Molecular insights into transgenerational non-genetic

Nature Reviews Genetics | AOP, published online 29 September 2015; doi:10.1038/nrg3964

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Molecular insights into transgenerational non-genetic inheritance of acquired behaviours Johannes Bohacek and Isabelle M. Mansuy

Abstract | 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. DNA methylation Involves the transfer of a methyl residue to cytosine (5-methylcytosine) in CpG dinucleotides.

DNA hydroxymethylation Results from oxidation of 5-methylcytosine into 5-hydroxymethylcytosine by ten-eleven translocation dioxygenases (TETs).

Post-translational modifications (PTMs). Covalent modifications such as methylation (mono, bi or tri), acetylation and phosphorylation on specific histone residues (primarily lysine, arginine and serine).

Laboratory of Neuroepigenetics, University of Zürich and ETH Zürich, Center for Neuroscience Zürich, Brain Research Institute, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland. Correspondence to I.M.M. e-mail: [email protected] doi:10.1038/nrg3964 Published online 29 September 2015

The classic view of biological inheritance of traits from parent to offspring implicates genetic factors, but it is now recognized that non-genetic factors — that is, factors that modify gene activity without changing the DNA sequence and that are sensitive to the environment — also contribute. Evidence for germline-dependent non-genetic inheritance of acquired traits in mammals has accumulated in neuroscience, behavioural neuroendocrinology, environmental toxicology and nutritional science1,2. This mode of inheritance is crucial for medical genetics because it helps to explain the aetiology, expression and heritability of prevalent neuropsychiatric diseases such as depression, personality disorders, anxiety and autism3. Identification of non-genetic marks and mechanisms might provide biomarkers for disease diagnosis and treatment monitoring, and possibly prevention4. In mammals, principal non-genetic modifications in germ and somatic cells include the epigenetic marks DNA methylation , DNA hydroxymethylation and posttranslational modifications (PTMs) of histones and protamines, and other processes that involve non-coding RNAs (ncRNAs), histone variants and nucleosome positioning5–8. These modifications (called epimutations when involving heritable changes in DNA methylation) are more frequent than genetic mutations9, and whereas genome repair and maintenance of genetic integrity are actively controlled, no specific machinery to repair aberrant non-genetic marks is known. In a given individual, non-genetic marks fluctuate throughout life and carry important information about previous experiences

and encountered environments and their effects on the organism. Some non-genetic marks can be transferred to daughter cells during mitosis and meiosis10. However, they are not always stable and can be randomly lost or modified. Inheritance of non-genetic marks is therefore non-Mendelian, and non-genetic traits do not segregate as in classic genetics. Some behavioural traits in mammals can be altered by environmental factors encountered acutely or chronically, and can be transmitted by various modes. Acquired traits can be transferred to the offspring independently of germ cells. In this case, they involve behavioural, social, physiological and/or hormonal parameters experienced pre- and/or postnatally and require exposure of each generation to the causative factor (or factors) for perpetuation (BOX 1). True inheritance of acquired traits depends on germ cells and on non-genetic modifications in these cells. These modifications are induced by environmental factors, and can be stable and be transferred to the embryo on fertilization. They can have functional consequences in the offspring during development and/or adulthood, and elicit specific behavioural manifestations that can be propagated to subsequent generations without the initial causative trigger (FIG. 1). In rodents, complex behaviours such as social interactions, behavioural despair, response to adversity or stress, addictive behaviours and cognitive functions can be transgenerationally affected by the environment. Transmission of acquired traits also occurs in humans (BOX 2). When inherited, non-genetic modifications and their consequences on behaviour can

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REVIEWS Box 1 | Germline-independent transmission: modes and assessment strategies in laboratory rodents Several routes of transmission of acquired traits that do not involve germ cells can operate during mating, and during embryonic and postnatal life in mammals. The composition of the seminal fluid, which is transferred to the female with sperm during mating, can change with the environment and influence the offspring independently of sperm93. Intrauterine signals such as hormones, immune factors, nutrients and odours, modified by the presence of a male during gestation, can also influence a fetus94,95. Postnatally, the amount and quality of maternal care can determine the offspring’s behaviour in adulthood. Poorly nurtured female pups become poor mothers, and have increased anxiety and epigenetic dysregulation in the brain96. Maternal factors including milk composition97, microbiota98 and odours99 can also transfer environmental cues from mother to offspring. Some of these confounding factors can be minimized by using patrilines, although a male’s fitness may affect maternal investment and introduce biases100. Other strategies are available to reduce confounding factors. Artificial insemination or in#vitro fertilization (IVF) can be used to exclude seminal fluid and parental interactions during mating. However, both methods involve superovulation, and IVF requires in#vitro culture conditions, which could affect epigenetic programming101. Embryo transfer can be used to avoid intrauterine and maternal care effects. Cross-fostering of pups to a control dam can be used to exclude the contribution of postnatal maternal factors and behaviours. Direct injection of molecules such as sperm RNAs into fertilized wild-type eggs can be used to exclude confounding parental variables and directly test the contribution of specific components21.

Non-coding RNAs (ncRNAs). Functional RNA molecules that are not translated into proteins. ncRNAs can regulate gene transcription by DNA binding and prevent translation by mRNA silencing or degradation. Major ncRNAs include the small ncRNAs (microRNAs, small interfering RNAs, PIWI-interacting RNAs and small nucleolar RNAs) and long ncRNAs.

