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Reprod Dom Anim 38, 276–289 (2003)
! 2003 Blackwell Verlag, Berlin
ISSN 0936-6768

Embryo-Maternal Communication in Bovine – Strategies for Deciphering

a Complex Cross-Talk
E Wolf1, GJ Arnold2, S Bauersachs1, HM Beier3, H Blum2, R Einspanier4, T Fröhlich2, A Herrler3, S Hiendleder1, S Kölle5,
K Prelle1, H-D Reichenbach6, M Stojkovic1, H Wenigerkind7 and F Sinowatz5
1
Institut für Molekulare Tierzucht und 2Laboratorium für Molekulare Biologie, Genzentrum der Ludwig-Maximilians-Universität München,
München; 3Institut für Anatomie und Reproduktionsbiologie, Universitätsklinikum der RWTH Aachen, Aachen; 4Institut für Physiologie,
Wissenschaftszentrum Weihenstephan, Technische Universität München, Freising; 5Institut für Tieranatomie, Ludwig-Maximilians-Universität
München, München; 6Institut für Tierzucht, Bayerische Landesanstalt für Landwirtschaft, Grub and 7Bayerisches Forschungszentrum für
Fortpflanzungsbiologie (BFZF), Oberschleissheim, Germany

Contents environment. The ability of embryonic interferon tau

Early embryonic development, implantation and maintenance
of a pregnancy are critically dependent on an intact embryo-
maternal communication. So far, only few signals involved in
this dialogue have been identified. In bovine and other
ruminants, interferon tau is the predominant embryonic
pregnancy recognition signal, exhibiting antiluteolytic activity.
However, this is just one aspect of the complex process of
embryo-maternal signalling, and a number of other systems
are more likely to be involved. To gain a more comprehensive
understanding of these important mechanisms, integrated
projects involving specialists in embryology, reproductive
biotechnology and functional genome research are necessary
to perform a systematic analysis of interactions between pre-
implantation stage embryos and oviduct or uterine epithelial
cells, respectively. State-of-the-art transcriptomic and proteo-
mic technologies will identify reciprocal signals between
embryos and their maternal environment and the respective
downstream reaction cascades. For in vivo studies, the use of
monozygotic twins as recipient animals provides elegant model
systems, thus eliminating genetic variability as a cause of
differential gene expression. In addition, suitable systems for
the co-culture of oviduct epithelial or endometrium cells with
the respective embryonic stages need to be established for
functional validation of candidate genes potentially involved in
the dialogue between embryos and their maternal environ-
ment. The knowledge of these mechanisms should help to
increase the pregnancy rate following embryo transfer and to
avoid embryonic losses. Candidate genes involved in embryo-
maternal communication will also be used to define new
quality criteria for the selection of embryos for transfer to
recipients. Another application is the supplementation of
embryotrophic factors or components of embryo-maternal
signalling in optimized formulations, such as bioartificial
matrices. As a long-term goal, signalling mechanisms identified
in bovine will also be functionally evaluated in other species,
including the human.
Scientific and Economic Implications of
Embryo-Maternal Communication in Bovine
In cattle, up to 40% of total embryonic losses occur
between days 8 and 17 of pregnancy, indicating that
early embryonic mortality is the main source of repro-
ductive wastage (Humblot 2001; Thatcher et al. 2001).
This high embryonic mortality rate has enormous
economic implications, increasing the number of days
open and retarding genetic progress.
The high failure rate to maintain a pregnancy is
generally assumed to be a consequence of insufficient
communication between the conceptus and the maternal
(IFNs) to inhibit uterine secretion of prostaglandin F2a
is critical to the establishment of pregnancy in cattle,
and the critical period for this signal appears to be
between days 15 and 17, when the endometrium in the
non-pregnant situation initiates a biochemical reaction
cascade finally resulting in luteolysis (Thatcher et al.
2001).
Although embryonic secretion of and uterine recep-
tivity for IFNs are undoubtedly key factors of preg-
nancy recognition and maintenance in cattle, other
factors may be involved that either modulate the IFNs
system or act independently of IFNs.
The advent of state-of-the-art transcriptomic and
proteomic technologies allows, for the first time, a
holistic analysis of mechanisms involved in signalling
between an embryo and its maternal environment
before implantation. This is undoubtedly one of the
most exciting processes in biology of reproduction and
has enormous practical implications. Reduction of
embryonic losses by either augmenting embryonic
pregnancy recognition signals or selecting the most
suitable recipient animal according to novel markers
of uterine receptivity would markedly reduce the cost
of cattle production (Binelli et al. 2001; Peterson and
Lee 2003). Moreover, identified signalling pathways
can be investigated in other species, such as the
human, where primary screens are hampered by the
lack of defined material. This review provides an
overview over known and candidate mechanisms of
embryo-maternal communication, introduces model
systems and techniques for a systematic study of
these processes, and discusses future perspectives for
scientific and practical exploitation of the expected
results.
Known Mechanisms of Pregnancy Recognition
in Ruminants
In ruminants, IFNs is well characterized as an import-
ant embryonic pregnancy recognition signal. IFNs is
encoded by multiple genes (Ealy et al. 2001) and is
expressed and secreted by trophectoderm cells of
blastocysts (reviewed in Roberts et al. 1990). Secretion
of IFNs by bovine blastocysts in vivo is highest between
days 15 and 17, whereas in a specific culture system for
bovine trophoblastic vesicles increasing IFNs secretion

