Gamete Biology

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

Abstract
The main objective of this 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 PCR-based cDNA subtraction and cDNA microarrays. Three subtractive libraries consisting of 3,456 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 of these clones were used for cDNA microarray preparation. Results from these microarray analysis 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 RT-PCR to confirm their preferential expression in the oocyte when compared to somatic tissues. The results obtained in this study confirmed that microarray analysis is a robust technique to identify true positives from the SSH experiment. Furthermore, by obtaining oocyte-specific genes from three species simultaneously, it 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.

Key words: Gamete Biology • Gene regulation • Oocyte development

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