Nucleosome positioning The location of nucleosomes on the chromatin. It is non-random and dynamic, and affects gene regulation.

Behavioural despair When an animal stops struggling to escape adverse conditions, such as being suspended by the tail (tail suspension test) or placed in a small container of cold water (forced swim test). Struggling or immobility can, however, sometimes reflect the survival response or adaptive learning depending on the task conditions.

Environmental enrichment The supplementation of an individual’s living conditions by social, sensory and physical stimuli.

be beneficial if the offspring encounters similar (matching) environments in which the modifications can make them better adapted, and improve well-being or survival11. However, they can be maladaptive if the environment has changed, and the acquired adaptations are no longer advantageous. In extreme cases of mismatch, susceptibility to psychiatric conditions, metabolic disorders and cancer may be increased2. In those circumstances, erasure of non-genetic changes might be desirable to correct such mismatch in subsequent generations. This Review outlines current evidence in mammals — specifically, rodents and humans — that environmental factors can influence complex behaviours across generations. The nature of the underlying nongenetic modifications in germ cells and the window of susceptibility for their induction are discussed, as well as potential mechanisms of induction and maintenance. Non-genetic germline inheritance of biological functions, such as physical features, metabolism, reproduction and longevity, in plants and invertebrates is well delineated1,12–14 but is not discussed except when providing relevant molecular insight for mammals.

Inheritance of acquired behaviours Social behaviours. In rodents and humans, social abilities are shaped by early experiences, particularly parental care, attachment and social support. Negative experiences can affect social behaviours across generations. In mice, chronic and unpredictable traumatic experiences in early postnatal life (postnatal day 1 (P1)–P14) and chronic social instability in adolescence (P27–P76) alter social recognition and interaction, respectively, across up to three generations15,16. The alterations are transmitted to the offspring by both females and males, even if in the case of males they themselves are asymptomatic. This finding suggests mechanisms of transgenerational inheritance in germ cells and the existence of ‘silent carrier’ parents who can transmit the symptoms without expressing them. Social behaviours can also be affected across several generations by intrauterine exposure to chemicals such as the endocrine disruptor bisphenol A, which is used in plastics17.

Depressive-like behaviours and anxiety. Mood and emotions modulate many behaviours, and their alteration can cause depression in humans, often within families. Some aspects of depressive behaviours such as behavioural despair can be triggered in animal models across generations by negative experiences. In mice, embryos exposed to maternal chronic variable stress in utero (embryonic day 1 (E1)–E7), or newborns exposed to unpredictable traumatic experiences (P1–P14), and their offspring show depressive-like behaviours in adulthood18,19. In the case of postnatal trauma, up to three generations are affected in a complex (mechanistically unclear) sex-dependent manner 19. These symptoms are independent of maternal factors, as they persist after cross-fostering 19,20 (BOX 1). Rather, transmission implicates non-genetic processes in germ cells15,21 (see section on mechanisms below). In male mice, repeated social stress during adolescence increases anxiety across two generations16, and chronic social defeat in adulthood induces behavioural despair and anxiety in the offspring 22. Notably, in this latter case, behavioural despair, but not the increase in anxiety, can also be transmitted by in vitro fertilization (IVF)22. Thus, all symptoms are not systematically passed to the progeny by sperm cells, suggesting that some germline non-genetic modifications might be corrected or erased during IVF, whereas others are maintained. Response to adversity or stress. The ability to respond appropriately and to cope with stress in challenging situations is another form of behaviour that is influenced by the environment in a heritable manner. In mice, postnatal traumatic experiences affect coping and stress responsiveness transgenerationally through both females and males19–21. Fetal exposure to alcohol or to vinclozolin, an endocrine disruptor that is used as a fungicide in agriculture, also increases stress sensitivity in exposed rats and their adult offspring across two or three generations23,24. Some of the inherited effects can be corrected by environmental enrichment in adolescent rat offspring 25, suggesting reversibility of symptoms.

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REVIEWS Addictive behaviours. Consumption of drugs of abuse and alcohol can result in inherited behavioural phenotypes. Whereas addiction in humans often runs in families, parental addiction in rodents can alter drug sensitivity in the offspring. In rats, chronic cocaine selfadministration before mating induces cocaine resistance in the male offspring 26. In adolescent females, repeated morphine injections increase the sensitivity of their male offspring to the analgesic effects of morphine27. Likewise, in mice, chronic exposure to ethanol vapour before mating reduces ethanol preference and consumption in the male offspring, and increases the animals’ sensitivity to the inhibitory effects of alcohol on anxiety 28. Thus, adaptive and counter-regulatory responses can develop in the offspring of addicted rodents, allowing better coping with drug exposure later in life. In humans, such adaptive responses might be obscured by psychosocial and/or socioeconomic factors associated with parental drug abuse and their impact on family dynamics.

Perseveration The repetition of the same response in the absence of any adjustment to changing requirements of a task.

Operant conditioning tasks Tasks that involve behavioural learning through repeated reinforcement or punishment.

