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Review 1
Bauersachs S et al. Transcriptome Profi ling of Endometrium … Exp Clin Endocrinol Diabetes 2008 ; 116: 1 – 14
ECED/898/23.5.2008/Macmillan
received 05.12.2007
fi rst decision 14.02.2008
accepted 11.04.2008
Bibliography
DOI 10.1055/s-2008-1076714
Exp Clin Endocrinol Diabetes
2008 ; 116: 1 – 14
© J. A. Barth Verlag in
Georg Thieme Verlag KG
Stuttgart · New York
ISSN 0947-7349
Correspondence
Prof. Dr. E. Wolf
Gene Center
Feodor-Lynen-Str. 25
81377 Munich
Germany
Tel.: + 49 / 89 / 2180 768 00
Fax: + 49 / 89 / 2180 768 49
ewolf@lmb.uni-muenchen.de
Key words
䊉
䉴
Bos taurus
䊉
䉴
transcriptome
䊉
䉴
uterus
䊉
䉴
embryo-maternal
communication
䊉
䉴
fertility
Transcriptome Studies of Bovine Endometrium Reveal
Molecular Profi les Characteristic for Specifi c Stages of
Estrous Cycle and Early Pregnancy
secretory cells, the latter producing estral mucus.
The uterine glands are elongated as a result of
the mucosal edematization [3] . Furthermore,
estradiol causes a rapid and high increase of
luteinizing hormone (LH) release [4] , which is
necessary for ovulation [5] and formation of the
corpus luteum. The LH surge is preceded by the
decrease of estradiol. Following ovulation pro-
gesterone levels increase slowly during the 3
days period of metestrus in the course of the for-
mation of the new corpus luteum (CL). Mucosal
edema and contractility of the smooth muscula-
ture are declining during metestrus. During die-
strus the surface epithelial cells are fl at and the
highly active and proliferating uterine glands
secrete uterine milk or histotroph [3] . The CL
evolves its complete function and produces pro-
gesterone and oxytocin. The prolonged action of
progesterone on the uterus leads to the down-
Introduction
&
During estrous cycle and pregnancy the bovine
endometrium undergoes marked morphological
and functional changes. Several hormones are
involved in these processes, most importantly
the steroid hormones progesterone (P4) and
estrogen [1] . On the basis of hormone profi les
and functional changes in the endometrium, the
bovine estrous cycle can be divided into four
stages: preestrus, estrus, metestrus and diestrus.
At preestrus and estrus, P4 levels are low and the
dominant follicle produces estrogens. This leads
to cell proliferation and increased ribosomal RNA
and DNA synthesis [2] . Furthermore, estradiol
increases blood circulation and edematization of
the mucosa, and the smooth musculature shows
high contractility. The highly cuboidal endome-
trial epithelium consists of both ciliated and
Authors S . B a u e r s a c h s
1
,
2
, K . M i t k o
1
, S . E . U l b r i c h
3
, H . B l u m
1
, E . W o l f
1
,
2
Affi liations
1
Laboratory for Functional Genome Analysis ( LAFUGA ), Gene Center, LMU Munich, Munich, Germany
2
Chair for Molecular Animal Breeding and Biotechnology, Gene Center, LMU Munich, Munich, Germany
3
Physiology Weihenstephan, TU Munich, Weihenstephan, Germany
Abstract
&
The endometrium undergoes marked functional
changes during estrous cycle and pregnancy. As
the adjacent environment of the conceptus, it
represents the maternal interface for embryo-
maternal communication, which is essential to
maintain pregnancy. Transcriptome studies pro-
vide the unique opportunity to assess molecular
profi les changing in response to endocrine or
metabolic stimuli or to embryonic pregnancy
recognition signals. Here we review the cur-
rent state of transcriptome profi ling techniques
and the results of a series of transciptome stud-
ies comparing bovine endometrium samples
during the estrous cycle or endometrium sam-
ples from pregnant vs. non-pregnant animals.
These studies revealed specifi c mRNA profi les
which are characteristic for the functional sta-
tus of the endometrium. Transcriptome studies
of endometrial samples recovered during the
pre-attachment period identifi ed many inter-
feron-stimulated genes, genes that are possibly
involved in embryo-maternal immune modula-
tion ( C1S, C1R , C4 , SERPING1 , UTMP , CD81 , IFITM1 ,
BST2 ), as well as genes affecting cell adhesion
( AGRN , CD81 , LGALS3BP , LGALS9 , GPLD1 , MFGE8 ,
and TGM2 ) and remodeling of the endometrium
( CLDN4 , MEP1B , LGMN , MMP19 , TIMP2 , TGM2 ,
MET , and EPSTI1) . The results of these transcrip-
tome studies were compared to those of similar
microarray analyses in human, mouse and Rhe-
sus monkey to identify similarities in endome-
trial biology between mammalian species and
species-specifi c differences. Future studies will
cover dynamic transcriptome changes between
different stages of early pregnancy, the relation-
ship between metabolic problems in dairy cows
and the functionality of reproductive tissues as
well as endometrium transcriptome profi les
in recipients of somatic cell nuclear transfer
embryos.
Review2
Bauersachs S et al. Transcriptome Profi ling of Endometrium … Exp Clin Endocrinol Diabetes 2008 ; 116: 1 – 14
ECED/898/23.5.2008/Macmillan
regulation of its own receptor in the luminal epithelium (LE) and
the superfi cial glandular epithelium (sGE) resulting in derepres-
sion of estrogen receptor alpha ( ESR1 ) and oxytocin receptor
( OXTR ) genes [1] . Binding of oxytocin to its receptor, which is
up-regulated in the LE of the endometrium from day 15 of the
estrous cycle, initiates the pulsatile secretion of prostaglandin
F
2
(PGF
2
). PGF
2
is produced from membrane phospholipids by
phospholipase A2, cyclooxygenase, and prostaglandin synthase
and is responsible for luteolysis [6, 7] . LH, in a paracrine manner,
also takes part in this process [8] . Progesterone levels decrease
in the course of luteolysis. The pituitary follicle-stimulating hor-
mone (FSH) causes growth of follicles on the ovary, which pro-
duce estrogens that in turn stimulate the production of FSH in a
positive feedback loop [4] . FSH together with LH then stimulates
one follicle to grow as dominant follicle, which releases inhibin
to inhibit growth of other subordinate follicles. The high level of
LH during this stage of the cycle primarily stimulates the matu-
ration of the dominant follicle, which produces high amounts of
estradiol responsible for the LH surge preceding the ovulation.
In case of a successful fertilization, trophectoderm cells of the
bovine blastocyst are secreting interferon tau (IFNT). The expres-
sion of IFNT rises dramatically in the course of the elongation of
the blastocyst with highest levels at day 17 of gestation. IFNT
acts on the endometrium via reducing the expression of uterine
estrogen and oxytocin receptors in the luminal epithelium, pre-
venting the pulsatile release of PGF
2
and luteolysis, thus facili-
tating the maintenance of pregnancy [9] .
