BACKGROUND
in mammals, early life starts with the formation of a zygote as a result of the fertilization of an oocyte.
sequential cleavage divisions lead to the formation of a morula stage embryo wherein a fluid-filled cavity emerges called the blastocoel.
two differentiated groups of cells can be distinguished in the embryo that is now called a blastocyst.
a group of cells adjacent to the blastocoel, the inner cell mass , is able to contribute to all cells of the three germ layers and is therefore referred to as being pluripotent.
the other group of cells, called the trophectoderm , forms an epithelium surrounding the blastocoel and the icm and is important for implantation within the uterus and contributes to the non-maternal part of the placenta.
in the icm further differentiation occurs by the formation of the epiblast, that will form the foetus, and the formation of extra-embryonic primitive endoderm  contributing to the yolk sac.
studies with mouse embryos have advanced our understanding of how a pluripotent cell population is established during pre-implantation development  <cit> .
during the first differentiation, the transcription factors cdx <dig> and oct <dig> are key regulators for the formation of respectively te and icm.
cdx <dig> represses the activity of oct <dig> in mouse te  <cit>  and is virtually absent in icm cells  <cit> .
oct <dig> in turn can counteract cdx <dig> activity in the inner cells of the morula.
the second differentiation is indicated by the expression of either nanog or gata <dig> in icm cells fated to become the epiblast or pe respectively  <cit> .
like for cdx <dig> and oct <dig> in the morula, nanog and gata <dig> inhibit each other’s transcription  <cit> .
whether the same genes and signalling pathways are also involved in the formation of a pluripotent cell population in other mammals remains to be established.
indeed, in contrast to the mouse, oct <dig> protein remains present in the te of bovine blastocysts even after transcription is down-regulated  <cit>  and its expression is not negatively regulated by cdx <dig>  <cit> .
in mouse embryos it has been established that gata6-stimulated fibroblast growth factor  signalling via the extracellular signal-regulated protein kinase  is responsible for nanog repression and thereby the formation of primitive endoderm  <cit> .
in bovine and human embryos however, although gata <dig> expression is specific for primitive endoderm, inhibition of erk signalling had a more moderate  or no  effect on the numbers of nanog and gata <dig> expressing cells suggesting that in these species other pathways are involved in the formation of the pluripotent cell population  <cit> .
these findings suggest species-specific mechanisms active in the specification of icm, te, epiblast and pe lineages and that further insight is needed into the molecular basis of cell sorting during the two first differentiation events.
when mouse icm cells are cultured under defined conditions, their pluripotent character can be maintained  <cit> .
however, the establishment of such embryonic stem  cells has only been successful for mice, non-human primates  <cit> , humans  <cit>  and rats  <cit> .
although pluripotency refers to the capacity to give rise to all embryonic and adult cell types, including the germ line, various states of pluripotency have been described.
these states are referred to as “naïve” and “primed”, with “primed” being more developmentally restricted  <cit> .
in mammals other than primates and rodents, the correct stages of embryos that contain pluripotent cells and culture conditions that maintain pluripotency have yet to be established  <cit> .
in order to identify genes that may be important for the acquisition and maintenance of pluripotency in bovine embryos a genome-wide gene expression analysis was performed in morulae, intact blastocysts, te and icm.
analyses of gene expression patterns in pre-implantation embryos to distinguish between pluripotent cells of the icm versus those of the te have previously made use of cell lines because of the technical difficulties of separating icm from te  <cit> .
here we have manually dissected individual icms from te.
as the late icm is composed of both pluripotent epiblast cells and the pe, the pluripotent character of the icm was enhanced by inhibition of the erk-pathway resulting in an increased percentage of icm cells that express nanog.
RESULTS
gene expression profile of pre-implantation embryos
to identify genes involved in bovine pluripotency, gene expression profiles of morula and blastocyst embryos, icm and te were generated using microarray analysis.
bovine cumulus oocyte complexes  were in vitro matured, fertilized and cultured for  <dig> or 9 days to obtain morula and blastocyst stage embryos, respectively .
in addition, icm and te were manually dissected from day  <dig> blastocysts .
from all samples rna was isolated and only those with a rna integrity number  ≥ <dig>  were used for further analysis.
to compensate for biological and technical errors, two biological replicates of each sample were labelled with either cy <dig> or cy <dig> and hybridized on the arrays with a common reference pool consisting of blastocysts so all samples could be compared .figure  <dig> microarray set-up and sample validation.
rna from morula , blastocyst , trophectoderm  and inner cell mass  was hybridized on microarrays.
a common reference sample composed of blastocysts was hybridized with each sample in duplicate in balanced dye-swap .
the heat map  illustrates pairwise clustering of microarray sample replicates.
yellow colour represents over-expressed probes and blue colour represents under-expressed probes as indicated by the colour legend .
representative pictures of immunofluorescent labelling of dissected icm  and te  for gata <dig>  and cdx <dig> ; double gata6-cdx <dig> nuclei appear yellow.
nuclear staining by dapi .
a 2d principal component analysis plot  with sample position indicating clustering of trophectoderm , blastocyst , inner cell mass  and morula  replicates.
samples were hybridized on a microarray slide containing almost  <dig>  probes per array coding for ~  <dig>  gene transcripts indicating that for a subset of genes more than one probe was present.
if the position of the probe is nearer to the 3’ end of the corresponding gene, signal intensity is expected to be higher  <cit>  and chance of incorrect signal by variations in rna integrity is smaller  <cit> .
therefore, the expression of the probes corresponding to the most 3’ ends of genes was used for the analysis  <cit> .
gene expression levels in morula, blastocyst, icm and te were determined and a hierarchical clustering analysis was performed.
the constructed heat map shows clear pairing of the morula, icm and te samples .
this is particularly important for the te and icm samples since these were manually dissected and confirms the reproducibility of the dissection.
we used mechanical isolation of icm from te using tungsten needles.
a selection of isolated icms was stained for cdx <dig> and gata <dig> to identify the contribution of te cells to the pooled microarray samples.
the isolated icms contained only ~20% cdx <dig> positive te .
since however some te cells remained attached to the icm, throughout the manuscript “icm” refers to the icm containing few te cells.
in the te samples all cells were cdx <dig> and gata <dig> positive .
blastocyst samples did not pair since their difference with the reference is minimal indicated by a near black appearance in the heat map.
a principal component analysis  further identified four categories according to cell type or developmental stage.
in a 2d plot, morula samples separate the farthest from the other samples.
te and icm samples are clearly separated from each other with the blastocyst replicates in between .
to further confirm the specificity of the samples, expression levels of genes that are known to be differentially expressed in bovine embryos were compared.
of the selected genes hmgb <dig>  sox <dig> and pou5f <dig>  are known to be expressed at relatively high levels in bovine morula embryos  <cit> , cdx <dig> and krt <dig> have the highest expression levels in te  <cit>  while fn <dig> and nanog are abundantly expressed in the icm  <cit> .
the relative expression levels of the selected genes as determined by microarray analysis in morula, blastocyst, icm and te were as expected, with highest levels of hmgb <dig> and sox <dig> expression in morulae, pou5f <dig> expression predominantly in morulae and icm, highest fn <dig> and nanog expression in the icm and cdx <dig> and krt <dig> expression at highest levels in te .
these selected genes and five additional genes  were also analysed for their expression levels by qrt-pcr, revealing a similar expression profile validating sample identity and demonstrating that the microarray data accurately reflect relative expression levels .figure  <dig> relative gene expression.
microarray data were analysed for selected genes known for their expression in morula , blastocyst , icm  and te  .
the same genes were analysed by qrt-pcr in 4– <dig> samples per embryo stage or cell type revealing the same pattern .