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Embryo-Maternal Communication in Bovine
was observed for a longer period (up to day 23 after
fertilization) (Stojkovic et al. 1995).
Experiments comparing the effects of intrauterine and
systemic application of recombinant IFNs in sheep
demonstrated that IFNs reduces the expression of
uterine oestrogen and oxytocin receptors via paracrine
mechanisms, thus preventing oxytocin-induced pulsatile
secretion of prostaglandin F2a, thereby inhibiting luteo-
lysis (Spencer et al. 1999). Consequently, progesterone
secretion is maintained, inducing – among other genes –
the expression of the extracellular matrix (ECM) protein
osteopontin. Osteopontin is thought to support attach-
ment of the embryo, probably by linking integrin
heterodimers which are expressed by trophectoderm
cells and by the luminal epithelium of the endometrium
(Johnson et al. 2001a).
IFNs induces the expression of a number of genes,
such as STAT (signal transducer and activator of
transcription) 1 and 2 (Stewart et al. 2001a), b2 micro-
globulin (Vallet et al. 1991), IFN-regulatory factor 1
(IRF-1) (Spencer et al. 1998), ubiquitin crossreactive
protein (Johnson et al. 1999), Mx protein (Ott et al.
1998), granulocyte-chemotactic protein-2 (Teixeira et al.
1997), and 2¢,5¢-oligoadenylate synthetase (OAS) (John-
son et al. 2001b). Further, IFNs stimulates the expres-
sion of granulocyte-macrophage colony-stimulating
factor, a cytokine with putative positive effects on the
conceptus, in stroma cells of the endometrium (Emond
et al. 2000). Other effects of IFNs in endometrium cells
include a reduction of oxytocin-induced cyclooxyge-
nase-2 and prostaglandin F-synthetase expression (Xiao
et al. 1999). Thus, IFNs supports the maintenance of a
pregnancy via multiple mechanisms.
The signal transduction cascade of IFNs via type I
IFN-receptors involves STATs and IRFs. When IFNs
acts on luminal epithelial cells of the endometrium,
STATs 1, 2, 3, 5a/b and 6 are tyrosine-phosphorylated
within 30 min and translocated into the nucleus
(Stewart et al. 2001a). Upon longer exposure to IFNs,
STATs 3, 5a/b, and 6 are rapidly dephosphorylated.
IFNs induces homodimerization of STAT 1 to form
the transcription factor GAF (gamma-activated factor)
as well as heterodimerization of STAT 1 and STAT 2.
This heterodimer associates with IRF-9, forming the
transcription factor complex ISGF3 (IFN-stimulated
gene factor 3). Whereas GAF binds to gamma-activa-
ted sequence elements in the promoter region of
specific IFN-stimulated genes (e.g. IRF-1), the
ISGF3-complex stimulates expression of other IFN-
induced genes (e.g. ISG17, Mx and OAS) by binding to
IFN-stimulated response elements (ISREs). Stimulation
of STAT 1, STAT 2, IRF-9 and IRF-1 expression has
also been shown to involve ISGF3 and GAF tran-
scription factor complexes (Stewart et al. 2001a,b). The
IRF-2 was found to play an important role with regard
to the localization of IFN-induced gene expression in
different compartments of the endometrium (Choi
et al. 2001).
Inadequate reaction of the endometrium to IFNs or
insufficient secretion of IFNs by the conceptus are major
reasons for early embryonic losses and pregnancy
failure. Therefore, the level of IFNs secretion has been
discussed as a parameter for the assessment of embryo
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277
quality (Hernandez-Ledezma et al. 1993). Serum-free
cultured hatched blastocysts were found to contain
higher levels of IFNs mRNA than control embryos
cultured in the presence of serum (Wrenzycki et al.
1999). In long-term culture experiments we observed
reduced IFNs secretion by bovine nuclear transfer
embryos vs in vivo-derived or in vitro-produced (IVP)
controls (Stojkovic et al. 1999). Furthermore, IFNs
secretion in vitro was found to be higher in female than
in male bovine embryos (Larson et al. 2001).
Although many experimental findings indicate a
pivotal role of IFNs in pregnancy recognition of
ruminants, a number of other systems may be involved
in the embryo-maternal dialogue.
Selected Local Factors May Orchestrate
a Paracrine Cross-Talk between Oocytes
or Embryos and Their Maternal Environment
A precise synchronization of all parts of the female
reproductive tract is the essential prerequisite for
successful oocyte maturation, fertilization and embry-
onic development. Under the influence of gonadotro-
phins the mammalian oocytes reach their final
developmental stages accompanied by remarkable chan-
ges in gene expression within the ovarian follicles in vivo
(review by Adashi 1994). Recent studies are abridged
here to approach the outcome of oocytes becoming
viable embryos by use of transcript and protein char-
acterization as well as through localizing selected factors
in reproductive tissues of different mammalian species.
Sexual steroid hormones (oestradiol and progester-
one) serve as peripheral conductors leading to local
tissue- and time-specific differentiation events in the
oviduct as well as in the endometrium, accordingly
supporting the ovulated egg and the development of
embryos. The mammalian oviduct is known to be under
the influence of these sexual steroids, and changes of
progesterone and oestrogens in the bovine oviduct
throughout the oestrous cycle have been well described
(Wijayagunawardane et al. 1998). Remarkable cellular
changes during the sexual cycle lead to well prepared
reproductive epithelia expecting the ovulated oocyte or
early embryo. Recently, distinct changes in the expres-
sion of corresponding progesterone receptor and both
oestradiol receptors directly depending on peripheral
gonadotrophins (FSH or LH) could be characterized in
a cycle-specific manner for the bovine oviduct (Ulbrich
et al. 2003a). Interestingly, a hyaluronan (HA)-synthes-
izing and -binding system was also present in bovine
oviducts, probably interacting with the mature oocyte
(Ulbrich et al. 2003b) and with cleavage stage embryos
(see below). In addition, peptide hormones and growth
factors, small proteins acting predominantly in a para-
crine fashion, may be involved in early embryo-maternal
interactions. Several interesting candidate systems are
discussed in detail below.
Growth hormone
Pituitary growth hormone (GH) is assumed to control
growth and differentiation processes after birth. As
hypophysectomized mouse, rat and rabbit foetuses