Transcriptome Profi ling – Current Techniques and
Future Developments
&
Mammalian genomes contain approximately 23,000 protein-
coding genes. The number of individual transcripts found in
mammalian transcriptomes is signifi cantly higher due to tran-
script diversity and the fast growing world of non-coding RNAs
[10] . Various analytical approaches have been developed to pro-
fi le transcriptomes [11] . Currently, the most powerful technolo-
gies are hybridization-based or sequencing-based, both able to
generate comprehensive genome-wide expression profi les. The
most widespread approach is currently the microarray technol-
ogy. Gene-specifi c nucleic acids are immobilized as probes on
solid supports and hybridized with labeled nucleic acids derived
from mRNA. The amount of bound label corresponds to the
concentration of the corresponding transcript in the biological
sample. There are various microarray platforms with different
strategies to analyze gene expression, each of them suited for
particular needs [12, 13] . Custom-made arrays based on spotted
cDNAs or oligonucleotides are well suited to study gene expres-
sion, especially if no commercial arrays are available. Commer-
cial genome-wide platforms offer measurement of expression of
all known genes of many model organisms and also comprehen-
sive arrays for several domestic animals. Despite the high per-
formance of microarray-based approaches, the reliability of
measurements was often challenged due to inconsistent results
that have been reported with different microarray platforms.
However, the MicroArray Quality Control (MAQC) project has
recently investigated a variety of microarray platforms and
alternative technology platforms [14] . Reproducible measure-
ment of gene expression at multiple test sites was achieved as
well as high concordance with different platforms. Detailed
analyses were done to compare performance of one- and two-
color systems [15] . Furthermore, the performance of fi ve com-
mercial microarray platforms and the impact of different
normalization methods for expression data were examined [16] .
In order to determine the reliability of the quantifi cation of gene
expression, DNA microarray results were evaluated with three
different quantitative gene expression platforms [17] . High cor-
relation was observed between quantitative gene expression
values and microarray platform results. The only shortcoming of
the different microarray platforms was the limited and variable
sensitivity for detection of rare transcripts, which interferes
with the reproducible measurement of differentially expressed
genes. Nevertheless, optimization and standardization of meth-
ods have enhanced the reproducibility of the results across plat-
forms [18] . In humans, the microarray technology has been
successfully used for the analysis of endometrium during the
window of implantation and has provided remarkable insight
into endometrial maturation and implantation [19] .
The resolution of microarray-based analyses of the transcrip-
tome was further enhanced by the development of exon arrays.
These arrays measure the expression of individual exons of each
gene and enable the genome-wide identifi cation of differential
splicing [20] . Detailed studies of mammalian transcriptomes
revealed, that they are much more complex than previously
assumed and contain a vast amount of non-coding RNAs (ncRNAs)
[21, 22] . In order to identify ncRNAs, a series of high-density til-
ing microarrays was constructed, which represent sense and
antisense strands of the entire non-repetitive sequence of the
human genome [23] . This unbiased approach for the genome-
wide study of the transcriptome detected more than ten
thousand of so far undiscovered transcribed sequences in
addition to transcripts of known and predicted genes. These
new technological developments will also enter the fi eld of
endometrial biology and lead to new insights into regulatory
mechanisms during the sexual cycle and the process of
embryo implantation.
The fi rst high-performance sequencing platform was Massive
Parallel Signature Sequencing (MPSS) [24] . A sensitivity of a few
mRNA molecules per cell was achieved by counting the frequen-
cies of millions of short mRNA-derived sequence tags. Despite
its excellent performance, the MPSS approach has never gained
broad acceptance, presumably due to its complexity and its spe-
cial requirements. In the meantime, important advances have
been made in speeding up DNA sequencing and cutting the
costs. The fi rst “ next generation ” system, the 454 Genome
Sequencer FLX is based on sequencing by synthesis using a pyro-
sequencing protocol optimized for solid support and picoliter-
scale volumes [25] . It achieves approximately a 100-fold increase
in throughput over the current Sanger sequencing technology.
ABI ’ s SOLiD system works with a high-density bead array and
sequencing by ligation and generates up to several gigabases per
run ( www.solid.appliedbiosystems.com ). Illumina ’ s 1G Genome
Analyzer uses solid phase amplifi cation to create an ultra-high
density sequencing fl ow cell with more than 30 million clusters.
These are sequenced by synthesis with reversible terminators
containing removable fl uorescence ( www.illumina.com ). A
short survey of current developments of next generation
sequencing systems is presented in [26] . Due to their high per-
formance, these new technologies are particularly suited for the
analysis of mammalian transcriptomes and will enable the
detection of rare transcripts in complex tissues like the
endometrium. For such domestic animals, where no commercial
arrays or large cDNA collections are available, the next-genera-
Review 3
Bauersachs S et al. Transcriptome Profi ling of Endometrium … Exp Clin Endocrinol Diabetes 2008 ; 116: 1 – 14
ECED/898/23.5.2008/Macmillan
tion sequencing technologies will be an attractive alternative to
microarrays and could provide sequence information for the
design of genome-wide microarrays as well.
Transcriptome changes in bovine endometrium
during the estrous cycle
&
Although the basic principles of hormonal regulations in the
endometrium during the estrous cycle are known, the detailed
molecular mechanisms are not well understood. To get a fi rst
insight into such mechanisms two analyses of transcriptome
changes in bovine intercaruncular endometrium during the
estrous cycle were performed. In the fi rst study late estrus
(day 0, low progesterone) and diestrus phase (day 12, high pro-
gesterone) were compared using a combination of subtracted
cDNA libraries and cDNA array hybridization [27] . This study
revealed 133 genes showing at least a two-fold change of their
mRNA abundance, 65 with higher levels at estrus and 68 with
higher expression at diestrus. In the second study endometrium
samples were analyzed derived from several stages of the estrous
cycle: estrus (day 0), metestrus (day 3.5), diestrus (day 12), late
diestrus (slaughtered at day 18, high serum progesterone levels),
and preestrus (slaughtered at day 18, low serum progesterone
levels) [28] . For the generation of mRNA expression profi les a
bovine oviduct and endometrium (BOE) array [29] was used,
which was developed based on a series of differential gene
expression studies in endometrium (different stages of the
estrous cycle, day 15 and day 18 pregnant vs. non-pregnant)
[27, 30, 31] and in oviduct epithelial cells (different stages of the
estrous cycle) [32, 33] . Analysis of expression data revealed 269
genes with signifi cant changes in their transcript levels during
the estrous cycle in distinct temporal patterns. Two major types
of expression profi les were observed that showed highest mRNA
levels during the estrous phase or highest levels during the
luteal phase, respectively. A minor group of genes exhibited
highest mRNA levels on day 3.5. The number of differentially
expressed genes during the estrous cycle was comparable to
those found in similar studies in mouse, Rhesus monkey and
human, which have been done with high-density cDNA or oligo-
nucleotide arrays, respectively [34 – 36] . Gene Ontology classifi -
cation of the genes with known function characterized the estrus
time by elevated expression of genes related to focal adhesion
formation, cell motility, cytoskeleton, extracellular matrix
(ECM), ECM remodeling, and cell growth.
