Establishment and characterization of functional sheep endometrial luminal epithelial organoids

Background The luminal epithelium of the sheep endometrium plays a pivotal role in embryo implantation. However, there is currently a lack of in vitro models that accurately mimic its physiological functions. Although endometrial organoid systems have been well established in humans and mice, comparable models for ruminants remain underdeveloped due to their unique reproductive physiology. Therefore, establishing an organoid model that faithfully recapitulates the characteristics of the sheep endometrial luminal epithelium is essential for advancing our understanding of embryo implantation in this species. Results In this study, we systematically optimized the culture system and found that the combined supplementation of WNT3A, CHIR99021, and Y-27632 significantly improved the efficiency of organoid formation from sheep endometrial luminal epithelial cells. Notably, the innovative introduction of an EPHA1 agonist further promoted the development of functional organoids with a stable diameter exceeding 100 µm and mature morphological features. These organoids exhibited typical luminal epithelial characteristics, including microvilli, tight junctions, and secretory vesicles, expressed key epithelial markers (KRT18, TROP2, and EPCAM), and achieved an apical-out polarity in up to 86% of structures—effectively simulating the in vivo implantation microenvironment. Upon hormonal stimulation, the organoids displayed responses consistent with those of native tissue. Transcriptomic analysis confirmed a high degree of similarity between the organoids and primary endometrial luminal epithelium. Functional co-culture experiments further demonstrated that these organoids significantly promoted blastocyst proliferation and achieved stable adhesion with trophoblast cells. Conclusion This study reports the first successful establishment of a stable and functional sheep endometrial luminal epithelial organoid model that closely mimics the structural and physiological features of the native tissue. This model provides a powerful in vitro platform for investigating the mechanisms of embryo implantation in ruminants and lays a solid foundation for the development of diagnostic and therapeutic strategies for reproductive disorders in livestock.