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,2
* Institute of Molecular Animal Breeding and Biotechnology, and
Laboratory for Functional Genome Analysis, Gene Center, Ludwig-Maximilians University Munich, D-81377 Munich, Germany
2 Corresponding author: ewolf{at}lmb.uni-muenchen.de
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ABSTRACT |
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Key Words: functional genomics • endometrium • oviduct • diagnostic array
The endometrium undergoes marked morphological and functional changes during the estrous cycle and during early pregnancy that are mainly regulated by the hormones progesterone, estradiol, and oxytocin (Spencer et al., 2004). To study these changes at the mRNA level, a series of studies using a combination of subtracted cDNA libraries and cDNA array hybridization has been performed to identify differentially expressed genes. In these studies, endometrial samples from heifers at d 0 (estrus) vs. d 12 (diestrus; Bauersachs et al., 2005) and samples from d 12 vs. d 20 (preestrus; unpublished data of S. Bauersachs) have been compared. Day 18 of pregnancy has been analyzed using a monozygotic twin model, in which pregnancy was achieved by transfer of embryos produced in vitro (Klein et al., 2006), and a second model, in which cyclic heifers were inseminated with semen or seminal plasma only and slaughtered on d 18 (Bauersachs et al., 2006). In addition, endometrial samples from d 15 of pregnancy have been compared with d-15 controls (unpublished data of S. Bauersachs). Furthermore, different studies of bovine oviduct epithelial cells during the estrous cycle have been conducted (Bauersachs et al., 2003, 2004). The genes expressed differently in the various studies of endometrial samples and in the oviduct epithelium represent an interesting set of candidates for evaluating the functional state of the female reproductive tract. Therefore, cDNA fragments of all genes identified in the different studies together with cDNA fragments or long oligonucleotides of candidate genes deduced from the literature were integrated on a single cDNA array, the bovine oviduct and endometrium (BOE) cDNA array version 1.
More than 20,000 cDNA clones of different subtracted libraries have been screened by microarray analyses in the studies that contributed to the genes on the BOE array. From these clones, 1,344 nonredundant cDNA clones were extracted, mainly consisting of those cDNA clones identified as differentially expressed and some control genes, which can be used as internal references. Fragments representing different parts of a transcript were included. Altogether, the BOE array covers approximately 950 different genes. The bovine cDNA clones were annotated based on comparisons to Gen-Bank sequences using the basic local alignment search tool (discontiguous Mega BLAST) at the National Center for Biotechnology Information (http://www.ncbi.nl-m.nih.gov/blast/blast.cgi), searches in the bovine genome (Ensembl; http://www.ensembl.org/Bos_taurus/blastview), and the assignment of corresponding expressed sequence tags to the UniGene database (http://www.ncbi.nlm.nih.gov/sites/entrez?db=unigene). The human orthologous genes, as far as they exist, were also identified to obtain more information regarding gene function.
Based on the generated list of cDNA clones, the templates for PCR amplification were rearranged from the original 190 to 14 new 96-well plates using a liquid handling station (Multiprobe II Plus EX, Perkin Elmer, Rodgau, Germany). The cDNA fragments were amplified via PCR using modified T7 and T3 primers that bind in the plasmid vector adjacent to the cDNA fragments. The PCR products were spotted on nylon membranes using a microarray robot (Omnigrid Accent, GeneMachines, San Carlos, CA ; Bauersachs et al., 2004). The identity of the cDNA clones was confirmed by DNA sequencing of randomly selected cDNA fragments. Based on the data for the orthologous human genes, a classification according to Gene Ontology (GO; http://geneontology.org/) categories was performed. The Affymetrix NetAffx Analysis Center (http://www.affymetrix.com) was used to get a summary of the most specific GO annotations. Data were further reduced to obtain a meaningful overview for the 3 major categories: biological process, molecular function, and cellular component (Table 1
). In addition to the GO analysis, the known genes were assigned to molecular pathways (http://www.genome.ad.jp/kegg/tool/search_pathway.html). Table 2 has the numbers of genes that were found in signaling, cell communication and interaction, and immune-related pathways.
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For a comparison of the BOE array with the existing Affymetrix bovine genome array, which contains approximately 24,000 probe sets, endometrial tissue samples derived from four 18-d pregnant animals (AI) and four 18-d controls were analyzed (for details of sample preparation, see Bauersachs et al., 2006). Raw signals of the BOE array were normalized with vsn (variance stabilization and calibration for microarray data, Bio-Conductor), and differentially expressed genes were identified with the Microsoft Excel add-in SAM (Microsoft Corp., Redmond, WA). Raw data from the Affymetrix arrays were normalized with robust multiarray average (RMA, BioConductor), and significance analysis was done with the SAM tool in siggenes (SAM and Efron’s empirical Bayes approaches, BioConductor). With the BOE array, 97 genes with at least 2-fold change were found at a false discovery rate (FDR) of 2%. On the Affymetrix array, 352 genes with at least 2-fold change were identified (FDR 2%).
Annotation of differentially expressed probe sets from Affymetrix was completed with Biomart (Ensembl), BLAST (http://www.ncbi.nlm.nih.gov/blast/blast.cgi) analyses, and mapping of probe sets to the bovine genome sequence to allow a comparison with the data from the BOE array. The overlap was 61 genes or 62.9%. All changes of gene expression were consistent between the BOE array and the Affymetrix array. Of the additional 36 genes from the BOE array, 20 were not present on the Affymetrix array based on the bovine Entrez Gene ID of the current annotation (March 2007). The remaining 16 probe sets revealed no significant changes on the Affymetrix array. Sensitivity seems to be similar to that of the Affymetrix array. The large number of additional genes obtained from the Affymetrix array showed mostly changes between 2-fold and 3-fold. Because the cDNA clones of the BOE array are derived from subtracted cDNA libraries, the BOE array is more enriched for genes that show larger gene expression differences.
The expression of 13 genes, which were identified by the BOE array, has been analyzed with reverse transcription-quantitative PCR (RT-qPCR) in 2 of our previous studies. The obtained changes in gene expression showed good correlation between RT-qPCR and array data (Table 3).
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In conclusion, reproducibility of cDNA array hybridization with the BOE array was demonstrated, results were consistent compared with the Affymetrix bovine genome array, and the results of the method were highly correlated with RT-qPCR data. There are manifold applications for the BOE array in basic and applied research for the analysis of endometrial transcriptome changes in normal reproductive physiology and in pathological conditions.
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FOOTNOTES |
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Received for publication February 21, 2007. Accepted for publication June 3, 2007.
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