Remodeling of extracellular matrix
Coordinated regulation of ECM remodeling can be inferred from
the expression profi les of matrix metallopeptidase 2 (gelatinase
A, 72 kDa gelatinase, 72 kDa type IV collagenase, MMP2 ) and
meprin A, beta ( MEP1B ) mRNAs coding for proteases involved in
ECM degradation [37] with highest levels at day 3.5 and during
the luteal phase, respectively. Expression profi les of TIMP metal-
lopeptidase inhibitors 1 and 2 ( TIMP1, TIMP2 ) mRNAs were
opposite to MMP2 with lowest levels at day 3.5. Messenger RNA
levels of most collagen genes and other constituents of the ECM
were similar from day 18P4L to day 3.5 and low at days 12 and
18P4H. As a potential regulator of ECM remodeling the HtrA ser-
ine peptidase 1 ( HTRA1 ) mRNA was identifi ed (expression pro-
fi le similar to mRNAs of fi brillar collagens), which codes for a
serine protease that is activated by C-propeptides of fi brillar col-
lagens [38] and has been shown to cleave transforming growth
factor-beta 1 (TGFB1) and other members of the TGF-beta family
[39] . In addition, expression of HTRA1 has been shown in human
placenta at the maternal-trophoblast interface [40] . The mRNA
expression profi les of all these genes suggest a complex regula-
tion of ECM remodeling in bovine endometrium during the
estrous cycle.
Regulation of invasive growth
In addition to genes related to ECM remodeling, a number of
genes with higher mRNA levels at estrus were identifi ed that
have been described in the context of positive regulation of
invasive processes, such as galectin-1 ( LGALS1 ), tenascin C ( TNC ),
osteonectin ( SPARC ), prohormone convertase 5 ( PCSK5 ), gastrin-
releasing peptide ( GRP ), annexin A2 ( ANXA2 ), and nephroblast-
oma overexpressed gene ( NOV ). The decreased mRNA levels of
these genes during the luteal phase may be associated with reg-
ulation of non-invasive implantation in cattle. Genes related to
the process of invasive growth that showed higher mRNA levels
in the bovine endometrium during the luteal phase were also
found. Some of these genes have been described as negative
regulators of invasive growth, e.g. tissue plasminogen activator
( PLAT ) [41] and cystatin M ( CST6 ) [42] .
Angiogenesis and regulation of blood fl ow
The process of angiogenesis and regulation of blood fl ow has
also an important role in the context of endometrial remodeling
during the estrous cycle and the specifi c functions of the
endometrium. Several genes related to this process were found
as differentially expressed, such as angiopoietin-like 2 ( ANGPTL2 ),
endothelial tyrosine kinase ( TEK , angiopoietin receptor TIE-2),
and neuropeptide Y ( NPY ) with higher mRNA levels at estrus and
ephrin-A1 ( EFNA1 ), endothelial differentiation, sphingolipid G-
protein-coupled receptor 3 ( EDG3 ), angiotensinogen (AGT ),
endothelial PAS domain protein 1 ( EPAS1 , Hypoxia-inducible
factor 2 alpha) and Kruppel-like factor 5 ( KLF5 ) with higher
mRNA levels during the luteal phase. ANGPTL2 is related to the
angiopoietins and has been described as regulator of endothelial
cell growth [43] . NPY is a potent angiogenic factor as well as a
stimulator of vascular smooth muscle proliferation [44] . AGT
codes for the progenitor of angiotensin II, which is a potent vaso-
constrictor and was described to regulate feto-placental angio-
genesis in the ovine placenta [45] . EPAS1 and KLF5 code for
transcription factors controlling the expression of VEGF (vascu-
lar endothelial growth factor), FLT1 ( VEGFR1 , vascular endothe-
lial growth factor receptor), KDR ( FLK1 / VEGFR2 , vascular
endothelial growth factor receptor 2), and TEK [46] and of PDGF ,
EGR1 (early growth response 1), SERPINE1 ( PAI1 , plasminogen
activator inhibitor type 1), NOS2A ( INOS , inducible nitric oxide
synthase 2A), and VEGF receptor genes [47] , respectively. KLF5 is
down-regulated with vascular development [47] which is in line
with the increased mRNA levels of KLF5 in bovine endometrium
during the luteal phase and its target TEK at estrus.
Metabolic and transport processes
During the luteal phase elevated concentrations of mRNAs
coding for a variety of proteins involved in metabolic and trans-
port processes were identifi ed. Elevated transport and metabo-
lism during the luteal phase may indicate increased secretion
of nutrients necessary for the development of an embryo [48] .
In this context elevated mRNA levels of mitochondrial aconi-
tase 2 ( ACO2 ), oxoglutarate dehydrogenase ( OGDH ), and isoci-
trate dehydrogenases 1 and 2 ( IDH1 , IDH2 ), which are involved
Review4
Bauersachs S et al. Transcriptome Profi ling of Endometrium … Exp Clin Endocrinol Diabetes 2008 ; 116: 1 – 14
ECED/898/23.5.2008/Macmillan
in the TCA-cycle, were observed during the luteal phase. Fur-
thermore, genes encoding different transporters were identi-
fi ed as upregulated during the luteal phase, such as the solute
carrier family members SLC1A1 , SLC11A2 , and SLC16A1 . The
expression of SLC1A1 , coding for a glutamate transporter, has
been detected in human placenta and suggested to be involved
in active transport of glutamate between the fetal and mater-
nal blood circulation [49] . In addition, elevated mRNA levels of
SLC1A1 have been found in human endometrium during the
secretory phase in two microarray studies ( Table 1 ). Likewise,
expression of SLC11A2 , encoding a metal ion transporter has
been observed in human placenta [50] . SLC16A1 codes for
monocarboxylate transporter 8 (MCT8) described as a power-
ful and specifi c thyroid hormone (TH) membrane transporter
with an important role in TH transport during fetal develop-
ment and a specifi c role in human placental development [51] .
Furthermore, mRNAs coding for selenoprotein P (SEPP1), a
selenium-supply protein [52] , and ectonucleotide pyrophos-
phatase / phosphodiesterase 1 (ENPP1), a regulator of extracel-
lular pyrophosphate concentrations [53] , were identifi ed.
Analysis of pathway databases, interaction networks, and litera-
ture data mining revealed physiological processes and signaling
cascades, which are potentially involved in the regulation of the
endometrial changes during the estrous cycle, such as
endometrium remodeling, regulation of angiogenesis, regula-
tion of invasive growth, cell adhesion, and embryo feeding. Two
signaling pathways, namely the TGF-beta signaling pathway and
retinoic acid signaling, are suggested by pathway analyses and
interaction networks to play a central role in the regulation of
endometrial remodeling during the estrous cycle. This is also in
line with current knowledge of regulation of endometrial func-
tions by TGF-beta during the cycle, implantation, and pregnancy
[54, 55] . Furthermore, induction of Tgfb1 mRNA and protein by
estrogen has been shown in epithelial cells of the mouse
endometrium [56] . In this context a regulatory role of retinoic
acid signaling together with progesterone and TGF-beta for
endometrial maturation has been proposed in humans [57] .