Cognitive functions. Rats fed a low-protein diet for more than 10 generations have cognitive deficits such as impaired home orienting and visual discrimination, which are more severe than when malnutrition endures for only one generation29,30. Whereas some deficits disappear when the rats return to a normal diet for two generations, others persist, suggesting complex regulation of transmission. Heritable germline effects on cognitive functions also occur after early life experiences. Postnatal environmental enrichment in mice improves memory in the direct offspring but not in subsequent offspring. Transmission is mediated by females and persists after cross-fostering 31. Conversely, unpredictable traumatic stress in male mice impairs social memory in the offspring across two generations15. Hippocampus-dependent spatial memory is also affected in exposed males and their direct offspring 32, and is associated with transcriptional changes and impaired synaptic plasticity in the hippocampus of the offspring. These aberrations persist after cross-fostering but are not transmitted to the grandoffspring, suggesting transient effects in the germ line32. In some cases, early adverse experiences can also favour cognitive functions in the offspring. Postnatal traumatic stress in male mice improves goal-directed behaviours and behavioural flexibility in the offspring, and reduces perseveration in operant conditioning tasks33. Some of these inherited behaviours are corrected in the offspring by the mineralocorticoid receptor antagonist spironolactone, demonstrating their reversibility. In adult male mice, odour conditioning can be passed to the offspring and can persist after crossfostering and IVF34. This is reminiscent of transmission of acquired odour preference in Caenorhabditis elegans larvae35, but such inheritance of memory acquired in adulthood needs confirmation in mammals. In all of these cases, the duration, nature and severity of the experience or exposure probably determine whether behaviours are affected transgenerationally. Delineation of the pathways activated by experience or exposure that cause non-genetic modifications in germ cells is necessary to gain insight into the mechanisms of transgenerational inheritance.

Converging effects of factors There might be several reasons as to why different environmental factors cause similar behavioural symptoms across generations. Such convergence could stem from the existence of common genomic regions that are susceptible to different environmental influences36. When disrupted in developing or mature germ cells, these regions could perturb similar signalling pathways. Epigenetic control regions that enable differentially methylated regions (DMRs) and long ncRNAs to distally control different genomic loci have been identified37 and could underlie environmental effects. Furthermore, different environmental factors could directly activate overlapping signalling pathways. For example, stressful experiences, endocrine disruptors and alcohol all involve glucocorticoid signalling. Through functional glucocorticoid receptors expressed on Sertoli cells38, spermatogonia39 and mature sperm40, corticosterone could affect germ cells at different developmental stages. Indeed, repeated injections of the glucocorticoid receptor agonist dexamethasone in adult mice alter DNA methylation in the sperm of injected males and the hippocampus and kidney of their male offspring 41. Different environmental factors might also regulate common gene networks by acting on ncRNAs with similar targets. Systematic comparison of transcriptional profiles after different experiences or exposures that elicit comparable behavioural symptoms could ultimately help to identify common pathways and associated genomic loci. Mechanisms of transmission Evidence in animals that acquired behaviours are heritable through non-genetic processes in gametes raises several questions. First, what kind of non-genetic modifications can be induced in germ cells by environmental factors? Second, when can germ cells be affected such that these alterations are inherited, and are there sensitive periods during development? Third, how can the modifications be induced and, once established, maintained to affect the offspring? To discuss these questions, we focus on male germ cells because they have been the most directly implicated in non-genetic germline inheritance in mammals. Female germ cells probably also contribute, but evidence is currently lacking owing to problematic confounding intrauterine and maternal factors (BOX 1), and the scarcity of oocytes. Which non-genetic modifications can be modulated? Many of the non-genetic mechanisms occurring in somatic cells also exist in germ cells5,42 (FIG. 2). DNA can be hyper- or hypomethylated by environmental factors at various loci in sperm, and the alterations can be passed to the offspring. Multiple DMRs have reduced DNA methylation in primordial germ cells (PGCs) and sperm after fetal vinclozolin treatment or caloric restriction43,44. By contrast, fetal alcohol exposure increases DNA methylation and decreases DNA hydroxymethylation at loci such as the pro-opiomelanocortin (Pomc) promoter in sperm23. Vinclozolin- and ethanol-induced DMRs persist in sperm across at least three generations23,45. Likewise, postnatal experiences alter DNA methylation at different genes



REVIEWS Germ cells

Pollution Environmental toxins

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REVIEWS ◀ Figure 1 | Environmental factors influence behaviour across generations. Exposure

to various environmental factors in early life or adulthood can induce non-genetic modifications in germline and somatic cells (F0 generation) (top panel). If these modifications are not transmitted to the embryo, or if they are erased or diluted during development by, for example, reprogramming mechanisms that establish totipotency in the zygote60, they do not affect behaviour in subsequent generations (left panel). If non-genetic modifications are transmitted on fertilization (F1 generation) and persist in somatic cells (such as brain cells) during development and adulthood, they can induce behavioural changes in the offspring throughout life (middle panel). If the non-genetic modifications are transmitted and persist in germ and somatic cells, they can affect subsequent generations without the initial trigger (right panel). Silent carriers (not shown) have non-genetic modifications in the germ line and transmit them to the offspring but are asymptomatic (no behavioural alteration in F1 but in F2). The figure illustrates transmission by males, but transmission by females can also occur16,20,27. The forced swim task and a classic maze are shown as representative behaviourial tasks. Similar dynamics probably apply to humans (BOX 2).

