# Establishment and Characterization of OFT and OFO Cell Lines from Olive Flounder (Paralichthys olivaceus) for Use as Feeder Cells

## TL;DR

Researchers developed testicular and ovarian cell lines from olive flounder to support germline stem cell culture, a key step for improving aquaculture.

## Contribution

Establishment of OFT and OFO cell lines as potential feeder cells for in vitro germline stem cell culture in olive flounder.

## Key findings

- OFT and OFO cell lines expressed gonadal somatic cell markers but not germ cell markers.
- SNP analysis indicated OFT originated from XY males and OFO from XX females.
- Coculture experiments showed GSCs adhered to both OFT and OFO cell lines.

## Abstract

Germline stem cells (GSCs) have applications in aquaculture. As a preliminary step toward the development of an in vitro GSC culture system for olive flounder (Paralichthys olivaceus), testicular and ovarian cell lines (OFT and OFO, respectively) of this species were established and characterized. Coculture of OFT or OFO with enriched P. olivaceus male GSCs showed that these cell lines were capable of maintaining male GSCs as feeder cells. Our research will contribute to the development of an in vitro GSC system for P. olivaceus.

Olive flounder (Paralichthys olivaceus) is a commercially important fish species in Japan, China, and the Republic of Korea. Despite numerous attempts to improve productivity, there have been no studies of in vitro germline stem cell (GSC) culture in this species. Here, olive flounder testicular and ovarian cell lines (OFT and OFO, respectively) were established and characterized. RT-PCR demonstrated that OFT and OFO expressed several gonadal somatic cell markers, including wt1 and fgf2, but lacked expression of germ cell markers, such as vasa, nanos2, and scp3. In addition, SNP analysis revealed that OFT originated from XY male P. olivaceus and OFO originated from XX female P. olivaceus. These results suggest that OFT was composed of Sertoli cells and OFO was composed of granulosa cells and theca cells. Finally, coculture of OFT or OFO with enriched male P. olivaceus GSCs isolated from the top 20% and 20–30% Percoll density gradient layers showed that GSCs were attached on both cell lines. In conclusion, we established P. olivaceus testicular and ovarian cell lines, which were expected to use for development of an in vitro GSC culture system.