normalization was performed with the reference genes encoding rpl <dig>  sdha and ywhaz.
y-axis depicts relative mean expression to sample with highest expression set at  <dig> per gene.
bars with different letters are significantly different  and error bars indicate standard deviation.
genes differentially expressed between morulae and blastocysts
most differences in gene expression were found between morulae and blastocysts.
when gene expression in morulae was compared with that in blastocysts using a >2-fold difference with p-value < <dig>  as cut-off,  <dig>  genes were differentially expressed.
in the blastocyst,  <dig>  genes were up-regulated, up to 107-fold, while  <dig>  genes were expressed at higher levels in morulae .
the  <dig> most differentially expressed genes were expressed with at least a 30-fold difference of which only  <dig> gene  was expressed at higher levels in the morula .figure  <dig> relative gene expression in blastocyst versus morula and inner cell mass versus trophectoderm samples.
all probes representing genes  were plotted for their expression levels.
black lines represent the 2-fold cut off.
axes are log <dig> transformed and depict the normalized expression signal in the indicated sample.
genes analysed in qrt-pcr are indicated.
genes with significant  higher expression levels in blastocyst or morula are indicated with green or purple dots, respectively.
genes with significant  higher expression levels in inner cell mass or trophectoderm are indicated with orange or blue dots, respectively.
list of the most differentially expressed genes between blastocyst and morula
rank
entrez gene name
gene discription
agrigo id
fc
the  <dig> most differentially expressed genes  between blastocyst and morula are listed.
fc = fold-change and positive values indicate higher expression in blastocyst and negative values have higher expression in morula.
to identify properties of the differentially expressed genes a parametric analysis of gene set enrichment  was performed using agrigo  <cit>  and a list of the five most-enriched gene ontology  terms per categorie was generated .
a large group of differentially expressed genes was involved in the lipid metabolic process  in the blastocyst.
a much smaller group of genes that function in pepsin a activity  was over-represented in blastocysts and indeed, a large group of genes up-regulated in the blastocyst located to the plasma membrane .
compared to the blastocyst, in the morula more genes were involved in the nucleobase-containing compound metabolic process  and more specifically in rna processing .
morula up-regulated genes were found in the enriched molecular function go terms nucleic acid binding  and transcription regulator activity .table  <dig>
agrigo parametric analysis of gene set enrichment  analysis
go_acc
ontology
description
genes #
p-value
fdr
lipid metabolic process
pepsin a activity
membrane
plasma membrane
membrane part
extracellular region part
integral to membrane
genes differentially expressed between blastocyst and morula were assessed for their properties described by gene ontology  terms in respect to their relative expression.
the five most significantly enriched go-terms  are listed for each go domain.
p = biological process, f = molecular function and c = cellular component.
bold descriptions indicate enrichment for blastocyst.
regular descriptions indicate enrichment for morula.
fdr = false discovery rate .
genes differentially expressed between icm and te
when gene expression in the icm was compared with that of the te,  <dig> genes were differentially expressed.
here, the majority  of the differentially expressed genes were expressed at higher levels in the icm.
of all genes, the difference in expression levels of nanog was the largest .
sox <dig> and pou5f <dig> that together with nanog form the core transcriptional regulatory circuitry in pluripotent cells were also over-expressed in the icm compared with te .table  <dig>
list of the most differentially expressed genes between inner cell mass and trophectoderm
rank
entrez gene name
gene description
agrigo id
fc
the  <dig> most differentially expressed genes  between inner cell mass and trophectoderm are listed according to their relative expression.
fc = fold change and positive values indicate higher expression in inner cell mass.
for human es cells, a network of nanog-, sox2- and pou5f1-target genes that encode transcription factors and chromatin modulators has been established  <cit> .
from the list of positively regulated genes in human es cells, apart from the core network only expression of stat <dig> and zic <dig> was significantly up-regulated in bovine icm versus te .
again, we performed a parametric analysis of gene set enrichment with the genes differentially expressed between icm and te in agrigo  <cit> .
with a false discovery rate  ≤ <dig> , enzyme linked receptor protein signaling pathway  and peptidyl-tyrosine phosphorylation  terms were enriched for the icm whereas for the te, genes were enriched in the sterol biosynthetic process  .
only genes up-regulated in respect to the te were used for a singular enrichment analysis  in order to identify characteristics specific for the cells of the icm .
in particular, terms containing genes involved in rna splicing  and regulation of gene expression  were enriched even to a more specific level of enriched child terms like nuclear mrna splicing  and chromatin silencing , respectively .table  <dig>
agrigo parametric analysis of gene set enrichment  analysis
go_acc
ontology
description
genes #
p-value
fdr
intracellular signaling pathway
peptidyl-tyrosine modification
peptidyl-tyrosine phosphorylation
enzyme linked receptor protein signaling pathway
all genes differentially expressed between icm and te were assessed for their properties described by gene ontology  terms in respect to their relative expression.
this reveals  <dig> enriched go-terms with a fdr ≤  <dig>  in the go domains biological process  and cellular component .
ranked according to z-score.
bold descriptions indicate enrichment for inner cell mass.
regular descriptions indicate enrichment for trophectoderm.
fdr = false discovery rate .
genes differentially expressed between mapk-inhibited and control icms
it has been described that culture of icm cells in the presence of an fgfr inhibitor, a mapk inhibitor and an inhibitor of gsk3β, the so-called 3i culture system, leads to stable es cell lines from non-permissive mouse strains  <cit>  and rat embryos  <cit> .
culture of mouse embryos in the presence of a mapk inhibitor resulted in all cells of the icm expressing nanog while the expression of gata <dig> was down-regulated  <cit> .
similarly, when bovine embryos were cultured in the presence of the mapk inhibitor pd <dig>  a larger percentage, although not all, of the icm cells expressed nanog  <cit> .
therefore, in order to identify nanog target genes and genes that may be important for pluripotency, icms were isolated from bovine embryos cultured in the presence of the mapk inhibitor pd <dig>  gene expression in these icms was compared with that from control icms using microarray analysis.
in total  <dig> genes were differentially regulated between control  and mapk-inhibited icms, ≥2-fold difference with p-value ≤  <dig>  as cut-off, with the expression of  <dig> genes up-regulated and the expression of  <dig> genes down-regulated .
as expected, nanog expression was up-regulated in the mapk-inhibited icm as detected by microarray analysis .
expression differences between control icm and icm from embryos exposed to the mapk inhibitor detected by qrt-pcr verified the microarray data .
furthermore, immunostaining showed an increase in the percentage of nanog expressing cells in the icm after mapk inhibition  as we had previously established  <cit> .
to our surprise several interferon coding genes were dramatically down-regulated after mapk inhibition such as ifnw <dig> and ifnt .
gene expression analysis by qrt-pcr confirmed the microarray results and further showed a decreased ifnt expression in te and to a greater extend in icm upon mapk inhibition .figure  <dig> expression in and go-analysis of mapk-inhibited and control inner cell mass.
all probes representing genes  were plotted for their expression levels .
black lines represent the 2-fold cut-off.
axes are log <dig> transformed and depict the normalized expression signal in the indicated sample.
genes discussed in text  are indicated.
genes with significant  higher expression levels in mapk-inhibited and control inner cell masses are indicated with red and green dots, respectively.
mapk-inhibited icm  gene expression of nanog, pou5f <dig>  sox <dig> and gata <dig> were determined by microarray  and qrt-pcr  presented relative to gene expression in control icm .
significant differences are indicated; * p <  <dig> ; ** p <  <dig> .
ifnt expression  was determined by qrt-pcr in control te , pd0325901-treated te  and pd0325901-treated icm  and presented relative to control icm .
normalization was performed with the reference genes rpl <dig>  sdha and ywhaz.
error bars indicate standard deviation and data bars with different letters are significantly different .
a venn diagram  shows the numbers of overrepresented  and underrepresented  go-terms in the mapk-inhibited inner cell mass.
