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RESEARCH ARTICLE
Transgenerational inheritance of behavioral
and metabolic effects of paternal exposure
to traumatic stress in early postnatal life:
evidence in the 4th generation
Gretchen van Steenwyk , Martin Roszkowski , Francesca Manuella,
Tamara B. Franklin
1
and Isabelle M. Mansuy *
Laboratory of Neuroepigenetics, Brain Research Institute, Faculty of Medicine, University of Zurich & Institute
for Neuroscience, Department of Health Science and Technology, ETH Zurich, Winterthurerstrasse 190, CH-8057
Zurich, Switzerland
*Correspondence address. Laboratory of Neuroepigenetics, Brain Research Institute, University of Zurich, Winterthurerstrasse 190, 8057 Zurich,
Switzerland. Tel: þ41 44 6353360; Fax: þ41 44 6353303; Email: mansuy@hifo.uzh.ch
1
Present address: Department of Psychology and Neuroscience, Dalhousie University, Life Sciences Centre, 1355 Oxford Street, P.O. Box 15000, Halifax,
NS B3H 4R2, Canada
Managing Editor: Mike Skinner
Abstract
In the past decades, evidence supporting the transmission of acquired traits across generations has reshaped the field of
genetics and the understanding of disease susceptibility. In humans, pioneer studies showed that exposure to famine,
endocrine disruptors or trauma can affect descendants, and has led to a paradigm shift in thinking about heredity. Studies in
humans have however been limited by the low number of successive generations, the different conditions that can be
examined, and the lack of mechanistic insight they can provide. Animal models have been instrumental to circumvent these
limitations and allowed studies on the mechanisms of inheritance of environmentally induced traits across generations in con-
trolled and reproducible settings. However, most models available today are only intergenerational and do not demonstrate
transmission beyond the direct offspring of exposed individuals. Here, we report transgenerational transmission of behavioral
and metabolic phenotypes up to the 4th generation in a mouse model of paternal postnatal trauma (MSUS). Based on large ani-
mal numbers (up to 124 per group) from several independent breedings conducted 10 years apart by different experimenters,
we show that depressive-like behaviors are transmitted to the offspring until the third generation, and risk-taking and glucose
dysregulation until the fourth generation via males. The symptoms are consistent and reproducible, and persist with similar se-
verity across generations. These results provide strong evidence that adverse conditions in early postnatal life can have trans-
generational effects, and highlight the validity of MSUS as a solid model of transgenerational epigenetic inheritance.
Key words: transgenerational; epigenetic inheritance; mouse model; early-life trauma; behavior; 3rd and 4th generation
Received 22 May 2018; revised 16 July 2018; accepted 22 August 2018
V
CThe Author(s) 2018. Published by Oxford University Press.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/
licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited.
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1
Environmental Epigenetics, 2018, 1–8
doi: 10.1093/eep/dvy023
Research Article
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Introduction
The concept of transgenerational epigenetic inheritance implies
that epigenetic signatures induced by exposure can be maintained
across generations and may be responsible for the manifestation
of phenotypes in parents and their offspring. This stands in
contrast to the Mendelian model of inheritance by which genetic
factors are the sole hereditary vectors of trait variation.
Transgenerational epigenetic inheritance has major implications
for disease etiology in humans and helps explain complex condi-
tions such as neuropsychiatric, metabolic and immunological dis-
orders whose heritability cannot be explained only by genetic
factors [1]. Although the concept initially met reservation due to
conceptual limitations such as the fact that the epigenome is
reprogrammed in developing germ cells and the early embryo, evi-
dence has accumulated to indicate that environmentally induced
epigenetic alterations and phenotypes can indeed be transmitted
across generations in mammals (for recent reviews see Refs. 2–5).
Epidemiological studies in humans have implicated grand-
parental and parental environmental conditions such as nutri-
ent availability, exposure to endocrine disruptors or traumatic
experiences in disease susceptibility in descendants [6,7].
