Embryonic diapause is conserved across mammals.

Abstract

Embryonic diapause (ED) is a temporary arrest of embryo development and is characterized by delayed implantation in the uterus. ED occurs in blastocysts of less than 2% of mammalian species, including the mouse (Mus musculus). If ED were an evolutionarily conserved phenomenon, then it should be inducible in blastocysts of normally non-diapausing mammals, such as domestic species. To prove this hypothesis, we examined whether blastocysts from domestic sheep (Ovis aries) could enter into diapause following their transfer into mouse uteri in which diapause conditions were induced. Sheep blastocysts entered into diapause, as demonstrated by growth arrest, viability maintenance and their ED-specific pattern of gene expression. Seven days after transfer, diapausing ovine blastocysts were able to resume growth in vitro and, after transfer to surrogate ewe recipients, to develop into normal lambs. The finding that non-diapausing ovine embryos can enter into diapause implies that this phenomenon is phylogenetically conserved and not secondarily acquired by embryos of diapausing species. Our study questions the current model of independent evolution of ED in different mammalian orders.

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Embryonic Diapause Is Conserved across Mammals
Grazyna E. Ptak1,2*, Emanuela Tacconi1, Marta Czernik1, Paola Toschi1, Jacek A. Modlinski2,
Pasqualino Loi1
1Department of Comparative Biomedical Sciences, University of Teramo, Teramo, Italy, 2Department of Experimental Embryology, Institute of Genetics and Animal
Breeding of the Polish Academy of Sciences, Jastrzebiec, Poland
Abstract
Embryonic diapause (ED) is a temporary arrest of embryo development and is characterized by delayed implantation in the
uterus. ED occurs in blastocysts of less than 2% of mammalian species, including the mouse (Mus musculus). If ED were an
evolutionarily conserved phenomenon, then it should be inducible in blastocysts of normally non-diapausing mammals,
such as domestic species. To prove this hypothesis, we examined whether blastocysts from domestic sheep (Ovis aries)
could enter into diapause following their transfer into mouse uteri in which diapause conditions were induced. Sheep
blastocysts entered into diapause, as demonstrated by growth arrest, viability maintenance and their ED-specific pattern of
gene expression. Seven days after transfer, diapausing ovine blastocysts were able to resume growth in vitro and, after
transfer to surrogate ewe recipients, to develop into normal lambs. The finding that non-diapausing ovine embryos can
enter into diapause implies that this phenomenon is phylogenetically conserved and not secondarily acquired by embryos
of diapausing species. Our study questions the current model of independent evolution of ED in different mammalian
orders.
Citation: Ptak GE, Tacconi E, Czernik M, Toschi P, Modlinski JA, et al. (2012) Embryonic Diapause Is Conserved across Mammals. PLoS ONE 7(3): e33027.
doi:10.1371/journal.pone.0033027
Editor: Wendy Dean, The Babraham Institute, United Kingdom
Received August 18, 2011; Accepted February 3, 2012; Published March 12, 2012
Copyright: ? 2012 Ptak et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by the European Research Council (FP7/2007–2013)/Programme IDEAS GA nu210103 to GEP. The funders had no role in study
design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: gptak@unite.it
Introduction
Embryonic diapause (ED), or temporary arrest of embryo
development, is a widespread phenomenon in the plant and
animal kingdoms. ED is very ‘‘useful’’ in situations when further
embryo development is risky, for example in harsh climates, in
case of temperature or precipitation fluctuation, or due to
maternally driven stimuli, such as during lactation [1]. In
mammals, this protective phenomenon is maternally controlled
[2] and can be experimentally induced in mice by ovariectomy,
which prevents the ovarian estradiol surge that is necessary for
implantation [3,4]. ED occurrence is widespread in insects, fishes,
birds and marsupials. Among placental mammals (Eutheria), it has
been described in species that belong to all the highly represented
orders (Rodentia, Insectivora, Carnivora, Chiroptera, Edentata and
Artiodactyla), but not in Primates, in which the occurrence of this
phenomenon was not explicitly investigated. In the largest
mammalian order, Rodentia, ED was confirmed or surmised in a
considerable number of species [1,5]. In Rodentia and Insectivora the
occurrence of post-partum oestrus that leads to pregnancy
concomitantly with lactation may cause the induction of the so
called ‘facultative’ ED. The other form of ED (‘obligate’ ED,
because it occurs in all pregnancies) was described to be induced
by environmental factors, for example photoperiod. In both forms,
the physiological mechanism was recognized to be the same [6].