<dig> go-terms are in common.
genes up-regulated in the pd-treated or in the control icms were enriched in  <dig> of the same go terms after a singular enrichment analysis containing at least two genes and a fdr ≤ <dig>  .
when the relative gene expression was taken into account only  <dig> go terms were found to be enriched with a fdr <  <dig> , of which  <dig> were under-represented in the pd-treated icms .
the only function-describing term was receptor binding  and this term was under-represented in the pd group.table  <dig>
agrigo parametric analysis of gene set enrichment  analysis
go_acc
ontology
description
genes #
p-value
fdr
intracellular non-membrane-bounded organelle
non-membrane-bounded organelle
all genes differentially expressed between mapk-inhibited and control icms were assessed for their properties described by gene ontology  terms in respect to their relative expression.
this revealed  <dig> enriched go-terms with a fdr ≤  <dig>  in the go domains molecular function  and cellular component .
bold descriptions indicate enrichment in mapk inhibited icm.
regular descriptions indicate enrichment in control icm.
cross-comparisons
the  <dig> genes differentially expressed in the icm versus te and the  <dig> differentially expressed genes in the mapk inhibited versus control icms were compared revealing that  <dig> genes were shared .
of these genes  <dig> were both up-regulated in icm and after mapk inhibition while  <dig> were contra-regulated .
only prph was expressed at lower levels in icm than in te and  <dig> genes were down-regulated after mapk inhibition and the expression of  <dig> genes was further up-regulated .
although it has been described that nanog activates pou5f <dig> and sox <dig> transcription, expression of these genes was not significantly altered after up-regulation of nanog expression by mapk inhibition.table  <dig>
list of genes differentially expressed in icm versus te and pd treated versus control icms
gene name
fc
fc
alphabetical ranking of differentially expressed genes  in inner cell mass versus trophectoderm  and mapk-inhibited versus control inner cell masses .
fc = fold change in respect to icm or pd.
the expression of gata <dig> was significantly down-regulated in the icm after mapk inhibition, although the difference was less than 2-fold and was not significant in the qrt-pcr analysis.
the expression of  <dig> other icm-specific genes was significantly down-regulated upon mapk inhibition suggesting that these genes are involved in primitive endoderm formation .
expression of a number of the genes was also analysed by qrt-pcr showing a similar pattern and validating the microarray data .
indeed mouse follistatin, coded by fst, has been implicated as a marker for primitive endoderm derivatives  <cit> .
other genes down-regulated in the mapk inhibited bovine icms themselves code for negative regulators of mapk activity such as dusp <dig> and spry <dig>
the  <dig> shared genes were analyzed for their properties by gene ontology analysis.
a page analysis in agrigo revealed  <dig> enriched go-terms of which none had a fdr <  <dig> .
when the sea was performed using either icm up-regulated, pd up-regulated or pd down-regulated genes , a venn diagram with the enriched terms  revealed  <dig> enriched go-terms shared between the up-regulated gene comparisons .
discussion
during bovine pre-implantation development several cell types display a pluripotent character.
the failure in generating true pluripotent es cell lines from bos taurus embryos however indicates that, compared with murine and human embryos, other genes are involved in maintenance of pluripotency, that the correct embryonic stage with pluripotent cells has not been used, or that the culture conditions employed did not sufficiently inhibit differentiation.
interestingly, induced pluripotent stem  cells generated from bovine cells also behave differently than mouse ips cells.
similar to porcine ips cells, the introduced transgenes are not silenced in the currently used culture conditions but remain expressed in these cells suggesting that other factors are needed for maintenance of pluripotency  <cit> .
we performed a microarray analysis comparing morula, blastocyst, icm and te gene expression profiles to identify genes possibly involved in pluripotency.
further enrichment of the pluripotent character of the icm was achieved by inhibiting the mapk pathway through exposure to the mek inhibitor pd <dig> during in vitro culture thereby increasing the percentage of nanog expressing cells in the icm/epiblast  <cit> .
to obtain samples for the microarray analysis embryos were cultured up to the morula stage or blastocyst stage.
blastocysts were dissected manually to separate icm and te.
the advantage of this technique is that icm and te are isolated from the same embryo, in contrast to for example a technique like immunosurgery.
manually dissecting blastocysts is challenging however and it is unavoidable that few te cells remain attached to the icm.
we therefore verified that the separation of the two cell types was successful.
for separation of icm from te, nagatomo et al.
have used either a micromanipulator or mild trypsin treatment to separate icm  <cit> .
icms isolated using the micromanipulator still contained  <dig> % te cells  <cit> .
in our hands the percentage of te cells remaining in the icm isolates was ~20% as determined by cdx <dig> expression, indicating a low contribution of te cells to the icm transcriptome.
the disadvantage of mild trypsin treatment to isolate ‘pure’ icm cells, is that te and icm cannot be compared from the same embryo and that the trypsin treatment by itself may cause a difference in gene expression.
the observation that duplicate samples paired together and that te and icm clustered apart from each other together with the expected expression patterns of known te- and icm-specific genes in the microarray as well as by qrt-pcr indicates that indeed the separation was specific and reproducible.
several genes were represented on the array by multiple probes and in those cases we only used the expression data of the most 3’-located probe.
unfortunately, the bos taurus genome is not completely annotated  <cit>  and indeed approximately 5% of the probes representing genes differentially expressed between blastocyst and morula could not be identified.
the other comparisons could be made with all probes linked to a known differentially expressed gene.
for the gene ontology analysis genes need to be associated with a go-term.
not all genes are associated with a go-term and therefore  <dig> % –  <dig> % of the genes could not be analysed in the agrigo gene ontology analysis.
in vitro derived embryos were used as this enabled us to generate the numbers needed for rna extraction.
particularly for the icm and te samples large numbers of embryos were needed to obtain sufficient amounts of rna for hybridization .
although a significant difference in gene expression between in vitro and in vivo derived embryos has been demonstrated  <cit>  the birth of healthy animals from in vitro derived embryos indicates that the pathways for pluripotency are functional in these embryos.
when gene expression was compared between different stages of in vivo derived bovine embryos most genes were found to be differentially expressed between early development  and later stages   <cit> .
most likely these differences in gene expression are caused by embryonic genome activation around the 8-cell stage  <cit> .
a larger number of genes  was expressed in in vivo derived oocytes compared with in vitro matured oocytes  <cit> , indicating that in our study with in vitro derived embryos important genes may not have been detected.
however, since in vitro derived embryos are commonly used for embryo transfer and give rise to healthy animals, it can be expected that genes important for pluripotency are sufficiently expressed and the pathways for pluripotency are functional in in vitro derived embryos.
we started our analysis by comparing gene expression in blastocysts with that in morulae.
this indicated that most differentially expressed genes are expressed at higher levels in the blastocyst but a go-analysis revealed that most genes expressed at higher levels in morulae are involved in gene transcription.
this might be a result of the embryonic genome activation initiated during the 8- <dig> cell stage in cattle embryos preceding the morula stage  <cit> .
next we tried to identify genes involved in pluripotency by comparing gene expressions in icm and te.
mouse and human es cell pluripotency is regulated by nanog, sox <dig> and oct <dig>  and these factors enhance each other’s transcription  <cit> .
indeed, their gene expression levels were found higher in the bovine icm samples compared with te.
remarkably, in the comparison of the icm with the te, expression of genes in the go-category rna splicing was specifically up-regulated in the icm.