278
display almost normal intrauterine growth (Gluckmann
et al. 1981), embryonic development has, for a long
time, been considered to be independent of GH. Recent
studies, however, have shown that GH exerts distinct
effects on development, differentiation and metabolism
of pre-implantation embryos. The development of
mouse and bovine embryos during in vitro culture was
clearly improved by supplementation of the medium
with GH (Drakakis et al. 1995; Izadyar et al. 1996;
Fukaya et al. 1998; Izadyar et al. 2000; Stojkovic et al.
2000; Moreira et al. 2002). Blastocysts cultured with
GH revealed increased cell numbers both in the inner
cell mass and trophectoderm (Kölle et al. 2002). The
potential role of GH as a factor of embryo-maternal
communication is substantiated by the fact that pre-
implantation embryos synthesize GH and its receptor
(GHR) in a stage-specific pattern. In the bovine, GHR
mRNA was detectable from the second day of embry-
ogenesis onwards (Kölle et al. 1998; Kölle et al. 2001a).
Interestingly, the highest levels of GHR mRNA were
found in morulae (day 6) when a functional GHR was
shown to be present (Kölle et al. 2001a). Supplementa-
tion of GH during in vitro culture significantly reduced
the number of apoptotic cells in 6–8-day-old embryos
(Kölle et al. 2002). Further, GH was found to be
involved in the regulation of the embryonic metabolism.
Bovine blastocysts cultured in serum-containing
medium are characterized by huge accumulations of
glycogen in the embryonic cells and in the intercellular
spaces. After culture with GH, glycogen was completely
eliminated from the embryonic cells. Furthermore, GH-
treated embryos displayed an increased exocytosis of
lipids (Kölle et al. 2001a). Altogether, the morphology
of bovine embryos cultured in vitro with GH was similar
to that of ex vivo embryos. This was also seen at the
ultrastructural level. Transmission electron microscopic
studies showed that embryos treated with GH – similar
to ex vivo embryos – mainly possess the typical
embryonic mitochondria with few cristae. In contrast,
embryos cultured in synthetic oviduct fluid medium
supplemented with polyvinylalcohol revealed predom-
inantly mature mitochondria with numerous cristae. In
addition, GH was shown to affect the number, size, form
and depth of zona pellucida pores (Kölle et al. 2001b).
Growth hormone itself is generally expressed in later
embryonic stages compared with GHR. GH mRNA was
detected in bovine blastocysts at day 8 (Kölle et al.
2001a). This implies that in the first days of embryo-
genesis the embryonic GHR is activated by maternal
GH, further supporting a role of the GH system in
embryo-maternal communication.
Insulin-like growth factors
The insulin-like growth factor (IGF) system comprises
an increasingly complex network of ligands (IGF-I and
IGF-II), receptors (type I IGF receptor ¼ IGF-IR,
type II IGF/cation-independent mannose-6-phosphate
receptor ¼ IGF-IIR), high-affinity binding proteins
(IGFBP-1 to -6), and specific proteases affecting their
biological activity, i.e. regulation of cell growth and
differentiation (for review, see Jones and Clemmons
1995; Rajaram et al. 1997). Components of the IGF
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E Wolf et al.
system are widely expressed in the female reproductive
tract, with maximum expression in the oviduct of IGF-
I, IGF-II, IGF-IR and IGFBP-3 mRNAs in the period
when gametes and embryos are in transit, eventually
creating an optimum environment for early embryonic
growth and metabolism (for review, see Watson et al.
1999).
Furthermore, expression of components of the IGF
system has been detected in pre-implantation embryos
from a variety of mammalian species, including mouse,
cattle and water buffalo (reviewed in Prelle et al. 2001).
Targeted inactivation of the genes coding for IGF-I
(Igf1), IGF-II (Igf2) and IGF-IR (Igf1r) in mice resulted
in an obvious phenotype (foetal growth retardation)
only in the second half of pregnancy (reviewed in
Efstratiadis 1998). These findings suggest that IGF-I
and IGF-II are not absolutely required during early
embryonic development, although detailed analyses for
quantitative effects have not been conducted.
Numerous studies evaluated effects of IGF supple-
mentation of culture media on early embryos from
various species (reviewed in Prelle et al. 2001). For
bovine embryos, variable effects of IGF peptides have
been described, depending on dose and on the respective
culture system used. Whereas Flood et al. (1993) could
not show a positive effect of either long R3IGF-I (LR3)
or IGF-II in bovine embryo culture, Herrler et al. (1992)
observed an increased proportion of development to
morula and blastocyst stages in a cumulus cell co-culture
system supplemented with 20% oestrous cow serum
(ECS) and with additional 50 ng IGF-I/ml. Similarly,
supplementation of serum (10% ECS)-containing
embryo culture medium with 100 ng IGF-I/ml stimula-
ted development and hatching of IVP bovine blasto-
cysts. However, IGF-I was not sufficient to replace the
effects of cumulus cell co-culture or serum supplemen-
tation (Palma et al. 1997).
In many biological systems, bioavailability and effects
of IGFs are modulated by IGFBPs, e.g. by competition
with IGF receptors for IGFs (for review, see Rajaram
et al. 1997; Schneider et al. 2000). Expression of
mRNAs coding for IGFBP-2, -3 and -4 was detected
by reverse transcriptase-polymerase chain reaction
(RT-PCR) analysis of large pools (n > 50) of bovine
embryos throughout development to blastocyst stages,
whereas IGFBP-5 transcripts were only detectable in
blastocysts. In contrast, IGFBP-1 and -6 mRNAs were
not detectable in early bovine embryos (Winger et al.
1997). This observation invited the concept that effects
of maternally derived IGFs might be modulated by
embryonic IGFBPs to support bovine pre-implantation
development (Winger et al. 1997). Using real time
RT-PCR assays, we were able to determine the abun-
dance of transcripts for IGFBPs and IGF-IR in pools of
only two blastocysts. These experiments demonstrated
that the mRNA levels for IGFBP-2 and IGFBP-5 as
well as for IGF-IR are regulated by exogenous IGF-I
peptides in pre-implantation embryos (Prelle et al.
2001). The present findings support the hypothesis that
pre-implantation bovine embryos modulate effects of
maternal IGFs by expression of IGFBPs and IGF-
IR, suggesting that the IGF system is a component of
embryo-maternal interactions.