in sperm. For example, unpredictable traumatic stress induces hypomethylation at the promoter CpG island of corticotropin-releasing factor receptor 2 (Crfr2; also known as Crhr2) and of protein kinase C gamma (Prkcg), but also hypermethylation at the promoters of methylCpG-binding protein 2 (Mecp2) and Nr3c2 (which encodes a mineralocorticoid receptor) in adult mouse sperm19,32,33. Similar aberrations in DNA methylation affect both sperm and the brain of the offspring. Although in theory any part of the genome could be differentially methylated in response to experiences or exposure, some regions are more prone to epimutations than others. Work on environmental toxins has identified genomic sequences and DNA-binding motifs (such as those bound by zinc-finger proteins) that are over-represented in environment-sensitive regions in germ and Sertoli cells36. Each toxicant affects different DMRs, but similar DMRs are systematically targeted by a given toxicant 46. Notably, these DMRs are within CpG desert regions, which contribute to gene regulation and may therefore affect gene activity. Furthermore, genomic regions in sperm that contain nucleosomes are more accessible than regions packaged with protamines, and sequences such as CTCF transcription factor-binding sites are over-represented in these regions and could favour the recruitment of epigenetic enzymes47. Intrauterine undernutrition widely decreases DNA methylation in sperm during adulthood, indeed, particularly in regions that retain nucleosomes44. The presence of similar non-genetic modifications in sperm of both father and son, and in relevant organs in the offspring, is a strong indication of transgenerational inheritance. However, for direct proof, it is necessary to demonstrate that non-genetic modifications are required for the phenotypic expression and transmission of specific traits, which is conceptually and technically challenging. Currently, the most direct evidence involves intracisternal A particle (IAP) retrotransposons at the Agouti and axin fused (AxinFu) genes in mice, for which the level of DNA methylation directly modulates gene expression and causes specific phenotypes48. However, in most cases of transgenerational transmission, several loci or genes are affected, making it difficult to assign specific modifications to a given phenotype. Alterations

in sperm DNA methylation are usually small (a few per cent) and hard to detect when measured in sperm cell populations. Small changes at an individual CpG suggest that only a few sperm cells carry the change, making it unlikely to be relevant for inheritance. However, when multiple CpGs are affected within a given locus even by only a few per cent, the chance is higher that many sperm cells carry at least one or several of the altered CpGs rather than a few cells carrying all altered CpGs, which may have a functional impact and be relevant for inheritance. Causal evidence is also limited by the difficulty of linking DNA methylation to gene expression in sperm cells, as these cells are transcriptionally quiescent. Epimutations that are non-functional in sperm could be maintained and/or relayed by other marks to modulate gene activity later in development or adulthood. Assigning DNA methylation changes to gene activity is also problematic when methylation occurs in distal regions with no obvious physical link to the gene. In this respect, confirming that epimutations are functional requires identification of the target genes, cells and time windows in which they control gene activity. Although epimutations transmitted by sperm should theoretically affect every cell in the progeny, they may be present in only some cells if corrected or inefficiently maintained during development. Ultimate proof of their causal contribution to transmission would require mimicking them at individual loci or even CpGs in germ cells, which may become feasible in the future with new techniques such as CRISPR–Cas9 (clustered regularly interspaced short palindromic repeats–CRISPR-associated 9)49. The level of histones and their PTMs can also be modulated by environmental factors. H3 acetylation is increased at the brain-derived neurotrophic factor (Bdnf) promoter in the sperm of cocaine-treated adult rats and the brain of their male progeny 26. Histone PTMs at the promoter of Nr3c2 are altered in the offspring of mice exposed to unpredictable traumatic experiences and are linked to reduced Nr3c2 expression33. Furthermore, H3 lysine 27 trimethylation (H3K27me3) is decreased at specific genetic loci in sperm in response to a low-protein diet in early life50. H3K27me3 is lower and the occurrence of the H2A.Z variant is increased at the promoter of peroxisome proliferator-activated receptor gamma (Pparg) in the sperm of adult rats exposed to a hepatotoxin compared with control rats51. Histone PTMs and histone variants might also modulate DNA methylation and thereby relay some epigenetic information. Increased levels of H2A.Z could cause DNA hypomethylation, as H2A.Z and methylated DNA are mutually exclusive44,52. Notably, histone PTMs and their associated proteins, such as Polycomb repressive complex 2 (PRC2), have been implicated in transgenerational epigenetic inheritance in Drosophila melanogaster and C. elegans 13. However, in rodents, because mature sperm contains only 1–5% histones and lacks some PTMs such as H3 acetylation53, it is uncertain whether sperm histone PTMs are sufficient to convey instructive information for inheritance. The level and composition of ncRNAs in germ cells vary with changing environments. Postnatal chronic



REVIEWS Box 2 | The case for non-genetic germline inheritance in humans In humans, the best evidence for transgenerational inheritance induced by environmental factors relates to the effects of diet and food availability in early life. Children, in particular boys, who were exposed to intrauterine famine — that is, the 5-month Dutch famine in 1944–1945 — and their own children, develop obesity, glucose intolerance and coronary heart disease in adulthood102–104. In some subjects, the symptoms were associated with altered DNA methylation in blood 60 years later105. Risk is highest when gestational famine is followed by consumption of a calorie-rich diet later in life, suggesting a mismatch effect106. Furthermore, whereas abundant food in paternal grandparents during pre-puberty increases mortality risk in grandoffspring65, reduced food availability reduces mortality risk107,108. However, results on the offspring’s birth weight are variable, suggesting non-homogeneous cohorts or inconsistent effects109. Seasonal diet variation, nutritional factors and parental stress also affect health and, in some cases, DNA methylation in the offspring110–113. Importantly, studies in humans are confounded by genetic heterogeneity, family dynamics, living and seasonal conditions, and limited experimental control, and need careful interpretation.

