Gene expression profiling of bovine endometrium during the oestrous cycle: detection of molecular pathways involved in functional changes

in Journal of Molecular Endocrinology

The endometrium plays a central role among the reproductive tissues in the context of early embryo–maternal communication and pregnancy. It undergoes typical changes during the sexual/oestrous cycle, which are regulated by the ovarian hormones progesterone and oestrogen. To identify the underlying molecular mechanisms we have performed the first holistic screen of transcriptome changes in bovine intercaruncular endometrium at two stages of the cycle – end of day 0 (late oestrus, low progesterone) and day 12 (dioestrus, high progesterone). A combination of subtracted cDNA libraries and cDNA array hybridisation revealed 133 genes showing at least a 2-fold change of their mRNA abundance, 65 with higher levels at oestrus and 68 at dioestrus. Interestingly, genes were identified which showed differential expression between different uterine sections as well. The most prominent example was the UTMP (uterine milk protein) mRNA, which was markedly upregulated in the cranial part of the ipsilateral uterine horn at oestrus. A Gene Ontology classification of the genes with known function characterised the oestrus time by elevated expression of genes, for example related to cell adhesion, cell motility and extracellular matrix and the dioestrus time by higher expression of mRNAs encoding for a variety of enzymes and transport proteins, in particular ion channels. Searching in pathway databases and literature data-mining revealed physiological processes and signalling cascades, e.g. the transforming growth factor-β signalling pathway and retinoic acid signalling, which are potentially involved in the regulation of changes of the endometrium during the oestrous cycle.

Abstract

The endometrium plays a central role among the reproductive tissues in the context of early embryo–maternal communication and pregnancy. It undergoes typical changes during the sexual/oestrous cycle, which are regulated by the ovarian hormones progesterone and oestrogen. To identify the underlying molecular mechanisms we have performed the first holistic screen of transcriptome changes in bovine intercaruncular endometrium at two stages of the cycle – end of day 0 (late oestrus, low progesterone) and day 12 (dioestrus, high progesterone). A combination of subtracted cDNA libraries and cDNA array hybridisation revealed 133 genes showing at least a 2-fold change of their mRNA abundance, 65 with higher levels at oestrus and 68 at dioestrus. Interestingly, genes were identified which showed differential expression between different uterine sections as well. The most prominent example was the UTMP (uterine milk protein) mRNA, which was markedly upregulated in the cranial part of the ipsilateral uterine horn at oestrus. A Gene Ontology classification of the genes with known function characterised the oestrus time by elevated expression of genes, for example related to cell adhesion, cell motility and extracellular matrix and the dioestrus time by higher expression of mRNAs encoding for a variety of enzymes and transport proteins, in particular ion channels. Searching in pathway databases and literature data-mining revealed physiological processes and signalling cascades, e.g. the transforming growth factor-β signalling pathway and retinoic acid signalling, which are potentially involved in the regulation of changes of the endometrium during the oestrous cycle.

Introduction

During the sexual/oestrous cycle characteristic changes occur in the bovine endometrium regarding its composition and differentiation status. These changes are mainly regulated by the hormones progesterone (P4), oestradiol and oxytocin (for review see Spencer et al. 2004). At oestrus, levels are low and increase after P4 ovulation until the highest levels at dioestrus. The increase of P4 is associated with the downregulation of its own receptor in the endometrial epithelium. Via oxytocin, luteolytic pulses of prostaglandin-F are released leading to the regression of the ovarian corpus luteum, termed luteolysis (for review see Goff 2004). This results in the entry into a new ovarian cycle. In contrast, if an embryo is present and produces specific factors (e.g. interferon-τ) the luteolytic mechanism in the endometrium is blocked and the functional corpus luteum is maintained, providing the basis for further development and the implantation of the conceptus. Although these basic means of hormonal regulations in the endometrium during the oestrous cycle are known, the detailed molecular mechanisms are not well understood.

In humans several studies using microarray analyses have been done investigating gene expression changes in the endometrium during the menstrual cycle. In two studies the proliferative phase was compared with the ‘window of implantation’ time (Kao et al. 2002, Borthwick et al. 2003) and in another two studies gene expression differences between the early secretory phase (2–4 days after the luteinising hormone (LH) surge) and the receptive phase (7–9 days after the LH surge) were investigated (Carson et al. 2002, Riesewijk et al. 2003). Two recent studies also provided first insights into changes of gene expression in the endometrium during the oestrous cycle comparing the proliferative vs secretory phase in the mouse (Tan et al. 2003) and the Rhesus monkey (Ace & Okulicz 2004). For ruminants no comparable study has been done so far. Recently, we successfully applied a combination of subtracted cDNA libraries and cDNA array hybridisation to compare the mRNA expression profiles of bovine epithelial cells of the ipsilateral vs the contralateral oviduct (Bauersachs et al. 2003) and of cells from the ipsilateral oviduct at oestrus and dioestrus (Bauersachs et al. 2004) respectively. In the present study a similar approach was applied to analyse differential gene expression in the bovine intercaruncular endometrium at two important phases of the oestrous cycle – the time around ovulation (late oestrus) and the time of high P4 (dioestrus) when the endometrium is prepared for communication with an embryo. Based on the results of bioinformatic analyses and literature data-mining, molecular pathways involved in the regulation of specific functions of the bovine endometrium during the oestrous cycle were identified.

Materials and methods

Synchronisation of oestrous cycle and collection of endometrial tissue samples

Six cyclic heifers (Deutsches Fleckvieh) between 18 and 24 months old were cycle synchronised by injecting i.m. a single dose of 500 μg cloprostenol (Estrumate; Essex Tierarznei, Munich, Germany) at dioestrus. Animals were observed for sexual behaviour (i.e. toleration, sweating, vaginal mucus) to determine standing heat, which occurred around 60 h after Estrumate injection. All animals were checked by ultrasound-guided follicle monitoring starting 48 h after Estrumate application at intervals of 6 h. Blood samples were taken at day 20 and day 0 of the oestrous cycle every 6–9 h to determine serum LH levels (Schams & Karg 1969) and just before slaughtering to determine serum P4 levels (Prakash et al. 1987). Three animals were slaughtered the morning after standing heat occurred within 8 h after the LH surge and three animals 12 days after oestrus; the former group displayed low serum P4 levels (<1.0 ng/ml) and the latter had high serum P4 levels (>6.0 ng/ml). The uterus was removed, opened longitudinally and divided into seven sections: corpus plus caudal, middle and cranial parts of the ipsilateral and the contralateral uterine horns (see Fig. 1a). Samples were carefully cut out from the lamina propria of the intercaruncular endometrium with a scalpel and immediately transferred into cryo-tubes and frozen in liquid nitrogen or on dry ice. Samples were stored at −80 °C until further processing. Tissue samples for in situ hybridisation were also taken from the same animals. All experiments with animals were conducted with permission from the local veterinary authorities and in accordance with accepted standards of humane animal care.

Generation of subtracted cDNA libraries and cDNA array hybridisation

The production of subtracted libraries was done according to the suppression subtractive hybridisation (SSH) method (Diatchenko et al. 1996). Total RNA from endometrial tissue samples was isolated using Trizol reagent (Invitrogen) according to the manufacturer’s instructions. Equal amounts of RNA of all seven sections of the uterus were pooled for the preparation of subtracted cDNA libraries for oestrus and dioestrus. The construction of the libraries was done as previously described (Bauersachs et al. 2003, 2004). For every library, 1536 cDNA clones were randomly picked, the cDNA fragments amplified via PCR, spotted by a spotting robot onto small nylon membranes and analysed by array hybridisation (for details see Bauersachs et al. 2004). The cDNA arrays were hybridised with radioactively labelled probes derived from 42 tissue samples corresponding to the six animals. Up to 12 probes were hybridised in parallel. All hybridisations that were directly compared in the data analysis were processed simultaneously under equal conditions.

Analysis of array data

Array evaluation was done using AIDA Image Analyzer Software (Version 3.41; Raytest, Straubenhardt, Germany). Background was subtracted with the ‘Weighted image regions’ function. Raw data obtained by AIDA Array software were exported to Microsoft Excel and normalised to the mean signals of internal reference cDNAs of each array. Normalised data were compared pair-wise (oestrus vs dioestrus) using the datasets derived from the hybridisation experiments that were processed simultaneously. cDNA clones were set as differentially expressed between oestrus and dioestrus if they showed at least a 2-fold up- or downregulation in every oestrus–dioestrus pair in at least one section of the uterus. In Tables 1 and 2 the means of the ratios of the single uterine sections and the three pair-wise comparisons are shown. The ratios of the uterine sections were very similar for almost all genes. The coefficient of variation (CV) values for the three comparisons are also shown. If a gene was represented on the array by more than one cDNA fragment the mean expression difference was calculated. Differential expression across the uterine segments (uterine horn cranial to corpus) was analysed by ANOVA (parametric test, Benjamini & Hochberg false discovery rate) and a post-hoc test (Student–Newman–Keuls) using GeneSpring software Version 6.1 (Silicon Genetics, Redwood City, CA, USA). The analysis was done separately for the two sides of the uterus and the two times of the cycle. The four values of every cDNA derived from the cranial, middle and caudal uterine horn, and the corpus over the mean of all four values were used to consider the relative changes between these four uterine sections.

Sequencing of cDNAs with differential hybridisation signals and data analysis

cDNA fragments showing differential hybridisation signals were sequenced directly from spotting solutions by automated DNA sequencing (3100-Avant Genetic Analyzer; Applied Biosystems, Langen, Germany). Resulting sequences were compared with public sequence databases using the basic local alignment search tool at the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov/blast/blast.cgi). cDNAs without similar entries in the ‘nr’ database were in addition compared with the ‘est’ database or the raw version of the bovine genome (http://pre.ensembl.org/Multi/blastview?species=Bos_taurus). Based on the human homologues simplified GOs were built using the GeneSpring software. Resulting data were supplemented with additional information from LocusLink (www.ncbi.nlm.nih.gov/locusLink/) and Entrez Gene (www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=gene), and from the literature. Pathway analyses were done searching the KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway database (www.genome.ad.jp/kegg/pathway.html) and the literature (PubMed abstracts).