Embryo-Maternal Communication in Bovine
Other growth factors
The bovine oviduct secretes many other growth factors,
including fibroblast growth factors (FGFs) (Gabler
et al. 1997) and vascular endothelial growth factor
(VEGF) (Gabler et al. 1999). Concentrations of FGF-
1 in oviductal flushings were found to be significantly
higher at ovulation than during the luteal phase, and
concentrations of FGF-2 were higher at the pre-ovula-
tory than at the post-ovulatory stage (Gabler et al.
1997). For VEGF, highest concentrations were found
during the preovulatory phase, which is in line with the
concept that VEGF may be involved in creating an
optimal local environment for fertilization and for the
developing embryo, e.g. by increasing vascular per-
meability (Gabler et al. 1999).
To evaluate the functional role of these growth factors
to support early embryonic development, they were used
as supplements for the culture of bovine embryos. FGF-
2 and transforming growth factor beta (TGFb) were
shown to act synergistically promoting bovine embryo
development in serum-free culture (Larson et al. 1992).
Supplementation of medium with VEGF improves the
in vitro maturation of bovine oocytes, acting synergis-
tically with FSH (Einspanier et al. 2002) and stimulates
initial embryonic development, depending on the pres-
ence of cumulus cells (Luo et al. 2002). These findings
suggest that the FGF and VEGF systems are involved in
early embryo-maternal interactions.
Hyaluronic acid system
The ECM polysaccharide hyaluronic acid (HA) is a
non-sulphated linear polymer of (1-b-4) D-glucuronic
acid and (1-b-3) N-acetyl-D-glucosamine. This glucos-
aminoglycan regulates cellular events such as signalling
and changes in gene expression, affecting proliferation,
differentiation, adhesion, migration, and morphogenesis
(reviewed in Fraser et al. 1997). These multiple effects
are mediated through interactions of HA with proteins,
including the HA-binding proteins or hyaladherins
(reviewed in Day and Prestwich 2002) as well as cell
surface and intracellular receptors: CD44, LYVE-1, and
CD168 or receptor for hyaluronic acid mediated
motility/intracellular HA-binding protein (RHAMM/
IHABP) (reviewed in Stojkovic et al. 2003).
As HA is a normal component of fluids of the
mammalian reproductive tract (reviewed in Stojkovic
et al. 2002), an important role for oocyte maturation,
fertilization, early embryogenesis, and the arrangement
of the reproductive tract for establishment of a preg-
nancy has been assumed. HA was shown to promote
differentiation of extraembryonic structures of mouse
embryos (Hamashima 1982), and when HA was added
to the culture medium the rates of implantation and
foetal development after transfer of mouse blastocysts
to recipients increased (Gardner et al. 1999). Accord-
ingly, positive effects of HA on development and cell
numbers of blastocysts were observed in bovine embryo
culture experiments (Stojkovic et al. 2002; Lane et al.
2003). In addition, large amounts of HA are synthesized
by cumulus cells (Stojkovic et al., unpublished data) and
pre-implantation bovine embryos. Newly synthesized
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279
HA was already detectable in two-cell stages, but
morula and, especially, hatching blastocyst stages were
found to secrete significantly more HA when compared
with other early embryonic stages (Stojkovic et al.
2003).
The effects of HA on early embryos may be mediated
by the principal HA receptors (CD44) which are
abundantly present in cumulus-oocyte-complexes and
pre-implantation embryos from several species
(reviewed in Stojkovic et al. 2003). In addition,
RHAMM/IHABP mRNA was detected and its abun-
dance was found to be regulated during bovine embryo
development in vitro (Stojkovic et al. 2003). The relative
levels of RHAMM/IHABP transcripts in bovine blasto-
cyst stages cultured in the presence of HA were higher
than in control embryos, suggesting that HA regulates
expression of RHAMM/IHABP. Increased RHAMM/
IHABP mRNA levels and HA secretion at the morula
stage could be related to the polarization and differen-
tiation processes finally resulting in trophectoderm and
inner cell mass cells of blastocysts. A marked increase in
HA synthesis at hatched blastocyst stage may be
involved in elongation of embryos, embryo-maternal
interaction and successful maintenance of pregnancy.
These mechanisms deserve further investigation.
In summary, a complex paracrine network of several
interacting factors is suggested to mediate the effects of
peripheral hormones through growth factors and com-
ponents of the ECM. These local regulatory networks
enable a coordinated cross-talk between gametes or
embryos, respectively, and their maternal environment,
thereby establishing the required conditions for success-
ful oocyte maturation, fertilization and early embryonic
development. Systematic functional dissection of such
paracrine events by appropriate molecular techniques
should gain the knowledge which is necessary to
improve protocols for assisted reproduction on both
the embryonic and the maternal side.
Compartmentalization of Embryo-Maternal
Interactions – the Mail Box Function of the
Extraembryonic Matrix
Initially, pre-implantation embryos are surrounded by
an extraembryonic matrix (EEM), generally known
as zona pellucida (Herrler and Beier 2000). Bovine
embryos hatch at 192–216 h post-insemination (hpi) and
thus spend about 50% of their pre-implantation devel-
opment within the zona pellucida (Betteridge 1995). The
embryonic genome becomes active already much earlier,
with a major wave of transcriptional activation at the
eight-cell stage (66–72 hpi). At that time the embryo is
still located in the oviduct, passing to the uterus by day
3.5. The embryo already synthesizes its own proteins
and seems to react to proteins secreted by the oviduct,
suggesting that embryo-maternal communication is
already established at this stage. Investigations demon-
strated, that the EEM is important for presenting,
accumulating and modulating signals between embryo
and maternal environment. Sonographic imaging stud-
ies of early embryos in utero revealed that the embryo
does not float in a recognizable volume of maternal

