Elsevier

Biological Psychiatry

Volume 68, Issue 5, 1 September 2010, Pages 408-415
Biological Psychiatry

Priority Communication
Epigenetic Transmission of the Impact of Early Stress Across Generations

https://doi.org/10.1016/j.biopsych.2010.05.036Get rights and content

Background

Traumatic experiences in early life are risk factors for the development of behavioral and emotional disorders. Such disorders can persist through adulthood and have often been reported to be transmitted across generations.

Methods

To investigate the transgenerational effect of early stress, mice were exposed to chronic and unpredictable maternal separation from postnatal day 1 to 14.

Results

We show that chronic and unpredictable maternal separation induces depressive-like behaviors and alters the behavioral response to aversive environments in the separated animals when adult. Most of the behavioral alterations are further expressed by the offspring of males subjected to maternal separation, despite the fact that these males are reared normally. Chronic and unpredictable maternal separation also alters the profile of DNA methylation in the promoter of several candidate genes in the germline of the separated males. Comparable changes in DNA methylation are also present in the brain of the offspring and are associated with altered gene expression.

Conclusions

These findings highlight the negative impact of early stress on behavioral responses across generations and on the regulation of DNA methylation in the germline.

Section snippets

Animals

C57Bl6/J females and males (2.5 months) were obtained from Elevage Janvier (Le Genest Saint Isle, France) and maintained in a temperature- and humidity-controlled facility on a 12 hour reversed light–dark cycle with food and water ad libitum. All procedures were carried out in accordance with Swiss cantonal regulations for animal experimentation.

Maternal Separation

Dams and litters were subjected to unpredictable maternal separation combined with unpredictable maternal stress (MSUS) for 3 hours daily from postnatal day 1 through 14 (PND 1–14) or were left undisturbed except for a cage change once a week (control) until weaning (PND21). Maternal behaviors were monitored during the first 2 weeks after delivery by noting the behavior occurring each minute during 30 min, three times per day (shortly before, shortly after, and 2–3 hours after separation; see

Behavioral Testing

In all tests, the experimenter was blind to treatment, and behaviors were monitored by direct observation and videotracking (Ethovision, Noldus Information Technology Wageningen, The Netherlands). Behaviors were assessed in adult F1, F2, or F3 animals (3–8 months old). Each animal was tested on a maximum of five tasks, 1 to 2 weeks apart, starting with the least aversive task, under indirect, dim red light. The forced swim test and sucrose consumption were used to assess depressive-like

DNA Methylation Assays

Pyrosequencing (Qiagen, Hilden, Germany) was used to quantify methylation of CpG islands (defined as ≥ 60% CG dinucleotides, an observed vs. expected CpG dinucleotide ratio of ≥ 60%, and sequence window ≥ 150 bp) (10, 11, 12) in candidate genes in sperm from F1 males.

Sample Preparation and Bisulfite Treatment

Genomic DNA was prepared from sperm collected from the caudal epididymis of F1 and F2 males, similar to Rakyan et al. (13), and cortex (∼ bregma –2.3) collected from F2 female offspring (DNeasy Blood and Tissue Kit, Qiagen). Purified DNA was processed by bisulfite modification (EZ DNA Methylation—Gold Kit, Zymo Research, Irvine, California).

Pyrosequencing

The percentage of methylated alleles at each CG site was quantified in bisulfite-converted DNA by pyrosequencing, a high-resolution method for quantitative analyses of the relative proportion of methylated versus unmethylated nucleotides (see Supplement 1, Supplementary Methods). Interassay variability of methylation values was less than 5%, consistent with that previously reported (14).

In Vitro Methylation Assays

Acute mouse brain slices were treated with the DNA methyltransferase (DNMT) inhibitor zebularine, a hypomethylating agent, and methylation levels were quantified with methylation-specific primers, as described in Levenson et al. (15) (see Supplement, 1 Supplementary Methods).

Quantitative Real-Time Reverse Transcription Polymerase Chain Reaction

DNaseI-treated RNA isolated from cortex (RNeasy Mini Kit; Qiagen) was used for reverse transcription (RT), using the SuperScript First-Strand Synthesis System II for RT-polymerase chain reaction (PCR; Invitrogen Carlsbad, California). Quantitative RT-PCR was performed in an ABI 7500 thermal cycler using TaqMan probes (Applied Biosystems Foster City, California; see Supplement 1, Supplementary Methods).