this indicates a higher transcriptional activity in icm cells than in te cells.
this is further reflected in the > <dig> genes up-regulated in icm compared with te.
using deep sequencing, ozawa et al.
examined genes differentially expressed between icm and te of day  <dig> in vitro derived embryos  <cit> .
all of  <dig> icm-characteristic genes that ozawa et al.
found were also up-regulated in our study, except for zc3hav <dig> and il6r.
expression of il6r was indeed significantly up-regulated in the icm but the difference was below the cut-off used .
these results confirm the specificity and reliability of the icm isolation and microarray analysis.
compared with our results ozawa et al.
found more genes  to be differentially expressed between icm and te, most likely because of the less stringent cut-off value used   <cit> .
by enhancing the overall nanog expression in the icm we had anticipated that sox <dig> and pou5f <dig> expression were similarly enhanced.
surprisingly however, in icms from embryos cultured in the presence of a mapk inhibitor, gene expression levels of nanog were up-regulated while those of pou5f <dig> and sox <dig> remained relatively unchanged.
these results suggest that in bovine cells nanog by itself is not sufficient in maintaining the core pluripotency network.
an unexpected result was the expression of several interferon-coding genes in the icm.
various reports have described exclusive ifnt expression in trophectoderm or te derived cell lines  <cit> .
we detected ifnt expression in the isolated icms at similar levels as in te however and the expression in the icm was down-regulated upon mapk inhibition even to a greater extent than in te.
in ungulates, interferon tau  expression by te is important for maternal pregnancy recognition  <cit> .
in bovine day  <dig> blastocysts interferon tau has been detected at varying intensity in the te and was concentrated at the border of the icm and te  <cit> .
by dissecting the icm, part if not all of the interferon tau-positive adjacent cells have been included in the icm samples accounting for the observed ifnt expression in the icm samples.
together with cdx <dig> predominantly expressed in te cells and capable of increasing ifnt transcription  <cit> , this might explain the greater expression reduction found in icm  than in te .
nevertheless, our results and the previously reported location of interferon tau expression  <cit>  do not exclude ifnt expression in the icm even though its function in the icm is unknown.
after exposure to pd <dig> expression of nanog in the icm was enhanced as compared to control icms.
in the mouse more than  <dig>  genes have been identified containing nanog binding sites  <cit> .
of the  <dig> genes identified to be differentially expressed in bovine icms and after mapk inhibition, only five were homologous to murine genes containing nanog binding sites.
of those genes only expression of nanog was up-regulated after mapk inhibition.
of the remaining four, cd8b, dusp <dig>  jam <dig> and spry <dig>  the expression was enhanced in the icm but their expression was down-regulated after mapk inhibition.
the role of the glycoprotein cd8b in early embryonic development, and more specifically in the icm, is unclear.
its expression can be regulated however by mapk signalling  <cit>  possibly accounting for the observed down-regulation after pd <dig> treatment.
dusp <dig> is suggested to function in the negative feedback control of mapk signalling specifically dephosphorylating erk1/ <dig>  <cit> .
also spry <dig> is known for its involvement in the mapk pathway by interacting with grb <dig> and gap <dig> and as such inhibiting ras activation  <cit>  and antagonizing fgf activity  <cit> .
therefore, the down regulation of dusp <dig> and spry <dig> expression by mapk inhibition is most likely a direct result of the mapk inhibition rather than result from the up-regulation of nanog expression.
jam <dig> is expressed in both embryonic and adult stem cell lines  <cit>  and its expression is enhanced in undifferentiated mouse es cells compared to early stages of differentiation.
since mouse es cells that genetically lack jam <dig> maintain pluripotency however, the function of jam <dig> in stem cells remains unknown  <cit> .
in mouse sertoli cells inhibition of erk activity did not affect jam <dig> transcription  <cit> , suggesting that the observed reduced jam <dig> expression resulted from increased nanog levels.
interestingly, jam <dig> expression was also down-regulated after oct <dig> had been exogenously introduced into human cells, suggesting that low levels of jam <dig> induce or indicate differentiation  <cit> .
in our bovine icms pou5f <dig> expression was however not significantly up-regulated after enhanced nanog expression.
surprisingly, no other genes that had been identified as overlapping nanog putative targets in mouse and human es cells  <cit>  appeared to be up- or down-regulated in bovine icms with enhanced nanog expression.
apart from the core pluripotency markers nanog, sox <dig> and oct <dig>  other transcription factors are reported to be involved in mouse or human pluripotency.
of all transcription factors differentially expressed between icm and te, otx <dig> ranked third and was  <dig> -fold higher expressed in icm.
in mouse es cells otx <dig> was reported to be required for the transition to a stable epiblast stem cell condition  <cit> .
recently, it was shown that otx <dig> is one of the earliest transcription factors to be activated during exit from a naïve ground state in mes cells  <cit> .
although the mapk pathway is important in cell differentiation  <cit>  and therefore might influence otx <dig> expression we did not detect a difference in otx <dig> expression in the pd treated bovine icms.
together, these findings suggest that the icms under investigation were already in a “primed” state.
transcription factors involved in the lif or bmp pathway were also amongst the genes with up-regulated expression levels in the icm.
although bmp <dig> was not differentially expressed, stat <dig> , id <dig>  and id <dig>  were expressed at higher levels in icm than in te.
stat <dig> is capable of suppressing mesoderm and endoderm commitment whereas id genes suppress neuroectoderm commitment in mes cells.
fibronectin, with expression levels almost 4-fold higher in icm, can induce id expression and also nanog is capable of activating stat <dig> and inducing id genes  <cit> .
up-regulated nanog expression did however not induce stat <dig> or id expression in mapk inhibited icms.
although the level of expression might not be high enough, the increased expression of stat <dig>  id <dig> and id <dig> suggests that, although in a primed state, differentiation is not initiated yet in the bovine day  <dig> icms.
the transcription factor prdm <dig> is implicated to act as a safeguard for maintaining pluripotency  <cit>  and is uniquely expressed in mouse compacted morula, icm, the early epiblast, primordial germ cells and es cells  <cit> .
indeed, the expression of prdm <dig> was found to be up-regulated in bovine morulae compared to blastocysts  and icm versus te .
it has been reported that in mouse es cells prdm <dig> attenuates fgf-induced differentiation  <cit> .
we did however not observe a difference in levels of prdm <dig> expression in mapk-inhibited icms , suggesting that the fgf- or mapk-signalling pathways do not repress prdm <dig> expression in bovine pluripotent cells.
expression of the zic gene family members zic <dig> and zic <dig> was also up-regulated in the icm .
zic <dig> and its orthologues are expressed in frog  <cit>  and zebrafish  <cit> , pregastrulation embryos and in mouse e <dig>  and icm of e <dig>  embryos  <cit> .
zic <dig> is implicated to play an important role in maintaining pluripotency in mouse es cells  <cit>  and contains oct <dig>  nanog and sox <dig> binding sites  <cit> .
indeed, after increased nanog expression by mapk inhibition zic <dig> expression increases  <dig>  fold  but zic <dig> expression decreased  <dig> -fold .
other reported genes to safeguard pluripotency such as parp <dig> and parp <dig>  <cit>  were expressed at higher levels in morula than in blastocyst , did not show a differential expression between icm and te and were not differentially expressed after mapk inhibition.
all together, these findings indicate that, despite the increased zic <dig> and nanog expression, mapk inhibition by pd <dig> is insufficient to maintain a pluripotent state in bovine icm cells.
