48 Derivation and characterization of sheep endometrial organoids

Endometrial organoids (EOs) are crucial for advancing our understanding of the intricate interactions between maternal and embryonic biology, serving as a valuable tool for investigating reproductive processes, particularly embryo implantation. In sheep, the endometrium comprises two distinct compartments: the aglandular caruncular area and the glandular intercaruncular area. However, there have been no reports on sheep EOs to date. Therefore, this study aimed to establish EOs from the uterus of sheep (Harri breed). After euthanasia, uteri (n = 4) from diestrus sheep were collected, and endometrial tissues including both endometrial compartments were aseptically harvested and chopped into 1-mm fragments with a sterile scalpel. Following collagenase (0.1%) and trypsin-EDTA (0.25%) digestion and purification through a 70-µm cell strainer, endometrial cells were embedded in cooled, reduced growth factor Matrigel. Dome-shaped structures containing endometrial cells were cultured in petri dishes with organoid formation medium under 5% CO₂ at 37°C and visualized with a time-lapse microscopy. The medium included advanced Dulbecco’s Modified Eagle medium supplemented with a cocktail of inhibitors, growth supplements, and hormones (Saadeldin et al. 2024 Reproduction 167, e230429). To enhance long-term cryopreservation, a ROCK inhibitor (RI, Y-27632, 10 μM) was incorporated into the cryopreservation medium. Time-lapse imaging revealed that EOs progressively grew with a clockwise rotational behavior, formed the spherical vesicular shape by Day 4, and by Days 7–8 they were sized 150–300 µm. EOs could be sustained within Matrigel domes for up to 12 days, growing proportionally to ~500 µm in diameter. Immunofluorescence confirmed cytoplasmic expression of vimentin and β-catenin, and nuclear expression of lamin and Ki67, indicating that over 90% of organoid nuclei were proliferative. Fusion of EOs was observed within the domes, resulting in the formation of tubule-like structures when two or more organoids merged. This study optimizes EO derivation from sheep and provides a platform for investigating uterine organogenesis.