Establishment, Identification And Application Of Gonadal Cell Lines From Southern Catfish(Silurus Meridionalis Chen)

Fish cell lines have important role in the study of aquatic animal virology, environmental toxicology, fish genetic breeding and resources conservation, and fish endocrinology. Since the first permanent cell line of fish origin, RTG-2, is developed from the gonad of rainbow trout (Oncorhynchus mykiss), more than280different cell lines from various tissues of freshwater and marine fish are established. However, the number of fish cell lines originating from gonadal tissues is limited. Fish gonadal somatic cells, such as granulosa cells, Sertoli cells, etc., play an essential role in the process of sex determination and differentiation. Fourhtermore, culture of gonadal somatic cells in vitro is in great need owing to the popular issue of germline stem cells. It is reported that dissociated germ cells of Zebrafish (Danio rerio) could be co-cultured on a feeder layer of autologous gonadal cells for several months. Southern catfish (Silurus meridionalis) belongs to Siluridae, Siluriformes, and is widely distributed in the Yangtze River basin. Owing to its fast growth, high fecundity, nutritional values and resistance to diseases, Southern catfish is an important economic fish in China. More interestingly, the sex ratio of the feral Southern catfish is reported to be about1:1, while the fish obtained by artificial fertilization is always female. The mechanisms involved has been carried out for many years and remains elusive. In the present study, establishment of somatic cell lines from Southern catfish gonad might provide a powerful tool for the culture, proliferation and differentiation of germ cells (particularly germline stem cells), gene function study and the effect of environmental endocrine disruptors.In recent years, more and more reports show that the worsening environmental estrogens (EEs) pollution in waters around the world is attributed to the reproductive abnormality, cancer, metabolic disturbance of human and other animals. A real-time efficient monitoring system of EEs is the premise of prevention. EEs are varied and low content. Biological detection has an irreplaceable position and role in EEs detection. A highly sensitive EEs detection system by using pEGFP-ERa with expression of human ERa and pGL3-ERE4-luc with ERE4, respectively, to co-transfect mammal cell line has been established. However, there are significant differences in the celluar environment, the expression patterns of associated moleculars and the sensitivities to EEs between fish and mammal cells. Whether the above EEs monitoring system used in mammal cells could reflect the influence of EEs on fishes, which needs further investigation.In view of this, two aspects of the work was carried out in our study. On the one hand, ovarian and testicular somatic cell lines from Southern catfish have been established and characterized. On the other hand, pEGFP-ccERa which could stably express channel catfish estrogen receptor a (ERa) was successfully constructed, and then pEGFP-ccERa plus pGL3-ERE4-luc were used to co-transfect fish cell line and mammal cell line, respectively. The detail is as follows:1Establishment and characterization of ovarian and testicular cell lines from Southern catfish(1) Ovarian cell line of Southern catfishThe ovarian tissue from about3months female southern catfish was dissociated with collagenase IV and trypsin-EDTA solution. The pellets were cultured in Leibovitz’s L-15medium supplemented with25mM Hepes,15%FBS,500U/mL penicillin and streptomycin,10ng/mL recombinant basic fibroblast growth factor (bFGF),1ng/mL leukemia inhibitory factor (LIF),1%non-essential amino acid,0.5mM β-mercaptoethanol and5%autologous serum at28℃. The characterization of ovarian cell lines were analyzed by karyotype analysis, RT-PCR and gene transfection.Results in detail are as follows:1) A monolayer of primary cultures was obtained from the ovarian cells of Southern catfish after initial inoculation at15day. The ovarian cell cultures were fibroblast-like. Primary cultures were dissociated by treatment with0.25%trypsin-EDTA solution into single cells and transferred into another fresh flask at a split ratio of1:2. During the first8subcultures, the cells grew relatively slower and were subcultured every7-10days. From the passage9, the cells grew faster and were subcultured every3-6days. The cell line is successfully subcultured for more than90passages over18months and is designated as SCO1.2) SCO1cells were cultured at different mediums (DMEM, L-15, DMEM/F12), different temperature conditions (18℃,28℃,37℃), and different FBS concentrations (5%,15%,25%). The results showed that the optimum growth condition was cultured at28℃in L-15medium with15%FBS.3) By RT-PCR detecting, the SCO1cells expressed Foxl2, Sfl and Wtlb, but not Vasa, Wtla, Cypl9a and Dmrtl. Taken together, the gene expression patterns of SCO1indicate that it might originate from ovarian granulosa cells.4) Karyotype analysis revealed that62%of the SCO1cells possessed a diploid chromosome number of2n=58, and more than80%cells had the chromosome numbers ranged from50-65at passage20,30and70, which suggests that SCO1retain a normal karyotype.5) SCO1cells were transfected with pIRES-hrGFP-la by TransIT-2020, the expression of GFP could be detected as early as24h. Furthermore, the transfection efficiency was approximately30%at48h.(2) Testicular cell line of Southern catfishA testicular somatic cell line of Southern catfish was established as the above described. The cells were fibroblast-like and successfully subcultured for56passages over15months (hereafter designated as SCT1). Karyotype analysis revealed that52%of the SCT1cells possessed a diploid chromosome number of2n=58, and more than70%cells had the chromosome numbers ranged from50-65at passage19and43. SCT1expressed Foxl2and Sfl, but not Vasa, Wtla, Wtlb, Cypl9a and Dmrtl. After SCT1cells were transfected with pIRES-hrGFP-la by TransIT-2020, the expression of GFP could be detected as early as24h and the transfection efficiency was approximately25%at48h.2Detection system of Environment Estrogens(1) The full length cDNA of Estrogen receptor a was obtained from Channel catfish (Ictalurus punctatus) by RT-PCR (designated as ccERa), and then the expression vector pEGFP-ccERa was successfully constructed.(2) The pEGFP-ccERa plus pGL3-ERE4-Luc plasmid were co-transfected to HEK293and SCO1cells by TransIT-2020, respectively. Compared with the pEGFP-hERa, our results showed that:1) when pEGFP-hERa was used, the detection limit of estrogen (E2) was10-10mmol/L in HEK293and10-9mmol/L in SCO1;2) howevre, when pEGFP-ccERa was used, the detection limit of E2was10-9mmol/L in HEK293cells and10-10mmol/L in SCO1. Thus, pEGFP-ccERa has a higher sensitivity to the environmental estrogens in fish cells (SCO1) compared with pEGFP-hERa.(3) pEGFP-ccERa plus pGL3-ERE4-Luc was used to detect the relative luciferase activity of MT, EE2, E2, DES, BPA, OP and NP in SCO1for48h, which have different estrogen activity under the same concentration (10-5mmol/L). The results indicate that the relative luciferase activity is EE2> E2> DES> BPA> OP> NP> MT, which is in agree with the relative estrogen activity of these EEs. Taken together, our detection system can exactly detect the estrogen activity of EEs.In summary, ovarian and testicular somatic cell lines from Southern catfish are successfully established for the first time (designated as SCO1and SCT1, respectively). What’s more, a high sensitive environment estrogen detection system has been established in fish cell line. This research not only provides necessary platforms and tools for germ cells culture, gene function study and so on, but also plays an important role for the detection of EEs.