Recent work has demonstrated that environmental factors experienced by parents can affect their offspring across multiple generations, and that such transgenerational transmission can depend on the germline. Causal evidence for the involvement of germ cells is rare, however, and the underlying molecular mechanisms remain poorly understood. Further, studies often employ varying methods in experimental design and data interpretation. We provide a critical analysis of these issues and suggest possible solutions and guidelines for improving study design and generating reproducible and high-quality data.
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Bohacek, J. & Mansuy, I.M. Molecular insights into transgenerational non-genetic inheritance of acquired behaviours. Nat. Rev. Genet. 16, 641–652 (2015).
Wei, Y.P.Y., Schatten, H. & Sun, Q.-Y. Environmental epigenetic inheritance through gametes and implications for human reproduction. Hum. Reprod. Update 21, 194–208 (2015).
Heard, E. & Martienssen, R.A. Transgenerational epigenetic inheritance: myths and mechanisms. Cell 157, 95–109 (2014).
Nilsson, E.E. & Skinner, M.K. Environmentally induced epigenetic transgenerational inheritance of disease susceptibility. Transl. Res. 165, 12–17 (2015).
Grossniklaus, U., Kelly, W.G., Ferguson-Smith, A.C., Pembrey, M. & Lindquist, S. Transgenerational epigenetic inheritance: how important is it? Nat. Rev. Genet. 14, 228–235 (2013).
Whitelaw, E. Disputing Lamarckian epigenetic inheritance in mammals. Genome Biol. 16, 60 (2015).
Deans, C. & Maggert, K.A. What do you mean, “epigenetic”? Genetics 199, 887–896 (2015).
Bird, A. Perceptions of epigenetics. Nature 447, 396–398 (2007).
Mann, J.R. Epigenetics and memigenetics. Cell. Mol. Life Sci. 71, 1117–1122 (2014).
Franklin, T.B. et al. Epigenetic transmission of the impact of early stress across generations. Biol. Psychiatry 68, 408–415 (2010). First demonstration that traumatic experiences in early postnatal life can lead to transgenerational epigenetic inheritance in mammals, involving changes in epigenetic marks across generations.
Bohacek, J. et al. Pathological brain plasticity and cognition in the offspring of males subjected to postnatal traumatic stress. Mol. Psychiatry 20, 621–631 (2015).
Skinner, M.K. Endocrine disruptor induction of epigenetic transgenerational inheritance of disease. Mol. Cell. Endocrinol. 398, 4–12 (2014).
Gapp, K. et al. Early life stress in fathers improves behavioural flexibility in their offspring. Nat. Commun. 5, 5466 (2014).
Radford, E.J. et al. In utero effects. In utero undernourishment perturbs the adult sperm methylome and intergenerational metabolism. Science 345, 1255903 (2014).
Wei, Y. et al. Paternally induced transgenerational inheritance of susceptibility to diabetes in mammals. Proc. Natl. Acad. Sci. USA 111, 1873–1878 (2014).
Gapp, K. et al. Implication of sperm RNAs in transgenerational inheritance of the effects of early trauma in mice. Nat. Neurosci. 17, 667–669 (2014). First causal proof that RNAs from sperm are mediators of trauma-induced behavioral and molecular changes from father to offspring.
Vassoler, F.M., White, S.L., Schmidt, H.D., Sadri-Vakili, G. & Pierce, R.C. Epigenetic inheritance of a cocaine-resistance phenotype. Nat. Neurosci. 16, 42–47 (2013).
Zeybel, M. et al. Multigenerational epigenetic adaptation of the hepatic wound-healing response. Nat. Med. 18, 1369–1377 (2012). This study shows that circulating factors in blood can interact with sperm epimodifications and impact epigenetic inheritance.
Blake, G.E. & Watson, E.D. Unravelling the complex mechanisms of transgenerational epigenetic inheritance. Curr. Opin. Chem. Biol. 33, 101–107 (2016).
van Otterdijk, S.D. & Michels, K.B. Transgenerational epigenetic inheritance in mammals: how good is the evidence? FASEB J. 30, 2457–2465 (2016).
Holland, M.L. et al. Early-life nutrition modulates the epigenetic state of specific rDNA genetic variants in mice. Science 353, 495–498 (2016).
