SummaryGene-environment interactions are determining factors for the etiology of psychiatric disorders, diabetes and cancer, and are thought to contribute to disease inheritance across generations. Small non-coding RNAs (sncRNAs) are potential vectors at the interface between genes and environment. Here, we report that environmental conditions involving traumatic stress in early life in mice altered microRNAs (miRNAs) expression, and behavioral and metabolic responses in the progeny. Several miRNAs were affected in the serum and brain of both, the traumatized animals and their progeny when adult, but also in the sperm of traumatized males. Injection of sperm RNAs from these males into fertilized wild-type oocytes reproduced the behavioral and metabolic alterations in the resulting offspring. These results strongly suggest that sncRNAs are sensitive to environmental factors in early life, and contribute to the inheritance of trauma-induced phenotypes across generations. They may offer potential diagnostic markers for associated pathologies in humans.While the genetic make-up of an individual contributes to disease risk and heritability 1 , environmental factors, in particular, adverse and traumatic experiences in early life are also critical. How they mediate their influence is poorly understood but likely involves nongenetic mechanisms. Small non-coding RNAs (sncRNAs) are potential mediators of geneenvironment interactions that can relay signals from the environment to the genome and exert regulatory functions on gene activity 2 . They are implicated in gene dysregulation in many diseases including psychiatric and neurological conditions, cancer and metabolic disorders 2-4 . Recent studies in C. elegans 5,6 and mice 7,8 have suggested that sncRNAs can * Corresponding author: mansuy@hifo.uzh.ch. $ Current address: Neuroscience Center, University Geneva, Switzerland Authors' contribution K.G. did all RT-qPCRs, behavioral tests, metabolic measurements, sperm RNA preparation for sequencing libraries and for RNA injection into fertilized oocytes and part of the sequencing analyses. A.J. performed Western blots and cell culture experiments and assisted with metabolic measurements. J.B. carried out the MSUS procedures and produced MSUS animals. J.P. and P.S. did most RNA sequencing analyses. P.P. did the RNA injection experiments. E.M. and L.F. helped design RNA sequencing analysis. K.G. and I.M.M. designed the study, interpreted the results and wrote the manuscript.

Behavioural traits in mammals are influenced by environmental factors, which can interact with the genome and modulate its activity by complex molecular interplay. Environmental experiences can modify social, emotional and cognitive behaviours during an individual's lifetime, and result in acquired behavioural traits that can be transmitted to subsequent generations. This Review discusses the concept of, and experimental support for, non-genetic transgenerational inheritance of acquired traits involving the germ line in mammals. Possible mechanisms of induction and maintenance during development and adulthood are considered along with an interpretation of recent findings showing the involvement of epigenetic modifications and non-coding RNAs in male germ cells.

BackgroundThe concept that individual traits can be acquired and transmitted by the germline through epigenetic mechanisms has gained recognition in the past years. However, epigenetic marks in sperm have not been are not well identified.ResultsUsing a novel proteomic approach that combines peptide-based bottom-up and intact protein top-down tandem mass spectrometry, we report the identification of epigenetic marks on histones and protamines in adult mouse sperm. We identified a total of 26 post-translational modifications (PTMs) on specific residues of the core histones H2B, H3 and H4, and the linker histone H1, four of which had not been described previously in any tissue or cell line. We also detected 11 novel PTMs on the protamines PRM1 and PRM2 and observed that they are present in specific combinations on individual protamines.ConclusionsBoth histones and protamines carry multiple PTMs in the adult mouse sperm. On protamines, specific PTM combinations might form a ‘protamine code’ similar to the ‘histone code’. These findings suggest a potential role for PTMs on sperm histones and protamines in epigenetic signatures underlying transgenerational inheritance.

Traumatic experiences in childhood can alter behavioural responses and increase the risk for psychopathologies across life, not only in the exposed individuals but also in their progeny. In some conditions, such experiences can however be beneficial and facilitate the appraisal of adverse environments later in life. Here we expose newborn mice to unpredictable maternal separation combined with unpredictable maternal stress (MSUS) for 2 weeks and assess the impact on behaviour in the offspring when adult. We show that MSUS in male mice favours goal-directed behaviours and behavioural flexibility in the adult offspring. This effect is accompanied by epigenetic changes involving histone post-translational modifications at the mineralocorticoid receptor (MR) gene and decreased MR expression in the hippocampus. Mimicking these changes pharmacologically in vivo reproduces the behavioural phenotype. These findings highlight the beneficial impact that early adverse experiences can have in adulthood, and the implication of epigenetic modes of gene regulation.

