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A guide to designing germline-dependent epigenetic inheritance experiments in mammals

Nature Methods volume 14, pages 243–249 (2017)Cite this article

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Abstract

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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Figure 1: Experimental strategies to causally test germline dependence of inherited phenotypes.
Figure 2: Weaning strategies for rodent studies of germline epigenetic inheritance.

References

  1. Bohacek, J. & Mansuy, I.M. Molecular insights into transgenerational non-genetic inheritance of acquired behaviours. Nat. Rev. Genet. 16, 641–652 (2015).

    CAS  PubMed  Google Scholar 

  2. 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).

    CAS  PubMed  Google Scholar 

  3. Heard, E. & Martienssen, R.A. Transgenerational epigenetic inheritance: myths and mechanisms. Cell 157, 95–109 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. Nilsson, E.E. & Skinner, M.K. Environmentally induced epigenetic transgenerational inheritance of disease susceptibility. Transl. Res. 165, 12–17 (2015).

    CAS  PubMed  Google Scholar 

  5. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

  6. Whitelaw, E. Disputing Lamarckian epigenetic inheritance in mammals. Genome Biol. 16, 60 (2015).

    PubMed  PubMed Central  Google Scholar 

  7. Deans, C. & Maggert, K.A. What do you mean, “epigenetic”? Genetics 199, 887–896 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. Bird, A. Perceptions of epigenetics. Nature 447, 396–398 (2007).

    CAS  PubMed  Google Scholar 

  9. Mann, J.R. Epigenetics and memigenetics. Cell. Mol. Life Sci. 71, 1117–1122 (2014).

    CAS  PubMed  Google Scholar 

  10. 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.

    PubMed  Google Scholar 

  11. 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).

    CAS  PubMed  Google Scholar 

  12. Skinner, M.K. Endocrine disruptor induction of epigenetic transgenerational inheritance of disease. Mol. Cell. Endocrinol. 398, 4–12 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Gapp, K. et al. Early life stress in fathers improves behavioural flexibility in their offspring. Nat. Commun. 5, 5466 (2014).

    PubMed  Google Scholar 

  14. Radford, E.J. et al. In utero effects. In utero undernourishment perturbs the adult sperm methylome and intergenerational metabolism. Science 345, 1255903 (2014).

    PubMed  PubMed Central  Google Scholar 

  15. Wei, Y. et al. Paternally induced transgenerational inheritance of susceptibility to diabetes in mammals. Proc. Natl. Acad. Sci. USA 111, 1873–1878 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. 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.

    CAS  PubMed  PubMed Central  Google Scholar 

  17. 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).

    CAS  PubMed  Google Scholar 

  18. 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.

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Blake, G.E. & Watson, E.D. Unravelling the complex mechanisms of transgenerational epigenetic inheritance. Curr. Opin. Chem. Biol. 33, 101–107 (2016).

    CAS  PubMed  Google Scholar 

  20. van Otterdijk, S.D. & Michels, K.B. Transgenerational epigenetic inheritance in mammals: how good is the evidence? FASEB J. 30, 2457–2465 (2016).

    CAS  PubMed  Google Scholar 

  21. Holland, M.L. et al. Early-life nutrition modulates the epigenetic state of specific rDNA genetic variants in mice. Science 353, 495–498 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. 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).

    PubMed  PubMed Central  Google Scholar 

  23. Halfmann, R. & Lindquist, S. Epigenetics in the extreme: prions and the inheritance of environmentally acquired traits. Science 330, 629–632 (2010).

    CAS  PubMed  Google Scholar 

  24. 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).

    CAS  PubMed  Google Scholar 

  25. Youngson, N.A. & Whitelaw, E. Transgenerational epigenetic effects. Annu. Rev. Genomics Hum. Genet. 9, 233–257 (2008).

    CAS  PubMed  Google Scholar 

  26. Danchin, É. et al. Beyond DNA: integrating inclusive inheritance into an extended theory of evolution. Nat. Rev. Genet. 12, 475–486 (2011).

    CAS  PubMed  Google Scholar 

  27. 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).

    PubMed  Google Scholar 

  28. Adalsteinsson, B.T. & Ferguson-Smith, A.C. Epigenetic control of the genome-lessons from genomic imprinting. Genes (Basel) 5, 635–655 (2014).

    Google Scholar 

  29. Jimenez-Chillaron, J.C. et al. Intergenerational transmission of glucose intolerance and obesity by in utero undernutrition in mice. Diabetes 58, 460–468 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Saavedra-Rodríguez, L. & Feig, L.A. Chronic social instability induces anxiety and defective social interactions across generations. Biol. Psychiatry 73, 44–53 (2013).

    PubMed  Google Scholar 

  31. Weber-Stadlbauer, U. et al. Transgenerational transmission and modification of pathological traits induced by prenatal immune activation. Mol. Psychiatry 22, 102–112 (2017).

    CAS  PubMed  Google Scholar 

  32. 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).

    CAS  PubMed  Google Scholar 

  33. Weaver, I.C. et al. Epigenetic programming by maternal behavior. Nat. Neurosci. 7, 847–854 (2004).

    CAS  PubMed  Google Scholar 

  34. 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).

    CAS  PubMed  Google Scholar 

  35. 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.

    CAS  PubMed  Google Scholar 

  36. Dietz, D.M. et al. Paternal transmission of stress-induced pathologies. Biol. Psychiatry 70, 408–414 (2011).

    PubMed  PubMed Central  Google Scholar 

  37. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

  38. 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).

    PubMed  PubMed Central  Google Scholar 

  39. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. 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).

