Although testicular organoids have remarkable potential as testicular models in vitro, there have been few studies about testicular organoids in teleost fish. As a first step to establish a stable culture system for fish testicular organoids, we investigated the efficient conditions for an aggregate culture of dispersed testicular cells from adult marine medaka (Oryzias dancena) by evaluating the effects of culture methods and media composition on an aggregate culture. As the results, we found that culturing dispersed testicular cells in an ultra-low attachment 96 well without Matrigel was most effectively able to induce the formation of testicular cell aggregates among the five different methods tested. Subsequently, through media testing, we confirmed that the modified ESM2 was more optimal for this aggregate culture than the media conventionally used in porcine, human, and rat testicular aggregate cultures. Furthermore, we demonstrated that three supplements in the modified ESM2 including fish serum (FS), basic fibroblast growth factor (bFGF), and embryo extracts (EE) did not influence the number and size of the testicular aggregates formed, but fetal bovine serum and other supplements including β-mercaptoethanol, non-essential amino acids, sodium pyruvate, and sodium selenite were affected significantly. Nevertheless, the removal of three supplements (FS, bFGF, and EE) during culture negatively affected scp3 and sox9a expression levels, indicating their necessity. Finally, we identified that the sperms derived from in vitro cultured testicular aggregates were able to produce offspring after fertilization with naturally matured oocytes. The results from this study will provide fundamental information to develop the techniques for fish testicular organoid culture, which will eventually contribute to the development of reproductive biotechnology for aquaculture and the conservation of endangered fish species.

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.

Spermatogonial stem cells (SSCs) are pivotal in surrogate broodstock technology. However, species-specific protocols for the efficient enrichment and long-term preservation of SSCs in olive flounder (Paralichthys olivaceus) are not yet fully established. In this study, we evaluated and optimized methods for the isolation and cryopreservation of P. olivaceus SSCs. First, we compared two enrichment methods, including Percoll density gradient centrifugation (PDGC) and differential plating (DP). Although SSCs enriched by both methods showed increased expression of SSC-specific marker genes, PDGC resulted in significantly greater enrichment than DP. A combination of PDGC and DP did not further improve enrichment efficiency, suggesting that PDGC alone is sufficient in P. olivaceus. Second, we optimized cryopreservation conditions according to various cryoprotectants. Among the conditions, SSCs cryopreservation using 1.3 M propylene glycol (PG) as a permeating agent and 0.2 M raffinose (Raf) as a non-permeating cryoprotectant provided the highest cell viability (56.1%), demonstrating a synergistic protective effect. Finally, preliminary in vivo migration and localization ability of the cryopreserved SSCs was confirmed through xenotransplantation into zebrafish (Danio rerio) larvae. PKH26-labeled donor cells exhibited successful initial localization and short-term persistence within the presumptive gonadal ridge of the recipients at 5 days post-transplantation. These findings provide an optimized protocol for the handling and preservation of P. olivaceus germline resources, contributing to the technical advancement of surrogate reproduction strategies in this species.

In vitro 3D cell culture systems provide a physiologically relevant microenvironment that mimics native tissue architecture. However, establishing functional 3D testicular aggregates in teleosts remains challenging. In this study, we established a novel 3D co-culture aggregate system for olive flounder (Paralichthys olivaceus) primary testicular cells by incorporating the established olive flounder testicular cell (OFT) line. Primary testicular cells alone failed to form 3D aggregates within the first 24 h in ultra-low attachment plates. However, co-culturing with OFT cells rapidly initiated 3D aggregate assembly within 24 h. Furthermore, supplementation with a cocktail of sex hormones and growth factors maintained high cell viability without cytotoxic effects. Notably, quantitative real-time PCR analysis revealed that hormone and growth factor supplementation significantly upregulated the transcript levels of both plzf (2.15-fold) and scp3 (2.34-fold). These results demonstrate that the OFT cell line is essential for facilitating the assembly and incorporation of primary testicular cells into 3D aggregates, while sex hormones and growth factors promote spermatogonial stem cell maintenance and meiotic progression within the aggregates. Overall, this 3D co-culture platform serves as a valuable in vitro tool for fish spermatogonial stem cell research and reproductive biotechnology in aquaculture.