Establishment of a germline cell-containing ovarian cell line capable of oocyte generation in vitro from sterlet Acipenser ruthenus

Sturgeons (order Acipenseriformes) represent one of the most ancient lineages of Actinopterygii. Presently, most sturgeon species are classified as critically endangered. In this study, a novel ovarian cell line which contained germline cells derived from the sterlet (Acipenser ruthenus) was established, designated as the ARO cell line. Germline cells in the cell line were identified using marker gene analysis, alkaline phosphatase activity assay and immunofluorescence staining. Transmission electron microscope observation showed that previtellogenic oocytes existed in the ARO cell line. Flow cytometry analysis indicated the cell line maintained meiotic competence to generate DNA reduced ploidy (diploid) oocytes. Under 3D culture condition, germinal vesicle (GV) stage, germinal vesicle breakdown (GVBD) stage and meiosis I oocytes were found. Furthermore, the cell line exhibited sustained proliferation and self-organization into reconstituted ovaries (rOvaries). Transcriptome analysis of rOvaries revealed that rOvaries had formed cell junctions and normal intercellular signaling, and possessed normal endocrine function and oocyte-producing capacity. In summary, the established ARO cell line exhibited stable passaging over multiple generations in vitro, was capable of sustainably generating oocytes, and was expected to be applicable to the establishment of ovarian organoids.