Real-time RT-PCR

The same 42 RNA samples as for array hybridisation were used. One microgram of each sample of total RNA was reverse transcribed in a total volume of 60 μl, containing 1 × buffer (Promega), 0.5 mM dNTPs (Roche), 2.5 μM hexamer primers (Gibco BRL, Grand Island, NY, USA) and 200 U Superscript RT enzyme (Promega). Primers were designed to amplify specific fragments referring to selected regulated genes: CLDN10 (forward 5′-CATTTCATGCCAATCAGGG; reverse 5′-CGCTGGACGGTTACATCC (97 bp)), CPN1 (forward 5′-AGAGAGGCCCTGCCTGAC; reverse 5′-AGAAAGAATGCTTCTTCTGGTG (90 bp)), EPHX2 (forward 5′-ACTCCTTGTGAACACCCCAG; reverse 5′-TAGAGGCCCCCTGAAACC (109 bp)), INHBA (forward 5′-CGATGGGCAGAACATCATC; reverse 5′-GTCTTCTTTGGACCGTCTCG (113 bp)), MGP (forward 5′-CCCGGAGACACCATGAAG; reverse 5′-GAGGGAGAGGGGAGAATCC (161 bp), MS4A8B (forward 5′-CTGACCTGCTACCAAACCAAC; reverse 5′-GCCCAAACAAGGGAGGTATTC (191 bp)), PENK (forward 5′-TCTGGAATGTGAGGGGAAAC; reverse 5′-CTTCTTAGCAAGCAGGTGGC (142 bp)), PTHLH (forward 5′-TCCCCTAACTCCAAGCCTGC; reverse 5′-TTTTCTTTTTCTTGCCGGG (148 bp)), SOX4 (forward 5′-ATTTCGAGTTCCCGGACTAC; reverse 5′-CCTTCAGTAGGTGAAGACCAGG (101 bp)), UTMP (forward 5′-ATATCATCTTCTCCCCCATGG; reverse 5′-GTGCACATCCAACAGTTTGG (126 bp)), and the housekeeping gene Ubiquitin (forward 5′-AGATCCAGGATAAGGAAGGCAT; reverse 5′-GCTCCACCTCCAGGGTGAT (198 bp)) (referring to Neuvians et al. 2003). All amplified PCR fragments were sequenced with forward and reverse primers (3100-Avant Genetic Analyzer; Applied Biosystems) to verify the resulting PCR product. Thereafter the specific melting point of the amplified product served as verification of the product identity (CLDN10, CPN1 and MS4A8B 84 °C, EPHX2, PTHLH and MGP 85 °C, INHBA, PENK and UTMP 86 °C, SOX4 87 °C and Ubiquitin 88 °C) (Pfaffl et al. 2003). For each of the following real-time PCR reactions, 1 μl cDNA was used to amplify specific target genes. Quantitative real-time PCR reactions using the LightCycler DNA Master SYBR Green I protocol (Roche) were performed as described previously (Ulbrich et al. 2004). In each PCR reaction 17 ng/μl cDNA were introduced and amplified in a 10 μl reaction mixture (3 mM MgCl2, 0.4 μM primer forward and reverse each, 1 × Light Cycler DNA Master SYBR Green I; Roche) using a real-time LightCycler instrument (Roche). The annealing temperature was 60 °C for all reactions. To ensure an accurate quantification, a high temperature fluorescence measurement at 80 °C and 78 °C for EPHX2, INHBA, MGP, PENK, PTHLH and UTMP and CPN1, CLDN10, MS4A8B and Ubiquitin respectively, was undertaken in a fourth segment of the PCR. The cycle number required to achieve a definite SYBR Green fluorescence signal was calculated by the second derivative maximum method (crossing point, CP) (LightCycler software Version 3.5.28). The CP is correlated inversely with the logarithm of the initial template concentration. As negative controls, water instead of cDNA was used.

Data analysis of real-time RT-PCR

The CPs determined for the target genes were normalised against the housekeeping gene Ubiquitin. Results are presented as means (n=3) (see Table 4). Expression ratios are presented as x-fold regulation between oestrus and dioestrus relating to each section. Differences between day 0 and day 12 for each section were analysed using one-way ANOVA. The normal distribution was tested by the Kolmogorow–Smirnov method, followed by a Student’s t-test to find significant differences (Sigma-Stat, Version 2.03). The level of significance (P-value) is stated in Table 4.

In situ hybridisation

Formalin-fixed (3.7%), paraffin-embedded samples were used to localise transcripts of PENK (proenkephalin), PTHLH (parathyroid hormone-like hormone), SAL1 (salivary lipocalin), INHBA (inhibin beta A) and UTMP (uterine milk protein precursor) within bovine uterus tissue (middle section of the ipsilateral horn). All samples except those used for detection of PENK mRNA were recovered during the oestrous phase. For PENK, material harvested at day 12 of the oestrous cycle was used. The same animals were used as for array hybridisation and real-time RT-PCR. Buffers (50 mM Tris-buffered saline (TBS) and 0.1 M sodium phosphate buffer) were adjusted to pH 7.4 unless otherwise noted. All solutions for in situ hybridisation were prepared using diethyl pyrocarbonate-treated water and glassware sterilised at 200 °C. In addition, all steps prior to and during hybridisation were conducted under RNase-free conditions.

Sections were deparaffinised with xylene (3 × 10 min), immersed in isopropanol (2 × 5 min) and then allowed to air dry. Dried sections were submerged in 2 × saline sodium citrate (SSC, pH 7.0) and preheated in a Bain-Marie water bath (80 °C) for 10 min followed by gradual cooling off for 20 min at room temperature. Slides were then washed in distilled water (2 × 5 min), TBS (2 × 5 min) and permeabilised for 20 min with 0.05% proteinase E (VWR, Ismaning, Germany) in TBS at room temperature. Sections were relocated in TBS (2 × 5 min) followed by distilled water (2 × 5 min) and post-fixed for 10 min in 4% paraformaldehyde/PBS (pH 7.4). After washing in PBS (2 × 5 min) and distilled water, slides were dehydrated in an ascending graded series of ethanol and air-dried. Hybridisation was carried out by overlaying the dried sections with 40 μl of the corresponding biotinylated oligonucleotide probe (100 pmol/μl), diluted 1:20 in in situ hybridisation solution (DAKO, Munich, Germany) and incubating them in a humidified chamber (using cover slips to prevent drying-out) at 38 °C overnight. RNase-free hybridisation solution (DAKO) contained 60% formamide, 5 × SSC, hybridisation accelerator, RNase inhibitor and blocking reagents. Subsequently, slides were washed in 2 × SSC (2 × 15 min, preheated to 38 °C), distilled water (2 × 5 min) and TBS (2 × 5 min). Detection of hybridised probes was performed using horseradish peroxidase-labelled ABC kit reagents developed with 3,3′-diaminobenzidine (DAKO) according to the manufacturer’s instructions. Negative controls were done omitting the oligonucleotide probe and hybridisation with sense oligonucleotide probes. The sequences of the antisense oligonucleotides were as follows: INHBA: 5′-GATGATGTTCTGCCCATCG, UTMP: 5′-GTGCACATCCAACAGTTTGG, PTHLH: 5′-CAGGCTTGGAGTTAGGGGA, SAL1: 5′-GTAT CTGGTTCTCGGGCGT and PENK: 5′-GTTTCCC CTCACATTCCAGA.

Results

To identify differentially expressed genes in the bovine endometrium between the (late) oestrous and the dioestrous stage a combination of subtracted cDNA libraries and cDNA array hybridisation was applied. Two subtracted cDNA libraries were produced for oestrus and dioestrus respectively, starting from a pool of RNA samples derived from seven sections of the uterus (Fig. 1a). One thousand five hundred and thirty-six randomly picked cDNA clones of each library were analysed by cDNA array hybridisation with 42 samples of six animals (oestrus n=3, dioestrus n=3).

First, array hybridisation data were analysed for differences between oestrus and dioestrus. This analysis revealed 444 cDNA fragments showing at least 2-fold signal differences between oestrus and dioestrus in all three animal pairs (at least one uterine section). Figure 1b shows the hybridisation signals of one cDNA fragment of the UTMP (uterine milk protein) gene derived from the probes of one animal at oestrus and one at dioestrus as an example. The sequence analysis of the cDNA fragments revealed 65 different genes or mRNAs with higher expression levels at oestrus (see Table 1). Sixty-two genes corresponded to genes of known or inferred function, either the bovine gene or the likely human orthologue. The most pronounced differences in mRNA levels were found for CLDN10 (claudin 10), INHBA (inhibin beta A), MMP2 (matrix metalloproteinase 2), PCSK5 (proprotein convertase 5), RARRES1 (retinoic acid (RA) receptor responder 1), SAL1 (salivary gland protein), TNC (tenascin C), UTMP and one unknown gene. The expression difference of these genes was termed ‘on’, i.e. no or very weak signals were detected at dioestrus by array hybridisation. The cDNA fragments of 25 genes were found more than once among the 444 cDNA fragments, most frequently for SPARC (osteonectin, 31 cDNA fragments) and UTMP (29 cDNA fragments).

At dioestrus, 68 genes with increased expression were detected (Table 2). Fifty-six genes corresponded to genes of known or inferred function. The genes with the highest differences in mRNA levels were AGT (angiotensinogen), ATP1B2 (Na/K ATPase), CYP26A1 (cytochrome P450 family member), DGAT2 (diacylglycerol O-acyltransferase), LY6 G6C and LY6 G6E (lymphocyte antigen complex), PENK (proenkephalin), TDGF1 (teratocarcinoma-derived growth factor 1) and a cDNA of unknown function. Again, for 25 genes more than one cDNA fragment was detected, most frequently for PENK (28 cDNA fragments) and GM2A(similar to GM2 ganglioside activator protein) (12 cDNA fragments).

In addition to the comparison of oestrus and dioestrus, the expression along the uterine horns from cranial to the corpus was investigated at the ipsilateral and the contralateral side at oestrus and dioestrus respectively. A few genes showed indeed expression differences from the cranial uterine horn to the corpus (see Table 3). Most of the genes with such expression differences showed a lower expression only in the corpus, e.g. the expression of the majority of genes was very similar across the uterine horns. The most pronounced expression gradient was observed for the UTMP mRNA during oestrus at both sides of the uterus. Gene expression was also analysed between the corresponding ipsilateral and contralateral sections of the uterine horns. Our recent study of the bovine oviduct (Bauersachs et al. 2003) and two studies of the bovine endometrium (Malayer et al. 1988, Williams et al. 1992) indicated that there could also be gene expression differences between the two uterine horns. For a few genes there was a tendency for an expression difference between the two uterine horns, but it was not significant.