stress or a high-fat diet in adulthood deregulates several microRNAs (miRNAs) and PIWI-interacting RNAs (piRNAs) in sperm and testis across generations21,54,55. A causal link between sperm RNAs and non-genetic inheritance has emerged from animal models. In a paramutation mouse model, sperm RNAs can induce fur depigmentation in wild-type offspring of parents heterozygous for a Kit-null mutation56. Injection of total sperm RNA from Kit mutants or of miR-221 and miR-222, which specifically target Kit mRNAs, into fertilized wild-type eggs recapitulates depigmentation57. Importantly, sperm RNAs can mediate inheritance of acquired traits across generations. Injection of sperm RNAs from male mice subjected to postnatal trauma into wild-type fertilized oocytes reproduces behavioural and metabolic symptoms not only in mice arising from the RNA-injected eggs but also in their progeny 21. Even if the levels of ncRNAs are proportionally lower in sperm than in oocytes, their delivery at fertilization probably affects the developing embryo. In some cases, inhibiting a single sperm miRNA can impair development of the offspring 56, indicating the vital contribution of some sperm-borne mi RNAs. Furthermore, some sperm ncRNAs might predominate in the zygote compared with oocyte ncRNAs, as the levels of some miRNAs dramatically decline in oocytes before fertilization58.

Paramutation The transfer of epigenetic information from one allele of a gene to the other allele to establish a heritable state of gene expression.

When could germ cells be affected by environmental factors? In mammals, male gametes go through successive phases of development that begin during embryogenesis and continue throughout postnatal and adult life (BOX 3; FIG. 3). Each phase can potentially be affected, but prenatal and early postnatal phases are the most vulnerable, because they are phases of establishment and dynamic regulation of epigenetic marks59. Once established, marks are probably less susceptible to interference. Before fertilization, mature sperm and oocytes have methylated DNA and PTMs on protamines (sperm) and histones, and contain several populations of ncRNAs5 (FIG. 2). After fertilization, most epigenetic marks undergo reprogramming in the zygote. DNA is massively demethylated (from 70% to 30%), mostly by

passive dilution through DNA replication and active conversion of 5-methylcytosine to 5-hydroxymethylcytosine by ten-eleven translocation dioxygenases (TETs) in specific regions60. However, DNA methylation persists at imprinted genes, IAP and LINE1 retrotransposons, and other loci. After re-establishment, DNA methylation is erased again at E10.5–E13.5, and only 7–14% of parental DNA methylation remains by the time of sex determination60. At around E15.5, DNA methylation is rapidly re-acquired, and the germ cell-specific profile is fully established in pachytene spermatocytes after birth. This is recapitulated in each pachytene spermatocyte generated in postnatal and adult life. Further to DNA methylation, protamines and histones are dynamically regulated after fertilization. Sperm protamines are actively exchanged with maternally inherited histones to allow chromatin decondensation. Testis-specific histone variants are installed around pre-leptotene, and histones progressively acquire PTMs61 (FIG. 3). Finally, ncRNAs are also dynamically expressed in developing germ cells, mostly during meiosis62 but, unlike DNA methylation and histone PTMs, they are not subjected to reprogramming. ncRNAs can be transferred to the zygote at fertilization7. During embryogenesis, epigenetic marks in germ cells are strongly influenced by environmental factors. Exposure to endocrine disruptors, plastics, dioxin and jet fuels between E8 and E14, or to alcohol between E7 and E21 causes epimutations in sperm46, indicating that fetal germ cells are sensitive to multiple factors. The resulting embryonic DMRs can be perpetuated across generations. DMRs induced by vinclozolin treatment at E8–E14 persist in promoter regions in PGCs, prospermatogonia and mature sperm in the grandoffspring 63. Transmission probably depends on early exposure, as vinclozolin treatment at E15–E21 has no effect on testes64. Undernutrition from E12.5 to birth induces widespread epimutations in adult mouse sperm43,44. Some epimutations affect the liver 43, altering gene expression and metabolic functions of the progeny. However, protein restriction after weaning does not induce epimutations in adult sperm, consistent with lower susceptibility of germ cells to environmental factors later in life50. In all cases of intrauterine exposure, if PGCs are affected, the resulting gametes produced later in life probably carry a memory of exposure that can be maintained and transmitted to subsequent generations. Germ cells are also susceptible to environmental factors after birth, particularly in the early postnatal period. In humans, the prepuberty slow growth period (before 15 years of age) is highly sensitive to food supply, with durable effects across generations65 (BOX 2). In mice, unpredictable traumatic stress from P1 to P14 alters ncRNAs in the sperm, brain and serum in adulthood21, and affects sperm DNA methylation in exposed males and their offspring 19,32. A high-fat diet from weaning to 9–12 weeks alters sperm DNA methylation at metabolic genes across three generations54,66. The sensitivity of postnatal germ cells may result from their responsiveness to signalling molecules such as hormones, cytokines and growth factors possibly released locally (for example, by Sertoli cells) or systemically following environmental



REVIEWS exposure. Hormone, cytokine and growth factor receptors are expressed at various stages of each spermatogenic cycle67–69 (BOX 3). Furthermore, developing Sertoli cells are also vulnerable; for instance, their epigenome and transcriptome are altered by embryonic exposure to vinclozolin, with effects still evident in grandoffspring in rats70. Fully differentiated Sertoli cells do not proliferate or renew; therefore, any permanent functional alteration could have a lifetime impact.