Only ‘facultative’ diapause was described in Rodentia and both
forms in Insectivora. In other orders, the occurrence of an
exclusively ‘obligate’ form was postulated [6]. Since ED is
associated with unfavorable environmental conditions that may
harm the developing embryo, ED could have been more
widespread in the ancestors of currently living mammals, for
example during glaciations [6,7]. In particular, ‘facultative’
diapause was postulated to be a reminiscence of an ancestral
strategy of mammalian development [5]. Nowadays, ED is less
common in Eutheria, probably because this feature might be
dispensable for species living in mild environmental conditions
[8,6]. Indeed, in ruminants (order: Artiodactyla) that live in a
controlled, mild environment, ED has not been observed with the
exception of the roe deer (Capreolus capreolus) [9,10,11,12]. In this
species, ED lasts 5 months and thus it extends the total period of
pregnancy to 9 months, so that offspring are delivered in the
optimal season, the spring. In the roe deer, the occurrence of
‘obligate’ ED is under photoperiodic control, a predictable factor,
which is correlated with seasonal temperature variations and
which generally precludes the survival of offspring that might have
developed without the embryonic quiescence period. Nevertheless,
instances of ED absence have been recorded in some cases [12].
Indeed, and conversely to what is generally believed, in several
species characterized by ‘obligate’ diapause, its occurrence is
actually flexible and variable in time [13,14]. For example, the
occurrence and the extent of ED in mustelids are correlated with
their habitat, environmental temperature, litter size and female
mating period [15,16,17]. In some cases, ED occurrence is
restricted to subspecies that are geographically isolated [7]. ED
can be also be induced in normally non-diapausing mustelids, such
as the ferret (Mustela furo), by influencing their endocrine milieu
[18]. Such flexible occurrence of ED suggests that the use of the
term ‘obligate’ diapause may not be appropriate. Indeed, the
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optimal timing for reproduction may vary from year to year and
from place to place. The flexible occurrence of ED in the same
species supports the idea that ecological factors have a significant
role in the determination of this trait [13,14,19]. This should be
taken into account when classifying a species as diapausing or non-
diapausing. The current knowledge about the variations of this
trait in mammals is limited due to the difficulty of studying embryo
development in wildlife. It is also rather complicated to establish to
what extent ED occurs in mammals. Rodents are the most
commonly used mammalian models for reproductive physiology
studies and the flexible occurrence of ED was reported in lactating
rats already 120 years ago [20]. Many routes might lead to
induction of ED in a flexible fashion in rodents, such as pregnancy
concomitant with heavy lactation, pre-puberty, elevated environ-
mental temperature and even situations of social stress, such as
overcrowding or presence of strange males [21,22]. ED occur-
rence consequently to maternal stress has been hypothesized also
in human concepti [23].
Since 1854 ED was known to occur in only one species
belonging to Artiodactyla, the roe deer [9]. Much later, its
occurrence was postulated also in other species belonging to this
suborder (Cervidae), such as the Pe `re David’s deer (Elaphurus
davidianus) [24,1]. Also in other deer, gestation length was reported
to be highly variable and influenced by various environmental
factors [25]. Growing evidence of a much higher number of
diapausing species (also in other orders) than previously estimated
suggests that ED can be a basic reproductive phenomenon [26].
On the contrary, to date it is generally believed that ED has evolved
independently within different taxonomic groups of mammals [27,6,7,28].