For ten selected genes the expression in the endometrium was quantified by the use of real-time RT-PCR (Table 4). The same 42 RNA samples as for array hybridisation were used. All segments of the uterus were analysed for EPHX2 (epoxide hydrolase 2), MGP (matrix gla protein) and UTMP mRNA assuming a gradient along the uterine horns. For all other genes examined only the ipsi- and contralateral middle sections representing the total uterine horn were analysed. The mean crossing points (CP) normalised to the housekeeping gene Ubiquitin and the resulting expression ratios comparing oestrus and dioestrus are shown. The abundance of all investigated specific transcripts was remarkably different between the oestrus and dioestrus stage. The highest ratio between oestrus and dioestrus (150-fold) was found for the UTMP mRNA (cranial part of the ipsilateral uterine horn). The strong gradient of the UTMP mRNA abundance along the uterine horns at both sides of the uterus during oestrus was clearly confirmed, whereas for EPHX2 and MGP only a tendency for a slightly higher expression in the cranial parts of the uterine horns was found. Overall the results of quantitative RT-PCR and array hybridisation correlated very well.

For five selected genes (INHBA, PTHLH, SAL1, UTMP and PENK) in situ hybridisation with bovine endometrial tissue sections were done to localise the mRNA expression in this complex tissue. A specific pattern of mRNA distribution was found for each of these genes (Fig. 2a and b). The hybridisation signal was always confined to cells of the endometrium and was absent in the myometrium and the serosa. No specific signal was observed in sections hybridised with the sense strand (Fig. 2c) or in sections incubated with buffer only instead of the oligonucleotide probes. Considerable levels of the INHBA message were detected in the supranuclear area of the surface epithelium. The superficial uterine glands also showed a distinct staining of several cells within a cross-section of the gland. In the deep uterine glands adjacent to the myometrium, the INHBA message appeared somewhat reduced, but was still more pronounced in the apical area of the glandular epithelium. Most of the fibroblasts in the endometrium displayed a weak hybridisation signal. High levels of PTHLH mRNA were found in the uterine surface epithelium and in the deep uterine glands. Also the fibroblasts of the endometrium showed a distinct staining. In the superficial uterine glands, the hybridisation signal was less pronounced and appeared concentrated to the supranuclear area of the glands. Strong staining for SAL1 mRNA could be demonstrated in the uterine surface epithelium and in the supranuclear epithelial area of the superficial uterine glands. Deep uterine glands stained generally more weakly, with a distinct signal in the apical region of the glands. mRNA for UTMP was detected in dispersed epithelial cells of the surface epithelium but distinct staining was found in the epithelium of the superficial and deep uterine glands. PENK was weakly expressed in the surface epithelium but the signal intensity increased in the uterine glands.

To deduce a picture of the regulatory processes that proceed during the oestrous cycle in the endometrium from the changes at the mRNA level, two bioinformatic approaches were applied. At first, a functional classification of the identified genes was done by building a simplified Gene Ontology (GO) for genes upregulated either at oestrus (see Table 1, Ontology) or at dioestrus (see Table 2, Ontology). For a graphical illustration the numbers of genes assigned to the 19 functional groups at oestrus and dioestrus are compared in Fig. 3. Some biological processes or molecular functions clearly dominated either at oestrus or at dioestrus. At oestrus the functional groups ‘Cytoskeleton’, ‘Cell motility’, ‘Cell adhesion’, ‘Extracellular matrix (ECM) structural proteins’, ‘ECM remodelling’ and ‘Proliferation’ clearly prevail. At dioestrus, genes of several groups of enzymes and ‘Transport’ are more upregulated.

Furthermore, involved pathways and signalling cascades were identified by the analysis of KEGG pathways and a literature search. Thirteen pathways and the assigned genes at oestrus and dioestrus are listed in Table 5. The highest number of genes (17 genes) was assigned to the transforming growth factor-β (TGF-β) signalling pathway. Most of these genes (14 genes) were upregulated at oestrus and corresponded to target genes or to inhibitors of this pathway. In Fig. 4 a simplified TGF-β signalling pathway based on the Homo sapiens reference pathway derived from the KEGG pathway database with the assigned genes is shown. For the Prostaglandin metabolism (four genes) as well as for the Phosphoinositide signalling (four genes) only such genes could be assigned that were upregulated at dioestrus. In contrast, for the Phospholipase C pathway (three genes) and also for the Ribosome pathway (four genes) only such genes were found that were upregulated at oestrus.

Finally, a comparison of the genes found in this and other studies of the human, the non-human primate (Rhesus monkey), and the mouse system (Carson et al. 2002, Kao et al. 2002, Borthwick et al. 2003, Riesewijk et al. 2003, Tan et al. 2003, Ace & Okulicz 2004) was done (Table 6). The cycle stages that were compared in these studies are stated in the footnotes of Table 6. The overall overlap with these studies was 34 genes. Considering closely related genes of the same gene family the overlap was even higher (data not shown). Thirteen genes showed a similar regulation, the regulation of 17 genes was contrary, and four genes were mentioned as ‘expressed’ in the study of Borthwick et al.(2003).

Discussion

The aim of this study was the identification of differentially expressed genes in the bovine endometrium during the oestrous cycle, in particular between the late oestrus and the dioestrus stage to uncover regulatory pathways involved in the control of the complex cycle-dependent changes in this tissue. Therefore, a combination of SSH and cDNA microarrays was used. This resulted in a relatively large number of genes differentially expressed between oestrus and dioestrus (133 genes) and also a couple of genes with clear expression differences between the uterine sections. The number of differentially expressed genes between the oestrus and the dioestrus stage was comparable with that found in similar studies in the mouse, the Rhesus monkey and in humans, which have been done with high-density cDNA or oligonucleotide arrays respectively (Borthwick et al. 2003, Tan et al. 2003, Ace & Okulicz 2004).

Using quantitative real-time RT-PCR the results of the array hybridisation were clearly confirmed. The correlation of the data was very good, although microarray data usually do not provide exact quantitative expression differences as quantitative PCR. Reasons for minor deviations in expression ratios have already been discussed elsewhere (Bauersachs et al. 2004). The analysis of genes with high expression ratios between oestrus and dioestrus could be done very precisely by real-time RT-PCR, extending the qualitative array result ‘on’ referring to no signal at one cycle phase because of too low expression signals. For instance, the expression of UTMP mRNA in the ipsilateral cranial endometrium at oestrus was almost 150-fold higher than at dioestrus.

The classification of the identified genes based on GOs revealed that upregulation of mRNAs of distinct functional groups clearly dominated at oestrus or dioestrus. At oestrus the striking upregulation of mRNAs for proteins of the ECM and for proteins involved in ECM remodelling indicates changes in the composition of the connective tissue during the oestrous cycle as proposed by Curry & Osteen (2001). Furthermore, upregulation of mRNAs coding for proteins that negatively regulate cell growth and proliferation, and with apoptosis-related functions prevailed at oestrus. For proliferation-related/inhibiting genes these changes may reflect the end of the ‘proliferative phase’ of the endometrium since the animals representing day 0 were slaughtered at late oestrus.

At dioestrus, a number of transcripts for different enzymes and for proteins involved in transport processes are upregulated. The cyclic regulation of uterine fluids in the endometrium by differential expression of genes coding for ion channels was first shown by Chan et al.(2002). The identification of three mRNAs coding for ion channels (group ‘transport’) provides further evidence for this. The upregulation of many mRNAs for enzymes may be to some extent an indication for the increase of the prostaglandin metabolism at dioestrus (Goff 2004, Spencer et al. 2004).

The genes of known function were further analysed in the context of different pathways where they are involved. The most prominent pathway was the TGF-β signalling with 17 genes upregulated either at oestrus or at dioestrus. At oestrus, transcripts of target genes of this signalling pathway and transcripts for proteins that are shown to inhibit TGF-β signalling were upregulated. In contrast, at dioestrus only three genes were identified which are described as being involved in TGF-β signalling. The identification of this considerable number of genes involved in or regulated by TGF-β signalling is in line with current knowledge of regulation of the endometrium by TGF-β during the oestrous cycle, implantation and pregnancy (Godkin & Dore 1998, Tabibzadeh 2002). Furthermore, the induction of TGF-β mRNAs and proteins by oestrogen has been shown in epithelial cells of the mouse endometrium (Nelson et al. 1992). This fits very well with the results of our study, where at late oestrus (oestrogen levels low) target genes of TGF-β are still upregulated but negative regulators of this pathway are already present. Another pathway that could play a role in the regulation of the endometrial differentiation state is the RA signalling. In humans the regulatory role for endometrial maturation of RA signalling together with P4 and TGF-β has already been described (Osteen et al. 2003). At dioestrus two mRNAs (CYP26A1, RDHE2) are elevated for proteins involved in RA metabolism whereas at oestrus the expression of two genes, which are described in context with differentiation was higher. Furthermore, the upregulation of mRNAs coding for proteins participating in the prostaglandin metabolism is consistent with the literature (Goff 2004, Spencer et al. 2004).

The comparison of the identified genes with microarray studies in other species revealed an overlap of 29%, whereas half of the changes in mRNA concentrations for these genes were not in the same direction. For the interpretation of this result it has to be considered that the overlap between the human studies was also relatively small for different reasons (see Riesewijk et al. 2003) although the same microarray platform (Affymetrix) was used. Furthermore, there are substantial differences between species regarding (i) endometrial changes during the cycle (oestrous cycle lesser changes than during menstrual cycle) and (ii) the type of implantation of the embryo. In humans there is an early (at the blastocyst stage) and invasive implantation, whereas in the bovine species implantation is less invasive and follows an extended peri-implantation period (after day 18 of the cycle) when the trophoblast spans nearly the complete uterus. In this context the comparison of these different gene expression profiles shows that there are common regulations but also distinct differences between species.

This is further supported by the expression patterns and/or potential functions that were already described for a number of the identified genes. Most of the data in the literature, although mainly derived from studies in other species, were in line with the results of the present study. Cycle-dependent expression with a higher level in proliferative in comparison with secretory phase endometrium was shown for example for CTSK (cathepsin K) (Jokimaa et al. 2001), PRSS11 (serine protease 11, insulin-like growth factor (IGF) binding) (De Luca et al. 2003), PTHLH (parathyroid hormone-like hormone) (Hoshi et al. 2001), GJA1 (connexin 43) (Jahn et al. 1995), and TNC (tenascin C) mRNAs (Taguchi et al. 1999) in humans, and SPARC (osteonectin), COL5A2, COL6A3, LGALS1 (galectin 1) and SOX4 mRNAs in the Rhesus monkey (Ace & Okulicz 2004). In sheep, GRP (gastrin-releasing peptide) mRNA (Whitley et al. 1998) and IGFBP6 (IGF-binding protein-6) mRNA (Gadd et al. 2002) showed maximal concentrations in the endometrium around ovulation. In the present study of bovine endometrium all these genes revealed higher expression levels at oestrus than at dioestrus. For two of these genes induction of mRNA expression by oestrogen has been shown in the endometrium of the rat for GJA1 (Grummer et al. 1994) and in human endometrial stromal cells for PTHLH (Casey et al. 1993). UTMP mRNA expression in sheep endometrium was detected at day 15 of the oestrous cycle (Stewart et al. 2000) but at oestrus UTMP expression was not analysed. Here we show highest expression of UTMP mRNA in the bovine endometrium of the cranial uterine horns at oestrus.

