Establishment and Characterization of a Novel Ovarian Germline Cell Line with Oogonial Stem Cell-like Properties from the Japanese Eel (Anguilla japonica)

The Japanese eel (Anguilla japonica) is an economically important aquaculture species, and its reproductive development and artificial propagation have received considerable attention. However, the absence of a stable in vitro female germ cell culture system has hindered investigations into oogenesis, reproductive regulation, and germplasm conservation. In this study, cells isolated from immature Japanese eel ovaries were enriched through differential adhesion, Percoll density-gradient centrifugation, and single-cell cloning, followed by optimization of culture conditions. A stable oogonial stem cell-like cell line of A. japonica, designated AjOSCL, was established and characterized via morphological observation, proliferation assays, colony-forming assays, karyotyping, immunofluorescence staining, alkaline phosphatase detection, and transcriptomic sequencing. AjOSCL maintained stable growth and self-renewal capacity during continuous culture for over 35 passages in vitro, with a cumulative culture duration exceeding 12 months, and retained a predominant chromosome number of 2n = 38 (consistent with the chromosome number of A. japonica), suggesting chromosomal stability during long-term culture. AjOSCL exhibited strong alkaline phosphatase activity and expressed germline-related markers (e.g., Vasa and Nanos2), together with pluripotency-related genes or proteins such as Nanog and Oct4. Transcriptomic analysis further revealed that AjOSCL displayed a molecular profile compatible with a germline stem cell-like phenotype, with enrichment of pathways related to germ cell maintenance and oocyte development, including ECM–receptor interaction, TGF-β signaling, Wnt signaling, and oocyte meiosis. Collectively, these results indicate that AjOSCL may serve as a renewable in vitro model for studying oogonial maintenance, offering a foundation for future investigations into female germ cell biology and germplasm conservation in A. japonica.