A Modified Co-Culture System for Understanding Granulosa-Theca Cell Interactions in the Bovine Ovary.

A reliable co-culture of granulosa (GCs) and theca cells (TCs) has the potential to facilitate a more comprehensive understanding of the interactions, signaling pathways, and substrate exchange between both cell compartments in the bovine ovarian follicle. Utilizing commercially available cell culture inserts, a reproducible co-culture model of bovine GCs and TCs was established. It was observed that primary theca and granulosa cells require considerably more time to attach to the insert membrane compared to cell lines. Initially, the TCs were seeded onto the inverted insert membrane, with a truncated tube serving as an inoculation container to prevent medium leakage. Following a 72 h incubation period, the insert was inverted, allowing the GCs to be cultured on the opposite side of the membrane. This co-culture was then subjected to an additional 6 days of incubation at 37 °C and 5% CO2, with media exchange every other day. The expression of marker genes indicated cell-type-specific patterns, with CYP17A1 being highly abundant in TCs and not expressed in GCs. Conversely, CYP19A1 showed high levels in GCs and only low levels in TCs. The hormone analysis supported the presence of a physiological co-culture system, as evidenced by the synthesis of estradiol. This modified co-culture model supports reproducible studies of paracrine signaling and substrate transport between the somatic cells of the follicle.