Increased endometrial mRNA levels during the secretory phase were described for the RA catabolic enzyme CYP26A1 (RA 4-hydroxylase), for TGM2 (tissue transglutaminase) (Deng et al. 2003), and for HPGD (15-hydroxyprostaglandin dehydrogenase) (Kelly et al. 1994) in humans and the PENK mRNA in the rat (Jin et al. 1988). In mouse endometrium, positive regulation of PENK by P4 has been indicated by a study of Cheon et al.(2002). In contrast to these findings, PENK is upregulated during proliferative phase in the endometrium of primates including humans (Low et al. 1989, Carson et al. 2002, Riesewijk et al. 2003, Ace & Okulicz 2004). HPGD and TGM2 were also described to be upregulated by P4 (Fujimoto et al. 1996, Greenland et al. 2000). In sheep, TIMP2 (tissue inhibitor of metalloproteinase 2) mRNA (1.0 kb transcript) abundance in the endometrium has been shown to be stimulated by P4 and to be increased at day 10 of the cycle (Hampton et al. 1995). All these genes showed higher expression levels at dioestrus compared with oestrus in the bovine endometrium.

Furthermore, a role during pregnancy or the implantation process was shown or assumed for APOE (apolipoprotein E) (Overbergh et al. 1995), CLDN10 (claudin 10) (Wang et al. 2004), EFNA1 (ephrin A1) (Fujiwara et al. 2002), FBLN1 (fibulin 1) (Haendler et al. 2004), fibronectin (Rider et al. 1992), GJA1 (Gabriel et al. 2004), LGALS1 (Maquoi et al. 1997), MGP (matrix gla protein) (Spencer et al. 1999), MMP2 (matrix metalloproteinase 2) (Goffin et al. 2003), PENK (Jin et al. 1988), PLA2 G10 (phospholipase A2) (Goff 2004), SERPINA1 (Marshall & Braye 1987), TNC (Michie & Head 1994) and UTMP (Hansen 1998).

The endometrium is a relatively complex tissue composed of many different cell types. For the interpretation of differential gene expression the assignment to distinct cell types is important. Therefore, in situ hybridisations for five selected genes were done. All five genes were expressed specifically in the endometrium, mainly in the surface and the glandular epithelium. Nevertheless, there were distinct differences in their expression patterns, for example INHBA and PTHLH mRNAs were also detectable in fibroblasts of the stroma. The expression pattern of the UTMP mRNA in the bovine uterus at oestrus was similar to that found in sheep at day 19 of pregnancy (Stewart et al. 2000). For PENK the mRNA expression was highest in the deep glandular similar to the localisation in the mouse uterus (Rosen et al. 1990).

Our study is the first holistic screen for transcriptome changes in bovine endomtrium between oestrus and dioestrus. We identified a number of molecular pathways, most prominently the TGF-β pathway, involved in the marked functional changes of endometrium between these two stages. To complete the analysis of gene expression during the oestrus cycle further stages (e.g. preoestrus and metoestrus) will be investigated and the function of candidate genes, e.g. UTMP, will be characterised. Furthermore, the differentially regulated genes identified in this study represent an important basis for future studies, e.g. on embryo–maternal interactions during the pre-implantation period, and for differential diagnosis of fertility problems via array-based analysis of endometrial biopsies.

Table 1

Mean fold-change of all uterine sections for the genes upregulated at oestrus

Gene nameGenBankMeanCV (%)Ontology
Gene name: official gene name/symbol from Entrez Gene; On: no expression detected at dioestrus; 1 no expression detected in the corpus of one pair at dioestrus and oestrus; 2 inconsistent expression in the corpus; 3 no expression detected in the corpus of all animals; (−) negative regulation; (+) positive regulation
Gene/cDNA/homologue description
88128 MARC 1BOV B. taurus cDNA (UniGene Bt.5193, strongly similar to acid phosphatase 5, tartrate resistant (H. sapiens))ACP5AW4470453.1140ECM remodelling
B. taurus mRNA for similar to alpha 2 actinACTA2AB0987972.45Cytoskeleton
B. taurus β-actinACTBAY1419702.15Cell motility, cytoskeleton
H. sapiens actin, gamma 2, smooth muscle, entericACTG2NM_0016153.0232Cell motility, cytoskeleton
B. taurus annexin A2ANXA2NM_1747162.1220ECM remodelling, cell growth
B. taurus apolipoprotein EAPOENM_1739913.3127Transport, apoptosis-related function, cytoskeleton
B. taurus carbonic anhydrase IICA2NM_1785724.6247ECM remodelling
H. sapiens claudin 10CLDN10NM_006984On1Cell adhesion
B. taurus partial col1A1 gene for pro alpha 1(I) collagenCOL1A1AJ3121123.919ECM structural protein
B. taurus collagen, type I, alpha 2COL1A2NM_1745204.225ECM structural protein
H. sapiens collagen, type V, alpha 2COL5A2NM_0003932.815ECM structural protein
M. musculus procollagen, type VI, alpha 3COL6A3BC0579035.1231ECM, cell adhesion
B. taurus COL12 mRNA for type XII collagenCOL12A1AB0998825.7123ECM, cell adhesion
H. sapiens collagen, type XV, alpha 1COL15A1NM_0018553.721ECM structural protein
H. sapiens cathepsin K (pycnodysostosis)CTSKNM_0003964.2144ECM remodelling
B. taurus decorinDCNNM_1739061.9221Signal transduction, ECM remodelling
B. taurus mRNA for similar to elongation factor-1-γEEF1GAB0989332.2215Protein biosynthesis
H. sapiens fibulin 1FBLN1NM_0019963.941ECM, cell adhesion
M. musculus fibulin 5FBLN5NM_0118122.6117ECM, cell adhesion
B. taurus partial mRNA for fibronectin (V+I-10)- splice variantFN1AJ3205283.5219ECM, cell adhesion
B. taurus gap junction protein, α 1, 43 kDa (connexin 43)GJA1NM_1740683.519Signal transduction, cell
B. taurus gastrin-releasing peptideGRPNM_1783192.343Signal transduction, cell growth
H. sapiens IGFBP-6IGFBP6NM_0021782.08Signal transduction, cell growth (−), proliferation (−)
B. taurus inhibin, beta AINHBANM_174363On1Pregnancy, signal transduction, cell growth(−), proliferation (−), apoptosis-related function
B. taurus mRNA for similar to 40S ribosomal protein SA (P40) (laminin receptor 1)LAMR1AB0990111.8224Signal transduction, cell adhesion
B. taurus lectin, galactoside-binding, soluble, 1LGALS1NM_1757822.822Immune-related, signal transduction, ECM remodelling, cell growth (−),proliferation (−), apoptosis- related function, cell adhesion
H. sapiens melanoma antigen, family D, 1MAGED1NM_0069862.4111Apoptosis-related function
B. taurus microfibril-associated glycoprotein-2(MAGP2)MAGP2, MFAP5NM_1743863.342ECM structural protein
H. sapiens hypothetical protein MGC10540MGC10540NM_0323532.513
B. taurus matrix metalloproteinase 2 (72 kDa type IV collagenase)MMP2NM_174745On2ECM remodelling, pregnancy, hydrolase
H. sapiens mucin 16MUC16AF3614863.8241Immune-related
H. sapiens Norrie disease (pseudoglioma)NDPNM_0002664.811Signal transduction, cell growth, Proliferation
B. Taurus nephroblastoma overexpressed protein precursor geneNOVAF0550767.027Signal transduction, cell motility, cell adhesion, cell growth
H. sapiens nucleophosmin (nucleolar phosphoprotein B23, numatrin)NPM1NM_0025201.7222Proliferation, apoptosis-related function
B. taurus procollagen-proline, 2-oxoglutarate4-dioxygenase (proline 4-hydroxylase), beta polypeptideP4HBNM_1741352.710ECM remodelling
H. sapiens procollagen C-endopeptidase enhancerPCOLCENM_0025932.618ECM remodelling
H. sapiens proprotein convertase subtilisin/kexin type 5PCSK5NM_006200On1ECM remodelling, hydrolase
H. sapiens pyruvate kinase, musclePKM2NM_0026541.918Glycolysis
B. taurus peroxiredoxin 2PRDX2NM_1747632.119Oxidoreductase
H. sapiens protease, serine, 11 (IGF binding)PRSS11NM_0027752.6234Signal transduction, cell growth (−), hydrolase
H. sapiens patched homologue 2 (Drosophila)PTCH2AY4386642.5243Signal transduction, proliferation (−)
B. taurus parathyroid hormone-like hormonePTHLHNM_1747536.3151Signal transduction, proliferation, pregnancy
H. sapiens transcription factor RAM2RAM2NM_0187192.721Transcriptional regulation
H. sapiens RA receptor responder (tazarotene induced) 1RARRES1NM_206963OnProliferation (−), cell adhesion
H. sapiens regenerating islet-derived family, member 4REG4NM_0320442.227Proliferation
B. taurus mRNA for similar to ribosomal protein L18aRPL18AAB0989162.1215Protein biosynthesis
H. sapiens ribosomal protein L7aRPL7ANM_0009722.1219Protein biosynthesis
B. taurus mRNA for similar to S100 calcium-binding protein A11S100A11AB0990122.76Proliferation (−), cell motility
CES014220 B. taurus skin cDNA library B. taurus cDNA (UniGene Bt.13796)SAL1CF769307OnSignal transduction, transport, pregnancy
4059330 BARC 8BOV B. taurus cDNA (UniGene Bt.21836)SEC61A1CK8348032.228Transport
B. taurus serine (or cysteine) proteinase inhibitor, clade A, member 1SERPINA1NM_1738823.014Immune-related
B. taurus serine (or cysteine) proteinase inhibitor, clade F, member 1SERPINF1NM_1741402.411Proliferation, apoptosis-related function
H. sapiens serine (or cysteine) proteinase inhibitor, clade H (heat shock protein 47), member 1, (collagen binding protein 1)SERPINH1NM_0012354.3226ECM remodelling
H. sapiens SRY (sex determining region Y)-box 4SOX4NM_0031072.6112Transcriptional regulation
B. taurus secreted protein, acidic, cysteine-rich (osteonectin)SPARCNM_1744643.23ECM structural protein, ECM remodelling, cell motility, proliferation (−)
H. sapiens Tax interaction protein 1TAX1BP3NM_0146042.3113Signal transduction, proliferation (−)
B. taurus thrombospondinTHBS1NM_1741961.7135ECM, cell adhesion, cell motility, signal transduction
H. sapiens tenascin C (hexabrachion)TNCNM_002160On1Cell adhesion, ECM structural protein, immune-related
H. sapiens tubulin, alpha 1 (testis specific)TUBA1NM_0060002.126Cytoskeleton
H. sapiens beta 5-tubulin (OK/SW-cl.56)TUBBNM_1780142.4126Cytoskeleton
B. taurus uterine milk protein precursorUTMP, LOC286871NM_174797On3Immune-related, pregnancy
B. taurus vimentinVIMNM_1739692.0229Cytoskeleton, immune-related
H. sapiens zinc finger protein 564ZNF564BC0364812.5240Transcriptional regulation
4062746 BARC 8BOV B. taurus (UniGene Bt.13993)CK8374953.016
Genomic scaffold 246248AAFC01272447On
Table 2