Prenatal exposure to infectious or inflammatory insults is increasingly recognized to contribute to the etiology of psychiatric disorders with neurodevelopmental components, including schizophrenia, autism and bipolar disorder. It remains unknown, however, if such immune-mediated brain anomalies can be transmitted to subsequent generations. Using an established mouse model of prenatal immune activation by the viral mimetic poly(I:C), we show that reduced sociability and increased cued fear expression are similarly present in the first- and second-generation offspring of immune-challenged ancestors. We further demonstrate that sensorimotor gating impairments are confined to the direct descendants of infected mothers, whereas increased behavioral despair emerges as a novel phenotype in the second generation. These transgenerational effects are mediated via the paternal lineage and are stable until the third generation, demonstrating transgenerational non-genetic inheritance of pathological traits following in-utero immune activation. Next-generation sequencing further demonstrated unique and overlapping genome-wide transcriptional changes in first- and second-generation offspring of immune-challenged ancestors. These transcriptional effects mirror the transgenerational effects on behavior, showing that prenatal immune activation leads to a transgenerational transmission (presence of similar phenotypes across generations) and modification (presence of distinct phenotypes across generations) of pathological traits. Together, our study demonstrates for, we believe, the first time that prenatal immune activation can negatively affect brain and behavioral functions in multiple generations. These findings thus highlight a novel pathological aspect of this early-life adversity in shaping disease risk across generations.

Adverse experiences in early life are risk factors for the development of behavioral and physiological symptoms that can lead to psychiatric and cognitive disorders later in life. Some of these symptoms can be transmitted to the offspring, in some cases by non-genomic mechanisms involving germ cells. Using a mouse model of unpredictable maternal separation and maternal stress, we show that postnatal trauma alters coping behaviors in adverse conditions in exposed males when adult and in their adult male progeny. The behavioral changes are accompanied by increased glucocorticoid receptor (GR) expression and decreased DNA methylation of the GR promoter in the hippocampus. DNA methylation is also decreased in sperm cells of exposed males when adult. Transgenerational transmission of behavioral symptoms is prevented by paternal environmental enrichment, an effect associated with the reversal of alterations in GR gene expression and DNA methylation in the hippocampus of the male offspring. These findings highlight the influence of both negative and positive environmental factors on behavior across generations and the plasticity of the epigenome across life.

In the past decades, evidence supporting the transmission of acquired traits across generations has reshaped the field of genetics and the understanding of disease susceptibility. In humans, pioneer studies showed that exposure to famine, endocrine disruptors or trauma can affect descendants, and has led to a paradigm shift in thinking about heredity. Studies in humans have however been limited by the low number of successive generations, the different conditions that can be examined, and the lack of mechanistic insight they can provide. Animal models have been instrumental to circumvent these limitations and allowed studies on the mechanisms of inheritance of environmentally induced traits across generations in controlled and reproducible settings. However, most models available today are only intergenerational and do not demonstrate transmission beyond the direct offspring of exposed individuals. Here, we report transgenerational transmission of behavioral and metabolic phenotypes up to the 4th generation in a mouse model of paternal postnatal trauma (MSUS). Based on large animal numbers (up to 124 per group) from several independent breedings conducted 10 years apart by different experimenters, we show that depressive-like behaviors are transmitted to the offspring until the third generation, and risk-taking and glucose dysregulation until the fourth generation via males. The symptoms are consistent and reproducible, and persist with similar severity across generations. These results provide strong evidence that adverse conditions in early postnatal life can have transgenerational effects, and highlight the validity of MSUS as a solid model of transgenerational epigenetic inheritance.

Traumatic stress in early-life increases the risk for cognitive and neuropsychiatric disorders later in life. Such early stress can also impact the progeny even if not directly exposed, likely through epigenetic mechanisms. Here, we report in mice that the offspring of males subjected to postnatal traumatic stress have decreased gene expression in molecular pathways necessary for neuronal signaling, and altered synaptic plasticity when adult. Long-term potentiation is abolished and long-term depression is enhanced in the hippocampus, and these defects are associated with impaired long-term memory in both the exposed fathers and their offspring. The brain-specific gamma isoform of protein kinase C (Prkcc) is one of the affected signaling components in the hippocampus. Its expression is reduced in the offspring, and DNA methylation at its promoter is altered both in the hippocampus of the offspring and the sperm of fathers. These results suggest that postnatal traumatic stress in males can affect brain plasticity and cognitive functions in the adult progeny, possibly through epigenetic alterations in the male germline.