Submitted to: Molecular Reproduction and Development
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/23/2006
Publication Date: 10/10/2006
Citation: Ramachandra, R.K., Lankford, S.E., Weber, G.M., Rexroad III, C.E., Yao, J. 2006. Identification of OORP-T, a novel oocyte-specific gene encoding a protein with a conserved oxysterol binding protein domain in rainbow trout. Molecular Reproduction and Development 74(4) 502-11. Interpretive Summary: One approach to gaining a better understanding of the reproductive cycle of rainbow trout includes the identification and characterization of genes regulating the reproductive processes of oogenesis, ovarian folliculogenesis, fertilization and early embryonic development. Key oocyte-specific genes known to be vital in folliculogenesis include Factor In the Germline alpha (Figla), Growth Differentiation Factor 9 (GDF9) and Bone Morphogenetic Protein 15 (BMP-15). The essential roles of these genes in follicular development have been demonstrated primarily by their targeted deletion in mice and molecular genetic approaches in farm species. Efforts to determine and characterize the factors influencing folliculogenesis and embryonic development in fish have been sparse. In an attempt to characterize the oocyte transcriptome in rainbow trout, we have recently constructed a normalized cDNA library from rainbow trout oocytes and generated ~20,000 ESTs from this library. A significant number of novel EST sequences have been identified, of which some appear to be absent in other somatic libraries. Here, we report the identification and cloning of a novel gene specifically expressed in trout oocytes based on an EST clone derived from the library. The predicted protein contains a conserved oxysterol binding protein (OSBP) domain at the C terminus that is characteristic of OSBP-related proteins (ORPs) known to be involved in lipid metabolism. Based on the protein structure and the expression patterns of this novel gene, we propose an important role of this gene in yolk lipid storage, movement and usage during folliculogenesis and early embryonic development of rainbow trout.
Technical Abstract: Genes specifically expressed in oocytes are important for the development of oocytes and early embryos. By analyzing expressed sequence tags (ESTs) from a rainbow trout oocyte cDNA library, we identified a novel EST sequence that does not show homology to any sequences in the GenBank. Analysis of tissue distribution by RT-PCR revealed that this gene was only expressed in unfertilized oocytes. Sequencing of the EST clone identified a cDNA of 3,163 bp. Northern blot analysis showed the novel gene has a single transcript of 3.4 kb. Additional 5 sequence was obtained by 5 RACE, extending the novel cDNA to 3,333 bp. Analysis of the full-length cDNA identified an open reading frame (ORF) encoding a protein of 564 amino acids. The novel protein contains a conserved oxysterol binding protein (OSBP) domain at the C terminus that is characteristic of OSBP-related proteins (ORPs) implicated in lipid metabolism. Therefore, we named the novel gene as Oocyte-specific Oxysterol binding protein Related-Protein of Trout (OORP-T). In situ hybridization showed that the OORP-T mRNA appears to be confined to the cytoplasm of vitellogenic oocytes. Transcription of OORP-T appears to start during pre-vitellogenesis and increases steadily, reaching its peak in the late vitellogenic stage. OORP-T transcript is abundantly present in unfertilized eggs but the level drops significantly in day 2 embryos and continues to decline in day 7 embryos after which it remains low. We propose that OORP-T may play an important role in the utilization of yolk-derived lipid products during oocyte development and early stages of embryonic development in rainbow trout.