A Novel Dynamic Expression of vasa in Male Germ Cells during Spermatogenesis in the Chinese Soft-Shell Turtle...

vasa gene encodes a highly conserved DEAD-box RNA helicase, required for germ cell development across animal kingdom. Vasa mutations cause male infertility in mammals. It has been widely used as a biomarker for studying animal fertility or manipulating germ cells in organisms. However, in reptilians, the functions of vasa gene involved in germ cell differentiation are largely unclear; this hampers the development of biological techniques and the improvement of the productivity in these species. Here a vasa cDNA was isolated in Chinese soft-shell turtle and it predicts a protein of 691 amino acid residues, which is 72%, 69%, 58%, 59%, and 54-56% identical to its homolog from mouse, platypus, frog, chicken, and fish, respectively, and named as PsVasa. The Psvasa mRNA was detected exclusively in the gonads of both sexes by RT-PCR. Chromogenic RNA in situ hybridization revealed that the Psvasa mRNA was restricted to germ cells in the testis: The psvasa mRNA is undetectable in resting spermatogonia, appears in proliferating spermatogonia, and becomes abundant in spermatocytes and detectable in spermatozoa. Immunofluorescence staining demonstrated that the PsVasa in the testis is also restricted to the germ cells, rich in spermatocytes and elongated spermatids but hardly detectable in spermatogonia and spermatozoa. Taken together, Psvasa is potentially a reliable germ cell marker in the Chinese soft-shell turtle; its RNA expression could distinguish the different spermatogenic stages of germ cells. These findings shed new insights into understanding the evolutionary conservations and divergences of vasa gene's functions in male germ cell differentiation in metazoans.