Paternal stress exposure alters sperm microRNA content and reprograms offspring HPA stress axis regulation

Abstract

Neuropsychiatric disease frequently presents with an underlying hyporeactivity or hyperreactivity of the HPA stress axis, suggesting an exceptional vulnerability of this circuitry to external perturbations. Parental lifetime exposures to environmental challenges are associated with increased offspring neuropsychiatric disease risk, and likely contribute to stress dysregulation. While maternal influences have been extensively examined, much less is known regarding the specific role of paternal factors. To investigate the potential mechanisms by which paternal stress may contribute to offspring hypothalamic-pituitary-adrenal (HPA) axis dysregulation, we exposed mice to 6 weeks of chronic stress before breeding. As epidemiological studies support variation in paternal germ cell susceptibility to reprogramming across the lifespan, male stress exposure occurred either throughout puberty or in adulthood. Remarkably, offspring of sires from both paternal stress groups displayed significantly reduced HPA stress axis responsivity. Gene set enrichment analyses in offspring stress regulating brain regions, the paraventricular nucleus (PVN) and the bed nucleus of stria terminalis, revealed global pattern changes in transcription suggestive of epigenetic reprogramming and consistent with altered offspring stress responsivity, including increased expression of glucocorticoid-responsive genes in the PVN. In examining potential epigenetic mechanisms of germ cell transmission, we found robust changes in sperm microRNA (miR) content, where nine specific miRs were significantly increased in both paternal stress groups. Overall, these results demonstrate that paternal experience across the lifespan can induce germ cell epigenetic reprogramming and impact offspring HPA stress axis regulation, and may therefore offer novel insight into factors influencing neuropsychiatric disease risk.

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.