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1 Institute of Molecular Animal Breeding, Gene Center of the Ludwig-Maximilians University, Feodor-Lynen-Str. 25, 81377 Munich, Germany
2 Laboratory for Functional Genome Analysis (LAFUGA), Gene Centre of the Ludwig-Maximilians University, Munich, Germany
3 Physiology-Weihenstephan, Technical University of Munich, Freising, Germany
4 Institute of Veterinary Biochemistry, Free University of Berlin, Berlin, Germany
5 Bavarian Research Centre for Biology of Reproduction, Oberschleissheim, Germany
6 Institute of Veterinary Anatomy, Histology and Embryology, Ludwig-Maximilians University, Munich, Germany
(Requests for offprints should be addressed to E Wolf; Email: ewolf{at}lmb.uni-muenchen.de)
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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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-
) 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.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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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.
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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.
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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 x 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 x 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.
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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 x 10 min), immersed in isopropanol (2 x 5 min) and then allowed to air dry. Dried sections were submerged in 2 x 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 x 5 min), TBS (2 x 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 x 5 min) followed by distilled water (2 x 5 min) and post-fixed for 10 min in 4% paraformaldehyde/PBS (pH 7.4). After washing in PBS (2 x 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 x SSC, hybridisation accelerator, RNase inhibitor and blocking reagents. Subsequently, slides were washed in 2 x SSC (2 x 15 min, preheated to 38 °C), distilled water (2 x 5 min) and TBS (2 x 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.
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Results |
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). 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.
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). 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.
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, 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.
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. The highest number of genes (17 genes) was assigned to the transforming growth factor-ß (TGF-ß) 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-ß 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.
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). 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).
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Discussion |
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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-ß 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-ß signalling were upregulated. In contrast, at dioestrus only three genes were identified which are described as being involved in TGF-ß signalling. The identification of this considerable number of genes involved in or regulated by TGF-ß signalling is in line with current knowledge of regulation of the endometrium by TGF-ß during the oestrous cycle, implantation and pregnancy (Godkin & Dore 1998, Tabibzadeh 2002). Furthermore, the induction of TGF-ß 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-ß 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-ß 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-ß 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.
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References |
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Bauersachs S, Blum H, Mallok S, Wenigerkind H, Rief S, Prelle K & Wolf E 2003 Regulation of ipsilateral and contralateral bovine oviduct epithelial cell function in the postovulation period: a transcriptomics approach. Biology of Reproduction 68 1170–1177.
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Received 21 March 2005
Accepted 29 March 2005
Made available online as an Accepted Preprint 15 April 2005
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