In mammals, rodent models of coat color linked to the agouti
vy
locus and DNA methylation [8] or exposure to the endocrine dis-
ruptor vinclozolin [9], have shown altered phenotypes that are
stably transmitted to non-exposed offspring. Other exposure
models, including various dietary regimes [10–18], environmen-
tal toxins [19–26], postnatal trauma [27], prenatal glucocorti-
coids [28], prenatal immune activation [29], psychotropic
medication [30] or olfactory stimulation [31], have linked expo-
sure to altered traits in non-exposed offspring. However today,
most models are intergenerational and exhibit traits that are
transmitted only to the direct offspring of exposed individuals
but not to further generations. Moreover, most models use ex-
posure until breeding, and thus have effects in the progeny that
may be due to the acute exposure of sperm at the time of breed-
ing that may not persist beyond exposure, which is a limitation.
To gain understanding of the mechanisms of transgenera-
tional epigenetic inheritance, models with transmission up to the
3rd generation or possibly to further generations, are needed. Our
lab has developed a mouse model of early postnataltrauma based
on unpredictable maternal separation combined with unpredict-
able maternal stress (MSUS) in which multiple effects were docu-
mented in the offspring up to three generations. Mice exposed to
MSUS and their offspring have increased risk-taking behaviors
[27,32–37], depressive-like symptoms [27,32–37], altered social
recognition [38], memory deficits [35] and insulin/glucose dysre-
gulation [34]. They also show stress resilience [38]andimproved
behavioral flexibility [33] in some conditions. The symptoms are
transmitted through bothmales and females [27,32].
Because this model is robust and epigenetic changes have
been detected in the male germline and in tissues in the off-
spring [27,34] (female germline not tested), we examined
whether symptoms are also present in the 4th generation pro-
duced from the patriline. The results show that both behavioral
and metabolic symptoms induced by MSUS are present in mice
from the 4th generation, indicating that MSUS is a solid and re-
producible transgenerational model of early life adversity.
Results
MSUS Paradigm
The MSUS paradigm was designed to mimic in mice, exposure to
traumatic experiences during childhood in humans. MSUS is
based on the combination of adverse conditions subjected to
young mouse pups during early postnatal life and to their
mother during the same period (Fig. 1). The paradigm consists of
separating mouse pups (F1) from their mother (F0) unpredictably
each day for 3 hours from postnatal day 1 (PND1) to PND14
(Fig. 1A). In this paradigm, unpredictability is critical because it
avoids that mothers predict separation and compensate for their
absence by providing more maternal care before separation.
Instead, it leads to an overall decreased and disorganized mater-
nal care, especially between PND1 and PND7 [27]. Further to sep-
aration, dams are also exposed to an additional stressor
unpredictably (anytime during the 3 hours of separation)
(Fig. 1B). Here again, unpredictability is important as it increases
the severity of the stressor, and the combination of unpredictable
maternal separation with such unpredictable maternal stress
was shown to induce stronger behavioral phenotypes in the off-
spring than separation alone [32]. Breeding was conducted until
the 4th generation by mating adult males at each generation (F1,
F2 and F3) with naı¨ve primiparous control females (Fig. 1C).
Transgenerational Effects of MSUS are Robust and
Reproducible, and Observed Until the 4th Generation
The MSUS paradigm has been repeatedly demonstrated to cause be-
havioral and metabolic alterations in adulthood, not only in directly
exposed mice (F1) but also in their (unexposed) offspring (F2) [27,32,
34–36,38] and grand-offspring (F3) [27,38]. We consolidated these
findings by obtaining different cohorts of mice from several MSUS
breedings across several years. The mice were tested on different
tasks by different experimenters and the data were pooled to reach
large n(Supplementary Table S1). When tested on an elevated plus
maze, F1 and F2 MSUS males spent more time on the open arms of
the maze (Fig. 2A), confirming transmission of reduced aversion to
open space from father to offspring. The grand-offspring (F3) did not
show this phenotype. However, mice from F1, F2 and F3 generations
had decreased latency to first enter an open arm (Fig. 2B), suggesting
increased risk-taking behavior that is transgenerationally transmit-
ted to F3. Further to the elevated plus maze, F3 mice were also
tested on a forced swim test to assess passive coping, a trait charac-
teristic of depressive-like behaviors. Consistent with that observed
previously [27], F3 MSUS males spent more time floating than con-
trols (Fig. 3A), confirming that depressive-like symptoms are repro-
ducible in the 3rd generation.
