Transgenerational Inheritance Research Articles

Transmission of reduced levels of miR-34/449 from sperm to preimplantation embryos is a key step in the transgenerational epigenetic inheritance of the effects of paternal chronic social instability stress

ABSTRACT The transgenerational effects of exposing male mice to chronic social instability (CSI) stress are associated with decreased sperm levels of multiple members of the miR-34/449 family that persist after their mating through preimplantation embryo (PIE) development. Here we demonstrate the importance of these miRNA changes by showing that restoring miR-34c levels in PIEs derived from CSI stressed males prevents elevated anxiety and defective sociability normally found specifically in their adult female offspring. It also restores, at least partially, levels of sperm miR-34/449 normally reduced in their male offspring who transmit these sex-specific traits to their offspring. Strikingly, these experiments also revealed that inducing miR-34c levels in PIEs enhances the expression of its own gene and that of miR-449 in these cells. The same induction of embryo miR-34/449 gene expression likely occurs after sperm-derived miR-34c is introduced into oocytes upon fertilization. Thus, suppression of this miRNA amplification system when sperm miR-34c levels are reduced in CSI stressed mice can explain how a comparable fold-suppression of miR-34/449 levels can be found in PIEs derived from them, despite sperm containing ~50-fold lower levels of these miRNAs than those already present in PIEs. We previously found that men exposed to early life trauma also display reduced sperm levels of miR-34/449. And here we show that miR-34c can also increase the expression of its own gene, and that of miR-449 in human embryonic stem cells, suggesting that human PIEs derived from men with low sperm miR-34/449 levels may also contain this potentially harmful defect.

Epigenetic mechanisms linking early-life adversities and mental health.

Early-life adversities, whether prenatal or postnatal exposure, have been linked to adverse mental health outcomes later in life increasing the risk of several psychiatric disorders. Research on its neurobiological consequences demonstrated an association between exposure to adversities and persistent alterations in the structure, function, and connectivity of the brain. Consistent evidence supports the idea that regulation of gene expression through epigenetic mechanisms are involved in embedding the impact of early-life experiences in the genome and mediate between social environments and later behavioral phenotypes. In addition, studies from rodent models and humans suggest that these experiences and the acquired risk factors can be transmitted through epigenetic mechanisms to offspring and the following generations potentially contributing to a cycle of disease or disease risk. However, one of the important aspects of epigenetic mechanisms, unlike genetic sequences that are fixed and unchangeable, is that although the epigenetic markings are long-lasting, they are nevertheless potentially reversible. In this review, we summarize our current understanding of the epigenetic mechanisms involved in the mental health consequences derived from early-life exposure to malnutrition, maltreatment and poverty, adversities with huge and pervasive impact on mental health. We also discuss the evidence about transgenerational epigenetic inheritance in mammals and experimental data suggesting that suitable social and pharmacological interventions could reverse adverse epigenetic modifications induced by early-life negative social experiences. In this regard, these studies must be accompanied by efforts to determine the causes that promote these adversities and that result in health inequity in the population.

The Current State of Knowledge on Osteoporosis in Holocaust Survivors and Their Descendants.

Starvation in early life can cause poor bone health and metabolic aberrations in bone minerals, leading to abnormal bone development. Holocaust survivors have been exposed to starvation and malnutrition before and during World War II. This paper aims to provide the current state of knowledge on the osteoporosis risk in Holocaust survivors and their descendants. The PubMed and Scopus databases were searched. Papers that reported original data on the risk of osteoporosis in Holocaust survivors and in their offspring were included in the study. Ten studies were included in this review. The majority of studies were case-control ones (n=7) versus two self-reported and one longitudinal study. Despite the limited cohort numbers and the small number of studies in the literature, the data showed a potential increased risk of osteoporosis in Holocaust survivors and especially in their descendants. The review of these studies showed a higher prevalence of osteoporosis among Holocaust survivors and their offspring. Knowledge of the trans-generational inheritance of osteoporosis in the descendants of Holocaust survivors should increase the awareness of primary care health workers on osteoporosis screening and early diagnosis and implementation of preventive measures, including adequate vitamin D and calcium supplementation, and pharmacological treatment.

Moving toward the Inclusion of Epigenomics in Bacterial Genome Evolution: Perspectives and Challenges.

