A Novel Culture System for Inhibiting In Vitro Differentiation of Ovine Granulosa Cells

The in vitro granulosa cell (GC) model presents a valuable tool to explore antral follicle development. A full understanding of the reasons and blocking methods that occur during in vitro luteinization of sheep GCs, stimulated by serum culture, is a complex goal that has not been completely achieved. Herein, the phenomenon and causes of GC differentiation, as well as the methods for inhibiting luteinization in an in vitro culture system, were investigated by immunofluorescence, Western blot, RT-qPCR, and ELISA techniques. The results reveal that, when compared to fresh GCs, FSHR protein levels in primary GCs significantly decreased in serum-containing media, while STAR protein levels significantly increased, implying that sheep GCs can differentiate in serum-containing media. LH concentrations were significantly greater in serum-containing media compared to serum-free media. The LH receptor (LHR) mRNA expression in primary-generation GCs steadily increased with longer culture times, indicating that LH-LHR signaling leads to GC luteinization in vitro. In primary and second-generation GCs, 180 nmol/L BAY-899, an LHR-specific antagonist, significantly increased FSHR protein expression, reduced STAR protein synthesis, and inhibited P4 secretion within 48 h of in vitro culture compared to controls. BAY-899 showed no adverse effects on fifth-generation GCs growth, implying that BAY-899 can inhibit GC luteinization while not affecting cell proliferation. In conclusion, this study found that the LHR antagonist BAY-899 can preserve the features of sheep GCs in vitro by suppressing the spontaneous luteinization process caused by LH-LHR signaling, which has a key methodological implication for studying the mechanics of antral follicle formation in vivo.