Establishment and characterization of a gonad cell line from half-smooth tongue sole Cynoglossus semilaevis pseudomale.

A new cell line was established from half-smooth tongue sole Cynoglossus semilaevis pseudomale gonad (CSPMG). Primary culture was initiated from gonad tissues pieces, and the CSPMG cells were cultured at 24 °C in Dulbecco's modified Eagle medium/F12 medium (1:1) (pH7.0), supplemented with 20 % fetal bovine serum, basic fibroblast growth factor, epidermal growth factor, insulin-like growth factor-I, 2-mercaptoethanol, penicillin and streptomycin. The cultured CSPMG cells, in fibroblast shape, proliferated to 100 % confluency 10 days later and had been subcultured to passage 109. Chromosome analyses indicated that the CSPMG cells exhibited chromosomal aneuploidy with a modal chromosome number of 42, which displayed the normal diploid karyotype of half-smooth tongue sole (2n = 42t, NF = 42). Reverse transcription polymerase chain reaction revealed CSPMG cells could express gonad somatic cell functional genes Sox9a, Wt1a and weakly germ cell marker gene Vasa, but not male specific gene Dmrt1. Transfection experiment demonstrated that CSPMG cells transfected with pEGFP-N3 plasmid and small RNA could express green and red fluorescence signals with high transfection efficiency. In conclusion, a continuous CSPMG cell line has been established successfully. The cell line might serve as a valuable tool for studies on the mechanism of sex determination, sex reversal and gonad development in flatfish.

Polymorphism and Balancing Selection of MHC Class II DAB Gene in 7 Selective Flounder (Paralichthys olivaceus) Families.

In order to determine the genetic variation of the MHC class IIB exon2 allele in the offspring, 700 fry from seven families of Japanese flounder challenged with V. anguillarum were studied, and different mortality rates were found in those families. Five to ten surviving and dead fry from each of the seven families were selected to study the MHC class II B exon2 gene with PCR and a direct sequencing method. One hundred and sixteen different exon2 sequences were found and 116 different alleles were identified, while a minimum of four loci were revealed in the MHC class II B exon2 gene. The ratio (d(N)/d(S)) of nonsynonymous substitution (d(N)) to synonymous substitutions (d(S)) in the peptide-binding region (PBR) of the MHC class IIB gene was 6.234, which indicated that balancing selection is acting on the MHC class IIB genes. The MHC IIB alleles were thus being passed on to their progeny. Some alleles were significantly more frequent in surviving than dead individuals. All together our data suggested that the alleles Paol-DAB*4301, Paol-DAB*4601, Paol-DAB*4302, Paol-DAB*3803, and Paol-DAB*4101 were associated with resistance to V. anguillarum in flounder.