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 gene expression, a microarray analysis of endometrium tissue samples collected from 20 cyclic heifers at five stages of the estrous cycle (days 0, 3.5, 12, 18, 20) was performed. RNA was extracted from these tissue samples and analyzed with a custom-made bovine oviduct and endometrium (BOE) cDNA array. 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 1,440 cDNA fragments on the array. Twenty radioactively labeled cDNA samples (n=4 for each cycle stage) were hybridized with the BOE array. After normalization of raw data (vsn; BioConductor) and significance analysis (SAM, FDR 1%), 225 mRNAs were identified that showed alterations of their concentration during the estrous cycle. K-means clustering revealed different specific expression profiles. Most of the identified mRNAs showed their highest concentration either on day 12 (high serum progesterone level) or on days 20 and 0 (low serum progesterone). Furthermore, a number of genes showed highest mRNA levels at day 18, the day around embryo implantation. The least proportion of mRNAs showed the highest abundance at day 3.5. In relation to this classification co-regulated genes were assigned to Gene Ontology categories and molecular pathways to find key regulatory genes associated with the biological functions of the endometrium.