Identification, Isolation, and In Vitro Culture Trials of Ovarian Germ Stem Cells from Different Teleost Fish Species

Germ cell xenotransplantation is a promising tool for enhancing aquaculture production and supporting the conservation of declining fish species. A major limitation is the reliable identification of germ stem cells, which remains challenging due to the scarcity of validated molecular markers. This study assessed the suitability of different immunohistochemical approaches for identifying ovarian germ stem cells (OGSCs) in three Mediterranean species of commercial interest: European hake (Merluccius merluccius), meagre (Argyrosomus regius), and flathead grey mullet (Mugil cephalus). Among the antibodies tested (anti-OCT4, anti-VASA, anti-Sox2), only anti-VASA and anti-Sox2 labeled cells with morphological features consistent with OGSCs, although staining intensity, specificity, and cross-reactivity with early oocytes varied across species. In parallel, preliminary protocols for OGSC isolation and in vitro proliferation were developed for European hake and meagre. Enzymatic dissociation with collagenase, followed by Ficoll-Paque density separation and culture in Leibovitz's L-15 medium supplemented with 2% fetal bovine serum at 25 °C, resulted in the highest OGSC recovery and detectable increases in proliferating cells. A sequential pre-plating step enhanced enrichment in meagre but caused substantial cell loss in hake. Overall, this study provides a comparative evaluation of female germline stem cell markers in these species, contributing to future surrogate reproduction and germplasm conservation strategies.