CONCLUSIONS
we have identified whole genome expression profiles of different stages of bovine embryos and te and the pluripotent icm of blastocysts.
in addition, the transcriptome of icms with enhanced nanog expression after inhibition of mapk activity was established.
unfortunately, these expression profiles did not lead to  pathways or indications how to maintain pluripotency and possibly generate genuine bovine es cells.
furthermore, it became apparent that although mapk inhibition increased nanog and zic <dig> expression, this is insufficient to maintain pluripotency.
comparing transcription factor expression in the bovine icms used in the microarray with known expressions in mouse pluripotent cells indicates a “primed” or epiblast state.
therefore, the data presented in this paper can act as a starting point for further research on bovine pluripotency.

 in mammals, early life starts with the formation of a zygote as a result of the fertilization of an oocyte. sequential cleavage divisions lead to the formation of a morula stage embryo wherein a fluid-filled cavity emerges called the blastocoel. two differentiated groups of cells can be distinguished in the embryo that is now called a blastocyst. a group of cells adjacent to the blastocoel, the inner cell mass, is able to contribute to all cells of the three germ layers and is therefore referred to as being pluripotent. the other group of cells, called the trophectoderm, forms an epithelium surrounding the blastocoel and the icm and is important for implantation within the uterus and contributes to the non-maternal part of the placenta. in the icm further differentiation occurs by the formation of the epiblast, that will form the foetus, and the formation of extra-embryonic primitive endoderm contributing to the yolk sac. studies with mouse embryos have advanced our understanding of how a pluripotent cell population is established during pre-implantation development. during the first differentiation, the transcription factors cdx and oct are key regulators for the formation of respectively te and icm. cdx represses the activity of oct in mouse te and is virtually absent in icm cells. oct in turn can counteract cdx activity in the inner cells of the morula. the second differentiation is indicated by the expression of either nanog or gata in icm cells fated to become the epiblast or pe respectively. like for cdx and oct in the morula, nanog and gata inhibit each other’s transcription. whether the same genes and signalling pathways are also involved in the formation of a pluripotent cell population in other mammals remains to be established. indeed, in contrast to the mouse, oct protein remains present in the te of bovine blastocysts even after transcription is down-regulated and its expression is not negatively regulated by cdx. in mouse embryos it has been established that gata6-stimulated fibroblast growth factor signalling via the extracellular signal-regulated protein kinase is responsible for nanog repression and thereby the formation of primitive endoderm. in bovine and human embryos however, although gata expression is specific for primitive endoderm, inhibition of erk signalling had a more moderate or no effect on the numbers of nanog and gata expressing cells suggesting that in these species other pathways are involved in the formation of the pluripotent cell population. these findings suggest species-specific mechanisms active in the specification of icm, te, epiblast and pe lineages and that further insight is needed into the molecular basis of cell sorting during the two first differentiation events. when mouse icm cells are cultured under defined conditions, their pluripotent character can be maintained. however, the establishment of such embryonic stem cells has only been successful for mice, non-human primates, humans and rats. although pluripotency refers to the capacity to give rise to all embryonic and adult cell types, including the germ line, various states of pluripotency have been described. these states are referred to as “naïve” and “primed”, with “primed” being more developmentally restricted. in mammals other than primates and rodents, the correct stages of embryos that contain pluripotent cells and culture conditions that maintain pluripotency have yet to be established. in order to identify genes that may be important for the acquisition and maintenance of pluripotency in bovine embryos a genome-wide gene expression analysis was performed in morulae, intact blastocysts, te and icm. analyses of gene expression patterns in pre-implantation embryos to distinguish between pluripotent cells of the icm versus those of the te have previously made use of cell lines because of the technical difficulties of separating icm from te. here we have manually dissected individual icms from te. as the late icm is composed of both pluripotent epiblast cells and the pe, the pluripotent character of the icm was enhanced by inhibition of the erk-pathway resulting in an increased percentage of icm cells that express nanog.  gene expression profile of pre-implantation embryos to identify genes involved in bovine pluripotency, gene expression profiles of morula and blastocyst embryos, icm and te were generated using microarray analysis. bovine cumulus oocyte complexes were in vitro matured, fertilized and cultured for or 9 days to obtain morula and blastocyst stage embryos, respectively. in addition, icm and te were manually dissected from day blastocysts. from all samples rna was isolated and only those with a rna integrity number ≥ were used for further analysis. to compensate for biological and technical errors, two biological replicates of each sample were labelled with either cy or cy and hybridized on the arrays with a common reference pool consisting of blastocysts so all samples could be compared.figure microarray set-up and sample validation. rna from morula, blastocyst, trophectoderm and inner cell mass was hybridized on microarrays. a common reference sample composed of blastocysts was hybridized with each sample in duplicate in balanced dye-swap. the heat map illustrates pairwise clustering of microarray sample replicates. yellow colour represents over-expressed probes and blue colour represents under-expressed probes as indicated by the colour legend. representative pictures of immunofluorescent labelling of dissected icm and te for gata and cdx; double gata6-cdx nuclei appear yellow. nuclear staining by dapi. a 2d principal component analysis plot with sample position indicating clustering of trophectoderm, blastocyst, inner cell mass and morula replicates. samples were hybridized on a microarray slide containing almost probes per array coding for ~  gene transcripts indicating that for a subset of genes more than one probe was present. if the position of the probe is nearer to the 3’ end of the corresponding gene, signal intensity is expected to be higher and chance of incorrect signal by variations in rna integrity is smaller. therefore, the expression of the probes corresponding to the most 3’ ends of genes was used for the analysis. gene expression levels in morula, blastocyst, icm and te were determined and a hierarchical clustering analysis was performed. the constructed heat map shows clear pairing of the morula, icm and te samples. this is particularly important for the te and icm samples since these were manually dissected and confirms the reproducibility of the dissection. we used mechanical isolation of icm from te using tungsten needles. a selection of isolated icms was stained for cdx and gata to identify the contribution of te cells to the pooled microarray samples. the isolated icms contained only ~20% cdx positive te. since however some te cells remained attached to the icm, throughout the manuscript “icm” refers to the icm containing few te cells. in the te samples all cells were cdx and gata positive. blastocyst samples did not pair since their difference with the reference is minimal indicated by a near black appearance in the heat map. a principal component analysis further identified four categories according to cell type or developmental stage. in a 2d plot, morula samples separate the farthest from the other samples. te and icm samples are clearly separated from each other with the blastocyst replicates in between. to further confirm the specificity of the samples, expression levels of genes that are known to be differentially expressed in bovine embryos were compared. of the selected genes hmgb sox and pou5f are known to be expressed at relatively high levels in bovine morula embryos, cdx and krt have the highest expression levels in te while fn and nanog are abundantly expressed in the icm. the relative expression levels of the selected genes as determined by microarray analysis in morula, blastocyst, icm and te were as expected, with highest levels of hmgb and sox expression in morulae, pou5f expression predominantly in morulae and icm, highest fn and nanog expression in the icm and cdx and krt expression at highest levels in te. these selected genes and five additional genes were also analysed for their expression levels by qrt-pcr, revealing a similar expression profile validating sample identity and demonstrating that the microarray data accurately reflect relative expression levels.figure relative gene expression. microarray data were analysed for selected genes known for their expression in morula, blastocyst, icm and te. the same genes were analysed by qrt-pcr in 4– samples per embryo stage or cell type revealing the same pattern. normalization was performed with the reference genes encoding rpl sdha and ywhaz. y-axis depicts relative mean expression to sample with highest expression set at per gene. bars with different letters are significantly different and error bars indicate standard deviation. genes differentially expressed between morulae