Species-specifi c and conserved mechanisms
The comparison of the identifi ed genes with microarray studies
in human, mouse, and Rhesus monkey [34 – 36, 58 – 60] revealed
an overlap of 70 genes (differentially expressed in bovine study
and in at least one of the other studies) ( Table 1 ). The changes of
mRNA levels were similar for 38 genes, contrary for 29 genes
and contrary within the compared studies for three genes,
regarding the expression profi le during the sexual cycle or the
regulation by estrogen, respectively. This fi nding refl ects the dif-
ferences between ruminant species and primates and rodents
regarding i) histological changes in the endometrium during the
cycle (estrous cycle less changes compared to menstrual cycle)
and ii) the type of implantation of the embryo (delayed and non-
invasive type in ruminants). However, similar gene expression
profi les suggest that there are also some common regulatory
mechanisms between mammalian species.
For example, the mRNA for mucin 16 ( MUC16 ) is downregulated
during the luteal phase in bovine endometrium. In human
endometrium MUC16 protein has been shown to prevent initial
trophoblast cell adhesion and expression is lost during the
receptive phase [61] , suggesting a common regulation of tro-
phoblast cell adhesion via mucins in different mammalian spe-
cies. Another example for similar gene expression between
mammalian species is claudin 4 ( CLDN4 ). In line with the up-
regulation in bovine endometrium during early pregnancy [30]
and during the luteal phase of the estrous cycle [28] , CLDN4
mRNA was found elevated at the window-of-implantation time
in four microarray studies of human endometrium [35, 58 – 60] .
Claudins are major cell adhesion molecules in tight junctions
involved in intercellular sealing in simple and stratifi ed epithelia
[62] . Claudin 4 has been found to selectively decrease Na
+
per-
meability in tight junctions. In contrast, the mRNA level of
CLDN10 is strongly up-regulated in bovine endometrium at day
0 of the estrous cycle [27] . These fi ndings suggest a role for tight
junctions in preparation of the endometrium for implantation.
One example for species-specifi c regulation is PCSK5 (proprotein
convertase subtilisin / kexin type 5). For the PCSK5 protein an
essential role in preparing the uterus for embryo implantation
in the mouse and in primates has been shown [63] . In contrast,
PCSK5 mRNA is downregulated in bovine endometrium at die-
strus supporting the hypothesis that PCSK5 positively regulates
invasive embryo implantation and thus has to decrease in bovine
endometrium during the luteal phase. Similar to PCSK5 , galec-
tin-1 ( LGALS1 ) mRNA concentration was increased at estrus. The
LGALS1 protein, a multifunctional secreted member of the galec-
tin family, plays a pivotal role in the modulation of cell adhesion,
cell growth, infl ammation, and angiogenesis [64 – 66] . In human
endometrium LGALS1 protein has been mainly localized in stro-
mal cells with increased expression in the late secretory phase
and in decidual tissue [64] , whereas in situ hybridization studies
of bovine endometrium revealed pronounced expression in the
superfi cial epithelial glands and weak to moderate expression in
other cells. The different expression of LGALS1 in bovine and
human endometrium, i.e. between species with different types
of placentation, suggests this gene as a further interesting candi-
date for regulation of embryo implantation.
The bovine proenkephalin ( PENK ) mRNA, which is increased
during diestrus, has been found as up-regulated in the rat
endometrium during secretory phase [67] and positively regu-
lated by progesterone in the endometrium of mice [68] . In con-
trast, PENK mRNA is up-regulated during the proliferative phase
in the endometrium of primates including humans [36, 58, 60, 69] .
For this gene the mRNA pattern is similar in ruminant species
and rodents, but different in primates.
Transcriptome changes in the endometrium
during the pre-implantation phase
&
Transcriptome analyses of bovine endometrium comparing
samples recovered from day 18 pregnant animals and corre-
sponding non-pregnant controls were performed using two dif-
ferent experimental models. In the fi rst model monozygotic
twin cows were used, where one twin received two in vitro-pro-
duced embryos and the corresponding twin a sham transfer at
day 7 of the estrous cycle [31] (
䊉
䉴
F i g . 1 ). Endometrial tissue
samples were recovered on day 18 of pregnancy and the estrous
cycle, respectively. Using this genetically defi ned model system,
87 different genes were identifi ed as upregulated in pregnant
animals. Almost half of these genes has been described as classi-
cal type I interferon-induced genes, i.e., is probably induced by
interferon tau (IFNT), the embryonic pregnancy recognition sig-
nal in ruminants. In the second model pregnancy was obtained
by artifi cial insemination of heifers. Control animals received a
sham insemination (sperm cells were removed by centrifuga-
tion). Endometrial tissue samples were recovered on day 18 of
Review 5
Bauersachs S et al. Transcriptome Profi ling of Endometrium … Exp Clin Endocrinol Diabetes 2008 ; 116: 1 – 14
ECED/898/23.5.2008/Macmillan
Table 1 Comparison with microarray studies in human, Rhesus monkey and mouse
Gene
symbol
Gene name hsa
Entrez
Gene ID
Fold up at day of
estrous cycle
1
day
18AI vs
18P4L
2
18ET
vs
18ST
3
hsa LH +
(6 – 8) vs
LH − (3 – 5)
4
hsa LH +
(7 – 9) vs
LH + (2 – 4)
5
hsa LH +
(8 – 10) vs
LH − (4 – 6)
6
hsa LH + 8
vs LH + 3
7
hsa LH + 7
vs LH + 2
8
mmul day
(21 – 23) vs
day13
9
mmu
implant
10
mmu
estrogen
induced
11
mmu
estrus vs
diestrus
12
Ana-
logy
ACTA2 actin, alpha 2, smooth
muscle, aorta
59 3.2 0 − 2.7 3.7 4.4 −
AHCY S-adenosylhomocysteine
hydrolase
191 2.1 2.3 +
ALPL alkaline phosphatase,
liver / bone / kidney
249 6.7 12 − 11.0 −
ANXA2 annexin A2 302 3.2 0 − 2.4 4.7 5.6 4.0 −
AP1GBP1 ap1 gamma subunit bind-
ing protein 1
11276 2.3 7.6 +
APOE apolipoprotein e 348 2.9 18P4L 100.0 −
B2M beta-2-microglobulin 567 2.2 12 2.8 3.0 3.4 +
BAIAP2 bai1-associated protein 2 10458 3.1 12 − 3.2 −
