygotes from both EV-treatment groups were transferred into the designated right or left side of the same naive foster females to assess offspring neurodevelopment at mid-gestation (E12.5, top), or into separate recipient females for assessment of an F1 stress phenotype in adulthood (bottom). b, c Gene set enrichment analysis (GSEA) was used to analyze RNA-sequencing of E12.5 brains, with significant enrichment of gene sets related to (b) synaptic signaling and (c) neurotransmitter transport in embryos from EVCort sperm compared with EVVeh sperm (N = 6 embryos/EV treatment, GSEA normalized enrichment score (NES) > |1.8|, FDR < 0.05). d The top three significant clusters of gene ontology (GO) terms enriched in EVCort E12.5 brains determined by GSEA and clustered under parent terms were related to synaptic signaling (N = 6 embryos/treatment, NES > |1.8| and FDR < 0.05). e EVCort adult F1 offspring showed the same phenotypic pattern of stress reactivity to an acute 15-min restraint (gray bar) as paternal stress F1 offspring, compared with EVVeh F1 offspring (two-way rmANOVA, interaction of EV treatment × time F(3,21) = 8.480, p = 0.0007, time (F(3,21) = 122.6, p = 1.8125−13), EV treatment (F(1,7) = 1.868, p = 0.2139, N = 4–5 offspring/EV treatment), with a significant reduction in the maximal rise of corticosterone at 30-min (Bonferroni’s post hoc test, t(28) = 2.733, *adjusted p = 0.043). Error bars represent mean ± SEM.

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