The universality of DNA methylation as an epigenetic regulatory mechanism belongs to all biological kingdoms. However, while eukaryotic systems have been the primary focus of DNA methylation studies, the molecular mechanisms in prokaryotes are less known. Nevertheless, DNA methylation in prokaryotes plays a pivotal role in many cellular processes such as defense systems against exogenous DNA, cell cycle dynamics, and gene expression, including virulence. Thanks to single-molecule DNA sequencing technologies, genome-wide identification of methylated DNA is becoming feasible on a large scale, providing the possibility to investigate more deeply the presence, variability, and roles of DNA methylation. Here, we present an overview of the multifaceted roles of DNA methylation in prokaryotes and suggest research directions and tools which can enable us to better understand the contribution of DNA methylation to prokaryotic genome evolution and adaptation. In particular, we emphasize the need to understand the presence and role of transgenerational inheritance, as well as the impact of epigenomic signatures on adaptation and genome evolution. Research directions and the importance of novel computational tools are underlined.

The Interplay between Endocrine-Disrupting Chemicals and the Epigenome towards Metabolic Dysfunction-Associated Steatotic Liver Disease: A Comprehensive Review.

Metabolic dysfunction-associated steatotic liver disease (MASLD), described as the most prominent cause of chronic liver disease worldwide, has emerged as a significant public health issue, posing a considerable challenge for most countries. Endocrine-disrupting chemicals (EDCs), commonly found in daily use items and foods, are able to interfere with nuclear receptors (NRs) and disturb hormonal signaling and mitochondrial function, leading, among other metabolic disorders, to MASLD. EDCs have also been proposed to cause transgenerationally inherited alterations leading to increased disease susceptibility. In this review, we are focusing on the most prominent linking pathways between EDCs and MASLD, their role in the induction of epigenetic transgenerational inheritance of the disease as well as up-to-date practices aimed at reducing their impact.

A natural bacterial pathogen of C. elegans uses a small RNA to induce transgenerational inheritance of learned avoidance.

C. elegans can learn to avoid pathogenic bacteria through several mechanisms, including bacterial small RNA-induced learned avoidance behavior, which can be inherited transgenerationally. Previously, we discovered that a small RNA from a clinical isolate of Pseudomonas aeruginosa, PA14, induces learned avoidance and transgenerational inheritance of that avoidance in C. elegans. Pseudomonas aeruginosa is an important human pathogen, and there are other Pseudomonads in C. elegans' natural habitat, but it is unclear whether C. elegans ever encounters PA14-like bacteria in the wild. Thus, it is not known if small RNAs from bacteria found in C. elegans' natural habitat can also regulate host behavior and produce heritable behavioral effects. Here we screened a set of wild habitat bacteria, and found that a pathogenic Pseudomonas vranovensis strain isolated from the C. elegans microbiota, GRb0427, regulates worm behavior: worms learn to avoid this pathogenic bacterium following exposure, and this learned avoidance is inherited for four generations. The learned response is entirely mediated by bacterially-produced small RNAs, which induce avoidance and transgenerational inheritance, providing further support that such mechanisms of learning and inheritance exist in the wild. We identified Pv1, a small RNA expressed in P. vranovensis, that has a 16-nucleotide match to an exon of the C. elegans gene maco-1. Pv1 is both necessary and sufficient to induce learned avoidance of Grb0427. However, Pv1 also results in avoidance of a beneficial microbiome strain, P. mendocina. Our findings suggest that bacterial small RNA-mediated regulation of host behavior and its transgenerational inheritance may be functional in C. elegans' natural environment, and that this potentially maladaptive response may favor reversal of the transgenerational memory after a few generations. Our data also suggest that different bacterial small RNA-mediated regulation systems evolved independently, but define shared molecular features of bacterial small RNAs that produce transgenerationally-inherited effects.

Calling the question: what is mammalian transgenerational epigenetic inheritance?

ABSTRACT While transgenerational epigenetic inheritance has been extensively documented in plants, nematodes, and fruit flies, its existence in mammals remains controversial. Several factors have contributed to this debate, including the lack of a clear distinction between intergenerational and transgenerational epigenetic inheritance (TEI), the inconsistency of some studies, the potential confounding effects of in-utero vs. epigenetic factors, and, most importantly, the biological challenge of epigenetic reprogramming. Two waves of epigenetic reprogramming occur: in the primordial germ cells and the developing embryo after fertilization, characterized by global erasure of DNA methylation and remodelling of histone modifications. Consequently, TEI can only occur if specific genetic regions evade this reprogramming and persist through embryonic development. These challenges have revived the long-standing debate about the possibility of inheriting acquired traits, which has been strongly contested since the Lamarckian and Darwinian eras. As a result, coupled with the absence of universally accepted criteria for transgenerational epigenetic studies, a vast body of literature has emerged claiming evidence of TEI. Therefore, the goal of this study is to advocate for establishing fundamental criteria that must be met for a study to qualify as evidence of TEI. We identified five criteria based on the consensus of studies that critically evaluated TEI. To assess whether published original research papers adhere to these criteria, we examined 80 studies that either claimed or were cited as supporting TEI. The findings of this analysis underscore the widespread confusion in this field and highlight the urgent need for a unified scientific consensus on TEI requirements.

