181 MESSENGER RNA PROFILING OF BOVINE ENDOMETRIUM DURING THE ESTROUS CYCLE USING A CUSTOM-MADE cDNA ARRAY

Abstract

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.