Most probably due to this assumption, experiments on ED
induction in mammals have not been carried out with the
exception of one mustelid species, the ferret [29]. We propose that
ED could be a fundamental, evolutionarily conserved phenome-
non that is however not used in many species because no longer
necessary. If this hypothesis is correct, embryonic diapause should
be inducible in blastocysts from non-diapausing mammals, such as
the domestic sheep, which is classified in the same suborder as the
roe deer, but only the latter is recognized as a diapausing species.
Here we show that diapause can be successfully induced in ovine
blastocysts.
Results
Induction of embryonic arrest in blastocysts from a
domestic species
To investigate whether ED could be induced in a domestic
species, 856 early ovine blastocysts (Group 1), 150 ovine
blastocysts (Group 2), 72 early mouse blastocysts (Group 3) and
27 mouse blastocysts (Group 4) were used (Figure 1). Seven days
after transfer of early ovine (Group 1) and mouse blastocysts
(Group 3) into uteri of pseudo-pregnant mice, in which diapause
conditions were induced, uterine flushing was performed. From
the inter-species (sheep-mouse) transfers (Group 1), 180/856 (21%)
viable blastocysts were recovered as indicated by their re-
expansion in culture within 1–2 hours. None of embryos had
elongated, as normally observed in ruminants, and their size,
according to the diameter of their zona pellucida, had not changed
during the seven days. From intra-species (mouse-mouse) embryo
transfers (Group 3), 35/72 (49%) of viable blastocysts were
recovered. Diapausing, viable ovine and mouse embryos showed
arrested DNA replication (assessed by BrdU incorporation), while
both negative controls, represented by ovine blastocysts cultured in
vitro (Group 2) and murine blastocysts flushed from intact mice (5.4
blastocysts/female) (Group 4), exhibited high level of DNA
synthesis (Figure 2a). The percentage of dead cells was lower in
diapausing ovine (P,0.018) and mouse (P,0.003) blastocysts than
in controls. Moreover, mouse blastocysts showed a more marked
decrease in the proliferation and cell death rate than ovine
embryos (P,0.0001). Further analysis revealed differential expres-
sion of ED markers between diapausing and active ovine
blastocysts (Figure 2b). Genes that positively regulate cell
proliferation (PCNA; Proliferating Cell Nuclear Antigen) and signaling
(HB-EGF; Heparin-binding EGF-like growth factor) were down-
regulated, whereas the anti-proliferative BTG1(B-cell Translocation
Gene 1) gene was strongly up-regulated. Conversely, the expression
of IGF2R (Insulin-like Growth Factor 2 Receptor) did not differ
significantly between diapausing and active ovine blastocysts.
CB1 (cannabinoid receptor type 1), which is normally down-regulated
before implantation, was highly expressed in diapausing ovine
blastocysts (Figure 2c).
Diapausing sheep embryos can develop to term
The induction of diapause in ovine blastocysts was fully
reversible (Figure 3). Diapausing ovine embryos restarted growing
in vitro even at higher rate than control ovine blastocysts (Group 2).
Furthermore, blastocysts hatched from their zona pellucida and after
transfer to recipient sheep they developed to term (8 lambs/18
transferred blastocysts) at a proportion statistically comparable to
control embryos (6 lambs/22 transferred blastocysts). The
pregnancy length following transfer of diapausing blastocysts was
similar to controls (147.4 vs. 148.1 days, respectively). All offspring
had normal birth weight (2.5–3.9 kg) and were healthy.
Discussion
These results show that embryos from a domestic mammal, the
sheep, can enter into diapause when adequate conditions are
created.