Signatures of metabolic diseases on spermatogenesis and testicular metabolism.

Diets leading to caloric overload are linked to metabolic disorders and reproductive function impairment. Metabolic and hormonal abnormalities stand out as defining features of metabolic disorders, and substantially affect the functionality of the testis. Metabolic disorders induce testicular metabolic dysfunction, chronic inflammation and oxidative stress. The disruption of gastrointestinal, pancreatic, adipose tissue and testicular hormonal regulation induced by metabolic disorders can also contribute to a state of compromised fertility. In this Review, we will delve into the effects of high-fat diets and metabolic disorders on testicular metabolism and spermatogenesis, which are crucial elements for male reproductive function. Moreover, metabolic disorders have been shown to influence the epigenome of male gametes and might have a potential role in transmitting phenotype traits across generations. However, the existing evidence strongly underscores the unmet need to understand the mechanisms responsible for transgenerational paternal inheritance of male reproductive function impairment related to metabolic disorders. This knowledge could be useful for developing targeted interventions to prevent, counteract, and most of all break the perpetuation chain of male reproductive dysfunction associated with metabolic disorders across generations.

Future in the past: paternal reprogramming of offspring phenotype and the epigenetic mechanisms.

That certain preconceptual paternal exposures reprogram the developmental phenotypic plasticity in future generation(s) has conceptualized the "paternal programming of offspring health" hypothesis. This transgenerational effect is transmitted primarily through sperm epigenetic mechanisms-DNA methylation, non-coding RNAs (ncRNAs) and associated RNA modifications, and histone modifications-and potentially through non-sperm-specific mechanisms-seminal plasma and circulating factors-that create 'imprinted' memory of ancestral information. The epigenetic landscape in sperm is highly responsive to environmental cues, due to, in part, the soma-to-germline communication mediated by epididymosomes. While human epidemiological studies and experimental animal studies have provided solid evidences in support of transgenerational epigenetic inheritance, how ancestral information is memorized as epigenetic codes for germline transmission is poorly understood. Particular elusive is what the downstream effector pathways that decode those epigenetic codes into persistent phenotypes. In this review, we discuss the paternal reprogramming of offspring phenotype and the possible underlying epigenetic mechanisms. Cracking these epigenetic mechanisms will lead to a better appreciation of "Paternal Origins of Health and Disease" and guide innovation of intervention algorithms to achieve 'healthier' outcomes in future generations. All this will revolutionize our understanding of human disease etiology.

Transgenerational Inheritance of Obesity Caused by Endocrine-Disrupting Chemicals

Transgenerational Inheritance of Obesity Caused by Endocrine-Disrupting Chemicals

Transgenerational Epigenetic Inheritance of Cardiovascular Diseases: A Network Medicine Perspective.

The ability to identify early epigenetic signatures underlying the inheritance of cardiovascular risk, including trans-and intergenerational effects, may help to stratify people before cardiac symptoms occur. Prospective and retrospective cohorts and case-control studies focusing on DNA methylation and maternal/paternal effects were searched in Pubmed from 1997 to 2023 by using the following keywords: DNA methylation, genomic imprinting, and network analysis in combination with transgenerational/intergenerational effects. Maternal and paternal exposures to traditional cardiovascular risk factors during critical temporal windows, including the preconceptional period or early pregnancy, may perturb the plasticity of the epigenome (mainly DNA methylation) of the developing fetus especially at imprinted loci, such as the insulin-like growth factor type 2 (IGF2) gene. Thus, the epigenome is akin to a "molecular archive" able to memorize parental environmental insults and predispose an individual to cardiovascular diseases onset inlater life. Direct evidence for human transgenerational epigenetic inheritance (at least three generations) of cardiovascular risk is lacking but it is supported by epidemiological studies. Several blood-based association studies showed potential intergenerational epigenetic effects (single-generation studies) which may mediate the transmittance of cardiovascular risk from parents to offspring. In this narrative review, we discuss some relevant examples of trans- and intergenerational epigenetic associations with cardiovascular risk. In our perspective, we propose three network-oriented approaches which may help to clarify the unsolved issues regarding transgenerational epigenetic inheritance of cardiovascular risk and provide potential early biomarkers for primary prevention.

