PLATFORM SESSION 11. GAMETE BIOLOGY AND GAMETOGENESIS II
Monday, August 2, 2004
2:00 PM–4:00 PM
Chair: John McCarrey
Co-Chair: Barbara Sotolongo
(299) VALIDATION AND APPLICATION OF LINEAR AMPLIFICATION PROCEDURES FOR STUDY OF OOCYTE GENOMICS: GENE EXPRESSION PROFILING OF BOVINE OOCYTE MATURATION.
Patel , Osman1, Bettegowda , Anilkumar 1, Yao, Jianbo5, Suchyta, Steve3, Ireland , James 2, 3, Coussens , Paul3, Smith , George1, 3, 4, 1 Laboratory of Mammalian Reproductive Biology and Genomics, East Lansing, MI5 Division of Animal and Veterinary Sciences, Morgantown, WV3 Center for Animal Functional Genomics, East Lansing, MI2 Molecular Reproductive Endocrinology Laboratory, East Lansing, MI4 Department of Physiology, East Lansing, MI
ABSTRACT- Discovery of genes important for oocyte competence and early embryo development is significantly hindered because RNA must be isolated from ten of thousands of oocytes to perform cDNA microarray analysis. Therefore, our objectives were to evaluate the fidelity of T7-based linear amplification technology for use in cDNA microarray experiments with minute amounts of starting material and to apply this technology for use in gene expression profiling of germinal vesicle (GV) and in vitro matured metaphase II (MII) bovine oocytes. In Study 1, aliquots of 2, 20 or 40 ng of RNA from bovine fetal ovaries and adult spleen were amplified, and 25 g of amplified (for each aliquot) and non-amplified RNA from fetal ovary versus spleen were subjected to cDNA microarray analysis. The results demonstrated that the hybridization intensity of each of the 7,000 spots per slide for amplified (aRNA) versus non-amplified RNA was highly correlated (2 ng = 0.84, 20 ng = 0.88, 40 ng = 0.90; P < 0.01). In Study 2, RNA was extracted from four pools of 20 oocytes at two different stages of maturation (GV versus MII). Each pool of RNA was amplified and 15 g aRNA per pool was subjected to microarray analysis using a cDNA array containing ESTs representing > 19,000 unique genes in the bovine genome. A total of 1,130 genes displaying greater abundance in MII oocytes and 1,096 genes displaying greater abundance in GV oocytes (Cye5/Cye3 > 2.0 or < 0.5) were detected (P < 0.05). Of the 2,226 differentially expressed genes, 800 were classified by hierarchal clustering into 1 of 10 gene ontology categories ranging from signal transduction (26%), cytoskeletal/structural proteins (10%) to immune modulators (1%). We conclude that linear amplification technologies provide a viable tool to facilitate gene expression profiling of oocytes at key stages of development. Gene expression profiling of GV and MII oocytes revealed potentially novel information on oocyte transcriptome composition at two stages of development with potential relevance to meiotic maturation and (or) early embryogenesis. (Supported by the Rackham Foundation and the Michigan Agricultural Experiment Station).
KEY WORDS: gene expression, bovine, oocyte, microarray