Mammalian embryos can develop independently until the
blastocyst stage and then they recognize uterine signals necessary
for their further development. If such signals are not sent by the
uterus, embryos will stop or retard their growth. ED can therefore
be explained as an adaptation of the embryo to environmental
constraints. In our study, DNA synthesis, albeit at an extremely
low level, was still observed in diapausing ovine blastocysts 7 days
after transfer into pseudo-pregnant mice in which diapause
conditions were induced. It is possible that blastocysts from sheep,
a species which belong to Artiodactyla, grow slowly but continuously
throughout the duration of diapause, as it occurs in its diapausing
relative, the roe deer [11]. Alternatively, sheep blastocysts might
need more time to completely stop proliferation. According to the
works by MacLaren [30] and Copp [31], during the first days of
diapause, murine blastocysts similarly slow down growth, while
only around 7 dpc they are completely arrested. In agreement
with these reports, no DNA synthesis was observed in diapausing
mouse embryos in our study. Absence of DNA synthesis was also
noted in diapausing rat, mink and fur seal embryos [32]. The
down-regulation of PCNA, a cell cycle regulatory gene, also was
observed in very distinct taxa: insects, mouse [33,34] and sheep
(present study). Also genes that regulate negatively cell prolifera-
tion, such as BTG1, are maximally expressed during ED both in
mouse [34] and sheep. On the other hand, in contrast to what was
reported in the mouse [34], the expression of IGF2R, which retards
cell proliferation when over-expressed, did not differ between
diapaused and active ovine blastocysts. Different regulation of
IGF2R in ovine and murine diapaused embryos may be related to
the different stage of ED in which the analysis were performed
(2.0–2.5 days of ED vs. 5–6 days, respectively). It was
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demonstrated in the mouse that embryos entering a quiescent state
convert only slightly (1%) their global gene expression pattern
[34]. HB-EGF expression, the earliest indicator of embryo
signaling to the uterus [35,36], is down-regulated both in
diapausing sheep (our study) and mouse blastocysts [34]. In active
blastocysts, the Cannabinoid Receptor 1 (CB1) is constantly down-
regulated, while high level of CB1 expression is observed during
embryonic diapause [37]. CB1 is functional in mouse and ovine
embryos [37,38] and the present finding shows that CB1 down-
regulation during ED is conserved in both species. Collectively, the
molecular control of ED appears similar in very different animal
species. Unfortunately, reports about molecular regulation of ED
are not available for other mammalian species, probably due to
the low interest in the subject and the experimental difficulties (low
availability of gene sequences and antibodies).
It is fascinating that growth arrest in diapausing embryos does
not lead to death. Even embryos which cannot implant (by
exposing the female to constant darkness or following ovariectomy)
are able to survive for long periods (up to 300 days in mustelids)
[17]. In our study, cell death in both mouse and sheep diapaused
blastocysts was even lower than in controls. An earlier study in
which the cell death index in diapausing and active mouse
embryos was compared did not reveal any differences in this value,
although the ED duration was not the same [31]. Higher cell
death in control ovine blastocysts may be attributed to the sub-
optimal in vitro conditions used to obtain this group of embryos.
However, a higher rate of cell death was also observed in control
mouse blastocysts developed in vivo. Since programmed cell death
regulates epiblast differentiation in actively growing blastocysts
[39,40], the decreased level of cell death in dormant blastocysts
should not be surprising. It has been recently suggested that cell
death is avoided during ED as there is no risk of oxidative stress
because opening of the mitochondrial permeability transition pore
and release of Cytochrome c do not occur [41]. Lower cell death
during ED could be also due to DNA repairing activity [42].
With the exception of studies in the ferret (Mustela furo) [29,18],
there is no available scientific data about the experimental
induction of ED in non-diapausing species. In ferret blastocysts,
the zona pellucida was retained during ED [17]. Conversely,
hatched blastocysts are observed during ED both in the mouse and
roe deer, the only diapausing ruminant [11,1]. This is because the
zona pellucida is retained in blastocysts of diapausing species with
inactive corpora lutea, such as mustelids or kangaroos, and not in
species with active corpora lutea, such as roe deer. The zona pellucida
provides physic-mechanical support for the embryos. Its retention
may also provide protection from the uterine immune response.
Zona pellucida was retained in diapausing ovine embryos in our
experiments, which may be indicative of its immune-protective
function in a situation in which embryos are placed in the uterus of
another species. On the other hand, it is worth noting that
immediately following flushing, broken zonae were observed in
some diapaused ovine blastocysts. It cannot be excluded that
Figure 1. Experimental design of embryonic diapause (ED) induction in ovine blastocysts by transfer into ovariectomised pseudo-
pregnant mice at 2.5 dpc. Following uterine flushing, diapausing ovine blastocysts were analyzed or transferred to foster ewes at day 6 after
oestrus for full term development. The timing indicated in the diagram refers to embryos.