BCAT1 branched chain ami-
notransferase 1, cytosolic
586 6.1 12 4.7 −
BST2 bone marrow stromal cell
antigen 2
684 8.3 18.1 − 10.0 −
C10orf10 chromosome 10 open
reading frame 10
11067 3.9 13.4 6.7 +
C10orf58 chromosome 10 open
reading frame 58
84293 6.6 18P4L 2,1 +
C1R complement component
1, r
715 3.8 18P4H 3.4 4.0 3.7 2.0 3.1 +
C1S complement component
1, s
716 3.8 3.0 +
CCNB1 cyclin b1 891 4.7 12 − 2.5 − 3.0 −
CD74 cd74 antigen (invariant
polypeptide of MHC class
II antigen-associated)
972 3.4 3.5 2.4 − 2.3 +
CLDN4 claudin 4 1364 3.3 18P4H 2.9 43.2 45.0 3.9 17.3 +
COL15A1 collagen, type XV, alpha 1 1306 3.3 18P4L − 4.2 3.6 −
COL1A1 collagen, type I, alpha 1 1277 4.7 0 − 4.4 4.1 +
COL1A2 collagen, type I, alpha 2 1278 4.0 0 − 3.2 − 2,4 −
COL3A1 collagen, type III, alpha 1 1281 4.0 0 − 3.1 − 3.1 2.4 − 2,2 ±
COL4A1 collagen, type IV, alpha 1 1282 2.6 18P4L 2.4 −
COL4A2 collagen, type IV, alpha 2 1284 − 3.5 − 2.9 +
COL5A2 collagen, type V, alpha 2 1290 2.8 0 − 2.0 +
COL6A2 collagen, type VI, alpha 2 1292 − 2.0 − 3.6 +
COL6A3 collagen, type VI, alpha 3 1293 3.9 0 − 5.8 − 8.0 +
CTGF connective tissue growth
factor
1490 3.2 − 2.9 −
Review6
Bauersachs S et al. Transcriptome Profi ling of Endometrium … Exp Clin Endocrinol Diabetes 2008 ; 116: 1 – 14
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Table 1 Continued
CXCR4 chemokine (c-x-c motif)
receptor 4
7852 2.2 18P4H − 3.7
−
CYP26A1 cytochrome p450, family
26, subfamily A1
1592 33.3 12 15.6 +
DCN decorin 1634 1.9 0 7.0 +
DIO2 deiodinase, iodothyro-
nine, type II
1734 4.5 3.5 − 2.3 − 2.4 +
DKK1 dickkopf homolog 1
(xenopus laevis)
22943 11.9 18.6 12.6 12.1 7.1 +
EDG3 endothelial differentia-
tion, sphingolipid g-pro-
tein-coupled receptor, 3
1903 2.4 12 − 2.6 +
EFNA1 ephrin-a1 1942 2.9 12 4.9 15.5 +
FBLN5 fi bulin 5 10516 2.6 0 6.0 −
FOXA2 forkhead box a2 3170 5.8 0 − 2.1 +
GABARA-
PL1
GABA(A) receptor-associ-
ated protein like 1
23710 2.5 12 2.7 3.8 +
GPLD1 glycosylphosphatidyli-
nositol specifi c phos-
pholipase d1
2822 5.4 − 3.6 −
HLA-A major histocompatibility
complex, class I, A
3105 2.7 2.6 2.0 +
HPGD hydroxyprostaglandin
dehydrogenase 15-(NAD)
3248 6.7 12 − 14.0 −
HSPA5 heat shock 70 kda protein
5 (glucose-regulated
protein, 78 kda)
3 3 0 9 − 2 . 6 1 . 4 −
IDH1 isocitrate dehydrogenase
1 (NADP + )
3417 5.3 12 2.7 5.3 − 3.4 ±
IFIT1 interferon-induced pro-
tein with tetratricopep-
tide repeats 1
3434 25.2 16.3 − 2.6 −
IGF1 insulin-like growth factor
1 (somatomedin C)
3479 − 2.4 − 3,9 −
IGFBP2 insulin-like growth factor
binding protein 2
3485 4.8 18P4L 2.6 2.7 −
IGFBP4 insulin-like growth factor
binding protein 4
3487 2.7 18P4L − 2.5 − 3,5 ±
IRF6 interferon regulatory
factor 6
3664 5.3 12 − 3.9 −
ITM2B integral membrane
protein 2b
9445 1.7 3.5 − 2.9 +
KLF5 kruppel-like factor 5
(intestinal)
688 2.6 12 11.5 +
LAMB1 laminin, beta 1 3912 − 2.1 2.6 −
RPSA ribosomal protein SA
(laminin receptor 1)
3921 1.8 0 − 2,2 −
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LDHA lactate dehydrogenase a 3939 − 2.0 2.3 −
LGALS1 lectin, galactoside-bind-
ing, soluble, 1 (galectin 1)
3956 4.0 0 − 2.6 +
MFGE8 milk fat globule-egf fac-
tor 8 protein
4240 6.9 2.6 +
NCOR2 nuclear receptor co-re-
pressor 2
9612 3.0 0 − 2.4 +
NDP norrie disease (pseudo-
glioma)
4693 7.3 0 − 2.2 +
NOV nephroblastoma over-
expressed gene
4856 14.5 0 5.5 −
NP nucleoside phospho-
rylase
4860 2.7 18P4L 2.6 2.4 −
NUPR1 nuclear protein 1 (p8
protein (candidate of
metastasis 1))
26471 4.0 12 8.2 2.7 4.5 7.2 +
PCBP2 poly(RC) binding
protein 2
5094 2.1 3.5 − 2.5 +
PCOLCE procollagen c-endopepti-
dase enhancer
5118 3.0 0 2.7 −
PCSK5 proprotein convertase
subtilisin / kexin type 5
5125 8.8 0 − 6.7 +
PENK proenkephalin 5179 45.1 18P4H 9.3 4.0 − 13.9 − 25.0 − 6.4 −
PLK2 polo-like kinase 2 (dro-
sophila)
10769 5.1 0 − 6.6 − 3.5 2.0 − 2,4 ±
PRDX6 peroxiredoxin 6 9588 2.5 12 − 2,2 +
RARRES1 retinoic acid receptor
responder (tazarotene
induced) 1
5918 5.8 0 − 2.4 5.1 −
RBP4 retinol binding protein 4,
plasma
5950 − 2.8 5.6 −
RNH1 ribonuclease / angiogenin
inhibitor 1
6050 2.5 2.2 +
RPL36A ribosomal protein L36a 6173 3.9 12 − 3.5 −
SAT spermidine / spermine
n1-acetyltransferase
6303 3.0 12 2.4 3.0 3,9 +
SDC2 syndecan 2 6383 4.3 0 − 2.6 − 2.1 −
SEPP1 selenoprotein p,
plasma, 1
6414 3.5 12 3.3 − 8.0 +
SER-
PINA1
serpin peptidase inhibi-
tor, clade a, member 1
5265 3.0 0 − 5.1 7,0 +
SERP-
ING1
serpin peptidase inhibi-
tor, clade g (c1 inhibitor),
member 1
710 5.8 12 4.4 2.6 7.0 9.0 4.7 − 3.6 +
SGK serum / glucocorticoid
regulated kinase
6446 2.1 12 4.9 3.6 +
SLC1A1 solute carrier family 1,
member A1
6505 4.1 18P4H 2.1 26.0 31.5 +
Review8
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Table 1 Continued
SOX4 sry (sex determining
region y)-box 4
6659 2.6 0 − 3.2 − 3.7 +
SPARC secreted protein, acidic,
cysteine-rich (osteonec-
tin)
6678 4.1 0 − 3.0 − 3.2 +
STAT1 signal transducer and
activator of transcription
1, 91 kDa
6772 2.4 4.6 − 2.1 − 2.4 ±
STC1 stanniocalcin 1 6781 3.4 0 − 2.5 14.8 −
TAP1 transporter 1, atp-bind-
ing cassette, sub-family b
(mdr / tap)
6890 3.6 5.9 2.1 +
TEK tek tyrosine kinase,
endothelial
7010 9.5 18P4L − 5.1 3.7 ±
TGM2 transglutaminase 2 7052 10.6 12 3.4 5.3 7.2 +
THBS2 thrombospondin 2 7058 − 2.5 9.4 −
TIMP1 timp metallopeptidase
inhibitor 1
7076 3.4 18P4L − 5.8 2.0 −
TIMP2 timp metallopeptidase
inhibitor 2
7077 8.9 12 3.8 − 2.7 −
TJP1 tight junction protein 1,
zona occludens 1
7082 3.5 12 2.6 − 2.6 +
TNC tenascin c (hexabrachion) 3371 12.5 0 − 13.5 − 5.9 − 3.9 1.6 +
TNFSF10 tumor necrosis factor
(ligand) superfamily,
member 10
8743 4.9 3.8 +
TSC22D3 tsc22 domain family,
member 3
1831 4.7 12 − 3.2 −
TUBA1 tubulin, alpha 1 (testis
specifi c)
7277 3.0 18P4L 3.4 16.1 − 2,6 −
TUBB tubulin, beta 203068 2.6 18P4L − 3.0 − 2,0 −
UNC5B unc-5 homolog b
(c. elegans)
219699 − 3.4 − 3.3 +
WFDC2 wap four-disulfi de core
domain 2
10406 3.2 3.5 27.5 +
1Bauersachs et al. 2005, Mitko et al. 2008; 2 Bauersachs et al. 2006; 3 Klein et al. 2006; 4 Borthwick et al.; 5 Carson et al.; 6 Kao et al.;7 Mirkin et al.; 8 Riesewijk et al.; 9 Ace et al.; 10 Reese et al.; 11 Hong et al.; 12 Tan et al.