Transcriptomic effects of paternal cocaine-seeking on the reward circuitry of male offspring

It has been previously established that paternal development of a strong incentive motivation for cocaine can predispose offspring to develop high cocaine-seeking behavior, as opposed to sole exposure to the drug that results in drug resistance in offspring. However, the adaptive changes of the reward circuitry have not been fully elucidated. To infer the key nuclei and possible hub genes that determine susceptibility to addiction in offspring, rats were randomly assigned to three groups, cocaine self-administration (CSA), yoked administration (Yoke), and saline self-administration (SSA), and used to generate F1. We conducted a comprehensive transcriptomic analysis of the male F1 offspring across seven relevant brain regions, both under drug-naïve conditions and after cocaine self-administration. Pairwise differentially expressed gene analysis revealed that the orbitofrontal cortex (OFC) exhibited more pronounced transcriptomic changes in response to cocaine exposure, while the dorsal hippocampus (dHip), dorsal striatum (dStr), and ventral tegmental area (VTA) exhibited changes that were more closely associated with the paternal voluntary cocaine-seeking behavior. Consistently, these nuclei showed decreased dopamine levels, elevated neuronal activation, and elevated between-nuclei correlations, indicating dopamine-centered rewiring of the midbrain circuit in the CSA offspring. To determine if possible regulatory cascades exist that drive the expression changes, we constructed co-expression networks induced by paternal drug addiction and identified three key clusters, primarily driven by transcriptional factors such as MYT1L, POU3F4, and NEUROD6, leading to changes of genes regulating axonogenesis, synapse organization, and membrane potential, respectively. Collectively, our data highlight vulnerable neurocircuitry and novel regulatory candidates with therapeutic potential for disrupting the transgenerational inheritance of vulnerability to cocaine addiction.

Exploring transgenerational inheritance in epigenotypes of DAT heterozygous rats: Circadian anomalies and attentional vulnerability

Dopamine (DA) is mainly involved in locomotor activity, reward processes and maternal behaviors. Rats with KO gene for dopamine transporter (DAT), coding for a truncated DAT protein, are in hyperdopaminergic conditions and thus develop stereotyped behaviors and hyperactivity. Our aim was to test the prior transgenerational modulation of wild and truncated alleles as expressed in heterozygous DAT rats: specifically, we addressed the possible sequelae due to genotype and gender of the ancestors, with regard to behavioral differences in F1, F2, F3 rats. We studied non-classical DAT heterozygotes (HETs) based on two specular lines, with putative grand-maternal vs. grand-paternal imprinting. MAT females (F1; offspring of KO male and WT female) mated with a KO male to generate MIX offspring (F2). Specularly, PAT females (F1; offspring of KO female and WT male) mated with a KO male to generate PIX offspring (F2). Similarly to PAT, we obtained MUX (F2; HET offspring of MAT sire and KO dam); we also observed the F3 (MYX: HET offspring of KO male and MUX female, thus with DAT-KO maternal grandmother like also for PIX). We studied their circadian cycle of locomotor activity and their behavior in the elevated-plus-maze (EPM). Locomotor hyper-activity occurs in F1, the opposite occurs in F2, with MYX rats appearing undistinguishable from WT ones. Open-arm preference emerged in PIX and MIX rats. Only MAT and MYX rats showed a significant vulnerability for ADHD-like inattentive symptoms (duration of rearing in the EPM; Viggiano et al., 2002). A risk-taking profile is evident in the F2 phenotype, while inattentiveness from F1 progeny tends to be transferred to F3. We hypothesize that DAT-related phenotypes result from effective inheritance through pedigree of imprints that are dependent on grandparents, suggesting a protective role for gestation within a hyperdopaminergic uterus. For major features, similar odd (F1, F3) generations appear opposed to even (F2) ones; for minor specific features, the phenotype transfer may affect the progenies with a male but not a female DAT-KO ancestor.