Mean fold-change of all uterine sections for the genes upregulated at dioestrus

Gene nameGenBankMeanCV (%)Ontology
Gene name: official gene name/symbol from Entrez Gene; On: no expression detected at oestrus; 1 inconsistent expression in the corpus; 2 no expression detected in all uterine sections of one animal pair at dioestrus and oestrus; (−) negative regulation; (+) positive regulation
Gene/cDNA/homologue description
H. sapiens ankyrin repeat and BTB (POZ) domain containing 1ABTB1NM_1720272.226Cell growth (−), proliferation (−)
Ovis ammon mRNA for angiotensinogenAGTD17520OnPregnancy
20 alpha-hydroxysteroid dehydrogenase (cattle)AKR1B1S549736.015Dehydrogenase
Alkaline phosphatase tissue non-specific isoform/TNS-AP (cattle)ALPLS816004.810ECM remodelling, hydrolase
B. taurus Rho GDP dissociation inhibitor (GDI) betaARHGDIBNM_1757975.99Signal transduction, immune-related, cell adhesion (−)
Human adult mRNA for Na/K ATPase beta 2 subunitATP1B2D87330OnTransport (ion channel)
503071 MARC 2BOV B. taurus cDNA (UniGene Bt.24822, moderately similar to NP_059345-BAI1-associated protein 2, isoform 2)BAIAP2BM0893513.1121Receptor activity
721820 MARC 6BOV B. taurus cDNA (UniGene Bt.17278, strongly similar to NP_005495 branched chain aminotransferase 1, cytosolic)BCAT1CB4618085.311Proliferation (+), transferase
H. sapiens chromosome 5 open reading frame 18C5orf18NM_0056693.63
H. sapiens cyclin B1CCNB1NM_0319663.313Cell cycle related (+)
H. sapiens chloride channel KaCLCNKANM_0040705.635Transport (ion channel)
H. sapiens CCR4-NOT transcription complex, subunit 1CNOT1NM_0162843.010
H. sapiens carboxypeptidase N, polypeptide 1, 50 kDaCPN1NM_0013084.716Proteolysis, hydrolase
H. sapiens cytochrome P450, family 26, subfamily A, polypeptide 1CYP26A1NM_000783OnPregnancy, transport
B. taurus DGAT2 gene for diacylglycerol O-acyltransferase 2DGAT2AJ534372OnTransferase
H. sapiens delta sleep inducing peptide, immunoreactorDSIPINM_1980575.112Immune-related, transcriptional regulation
H. sapiens embryonic ectoderm developmentEEDNM_1529912.831Signal transduction, transcriptional regulation, pregnancy
H. sapiens ephrin-A1EFNA1NM_0044283.243Signal transduction, kinase
H. sapiens epoxide hydrolase 2, cytoplasmicEPHX2NM_0019797.532Immune-related, hydrolase
H. sapiens RNA-binding proteinFLJ20273NM_0190272.912
603116 MARC 6BOV B. taurus cDNA (UniGene Bt.17182, moderately similar to NP_005259.1 gap junction protein, beta 5)GJB5CB4275313.137Transport (channel)
Similar to H. sapiens GM2 ganglioside activator proteinGM2ANM_0004059.843Glycolipid catabolism
H. sapiens helicase with zinc finger domainHELZNM_0148773.731Helicase activity, nucleic acid binding
Bubalus sp. mRNA for NAD+-dependent 15-hydroxyprostaglandin dehydrogenaseHPGDAJ2228376.740Pregnancy, oxidoreductase, dehydrogenase
B. taurus isocitrate dehydrogenase 1 (NADP+), solubleIDH1NM_1810124.036Oxidoreductase, dehydrogenase
B. taurus mRNA for immunoglobulin light chainIG G1X629178.158Immune-related (antibody)
H. sapiens integrin, beta 4ITGB4NM_0002132.816Cell adhesion, ECM remodelling, signal transduction, kinase
H. sapiens hypothetical protein BC001573LOC134147NM_1388094.241Hydrolase
971446 BARC 5BOV B. taurus cDNA (UniGene Bt.8371, moderately similar to low-density lipoprotein receptor-related protein 2)LRP2CK8486548.018Transport
148109 MARC 4BOV B. taurus cDNA (UniGene Bt.2346, moderately similar to NP_002337 lymphocyte antigen 6 complex, locus E)LY6EBE6640063.618Immune-related
H. sapiens lymphocyte antigen 6 complex, locus G6CLY6G6CNM_025261OnImmune-related
4080472 BARC 9BOV B. taurus cDNA clone (UniGene Bt.9683, similar to lymphocyte antigen 6 complex, locus G6E)LY6G6ECK965552On2Immune-related, signal transduction
H. sapiens cDNA FLJ37829 fis, weakly similar to H. sapiens mRNA for mucolipidin (UniGene Hs.535239 mucolipin 3)MCOLN3AK0951484.848Transport
H. sapiens hypothetical protein MGC15429MGC15429NM_0327502.49Catalytic activity
H. sapiens clone DNA176108 scavenger receptor hlg (UNQ2938)MGC45780AY35815021.19Receptor activity
B. taurus matrix Gla proteinMGPNM_1747074.519ECM structural protein, ECM remodelling
H. sapiens membrane-spanning 4-domains, subfamily A, member 8BMS4A8BNM_0314575.352Signal transduction
H. sapiens myotubularin related protein 3MTMR3NM_1530506.423Signal transduction
M. musculus N-myc downstream regulated 4NDRG4NM_1456024.938Cell growth (+), hydrolase
968490 MARC 4BOV B. taurus cDNA 3′ (UniGene Hs.5025, H. sapiens nebulette)NEBLCK8460366.713Actin-binding
4113753 BARC 9BOV B. taurus cDNA (UniGene Bt.14398, moderately similar to phosphatidylethanolamine-binding protein)PBPCK9403083.626Signal transduction
H. sapiens phosphatidylethanolamine-binding protein 4PEBP4NM_1449623.132
B. taurus proenkephalinPENKNM_174141OnSignal transduction, pregnancy
H. sapiens phospholipase A2, group XPLA2G10NM_0035615.212Lipid catabolism, hydrolase
725142 MARC 6BOV B. taurus cDNA (UniGene Bt.1286, moderately similar to NP_002880 retinoic acid receptor responder 2)RARRES2CB4639612.826Cell growth, proliferation
H. sapiens retinal short chain dehydrogenase reductaseRDHE2NM_1389694.940Dehydrogenase
4107201 BARC 9BOV B. taurus cDNA clone 9BOV33_L08 5′ (UniGene Hs.445030 Rho-related BTB domain containing 3)RHOBTB3CK9761672.530Signal transduction
B. taurus RTN3 (reticulon 3)RTN3BK0017973.934
H. sapiens secernin 1SCRN1NM_0147662.953Immune-related
B. taurus selenoprotein P-like protein precursorSEPP1NM_1744592.932Transport
H. sapiens serum/glucocorticoid regulated kinaseSGKNM_0056272.120Signal transduction, kinase, transferase, apoptosis, transport
H. sapiens solute carrier family 16 (monocarboxylic acid transporters), member 11SLC16A11NM_1533577.621Transport
H. sapiens SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily b, member 1SMARCB1NM_0030737.848Cell cycle related, transcriptional regulation
H. sapiens SRY (sex determining region Y)-box 17SOX17NM_0224543.025Transcription regulation, pregnancy
H. sapiens teratocarcinoma-derived growth factor 1TDGF1NM_003212OnSignal transduction, cell growth (+)
B. taurus transglutaminase 2TGM2NM_1775074.410Apoptosis related, transferase
B. taurus tissue inhibitor of metalloproteinase 2TIMP2NM_1744725.512ECM remodelling, pregnancy
H. sapiens transducin-like enhancer of split 1 (E(sp1) homologue, Drosophila)TLE1NM_0050773.213Signal transduction, transcriptional regulation
4070793 BARC 10BOV B. taurus cDNA (UniGene Bt.28503)CK9463144.928
4081167 BARC 9BOV B. taurus cDNA (UniGene Bt.36133)CK9657942.716
4082241 BARC 9BOV B. taurus cDNA (UniGene Bt.29337)CK9669604.834
4082241 BARC 9BOV B. taurus cDNA (UniGene Bt.29337)CK9669604.834
604955 MARC 6BOV B. taurus cDNA (UniGene Bt.24986)CB4291592.330
776530 MARC 6BOV B. taurus cDNA (UniGene Bt.12715)CB538542On
H. sapiens mRNA; cDNA DKFZp667B1913 (UniGene Hs.374451)AL8324033.028
H. sapiens mRNA; cDNA DKFZp761L1121 (UniGene Hs.171939)AL7136396.829
H. sapiens mRNA; cDNA DKFZp761M0111 (UniGene Hs.368337)AL1373462.422
Genomic Scaffold 110056AAFC011870786.137
Genomic Scaffold 289710AAFC010190203.875
Table 3

