Plant & Animal Genomes XIII Conference

January 15-19, 2005
Town & Country Convention Center
San Diego, CA

Comparison Of Differential Expression Patterns In Porcine Oocytes And Early Embryos By Using cDNA Microarrays

Kristin M Whitworth¹ , Cansu Agca¹ , Jin-Geol Kim¹ , Gordon K Springer² , Rahul Patel² , Nathan J Biven³ , Lawrence J Forrester³ , Nagappan Mathialagan⁴ , Jonathan A Green¹ , Randall S Prather¹

¹  University of Missouri-Columbia, Department of Animal Sciences,E225 ASRC,Columbia, MO 65211

²  University of Missouri-Columbia, Department of Computer Science,115 EBW,Columbia, MO 65211

³  University of Missouri-Columbia DNA Core Facility, 216 Life Sciences Center,Columbia, MO 65211

⁴  Monsanto Company, 800 North Lindbergh Boulevard St. Louis,MO,63167

Differential expression patterns were evaluated between germinal vesicle oocytes (GV), 4-cell (4C) and blastocyst stage embryos (BL) to determine key transcripts responsible for early embryonic development in the pig. GV were collected from aspirated follicles while 4C and BL were flushed directly from the pig reproductive tract. mRNA was purified from three pools of 50-100 embryos and oocytes, respectively, and amplified by using the Nugen’s Ovation aminoallyl system. Each pool was analyzed twice, resulting in three biological replicates and two technical replicates for each stage. Amplified cDNA was labeled with cy5 and compared to cDNA from a reference sample labeled with cy3. Labeled cDNA was hybridized to our in-house printed pig reproductive tissue-specific 19,968 spot cDNA microarrays. Microarrays were scanned by using a Genepix scanner, and files were loaded into Genespring 6.2.1 for analysis. Lowess normalization was performed on all good spots. Three comparisons were made: GV to 4C, 4C to BL, and GV to BL. ANOVA (p<0.05) was performed with the Benjamini and Hochberg False Discovery Rate multiple correction test on each comparison. A comparison of GV to 4C, 4C to BL and GV to BL resulted in 3214, 1989 and 4528 differentially detected cDNAs, respectively. Real-time PCR analysis on seven transcripts showed an identical pattern of changes in expression as observed on the microarrays (ZP3, ZP4, YWHAG, â-Actin, RPL23, PTMA, DLD) while one transcript deviated at a single cell stage (DMNT1). These results illustrate the complex mechanisms involved in pig early embryonic development.