The Study of the Relationship between Or8a1 (M71) Demethylation and Trans-generational Odor Fear Reaction

The transmission of changes in phenotype from the parental generation to their offspring after experiencing environmental stress is termed transgenerational memory. Trans-generational epigenetic inheritance (TEI), which involves DNA modification, histone modification, and miRNAs, is the cause of trans-generational memory. This is because they can regulate the expression of genes and relative phenotypes downstream. This article explores the mechanisms under trans-generational memory of an increase in acetophenone sensitivity in mice after the training of odor fear conditioning in the P0 generation. We designed a series of experiments to detect the demethylation status in olfactory sensory neurons (OSNs) and their precursor cells and an artificial edition of the demethylation status by CRISPR d-Cas9 system in either olfactory sensory neuron and sperms. We aim to gain some insights into whether the artificial demethylation of olfactory sensory neurons is sufficient to cause demethylation in sperms (from somatic to germ cells) and whether the artificial demethylation in sperms could lead to demethylation in OSNs in the following generations (from germ cells to somatic cells).

Genetic confounds of transgenerational epigenetic inheritance in mice.

Transgenerational epigenetic inheritance in mammals remains a controversial phenomenon. A recent study by Takahashi et al. provides evidence for this mode of inheritance in mice by using a CRISPR/Cas9-based epigenetic editing technique to modify DNA methylation levels at specific promoters and then demonstrating the inheritance of the gain in methylation in offspring. In this technical commentary, we argue that the method used in the original study inherently amplifies the likelihood of genetic changes that thereafter lead to the heritability of epigenetic changes. We provide evidence that genetic changes from multiple sources do indeed occur in these experiments and explore several avenues by which these changes could be causal to the apparent inheritance of epigenetic changes. We conclude a genetic basis of inheritance cannot be ruled out and thus transgenerational epigenetic inheritance has not been adequately established by the original study.

Transgenerational inheritance of methylmercury and vitamin A-induced toxicological effects in a Wistar rats environmental-based model

Mercury (Hg) and vitamin A (VitA) are two environmental factors with potential health impacts, especially during pregnancy and early childhood. Fish and seafood may present elevated levels of methylmercury (MeHg), the major Hg derivative, and VitA. This study aimed to evaluate the transgenerational effects of exposure to MeHg and/or VitA on epigenetic and toxicological parameters in a Wistar rat model. Our findings revealed persistent toxicological effects in generations F1 and F2 following low/mild doses of MeHg and/or VitA exposure during dams’ (F0) gestation and breastfeeding. Toxicological effects observed in F2 included chronic DNA damage, bone marrow toxicity, altered microglial content, reduced neuronal signal, and diminished male longevity. Sex-specific patterns were also observed. Co-exposure to MeHg and VitA showed both synergistic and antagonistic effects. Additionally, the study demonstrated that MeHg and VitA affected histone methylation and caused consistent effects in F2. While MeHg exposure has been associated with transgenerational inheritance effects in other organisms, this study provides the first evidence of transgenerational inheritance of MeHg and VitA-induced toxicological effects in rodents. Although the exact mechanism is not yet fully understood, these findings suggest that MeHg and VitA may perpetuate their impacts across generations. The study highlights the need for remedial policies and interventions to mitigate the potential health problems faced by future generations exposed to MeHg or VitA. Further research is warranted to investigate the transgenerational effects beyond F2 and determine the matrilineal or patrilineal inheritance patterns.

Invertebrate Immunity, Natural Transplantation Immunity, Somatic and Germ Cell Parasitism, and Transposon Defense.

While the vertebrate immune system consists of innate and adaptive branches, invertebrates only have innate immunity. This feature makes them an ideal model system for studying the cellular and molecular mechanisms of innate immunity sensu stricto without reciprocal interferences from adaptive immunity. Although invertebrate immunity is evolutionarily older and a precursor of vertebrate immunity, it is far from simple. Despite lacking lymphocytes and functional immunoglobulin, the invertebrate immune system has many sophisticated mechanisms and features, such as long-term immune memory, which, for decades, have been exclusively attributed to adaptive immunity. In this review, we describe the cellular and molecular aspects of invertebrate immunity, including the epigenetic foundation of innate memory, the transgenerational inheritance of immunity, genetic immunity against invading transposons, the mechanisms of self-recognition, natural transplantation, and germ/somatic cell parasitism.

