ESTABLISHMENT AND CHARACTERIZATION OF A TESTICULAR CELL LINE FROM CHINESE STURGEON (ACIPENSER SINENSIS)

The Chinese sturgeon, Acipenser sinensis, is a large anadromous fish, with an average 14 years for males and 21 years for females. It was listed as critically endangered by the International Union for Conservation of Nature (IUCN) since 2010, and its spawning activity was not observed during the past five spawning seasons. Therefore, there is an urgent need to preserve the genetic resources of Chinese sturgeon. To investigate the germplasm conservation of Chinese sturgeon and its spermatogonia stem cell culture in vitro, a new cell line (designated as AST) derived from the testis of Chinese sturgeon was established and characterized. The AST cell line are fibroblast-like cells, and have been stably subcultured for more than 80 passages. The optimal growth conditions for the AST cell line are DMEM medium, 25℃, and 15% FBS. After cryopreservation in liquid nitrogen, the viability of AST cells was about (81.36±1.13)%, as measured by trypan blue staining. Chromosome analysis of AST cells at the 30th passage showed that the number of chromosomes ranged from 142 to 310, and the modal number was 264. To detect the expression characterization of Sertoli cell-related genes (amh and gsdf), Leydig cell-related genes (cyp17a1), and germ cell-related genes (dazl, dnd and vasa) in AST cells by RT-PCR, all of the genes were found in the P0 and P1 cells, where their amounts were similar with those of in testis; however, in P15, P30, and P60 AST cells, only amh and vasa genes were detected with very low level, suggesting only a few number of Sertoli cells and germ cells in the late passage of AST cells. The signal of enhanced green florescence protein (GFP) were detected in a few of AST cells after transfection with pEGFP-N3 plasmid. The establishment of the AST cell line could provide important experimental material for the conservation genetic resources of Chinese sturgeon, the in vitro study of proliferation and differentiation of spermatogonia stem cell, and function analysis of testicular related genes.