Implication of sperm RNAs in transgenerational inheritance of the effects of early trauma in mice

Abstract

Small non-coding RNAs (sncRNAs) are potential vectors at the interface between genes and environment. We found that traumatic stress in early life altered mouse microRNA (miRNA) expression, and behavioral and metabolic responses in the progeny. Injection of sperm RNAs from traumatized males into fertilized wild-type oocytes reproduced the behavioral and metabolic alterations in the resulting offspring.

Figure 1. SncRNAs in adult sperm

Mapping of 15–44bp sequencing reads to a) the mouse reference genome, b) ribosomal RNAs, c) other non-coding RNAs and repeat regions and d) mitochondrial DNA, with multiple (black) or unique (grey) hits (n=16 mice, pooled in 4 samples). % total reads represents the proportion of reads with a given size mapping to the mouse genome or selected sequences over the total number of same-size reads. (e) Heatmap showing miRNAs (>100 reads) in control libraries which are altered by MSUS in adult sperm (n=3 (each pooled from 5 mice) in each group). The blue-to-yellow scale is the number of normalized reads of a given sample over the mean normalized reads of all control samples for each miRNA. Bioinformatic analyses were performed twice using two independent methods. Data are mean ± s.e.m.

Figure 2. Behavioral responses in MSUS males across generations, and in mice derived from RNA-injected oocytes

(a) Latency to first enter an open arm on an elevated plus maze in F1 (control, n=8; MSUS, n=18; t(24)=2.37) and F2 (control, n=30; MSUS, n=25; t(41.98)=3.74) mice. (b) Time spent in the bright compartment of the light dark box in F1 (control, n=16; MSUS, n=21; t(35)=−2.14) and F2 (control, n=33; MSUS, n=36; t(41.61)=−3) mice. (c) Time spent floating on the forced swim test in F1 (control n=14, MSUS n=16; t(28)=−2.34) and F2 (control n=19, MSUS n=20; t(37)=−2.36) mice. Results replicated in two independent experiments. (d) Insulin concentration in serum in F1 (control, n=5; MSUS, n=9; t(12)=0.28) and F2 (control, n=10; MSUS, n=10; t(18)=2.1) males. (e, f) Glucose level in blood in e) F2 MSUS (control, n=8; MSUS, n=7; F(1,13)=5.64) and f) Controls-RNAinj (n=8) and MSUS-RNAinj (n=8) (F(1,14)=9.72) males at baseline (time 0) and 15, 30 and 90 minutes after stress initiation. Data are mean ± s.e.m. *p<0,05, **p<0,01, ***p<0,001.

Figure 3. Molecular effects of MSUS in adult F1 and F2 mice

(a-f) RT-qPCR in (a) sperm of F1 control and MSUS adult males (miR-375-3p: control, n=10; MSUS, n=9; t(7.679)=−2.79. miR-375-5p: control, n=10; MSUS, n=10; t(18)=−3.19. miR-200b-3p: control, n=10; MSUS, n=10; t(11.17)=−2.46. miR-672-5p: control, n=10; MSUS, n=10; t(9.38)=−2.92. miR-466c-5p: control, n=10; MSUS, n=10 t(13.05)=−2.4), (b) serum of F1 control and MSUS adult males (miR-375-3p: control, n=8; MSUS, n=8; t(7.06)=−5.17. miR-375-5p: control, n=8; MSUS, n=8; t(7.01)=4.33. miR-200b-3p: control, n=8; MSUS, n=7; t(9.3)=0.90. miR-672-5p: control, n=8; MSUS, n=8; t(8.8)=2.24. miR-466c-5p: control, n=8; MSUS, n=7; t(7.90)=2.26), (c) hippocampus of F1 control and MSUS adult males (miR-375-3p: control, n=8; MSUS, n=6; t(12)=−2.34. miR-375-5p: control, n=8, MSUS, n=6; t(6.045)=0.59. miR-200b-3p: control, n=8; MSUS, n=6; t(5.8)=−1.1. miR-672-5p: control, n=8; MSUS, n=6; t(12)=−0.54. miR-466c-5p: control, n=8; MSUS, n=6; t(11)=−2.79), (d) serum of F2 control and MSUS adult males (miR-375-3p: control, n=6; MSUS, n=6; t(9)=0.93. miR-375-5p: control, n=5; MSUS, n=6; t(9)=0.93. miR-200b-3p: control, n=6; MSUS, n=6; t(10)=1.38. miR-672-5p: control, n=6; MSUS, n=6; t(5.29)=2.08. miR-466c-5p: control, n=6; MSUS, n=6; t(10)=2.21), (e) hippocampus of F2 control and MSUS adult males (miR-375-3p: control, n=7; MSUS, n=8; t(8.62)=−2.74. miR-375-5p: control, n=14; MSUS, n=15; t(17,89)=−2,14. miR-200b-3p: control, n=8; MSUS, n=8; t(14)=−1.47. miR-672-5p: control, n=7; MSUS, n=8; t(13)=−2.01). miR-466c-5p: control, n=7; MSUS, n=8; t(13)=−2.15), (f) sperm of F2 control and MSUS adult males (miR-375-3p: control, n=8; MSUS, n=8; t(14)=0.26; miR-375-5p: control, n=8; MSUS, n=8; t(14)=0.94; miR-200b-3p: control, n=4; MSUS, n=3; t(5)=0.44; miR-672-5p: control, n=4; MSUS, n=4; t(6)=−0.24; miR-466c-5p: control, n=4; MSUS, n=4; t(6)=−1.16). (g, h) Level of Ctnnb1 (g) mRNA (control n=7; MSUS n=7; t(12)=0.4) and (h) protein (control n=7; MSUS n=6; t(11)=3.26) in hippocampus of F2 control and MSUS males. Results replicated in an independent experiment using samples from a different batch of animals. Data are mean ± s.e.m. #p<0.1, *p≤0.05, **p<0.01, ***p≤0.001.