280
secretions, but is surrounded tightly by the endome-
trium. Therefore, the EEM represents the direct inter-
face between embryo and uterine epithelium, eventually
acting as a kind of ‘mail box’ of the embryo-maternal
communication (Herrler and Beier 2000).
After investigations on several species, it is known
that the EEM is not static, but evolves considerably
from the time of fertilization to hatching, especially in
rabbit and horse embryos (Dumont and Brummett
1985; Betteridge 1989; Denker 2000). The original zona
pellucida is formed as a three-dimensional network
structure (Sinowatz et al. 1998; Magerkurth et al. 1999).
By incorporating maternal and embryonic material, the
network gets tighter, but the meshes are still penetrable
for proteins/signals as big as 900 kDa (Hastings et al.
1972; Sellens and Jenkinson 1975; Guillomot and
Betteridge 1984). Therefore, the size of a signal molecule
is not limiting for its passage, but rather its hydrophobic
characteristics. Turner and Horobin (1997) demonstra-
ted that extremely hydrophilic proteins with low van der
Waals forces are not able to penetrate the EEM, while
lipophilic proteins with high van der Waals forces get
enriched in the matrix and proteins with a slightly
lipophilic or hydrophilic character and low van der
Waals forces easily penetrate the matrix. Consequently,
the simple detection of a protein in one compartment,
embryonic or maternal, does not directly mean that it is
able to penetrate the EEM and to act as a signal in the
embryo-maternal communication. Therefore, the speci-
fic characteristics and functions of the EEM have to be
considered as an intrinsic component of embryo-mater-
nal signalling mechanisms.
Investigating rabbit and horse extraembryonic
matrices it was realized, that the complexity of
embryo-maternal signalling increases dramatically as
development proceeds (Herrler and Beier 2000; Herrler
et al. 2002). At day 4.5 of rabbit embryo development,
the number of protein spots detectable by 2D poly-
acrylamide gel electrophoresis analysis of extraembry-
onic matrices increased by more than fourfold
compared with earlier stages. In the horse a steady
increase in the number of protein spots was detected
between days 8 and 12. Between 18 and 25% of those
proteins were of embryonic origin.
By Western blot analysis, several proteins were
identified. One of those proteins was IGFBP-3 which
was at least partly of embryonic origin (Herrler et al.
1997, 2002; Herrler and Beier 2000). As IGFBPs bind
IGFs with high affinity, IGFBP-3 in the EEM may
accumulate maternal IGFs in the immediate vicinity of
the embryo and promote its development by controlled
release of IGFs. The IGFs have been shown to be
survival factors supporting the development of preim-
plantation embryos (Herrler et al. 1998). In addition to
intact IGFBP-3, large amounts of IGFBP-3 fragments
were found in the EEM. Studies in sheep explained this
finding by the activity of maternal IGFBP-proteases
(Peterson et al. 1998), a known mechanism to release
IGFs into the embryonic microenvironment.
Another protein found in the rabbit EEM is maternal
haptoglobin (Herrler et al. 2001, 2002). Haptoglobin
has been shown to decrease the adhesion of leucocytes
to extracellular matrices (Nauta et al. 2002). Thereby,
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E Wolf et al.
maternal haptoglobin incorporated into the EEM may
cause maternal immunotolerance with regard to the pre-
implantation embryo.
Other proteins, such as heparin-binding epidermal
growth factor, P19 and uteroglobin, have also been
detected within the extraembryonic matrices of rabbit
and horse embryos (Herrler and Beier 2000; Herrler
et al. 2002) and may be involved in embryo-maternal
interactions.
Strategies for a Holistic Analysis
of Embryo-Maternal Communication
In vitro model systems
The milieu in the female reproductive tract is crucial
for mammalian reproduction. During pre-implantation
development the embryo is exposed to a markedly
changing environment (Leese 1988), and suitable in vitro
models of these processes are necessary to experiment-
ally address and understand the complex interactions
between embryos and their maternal environment.
Historically, in vitro cultured bovine embryos strug-
gled to pass the so-called ‘8–16-cell developmental
block’, a culture-related phenomenon during maternal-
to-zygotic transition (Wright and Bondioli 1981).
Oviducts from sheep (Eyestone et al. 1987) and rabbits
(Ellington et al. 1990) were used as temporary in vivo
culture system for IVP bovine embryos to overcome this
block. The general lack of understanding the develop-
mental block of bovine embryos led to the advent of
co-culture systems using somatic cells of the reproduc-
tive tract, typically oviduct epithelial cells (Gandolfi and
Moor 1987; Thibodeaux et al. 1992). Beneficial effects
have been attributed to the secretion of embryotrophic
factors as well as to a reduction in O2 tension and
glucose concentration (reviewed in Rief et al. 2002).
However, usually culture conditions are suboptimal,
resulting in dedifferentiation processes, i.e. loss of
morphological and functional properties of the oviduct
cells (reviewed in Reischl et al. 1999). Embryo co-culture
with oviduct cells has proved to be an inappropriate
system to produce bovine embryos for commercial
purposes, but it is a suitable model system for studying
embryo-maternal interactions. For this purpose, an
in vitro system has to mimic the in vivo environment
most closely and must meet the requirements of both
oviduct cells and co-cultured embryos. Dedifferentiation
of oviduct cells during culture can be inhibited in many
ways, including culture on matrigel (Joshi 1995) or
permeable cellulose nitrate matrices (Reischl et al.
1999). Furthermore, composition of medium and serum
supplementation have tremendous effects on cell mor-
phology and expression of cell type-specific marker
genes (Rief et al. 2002). As routine static culture in an
ordinary plastic Petri dish seems to be deleterious for
typical epithelial cell features, a perfusion culture system
using permeable cell supports (Reischl et al. 1999) was
established. Using sequential co-culture on oviduct
followed by endometrial cells the migration of the
embryo through the oviduct into the uterus before
implantation can be mimicked. Again, the most import-
ant prerequisite is to prevent dedifferentiation, i.e. loss
of physiological features. Culture of both uterine stroma

