Abstract
In vertebrate germ cell differentiation, gonadal somatic cells and germ cells are closely related. By analyzing this relationship, it has recently been reported in mammals that primordial germ cells (PGCs), induced from pluripotent stem cells and germline stem cells, can differentiate into functional gametes when co-cultured in vitro with fetal gonadal somatic cells. In some fish species, differentiation into functional sperm by reaggregation or co-culture of gonadal somatic cells and germ cells has also been reported; however, the relationship between gonadal somatic cells and germ cells in these species is not well-understood. Here, we report the transcriptional regulation of Müllerian inhibiting substance (MIS) and the establishment of a gonadal somatic cell line using mis-GFP transgenic fish, in medaka (Oryzias latipes)—a fish model which offers many advantages for molecular genetics. MIS is a glycoprotein belonging to the transforming growth factor β superfamily. In medaka, mis mRNA is expressed in gonadal somatic cells of both sexes before sex differentiation, and MIS regulates the proliferation of germ cells during this period. Using luciferase assays, we found that steroidogenic factor 1 (SF1) and liver receptor homolog 1 (LRH1) activate medaka mis gene transcription, probably by binding to the mis promoter. We also report that mis-GFP transgenic medaka emit GFP fluorescence specific to gonadal somatic cells in the gonads. By fusing Sertoli cells from transgenic medaka with a cell line derived from medaka hepatoma cancer, we produced a hybridoma cell line that expresses gonadal somatic cell-specific markers, including Sertoli and Leydig cell markers. Moreover, embryonic PGCs co-cultured with the established hybridoma, as feeder cells, proliferated and formed significant colonies after 1 week. PGCs cultured for 3 weeks expressed a germ cell marker dnd, as well as the meiotic markers sycp1 and sycp3. Thus, we here provide the first evidence in teleosts that we have successfully established a gonadal somatic cell-derived hybridoma that can induce both the proliferation and meiosis of germ cells.
Highlights
Germ cells are the only cell lineage that contributes to the generation
We found that steroidogenic factor 1 (SF1) and liver receptor homolog 1 (LRH1) significantly induced luciferase activity via the 3.1-kb mis promoter fragment (Figure 1B), but not via the mis promoter fragments (0.8 and 0.2-kb) that lack the three Ad4 sites (Figure 1C)
To investigate whether the Ad4 sites of the medaka mis promoter are indispensable for mis gene transcription in vivo, we established two of each mis-GFP transgenic medaka, using constructs containing either 3.1 or 0.8-kb fragments of the mis promoter fused to GFP
Summary
Germ cells are the only cell lineage that contributes to the generation. Germ cell differentiation is unique and precise, encompassing a dramatic differentiation from primordial germ cells (PGCs) to gametes. Studies on spermatogenesis using organ culture and in vitro culture have been reported in various species of fish, such as medaka (Oryzias latipes) [6], Japanese eel (Anguilla japonica) [7], zebrafish (Danio rerio) [8, 9], tilapia (Oreochromis niloticus) [10], and rainbow trout (Oncorhynchus mykiss) [11] These studies have shown that the mechanisms of differentiation and development of gonads, including germ cells in fish, can be directly evaluated by in vitro cultivation methods. Further evaluation of these relationships awaits the establishment of gonadal somatic cell lines and analysis of expression factors. Cell fusion could be used to immortalize gonadal somatic cells; to date no gonadal somatic hybridomas have been reported, due to a lack of selective media for screening and cloning
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