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pregnancy and the estrous cycle, respectively. In contrast to the
fi rst model, the control animals showed low serum progester-
one levels due to a shortened estrous cycle. Thus the differential
expression of the identifi ed genes could be based on embryonic
signals and / or hormonal differences. In this study 179 differen-
tially expressed genes were found, 109 with higher and 70 with
lower mRNA abundance in pregnant animals [30] . Among the
mRNAs with higher abundance in pregnant animals at least 41
are already described as induced by interferons.
ISG15ylation and ADP-ribosylation
In both studies a number of mRNAs encoding proteins that are
involved in specifi c protein modifi cation processes, such as
ISG15ylation and ADP-ribosylation, were identifi ed as upregu-
lated in endometrium samples of pregnant animals. The ISG15
ubiquitin-like modifi er has been hypothesized to be a critical
component of the microenvironment at the uterine-placental
interface during the progressive events of conceptus develop-
ment, implantation, and placentation [70] . ISG15 is an ubiquitin-
like protein that is conjugated to a number of target proteins
thereby regulating their functions. Messenger RNAs of fi ve new
potential members of the ISG15ylation system (DTX3L, IFITM1,
IFITM3, RNF213, and XAF1) were found at increased levels in
addition to mRNAs coding for the known components ISG15,
UBE1L, and BUBP43 (USP18). Another protein modifi cation proc-
ess, ADP-ribosylation, was represented by three up-regulated
mRNAs coding for members of the poly(ADP-ribose) polymerase
(PARP) superfamily ( PARP9 , PARP10 , and PARP12 ) [71] . This proc-
ess is involved, e.g., in the regulation of membrane traffi cking
and the actin cytoskeleton [71] . However, the function of the
three members identifi ed in our studies is yet not well defi ned.
Transcription factors
A number of mRNAs coding for a variety of transcription factors
was also identifi ed as upregulated in endometrium of pregnant
animals (
䊉
䉴
F i g . 2 ). NR2F2 (COUP-TFII), a nuclear orphan recep-
tor, has been shown to repress the human oxytocin gene pro-
moter in uterine epithelial cells [72] . In mice, NR2F2 has been
shown to regulate stromal cell differentiation (decidualization)
through the induction of BMP2 and indirectly regulates suppres-
sion of estrogen activity required for establishing a receptive
uterus [73] . A conditional knockout of Nr2f2 in the ovary and the
uterus led to severely impaired placental formation that results
in miscarriage [74] , whereas haploinsuffi ciency of Nr2f2 in the
mouse results in decreased progesterone synthesis in the CL
leading to reduced ability of the endometrium to support preg-
nancy [75] . A further interesting transcription factor gene in
context of placentation is EPAS1 , also known as HIF2A , which
codes for a basic -helix-loop-helix / PAS domain transcription
factor that has been shown to promote angiogenesis through
transactivation of the genes coding for vascular endothelial
growth factor, its receptors, and several other genes [46] .
Immune modulation
The modulation of the maternal immune system is essential to
prevent rejection of the conceptus, which represents a semi-
allograft. The transcriptome analyses of pregnant endometrium
revealed an up-regulation of several immune-related genes
(
䊉
䉴
F i g . 2 ). Four of them are involved in the complement system
(classical pathway), namely complement component genes C1S,
C1R , C4 , and the C1 inhibitor SERPING1 [30, 31] . In situ hybridiza-
tion experiments revealed specifi c expression of C1S, C1R , and
SERPING1 mRNA mainly in luminal and glandular epithelial cells
but also weak in stromal cells [31] . The simultaneous up-regula-
Fig. 1 Genetically standardized model for the systematic dissection of
mechanisms of embryo-maternal communication in bovine endometrium.
Fig. 2 Biological processes and involved genes which were found to be
differentially expressed in bovine endometrium in the pre-attachment
period. Solid boxes: up-regulated; dashed boxes: down-regulated.
Review10
Bauersachs S et al. Transcriptome Profi ling of Endometrium … Exp Clin Endocrinol Diabetes 2008 ; 116: 1 – 14
ECED/898/23.5.2008/Macmillan
tion of SERPING1 , encoding a protein known as C1 inhibitor [76] ,
in the luminal and glandular epithelial cells could be a mecha-
nism to protect the embryo against an attack by the complement
system. UTMP is another gene that may play a role in the modu-
lation of the maternal immune system. For ovine UTMP protein
inhibition of NK-like activity was shown and a role in protecting
the conceptus from maternal cytotoxic lymphocytes has been
suggested [77] . As in our study of pregnant cows, UTMP mRNA
has been found up-regulated in endometrium of pregnant sheep
[78] . During the estrous cycle UTMP mRNA showed highest lev-
els in bovine endometrium at estrus [27] . CD81 protein is
reported to infl uence activation, proliferation, and differentia-
tion of B-, T-, and other cells. Furthermore, association with
IFITM1 (Leu-13, 9 – 27) protein was shown in B-cells [79] . IFITM1
itself has been shown to suppress the activity of natural killer
cells [80] , suggesting a role in preventing maternal rejection of
the fetal semi-allograft. The product of the BST2 gene has been
suggested to be involved in pre-B-cell growth [81] . In situ hybrid-
ization with bovine endometrium derived from day 18 pregnant
animals revealed strongest expression in stromal cells [30] .