and blastocysts most differences in gene expression were found between morulae and blastocysts. when gene expression in morulae was compared with that in blastocysts using a >2-fold difference with p-value < as cut-off, genes were differentially expressed. in the blastocyst, genes were up-regulated, up to 107-fold, while genes were expressed at higher levels in morulae. the most differentially expressed genes were expressed with at least a 30-fold difference of which only gene was expressed at higher levels in the morula.figure relative gene expression in blastocyst versus morula and inner cell mass versus trophectoderm samples. all probes representing genes were plotted for their expression levels. black lines represent the 2-fold cut off. axes are log transformed and depict the normalized expression signal in the indicated sample. genes analysed in qrt-pcr are indicated. genes with significant higher expression levels in blastocyst or morula are indicated with green or purple dots, respectively. genes with significant higher expression levels in inner cell mass or trophectoderm are indicated with orange or blue dots, respectively. list of the most differentially expressed genes between blastocyst and morula rank entrez gene name gene discription agrigo id fc the most differentially expressed genes between blastocyst and morula are listed. fc = fold-change and positive values indicate higher expression in blastocyst and negative values have higher expression in morula. to identify properties of the differentially expressed genes a parametric analysis of gene set enrichment was performed using agrigo and a list of the five most-enriched gene ontology terms per categorie was generated. a large group of differentially expressed genes was involved in the lipid metabolic process in the blastocyst. a much smaller group of genes that function in pepsin a activity was over-represented in blastocysts and indeed, a large group of genes up-regulated in the blastocyst located to the plasma membrane. compared to the blastocyst, in the morula more genes were involved in the nucleobase-containing compound metabolic process and more specifically in rna processing. morula up-regulated genes were found in the enriched molecular function go terms nucleic acid binding and transcription regulator activity.table agrigo parametric analysis of gene set enrichment analysis go_acc ontology description genes # p-value fdr lipid metabolic process pepsin a activity membrane plasma membrane membrane part extracellular region part integral to membrane genes differentially expressed between blastocyst and morula were assessed for their properties described by gene ontology terms in respect to their relative expression. the five most significantly enriched go-terms are listed for each go domain. p = biological process, f = molecular function and c = cellular component. bold descriptions indicate enrichment for blastocyst. regular descriptions indicate enrichment for morula. fdr = false discovery rate. genes differentially expressed between icm and te when gene expression in the icm was compared with that of the te, genes were differentially expressed. here, the majority of the differentially expressed genes were expressed at higher levels in the icm. of all genes, the difference in expression levels of nanog was the largest. sox and pou5f that together with nanog form the core transcriptional regulatory circuitry in pluripotent cells were also over-expressed in the icm compared with te.table list of the most differentially expressed genes between inner cell mass and trophectoderm rank entrez gene name gene description agrigo id fc the most differentially expressed genes between inner cell mass and trophectoderm are listed according to their relative expression. fc = fold change and positive values indicate higher expression in inner cell mass. for human es cells, a network of nanog-, sox2- and pou5f1-target genes that encode transcription factors and chromatin modulators has been established. from the list of positively regulated genes in human es cells, apart from the core network only expression of stat and zic was significantly up-regulated in bovine icm versus te. again, we performed a parametric analysis of gene set enrichment with the genes differentially expressed between icm and te in agrigo. with a false discovery rate ≤, enzyme linked receptor protein signaling pathway and peptidyl-tyrosine phosphorylation terms were enriched for the icm whereas for the te, genes were enriched in the sterol biosynthetic process. only genes up-regulated in respect to the te were used for a singular enrichment analysis in order to identify characteristics specific for the cells of the icm. in particular, terms containing genes involved in rna splicing and regulation of gene expression were enriched even to a more specific level of enriched child terms like nuclear mrna splicing and chromatin silencing, respectively.table agrigo parametric analysis of gene set enrichment analysis go_acc ontology description genes # p-value fdr intracellular signaling pathway peptidyl-tyrosine modification peptidyl-tyrosine phosphorylation enzyme linked receptor protein signaling pathway all genes differentially expressed between icm and te were assessed for their properties described by gene ontology terms in respect to their relative expression. this reveals enriched go-terms with a fdr ≤  in the go domains biological process and cellular component. ranked according to z-score. bold descriptions indicate enrichment for inner cell mass. regular descriptions indicate enrichment for trophectoderm. fdr = false discovery rate. genes differentially expressed between mapk-inhibited and control icms it has been described that culture of icm cells in the presence of an fgfr inhibitor, a mapk inhibitor and an inhibitor of gsk3β, the so-called 3i culture system, leads to stable es cell lines from non-permissive mouse strains and rat embryos. culture of mouse embryos in the presence of a mapk inhibitor resulted in all cells of the icm expressing nanog while the expression of gata was down-regulated. similarly, when bovine embryos were cultured in the presence of the mapk inhibitor pd a larger percentage, although not all, of the icm cells expressed nanog. therefore, in order to identify nanog target genes and genes that may be important for pluripotency, icms were isolated from bovine embryos cultured in the presence of the mapk inhibitor pd gene expression in these icms was compared with that from control icms using microarray analysis. in total genes were differentially regulated between control and mapk-inhibited icms, ≥2-fold difference with p-value ≤  as cut-off, with the expression of genes up-regulated and the expression of genes down-regulated. as expected, nanog expression was up-regulated in the mapk-inhibited icm as detected by microarray analysis. expression differences between control icm and icm from embryos exposed to the mapk inhibitor detected by qrt-pcr verified the microarray data. furthermore, immunostaining showed an increase in the percentage of nanog expressing cells in the icm after mapk inhibition as we had previously established. to our surprise several interferon coding genes were dramatically down-regulated after mapk inhibition such as ifnw and ifnt. gene expression analysis by qrt-pcr confirmed the microarray results and further showed a decreased ifnt expression in te and to a greater extend in icm upon mapk inhibition.figure expression in and go-analysis of mapk-inhibited and control inner cell mass. all probes representing genes were plotted for their expression levels. black lines represent the 2-fold cut-off. axes are log transformed and depict the normalized expression signal in the indicated sample. genes discussed in text are indicated. genes with significant higher expression levels in mapk-inhibited and control inner cell masses are indicated with red and green dots, respectively. mapk-inhibited icm gene expression of nanog, pou5f sox and gata were determined by microarray and qrt-pcr presented relative to gene expression in control icm. significant differences are indicated; * p < ; ** p < . ifnt expression was determined by qrt-pcr in control te, pd0325901-treated te and pd0325901-treated icm and presented relative to control icm. normalization was performed with the reference genes rpl sdha and ywhaz. error bars indicate standard deviation and data bars with different letters are significantly different. a venn diagram shows the numbers of overrepresented and underrepresented go-terms in the mapk-inhibited inner cell mass. go-terms are in common. genes up-regulated in the pd-treated or in the control icms were enriched in of the same go terms after a singular enrichment analysis containing at least two genes and a fdr ≤. when the relative gene expression was taken into account only go terms were found to be enriched with a fdr < , of which were under-represented in the pd-treated icms. the only function-describing term was receptor binding and this term was under-represented in the pd group.table agrigo parametric analysis of gene set enrichment analysis go_acc ontology description genes # p-value fdr intracellular non-membrane-bounded organelle non-membrane-bounded organelle all genes differentially expressed between mapk-inhibited and control icms were assessed for their properties described by gene ontology terms in respect to their relative expression. this revealed enriched go-terms with a fdr ≤  in the go domains molecular function and cellular component. bold descriptions indicate enrichment in mapk inhibited icm. regular descriptions indicate enrichment in control icm. cross-comparisons the genes differentially expressed in the icm versus te and the differentially expressed genes in the mapk inhibited versus control icms were compared revealing that genes were