Oey, H., Isbel, L., Hickey, P., Ebaid, B. & Whitelaw, E. Genetic and epigenetic variation among inbred mouse littermates: identification of inter-individual differentially methylated regions. Epigenetics Chromatin 8, 54 (2015).
Halfmann, R. & Lindquist, S. Epigenetics in the extreme: prions and the inheritance of environmentally acquired traits. Science 330, 629–632 (2010).
Stilling, R.M., Dinan, T.G. & Cryan, J.F. Microbial genes, brain & behaviour – epigenetic regulation of the gut–brain axis. Genes Brain Behav. 13, 69–86 (2014).
Youngson, N.A. & Whitelaw, E. Transgenerational epigenetic effects. Annu. Rev. Genomics Hum. Genet. 9, 233–257 (2008).
Danchin, É. et al. Beyond DNA: integrating inclusive inheritance into an extended theory of evolution. Nat. Rev. Genet. 12, 475–486 (2011).
Weaver, I.C. Epigenetic programming by maternal behavior and pharmacological intervention. Nature versus nurture: let's call the whole thing off. Epigenetics 2, 22–28 (2007).
Adalsteinsson, B.T. & Ferguson-Smith, A.C. Epigenetic control of the genome-lessons from genomic imprinting. Genes (Basel) 5, 635–655 (2014).
Jimenez-Chillaron, J.C. et al. Intergenerational transmission of glucose intolerance and obesity by in utero undernutrition in mice. Diabetes 58, 460–468 (2009).
Saavedra-Rodríguez, L. & Feig, L.A. Chronic social instability induces anxiety and defective social interactions across generations. Biol. Psychiatry 73, 44–53 (2013).
Weber-Stadlbauer, U. et al. Transgenerational transmission and modification of pathological traits induced by prenatal immune activation. Mol. Psychiatry 22, 102–112 (2017).
Drickamer, L.C., Gowaty, P.A. & Holmes, C.M. Free female mate choice in house mice affects reproductive success and offspring viability and performance. Anim. Behav. 59, 371–378 (2000).
Weaver, I.C. et al. Epigenetic programming by maternal behavior. Nat. Neurosci. 7, 847–854 (2004).
Champagne, F.A., Francis, D.D., Mar, A. & Meaney, M.J. Variations in maternal care in the rat as a mediating influence for the effects of environment on development. Physiol. Behav. 79, 359–371 (2003).
Anway, M.D., Cupp, A.S., Uzumcu, M. & Skinner, M.K. Epigenetic transgenerational actions of endocrine disruptors and male fertility. Science 308, 1466–1469 (2005). First demonstration in rat that environmentally induced phenotypes can be passed across multiple generations and likely involve the germline epigenome.
Dietz, D.M. et al. Paternal transmission of stress-induced pathologies. Biol. Psychiatry 70, 408–414 (2011).
Rodgers, A.B., Morgan, C.P., Bronson, S.L., Revello, S. & Bale, T.L. Paternal stress exposure alters sperm microRNA content and reprograms offspring HPA stress axis regulation. J. Neurosci. 33, 9003–9012 (2013).
Finegersh, A. & Homanics, G.E. Paternal alcohol exposure reduces alcohol drinking and increases behavioral sensitivity to alcohol selectively in male offspring. PLoS One 9, e99078 (2014).
Govorko, D., Bekdash, R.A., Zhang, C. & Sarkar, D.K. Male germline transmits fetal alcohol adverse effect on hypothalamic proopiomelanocortin gene across generations. Biol. Psychiatry 72, 378–388 (2012).
Ng, S.-F.F. et al. Chronic high-fat diet in fathers programs β-cell dysfunction in female rat offspring. Nature 467, 963–966 (2010).
Carone, B.R. et al. Paternally induced transgenerational environmental reprogramming of metabolic gene expression in mammals. Cell 143, 1084–1096 (2010).
Fullston, T. et al. Paternal obesity initiates metabolic disturbances in two generations of mice with incomplete penetrance to the F2 generation and alters the transcriptional profile of testis and sperm microRNA content. FASEB J. 27, 4226–4243 (2013).
Mashoodh, R., Franks, B., Curley, J.P. & Champagne, F.A. Paternal social enrichment effects on maternal behavior and offspring growth. Proc. Natl. Acad. Sci. USA 109, 17232–17238 (2012).