    CAS  PubMed  Google Scholar 

  41. Carone, B.R. et al. Paternally induced transgenerational environmental reprogramming of metabolic gene expression in mammals. Cell 143, 1084–1096 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. 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).

    CAS  PubMed  Google Scholar 

  43. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

  44. 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).

    Google Scholar 

  45. Curley, J.P., Mashoodh, R. & Champagne, F.A. Epigenetics and the origins of paternal effects. Horm. Behav. 59, 306–314 (2011).

    PubMed  Google Scholar 

  46. Marsden, H.M. & Bronson, F.H. Estrous synchrony in mice: alteration by exposure to male urine. Science 144, 1469 (1964).

    CAS  PubMed  Google Scholar 

  47. 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).

    CAS  PubMed  Google Scholar 

  48. 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).

    PubMed  PubMed Central  Google Scholar 

  49. Whitten, W.K. Occurrence of anoestrus in mice caged in groups. J. Endocrinol. 18, 102–107 (1959).

    CAS  PubMed  Google Scholar 

  50. 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).

    CAS  PubMed  Google Scholar 

  51. Koike, H. et al. Behavioral abnormality and pharmacologic response in social isolation-reared mice. Behav. Brain Res. 202, 114–121 (2009).

    CAS  PubMed  Google Scholar 

  52. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

  53. Dias, B.G. & Ressler, K.J. Parental olfactory experience influences behavior and neural structure in subsequent generations. Nat. Neurosci. 17, 89–96 (2014).

    CAS  PubMed  Google Scholar 

  54. Wu, L. et al. Paternal psychological stress reprograms hepatic gluconeogenesis in offspring. Cell Metab. 23, 735–743 (2016).

    CAS  PubMed  Google Scholar 

  55. Wagner, K.D. et al. RNA induction and inheritance of epigenetic cardiac hypertrophy in the mouse. Dev. Cell 14, 962–969 (2008).

    CAS  PubMed  Google Scholar 

  56. 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.

    CAS  PubMed  Google Scholar 

  57. 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).

    CAS  PubMed  Google Scholar 

  58. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

  59. 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).

    CAS  PubMed  Google Scholar 

  60. Wei, Y. et al. Enriched environment-induced maternal weight loss reprograms metabolic gene expression in mouse offspring. J. Biol. Chem. 290, 4604–4619 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  61. Mitchell, E. et al. Behavioural traits propagate across generations via segregated iterative-somatic and gametic epigenetic mechanisms. Nat. Commun. 7, 11492 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  62. Padmanabhan, N. et al. Mutation in folate metabolism causes epigenetic instability and transgenerational effects on development. Cell 155, 81–93 (2013).

    CAS  PubMed  Google Scholar 

  63. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

  64. Cui, L. et al. Transcervical embryo transfer in mice. J. Am. Assoc. Lab. Anim. Sci. 53, 228–231 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  65. 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).

    CAS  PubMed  Google Scholar 

  66. Krishnan, V. et al. Molecular adaptations underlying susceptibility and resistance to social defeat in brain reward regions. Cell 131, 391–404 (2007).

    CAS  PubMed  Google Scholar 

  67. Franklin, T.B., Saab, B.J. & Mansuy, I.M. Neural mechanisms of stress resilience and vulnerability. Neuron 75, 747–761 (2012).

    CAS  PubMed  Google Scholar 

  68. Alter, M.D. et al. Paternal transmission of complex phenotypes in inbred mice. Biol. Psychiatry 66, 1061–1066 (2009).

    PubMed  PubMed Central  Google Scholar 

  69. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

  70. 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).

    PubMed  Google Scholar 

  71. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

  72. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

  73. Siklenka, K. et al. Disruption of histone methylation in developing sperm impairs offspring health transgenerationally. Science 350, aab2006 (2015).

    PubMed  Google Scholar 

  74. 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).

    PubMed  PubMed Central  Google Scholar 

  75. 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).

    CAS  PubMed  Google Scholar 

  76. 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).

    CAS  PubMed  Google Scholar 

  77. Noordzij, M. et al. Sample size calculations: basic principles and common pitfalls. Nephrol. Dial. Transplant. 25, 1388–1393 (2010).

    PubMed  Google Scholar 

  78. Holson, R.R. & Pearce, B. Principles and pitfalls in the analysis of prenatal treatment effects in multiparous species. Neurotoxicol. Teratol. 14, 221–228 (1992).

    CAS  PubMed  Google Scholar 

  79. Bohacek, J. & Mansuy, I.M. Epigenetic inheritance of disease and disease risk. Neuropsychopharmacology 38, 220–236 (2013).

    CAS  PubMed  Google Scholar 

  80. Chapman, K.M.M. et al. Targeted germline modifications in rats using CRISPR/Cas9 and spermatogonial stem cells. Cell Rep. 10, 1828–1835 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  81. 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.

    CAS  PubMed  PubMed Central  Google Scholar 

  82. Gemma, C. et al. Inactive or moderately active human promoters are enriched for inter-individual epialleles. Genome Biol. 14, R43 (2013).

    PubMed  PubMed Central  Google Scholar 

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Acknowledgements

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.

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  1. Medical Faculty of the University of Zürich and Department of Health Sciences and Technology of the ETH Zürich, Laboratory of Neuroepigenetics, Brain Research Institute, Neuroscience Center Zürich, Zürich, Switzerland

    Johannes Bohacek & Isabelle M Mansuy

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  1. Johannes Bohacek
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Correspondence to Johannes Bohacek.

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Integrated supplementary information

Supplementary Figure 1 Male germ cell development and spermatogenesis in mice.

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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