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Dad's microRNA affects his baby's brain
Scientists observe more and more often that DNA is not the only way a parent passes on traits to its children. Now, University of Pennsylvania researchers have found that stress changes a mouse's sperm in a way that affects his own pups' response to stress.
This change is transmitted epigenetically, or by means other than the DNA code, through molecules called microRNAs, or miRs.
Work led by Tracy L. Bale, Professor of Neuroscience in the University of Pennsylvania, School of Veterinary Medicine and the Perelman School of Medicine, gives important clues to understanding how a father can affect his children's brain development and mental health through purely biological means.
"It's remarkable to me that seemingly mild stress in a male mouse would trigger this massive change in microRNA response and would get wired into the course of his offspring's development."
Tracy L. Bale PhD, Department of Biomedical Sciences, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA
Bale collaborated on the work with graduate students Ali B. Rogers and Christopher P. Morgan and research specialist N. Adrian Leu of the School of Veterinary Medicine, University of Pennsylvania. The paper appears in Proceedings of the National Academy of Sciences also known as PNAS.
In earlier research, Bale's lab had shown that male mice stressed prior to being bred, by changing cages or exposing them to the odor of a predator via fox urine, had offspring with a dampened response to stress. When they compared sperm from the stressed fathers to unstressed fathers, they found increased expression of nine miRs in the stressed mice.
Unlike some other types of RNA, miRs do not create protein. They silence or degrade specific messenger RNA to prevent it from being translated into proteins.
"Just showing that the [miR] levels were different doesn't make it relevant or interesting," Bale said. "We wanted to find out whether they were playing a causal role."
To find out, the team microinjected the nine miRs into mouse zygotes — a zygote is the end result of the fusion of an egg and sperm and marks the end of fertilization. Zygotes were then implanted into normal female mice who carried them as surrogate mothers. A control group of zygotes received either a fake injection or an injection of only a single miR and not the full 9. When those offspring became adults, their response to stress was measured using the same methods as in the 2013 study.
Subjected to a mild stress, being restrained briefly, the offspring from the zygotes receiving 9 miR injections had lower cortisone levels compared to offspring in the control group.
The mice in the 9 miR injection group also had significant changes in hundreds of genes in their paraventricular nucleus — a tiny brain region involved in stress regulation and located just above the hypothalamus. These changes suggest wide-spread changes in early neurodevelopment.
Finally, researchers wanted to determine how miRs carry out their effects after fertilization. Because miRs are known to target and degrade mRNA, the team examined maternal mRNA found in the zygote. Maternal mRNA exists only to about the four-cell stage of development, specifically to direct the zygote.
Bale: "People used to think that because stored maternal mRNA gets translated during initial two-cell and four-cell development, the mom gets priority in those early stages and the dad gets none. But we thought "perhaps these sperm miRs might be attacking that maternal mRNA and directing which mRNAs get translated."
Researchers again injected miRs into zygotes and performed control injections, but this time they incubated the zygotes for eight hours and then increased the RNA in each single cell to look for level of gene expression. They found that the multi-miR injection appeared to be attacking maternal mRNA, reducing mRNA levels when compared to control injections. Specifically affected were genes involved in chromatin remodeling. [Chromatin is made up of macromolecules consisting of DNA, protein and RNA. The primary functions of chromatin are: 1) to package DNA into a smaller volume to fit within a cell, 2) to reinforce the DNA macromolecule to allow mitosis, 3) to prevent DNA damage, and 4) to control gene expression and DNA replication. Source: Wikipedia]
Bale suspects that stress may trigger the release of miRs from the epithelial lining of the epididymis — located between the testes and the vas deferens and where sperm mature and are stored.
Up next, the group is examining what factors could lead to exosome and miR release — and then whether any intervention might prevent them from passing on an abnormal stress to the next generation. Interventions such as providing a stressed male with enrichment or reward.
They also hope to study the role of miRs in humans to figure out whether, as found in mice, some miRs may vary in response to stress.
Significance
Studies examining paternal exposure to diverse environmental stimuli propose that epigenetic marks in germ cells, including small noncoding RNAs such as microRNA (miR), transmit experience-dependent information from parent to offspring. However, these nongenetic mechanisms of transgenerational inheritance are poorly understood, specifically how these germ-cell marks may act postfertilization to enact long-term changes in offspring behavior or physiology. In this study, through zygote microinjection of nine specific sperm miRs previously identified in our paternal stress mouse model, we demonstrate that sperm miRs function to reduce maternal mRNA stores in early zygotes, ultimately reprogramming gene expression in the offspring hypothalamus and recapitulating the offspring stress dysregulation phenotype.
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.
The authors declare no conflict of interest.
The work was supported by the National Institute of Mental Health.
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