Development of a 3D in vitro model to study corpus luteum of felids based on luteinized cells from antral follicles

The study aimed to establish a long-term 3D cell culture model using luteinized follicular cells to investigate the functionality and life cycle of the CL in felids. A mixture of cell types from antral follicles was luteinized in vitro and cultured for up to 23 days. The method, initially applied to the domestic cat, was later extended to Persian and Clouded leopards. Antral follicles were isolated and digested with enzymes; then, the cells were subjected to culture. Experimental subsets were treated with/without 1 µg/mL cloprostenol to validate the cell culture model's suitability for functional studies. In domestic cat samples, microscopic evaluation indicated luteinization, which was confirmed by increased progestagen concentrations and IHC staining for HSD3B and CYP11A1. The gene expression of selected steroidogenic factors (HSD3B1, STAR, CYP11A1) and hormone receptors (LHCGR, PTGFR, PRLR) significantly increased, while CYP17A1 expression decreased. Cloprostenol treatment resulted in reduction of steroidogenic activity, proving its suitability for functional studies. Persian and Clouded leopards' cell cultures exhibited similar patterns in progestagen secretion and gene expression, compared to domestic cats. This model, with its defined luteinization, as well as high and stable progestagen production, allows future investigation of factors regulating CL life cycle and function.