shared. of these genes were both up-regulated in icm and after mapk inhibition while were contra-regulated. only prph was expressed at lower levels in icm than in te and genes were down-regulated after mapk inhibition and the expression of genes was further up-regulated. although it has been described that nanog activates pou5f and sox transcription, expression of these genes was not significantly altered after up-regulation of nanog expression by mapk inhibition.table list of genes differentially expressed in icm versus te and pd treated versus control icms gene name fc fc alphabetical ranking of differentially expressed genes in inner cell mass versus trophectoderm and mapk-inhibited versus control inner cell masses. fc = fold change in respect to icm or pd. the expression of gata was significantly down-regulated in the icm after mapk inhibition, although the difference was less than 2-fold and was not significant in the qrt-pcr analysis. the expression of other icm-specific genes was significantly down-regulated upon mapk inhibition suggesting that these genes are involved in primitive endoderm formation. expression of a number of the genes was also analysed by qrt-pcr showing a similar pattern and validating the microarray data. indeed mouse follistatin, coded by fst, has been implicated as a marker for primitive endoderm derivatives. other genes down-regulated in the mapk inhibited bovine icms themselves code for negative regulators of mapk activity such as dusp and spry the shared genes were analyzed for their properties by gene ontology analysis. a page analysis in agrigo revealed enriched go-terms of which none had a fdr < . when the sea was performed using either icm up-regulated, pd up-regulated or pd down-regulated genes, a venn diagram with the enriched terms revealed enriched go-terms shared between the up-regulated gene comparisons. discussion during bovine pre-implantation development several cell types display a pluripotent character. the failure in generating true pluripotent es cell lines from bos taurus embryos however indicates that, compared with murine and human embryos, other genes are involved in maintenance of pluripotency, that the correct embryonic stage with pluripotent cells has not been used, or that the culture conditions employed did not sufficiently inhibit differentiation. interestingly, induced pluripotent stem cells generated from bovine cells also behave differently than mouse ips cells. similar to porcine ips cells, the introduced transgenes are not silenced in the currently used culture conditions but remain expressed in these cells suggesting that other factors are needed for maintenance of pluripotency. we performed a microarray analysis comparing morula, blastocyst, icm and te gene expression profiles to identify genes possibly involved in pluripotency. further enrichment of the pluripotent character of the icm was achieved by inhibiting the mapk pathway through exposure to the mek inhibitor pd during in vitro culture thereby increasing the percentage of nanog expressing cells in the icm/epiblast. to obtain samples for the microarray analysis embryos were cultured up to the morula stage or blastocyst stage. blastocysts were dissected manually to separate icm and te. the advantage of this technique is that icm and te are isolated from the same embryo, in contrast to for example a technique like immunosurgery. manually dissecting blastocysts is challenging however and it is unavoidable that few te cells remain attached to the icm. we therefore verified that the separation of the two cell types was successful. for separation of icm from te, nagatomo et al. have used either a micromanipulator or mild trypsin treatment to separate icm. icms isolated using the micromanipulator still contained % te cells. in our hands the percentage of te cells remaining in the icm isolates was ~20% as determined by cdx expression, indicating a low contribution of te cells to the icm transcriptome. the disadvantage of mild trypsin treatment to isolate ‘pure’ icm cells, is that te and icm cannot be compared from the same embryo and that the trypsin treatment by itself may cause a difference in gene expression. the observation that duplicate samples paired together and that te and icm clustered apart from each other together with the expected expression patterns of known te- and icm-specific genes in the microarray as well as by qrt-pcr indicates that indeed the separation was specific and reproducible. several genes were represented on the array by multiple probes and in those cases we only used the expression data of the most 3’-located probe. unfortunately, the bos taurus genome is not completely annotated and indeed approximately 5% of the probes representing genes differentially expressed between blastocyst and morula could not be identified. the other comparisons could be made with all probes linked to a known differentially expressed gene. for the gene ontology analysis genes need to be associated with a go-term. not all genes are associated with a go-term and therefore % – % of the genes could not be analysed in the agrigo gene ontology analysis. in vitro derived embryos were used as this enabled us to generate the numbers needed for rna extraction. particularly for the icm and te samples large numbers of embryos were needed to obtain sufficient amounts of rna for hybridization. although a significant difference in gene expression between in vitro and in vivo derived embryos has been demonstrated the birth of healthy animals from in vitro derived embryos indicates that the pathways for pluripotency are functional in these embryos. when gene expression was compared between different stages of in vivo derived bovine embryos most genes were found to be differentially expressed between early development and later stages. most likely these differences in gene expression are caused by embryonic genome activation around the 8-cell stage. a larger number of genes was expressed in in vivo derived oocytes compared with in vitro matured oocytes, indicating that in our study with in vitro derived embryos important genes may not have been detected. however, since in vitro derived embryos are commonly used for embryo transfer and give rise to healthy animals, it can be expected that genes important for pluripotency are sufficiently expressed and the pathways for pluripotency are functional in in vitro derived embryos. we started our analysis by comparing gene expression in blastocysts with that in morulae. this indicated that most differentially expressed genes are expressed at higher levels in the blastocyst but a go-analysis revealed that most genes expressed at higher levels in morulae are involved in gene transcription. this might be a result of the embryonic genome activation initiated during the 8- cell stage in cattle embryos preceding the morula stage. next we tried to identify genes involved in pluripotency by comparing gene expressions in icm and te. mouse and human es cell pluripotency is regulated by nanog, sox and oct and these factors enhance each other’s transcription. indeed, their gene expression levels were found higher in the bovine icm samples compared with te. remarkably, in the comparison of the icm with the te, expression of genes in the go-category rna splicing was specifically up-regulated in the icm. this indicates a higher transcriptional activity in icm cells than in te cells. this is further reflected in the > genes up-regulated in icm compared with te. using deep sequencing, ozawa et al. examined genes differentially expressed between icm and te of day in vitro derived embryos. all of icm-characteristic genes that ozawa et al. found were also up-regulated in our study, except for zc3hav and il6r. expression of il6r was indeed significantly up-regulated in the icm but the difference was below the cut-off used. these results confirm the specificity and reliability of the icm isolation and microarray analysis. compared with our results ozawa et al. found more genes to be differentially expressed between icm and te, most likely because of the less stringent cut-off value used. by enhancing the overall nanog expression in the icm we had anticipated that sox and pou5f expression were similarly enhanced. surprisingly however, in icms from embryos cultured in the presence of a mapk inhibitor, gene expression levels of nanog were up-regulated while those of pou5f and sox remained relatively unchanged. these results suggest that in bovine cells nanog by itself is not sufficient in maintaining the core pluripotency network. an unexpected result was the expression of several interferon-coding genes in the icm. various reports have described exclusive ifnt expression in trophectoderm or te derived cell lines. we detected ifnt expression in the isolated icms at similar levels as in te however and the expression in the icm was down-regulated upon mapk inhibition even to a greater extent than in te. in ungulates, interferon tau expression by te is important for maternal pregnancy recognition. in bovine day blastocysts interferon tau has been detected at varying intensity in the te and was concentrated at the border of the icm and te. by dissecting the icm, part if not all of the interferon tau-positive adjacent cells have been included in the icm samples accounting for the observed ifnt expression in the icm samples. together with cdx predominantly expressed in te cells and capable of increasing ifnt transcription, this might explain the greater expression reduction found in icm than in te. nevertheless, our results and the previously reported location of interferon tau expression do not exclude ifnt expression in the icm even though its function in the icm is unknown. after exposure to pd expression of nanog in the icm was enhanced as compared to control icms. in the mouse more than genes have been identified containing