Embryo-Maternal Communication in Bovine
and epithelial cells together reflects the in vivo situation
more closely than separation of these cell types (Findlay
et al. 1990). In addition to morphological criteria,
expression profiling and functional studies are necessary
to monitor the physiological status of cultured oviduct
or endometrial cells, evaluating their suitability for
studying embryo-maternal interactions. Complementary
to suitable in vivo models, cell culture systems are
essential for functional studies of candidate genes and
their products which may be involved in the embryo-
maternal dialogue.
In vivo model systems
Embryo cloning techniques provide excellent possibilit-
ies to produce identical genotypes, improving animal
experimentation by eliminating genetic variation (Brem
1986). Significant advantages of using clones rather than
unrelated animals are the increase in power of statistical
tests and the reduction in the number of experimental
animals needed (Lamberson 1994). Thus, clones provide
an excellent in vivo model for holistic analyses of
embryo-maternal interactions.
The classical method for producing clones in mam-
mals is embryo splitting at the morula or blastocyst
stage. Monozygotic twins produced by this technique
are identical with respect to both nuclear and mitoch-
ondrial (MT) DNA. Phenotypic discordance is attrib-
utable to the uterine environment of different recipients
and to environmental influences. Overall, a high simi-
larity was observed in pairs of twins produced by
embryo splitting and transfer of the halves to different
recipients (Gärtner et al. 1991).
Nuclear transfer cloning using embryonic, foetal or
post-natal cells as nuclear donors allows for the
production of larger numbers of genomic copies (review
by Wolf et al. 1998). Although these animals share the
same nuclear DNA, their mtDNA may be different as it
is almost exclusively derived from the recipient cytoplast
(Steinborn et al. 2000; Hiendleder et al. 2003). This
problem can be overcome by using oocytes recovered
from maternally related donors, e.g. by ovum pick-up
(Brüggerhoff et al. 2002). Nevertheless, phenotypic
characteristics may be rather different between clone
siblings produced by nuclear transfer (Gärtner et al.
3 × 3 monozygous twin pairs, estrous cycle synchronized
Oviduct phase
(Day 3)
⇐ ⇒
Control Transfer
Fig. 1. In vivo model for the study
of embryo-maternal communica- Sampling, RNA extraction, production of subtracted cDNA libraries
tion in bovine
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281
1998; Renard et al. 2002). This is most likely due to
abnormal epigenetic reprogramming upon transfer of
the donor nucleus into the recipient cytoplast. Epigenetic
reprogramming involves changes in DNA methylation
(Dean et al. 2001) and many other mechanisms affecting
chromatin structure and functionality (reviewed by Shi
et al. 2003). Thus, true clones produced by embryo
splitting appear to be more suitable as in vivo models for
studying embryo-maternal communication.
For investigating developmentally important stages,
such as the oviduct phase (day 3), the early uterine phase
(day 8) or the peri-implantation period (day 18),
embryos can be transferred into the ipsilateral oviduct
or uterine horn, respectively, of a synchronized twin,
whereas the other twin, which is also synchronized,
receives a sham transfer and serves as control. After a
defined time period, the recipients are slaughtered, and
embryos as well as oviduct and uterine cells are
harvested and preserved for transcriptomic and prote-
omic studies as well as for histological investigation.
Comparisons are made between the ipsi- and contra-
lateral side within one recipient and between the
pregnant and the non-pregnant recipient of a twin pair.
Figure 1 shows an experimental outline for studying
embryo-maternal communication using monozygotic
twins as an in vivo model.
Transcriptomics
The biosynthesis of each protein starts with the trans-
lation of its coding mRNA. The methods currently
available to analyse gene expression at the level of
mRNA are much more sensitive than those used for
analyses at the protein level. Transcriptomics permits to
work with limited amounts of biological material and to
investigate the role of regulatory elements like tran-
scription factors which are mostly expressed at low
levels. Especially, the activity of rarely transcribed genes
is, in most cases, difficult to detect or not detectable by
the analysis of protein patterns.
Therefore, we focus on the investigation of changes in
gene expression at the level of the mRNA in the bovine
reproductive tract during early embryonic development
to identify genes which are involved in embryo-maternal
communication.
Early uterine phase Peri-implantation phase
(Day 8) (Day 18)
Control Transfer Control Transfer
 14390531, 2003, 4, Downloaded from https://onlinelibrary.wiley.com/doi/10.1046/j.1439-0531.2003.00435.x by University Of Missouri Columbia, Wiley Online Library on [13/12/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
282 E Wolf et al.

In bovine, where only few mRNA sequences are

known until now, systematic gene expression analyses
are more difficult than in human or mouse. Microarray
analyses using commercial arrays (Reese et al. 2001) or
cDNA collections like the UniGene collection are not
feasible, and digital methods such as Serial Analysis of
Gene Expression (SAGE) (Velculescu et al. 1995) are
likewise restricted to known genes or mRNA sequences.
To circumvent this problem in our studies of embryo-
maternal communication, a combination of subtracted
cDNA libraries and cDNA array hybridization was used
(Fig. 2). This approach reveals cDNA fragments of
differentially expressed genes which can be identified by
their sequence homology with known human or mouse
genes and permit the cloning of the bovine full-length
cDNA as well. The major advantage of using subtracted
libraries as the basis for cDNA array hybridization is
the enrichment of differentially expressed genes, redu-
cing the number of cDNA clones that need to be
analysed.
First of all, oviduct epithelial or endometrium cells
are isolated at defined stages of the sexual cycle. These
samples are used to prepare cDNA and to enrich
differentially expressed genes by Suppression Subtrac-
tive Hybridization (Diatchenko et al. 1996). This pro- Fig. 2. Workflow for transcriptome analysis of embryo-maternal
communication
cedure is based on a so-called suppressor PCR and
requires 0.5–1 lg mRNA. Approximately, 1500–3000
cDNA clones for each subtracted cDNA library are the generation of extensive gene expression profiles over
PCR-amplified and spotted onto nylon membranes. different time points during early development of the
Differentially expressed genes are identified by hybrid- bovine embryo and in different sections of the repro-
ization of these arrays with radioactively labelled ductive tract.
complex cDNA probes, comparison of signal intensities, In order to study mechanisms of embryo-maternal
and determination of the corresponding nucleotide communication in various important phases of devel-
sequences (Bauersachs et al. 2003). Database analyses opment we analyse gene expression profiles in samples
of the obtained sequences may provide information of oviduct epithelium (day 3) or endometrium (days 8
about the functions of the identified genes as far as they and 18) of twins from which one harbours an embryo
are known. Based on these results, an oviduct-/uterus- whereas the other serves as non-pregnant control (see
specific cDNA array is created and serves as a basis for above).