While transgenerational transmission across 3 generations
has been reported twice with different behavioral tests in MSUS
mice [27,38], transmission to the 4th generation has not yet
been examined. We produced a 4th generation of MSUS and
control mice by breeding F3 males to control females and gener-
ated 2 batches of mice, 10 years apart (2007 and 2017;
Supplementary Table S2) then tested the male offspring when
adult (different experimenters). When tested on the elevated
plus maze, F4 males from both batches spent significantly more
time in the open arms of the maze and had significantly shorter
latency to first enter an open arm, similar to F1, F2 and F3 males
(Fig. 3B). Total distance covered was not changed, as expected
(Supplementary Fig. S1A). Then, when tested on the forced
swim test, F4 MSUS males (Batch 2 only, Batch 1 not tested) spent
a similar amount of time floating to controls (Fig. 3C),
suggesting no depressive-like symptoms thus no apparent
transmission of this trait beyond F3.
To examine if these effects affect both sexes, we also tested F4
MSUS and control females. Similar to males, F4 MSUS females
spent more time in the open arms and had shorter latency to first
enter an open arm on the elevated plus maze compared to F4
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controls (Fig. 4B), but had normal locomotor activity
(Supplementary Fig. S1B). F4 females spent a similar amount of
time floating as control females in a forced swim test (Fig. 4A),
suggesting no depressive-like symptoms, similar to F4 MSUS
males. These findings establish that the effects of paternal MSUS
persist across generations, some until the F3 and F4 generations.
Furthermore, they highlight risk-taking behavior as a highly
consistent trait that similarly affects males and females up to the
4th generation.
Metabolic Effects of MSUS are Transmitted
Transgenerationally
MSUS has been shown to dysregulate glucose and insulin levels
in F1 mice and their offspring [34]. We examined if these
A
C
B
Figure 1: MSUS paradigm. MSUS consists of (A) separating mouse pups (F1) from their mother (F0, naı¨veprimiparous control females mated with naı¨ve males) daily for
3 hours per day at an unpredictable time during the 12 hours active cycle, starting 1 day after birth (postnatal day 1, PND1) until PND14. (B) During separation, dams are
exposed to an additional unpredictable stressor by being subjected to either, a forced swim in 18C water for 5 minutes or a 20-minute physical restraint in a tube, any-
time (unpredictably) during the 3 hours. From PND15, mice are left undisturbed with their mother until PND21 (no further MSUS), are then weaned at PND21 and raised
normally until adulthood (C). Control litters are raised normally (left). Males used to generate the pups are removed from the breeding cage shortly after mating thus,
fathers never encounter their offspring and do not contribute to their rearing. When adult (3–8 months of age), F1 males are paired with naı¨ve primiparous control
females to sire the F2 generation, then F2 and F3 males are bred with naı¨ve primiparous control females to generate an F3 and F4 offspring, respectively. Males from
each generation are tested on the elevated plus maze, forced swim test, weight measurements and glucose response after physical restraint. MSUS is applied only to
F1 mice, mice from F2, F3 and F4 generations are not exposed to any manipulation. Phenotypes transmitted from father to offspring are intergenerational, phenotypes
that persist from father to offspring then grand-offspring or great grand-offspring are transgenerational
AB
Figure 2: persistent behavioral effects of MSUS across 3 generations on the elevated plus maze. MSUS treatment (A) increases the amount of time spent on the open
arms of an elevated plus maze in F1 and F2 mice but not F3 mice (F1 control n¼124, MSUS n¼118, t
240
¼2.26 P¼0.025; F2 control n¼49, MSUS n¼45, t
92
¼2.096 P¼
0.039; F3 control n¼38, MSUS n¼57, t
93
¼1.244 P¼0.217) and (B) decreases the latency to first enter an open arm in F1, F2 and F3 mice (F1 control n¼111, MSUS n¼
101, t
210
¼5.298 P<0.0001; F2 control n¼41, MSUS n¼39, t
78
¼4.353 P<0.0001; F3 control n¼40, MSUS n¼59, t
97
¼3.51 P¼0.0007). Data represent median 6whiskers.