Sperm

As spermatogenesis continues throughout life, adults retain immature germ cells (BOX 3) that may be affected by environmental factors depending on their nature, saliency and the duration of exposure. Exposure spanning one or more spermatogenic cycles could theoretically affect every cellular stage but, unless spermatogonia are affected, cells from subsequent cycles are normal. When in the germinal epithelium of seminiferous tubules, germ cells are protected to a certain degree by the

Oocyte ncRNAs ncRNAs

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Figure 2 | Major non-genetic marks in mammalian germ cells. Mature sperm cells and oocytes carry multiple non-genetic marks, some of which are implicated in transgenerational transmission of acquired traits. DNA methylation is prominent in sperm cells (80–90% overall CpGs) and has a unique profile that is distinct from that in somatic cells. Intergenic regions, CpG islands, and centric and pericentric chromosomal regions are differentially methylated such that genes involved in spermatogenesis are activated and genes involved in pluripotency and somatic functions are repressed. DNA methylation also occurs in oocytes (~40%). Chromatin contains canonical histones and histone variants in sperm and oocytes, but in sperm most histones are replaced by protamines, which are histone-like proteins that form toroid structures to enable genome compaction and carry post-translational modifications (PTMs)53. Only 1–5% of histones remain in mouse sperm (5–10% in human) and form nucleosomes. Retained histones are found in some cases in

C C G C G G C G C G

Hydroxymethyl

hypomethylated regions, CpG islands and gene promoters (including at developmental genes and microRNA clusters), but in other cases they are found in gene-poor regions. These discrepancies are attributed partly to differences in chromatin immunoprecipitation analyses5. Histones in sperm and oocytes show complex patterns of PTMs61. Histone variants can modulate nucleosomal dynamics and gene expression by promoting interactions with chromatin remodelling complexes. Their active incorporation or depletion can directly affect gene transcription6. Sperm cells contain various types of non-coding RNA (ncRNA) in germ granules, including small ncRNAs (microRNAs, PIWI-interacting RNAs, small interfering RNAs, small nucleolar RNAs and mature sperm-enriched tRNA-derived small RNAs) and long ncRNAs, and mRNAs. As sperm cells are transcriptionally quiescent, these RNAs are probably carried over from previous cellular stages. They are required for spermatogenesis, fertility, genome control (that is, transposon silencing) and early development7.



REVIEWS blood–epididymis barrier 71, but mature sperm cells can become vulnerable when they leave the epithelium and can be affected by environmental factors. For example, DNA is globally hypomethylated in testes and spermatids after a 10-week high-fat diet starting in adolescence in mice, and mRNA and miRNA expression is altered in testes and sperm, with metabolic consequences across two generations54. However, longer exposure (16 weeks) triggers DNA damage in sperm; this is in part due to lower levels of the histone deacetylase sirtuin 6 (SIRT6), which is involved in DNA repair 72. Paternal stress from adolescence to adulthood changes sperm miRNAs55, whereas 4 weeks of hepatotoxin treatment modifies histone composition51, and 8 weeks of cocaine administration alters histone PTMs in sperm26. Environmental insults in advanced paternal age may be more detrimental, as the sperm epigenome becomes less stable during ageing 42. How could modifications be induced and maintained? Non-genetic modifications induced by environmental factors in germ cells probably recruit endogenous mechanisms that normally operate in these cells. These mechanisms could vary depending on the time of induction. Epimutations involving hypomethylation at

DMRs during mid-to-late embryogenesis might result from impaired re-acquisition of DNA methylation patterns during PGC programming 44. In postnatal or adult life, hypomethylation might result from deregulation of DNA methyltransferases (DNMTs) or associated proteins. Conversely, hypermethylated DMRs might involve imprinting-like protection mechanisms, such as H3 and H4 acetylation73 or trans-acting maintenance factors74, which prevent reprogramming. During development, DNA methylation and histone PTMs are influenced by diet- or stress-related hormones such as oestrogen, leptin, insulin, corticosterone and environmental toxins that circulate in the blood and/or accumulate in scrotal fat. These factors might activate signalling pathways that lead to transcriptional deregulation of DNMTs or histone-modifying enzymes. For instance, spermatocytes carry androgen and oestrogen receptors that can modulate DNMT1 activity at DMRs67,75. Oestrogens can also regulate chromatin remodelling directly and/or indirectly through follicle-stimulating hormone76. Other receptors, including transforming growth factor β1 (TGFβ1) and TGFβ2 receptors, and noradrenergic receptors expressed on spermatogonia, round spermatids and mature sperm cells, could also contribute69.