Differential susceptibility of male and female germ cells to glucocorticoid-mediated signaling

While physiologic stress has long been known to impair mammalian reproductive capacity through hormonal dysregulation, mounting evidence now suggests that stress experienced prior to or during gestation may also negatively impact the health of future offspring. Rodent models of gestational physiologic stress can induce neurologic and behavioral changes that persist for up to three generations, suggesting that stress signals can induce lasting epigenetic changes in the germline. Treatment with glucocorticoid stress hormones is sufficient to recapitulate the transgenerational changes seen in physiologic stress models. These hormones are known to bind and activate the glucocorticoid receptor (GR), a ligand-inducible transcription factor, thus implicating GR-mediated signaling as a potential contributor to the transgenerational inheritance of stress-induced phenotypes. Here, we demonstrate dynamic spatiotemporal regulation of GR expression in the mouse germline, showing expression in the fetal oocyte as well as the perinatal and adult spermatogonia. Functionally, we find that fetal oocytes are intrinsically buffered against changes in GR signaling, as neither genetic deletion of GR nor GR agonism with dexamethasone altered the transcriptional landscape or the progression of fetal oocytes through meiosis. In contrast, our studies revealed that the male germline is susceptible to glucocorticoid-mediated signaling, specifically by regulating RNA splicing within the spermatogonia, although this does not abrogate fertility. Together, our work suggests a sexually dimorphic function for GR in the germline, and represents an important step towards understanding the mechanisms by which stress can modulate the transmission of genetic information through the germline.

Differential susceptibility of male and female germ cells to glucocorticoid-mediated signaling.

While physiologic stress has long been known to impair mammalian reproductive capacity through hormonal dysregulation, mounting evidence now suggests that stress experienced prior to or during gestation may also negatively impact the health of future offspring. Rodent models of gestational physiologic stress can induce neurologic and behavioral changes that persist for up to three generations, suggesting that stress signals can induce lasting epigenetic changes in the germline. Treatment with glucocorticoid stress hormones is sufficient to recapitulate the transgenerational changes seen in physiologic stress models. These hormones are known to bind and activate the glucocorticoid receptor (GR), a ligand-inducible transcription factor, thus implicating GR-mediated signaling as a potential contributor to the transgenerational inheritance of stress-induced phenotypes. Here, we demonstrate dynamic spatiotemporal regulation of GR expression in the mouse germline, showing expression in the fetal oocyte as well as the perinatal and adult spermatogonia. Functionally, we find that fetal oocytes are intrinsically buffered against changes in GR signaling, as neither genetic deletion of GR nor GR agonism with dexamethasone altered the transcriptional landscape or the progression of fetal oocytes through meiosis. In contrast, our studies revealed that the male germline is susceptible to glucocorticoid-mediated signaling, specifically by regulating RNA splicing within the spermatogonia, although this does not abrogate fertility. Together, our work suggests a sexually dimorphic function for GR in the germline, and represents an important step towards understanding the mechanisms by which stress can modulate the transmission of genetic information through the germline.

Paternal Prenatal and Lactation Exposure to a High-Calorie Diet Shapes Transgenerational Brain Macro- and Microstructure Defects, Impacting Anxiety-Like Behavior in Male Offspring Rats.

Prenatal exposure to high-energy diets (HED) increases the susceptibility to behavioral alterations in the male offspring. We addressed whether prenatal HED primes the transgenerational inheritance of structural brain changes impacting anxiety/depression-like behavior in the offspring. For this, we used female Wistar rats exposed to a HED [cafeteria (CAF) diet, n = 6] or chow [control (CON) n = 6] during development. Anxiety and depression-like behavior were evaluated in filial 1 (F1), filial 2 (F2), and filial 3 (F3) male offspring using the open field (OFT), elevated plus maze, novelty suppressed feeding (NSFT), tail suspension (TST), and forced swimming tests. Structural brain changes were identified by deformation-based morphometry (DBM) and diffusion tensor imaging using ex vivo MRI. We found that the F1, F2, and F3 offspring exposed to CAF diet displayed higher anxious scores including longer feeding latency during the NSFT, and in the closed arms, only F1 offspring showed longer stay on edges during the OFT versus control offspring. DBM analysis revealed that CAF offspring exhibited altered volume in the cerebellum, hypothalamus, amygdala, and hippocampus preserved up to the F3 generation of anxious individuals. Also, F3 CAF anxious exhibited greater fractional anisotropy and axial diffusivity (AD) in the amygdala, greater apparent diffusion coefficient in the corpus callosum, and greater AD in the hippocampus with respect to the control. Our results suggest that prenatal and lactation exposure to HED programs the transgenerational inheritance of structural brain changes related to anxiety-like behavior in the male offspring.