Results

To evaluate the extent to which early chronic stress constitutes a risk factor for persistent behavioral alterations, we established a model of unpredictable postnatal stress in mice (Figure 1A). Primiparous C57Bl/6 females (F0) and males were bred, and their litters (F1) were subjected to unpredictable maternal separation combined with unpredictable maternal stress (MSUS) for 3 hours daily from PND 1 to 14. We assessed the impact of the manipulation on the subsequent offspring by breeding

Discussion

Our results provide evidence that chronic and unpredictable stress during early postnatal life leads to depressive-like behaviors and alters the response to novel and aversive environments in adult mice. They show that these traits are in part transmitted to the subsequent generations. Transmission occurs through males and affects the offspring in a sex-dependent manner. The data also show that early stress alters DNA methylation in the germline of the stressed males, with either increased or

References (68)

  • J.M. Trasler

    Epigenetics in spermatogenesis

    Mol Cell Endocrinol

    (2009)
  • I. Roemer et al.

    Epigenetic inheritance in the mouse

    Curr Biol

    (1997)
  • R.G. Urdinguio et al.

    Epigenetic mechanisms in neurological diseases: Genes, syndromes, and therapies

    Lancet Neurol

    (2009)
  • J. Savitz et al.

    5-HT(1A) receptor function in major depressive disorder

    Prog Neurobiol

    (2009)
  • M.A. Pelleymounter et al.

    Behavioral and neuroendocrine effects of the selective CRF2 receptor agonists urocortin II and urocortin III

    Peptides

    (2004)
  • B.S. McEwen

    Understanding the potency of stressful early life experiences on brain and body function

    Metabolism

    (2008)
  • F. Cirulli et al.

    Risk factors for mental health: Translational models from behavioural neuroscience

    Neurosci Biobehav Rev

    (2009)
  • E.M. Marco et al.

    Detrimental psychophysiological effects of early maternal deprivation in adolescent and adult rodents: Altered responses to cannabinoid exposure

    Neurosci Biobehav Rev

    (2009)
  • R.A. Millstein et al.

    Effects of repeated maternal separation on anxiety- and depression-related phenotypes in different mouse strains

    Neurosci Biobehav Rev

    (2007)
  • S.T. Mani et al.

    In the cerebral cortex of female and male mice, amyloid precursor protein (APP) promoter methylation is higher in females and differentially regulated by sex steroids

    Brain Res

    (2006)
  • S. Chong et al.

    Epigenetic germline inheritance

    Curr Opin Genet Dev

    (2004)
  • A.S. Wilson et al.

    DNA hypomethylation and human diseases

    Biochim Biophys Acta

    (2007)
  • C. Kurkjian et al.

    DNA methylation: Its role in cancer development and therapy

    Curr Probl Cancer

    (2008)
  • T.L. Roth et al.

    Lasting epigenetic influence of early-life adversity on the BDNF Gene

    Biol Psychiatry

    (2009)
  • M.M. Sanchez et al.

    Early adverse experience as a developmental risk factor for later psychopathology: Evidence from rodent and primate models

    Dev Psychopathol

    (2001)
  • A. Bifulco et al.

    Childhood adversity, parental vulnerability and disorder: Examining inter-generational transmission of risk

    J Child Psychol Psychiatry

    (2002)
  • L.V. Harper

    Epigenetic inheritance and the intergenerational transfer of experience

    Psychol Bull

    (2005)
  • H.K. Kim et al.

    Intergenerational transmission of internalising and externalising behaviours across three generations: Gender-specific pathways

    Crim Behav Ment Health

    (2009)
  • S.K. Sterba et al.

    Trajectories of internalizing problems across childhood: Heterogeneity, external validity, and gender differences

    Dev Psychopathol

    (2007)
  • N.M. Cameron et al.

    Epigenetic programming of phenotypic variations in reproductive strategies in the rat through maternal care

    J Neuroendocrinol

    (2008)
  • I.C. Weaver et al.

    Epigenetic programming by maternal behavior

    Nat Neurosci

    (2004)
  • M.J. Fazzari et al.

    Epigenomics: Beyond CpG islands

    Nat Rev Genet

    (2004)
  • M. Weber et al.

    Distribution, silencing potential and evolutionary impact of promoter DNA methylation in the human genome

    Nat Genet

    (2007)
  • V.K. Rakyan et al.

    Transgenerational inheritance of epigenetic states at the murine Axin(Fu) allele occurs after maternal and paternal transmission

    Proc Natl Acad Sci U S A

    (2003)

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    Authors TBF and HR contributed equally to this work.

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