Gradients of gene expression from the cranial uterine horn to the corpus

Gene nameGenBankGradientcranmidcaudcorpPattern
Gradient: Es ip: oestrus, ipsilateral; Es co: oestrus contralateral; Di ip: dioestrus, ipsilateral; Di co: dioestrus contralateral; Pattern: pattern represents significance of signal differences revealed by the post-hoc test
Gene/cDNA description
88128 MARC 1BOV B. taurus cDNAACP5AW447045Es ip4.31.91.51.02111
(UniGene Bt.5193, strongly similar to acid phosphatase 5, tartrate resistant (H. sapiens))Es co4.12.71.51.03211
Ovis ammon mRNA for angiotensinogenAGTD17520Di co3.11.71.81.02111
H. sapiens epoxide hydrolase 2, cytoplasmicEPHX2NM_001979Di ip2.31.82.01.02221
qPCRDi ip1.61.11.41.0
Di co2.72.01.61.03221
qPCRDi co1.61.21.31.0
Similar to H. sapiens GM2 gangliosideGM2ANM_000405Di ip3.22.52.81.02221
activator proteinDi co3.22.72.21.02221
B. taurus IGFBP-4IGFBP4NM_174557Es ip3.92.22.21.03221
Es co2.81.91.91.02221
H. sapiens integrin, beta 4ITGB4NM_000213Di co2.51.81.61.03221
M. musculus N-myc downstream regulated 4NDRG4NM_145602Di ip2.82.62.71.02221
Di co3.93.42.81.02221
H. sapiens phosphorylase kinase, gamma 2PHKG2NM_000294Di ip3.23.03.41.02221
B. taurus uterine milk protein precursorUTMPNM_174797Es ip30.66.73.21.02111
qPCREs ip34.59.12.31.0
Es co20.56.63.71.04321
qPCREs co20.511.45.31.0
4100874 BARC 10BOV B. taurus cDNA clone 10BOV7_D17 (UniGene Bt.20233)CK960198Di ip3.12.83.01.02221
B. indicus BoLA DQB gene for first domain of MHC class 2 molecule, beta chainZ48207Es ip2.72.22.21.02221
Es co2.52.52.41.02221
Di co3.11.91.31.02111
Table 4

Results of quantitative real-time RT-PCR

Mean crossing points (n=3) normalised to ubiquitin
OestrusDioestrus
i-cri-mi-cac-crc-mc-caCi-cri-mi-cac-crc-mc-caC
Upregulated at oestrus
CLDN20.920.826.226.0
INHBA22.922.728.127.7
PTHLH23.323.327.227.0
SOX422.622.724.624.6
UTMP19.621.523.520.321.222.324.726.827.427.426.627.127.427.4
Upregulated at dioestrus
CPN128.628.726.826.7
EPHX226.626.626.926.626.626.726.722.823.222.922.723.123.023.4
MGP26.826.826.726.726.827.027.024.424.424.324.024.224.124.7
MS4A8B23.323.321.321.5
PENK24.925.220.019.9
Housekeeping gene
Ubiquitin18.318.318.318.018.218.318.718.017.817.918.018.018.117.9
Expression ratios between oestrus and dioestrus
i-cri-mi-cac-crc-mc-caC
FCPFCPFCPFCPFCPFCPFCP
i-cr, ipsilateral cranial; i-m, ipsilateral middle; i-ca, ipsilateral caudal; c-cr, contralateral cranial; c-m, contralateral middle; c-ca, contralateral caudal; C, corpus; FC: fold-change; P, P-value
Upregulated at oestrus
CLDN39.2<0.0137.2<0.01
INHBA36.8<0.0132.4<0.01
PTHLH15.0<0.0113.6<0.01
SOX44.2<0.013.6<0.01
UTMP149.4<0.0161.2<0.0115.40.0481.0<0.0162.4<0.0135.9<0.016.80.17
Upregulated at dioestrus
CPN13.5<0.014.0<0.01
EPHX214.2<0.0110.6<0.0115.6<0.0114.6<0.0111.6<0.0112.9<0.0110.3<0.01
MGP5.30.065.30.035.40.036.40.046.10.027.40.025.00.03
MS4A8B3.9<0.013.6<0.01
PENK30.2<0.0138.0<0.01
Housekeeping gene
Ubiquitin1.21.51.41.71.21.21.0
Table 5

Assignment to cellular pathways

Upregulated at oestrusFunctionUpregulated at dioestrusFunction
1 ID of the H. sapiens reference pathway
Pathway
TGF-beta signalling pathway (hsa04350)1DCNInhibition (hsa04350)LRP2Endocytosis, downregulated by TGFB1
INHBAInhibition (hsa04350)MGPBinding protein of BMP2
THBS1Inhibition (hsa04350)TDGF1Blocks INHBA signalling; ligand and coreceptor in nodal signalling pathway
COL1A1/2, 5A2, 6A3Target genes
FBLN5Target gene
FN1Regulated by TGF-β in endometrial cells
PRSS11Inhibition of TGFB1 and BMP
S100A11Growth inhibition
SPARCCoupled to TGFBR- and Smad2/3-dependent pathway
TNCTarget gene
UTMPBinding of INHBA
MAPK signalling pathway (hsa04010)GJA1Inhibited by LH via both PKA and MAPK cascadesAKR1B1Upregulated by oxidative stress via EGFR-ERK pathway/p38 kinase pathway
LAMR1MAPK are involved in the laminin receptor signal transduction pathwayDSIPIInhibits Raf-1 phosphorylation resulting in suppression of MEK/ERK-1/2 phosphorylation and AP1-dependent transcription
PTHLHPI-3-kinase pathway may be involved in PTHrP stimulation of Ras, MAPK pathway, upregulation of COL1EFNA1Activation of Eck (EPHA2)
DCNSuppressed by activation of the ERK1,2 signalling pathway in fibroblastsPBPInhibitor of the Raf/MAP kinase signalling cascade (Raf-1/MEK/ERK pathway)
PLA2G10MAPK signalling pathway (hsa04010)
RA signalling/metabolismLGALS1RA-induced differentiationCYP26A1Inactivates RA
RARRES1Induced by RARARRES2Induced by RA
SERPINH1Induced by RARDHE2RA metabolism
TGM2RA signalling
cAMP signalling pathwayCOL1A1;Inhibited by parathyroidRARRES2Ligand of GPCR CHEMR23
COL1A2hormone via the cAMP signalling pathway(CMKLR1)
INHBAComponent of the cAMP pathway leading to endometrial stromal decidualisationSGKRegulated by multiple protein kinases, including PKA
PTHLHcAMP signalling pathway
Wnt signalling pathway (hsa04310)NDPActivation of the classical Wnt pathwaySOX17Interaction with beta-catenin
TAX1BP3Negative regulation of cell proliferationTLE1Wnt/Wg signalling
VIMTarget of the beta-catenin/TCF pathway
ImmunoregulationLGALS1Regulator of inflammatory responseCPN1Inactivates C3a and C4a and reduces C5a
UTMPImmunosuppressionDSIPIGlucocorticoid hormone-mediated immunosuppression
SERPINA1Complement and coagulation cascades (hsa04610)
IGF systemIGFBP6IGF-binding proteinBAIAP2Insulin/IGF-I signalling
PCSK5Cleavage of IGF-I
PRSS11Cleavage of IGFBP-5
Prostaglandin and leukotriene metabolism (hsa00590)AKR1B1Glycerolipid metabolism (hsa00561)
PLA2G10hsa00590
EPHX2Prostaglandin metabolism
HPGDProstaglandin metabolism
Ribosome (hsa03010)RPL7Ahsa03010
RPL18Ahsa03010
LAMR1hsa03010
NPM1Ribosome biogenesis pathway
EGFR pathwayTNCNegative regulation ofTIMP2Suppresses EGF-mediated mitogenic signalling
Rho-ROCK pathway via EGFR pathway
AKR1B1Upregulated by oxidative stress via
EGFR-ERK/p38 kinase pathway
Sonic hedgehog signallingPTCH2Sonic hedgehog/PTCH signalling pathwayLRP2Regulatory component
PTHLHHedgehog signalling pathway
Phospholipase C pathwayGRPStimulation of the GRP receptor activates phospholipase C pathway
MMP2Generation is mediated by activation of phospholipase A(2)
PTHLHGq-mediated phospholipase C/Ca2+ signalling, apoptosis
Phosphoinositide signalling pathwaySGKComponent of PI-3-kinase signalling pathway
EFNA1Increased PI-3-kinase activity
MTMR3Phosphoinositide signalling
Table 6

Comparison with similar studies in humans, Rhesus monkeys and mice

Gene nameGene IDOe:Di1Ace &Okulicz2Borthwicket al.3Carsonet al.4Kaoet al.5Riesewijket al.6Tanet al.7
Gene name, gene ID derived from Entrez Gene; on, no expression detectable at oestrus or dioestrus respectively; e, expression detected; 1 expression ratios between oestrus and dioestrus; 2Ace et al. 2004 (rhesus monkey): expression ratios between sexual cycle day 13 and days 21–23; 3Borthwick et al. 2003 (human): sexual cycle days 9–11 and 6–8 days after LH surge; 4Carson et al. 2002 (human): 2–4 days and 7–9 days after LH surge; 5Kao et al. 2002 (human): sexual cycle days 8–10 and 8–10 days after LH surge; 6Riesewijk et al. 2003 (human): 2 days and 7 days after LH surge; 7Tan et al. 2003 (mouse): oestrus and dioestrus
Gene description
Actin, alpha 2ACTA2592.4−4
Actin, betaACTB602.1e
Annexin A2ANXA23022.1e−4.7−4
Apolipoprotein EAPOE3483.3−100
Collagen, type I, alpha 2COL1A212784.2e−2.4
Collagen, type V, alpha 2COL5A212902.82.0
Collagen, type VI, alpha 3COL6A312935.18.0
Eukaryotic translation elongation factor 1 gammaEEF1G19372.2e
Fibulin 5FBLN5105162.6−6
Laminin receptor 1 (ribosomal protein SA, 67 kDa)LAMR139211.8−2.2
Lectin, galactoside-binding, soluble, 1 (galectin 1)LGALS139562.82.6e
Nephroblastoma overexpressed geneNOV48567.0−5.5
Procollagen-proline, 2-oxoglutarate 4-dioxygenase (proline 4-hydroxylase), betaP4HB50342.7e
Procollagen C-endopeptidase enhancerPCOLCE51182.6−2.7
Proprotein convertase subtilisin/kexin type 5PCSK55125Oe on6.7
RA receptor responder 1RARRES15918Oe on−5
Serine (or cysteine) proteinase inhibitor, clade A, member 1SERPINA152653.05.17.0
SRY (sex determining region Y)-box 4SOX466592.63.7
Secreted protein, acidic, cysteine-rich (osteonectin)SPARC66783.23.2e
Tenascin C (hexabrachion)TNC3371Oe on5.93.9
Tubulin, alpha 1TUBA172772.1−3.4−16−2.6
Tubulin, beta polypeptideTUBB72802.4−2.0
VimentinVIM74312.0e
Alkaline phosphataseALPL249−4.811
BAI1-associated protein 2BAIAP210458−3.13.2
Cyclin B1CCNB1891−3.32.53
Cytochrome P450, family 26, subfamily A, polypeptide 1CYP26A11592Di on−15.6
Ephrin-A1EFNA11942−3.2−4.9−16
Hydroxyprostaglandin dehydrogenase 15-(NAD)HPGD3248−6.714
Isocitrate dehydrogenase 1 (NADP+), solubleIDH13417−4.0−5.33
ProenkephalinPENK5179−38.06.413.925
Serum/glucocorticoid-regulated kinaseSGK6446−2.1−4.9−3.6
Transglutaminase 2TGM27052−4.4−7
Tissue inhibitor of metalloproteinase 2TIMP27077−5.52.7
Figure 1

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Figure 1

(a) Uterine sections. Endometrial tissue samples were collected from seven sections of the uterus: corpus (c) as well as caudal (ca), middle (m) and cranial (cr) parts of the ipsilateral and the contralateral uterine horns. (b) Array hybridisation. Radioactively labelled cDNA probes were prepared from the seven tissue samples of every animal (oestrus n=3, dioestrus n=3). These 42 probes were hybridised with randomly picked clones of two subtracted libraries, one for genes upregulated at oestrus and one for genes upregulated at dioestrus. Signals of one cDNA fragment of the UTMP gene derived from the probes of one animal at oestrus and one at dioestrus are shown.

