The molecular regulation mechanism of dmrt1-based on the establishment of the testis cell line derived from two-spot puffer Takifugu bimaculatus.

The establishment of fish cell lines can provide an important in vitro model for developmental biology, pathology, and genetics and also an effective tool to investigate the interactions and related functions of genes. Two-spot puffer Takifugu bimaculatus is a high economic and nutritional value marine fish in Fujian in recent years. Nevertheless, dmrt1 plays a key role in the male differentiation from invertebrates to vertebrates. To understand the molecular regulatory mechanisms of dmrt1 in T. bimaculatus, a testis cell line called TBTc from a juvenile testis of this organism was established with modified Leibovitz's L-15 medium supplemented with 20% FBS, fish serum, embryo extract, and other growth factors. The TBTc with a stable karyotype can be passaged continuously, which was composed of fibroblast-like cells and expressed the marker genes of male-special cells, dmrt1, and amh, and the absence of vasa expression may rule out the possibility of the presence of germ cells. Therefore, TBTc appeared to consist of the mixture of the Sertoli cell and germ cell of the testis. The dmrt1 was significantly expressed in the testes and slightly expressed in the late embryonic development, illustrating that the dmrt1 may participate in the molecular regulation of gonads development and sex differentiation. With the high transfection efficiency of TBTc by electroporation, the cell lines could be used effectively in the study for the expression of exogenous and endogenous genes. Meanwhile, after the knockdown of dmrt1, the morphological changes and survival rates of cells proved that dmrt1 could affect the growth of testicular cells. Furthermore, with the loss of dmrt1, the expression of male-bias genes amh, sox9, and cyp11a was significantly decreased, and the expression of female-bias genes foxl2, sox3, and cyp19a was increased, which suggested that dmrt1 upregulates amh, sox9, and cyp11a and downregulates foxl2, sox3, and cyp19a to participate in the testis development. As a first fish gonadal cell lines of T. bimaculatus, which can be a more convenient, efficient, and rapid model for the investigation of the expression and function of genes, the results will lay a foundation for the next study of the molecular regulation mechanism in gonadal development and sex determination of fish in the future.