Parental Stress Effects on Offspring

Effects of parental environments on offspring behavior and gene expression and DNA methylation.

We assess whether chronic stress is passed, transgenerationally, from parents to their offspring and, if so, how far stress is passed across subsequent generations. To test this, we exposed males and females to either a control environment or predatory cues for 30 days (Figure 1A), used a cross-breeding design to produce three distinct lineages with offspring from: control parents, stressed mothers, and stressed parents (Figure 1B). First, we evaluated the effects of parental stress on the stress behaviors of offspring across four generations. Second, we analyzed the effects of parental stress on the mRNA expression of two genes associated with the HPA axis (CRH and Nr3c1) in all four generations of offspring. Third, we analyzed the effects of parental stress on the DNA methylation of the promotor regions of CRH and Nr3c1 across all generations. Finally, we assessed multiple costs and a potential benefit of descending from stressed parents in the F1 and F4 generations. The outcome of these experiments allows us to assess whether exposure to stress is passed down from the mother only via pregnancy (i.e., effects would only be found through in utero exposure to the F1 and their germ-line (F2)), through changes in germline (i.e., effects would be found across all subsequent generations), or some combination of in utero effects during pregnancy and changes to the germline of the F0 parents themselves.

F1 and F2 offspring of stressed adults display more stressed behaviors in absence of stimulus.

At 10 days old, we recorded the stress behavior of offspring in groups of 3 individuals originating from the same brood in four consecutive generations. Offspring of stressed adults in the F1 generation showed more stressed behaviors than offspring of control parents (Figure 2A). When compared to the control group, offspring of both stressed mothers and stressed parents tended to travel less distance, move slower, took longer to enter and spent less time in the white zone, had less distance between individuals, spent more time immobile, and spent more time near the edge of the test arena. F2 generation offspring showed similar stress behaviors, with offspring of stressed parents exhibiting more stressed behaviors than offspring of control parents. Like their F1 parents, F2 offspring of stressed mothers and stressed parents display the same stressed behaviors of traveling less distance, move slower, taking longer to enter and spend less time in the white zone, and spent more time near the edge of the test arena when compared to offspring of control parents. However, offspring of stressed mothers tended to relax in their immobility behaviors (i.e., less frequently immobile and less time being immobile) when compared to the F1 generation. While offspring of stressed parents maintained their immobility behaviors but relaxed in their shoaling behavior (i.e., they had more distance between individuals) when compared to their F1 counterparts. We found no significant difference in stress behaviors across any of the treatments in generations F3 and F4 suggesting that there is no longer any transmission of stressed behaviors from the F0 generation.

F1 and F2 offspring of stressed adults have more CRH expression and less Nr3c1 expression.

We dissected brains from all offspring at 40 days old to analyze the mRNA expression of two genes associated with the Hypothalamus-Pituitary-Adrenal axis (HPA): Corticotropin-releasing hormone (CRH) and Glucocorticoid receptor (Nr3c1). In the F1 generation, the treatment an offspring’s parents were from had a significant effect on expression of the CRH gene, with offspring of stressed parents having significantly more gene expression compared to offspring of control parents (Figure 2B). The stress a parent experienced also influenced the expression of the Nr3c1 gene, with offspring of control parents having significantly more expression when compared to offspring of stressed mothers or stressed parents (Figure 2C). Similar results were found in the F2 generation, the stress treatment an offspring’s parents were from had a significant effect on CRH expression, with offspring from control parents having significantly less expression than offspring from stressed parents. There was also an effect of treatment on Nr3c1 expression, with offspring from control parents having significantly greater expression than offspring of stressed mothers and stressed parents. There was no significant effect of treatment on F3 offspring.

F1 and F2 offspring of stressed adults have less CRH methylation and more Nr3c1 methylation.

Using a candidate gene approach, we examined the methylation of the promotor regions of CRH and Nr3c1 with bisulfite sequencing. We found that treatment an offspring’s parents were exposed to had a significant effect on methylation of the CRH gene, with offspring of stressed parents tending to have less methylation in the promotor compared to offspring of control parents (Figure 2D). The stress a parent experienced also influenced the methylation of the Nr3c1 gene, with offspring of control parents having significantly less sites methylated when compared to offspring of stressed mothers or stressed parents (Figure 2E). Similar methylation patterns were found in the F2 generation, the stress treatment an offspring’s parents were from had significantly effect on CRH methylation, with offspring from control parents tended to have more methylated sites than offspring from stressed mothers or stressed parents. Nr3c1 expression was also influenced by the parental stress treatments, with offspring from control parents having significantly less methylated sites than offspring of stressed mothers and stressed parents. While there was no significant effect of treatment on the number of methylated sites on the CRH promoter in the F3 generation, there was a significant effect of treatment on the Nr3c1 promoter. This was mostly driven by offspring from stressed parents having more methylation than offspring from control parents and tended to have more methylation than offspring of stressed mothers. There was no significant effect of treatment on the number of methylated sites on either gene in F4 offspring .

F1 offspring of stressed parents survive more often than control offspring but at a cost, with these costs and benefit disappearing in the F4 generation.

To analyze potential costs and benefits we examined only the most distant generations (F1 and F4) and only the most distinct stress treatments (control parents and stressed parents). We measured three potential costs of having stressed parents: 1) the effects it may have on growth rates, and 2) the effects it may have on growth and development gene expression. When evaluating the effects of having stressed parents on growth rates, we found that there was a significant effect of period of measure on growth rate in the F1 generation, with the period from birth to 10 days old being the fastest period of growth compared to 10-20 days, 20-30 days, or 30-40 days. Ten to 20 days old was the next fastest growth period, with offspring growing faster between 10 and 20 days old compared to 20-30 days and 30-40 days. The stress treatment an offspring’s parents were exposed to also had a significant effect of growth rate, with offspring from control parents growing faster than offspring from stressed parents (Figure 3A). There was also a significant interaction between the growth period, the treatment the parents were exposed to, and the sex of the offspring, with females from stressed parents growing faster than males between 20-30 days. In the F4 generation still had the same significant effect of period of measure on growth rate, with birth to 10 days old being the fastest compared to 10-20 days, 20-30 days, and 30-40 days. However, the effect of the stress treatment a parent was from had no significant effect on growth rate. We also examined the potential cost of having stressed parents on two growth related genes, BDNF and IGF2. We found in the F1 generation that BDNF expression was significantly influenced by the treatment an offspring’s parents were from, with offspring from control parents having more gene expression than offspring from stressed parents (Figure 3B). Similarly, treatment had a significant effect on IGF2 expression, with offspring from control parents having higher expression than offspring from stressed parents (Figure 3C). In the F4 generation, we found no significant difference between treatments on gene expression of either BDNF or IGF2. Finally, we investigated a potential benefit of having stress parents by looking at an offspring’s survival rate when encountering a predator. We found that offspring from stressed parents in the F1 generation were almost twice more likely to survive a predator attack than offspring from control parents (Figure 3D). This advantage that offspring from stressed parents had was gone in the F4 generation.

This research was supported via a Postdoctoral fellowship from the Hector Fellow Academy and completed in collaboration with Dr. Axel Meyer and Dr. Thomas Elbert at the University of Konstanz.