Reported nrepresents data after outlier removal using the ROUT test at Q¼5%. *P<0.05, ***P<0.001, ****P<0.0001
Evidence in the 4th generation |3
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symptoms are also present in mice from the F3 and F4 genera-
tions. In both F3 and F4 MSUS males, there was a trend for in-
creased glucose at baseline compared to control males (Fig. 5A),
unlike F2 MSUS males which had decreased glucose levels [34].
However, during a physical restraint challenge, the mounting of
glucose response was modestly but significantly attenuated in F4
MSUS males compared to controls, similar to that observed in F2
MSUS males [34](Fig. 5B). This effect was not observed in F3
MSUS males (Supplementary Fig. S2). Interestingly in F4 MSUS
males, body weight was lower than controls at PND21 but slightly
increased in adulthood (Fig. 5C), suggesting a rebound response.
This response may be due to increased food consumption as in-
dicated by higher food intake in MSUS mice (Fig. 5D), but does
not result from an inherently larger body size since tail length
was normal in MSUS mice (Fig. 5E). Body weight and food intake
in F4 females was unaffected by MSUS (Supplementary Fig. S3).
Discussion
This study provides evidence that exposure to traumatic stress
in early postnatal life in mice induces several behavioral
alterations that are transmitted across several successive gen-
erations. While increased risk-taking and glucose dysregulation
affect mice up to the 4th generation, depressive-like behaviors
affect F3 but not F4 MSUS males. This indicates that risk-taking
is a robust trait that perpetuates in descendants, suggesting
that it may be more penetrant than other traits. This may be be-
cause, although disadvantageous in some conditions, it may be
beneficial in challenging situations and provide a form of active
coping advantage. In contrast, passive coping associated with
behavioral despair as observed on the forced swim test was not
expressed by F4 MSUS mice. This however does not mean that
this trait has disappeared, since we observed in the past that
some F2 MSUS males did not show any depressive-like symp-
toms (were asymptomatic) but were still able to transmit this
trait to their offspring [27]. The manifestation of both risk-
taking and depressive-like behaviors in MSUS mice is interest-
ing because, together with antisocial behaviors observed in F3
males [38], these traits are typical of a common psychiatric dis-
order, borderline personality disorder (BPD), which has a life-
time prevalence of 6.4% in the population [39]. BPD is a severe
A
C
B
Figure 3: reproducible behavioral alterations by MSUS in F3 and F4 generations. Depressive-like symptoms shown by increased time spent floating on a forced swimtestin
MSUS males from (A) F3 generation but not from (C) F4 generation (F3: control n¼20, MSUS n¼19 t
37
¼3.37 P¼0.0018; F4: Batch 2 control n¼24, MSUS n¼26 t
48
¼0.424 P¼
0.6732). In (B), separate batches of F4 males (Batches 1 and 2) were tested on the elevated plus maze. Time spent on the open arms and latency to first enter an open arm are
similarly altered in both batches. (Batch 1 for time spent on open arms: control n¼22, MSUS n¼19, t
52
¼2.49 P¼0.0161; Batch 1 for latency to enter an open arm: control n¼
22, MSUS n¼19, t
51
¼2.432 P¼0.019; Batch 2 for spent time on open arms: control n¼27, MSUS n¼27, t
39
¼2.159 P¼0.037; Batch 2 latency to first enter an open arm: control n
¼28, MSUS n¼25, t
39
¼2.209 P¼0.033). Data represent median 6whiskers. Reported nrepresents data after outlier removal using the ROUT test at Q¼5%. *P<0.05, **P<0.01
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condition characterized by impulsive behaviors leading to