Fig. 3. Genes differentially regula-

ted in the ipsi- vs contra-lateral
bovine oviduct at day 3.5 after
standing heat (Bauersachs et al.
2003)

Embryo-Maternal Communication in Bovine
As a first proof of concept, the bovine oviduct
epithelium was analysed at day 3.5 of the oestrous cycle
and searched for genes differentially regulated in the ipsi-
and contra-lateral oviduct. More than 30 differentially
expressed genes were identified, which could be assigned
to different functional categories (Fig. 3, Bauersachs
et al. 2003). This study demonstrates the feasibility of
our approach. Investigations of gene expression profiles
in oviduct or endometrium cells isolated at oestrus vs
diestrus are underway.
Proteomics
The analysis of mRNA levels in cells is one of the most
sensitive strategies to explore complex biochemical
networks of biological systems. However, it has to be
considered that mRNA molecules are intermediate
products in the synthesis of functional proteins. It has
Tissue of
pregnant animals
Solubilization of proteins
comparison of the gels
Fig. 4. General strategy for 2-D
gel-based proteomics
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283
been shown that the correlation between mRNA level
and protein level is not sufficient to predict the level
of active proteins. Furthermore, proteins frequently
undergo post-translational modifications (PTMs) which
are crucial for their function, stability and activity.
Protein–protein interactions and PTMs are not at all
reflected at the mRNA level (Gygi et al. 1999b).
Therefore, besides transcriptional analysis, quantitation
of protein content in cells is indispensable to assess
biochemical processes of high complexity. The global
analysis of cellular proteins, including PTMs, is referred
to as proteomics. The focus of the present work is the
identification of proteins involved in embryo-maternal
communication. For this purpose we combined three
independent proteomic approaches. Two of them are
holistic, meaning that the overall protein composition of
tissues with relevance for embryo-maternal communi-
cation will be investigated de novo, i.e. without the need
Tissue of non-
pregnant animals
from tissue
2D-gel
electrophoresis
and computational
Isolation of spots
of interest and
digestion of the proteins
Identification of proteins
with MS-based techniques

284
for any biochemical pre-information. In contrast,
the third approach makes use of the transcriptional
pre-information achieved in the extremely sensitive
transcriptome analysis and addresses the proteins cor-
responding to significantly altered mRNAs in a well-
directed manner.
The first approach uses highly sophisticated proteo-
mics techniques to analyze and compare the entire
protein composition of cells in reproductive tissue
samples of pregnant and non-pregnant animals. We
apply classic techniques of proteomics, such as 2-D gel
electrophoresis (so far the only method that is able to
separate up to 10 000 proteins and their modifications)
(Klose 1975; O’Farrell 1975) in combination with the
identification of proteins by mass spectroscopy (MS).
The overall strategy is outlined in Fig. 4. In order to
obtain a maximum yield of separated proteins paired
with highest reproducibility and sensitivity in the quan-
titation of each protein spot, experimental protocols for
each type of tissue are optimized, especially the solubi-
lization and separation parameters. A set of 2-D gels of
the reproductive tissue samples from each pregnant and
non-pregnant animal (at least five gels per sample and
biological stage) is produced. The gels are computa-
tionally analysed in order to identify differences in both
the spot positions and intensities. Protein spots of
interest are excised and digested with a proteolytic
enzyme to produce a set of peptides unique for each
protein. The corresponding protein is then identified
with a technique called Peptide Mass Fingerprint. This
technique is based on exact measurement of the peptide
masses of the digested protein by MS, followed by a
comparison of these values with theoretical peptide
masses in the databases of known proteins (for a more
detailed description of the technique, see Gevaert and
Vandekerckhove 2000). A problem of this very fast and
effective technique concerning bovine proteins is the
fact, that only about 1400 bovine proteins are known so
far (Swiss-Prot Release 40.42, 31-Jan-2003). As a
consequence, in case of unknown proteins, we will
separate the peptides by microscale capillary chroma-
tography and sequence them by electrospray ionization
tandem mass spectrometry (ESI-MS/MS) (Metzger
et al. 1994) to obtain sequence tags suitable for identi-
fication.
In a second holistic approach, a recently developed
method called Isotope-Coded Affinity Tag (ICAT)
(Gygi et al. 1999a) is used. This method combines the
MS-based identification with accurate MS-based quan-
titative comparison of the protein patterns of two
complex mixtures. The method consists of four steps
(Fig. 5). First, one protein mixture (e.g. sample from a
pregnant animal) is labelled with ICAT reagent (consists
of biotin, a linker and a thiol-specific reactive group),
while the other mixture (e.g. sample from a non-
pregnant animal) is labelled with the ICAT reagent
containing different isotopes (e.g. 2H vs 1H or 13C vs
12
C). Secondly, the mixtures are pooled and proteins are
digested using a proteolytic enzyme to produce small
fragments. Thirdly, the labelled peptides are recovered
by affinity chromatography (with avidin columns) and
finally separated and analysed by ESI-MS/MS. The
relative quantitation is carried out by comparing the
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E Wolf et al.
Proteins of Proteins of
tissue 1 tissue 2
ICAT label He
1
H) av
h t( y( 2
Lig H)
Combine
Proteolytic Affinity
cleavage chromatography
LC-MS/MS
∆ ∆
m/z
Pair finding and quantitation
Fig. 5. Principle of the Isotope-Coded Affinity Tag (ICAT) technology
intensities of corresponding MS signals of the light and
the heavy version of labelled peptides. With the two
methodologies, classical 2-D gels and ICAT technique,
it is intended to find the differences in protein expression
in the reproductive tissues due to biochemical signals
between embryos and their maternal environment.
The third approach is able to link results obtained at
the transcriptional level to the protein level for individ-
ual proteins. The position of a protein on 2-D gels
cannot be predicted from genomic data due to process-
ing and unknown PTMs. As a consequence, to link
transcriptomic data to proteomic data, a specific probe
for the efficient detection of each protein of interest is
necessary. The most specific and sensitive probes to
detect proteins are antibodies. However, the main
problem of the antibody production is that, in most
cases, the corresponding protein for immunization
purposes is not available. To avoid this problem the
so-called peptide-induced antibodies (PIAs) (Arnold
and Fröhlich 1998; Fröhlich and Arnold 1999) are
generated against proteins coded by genes found differ-
entially expressed in the maternal environment during
early embryonic development. The general strategy of
PIA production is outlined in Fig. 6. In the first step
amino acid sequences are deduced from the correspond-
ing gene or cDNA sequence and unique peptides
containing appropriate antigenic determinants are
 14390531, 2003, 4, Downloaded from https://onlinelibrary.wiley.com/doi/10.1046/j.1439-0531.2003.00435.x by University Of Missouri Columbia, Wiley Online Library on [13/12/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Embryo-Maternal Communication in Bovine 285