Cell adhesion
The process of embryo attachment is essentially dependent on
cell adhesion processes. A functional classifi cation of the up-
regulated genes revealed a number of genes, which may be
involved in the process of cell adhesion between embryonic tro-
phoblast cells and cells of the luminal epithelium: AGRN , CD81 ,
LGALS3BP , and LGALS9 (
䊉
䉴
F i g . 2 ). In situ hybridization for AGRN ,
LGALS3BP , and LGALS9 showed strong staining in the luminal
epithelium in pregnant animals [30] . Agrin is a heparan sulphate
proteoglycan that has been shown to be involved in the forma-
tion of synapses of the neuromuscular junction and the immu-
nological synapse [82] . Thus, agrin may play a general role in the
formation of very close cell contacts and subsequent signaling
between the attached cells. The interferon-stimulated gene
LGALS3BP codes for a cell-adhesive protein of the extracellular
matrix, which self-assembles into ring-like structures and binds
beta1 integrins, collagens and fi bronectin [83] . LGALS9 is also an
interferon-stimulated gene and the encoded protein (galectin 9)
has been shown to mediate cell aggregation and cell adhesion
[84] . In sheep, galectin 15 has been suggested to be an attach-
ment factor important for peri-implantation blastocyst elonga-
tion [85] . Since the bovine LGALS15 gene does exist but is not
expressed, galectin 9 in cattle might have a similar role as galec-
tin 15 in the sheep. CD81 is a member of the transmembrane 4
superfamily (tetraspanin family). The encoded protein interacts
with integrins, suggesting a role in cell adhesion processes
[79, 86] . Overexpression of CD81 in tumor cell lines results in
reduced invasive growth [87] , suggesting a possible regulatory
role of CD81 protein in context of the non-invasive implantation
of the bovine embryo.
Remodeling of the endometrium
A number of additional candidates for endometrium remodeling
were found, such as MEP1B , legumain ( LGMN ), MMP19 , TIMP2 ,
transglutaminase 2 ( TGM2 ), met proto-oncogene (hepatocyte
growth factor receptor, MET ), and epithelial stromal interaction
1 ( EPSTI1 ) (
䊉
䉴
F i g . 2 ). The asparaginyl-specifi c cysteine protein-
ase LGMN may play a role in regulation of ECM remodeling as it
has been shown to activate MMP2 in vitro and in cultured cells
[88] . With MMP19 and TIMP2 two genes coding for components
of the matrix metalloproteinase system were identifi ed. A study
in normal breast tissue and mammary gland tumors revealed
strong expression of MMP19 protein in all tumor cells of benign
lesions, whereas the progression towards an invasive phenotype
and neoplastic dedifferentiation led to the disappearance of
MMP19 and a concomitant rise in the levels of MMP2 [89] . In
addition to the upregulation of TIMP2 mRNA at day 18 of preg-
nancy in bovine endometrium, TIMP2 mRNA levels are higher
at day 12 compared to day 0 of the estrous cycle [27] . These
fi ndings suggest an important role of MMP19 and TIMP2 , the
inhibitor of MMP2 [90] , for the regulation of attachment of the
conceptus. Furthermore, mRNA of TIMP1 , coding for a tissue
inhibitor of metalloproteinases, was decreased in endometrium
of day 18 pregnant animals compared to the control group [30] .
Salamonsen et al. discussed the important contribution of MMPs
and TIMPs to the marked endometrial remodeling associated
with early placentation in sheep [91] . TGM2 has been shown to
be involved in a variety of processes, including wound healing
and angiogenesis. Assembly and remodeling of the ECM in dif-
ferent tissues is mediated by cross-linking various ECM proteins
such as fi bronectin, proteoglycans, collagen V, osteonectin, lam-
inin, nidogen, and osteopontin and the covalent modifi cation
and activation of several growth factors [92] . In bovine
endometrium TGM2 mRNA is up-regulated at day 12 compared
to day 0 of the estrous cycle [27] and at day 18 of pregnancy [30] .
In the ovine endometrium expression of the hepatocyte growth
factor ( HGF ) mRNA has been detected in stromal cells and the
mRNA of its receptor ( MET ) exclusively in luminal and glandular
epithelial cells [93] . Hepatocyte growth factor has been sug-
gested to stimulate epithelial morphogenesis in preparation for
establishment and maintenance of pregnancy, conceptus
implantation, and placentation. In bovine endometrium, MET
mRNA levels were also elevated in endometrium of day 18 preg-
nant animals [30] . EPSTI1 has been found as highly upregulated
in invasive breast carcinomas compared with normal breast tis-
sue. However, in a tissue mRNA panel the most prominent ex-
pression of EPSTI1 was found in placenta. Expression of this gene
has been supposed to be crucial for invasion and metastasis of
cancer [94] . The up-regulation of EPSTI1 mRNA in bovine
endometrium at day 18 of pregnancy suggests a role of this gene
in endometrial remodeling prior to attachment of the embryo.
Species-specifi c and conserved mechanisms
Despite the distinct differences in the biology of reproduction
between mammalian species some genes were identifi ed, which
are regulated similarly in bovine endometrium of day 18 preg-
nant animals compared to endometrium of humans and the
Rhesus monkey during the putative window of implantation or
of the mouse at the implantation stage, respectively. Therefore,
some common regulatory processes in these species can be sug-
gested. For example DKK1 mRNA, coding for an inhibitor of WNT
signaling [95] , has been found as induced in four human studies
at the window of implantation time [35, 58 – 60] and in bovine
endometrium of day 18 pregnant animals [30] ( Table 1 ). Like-
wise, for CLDN4 higher mRNA levels were detected in human
endometrial tissue samples in four studies and in bovine
endometrium. Elevated mRNA levels were also found in two
human studies for nuclear protein 1 ( NUPR1 , candidate of metas-
tasis 1, P8), SLC1A1 and decidual protein induced by progester-
one ( C10orf10 ). There seem to be also some common regulatory
mechanisms of the maternal immune systems as indicated by
the similar regulation of C1R , SERPING1 , and TAP1 . Furthermore,
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Bauersachs S et al. Transcriptome Profi ling of Endometrium … Exp Clin Endocrinol Diabetes 2008 ; 116: 1 – 14
ECED/898/23.5.2008/Macmillan
TGM2 mRNA was shown to be present at higher levels in human
endometrium during the window of implantation.
Our recent studies of transcriptome changes in bovine
endometrium at the pre-implantation stage revealed a set of
very promising candidate genes involved in crucial biological
processes like cell adhesion, endometrial remodeling, regulation
of the maternal immune system, and the response to interferon
tau, the embryonic pregnancy recognition signal (
䊉
䉴
F i g . 2 ) . F u r -
thermore, with the set of genes identifi ed in these and related
studies pregnancy signaling in bovine endometrium can be
compared also between normal and pathological states or after
transfer of in vivo derived vs. in vitro produced embryos.
Relationship between metabolic disturbances and
functionality of the endometrium
&
The increase of the milk yield per cow and lactation reached in
the last decade is in negative correlation with parameters of
reproductive performance [96] , although a proportion of high
yielding cows does not exhibit fertility problems. Currently it is
not known whether (i) reduced reproductive performance is a
direct consequence of high milk yield, negative energy balance
and associated metabolic problems ( “ the physiological hypoth-
esis ” ), or whether (ii) so far unknown gene variants with a nega-
tive effect on fertility have been accumulated in the course of
effi cient selection for high milk yield, while selection for repro-
ductive performance was rather ineffi cient ( “ the genetic hypoth-
esis ” ). Most likely both effects play a role, but their respective
importance remains to be determined. Interestingly, a consider-
able inter-individual variance exists with regard to the simulta-
neous expression of a high milk yield and high fertility under
the same environmental conditions. Furthermore, potential dif-
ferences between breeds require specifi c attention.
