CLONING AND EXPRESSION ANALYSIS OF VASA IN CHINESE SOFT-SHELL TURTLE OOCYTES

vasa encodes a DEAD-box RNA helicase and is a highly conserved germ cell marker across animal phyla. vasa is necessary for germ cell development during embryogenesis and gametogenesis. The Chinese soft-shell turtle (Pelidiscus sinensis) was used as a model to study germ cell development and differentiation in reptiles. Here a full vasa cDNA was cloned, 3865 bp in total, containing a 5'-UTR of 90 bp, a 3'-UTR of 1699 bp and an open reading frame (ORF) of 2076 bp, encoding a protein of 691 aa. The deduced amino acid sequence contains 8 conserved motifs of DEAD-box family protein, and possesses 4 RGG repeats and 2 GG repeats. The predicted protein is 72% identical in sequence to its homologs from mouse. The vasa mRNA was detected exclusively in the gonads of both sexes in soft-shell turtle. Chromogenic and fluorescent RNA in situ hybridization revealed that the vasa mRNA was restricted to germ cells:It was highly expressed from primary oocyte to early growing oocyte and uniformly distributed in oocyte cytoplasm. In oocytes at the late stage, the vasa mRNA was perinuclear distributed and decreased in oocytes with the vitellogenesis proceeding. The vasa mRNA signal was undetectable in the mature oocytes. The findings indicated that vasa gene also played an important role in oogenesis in the Chinese soft-shell turtle. Hence turtle vasa is a reliable germ cell marker to identify female germ cells at different stages during oogenesis. These results provide a theoretical basis to study the germ cell development and differentiation during embryogenesis and/or gametogenesis, especially to investigate the initiation and migration of primordial germ cells (PGCs) in Chinese soft-shell turtle and reptiles.