254 IDENTIFICATION OF OOCYTE SPECIFIC GENES USING SSH AND MICROARRAY ANALYSIS

Abstract

The main objective of this project was to isolate and identify important genes specifically expressed in oocytes. These genes are characterized and may uncover the molecular mechanisms related to the unique functions found in the oocyte. Total RNA (1 μg) from denuded germinal vesicle-stage (GV) oocytes and somatic tissues were used for cDNA production. The mRNAs were reverse-transcribed and the cDNAs were amplified using the Smart cDNA amplification kit (Clontech, BD Biosciences, San Jose, CA, USA). SSH was performed with the PCR-Select cDNA Subtraction Kit (Clontech). Briefly, pools consisted of bovine oocytes for the tester and bovine somatic tissues for the driver. The same procedure was repeated with mouse and xenopus tissues. All of the 3500 subtracted PCR products generated by SSH were cloned, PCR amplified and sequenced. The resulting sequences were compared against the GenBank database using online computer BLASTn program. For microarray analysis, purified PCR products were spotted on GAPS II glass slides (Corning, Corning, NY, USA) using a VersArray Chip WriterPro robot (BioRad, Hercules, CA, USA). Forward- and reverse-subtracted PCR products were used as probes labeled with Cy-3 and Cy-5 dyes (Amersham, Piscataway, NJ, USA) using the Amino Allyle cDNA Labeling Kit (Ambion, Austin, TX, USA). Slides were scanned and analyzed using the ChipReader and ArrayPro Analyser software (Media Cybernetics, Carlsbad, CA, USA). Detection of the oocyte-specific zona pellucida (ZP), growth differentiation factor-9 (GDF-9), bone morphogenetic protein 15 (BMP15), H1 histone family, member O, oocyte-specific (H1oo), and cyclin B1 transcript in the oocyte subtracted library increased confidence in the validity of the subtraction procedure. All of these transcripts account for 12 % of the clones for the mouse subtracted oocyte library. Microarray analysis performed with the mouse arrays revealed that 33% (382) of the clones were differentially expressed (ratio > 10) in the oocyte for the mouse library. Of the 139 clones (12%) that seemed to be present only in the oocyte, 65 were found to be expressed in both the bovine and the xenopus subtracted oocyte library. The most interesting clones to date are #1518, a gene associated with pluripotency;; #1776, a cDNA associated with stem cells in mouse, and #1906, a protein that interacts with chromatin. Since they are conserved across species, the chances that their function is important are quite high. A validation step with RT-PCR analysis will need to be performed on genes identified as oocyte-specific, and the functionality of these genes will be determined with the RNAi technique. WEB page http://www.begc.crbr.ulaval.ca/ (Supported by NSERC).