Box 3 | Anatomy and cellular organization of developing germ cells in the adult rodent testis The figure shows developing germ cells at different stages of maturation in seminiferous tubules of the testis. Seminiferous tubules are composed of Sertoli cells, which divide the tubules into basal and apical compartments. Sertoli cells tightly control spermatogenesis and exert paraendocrine control over spermatogonial stem cells (SSCs)114. SSC division occurs in the basal compartment, and after pre-leptotene meiosis, developing spermatocytes cross the blood–testis barrier. Spermiogenesis continues in the apical compartment, and mature sperm is released into the lumen of the tubules during spermiation. Afterwards, spermatozoa travel through the epididymis for storage and maturation. SSC self-renewal and spermatogenic development are recapitulated in the testis throughout adulthood. Vas deferens

Spermat ogenesis requires approximat ely 40 days in mice115 and approximately 65 days in rats116. Multiple spermatogenesis cycles occur simultaneously and sequentially. Throughout this time, environmental factors can affect the epigenome of developing sperm, for example, by perturbing hormones that dynamically control spermatogenesis, such as testosterone, gonadotrophins and corticosterone. Developing germ cells express various receptors at different stages of maturation at the membrane or nuclear surface68. Receptors and ligands are represented in blue, green and red. Germ cells are subjected to natural epigenomic variability during spermatogenesis117. P-SPC, pachytene spermatocyte; PL-SPC, pre-leptotene spermatocyte.

Epididymis

Basement membrane

Testis Blood– testis barrier

Seminiferous tubules Spermatids P-SPC

PL-SPC

SSC

Spermatozoon

Cauda epididymis

Receptor and ligand Lumen Sertoli cell Sertoli nucleus Lumen


Apical compartment

Basal compartment


REVIEWS Non-genetic modifications in sperm may also be triggered by soluble blood factors. The injection of serum from adult rats with chronic hepatic injury into control rats can modulate H3K27me3 and H2A.Z at specific genes in sperm51. These secreted factors originate from rat myofibroblasts that have differentiated from hepatic stellate cells. They are also produced by cultured human hepatic stellate cells obtained from patients with liver disease51. Although most of the factors involved remain unknown, some of them have been identified as circulating RNAs in human melanomas77. Circulating RNAs are abundant in blood, and are protected from RNases by associating with high- or low-density lipoproteins78 and RNA-binding proteins including Argonaute 2

(REF. 79),

or by packaging in exosomes, microvesicles or apoptotic bodies80,81. Exosomes may transport RNAs produced from somatic cells to the germ line, crossing the blood–testis barrier (also known as the Sertoli cell barrier). Through receptor binding, endocytosis or membrane fusion, they could deliver RNAs into germ or Sertoli cells78. In mature sperm, circulating RNAs could be stabilized by endogenous RNA stabilization mechanisms such as binding to the protein MIWI, the mouse homologue of human PIWI. In human semen, exosomes are enriched for miRNAs, particularly in seminal fluid82. Tracing exosomes with markers would help to determine their cellular origin and destination. Finally, transporters that allow trafficking Spermatogenesis Spermiogenesis Metaphase I Metaphase II meiosis meiosis

Birth

PGC precursor

PGC

Prospermatogonium

Preleptotene

SSC

Blood– testis Pachytene barrier

Primary spermatocytes

Fertilization

Zygote Blastocyst E3.5

E6.25

E7.25

E10.5

E13.5

Testis

P8–P19

Secondary Elongating Sperm spermato- Round cytes spermatids spermatids cells

P20

P28

P35

Post-implantation embryo

Demethylation Remethylation

Demethylation

Maternal histones replace protamines

Remethylation Installation of testis-specific histone variants

Transition proteins replace histones

Protamines replace transition proteins

P-bodies Intermitochondrial cement

DNA methylation Chromatin RNA granules

Figure 3 | Timeline and dynamics of germ cell development in rodents. Male germ cell development during embryogenesis and early postnatal life. Germ cells start developing shortly after fertilization when a population of pluripotent cells gives rise to primordial germ cell (PGC) precursors in the epiblast (embryonic day 3.5 (E3.5)–E6.25). PGCs emerge from the epiblast at E7.25 as clusters of about 20 cells that settle in the fetal gonads. They rapidly proliferate and migrate to the genital ridge, where sex determination occurs at E12.5. PGCs stop dividing at E13.5 and give rise to mitotically arrested prospermatogonia (gonocytes). The first wave of spermatogenesisbeginsshortly after birth in the testisand lasts~35daysin rodents. Prospermatogonia develop into spermatogonial stem cells (SSCs), which are self-renewing cells that are maintained throughout life. SSCs proliferate by consecutive mitotic divisions and mature into spermatocytes during the first postnatal week. Spermatocytes then enter meiosis until around postnatal day 19 (P19)118 and cross the blood–testis barrier

Chromatoid body

established by Sertoli cells. In rodents, the blood–testis barrier is functional around P20 (REF. 119). Round spermatids, the earliest postmeiotic cells, are produced around weaning (P20–P22). They turn into elongating spermatids within 2–3weeks, during which most histones (90–95%in humans, 95–99% in mice) are replaced by transition proteins, and transition proteins are then replaced by protamines for chromatin compaction. Spermatids mature into sperm cells, which are released into the lumen of the seminiferous tubules from approximately P35 in mice (P44 in rats). Whereas late spermatocytes and round spermatids are transcriptionally active, elongating spermatids and sperm cells are inactive. Bars at the bottom indicate chromatin remodelling and cytoplasmic RNA granules including processing bodies (P-bodies) and chromatoid bodies present in developing germ cells throughout spermatogenesis. The composition of these granules remains largely unknown but includes RNAs and proteins that are critical for sperm development62.



