Early prenatal stress epigenetically programs dysmasculinization in second-generation offspring via the paternal lineage

Abstract

Studies have linked sex-biased neurodevelopmental disorders, including autism and schizophrenia, with fetal antecedents such as prenatal stress. Further, these outcomes can persist into subsequent generations, raising the possibility that aspects of heritability in these diseases involve epigenetic mechanisms. Utilizing a mouse model in which we previously identified a period in early gestation when stress results in dysmasculinized and stress-sensitive male offspring, we have examined programming effects in second-generation offspring of prenatally stressed (F2-S) or control (F2-C) sires. Examination of gene expression patterns during the perinatal sensitive period, when organizational gonadal hormones establish the sexually dimorphic brain, confirmed dysmasculinization in F2-S males, where genes important in neurodevelopment showed a female-like pattern. Analyses of the epigenomic miRNA environment detected significant reductions in miR-322, miR-574, and miR-873 in the F2-S male brain, levels that were again more similar to those of control females. Increased expression of a common gene target for these three miRNAs, β-glycan, was confirmed in these males. These developmental effects were associated with the transmission of a stress-sensitive phenotype and shortened anogenital distance in adult F2-S males. As confirmation that the miRNA environment is responsive to organizational testosterone, neonatal males administered the aromatase inhibitor formestane exhibited dramatic changes in brain miRNA patterns, suggesting that miRNAs may serve a previously unappreciated role in organizing the sexually dimorphic brain. Overall, these data support the existence of a sensitive period of early gestation when epigenetic programming of the male germline can occur, permitting transmission of specific phenotypes into subsequent generations.

Figure 1.

Second-generation males from the paternal stress lineage (F2-S) show dysmasculinized brain gene expression and miRNA expression patterns on PN1. A, Heat map illustration of custom Taqman qRT-PCR Array results demonstrating a broad shift in gene expression in the PN1 brain of F2-S male mice from a male-typical (F2-C♂) to a more female-typical (F2-C♀) pattern. B, Statistical analyses for sex differences detected 17 genes in the PN1 brain from our custom Taqman Array. In F2-S male PN1 brains, 13 of these 17 genes displayed expression levels closer to F2-C females than to F2-C male levels. C, As F2-S males show a reduced organizational masculinization, we examined gene expression for central estrogen programming targets: aromatase, and estrogen receptor α (ERα) and β (ERβ). ERβ was significantly increased in the F2-S male PN1 brain compared to F2-C male. D, Examination of the miRNA environment in F2 PN1 brain was examined using a miRNA Array. miR-322, miR-574–3p, and miR-873 expression were dysmasculinized in F2-S male mice. A single predicted shared gene target of these three miRs, β-glycan (TGFβr3), was identified by the database miRDB.org and examined in F2 PN1 brain. Where we found a reduction in miR expression in F2-S male mice, we detected an expected increase in expression of β-glycan. All data are mean per group ± SEM, n = 3–5 litters/group, *p < 0.05.

Figure 2.

Analyses of physiological and behavioral measures in adult second-generation males from the paternal stress lineage (F2-S♂) show a similar dysmasculinized physiology and stress-sensitive phenotype as their sires. As further evidence of dysmasculinization programmed during the perinatal period by testosterone, adult F2-S males showed a significant reduction in both anogenital distance lengths (A) and testis weights (B) compared to control males (F2-C♂). Behaviorally, while not all aspects of the first-generation stress-sensitive phenotype were transmitted along the paternal lineage to second-generation (F2) male offspring, we did detect increased immobility in a tail suspension test in F2-S male mice (C). Similar to first-generation findings, no further increase in immobility was observed in F2-S female offspring (F2-S♀) compared to control females (F2-C♀). E, While there was no statistically significant effect of F2-S in latency to locate the target in the Barnes maze spatial learning task, the direction of effect in F2-S males was similar to that previously reported for first-generation offspring. F, No differences were detected in latencies in F2 control and stress females. No main effects of F2-S in either male (G) or female (H) offspring were detected for corticosterone levels in response to an acute restraint stress. All data are mean per group ± SEM, n = 6–9 litters/group, *p < 0.05.

Figure 3.

Aromatase inhibition dramatically dysmasculinizes the neonatal brain miRNA environment during the perinatal sensitive period. Administration of a single injection of the aromatase inhibitor, formestane (20 μg), on PN1 produced a profound effect on the brain miRNA environment as analyzed by miRNA Taqman qRT-PCR Array. Pearson Correlational Hierarchical Clustering analysis of miRNA expression patterns was unable to distinguish between control female and formestane-treated males, while completely segregating control male samples from these groups, supporting a novel role of miRNA in organizing the sexually dimorphic brain. All data are mean per group ± SEM, n = 7–8 litters/group.