Article Figures & Data
Figures
- Figure 1.
Paternal stress experienced throughout puberty or in adulthood elicited stress dysregulation in offspring. A, B, Both male (A) and female (B) offspring of sires that had been exposed to chronic stress throughout puberty or only in adulthood produced less corticosterone relative to offspring of control sires following a 15 min restraint (shaded column). Total AUC of corticosterone production (shown in the inset) showed a significant decrease in both stress groups relative to controls. An expected sex difference in corticosterone production was observed, with females showing a greater response than males. Data are presented as mean ± SEM. n = 6–9 litters per group. *p < 0.05.
- Figure 2.
Paternal stress does not alter offspring performance in behavioral assessments or HPA response to an SSRI. A, B, Neither male (A) nor female (B) offspring of stressed sires showed altered PPI of the acoustic startle response compared to control offspring. C, PPI max startle was also unchanged by paternal stress in both sexes. D, No significant differences in time spent immobile on the tail suspension test were observed between control and paternally stressed male or female offspring. E, F, In the Barnes maze spatial learning and memory task, no differences were detected in the latency to learn the task. G, H, In the light–dark box, no treatment group differences were detected for time spent in the light or transitions between the compartments. There was an overall effect of females spending more time in the light compartment than males. I, J, Following administration of an SSRI, citalopram, at time 0 (black arrow), male and female offspring of stressed or control sires exhibited similar stress reactivity. Total AUC of corticosterone production (shown in the inset) did not vary with sex or paternal stress group. Data are presented as mean ± SEM. n = 5–9 litters per group. *p < 0.05.
- Figure 3.
Stress axis-related gene expression in offspring pituitary and adrenal glands was not significantly different between control (C), pubertal stress (P), or adult stress (A) groups. A–D, Real-time PCR analysis revealed no effect of paternal stress on pituitary CRFr1 (A), POMC (B), adrenal Mc2r (C), or 11βHSD-1 (D). Sex differences were observed, with males showing higher expression of CRFr1 and 11βHSD-1 and lower expression of Mc2r and POMC relative to females. Data are presented as mean ± SEM. n = 6–8 litters per group. **p < 0.001; ***p < 0.0001.
- Figure 4.
Paternal stress experienced throughout puberty or in adulthood produced robust changes in sperm miR content. A, Hierarchical clustering of sperm miR expression isolated a single clade containing all pubertal and adult stress males, and no controls (bolded red line). n = 4 per group. B, Analyses of significant differences in expression of specific miRs revealed nine that were significantly increased in both paternal stress groups. Data are presented as mean ± SEM. *p < 0.05, different from controls; #p < 0.05, different from pubertal stress.
Tables
- Table 1.
Summary of gene sets meeting GSEA enrichment criteria of FDR ≤ 0.25, p ≤ 0.01, and NES ≥ 1.6
Total PVN (enriched) BNST (enriched) c2 (curated) 305 0 (0.0%) 0 (0.0%) c3 (motif) 776 16 (2.1%) 114 (14.7%) c5 (gene ontology) 969 0 (0.0%) 6 (<1%) Gene set Type Size NES p FDR NR3CI (GR) c3 107 −1.683 0.008 0.235 CEBPdelta c3 212 −1.682 0.001 0.220 FOXD3 c3 178 −1.682 0.006 0.207 miR-522 c3 144 −1.678 0.008 0.203 NKX2–5 c3 109 −1.665 0.008 0.196 PAX8 c3 88 −1.661 0.004 0.194 TITF1 c3 212 −1.657 0.002 0.194 PIT1 c3 195 −1.649 0.010 0.201 DDIT3 c3 209 −1.641 0.004 0.200 NFI c3 226 −1.633 0.001 0.193 FOXI3 c3 165 −1.625 0.010 0.193 IPFI c3 220 −1.617 0.001 0.190 AR c3 227 −1.606 0.002 0.203 Members of c3 gene sets share a binding motif for the designated transcription factor or miR. Gene sets defined by conserved sequence only and those not meeting the criteria NES ≥ 1.6, p ≤ 0.01, and FDR ≤ 0.25 were omitted.
Table 3.Gene sets enriched in offspring BNST following paternal stress
Members of c3 gene sets share a binding motif for designated transcription factor or miRNA. c3 gene sets sharing the same name represent distinct binding sites for the transcription factor or miR given. Members of c5 gene sets share common ontolgy and do not necessarily represent coregulated genes. Gene sets defined by conserved sequence only and those not meeting the criteria NES ≥ 1.6, p ≤ 0.01, and FDR ≤ 0.25 were omitted. HAT, Histone acetyltransferase.
- Table 4.
Common predicted gene targets of the nine identified sperm miRNAs
Gray boxes indicate that the given gene was a predicted miR target in miRanda, miRDB, miRwalk, and TargetScan alorigthms. Asterisks denote a predicted function in chromatin regulation, DNA methylation, or miR processing.
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