Inter-order transplanted rice field eel female germline stem cells differentiate into male meiotic spermatogenic cells in zebrafish

Germ cell transplantation depends on high-quality donor germline stem cells and suitable recipients. Here, we established an inter-order xenogeneic transplantation system using female germline stem cells (fGSCs) from rice field eel ( Monopterus albus ) and germ cell-deficient zebrafish ( Danio rerio ). Donor fGSCs were enriched from juvenile ovaries by Percoll density-gradient centrifugation and characterized by RT-PCR, alkaline phosphatase staining, and Vasa/PCNA immunofluorescence. Sterile recipients were generated by dnd1 morpholino knockdown in Tg(piwil1:eGFP) embryos, followed by transplantation of PKH26-labeled donor cells near the gonadal ridge. The transplanted cells survived, migrated toward, and colonized the recipient gonadal region. At sexual maturity, transplanted fish exhibited remodeled gonadal structures, whereas dnd1 -depleted controls remained largely germ cell-deficient. Rice field eel-specific germline transcripts and Vasa-positive cells were detected in chimeric gonads, confirming donor-cell persistence and integration. Moreover, after in vitro induction, chimeric gonadal cultures expressed the rice field eel meiotic genes dmc1 and rec8 and displayed progressive morphological changes from spermatogonia-like to spermatozoa-like stages. These findings demonstrate that rice field eel fGSCs can persist within a distantly related teleost recipient, retain germline identity, and exhibit male germ cell-like developmental potential, providing a foundation for germplasm conservation and surrogate reproduction in rice field eel.