In theory, malfunctions of the ovaries (prolonged interval from
parturition to cyclicity, disturbed folliculogenesis and oocyte
maturation, ovarian cysts, abnormal progesterone profi les) as
well as problems related to oviduct and uterus physiology (e.g.
disturbed uterine receptivity leading to early embryonic mortal-
ity) may be responsible for the overall alarming fertility prob-
lems in high yielding dairy cows.
It has been shown that stimulation of milk production by
increased intake of dietary protein leads to an increase in blood
urea nitrogen (BUN) and is often associated with decreased fer-
tility [96] . Early studies of dairy cows involving embryo collec-
tion and evaluation did not provide evidence that high dietary
protein has an impact on ovarian follicular development, ovula-
tion, or fertilization of oocytes [97, 98] . Thus, disturbances of
early embryonic development and embryo-maternal interac-
tions are more likely an explanation for fertility problems asso-
ciated with high yield and protein feeding. Although it is not
clear whether the embryonic or the maternal compartment is
more critical, there is evidence that high protein diet and the
associated increase in BUN may change the uterine milieu, e.g.
the uterine pH, which may have important implications for the
transcriptional activity and functions of the endometrium. Inter-
estingly, bovine endometrial cells in culture are known to
respond directly to increasing urea concentrations with
increased secretion of PGF
2
[96] , which induces luteolysis in
vivo [9] . Moreover, there is evidence for embryotoxic effects of
PGF
2
in the pre-implantation period [99] . These observations
provide a plausible link between elevated plasma urea nitrogen
concentrations and decreased fertility. However, the molecular
mechanisms underlying the effects of increased BUN or other
metabolic changes on endometrial functions are incompletely
understood and need to be studied in vivo . A fi rst step could be a
transcriptome profi ling of endometrial biopsies from metaboli-
cally well-characterized cows in different situations of milk
yield and energy balance. These studies would clarify metabolic
infl uences on the functionality of the endometrium and eventu-
ally make an important contribution to the fertility monitoring
in high-yielding cows.
Evaluation of endometrium transcriptome
changes in response to embryos derived by
assisted reproduction techniques (ART)
&
Assisted reproduction techniques (ART) are becoming increas-
ingly important in human reproductive medicine and in animal
breeding and biotechnology as well. Although in vitro fertiliza-
tion and intracytoplasmic sperm injection have been established
to the level of clinical application, there are recent reports of a
higher frequency of epigenetic abnormalities in offspring
derived by ART as compared to natural reproduction [100] .
Somatic cell nuclear transfer cloning, which has been successful
in a number of species, is particularly critical with respect to
epigenetic abnormalities of the resulting embryos, fetuses and
offspring (reviewed in [101] ). For instance, we observed prema-
ture DNA methylation in a signifi cant proportion of bovine
somatic cell nuclear transfer embryos [102, 103] . DNA hyper-
methylation was observed in some tissues of cloned bovine
fetuses, but – to a lesser extent – also in fetuses derived from in
vitro -produced embryos [104, 105] . However, it is largely unclear
whether and how epigenetic changes cause developmental
abnormalities and abortions of cloned embryos or fetuses. A
number of studies revealed placental abnormalities as primary
cause of pregnancy loss after transfer of bovine SCNT embryos.
Placental changes include a reduced number, but increased size
of placentomes [106, 107] . Furthermore, we observed transpla-
cental leakage of maternal cells into the circulation of fetuses
derived by SCNT, but not in IVF-derived fetuses [108] . These
fi ndings raise the question, how and when these changes of pla-
cental functionality are induced. The fact that genes which may
be involved in placenta formation were found to be abnormally
expressed in SCNT embryos [109] invited the concept that pla-
cental abnormalities may have their origin in abnormal embryo-
maternal communication already at the pre-implantation
period. To clarify this hypothesis, we initiated a study evaluating
transcriptome profi les of endometrium samples in response to
SCNT embryos vs. embryos derived by in vitro- fertilization.
Importantly, several different nuclear donor cell lines were used
for SCNT to have a similar genetic variation in the SCNT and the
IVP groups, excluding specifi c effects of a particular embryonic
genotype as cause for transcriptome differences in the corre-
sponding endometrium samples (
䊉
䉴
F i g . 3 ). IVP and SCNT
embryos were generated and transferred to recipients as
described before [110, 111] . Endometrium samples from preg-
nant recipients carrying IVP or SCNT embryos were recovered
according to Bauersachs et al. [27] and Klein et al. [31] and proc-
essed for transcriptome profi ling using the Bovine Oviduct and
Endometrium (BOE) array version 1 [29] . Currently, the results
of the array hybridization experiment are under detailed analy-
sis. The analysis of abnormalities in embryo-maternal commu-
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Bauersachs S et al. Transcriptome Profi ling of Endometrium … Exp Clin Endocrinol Diabetes 2008 ; 116: 1 – 14
ECED/898/23.5.2008/Macmillan
nication after transfer of embryos derived by ART may be helpful
as an early readout for studies aiming at the improvement of
these reproduction technologies.
Conclusions and Perspectives
&
Transcriptome profi ling is a fi rst step for identifying molecular
mechanisms underlying the functional changes of endometrium
during the estrous cycle and during pregnancy. Our previous
studies identifi ed transcriptome profi les which are characteris-
tic for pregnant vs. non-pregnant animals or for different stages
of the estrous cycle. Future RNA expression studies should also
take non-coding RNAs and microRNAs, important regulators of
translation, into account. In addition to profi ling at the RNA level
proteomics studies of endometrium and of the uterine fl uid will
be essential to identify biologically relevant protein candidates.
Our fi rst study of endometrium proteome changes in pregnant
animals (day 18 = pre-attachment period) revealed four proteins
with higher abundance in pregnant endometrium, which have
previously not been known to be regulated in this period in
bovine [112] . Novel proteomics techniques, such as highly sensi-
tive saturation labeling [113] , will further increase the analytical
depth of proteome studies and facilitate analyses of microdis-
sected tissues.
In addition to questions related to basic research, endometrial
transcriptome profi les may have important implications for the
cattle breeding industry. At the moment improvement of fertil-
ity by genetic selection is hampered by the low heritability of
currently recorded fertility traits, e.g., the non-return rate 90
days after artifi cial insemination. Future studies will clarify
whether transcriptome profi les of endometrium biopsies taken
at a specifi c stage of the estrous cycle are indicative of the fertil-
ity status. If this turns out to be the case, detailed molecular
phenotyping could be combined with genotyping using a high-
density marker set. Thus associations between a favorable
endometrial transcriptome profi le and specifi c genetic markers
could be established, eventually realizing the concept of genomic
selection [114] for fertility.
Acknowledgments
&
Our studies are funded by the Deutsche Forschungsgemein-
schaft (FOR 478: “ Mechanisms of embryo-maternal communi-
cation ” ) and by the Bundesministerium f ü r Bildung und
Forschung (Fertilink: Functional genome research for the
improvement of fertility). The authors are part of the EU cost
action GEMINI.
Confl ict of interest : N o n e .
R e f e r e n c e s
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