Establishment and application in ovary research of porcine three dimensional ovarian organoids.

The porcine ovary is an invaluable model for human ovarian research and critical for agricultural reproductive efficiency. Traditional two-dimensional (2D) models fail to recapitulate the native ovary's 3D structure and functions, limiting their utility. This study aimed to establish a stable 3D porcine ovarian organoid model, evaluate its utility, and explore melatonin's regulatory effects. Porcine ovarian tissues from prepubertal gilts were digested, cultured in Matrigel® with optimized medium. Organoids were characterized via microscopy, qRT-PCR, and H&E/IF staining. Lentivirus-GFP was used to verify infectivity; while melatonin (1-20 μM) effects were tested via Cell Titer-Glo and qRT-PCR analysis. Typical round organoids formed on day 7, maintaining stability up to passage 10, expressing ovarian biomarkers (EPACM, CDH1, FSH, DDX4, etc.), and mimicking in vivo histology. Lentivirus infected organoids dose-dependently. Melatonin enhanced viability/size and upregulated FOXL2, FIGLA, and AMH genes in a dose-dependent manner. Thus, a stable and functional 3D porcine ovarian organoid model was established for gene perturbation. Melatonin benefits organoid growth via key gene upregulation, providing a novel tool for ovarian research and translational studies.