Transgenerational epigenetic programming via sperm microRNA recapitulates effects of paternal stress

Abstract

Epigenetic signatures in germ cells, capable of both responding to the parental environment and shaping offspring neurodevelopment, are uniquely positioned to mediate transgenerational outcomes. However, molecular mechanisms by which these marks may communicate experience-dependent information across generations are currently unknown. In our model of chronic paternal stress, we previously identified nine microRNAs (miRs) that were increased in the sperm of stressed sires and associated with reduced hypothalamic-pituitary-adrenal (HPA) stress axis reactivity in offspring. In the current study, we rigorously examine the hypothesis that these sperm miRs function postfertilization to alter offspring stress responsivity and, using zygote microinjection of the nine specific miRs, demonstrated a remarkable recapitulation of the offspring stress dysregulation phenotype. Further, we associated long-term reprogramming of the hypothalamic transcriptome with HPA axis dysfunction, noting a marked decreased in the expression of extracellular matrix and collagen gene sets that may reflect an underlying change in blood-brain barrier permeability. We conclude by investigating the developmental impact of sperm miRs in early zygotes with single-cell amplification technology, identifying the targeted degradation of stored maternal mRNA transcripts including sirtuin 1 and ubiquitin protein ligase E3a, two genes with established function in chromatin remodeling, and this potent regulatory function of miRs postfertilization likely initiates a cascade of molecular events that eventually alters stress reactivity. Overall, these findings demonstrate a clear mechanistic role for sperm miRs in the transgenerational transmission of paternal lifetime experiences.

Keywords: epigenetic; microRNA; paternal; stress; transgenerational.

Fig. 1.

Zygote microinjection of nine sperm miRs recapitulated the HPA axis phenotype. (A) Schematic of experimental design in which single-cell mouse zygotes were injected with nine miRs (multi-miR; 1 ng/μL final concentration of miR-29c, miR-30a, miR-30c, miR-32, miR-193-5p, miR-204, miR-375, miR-532-3p, and miR-698) or with one of two control conditions: an injection control (PBS only) or a control for the total amount of injected miRs (single miR; 1 ng/μL final concentration miR-193-5p alone) before implantation into surrogate females or preparation for later analyses. (B and D) Corticosterone levels in male and female adult mice were significantly reduced in the multi-miR group relative to PBS controls in response to a 15-min restraint from t = 0–15 min. (C and E) Total AUC of corticosterone production over 120 min was significantly decreased in the multi-miR group compared with PBS controls, and expected sex differences were observed. Data are presented as mean ± SEM n = 6–10 mice per group. *P < 0.05 multi-miR vs. PBS. (F) Model demonstrating the similarity in the shape of the male corticosterone response curve in the multi-miR–injected group with the outcome previously reported in offspring following paternal stress (11).

Fig. 2.

Altered transcriptome profile in adult PVN from multi-miR–injected zygotes supports a lasting epigenetic modification. (A–C) GSEA of RNA-seq data identified three significantly enriched C2 gene sets with normalized enrichment score (NES) > 1.8 and FDR < 0.05, illustrating a decrease in collagen formation and extracellular matrix genes in the multi-miR–injected group. (D) Differential expression analysis identified 298 (10 up, 288 down) genes significantly changed by at least 1.5 fold (log2 fold change ≥ |0.585|, dotted lines). Genes are presented in ranked order of expression from low to high. P < 0.05, FDR < 0.05. (E) Functional annotation clustering of dysregulated genes in the multi-miR group according to GO annotation (DAVID tools, ES = 1.3 equivalent to α = 0.05, gray bar indicates P < 0.05, and FDR < 0.05 for unique GO terms). (F) Significantly decreased expression of 17 collagen genes found in top extracellular matrix-enriched cluster. P < 0.05, FDR < 0.05. Data are shown relative to PBS-injected zygotes and presented as mean ± SEM, n = 6 biological replicates per group. ES, cluster enrichment score.

Fig. S1.

No change in PVN expression of the nine injected miRs or their mRNA targets. (A) qRT-PCR analysis of microRNA in the adult PVN in the multi-miR–injected group revealed no difference in the expression of miR-29c, miR-30a, miR-32, miR-193-5p, miR-204, miR-375, miR-532-3p, or miR-698. (B) PVN expression of the eight targeted maternal mRNA was similarly unchanged in the adult PVN. Expression in RPKM for Sirt1, Ube3a, Srsf2, IL6st, Ncl, Aara, Agfg1, and Ralbp1 was obtained by RNA-seq analysis. All data are presented as mean ± SEM, n = 6 mice per group. RPKM, reads per kilobase of transcript per million.

Fig. 3.

No differences in adult expression of stress-regulatory genes in the HPA axis. (A) Markers of major neuronal and nonneuronal cell populations in the PVN were assessed by qRT-PCR and were unchanged in the multi-miR–injected group: Avp, arginine vasopressin; Ot, oxytocin; Crf, corticotropin releasing factor; Cd11b, integrin alpha M; Gfap, glial fibrillary acidic protein. n = 6 mice per group. (B–E) Stress-axis related gene expression in the pituitary and adrenal glands was not significantly different between PBS and multi-miR–injected animals. No effect was observed for pituitary Crfr1, pituitary Pomc, adrenal Mc2r, or adrenal 11βhsd-1. Data presented as mean ± SEM, n = 6–9 mice per group. *P < 0.05.

Fig. 4.

Single-cell amplification studies confirmed sperm miR targeting of stored maternal mRNA. (A) Expression of many more maternal mRNA candidate genes in zygotes was reduced by multi-miR injection than by the single-miR injection. P < 0.005. (B–I) Expression of 8 of the 75 candidate genes—Sirt1, Ube3a, Srsf2, IL6st, Ncl, Aars, Agfg1, and Ralbp1—was significantly reduced by the multi-miR injection, with expression levels measuring log2 fold change > |1| (dotted lines) relative to PBS controls. Expression of these genes was unaffected in single-miR injection controls. Data are shown relative to PBS-injected zygotes and presented as mean ± SEM, n = 10–13 zygotes per group. *P < 0.05 multi-miR vs. PBS, P < 0.05 multi-miR vs. single miR.