Drickamer, L.C., Gowaty, P.A. & Wagner, D.M. Free mutual mate preferences in house mice affect reproductive success and offspring performance. Anim. Behav. 65, 105–114 (2003).
Curley, J.P., Mashoodh, R. & Champagne, F.A. Epigenetics and the origins of paternal effects. Horm. Behav. 59, 306–314 (2011).
Marsden, H.M. & Bronson, F.H. Estrous synchrony in mice: alteration by exposure to male urine. Science 144, 1469 (1964).
Whitten, W.K., Bronson, F.H. & Greenstein, J.A. Estrus-inducing pheromone of male mice: transport by movement of air. Science 161, 584–585 (1968).
Bohacek, J., von Werdt, S. & Mansuy, I.M. Probing the germline-dependence of epigenetic inheritance using artificial insemination in mice. Environ. Epigenet. 2, dvv015 (2016).
Whitten, W.K. Occurrence of anoestrus in mice caged in groups. J. Endocrinol. 18, 102–107 (1959).
Martin, A.L. & Brown, R.E. The lonely mouse: verification of a separation-induced model of depression in female mice. Behav. Brain Res. 207, 196–207 (2010).
Koike, H. et al. Behavioral abnormality and pharmacologic response in social isolation-reared mice. Behav. Brain Res. 202, 114–121 (2009).
Hickman, D.L. & Swan, M.P. Effects of age of pups and removal of existing litter on pup survival during cross-fostering between multiparous outbred mice. J. Am. Assoc. Lab. Anim. Sci. 50, 641–646 (2011).
Dias, B.G. & Ressler, K.J. Parental olfactory experience influences behavior and neural structure in subsequent generations. Nat. Neurosci. 17, 89–96 (2014).
Wu, L. et al. Paternal psychological stress reprograms hepatic gluconeogenesis in offspring. Cell Metab. 23, 735–743 (2016).
Wagner, K.D. et al. RNA induction and inheritance of epigenetic cardiac hypertrophy in the mouse. Dev. Cell 14, 962–969 (2008).
Rassoulzadegan, M. et al. RNA-mediated non-mendelian inheritance of an epigenetic change in the mouse. Nature 441, 469–474 (2006). First demonstration that RNAs contained in sperm can contribute to the inheritance of features in the offspring.
Denomme, M.M. & Mann, M.R.W. Genomic imprints as a model for the analysis of epigenetic stability during assisted reproductive technologies. Reproduction 144, 393–409 (2012).
Stone, B.J., Steele, K.H. & Fath-Goodin, A. A rapid and effective nonsurgical artificial insemination protocol using the NSET™ device for sperm transfer in mice without anesthesia. Transgenic Res. 24, 775–781 (2015).
Morgan, H.D., Sutherland, H.G., Martin, D.I. & Whitelaw, E. Epigenetic inheritance at the agouti locus in the mouse. Nat. Genet. 23, 314–318 (1999).
Wei, Y. et al. Enriched environment-induced maternal weight loss reprograms metabolic gene expression in mouse offspring. J. Biol. Chem. 290, 4604–4619 (2015).
Mitchell, E. et al. Behavioural traits propagate across generations via segregated iterative-somatic and gametic epigenetic mechanisms. Nat. Commun. 7, 11492 (2016).
Padmanabhan, N. et al. Mutation in folate metabolism causes epigenetic instability and transgenerational effects on development. Cell 155, 81–93 (2013).
Green, M., Bass, S. & Spear, B. A device for the simple and rapid transcervical transfer of mouse embryos eliminates the need for surgery and potential post-operative complications. Biotechniques 47, 919–924 (2009).
Cui, L. et al. Transcervical embryo transfer in mice. J. Am. Assoc. Lab. Anim. Sci. 53, 228–231 (2014).
Francis, D.D., Szegda, K., Campbell, G., Martin, W.D. & Insel, T.R. Epigenetic sources of behavioral differences in mice. Nat. Neurosci. 6, 445–446 (2003).
Krishnan, V. et al. Molecular adaptations underlying susceptibility and resistance to social defeat in brain reward regions. Cell 131, 391–404 (2007).
Franklin, T.B., Saab, B.J. & Mansuy, I.M. Neural mechanisms of stress resilience and vulnerability. Neuron 75, 747–761 (2012).
