Spheroid formation and luteinization of granulosa cells of felids in a long-term 3D culture

In the present study, granulosa cells (GCs) from domestic cats and Persian leopard were cultured and characterized from selected days. The culture period was divided into two phases: maintenance, which lasted for 7 days, and luteinization, which followed for up to 11 days. Luteinization was performed on ultra-low attachment plates, supporting the formation of spheroids in a medium supplemented with insulin, forskolin, and luteinizing hormone (LH). GCs of domestic cat produced estradiol (E2) and progesterone (P4) during the maintenance phase. The gene expressions of some proteins involved in steroidogenesis were stable (STAR, HSD3B1) or decreased over time (CYP11A1, HSD17B1, CYP17A1, and CYP19A1), which was similar to the expressions of gonatropin receptors (LHCGR and FSHR). During the luteinization phase, P4 concentration significantly increased (P < 0.05), and E2, in contrast to the proliferation phase, was below detection range. The expression of genes of proteins involved in steroidogenesis (STAR, CYP11A1, HSD3B1, HSD17B1, CYP17A1, and CYP19A1) and of gonadotropin receptors (LHCGR and FSHR) significantly increased during the luteinization period, but some expressions exhibited a decrease at the end of the phase (LHCGR, FSHR, HSD17B1, CYP19A1). The morphology of the luteinized GCs of domestic cat resembled large luteal cells and had numerous vacuole-like structures. Also, the GCs of Persian leopard underwent luteinization, shown by increasing P4 production and HSD3B1 expression. This study confirms that GCs from felids can be luteinized in a 3D spheroid system which can be a basis for further studies on luteal cell function of felids. Additionally, we could show that the domestic cat can serve as a model species for establishing cell culture methods which can be transferred to other felids.