During the oestrous cycle, the bovine endometrium exhibits characteristic morphological and functional changes, which are mainly induced by progesterone (P 4 ), oestrogens and oxytocin. We studied the response of the endometrium to this changing hormonal environment at the transcriptome level using a custom-made cDNA microarray. Endometrium samples were recovered from Simmental heifers on days 0 (oestrus), 3.5 (metoestrus), 12 (dioestrus) and 18. The latter group was divided into animals with high (late dioestrus) and low P 4 levels (preoestrus). Significance analysis of microarrays revealed 269 genes exhibiting significant changes in their transcript levels during the oestrous cycle in distinct temporal patterns. Two major types of expression profiles were observed, which showed the highest mRNA levels during the oestrus phase or the highest levels during the luteal phase respectively. A minor group of genes exhibited the highest mRNA levels on day 3.5. Gene ontology (GO) analyses revealed GO categories related to extracellular matrix remodelling, transport, and cell growth and morphogenesis enriched at oestrus, whereas immune response and particular metabolic pathways were overrepresented at dioestrus.

The endometrium undergoes marked functional changes during estrous cycle and pregnancy. As the adjacent environment of the conceptus, it represents the maternal interface for embryo-maternal communication, which is essential to maintain pregnancy. Transcriptome studies provide the unique opportunity to assess molecular profiles changing in response to endocrine or metabolic stimuli or to embryonic pregnancy recognition signals. Here we review the current state of transcriptome profiling techniques and the results of a series of transciptome studies comparing bovine endometrium samples during the estrous cycle or endometrium samples from pregnant vs. non-pregnant animals. These studies revealed specific mRNA profiles which are characteristic for the functional status of the endometrium. Transcriptome studies of endometrial samples recovered during the pre-attachment period identified many interferon-stimulated genes, genes that are possibly involved in embryo-maternal immune modulation ( C1S, C1R, C4, SERPING1, UTMP, CD81, IFITM1, BST2), as well as genes affecting cell adhesion ( AGRN, CD81, LGALS3BP, LGALS9, GPLD1, MFGE8, and TGM2) and remodeling of the endometrium ( CLDN4, MEP1B, LGMN, MMP19, TIMP2, TGM2, MET, and EPSTI1). The results of these transcriptome studies were compared to those of similar microarray analyses in human, mouse and Rhesus monkey to identify similarities in endometrial biology between mammalian species and species-specific differences. Future studies will cover dynamic transcriptome changes between different stages of early pregnancy, the relationship between metabolic problems in dairy cows and the functionality of reproductive tissues as well as endometrium transcriptome profiles in recipients of somatic cell nuclear transfer embryos.

Fertility problems are the main reason for slaughter of high-performance milk cows, because elongated calving intervals result in financial losses for the farmer and retard genetic progress. Genetic improvement of fertility would be of great benefit, but functional traits for effective selection are missing. Recent advances in functional genomics tools like DNA microarrays could be the key to identify gene expression patterns in the endometrium that correlate with maternal fertility. Therefore, a first version of a bovine oviduct and endometrium cDNA array was established that contains a set of 1,440 cDNA clones and long oligonucleotides representing 950 different genes. The major part of these genes has been identified in a series of differential gene expression studies in endometrium (different stages of the estrous cycle, d 18 pregnant vs. nonpregnant) and in oviduct epithelial cells (different stages of the estrous cycle) using a combination of subtracted cDNA libraries and cDNA array hybridization. Furthermore, cDNA clones of genes, which showed no changes in their mRNA levels in the analyzed tissues, were added as controls. Reproducibility of the array hybridization, a comparison with the Affymetrix bovine genome array, and confirmation of differential gene expression with reverse transcription-quantitative PCR is shown. Potential future applications include systematic studies of interactions between metabolic status and functionality of the endometrium to identify genes that could be used for differential diagnosis of fertility problems. Further, endometrium transcriptome profiles may serve as novel traits to improve fertility by genetic selection.

The endometrium provides the optimal conditions for the transport of sperm to the oviduct, to the site of fertilization, and later on for the reception of the embryo. This is reflected by specific morphological and functional changes during the estrous cycle, which are mainly regulated by the hormones progesterone, estradiol, and oxytocin. To study these changes on the level of messenger RNA, a microarray analysis of endometrial tissue samples was performed. Tissue samples were collected from 20 cyclic heifers (Deutsches Fleckvieh, between 17 and 31 months of age) after slaughter at Days 0, 3.5, 12, and 18 of the estrous cycle. The Day 18 group split into two subgroups, one with high and one with low progesterone levels in peripheral blood. Altogether there were 4 heifers in each experimental group. RNA was extracted from these tissue samples and analyzed with a custom-made bovine oviduct and endometrium (BOE) cDNA array (Bauersachs et al. 2007 J. Dairy Sci. 90, in press). The cDNAs present on the array were derived from several previously conducted differential gene expression studies of bovine endometrium between different stages of the estrous cycle, during early pregnancy, and from studies of bovine oviduct epithelial cells. In all of these studies, cDNAs of differentially expressed genes were identified using a combination of subtracted cDNA libraries and cDNA array hybridization. Redundant cDNA clones were removed, resulting in 1440 cDNA fragments on the array, representing 950 different genes. Twenty radioactively (33P) labeled cDNA samples (n = 4 for each cycle stage) were hybridized with the BOE array. After normalization of raw data (using BioConductor open source software) and significance analysis (SAM, FDR 1%), 272 mRNAs were identified that showed alterations of their concentration during the estrous cycle. Expression data from cDNAs with significant changes during the estrous cycle were used for cluster analyses with MultiExperiment Viewer 4.0 (Saeed et al. 2003 Biotechniques 34, 374–378). The main clusters represented genes upregulated either during the estrus or during the diestrus phase. Quantitatively enriched Gene Ontology (GO) categories were identified to find relevant functional groups and prominent biological processes. At estrus, e.g., GO categories extracellular matrix, cytoskeleton organization, and growth factor activity indicate changes in the composition of the endometrium during the estrous cycle. At diestrus, only a few overrepresented GO categories were found, mostly related to immune response and metabolism. The genes of known function were further analyzed in the context of interaction and regulatory networks. One of a number of central factors was TGF-β, which controls the expression of 12 genes upregulated at estrus and 8 at diestrus. In conclusion, the present study extended the results of our previously conducted analysis of bovine endometrium between the estrus and diestrus stages (Bauersachs et al. 2005 J. Mol. Endocrinol. 32, 449–466), revealed distinct temporal expression profiles, and identified additional genes differentially expressed during the estrous cycle. This work was supported by Grant BMBF FUGATO-Fertilink.

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