REVIEWS between postmeiotic spermatids and Sertoli cells may also permit specific signals to reach the germ line71. Once induced, non-genetic modifications in germ cells might be maintained by DNA-binding and DNMTtargeting proteins and specific sequence motifs, such as CTG CAG repeats, that favour or prevent DNA accessibility by DNMTs and associated enzymes 74. Protective proteins can preserve 5-methylcytosine from TET3-dependent oxidative demethylation by binding to H3K9me2 (REF. 83). PIWI proteins MILI and MIWI2, which interact with piRNAs that are highly abundant in the testis to maintain transposon silencing, can also help to preserve DNA methylation84,85. Conversely, TET1 and TET2, which erase DNA methylation in PGCs, might also be recruited86. Altered ncRNA composition may be maintained by epigenetic regulation of ncRNA genes through DNA methylation and/or histone PTMs. Once produced, ncRNAs might be stabilized by, for instance, methylation by RNA methyltransferases such as DNMT2, or they might have an altered turnover. Furthermore, ncRNA changes might also be relayed by other non-genetic mechanisms in the offspring, and may function only as initial triggers21. Because crosstalk occurs between DNA methylation, histone and/or protamine PTMs and ncRNAs to regulate the genome, these mechanisms probably function together to maintain non-genetic regulation of germ cells. Once established, non-genetic modifications may act by perturbing gene activity in the developing and/or adult individual. Aberrant DNA methylation and histone PTMs might affect gene expression directly or by altering chromatin composition. DNA methylation established in the early stages of development is known to prevent nucleosome retention during spermiogenesis, causing histone enrichment at hypomethylated CpG regions in sperm5. DMRs involving hypo- or hypermethylation could therefore modulate nucleosome content. Active H3K4me3 and repressive H3K27me3 that are not erased during development could also affect nucleosomes in mature sperm and possibly modulate the insertion of histone variants during repackaging of the haploid genome. Altered ncRNAs may affect the DNA structure, chromatin conformation, and transcriptional and posttranscriptional processes, depending on which population is affected. For instance, long ncRNAs may modify nuclear organization, miRNAs may silence specific genes, and piRNAs may affect retrotransposon sequences87–89. How these processes in germ cells influence transgenerational inheritance of behavioural traits remains unknown. But, importantly, similar induction and maintenance of nongenetic modifications probably operate in parallel cells and tissues, including germ cells, neurons and blood cells, and contribute to behavioural and other phenotypic changes in the exposed animal and its progeny.

Outlook and conclusions The idea that experiences and environmental factors can lead to heritable changes in traits and behaviours has gained solid experimental support, against past controversies about the existence of such modes of inheritance. Initial evidence implicating non-genetic modifications in

germ cells has accumulated, and the way in which these modifications are induced and maintained in germ cells is beginning to be delineated. However, several outstanding questions and challenges remain. First, the ensemble of non-genetic modifications induced by environmental factors and potential crosstalk between them need to be identified in male and female germ cells. This is not trivial considering the wide range of non-genetic modifications (some of which are probably still to be discovered), the low amount of material (that is, 3–4 pg of DNA and 10–20 fg of RNA per sperm cell in mice) and the necessity of ideally working with individual cells. Advances in high-throughput and single-cell methylome and RNA sequencing, and in methodologies to measure less explored marks such as DNA hydroxymethylation, non-CpG methylation, long ncRNAs, piRNAs, circular RNAs and protamine PTMs should help. Second, causal evidence for the implication of the identified non-genetic modifications is needed. This requires convincing demonstration that these modifications are functionally relevant and affect gene activity during development and/or adulthood in a consequential way for the observed phenotypes. Furthermore, the signalling molecules that trigger non-genetic modifications in developing germ cells and the mechanisms preserving or relaying them during development and adulthood should be identified. For all of these points, considering the fundamental importance of the concept of transgenerational inheritance for biology, the highest standards of theoretical and experimental models are required, including rigorous distinction between non-germline and germline inheritance, confirmation of germline inheritance across at least two generations, confirmation in independent cohorts of animals, strict validation of results and exclusion of non-germline factors (BOX 1). Examination of non-genetic modifications in humans — that is, traumatized populations — using accessible biological material such as blood, sperm and saliva will determine the relevance of findings in animals in the future. Finally, because non-genetic germline inheritance allows the transfer of important environmental information to future generations, it probably affects evolutionary processes, a challenging and somewhat disputed idea. It may provide an adaptive benefit in rapidly changing environments by broadening the range of behavioural responses, but it may also create maladaptive and pathological states that are not selected out if they do not affect survival. Non-genetic modifications could also contribute to genomic evolution by introducing genetic variability, as they can promote DNA mutations, recombination and transposition, and alter genome stability. Their complex interplay with genetic factors may affect outbred populations; for example, domestication modifies DNA methylation and non-Mendelian patterns of inheritance90, and mate preference and natural selection are influenced transgenerationally by non-genetic marks91. The integration of non-genetic germline inheritance and molecular aspects of genetic inheritance is essential and offers a unified theory of evolution that reconciles the contribution of environmental and genetic factors in a dual neo-Lamarckian and neo-Darwinian view 92.



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5. 6.

7.

8. 9.

10. 11.

12. 13. 14. 15.

16.

17.

18.

19.

20.

21.

22.

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Acknowle d ge m e nt s

The Mansuy laboratory is supported primarily by the University of Zürich, ETH Zürich and the Swiss National Science Foundation. The authors thank S. Steinbacher for help with drafting illustrations, K. Gapp for critically reading the manuscript and the anonymous reviewers for their constructive comments.

Co m p e t ing int e re s t s s t a t e m e nt

The authors declare no competing interests.
