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Herein, we established an efficient in vitro culture system (termed L15SP) for OSCs derived from Paralichthys olivaceus, consisting of L-15 medium supplemented with 15% fetal bovine serum, 2\u00a0\u03bcg\/L basic fibroblast growth factor, 2\u00a0\u03bcg\/L leukemia inhibitory factor, 50\u00a0\u03bcmol\/L \u03b2-mercaptoethanol, 1% fish serum, and 18\u00a0g\/L embryo extract protein, cultured at 23\u00b0C. This system enabled stable long-term passage of P. olivaceus OSCs. Further investigations revealed that interferon-induced transmembrane protein 3 (Ifitm3) was highly expressed on the membrane of P. olivaceus OSCs. Functional assays demonstrated that Ifitm3 significantly enhanced proliferation, migration, and stemness maintenance of long-term cultured OSCs. Ifitm3 positively modulated the expression of key PI3K-AKT signaling components, including pik3cb, pik3r1, and akt2, to regulate downstream OSC functional genes. Upon pathway activation, Ifitm3 facilitated PIP3 enrichment at the OSC plasma membrane, thereby amplifying PI3K-AKT signaling. In conclusion, Ifitm3 governed OSC biological functions by potentiating PI3K-AKT signal transduction. This study establishes a reliable long-term culture system for fish OSCs and identifies a novel functional marker for OSC identification, providing valuable support for fish germplasm preservation and genetic breeding.<\/jats:p>","DOI":"10.1093\/biolre\/ioag172","type":"journal-article","created":{"date-parts":[[2026,8,10]],"date-time":"2026-08-10T11:22:30Z","timestamp":1786360950000},"page":"1166-1183","source":"Crossref","is-referenced-by-count":0,"title":["Ifitm3 positively regulates homeostasis of long-term cultured oogonial stem cells derived from\n                    <i>Paralichthys olivaceus<\/i>"],"prefix":"10.1093","volume":"115","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-1572-8371","authenticated-orcid":false,"given":"Yuqin","family":"Ren","sequence":"first","affiliation":[{"name":"State Key Laboratory of Mariculture Biobreeding and Sustainable Goods, Beidaihe Central Experiment Station, Chinese Academy of Fishery Sciences , Qinhuangdao 066100 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