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hatched blastocysts were lost during flushing due to their fragility.
Indeed, we and others [22] remarked that diapaused murine
blastocysts are very fragile and they easily collapse during flushing.
The two-fold lower recovery rate of diapausing ovine blastocysts in
comparison to mouse blastocysts may be due to the ongoing
immune attack against foreign embryos present in the uterus. In
support to this suggestion, our preliminary control experiment
(data not shown) demonstrated no embryos or sign of implantation
in uteri of non-ovariectomised, pseudo-pregnant mice (at day 9.5
p.c.) in which ovine blastocysts were transferred for 7 days.
The phenotype of any diapausing embryo, also the ones
described in this work, is similar among the mammalian species
studied. First, no physiological differences of ED are observed among
embryos of different orders of mammals. Second, the dormancy
state is entered at the same embryonic stage, the blastocyst, in all
mammalian species, notwithstanding the species-related differenc-
es in the subsequent development. For example, in mouse,
dormant blastocysts will directly implant after activation, while in
roe deer, blastocysts will necessitate a further week of development
before implantation. Third, we show the lack of species-specificity of the
uterine conditions responsible for ED as they can delay the development
of naturally non-diapausing ovine embryos. The lack of species-
specificity in the uterine conditions that regulate ED and
implantation was previously demonstrated by inter-specific
transfer of two diapausing rodents: rats and mice [43]. It was
also demonstrated in Carnivora, in a work in which ferret embryos
entered into diapause (although they normally do not) after
transfer into the uterus of the mink (Mustela vison), a closely related
diapausing mustelid [29]. Our study demonstrates that the uterus
of Rodentia, in which ED is known to be ‘facultative’, can induced
diapause in embryos of Artiodactyla, in which ED, when present, is
classified as ‘obligate’. This finding supports the idea that similar
mechanisms are involved in both ‘facultative’ and ‘obligate’ ED.
In mammals, the embryo-maternal cross-talk starts at the
blastocyst stage. The metabolic activity is sharply reduced in
developed blastocysts in comparison to earlier stage embryos [44],
and there is essentially no need for maternal nutrition. Therefore,
this stage represents the ideal moment for developmental arrest if
the environmental conditions (including the maternal energy
reserves) are inadequate for straightforward progression of
pregnancy. The lack of maternal signaling has been suggested to
be a cause of diapause entry [45]. Such view is supported by our
experiments and also by other works in mice, where surgical
removal of the ovaries precludes the occurrence of the estradiol
surge, necessary for uterine receptivity [3]. Indeed, a maternal
trigger is necessary to release the blastocyst from diapause and for
its progression through development rather than to halt embryo
development, as it occurs in birds, where the embryo proceeds to
the blastodisc stage and arrests its development until incubation
time (i.e. maternal signal). Indeed, embryonic dormancy and
inability to develop further are related to the absence of the uterine
signal for implantation. Such deficiency of embryo-uterine cross-
talk takes place when mouse blastocysts are in a uterus in which
LIF (which signals uterine receptivity) is not expressed in
endometrium [46,47]. As a consequence, embryos enter into
diapause, but they maintain the ability to develop to term once
Figure 2. Confirmation of ED in ovine embryos. (a) Proportion of BrdU- and TUNEL-positive cells in diapausing ovine and murine blastocysts
flushed from ovariectomised mouse uteri and in controls (b) qRT-PCR analysis of genes involved in ED control. Genes that positively regulate cell
proliferation (PCNA) and signaling (HB-EGF) were not expressed in diapausing ovine blastocysts, while the anti-proliferative gene BTG1 was
significantly over-expressed. IGF2R mRNA expression did not differ statistically between diapausing and control blastocysts. (c) Immunolocalization of
CB1 (green) in diapausing (middle panel) and control ovine blastocysts (upper panel). Nuclei (red) were visualized with propidium iodide. CB1
expression is higher in diapausing ovine blastocysts. Lower panel: ovine blastocysts incubated with neutralized anti-CB1 antibody showing no
positive signal. For each experiment $5 blastocysts were used and it was repeated 3–5 times. Results are mean 6 S.E.M. *** p,0.0001, ** p,0.003,
*p,0.03.
