Cloning and characterization of a novel oocyte-specific gene zorg in zebrafish

Genes specifically expressed in oocytes are important for the development of both germ cells and embryos. Using the digital differential display program from the NCBI, we identified a novel EST sequence (no. DT881820) from zebrafish ovary libraries in the GenBank. The full-length cDNA of this transcript was obtained by performing 3′- and 5′-RACE and was confirmed by PCR and direct sequencing methods. The cDNA clone for this novel gene consisted of 894 base pairs and encoded a protein with 215 amino acids. Since this protein shared no significant homology with any other known proteins, we have named this gene zorg (zebrafish oogenesis related gene). Based on RT-PCR analysis, this gene was specially expressed in the ovary and was abundantly present at the blastula stage. However, the level of expression decreased significantly during the early gastrula periods. Based on whole-mount in situ hybridization studies, zorg transcripts were uniformly distributed in the cleavage stage through to the blastula stage. In addition, during early gastrulation, zorg transcripts appeared in the dorsal region, but became restricted to the primordial germ cells (PGC) in early somitogenesis and remained visible in PGC as they migrated towards the region of the gonad of embryos 24 h post-fertilization. In situ hybridization of sectioned ovaries demonstrated that zorg mRNAs appeared in the cytoplasm of stage I and stage II oocytes and localized in the cortex of stage III and stage IV oocytes during oogenesis. We hypothesized that the protein expressed from the zebrafish zorg gene has roles in the formation of the female germ cells and in early embryonic development in zebrafish.