Sequence of differentially Tissue

expressed gene
atggcctggt ttgccctcta cctcctgagc cttctctggg
ctacagctgg gactagtacc cagacccaga gttcatgctc
cgttccctca gcacaggagc ccttggtcaa tggaatacaa
.......

Selection of
In silico translation antigenic peptide

MAWFALYLLSLLWATAGTSTQTQSSCSVPSAQEPLVNGIQ Protein extraction

MNLAGAYNLKAQKLLTYQLMSSDNNDLTIGHLGLTIMALT
APSSPNAEASAFYGPSLAILALCQKNSEATLPIAVRFAKT
........

Synthesis of
the antigen

IM WATAGTSTQTQS

Immunization 2D-electrophoresis
of mice

2D-Western blot

Fig. 6. Concept of transcriptome

to proteome linkage

selected. In the next step we synthesize these peptides
coupled to antigenic immunomodulators and produce
antisera by immunization of BALB/c mice. Antisera are
tested against the antigen in an ELISA assay and the
corresponding protein is identified on the Western blot
of a standardized 2D gel of proteins from the appro-
priate bovine tissue. To confirm the correct identity of
the protein the detected spot is excised and analysed
with MS-based techniques. With these antibodies the
corresponding proteins are detected in a very fast
(ELISA-analysis and 1D Western blotting) and sensitive
(lower fmol range) way, and the presence of different
protein amounts is verified in the reproductive tissue
samples of pregnant and non-pregnant animals. Finally,
the antisera can be used for other studies e.g. for the
subcellular localization by immunohistochemical meth-
ods or to enrich the corresponding protein by immuno-
affinity purification or immunoprecipitation.
Future Perspectives
The proposed combination of in vitro and in vivo
systems with state-of-the-art analytical tools of tran-
scriptomic and proteomic research should reveal a
plethora of candidate genes and gene products which
may be involved in embryo-maternal interactions. The
major challenge will then be to select the functionally
relevant candidate genes and gene products and exploit
them scientifically and economically.
For target validation, a particular candidate gene
needs to be overexpressed or its expression needs to be
knocked down, depending on the direction of gene
regulation observed during embryo-maternal interac-
tion. The advent of suitable in vitro systems (e.g. Rief
et al. 2002) together with recent developments in vector
design (review by Kaufman 2000) as well as non-viral
(review by Davis 2002) and viral gene transfer methods
(review by Palu et al. 2000) will facilitate this target
validation process. The functional knock-down of spe-
cific genes by using RNA interference (RNAi) is
becoming an increasingly important tool for the study
of gene functions in mammalian cells (review by
Hannon 2002).
As a long-term goal, signalling mechanisms identified
in bovine will also be functionally evaluated in other
species, including the human. This will identify con-
served pathways of embryo-maternal communication
and unravel species-specific differences.
Biotechnological applications in animal breeding
include strategies to augment important signals in the
embryo-maternal dialogue, increasing the pregnancy
rate following embryo transfer and avoiding embryonic
losses. Important embryonic pregnancy recognition
signals could be expressed as recombinant proteins
and added to the transfer medium. Recombinant IFNs
administered locally into the uterus of cows prolonged
the life span of the corpus luteum (Meyer et al. 1995).
However, studies on the effect of this protein in a larger

286
number of cows, and under practical conditions, need to
be carried out (Binelli et al. 2001).
Candidate genes involved in embryo-maternal com-
munication may also be used to improve the conditions
for in vitro production of embryos, thereby avoiding
culture artefacts which may lead to large offspring
syndrome and other pathological changes associated
with embryo culture (reviewed in McEvoy et al. 2000).
The role of epigenetic changes induced by inappropriate
culture conditions and the consequences for embryo
development are an increasingly important research
topic (reviewed in Shi et al. 2003).
Gene products which are important for embryo-
maternal communication may also be used to define new
quality criteria for the selection of embryos for transfer
to recipients. A potential scenario could be the recovery
of an embryo biopsy at the blastocyst stage followed by
RNA extraction, reverse transcription, amplification
and labeling of cDNAs which are then analysed by array
hybridization. A feasibility study has recently been per-
formed using mouse embryos (Brambrink et al. 2002).
In summary, the holistic analysis of embryo-maternal
interactions in the preimplantation period has a plethora
of implications for basic science and biotechnology. This
field is more likely to evolve to one of the most
important topics in biology of reproduction.
Acknowledgements
Our studies on embryo-maternal communication are suppor-
ted by the Deutsche Forschungsgemeinschaft (Research Unit
‘Mechanisms of embryo-maternal communication’; FOR
478/1).
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Submitted: 22.02.2003
Author’s address (for correspondence): Eckhard Wolf, Institut für
Molekulare Tierzucht, Genzentrum der Ludwig Maximilians-Univer-
sität, Feodor-Lynen-Str. 25, 81377 München, Germany. E-mail:
ewolf@lmb.uni-muenchen.de