Citation: Journal of Molecular Endocrinology 34, 3; 10.1677/jme.1.01799

Figure 2

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Figure 2

In situ hybridisation. Tissue samples of the middle part of the ipsilateral uterine horn were used from animals slaughtered at oestrus for the detection of INHBA, PTHLH, SAL1 and UTMP mRNAs or at dioestrus for PENK mRNA. Endometrial sections near the epithelial surface (a), of the deep uterine glands (b), and hybridised with the corresponding sense oligonucleotide (c) are shown.

Citation: Journal of Molecular Endocrinology 34, 3; 10.1677/jme.1.01799

Figure 3

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Figure 3

Comparison of the simplified GO at oestrus and dioestrus. For the differentially expressed genes of known or inferred function a simplified GO was built. The number of genes is shown for every GO group at oestrus (black bars) and at dioestrus (grey bars) respectively.

Citation: Journal of Molecular Endocrinology 34, 3; 10.1677/jme.1.01799

Figure 4

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Figure 4

Genes involved in the TGF-β signalling pathway. Differentially expressed genes of known or inferred function were assigned to the TGF-β signalling pathway (based on the H. sapiens reference pathway hsa04350) by direct searching this pathway and by a literature search. Grey boxes: genes of the original pathway; white boxes/solid line: genes directly assigned to the pathway; white boxes/dashed line: genes assigned by literature search; bold style: upregulated at oestrus; italics: upregulated at dioestrus; for gene symbols in grey boxes see Entrez Gene (www.ncbi.nlm.nih.gov/entrez/query.fcgi).

Citation: Journal of Molecular Endocrinology 34, 3; 10.1677/jme.1.01799

We thank Susanne Rehfeld, Peter Rieblinger, Christian Erdle and Myriam Weppert for excellent animal and sample management. This study was supported by the German Research Foundation (FOR 478/1). The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.

References

  • AceCI & Okulicz WC 2004 Microarray profiling of progesterone-regulated endometrial genes during the rhesus monkey secretory phase. Reproductive Biology and Endocrinology254.

  • BauersachsS2003 Regulation of ipsilateral and contralateral bovine oviduct epithelial cell function in the postovulation period: a transcriptomics approach. Biology of Reproduction681170–1177.

  • BauersachsS2004 Monitoring gene expression changes in bovine oviduct epithelial cells during the oestrous cycle. Journal of Molecular Endocrinology32449–466.

  • BorthwickJM2003 Determination of the transcript profile of human endometrium. Molecular Human Reproduction919–33.

  • CarsonDD2002 Changes in gene expression during the early to mid-luteal (receptive phase) transition in human endometrium detected by high-density microarray screening. Molecular Human Reproduction8871–879.

  • CaseyML1993 Regulation of parathyroid hormone-related protein gene expression in human endometrial stromal cells in culture. Journal of Clinical Endocrinology and Metabolism77188–194.

  • ChanLN2002 Distribution and regulation of ENaC subunit and CFTR mRNA expression in murine female reproductive tract. Journal of Membrane Biology185165–176.

  • CheonYP2002 A genomic approach to identify novel progesterone receptor regulated pathways in the uterus during implantation. Molecular Endocrinology162853–2871.

  • CurryTE Jr & Osteen KG 2001 Cyclic changes in the matrix metalloproteinase system in the ovary and uterus. Biology of Reproduction641285–1296.

  • De LucaA2003 Distribution of the serine protease HtrA1 in normal human tissues. Journal of Histochemistry and Cytochemistry511279–1284.

  • DengL2003 Coordinate regulation of the production and signaling of retinoic acid by estrogen in the human endometrium. Journal of Clinical Endocrinology and Metabolism882157–2163.

  • DiatchenkoLet al.1996 Suppression subtractive hybridization: a method for generating differentially regulated or tissue-specific cDNA probes and libraries. PNAS936025–6030.

  • FujimotoM1996 Requirement for transglutaminase in progesterone-induced decidualization of human endometrial stromal cells. Endocrinology1371096–1101.

  • FujiwaraH2002 Human endometrial epithelial cells express ephrin A1: possible interaction between human blastocysts and endometrium via Eph-ephrin system. Journal of Clinical Endocrinology and Metabolism875801–5807.

  • GabrielS2004 Modulation of connexin expression in sheep endometrium in response to pregnancy. Placenta25287–296.

  • GaddTS2002 Regulation of insulin-like growth factor binding protein-6 expression in the reproductive tract throughout the estrous cycle and during the development of the placenta in the ewe. Biology of Reproduction671756–1762.

  • GodkinJD & Dore JJ 1998 Transforming growth factor beta and the endometrium. Reviews of Reproduction31–6.

  • GoffAK2004 Steroid hormone modulation of prostaglandin secretion in the ruminant endometrium during the estrous cycle. Biology of Reproduction7111–16.

  • GoffinF2003 Expression pattern of metalloproteinases and tissue inhibitors of matrix-metalloproteinases in cycling human endometrium. Biology of Reproduction69976–984.

  • GreenlandKJ2000 The human NAD+-dependent 15-hydroxyprostaglandin dehydrogenase gene promoter is controlled by Ets and activating protein-1 transcription factors and progesterone. Endocrinology141581–597.

  • GrummerR1994 Regulation of connexin26 and connexin43 expression in rat endometrium by ovarian steroid hormones. Biology of Reproduction511109–1116.

  • HaendlerB2004 Cycle-dependent endometrial expression and hormonal regulation of the fibulin-1 gene. Molecular Reproduction and Development68279–287.

  • HamptonAL1995 Tissue inhibitors of metalloproteinases in endometrium of ovariectomized steroid-treated ewes and during the estrous cycle and early pregnancy. Biology of Reproduction53302–311.

  • HansenPJ1998 Regulation of uterine immune function by progesterone – lessons from the sheep. Journal of Reproductive Immunology4063–79.

  • HoshiS2001 PTHrP and PTH/PTHrP receptor expressions in human endometrium. Endocrine Journal48219–225.

  • JahnE1995 Expression of gap junction connexins in the human endometrium throughout the menstrual cycle. Human Reproduction102666–2670.

  • JinDF1988 Estrous cycle- and pregnancy-related differences in expression of the proenkephalin and proopiomelanocortin genes in the ovary and uterus. Endocrinology1221466–1471.

  • JokimaaV2001Molecular Human Reproduction773–78.

  • KaoLC2002 Global gene profiling in human endometrium during the window of implantation. Endocrinology1432119–2138.

  • KellyRW1994 Prostaglandin inactivation is increased in endometrium after exposure to clomiphene. 50235–238.

  • LowKG1989 Proenkephalin gene expression in the primate uterus: regulation by estradiol in the endometrium. Molecular Endocrinology3852–857.

  • MalayerJR1988in vitro protein secretion by bovine endometrium. Journal of Reproduction and Fertility84567–578.

  • MaquoiE1997 Changes in the distribution pattern of galectin-1 and galectin-3 in human placenta correlates with the differentiation pathways of trophoblasts. Placenta18433–439.

  • MarshallRJ & Braye SG 1987 Immunohistochemical demonstration of alpha-1-antitrypsin and alpha-1-antichymotrypsin in normal human endometrium. International Journal of Gynecological Pathology649–54.

  • MichieHJ & Head JR 1994 Tenascin in pregnant and non-pregnant rat uterus: unique spatio-temporal expression during decidualization. Biology of Reproduction501277–1286.

  • NelsonKG1992 Transforming growth factor-alpha is a potential mediator of estrogen action in the mouse uterus. Endocrinology1311657–1664.

  • NeuviansTP2003Endocrine2293–100.

  • OsteenKG2003 Progesterone action in the human endometrium: induction of a unique tissue environment which limits matrix metalloproteinase (MMP) expression. Frontiers in Bioscience8d78–d86.

  • OverberghL1995Journal of Lipid Research361774–1786.

  • PfafflMW2003Journal of Steroid Biochemistry and Molecular Biology84159–166.

  • PrakashBS1987 Development of a sensitive enzyme immunoassay (EIA) for progesterone determination in unextracted bovine plasma using the second antibody technique. Journal of Steroid Biochemistry28623–627.

  • RiderV1992 Uterine fibronectin mRNA content and localization are modulated during implantation. Developmental Dynamics1951–14.

  • RiesewijkA2003 Gene expression profiling of human endometrial receptivity on days LH+2versus LH+7by microarray technology. Molecular Human Reproduction9253–264.

  • RosenH1990 Local regulation within the female reproductive system and upon embryonic implantation: identification of cells expressing proenkephalin A. Molecular Endocrinology4146–154.

  • SchamsD & Karg H 1969Acta Endocrinologica6196–103.

  • SpencerTE1999 Discovery and characterization of endometrial epithelial messenger ribonucleic acids using the ovine uterine gland knockout model. Endocrinology1404070–4080.

  • SpencerTE2004 Progesterone and placental hormone actions on the uterus: insights from domestic animals. Biology of Reproduction712–10.

  • StewartMD2000 Prolactin receptor and uterine milk protein expression in the ovine endometrium during the estrous cycle and pregnancy. Biology of Reproduction621779–1789.

  • TabibzadehS2002 Homeostasis of extracellular matrix by TGF-beta and lefty. Frontiers in Bioscience7d1231–d1246.

  • TaguchiM1999 Immunohistochemical localization of tenascin and Ki-67 nuclear antigen in human endometrium throughout the normal menstrual cycle. Journal of Medical and Dental Sciences467–12.

  • TanYF2003 Global gene profiling analysis of mouse uterus during the oestrous cycle. Reproduction126171–182.