Alter, M.D. et al. Paternal transmission of complex phenotypes in inbred mice. Biol. Psychiatry 66, 1061–1066 (2009).
Guerrero-Bosagna, C. et al. Epigenetic transgenerational inheritance of vinclozolin induced mouse adult onset disease and associated sperm epigenome biomarkers. Reprod. Toxicol. 34, 694–707 (2012).
Martínez, D. et al. In utero undernutrition in male mice programs liver lipid metabolism in the second-generation offspring involving altered Lxra DNA methylation. Cell Metab. 19, 941–951 (2014).
Rakyan, V.K. et al. Transgenerational inheritance of epigenetic states at the murine AxinFu allele occurs after maternal and paternal transmission. Proc. Natl. Acad. Sci. USA 100, 2538–2543 (2003).
Morgan, C.P. & Bale, T.L. Early prenatal stress epigenetically programs dysmasculinization in second-generation offspring via the paternal lineage. J. Neurosci. 31, 11748–11755 (2011).
Siklenka, K. et al. Disruption of histone methylation in developing sperm impairs offspring health transgenerationally. Science 350, aab2006 (2015).
Lazic, S.E. & Essioux, L. Improving basic and translational science by accounting for litter-to-litter variation in animal models. BMC Neurosci. 14, 37 (2013).
Deloris Alexander, A. et al. Quantitative PCR assays for mouse enteric flora reveal strain-dependent differences in composition that are influenced by the microenvironment. Mamm. Genome 17, 1093–1104 (2006).
Van Loo, P.L.P., Mol, J.A., Koolhaas, J.M., Van Zutphen, B.F.M. & Baumans, V. Modulation of aggression in male mice: influence of group size and cage size. Physiol. Behav. 72, 675–683 (2001).
Noordzij, M. et al. Sample size calculations: basic principles and common pitfalls. Nephrol. Dial. Transplant. 25, 1388–1393 (2010).
Holson, R.R. & Pearce, B. Principles and pitfalls in the analysis of prenatal treatment effects in multiparous species. Neurotoxicol. Teratol. 14, 221–228 (1992).
Bohacek, J. & Mansuy, I.M. Epigenetic inheritance of disease and disease risk. Neuropsychopharmacology 38, 220–236 (2013).
Chapman, K.M.M. et al. Targeted germline modifications in rats using CRISPR/Cas9 and spermatogonial stem cells. Cell Rep. 10, 1828–1835 (2015).
Hilton, I.B. et al. Epigenome editing by a CRISPR-Cas9-based acetyltransferase activates genes from promoters and enhancers. Nat. Biotechnol. 33, 510–517 (2015). An important technical advance to allow epigenome editing using CRISPR-Cas9.
Gemma, C. et al. Inactive or moderately active human promoters are enriched for inter-individual epialleles. Genome Biol. 14, R43 (2013).
The lab of IMM is funded by the University of Zurich, the ETH Zurich, the Swiss National Science Foundation, the ETHZ Foundation, Roche and private sponsors. J.B. received funding from the Forschungskredit of the University of Zurich (grant no. FK-15-035), the Vontobel Foundation, the Betty and David Koetser Foundation for Brain Research, and the EMDO Foundation. We thank G. van Steenwyk for critical reading of the manuscript and S. Steinbacher for illustrations.
The authors declare no competing financial interests.
Integrated supplementary information
Male germ cell development begins prenatally and continues throughout life in the testes. Epigenetic modifications involved in germline epigenetic inheritance can be studied in mature sperm cells that are stored for release in the cauda epididymis (A). Epigenetic modifications can already be induced and detected during early developmental stages, affecting primordial germ cells (PGCs, B) and/or spermatogonial stem cells (SSCs, C). Environmental factors can also impact Sertoli cells (D) or the epididymal duct (E), thus potentially affecting developing sperm cells upon transit through these structures. To unveil the mechanisms of germline epigenetic inheritance, future studies should aim to identify epigenetic modifications in some of these structures. SPCs = spermatocytes; PL=Pre-leptotene; P=Pachytene
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Bohacek, J., Mansuy, I. A guide to designing germline-dependent epigenetic inheritance experiments in mammals. Nat Methods 14, 243–249 (2017). https://doi.org/10.1038/nmeth.4181
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