Ifitm3 positively regulates homeostasis of long-term cultured oogonial stem cells derived from Paralichthys olivaceus†.

Fish oogonial stem cells (OSCs) are vital for fish reproduction research and germplasm conservation, yet stable long-term in vitro culture of fish OSCs remains a major technical bottleneck. Herein, we established an efficient in vitro culture system (termed L15SP) for OSCs derived from Paralichthys olivaceus, consisting of L-15 medium supplemented with15% fetal bovine serum (FBS), 2 μg/L basic fibroblast growth factor (bFGF), 2 μg/L leukemia inhibitory factor (LIF), 50 μmol/L β-mercaptoethanol (β-ME), 1% fish serum, and 18 g/L embryo extract protein, cultured at 23 °C. 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.