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Identification of Novel and Known Oocyte-Specific Genes Using Complementary DNA Subtraction and Microarray Analysis in Three Different Species¹

Centre de Recherche en Biologie de la Reproduction,³ Département des Sciences Animales, Université Laval, Québec, Canada G1K 7P4 Dairy and Swine Research and Development Center,⁴ Agriculture and Agri-Food Canada, Lennoxville, Québec, Canada J1M 1Z3 Departments of Biological Sciences & Computing Science,⁵ University of Alberta, Edmonton, Alberta, Canada T6G 2E8

The main objective of the present study was to identify novel oocyte-specific genes in three different species: bovine, mouse, and Xenopus laevis. To achieve this goal, two powerful technologies were combined: a polymerase chain reaction (PCR)-based cDNA subtraction, and cDNA microarrays. Three subtractive libraries consisting of 3456 clones were established and enriched for oocyte-specific transcripts. Sequencing analysis of the positive insert-containing clones resulted in the following classification: 53% of the clones corresponded to known cDNAs, 26% were classified as uncharacterized cDNAs, and a final 9% were classified as novel sequences. All these clones were used for cDNA microarray preparation. Results from these microarray analyses revealed that in addition to already known oocyte-specific genes, such as GDF9, BMP15, and ZP, known genes with unknown function in the oocyte were identified, such as a MLF1-interacting protein (MLF1IP), B-cell translocation gene 4 (BTG4), and phosphotyrosine-binding protein (xPTB). Furthermore, 15 novel oocyte-specific genes were validated by reverse transcription-PCR to confirm their preferential expression in the oocyte compared to somatic tissues. The results obtained in the present study confirmed that microarray analysis is a robust technique to identify true positives from the suppressive subtractive hybridization experiment. Furthermore, obtaining oocyte-specific genes from three species simultaneously allowed us to look at important genes that are conserved across species. Further characterization of these novel oocyte-specific genes will lead to a better understanding of the molecular mechanisms related to the unique functions found in the oocyte.

gamete biology, gene regulation, oocyte development

² Correspondence: Marc-André Sirard, Centre de Recherche en Biologie de la Reproduction, Department of Animal Sciences, Laval University, PQ G1K 7P4, Canada. FAX: 418 656 3766; marc-andre.sirard{at}crbr.ulaval.ca

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