  • UlbrichSE2004 Hyaluronan in the bovine oviduct–modulation of synthases and receptors during the estrous cycle. Molecular and Cellular Endocrinology2149–18.

  • WangX2004 Embryonic signals direct the formation of tight junctional permeability barrier in the decidualizing stroma during embryo implantation. Journal of Cell Science11753–62.

  • WhitleyJC1998 Temporal expression and cellular localization of a gastrin-releasing peptide-related gene in ovine uterus during the oestrous cycle and pregnancy. Journal of Endocrinology157139–148.

  • WilliamsBL1992in vitro bovine endometrial secretions. Journal of Dairy Science752112–2118.

 

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Figures

  • View in gallery

    (a) Uterine sections. Endometrial tissue samples were collected from seven sections of the uterus: corpus (c) as well as caudal (ca), middle (m) and cranial (cr) parts of the ipsilateral and the contralateral uterine horns. (b) Array hybridisation. Radioactively labelled cDNA probes were prepared from the seven tissue samples of every animal (oestrus n=3, dioestrus n=3). These 42 probes were hybridised with randomly picked clones of two subtracted libraries, one for genes upregulated at oestrus and one for genes upregulated at dioestrus. Signals of one cDNA fragment of the UTMP gene derived from the probes of one animal at oestrus and one at dioestrus are shown.

  • View in gallery

    In situ hybridisation. Tissue samples of the middle part of the ipsilateral uterine horn were used from animals slaughtered at oestrus for the detection of INHBA, PTHLH, SAL1 and UTMP mRNAs or at dioestrus for PENK mRNA. Endometrial sections near the epithelial surface (a), of the deep uterine glands (b), and hybridised with the corresponding sense oligonucleotide (c) are shown.

  • View in gallery

    Comparison of the simplified GO at oestrus and dioestrus. For the differentially expressed genes of known or inferred function a simplified GO was built. The number of genes is shown for every GO group at oestrus (black bars) and at dioestrus (grey bars) respectively.

  • View in gallery

    Genes involved in the TGF-β signalling pathway. Differentially expressed genes of known or inferred function were assigned to the TGF-β signalling pathway (based on the H. sapiens reference pathway hsa04350) by direct searching this pathway and by a literature search. Grey boxes: genes of the original pathway; white boxes/solid line: genes directly assigned to the pathway; white boxes/dashed line: genes assigned by literature search; bold style: upregulated at oestrus; italics: upregulated at dioestrus; for gene symbols in grey boxes see Entrez Gene (www.ncbi.nlm.nih.gov/entrez/query.fcgi).

References

AceCI & Okulicz WC 2004 Microarray profiling of progesterone-regulated endometrial genes during the rhesus monkey secretory phase. Reproductive Biology and Endocrinology254.

BauersachsS2003 Regulation of ipsilateral and contralateral bovine oviduct epithelial cell function in the postovulation period: a transcriptomics approach. Biology of Reproduction681170–1177.

BauersachsS2004 Monitoring gene expression changes in bovine oviduct epithelial cells during the oestrous cycle. Journal of Molecular Endocrinology32449–466.

BorthwickJM2003 Determination of the transcript profile of human endometrium. Molecular Human Reproduction919–33.

CarsonDD2002 Changes in gene expression during the early to mid-luteal (receptive phase) transition in human endometrium detected by high-density microarray screening. Molecular Human Reproduction8871–879.

CaseyML1993 Regulation of parathyroid hormone-related protein gene expression in human endometrial stromal cells in culture. Journal of Clinical Endocrinology and Metabolism77188–194.

ChanLN2002 Distribution and regulation of ENaC subunit and CFTR mRNA expression in murine female reproductive tract. Journal of Membrane Biology185165–176.

CheonYP2002 A genomic approach to identify novel progesterone receptor regulated pathways in the uterus during implantation. Molecular Endocrinology162853–2871.

CurryTE Jr & Osteen KG 2001 Cyclic changes in the matrix metalloproteinase system in the ovary and uterus. Biology of Reproduction641285–1296.

De LucaA2003 Distribution of the serine protease HtrA1 in normal human tissues. Journal of Histochemistry and Cytochemistry511279–1284.

DengL2003 Coordinate regulation of the production and signaling of retinoic acid by estrogen in the human endometrium. Journal of Clinical Endocrinology and Metabolism882157–2163.

DiatchenkoLet al.1996 Suppression subtractive hybridization: a method for generating differentially regulated or tissue-specific cDNA probes and libraries. PNAS936025–6030.

FujimotoM1996 Requirement for transglutaminase in progesterone-induced decidualization of human endometrial stromal cells. Endocrinology1371096–1101.

FujiwaraH2002 Human endometrial epithelial cells express ephrin A1: possible interaction between human blastocysts and endometrium via Eph-ephrin system. Journal of Clinical Endocrinology and Metabolism875801–5807.

GabrielS2004 Modulation of connexin expression in sheep endometrium in response to pregnancy. Placenta25287–296.

GaddTS2002 Regulation of insulin-like growth factor binding protein-6 expression in the reproductive tract throughout the estrous cycle and during the development of the placenta in the ewe. Biology of Reproduction671756–1762.

GodkinJD & Dore JJ 1998 Transforming growth factor beta and the endometrium. Reviews of Reproduction31–6.

GoffAK2004 Steroid hormone modulation of prostaglandin secretion in the ruminant endometrium during the estrous cycle. Biology of Reproduction7111–16.

GoffinF2003 Expression pattern of metalloproteinases and tissue inhibitors of matrix-metalloproteinases in cycling human endometrium. Biology of Reproduction69976–984.

GreenlandKJ2000 The human NAD+-dependent 15-hydroxyprostaglandin dehydrogenase gene promoter is controlled by Ets and activating protein-1 transcription factors and progesterone. Endocrinology141581–597.

GrummerR1994 Regulation of connexin26 and connexin43 expression in rat endometrium by ovarian steroid hormones. Biology of Reproduction511109–1116.

HaendlerB2004 Cycle-dependent endometrial expression and hormonal regulation of the fibulin-1 gene. Molecular Reproduction and Development68279–287.

HamptonAL1995 Tissue inhibitors of metalloproteinases in endometrium of ovariectomized steroid-treated ewes and during the estrous cycle and early pregnancy. Biology of Reproduction53302–311.

HansenPJ1998 Regulation of uterine immune function by progesterone – lessons from the sheep. Journal of Reproductive Immunology4063–79.

HoshiS2001 PTHrP and PTH/PTHrP receptor expressions in human endometrium. Endocrine Journal48219–225.

JahnE1995 Expression of gap junction connexins in the human endometrium throughout the menstrual cycle. Human Reproduction102666–2670.

JinDF1988 Estrous cycle- and pregnancy-related differences in expression of the proenkephalin and proopiomelanocortin genes in the ovary and uterus. Endocrinology1221466–1471.

JokimaaV2001Molecular Human Reproduction773–78.

KaoLC2002 Global gene profiling in human endometrium during the window of implantation. Endocrinology1432119–2138.

KellyRW1994 Prostaglandin inactivation is increased in endometrium after exposure to clomiphene. 50235–238.

LowKG1989 Proenkephalin gene expression in the primate uterus: regulation by estradiol in the endometrium. Molecular Endocrinology3852–857.

MalayerJR1988in vitro protein secretion by bovine endometrium. Journal of Reproduction and Fertility84567–578.

MaquoiE1997 Changes in the distribution pattern of galectin-1 and galectin-3 in human placenta correlates with the differentiation pathways of trophoblasts. Placenta18433–439.

MarshallRJ & Braye SG 1987 Immunohistochemical demonstration of alpha-1-antitrypsin and alpha-1-antichymotrypsin in normal human endometrium. International Journal of Gynecological Pathology649–54.

MichieHJ & Head JR 1994 Tenascin in pregnant and non-pregnant rat uterus: unique spatio-temporal expression during decidualization. Biology of Reproduction501277–1286.

NelsonKG1992 Transforming growth factor-alpha is a potential mediator of estrogen action in the mouse uterus. Endocrinology1311657–1664.

NeuviansTP2003Endocrine2293–100.

OsteenKG2003 Progesterone action in the human endometrium: induction of a unique tissue environment which limits matrix metalloproteinase (MMP) expression. Frontiers in Bioscience8d78–d86.

OverberghL1995Journal of Lipid Research361774–1786.

PfafflMW2003Journal of Steroid Biochemistry and Molecular Biology84159–166.

PrakashBS1987 Development of a sensitive enzyme immunoassay (EIA) for progesterone determination in unextracted bovine plasma using the second antibody technique. Journal of Steroid Biochemistry28623–627.

RiderV1992 Uterine fibronectin mRNA content and localization are modulated during implantation. Developmental Dynamics1951–14.

RiesewijkA2003 Gene expression profiling of human endometrial receptivity on days LH+2versus LH+7by microarray technology. Molecular Human Reproduction9253–264.

RosenH1990 Local regulation within the female reproductive system and upon embryonic implantation: identification of cells expressing proenkephalin A. Molecular Endocrinology4146–154.

SchamsD & Karg H 1969Acta Endocrinologica6196–103.

SpencerTE1999 Discovery and characterization of endometrial epithelial messenger ribonucleic acids using the ovine uterine gland knockout model. Endocrinology1404070–4080.

SpencerTE2004 Progesterone and placental hormone actions on the uterus: insights from domestic animals. Biology of Reproduction712–10.

StewartMD2000 Prolactin receptor and uterine milk protein expression in the ovine endometrium during the estrous cycle and pregnancy. Biology of Reproduction621779–1789.

TabibzadehS2002 Homeostasis of extracellular matrix by TGF-beta and lefty. Frontiers in Bioscience7d1231–d1246.

TaguchiM1999 Immunohistochemical localization of tenascin and Ki-67 nuclear antigen in human endometrium throughout the normal menstrual cycle. Journal of Medical and Dental Sciences467–12.

TanYF2003 Global gene profiling analysis of mouse uterus during the oestrous cycle. Reproduction126171–182.

UlbrichSE2004 Hyaluronan in the bovine oviduct–modulation of synthases and receptors during the estrous cycle. Molecular and Cellular Endocrinology2149–18.

WangX2004 Embryonic signals direct the formation of tight junctional permeability barrier in the decidualizing stroma during embryo implantation. Journal of Cell Science11753–62.

WhitleyJC1998 Temporal expression and cellular localization of a gastrin-releasing peptide-related gene in ovine uterus during the oestrous cycle and pregnancy. Journal of Endocrinology157139–148.

WilliamsBL1992in vitro bovine endometrial secretions. Journal of Dairy Science752112–2118.

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