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transferred to wild type, pseudo-pregnant mice. Interestingly, the
only report about ovine embryos that were rendered unable to
implant concerns blastocysts developed in the uterus of females
lacking the endometrial glands [48]. These embryos hatched and
remained viable, but could not grow and elongate. Although they
were not transferred to recipients in order to check whether they
could be activated and develop further, our experiment demon-
strates that diapausing ovine embryos are fully able to restart and
proceed normally through development once placed in the
receptive uterus of synchronized ewes.
Plausibly, ED is conserved in mammalian embryos and the
conditions necessary to induce this state by non-diapausing
females may not be difficult to put in place. It would be interesting
to check if ‘‘older’’ embryos asynchronously transferred to
‘‘younger’’ uterus enter into diapause. The development of
‘‘older’’ embryos placed in ‘‘younger’’ uterus is retarded (rat:
[49]; sheep: [50]; rabbit: [51]; horse: [52]). Notwithstanding of this
retard, they develop more successfully than their synchronized
counterparts, as demonstrated in mouse [53;54] and also in non-
diapausing species (pig: [55]; rabbit: [56]). Other developmental
benefits of the diapause for murine embryos includethe DNA
repair of lethally irradiated embryos [42] and an extended survival
of parthenogenetic embryos [57]. Furthermore, ED is considered
to be a permissive state for embryonic stem cell derivation [58,59].
Our findings might be of relevance for the development of
alternative strategies for the isolation of embryonic stem cells from
large animals. Inner cells mass can be isolated from diapausing
sheep embryos and cultured in conditions favoring the mainte-
nance of pluripotency, although different from mouse develop-
mental stage and ensuing cells signaling should be taken into
account. In mouse, the presence of LIF in culture is indispensable
to derive embryonic stem cells (ESC) from undifferentiated inner
cell mass. However, unlike in mouse, diapause entry in sheep
occurs at more developmentally advanced blastocyst, in which the
epiblast is already formed (just before gastrulation). Molecular
signaling which trigger stem cells derivation and maintenance vary
depending on the developmental stage of the embryo. Mouse
embryonic stem cells may be also successfully derived using late
epiblast [60]. For those cells, activin/Nodal pathway appears
indispensable. Epiblast-derived pluripotent cell lines (EpiSC) from
various species, such as mouse, rat or human are all functionally
similar and independent on LIF/GP130 signaling [60,61]. The
establishment of embryonic stem cells from diapausing sheep
blastocyst using chemically defined activin containing culture
medium could be more advantageous than so far used unsuccessful
approaches. Regardless of success of such strategies, basic studies
on epi/genetic mechanism ensuring a better survival of the
embryo entering diapause is highly needed.
The induction of ED in non-diapausing ovine embryos
questions the current model about the independent evolution of
diapause in different mammalian orders. This study provides a
starting point to verify the flexible occurrence of ED in mammals
Figure 3. Reversibility of growth arrest in ovine embryos following flushing from the uterus of ovariectomised mice. (a) Ovine
blastocysts before (i) and immediately after (ii) transfer to mouse uteri, and following 12 hours of culture in vitro (iii). (b) Percentage of BrdU-positive,
proliferating cells and of embryos hatching from the zona pellucida in diapaused ovine blastocysts after 48 hours in culture and number of offspring
developed from diapaused ovine blastocysts following their transfer into receptive uteri of foster ewes. Controls were in vitro cultured ovine
blastocysts (day 6.5). For each experiment $5 blastocysts were used and it was repeated 3–5 times. Results are mean 6 S.E. M *p,0.05.
doi:10.1371/journal.pone.0033027.g003
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