Gsdf is a key gene for testicular differentiation in teleost. However, little is known about the function of Gsdf in Chinese tongue sole (Cynoglossus semilaevis). In this study, we obtained the full-length Gsdf gene (CS-Gsdf), and functional characterization revealed its potential participation during germ cell differentiation in testes. CS-Gsdf transcription was predominantly detected in gonads, while the levels in testes were significantly higher than those in ovaries. During the different developmental stages in male gonads, the mRNA level was significantly upregulated at 86 dph, and a peak appeared at 120 dph; then, the level decreased at 1 and 2 yph. In situ hybridization revealed that CS-Gsdf mRNA was mainly localized in the Sertoli cells, spermatogonia, and spermatids in mature testes. After CS-Gsdf knockdown in the male testes cell line by RNA interference, a series of sex-related genes was influenced, including several sex differentiation genes, CS-Wnt4a, CS-Cyp19a1a and CS-Star. Based on these data, we speculated that CS-Gsdf may play a positive role in germ differentiation and proliferation via influencing genes related to sex differentiation.
In order to provide an applicable cell platform to study fish pathology and skin pigmentation, two cell lines derived from skin tissues of wild-type and albino Japanese flounder were established and named JFSK_wt and JFSK_alb, respectively. These two cell lines were cultured for 45 passages within approximately 300 days. JFSK_wt and JFSK_alb cells were maintained in Dulbecco's Modified Eagle's Medium and Ham's F-12 Nutrient Mixture (DMEM/F12) supplemented with antibiotics, fetal bovine serum (FBS), 2-mercaptoethanol (2-Me), N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), and basic fibroblast growth factor (bFGF). The optimal growth temperature for JFSK_wt and JFSK_alb cells was 24 °C, and microscopically, the two cell lines were composed of fibroblast-like cells. Chromosomal analysis revealed that JFSK_wt and JFSK_alb cells had an identical diploid karyotype with 2n = 48t. Results of viral inoculation assays revealed that both cell lines shared similar patterns of viral susceptibility to nervous necrosis virus (NNV). High transfection efficiency was observed in JFSK_wt and JFSK_alb cells transfected with a pEGFP-N3 reporter plasmid and Cy3-siRNA. The detection of dermal marker Dermo-1 showed that these two cells were both derived from the dermis. Finally, three genes involved in the melanogenesis pathway, including adenylate cyclase type 5 (adcy5), microphthalmia-associated transcription factor (mitf), and endothelin B receptor (ednrb), were downregulated in JFSK_alb versus JFSK_wt cells. Thus, the two cell lines, sampled from skin tissue of wild-type and albino Japanese flounder will be not only helpful for fish pathogen research but also beneficial for albinism-related gene function studies.
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