risky and potentially life-threatening conduct [40], emotional
liability including depressed mood [40] and impaired social
functioning [41,42]. MSUS mice also have memory deficits [35]
and stress-induced analgesia (unpublished data), which are
other prominent BPD symptoms [43,44]. Notably, BPD has a
strong heritability component that cannot be explained by ge-
netic factors alone [45], and instead, the risk to develop the dis-
order has been associated with adverse childhood experiences
[46,47]. Traumatic experiences in humans are known to result
in maladaptive coping strategies [48] and in increased risk-
taking behavior when occurring in childhood [49]. The
AB
Figure 4: behavioral phenotypes in F4 female progeny. (A) F4 MSUS females do not significantly differ from control females in time spent floating during the forced
swim test (control n¼20, MSUS n¼14, t
32
¼0.918 P¼0.366). (B) Time spent in the open arms of the elevated plus maze was increased (control n¼24, MSUS n¼20,
t
42
¼2.09 P¼0.043), while latency to first enter an open arm was decreased in F4 MSUS females (control n¼20, MSUS n¼16, t
34
¼3.01 P¼0.005). Data represent median
6whiskers. Reported nrepresents data after outlier removal using the ROUT test at Q¼5%. *P<0.05, **P<0.01
AC
BDE
Figure 5: transgenerational effects of MSUS treatment on glucose level. (A) Baseline glucose was measured in whole blood following tail prick in F3 (left) and F4 males
(right) (F3 control n¼13, MSUS n¼18, t
29
¼1.891 P¼0.069; F4 control n¼8, MSUS n¼8, t
14
¼1.84 P¼0.087). Continuing from (A), glucose concentrations in F4 blood
(B) was measured at 15-minute intervals during a 30-minute physical restraint challenge, and 60 minutes after release from the restraint tube (control n¼8, MSUS
n¼8, for interaction F
3, 42
¼2.99 P¼0.042). (C) Body weight of F4 males was measured at PND1, PND21 and in adulthood (PND1: control n¼48, MSUS n¼52, t
99
¼1.29
P¼0.199; PND21: control n¼46, MSUS n¼50, t
94
¼2.27 P¼0.025; adult: control n¼46, MSUS n¼51, t
95
¼1.86 P¼0.065). (D) Food intake (control n¼11, MSUS n¼12,
t
21
¼2.185 P¼0.04; nrepresents number of cages) and (E) tail length were measured in adult mice (control n¼46, MSUS n¼51, t
92
¼1.38 P¼0.172). Data represent me-
dian 6whiskers, except for (B) which represent mean 6s.e.m. Reported nrepresents data after outlier removal. #P<0.1, *P<0.05
Evidence in the 4th generation |5
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environmental etiology of BPD and its known heritability sug-
gest that it likely involves epigenetic factors, possibly in the
germline. While in humans, germline-dependent inheritance is
difficult to prove and cannot easily be distinguished from social
and rearing factors e.g. being raised by a parent with a psychiat-
ric illness can predispose a child to psychiatric illness [50],
germline-dependent inheritance implicating sperm RNA has
been causally demonstrated in our MSUS model [34].
Further to behavioral deficits, MSUS also causes metabolic
alterations across generations. Metabolic symptoms have been
reported in humans exposed to trauma, and metabolic syn-
drome can develop in response to prolonged stress [51] and in
people suffering from BPD [52] and post-traumatic stress disor-
der [53]. Notably, the effects of MSUS on metabolism are
expressed differently across generations. While in F2 MSUS
males, baseline glucose is downregulated [34], it is slightly upre-
gulated in F3 and F4 MSUS mice. This is in contrast to behavioral
traits which are similarly expressed across generations but is a
phenomenon already observed in other transgenerational mod-
els [9,17]. Differential expression of phenotypes across genera-
tions has been reported in other models of transgenerational
epigenetic inheritance [54].
