Psychiatric diseases have a strong heritable component known to not be restricted to DNA sequence-based genetic inheritance alone but to also involve epigenetic factors in germ cells. Initial evidence suggested that sperm RNA is causally linked to the transmission of symptoms induced by traumatic experiences. Here, we show that alterations in long RNA in sperm contribute to the inheritance of specific trauma symptoms. Injection of long RNA fraction from sperm of males exposed to postnatal trauma recapitulates the effects on food intake, glucose response to insulin and risk-taking in adulthood whereas the small RNA fraction alters body weight and behavioural despair. Alterations in long RNA are maintained after fertilization, suggesting a direct link between sperm and embryo RNA.

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.

30Psychiatric diseases have a strong heritable component known to not be restricted to 31 DNA sequence-based genetic inheritance alone but to also involve epigenetic factors in 32 germ cells 1,2 . Initial evidence suggested that sperm RNA is causally linked 2,3 to the 33 transmission of symptoms induced by traumatic experiences. Here we show that 34 alterations in long RNA in sperm contribute to the inheritance of specific trauma 35 symptoms. Injection of long RNA fraction from sperm of males exposed to postnatal 36 trauma recapitulates the effects on food intake, glucose response to insulin and risk-37 taking in adulthood whereas the small RNA fraction alters body weight and behavioral 38 despair. Alterations in long RNA are maintained after fertilization, suggesting a direct 39 link between sperm and embryo RNA. 40 41 42 43 Adverse experiences can have long-lasting transgenerational effects on mental and 46 physical health, and often increase disease risk 4,5 . Traumatic stress in early life in 47 particular, can induce pathologies like psychosis, depression and metabolic 48 dysfunctions in adulthood across generations 6 . To examine the biological factors 49 involved, we recapitulated heritable behavioural and metabolic effects of postnatal 50 trauma across several generations using a previously established model of 51 unpredictable maternal separation combined with unpredictable maternal stress 52(MSUS) in the mouse, that shows symptoms through up to three generations ( Fig.1) 2,7-53 13 . We have shown that such postnatal trauma alters small RNA in sperm and that 54 injection of total sperm RNA from exposed male mice into naïve fertilized oocytes elicits 55 symptoms reminiscent of those observed in natural offspring of exposed fathers 2 . Other 56 studies have demonstrated that adult stress 14,15 and environmental insults like altered 57 diet or vinclozolin exposure, or positive factors such as exercise or environmental 58 enrichment can affect small RNA in sperm 16-21 and somatic tissues 22 in the offspring.59Recently, tRNA fragments and their modifications were also found to be affected by 60 nutritional insult, and unmodified or modified sperm small RNA injected into fertilized 61 oocytes could mimic metabolic changes resulting from altered parental diet in the 62 progeny 18,23,24 . These studies therefore suggest that small RNA in sperm can be 63 carrier of heritable information. Here we sought to determine whether long RNA in 64 sperm also contributes to the transmission of the effects of previous exposure. 66 Materials and Methods 68Mice. C57BL/6J mice were housed in a temperature and humidity-controlled facility 69 under a reverse light-dark cycle, and food and water were provided ad libitum. 70 Experimental procedures were performed during the animals' active cycle. All71 experiments were approved by the cantonal veterinary office, Zurich (license 55/12 then 72 57/15).73 74 MSUS paradigm. C57BL/6J primiparous females and males were mated at 2.5-3 75 months of age. Randomly selected dams and litt...

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Summary: Stress-related disorders such as PTSD and depression are more prevalent in women than men. One reason for such discordance may be that brain regions involved in stress responses are more sensitive to stress in females. Here, we compared the effects of acute stress on gene transcription in the hippocampus of female and male mice, and also examined the involvement of two key stress-related hormones, corticosterone and corticotropin releasing hormone (Crh). Using quantitative reverse transcription polymerase chain reaction (RT-qPCR), we measured gene expression of Fos, Per1 and Sgk1 45 min after exposure to brief cold swim stress. Stress induced a stronger increase in Fos and Per1 expression in females than males. The handling control procedure increased Fos in both sexes, but occluded the effects of stress in males. Further, handling increased Per1 only in males. Sgk1 was insensitive to handling, and increased in response to stress similarly in males and females. The transcriptional changes observed after swim stress were not mimicked by corticosterone injections, and the stress-induced increase in Fos, Per1 and Sgk1 could neither be prevented by pharmacologically blocking glucocorticoid receptor (GR) nor by blocking Crh receptor 1 (Crhr1) before stress exposure. Finally, we demonstrate that the effects are stressor-specific, as the expression of target genes could not be increased by brief restraint stress in either sex. In summary, we find strong effects of acute swim stress on hippocampal gene expression, complex interactions between handling and sex, and a remarkably unique response pattern for each gene. Overall, females respond to a cold swim challenge with stronger hippocampal gene transcription than males, independent of two classic mediators of the stress response, corticosterone and Crh. These findings may have important implications for understanding the higher vulnerability of women to certain stress-related neuropsychiatric diseases. In summary, we find strong effects of acute swim stress on hippocampal gene expression, complex interactions between handling and sex, and a remarkably unique response pattern for each gene. Overall, females respond to a cold swim challenge with stronger hippocampal gene transcription than males, independent of two classic mediators of the stress response, corticosterone and Crh. These findings may have important implications for understanding the higher vulnerability of women to certain stress-related neuropsychiatric diseases.

Environmental factors can change phenotypes in exposed individuals and offspring and involve the germline, likely via biological signals in the periphery that communicate with germ cells. Here, using a mouse model of paternal exposure to traumatic stress, we identify circulating factors involving peroxisome proliferatoractivated receptor (PPAR) pathways in the effects of exposure to the germline. We show that exposure alters metabolic functions and pathways, particularly lipid-derived metabolites, in exposed fathers and their offspring. We collected data in a human cohort exposed to childhood trauma and observed similar metabolic alterations in circulation, suggesting conserved effects. Chronic injection of serum from trauma-exposed males into controls recapitulates metabolic phenotypes in the offspring. We identify lipid-activated nuclear receptors PPARs as potential mediators of the effects from father to offspring. Pharmacological PPAR activation in vivo reproduces metabolic dysfunctions in the offspring and grand-offspring of injected males and affects the sperm transcriptome in fathers and sons. In germ-like cells in vitro, both serum and PPAR agonist induce PPAR activation. Together, these results highlight the role of circulating factors as potential communication vectors between the periphery and the germline.