nanog binding sites. of the genes identified to be differentially expressed in bovine icms and after mapk inhibition, only five were homologous to murine genes containing nanog binding sites. of those genes only expression of nanog was up-regulated after mapk inhibition. of the remaining four, cd8b, dusp jam and spry the expression was enhanced in the icm but their expression was down-regulated after mapk inhibition. the role of the glycoprotein cd8b in early embryonic development, and more specifically in the icm, is unclear. its expression can be regulated however by mapk signalling possibly accounting for the observed down-regulation after pd treatment. dusp is suggested to function in the negative feedback control of mapk signalling specifically dephosphorylating erk1/. also spry is known for its involvement in the mapk pathway by interacting with grb and gap and as such inhibiting ras activation and antagonizing fgf activity. therefore, the down regulation of dusp and spry expression by mapk inhibition is most likely a direct result of the mapk inhibition rather than result from the up-regulation of nanog expression. jam is expressed in both embryonic and adult stem cell lines and its expression is enhanced in undifferentiated mouse es cells compared to early stages of differentiation. since mouse es cells that genetically lack jam maintain pluripotency however, the function of jam in stem cells remains unknown. in mouse sertoli cells inhibition of erk activity did not affect jam transcription, suggesting that the observed reduced jam expression resulted from increased nanog levels. interestingly, jam expression was also down-regulated after oct had been exogenously introduced into human cells, suggesting that low levels of jam induce or indicate differentiation. in our bovine icms pou5f expression was however not significantly up-regulated after enhanced nanog expression. surprisingly, no other genes that had been identified as overlapping nanog putative targets in mouse and human es cells appeared to be up- or down-regulated in bovine icms with enhanced nanog expression. apart from the core pluripotency markers nanog, sox and oct other transcription factors are reported to be involved in mouse or human pluripotency. of all transcription factors differentially expressed between icm and te, otx ranked third and was -fold higher expressed in icm. in mouse es cells otx was reported to be required for the transition to a stable epiblast stem cell condition. recently, it was shown that otx is one of the earliest transcription factors to be activated during exit from a naïve ground state in mes cells. although the mapk pathway is important in cell differentiation and therefore might influence otx expression we did not detect a difference in otx expression in the pd treated bovine icms. together, these findings suggest that the icms under investigation were already in a “primed” state. transcription factors involved in the lif or bmp pathway were also amongst the genes with up-regulated expression levels in the icm. although bmp was not differentially expressed, stat, id and id were expressed at higher levels in icm than in te. stat is capable of suppressing mesoderm and endoderm commitment whereas id genes suppress neuroectoderm commitment in mes cells. fibronectin, with expression levels almost 4-fold higher in icm, can induce id expression and also nanog is capable of activating stat and inducing id genes. up-regulated nanog expression did however not induce stat or id expression in mapk inhibited icms. although the level of expression might not be high enough, the increased expression of stat id and id suggests that, although in a primed state, differentiation is not initiated yet in the bovine day icms. the transcription factor prdm is implicated to act as a safeguard for maintaining pluripotency and is uniquely expressed in mouse compacted morula, icm, the early epiblast, primordial germ cells and es cells. indeed, the expression of prdm was found to be up-regulated in bovine morulae compared to blastocysts and icm versus te. it has been reported that in mouse es cells prdm attenuates fgf-induced differentiation. we did however not observe a difference in levels of prdm expression in mapk-inhibited icms, suggesting that the fgf- or mapk-signalling pathways do not repress prdm expression in bovine pluripotent cells. expression of the zic gene family members zic and zic was also up-regulated in the icm. zic and its orthologues are expressed in frog and zebrafish, pregastrulation embryos and in mouse e and icm of e embryos. zic is implicated to play an important role in maintaining pluripotency in mouse es cells and contains oct nanog and sox binding sites. indeed, after increased nanog expression by mapk inhibition zic expression increases fold but zic expression decreased -fold. other reported genes to safeguard pluripotency such as parp and parp were expressed at higher levels in morula than in blastocyst, did not show a differential expression between icm and te and were not differentially expressed after mapk inhibition. all together, these findings indicate that, despite the increased zic and nanog expression, mapk inhibition by pd is insufficient to maintain a pluripotent state in bovine icm cells.  we have identified whole genome expression profiles of different stages of bovine embryos and te and the pluripotent icm of blastocysts. in addition, the transcriptome of icms with enhanced nanog expression after inhibition of mapk activity was established. unfortunately, these expression profiles did not lead to pathways or indications how to maintain pluripotency and possibly generate genuine bovine es cells. furthermore, it became apparent that although mapk inhibition increased nanog and zic expression, this is insufficient to maintain pluripotency. comparing transcription factor expression in the bovine icms used in the microarray with known expressions in mouse pluripotent cells indicates a “primed” or epiblast state. therefore, the data presented in this paper can act as a starting point for further research on bovine pluripotency. 
the inability to culture icm cells as stem cells in the presence of an inhibitor of mapk activity together with the reported data indicates that mapk inhibition alone is not sufficient to maintain a pluripotent character in bovine cells. to investigate which genes are involved in bovine pluripotency, expression profiles were generated from morula, blastocyst, trophectoderm and inner cell mass samples using microarray analysis. as mapk inhibition can increase the nanog/gata ratio in the inner cell mass, additionally blastocysts were cultured in the presence of a mapk inhibitor and changes in gene expression in the inner cell mass were analysed. although nanog expression was elevated upon mapk inhibition, sox and pou5f expression showed little increase. expression of other genes in the mapk pathway including dusp and spry or influenced by mapk inhibition such as ifnt, was down-regulated. genes expressed at higher levels in the icm were enriched in the rna splicing and regulation of gene expression go term. the data obtained from the microarray studies provide further insight in gene expression during bovine embryonic development. they show an expression profile in pluripotent cells that indicates a pluripotent, epiblast-like state. genes with higher expression levels in morulae were enriched in the rna processing go term.
 genes and signalling pathways involved in pluripotency have been studied extensively in mouse and human pre-implantation embryos and embryonic stem cells. the unsuccessful attempts to generate es cell lines from other species including cattle suggests that other genes and pathways are involved in maintaining pluripotency in these species. to investigate which genes are involved in bovine pluripotency, expression profiles were generated from morula, blastocyst, trophectoderm and inner cell mass samples using microarray analysis. as mapk inhibition can increase the nanog/gata ratio in the inner cell mass, additionally blastocysts were cultured in the presence of a mapk inhibitor and changes in gene expression in the inner cell mass were analysed.  between morula and blastocyst genes were differentially expressed and the largest differences were found in blastocyst up-regulated genes. gene ontology analysis shows lipid metabolic process as the term most enriched with genes expressed at higher levels in blastocysts. genes with higher expression levels in morulae were enriched in the rna processing go term. of the differentially expressed genes comparing icm and te, the expression of nanog, sox and pou5f was increased in the icm confirming their evolutionary preserved role in pluripotency. several genes implicated to be involved in differentiation or fate determination were also expressed at higher levels in the icm. genes expressed at higher levels in the icm were enriched in the rna splicing and regulation of gene expression go term. although nanog expression was elevated upon mapk inhibition, sox and pou5f expression showed little increase. expression of other genes in the mapk pathway including dusp and spry or influenced by mapk inhibition such as ifnt, was down-regulated.  the data obtained from the microarray studies provide further insight in gene expression during bovine embryonic development. they show an expression profile in pluripotent cells that indicates a pluripotent, epiblast-like state. the inability to culture icm cells as stem cells in the presence of an inhibitor of mapk activity together with the reported data indicates that mapk inhibition alone is not sufficient to maintain a pluripotent character in bovine cells. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords pluripotencycattlemorulablastocysticmtrophectodermnanogmapkissue-copyright-statement© the author 2015 