In addition to male phenotypes, we also extend the previ-
ously reported transgenerational effects to MSUS females [27]
until the fourth generation. Behavioral differences in F4 MSUS
females are directly comparable to F4 MSUS males, suggesting
similar trait penetrance through the patriline in females.
However, this is not the case for metabolic phenotypes, suggest-
ing different mechanisms of transmission depending on the
phenotype. Regarding potential mechanisms of transmission,
our past work demonstrated a causal role for sperm RNA in the
transfer of phenotypes [34], and correlated changes in DNA
methylation in sperm with transgenerational phenotypes [27],
suggesting that several epigenetic factors are likely implicated.
Others factors or mechanisms may also be involved [55,56].
A distinct and unique feature of the MSUS paradigm is that
it is postnatal, which provides a significant advantage over
prenatal models because it avoids interference with gestational
developmental processes and epigenetic reprogramming occur-
ring during embryogenesis [2]. MSUS exposure is also brief and
limited to a specific time window between PND1 and PND14,
allowing for easier identification of the cells affected during
that time, in particular in developing gonads which have only a
limited number of different cell types at this stage. Most other
models have exposure extending from before conception
throughout embryogenesis and postnatal development until
adulthood [10,13–15,31], making interpretation more difficult
as each developmental stage is likely to be affected, producing
cumulated effects. Furthermore, in models with adult exposure
extending until breeding, the effects can result from acute fac-
tor(s) in gonads, supporting cells or seminal fluid at the time of
conception, and may not just implicate germ cells. With MSUS,
breeding occurs many months after exposure, eliminating any
acute changes and selecting for effects that persist until adult-
hood. This persistence suggests that spermatogonial cells may
be affected. Another unique advantage of MSUS is that it does
not involve any drug, chemical, nutritional insult or invasive
manipulation and relies on “natural” aspects of childhood mis-
treatment such as neglect, attachment disruption and abuse.
Other physiological parameters like altered maternal milk com-
position or lower body temperature due to separation may also
be implicated.
The use of a battery of behavioral tasks including the ele-
vated plus maze, forced swim test and in previous studies, the
open field test, light-dark box, emergence test, fear condition-
ing, social interaction task, operant conditioning, object recog-
nition and social defeat, and of several parameters on some of
the tests e.g. latency to enter and time spent in open arms on
the elevated plus maze, generated a comprehensive and
thorough behavioral profiling of MSUS mice across generations.
After conducting MSUS treatment in 30 independent experi-
ments since 2001 with up to 40 breeding pairs each time, this
study identifies the elevated plus maze and forced swim as reli-
able tests to validate the effects of MSUS across generations.
The effects observed on these tasks are robust, consistent and
highly reproducible. MSUS is currently one of the few available
mouse models of transgenerational epigenetic inheritance with
transmission up to the 4th generation, a depth of inheritance
previously demonstrated in rodents with prenatal stress [57],
toxicants [9,24], drugs [30,58] or genetic mutation (Mtrr hypo-
morphic) [59]. Studies of the mechanisms of transgenerational
inheritance are expected to have important implications for
public health in the future.
Acknowledgements
We thank the University of Zurich, the Swiss Federal
Institute of Technology in Zurich, the Swiss National
Science Foundation, Roche Research Foundation and
Novartis Foundation for Medical-Biological Research for
supporting this research. We thank Irina Lazar-Contes,
Katharina Gapp and Johannes Bohacek for help with the
MSUS breedings, Irina Lazar-Contes, Chiara Boscardin and
Emily Berry for assisting with logistic support during experi-
mentation, Silvia Schelbert for lab organization, Gorjan
Slokar for participating to the first draft of the manuscript,
Deepak Tanwar for discussing statistical matters, Oliver
Sturman for technical help with behavioral equipment and
Yvonne Zipfel, for excellent animal care and help with
health and housing matters.
Supplementary data
Supplementary data are available at EnvEpig online.
Conflict of interest statement. None declared.
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