Comparative studies between freshly isolated and spontaneously immortalized bovine granulosa cells: Protein secretion, steroid metabolism, and responsiveness to growth factors

Abstract

A bovine granulosa cell line (BGC-1) has been obtained by spontaneous immortalization of primary cultures. BGC-1 cells have retained some characteristics of primary cultures, such as the hormonal regulation of fibronectin biosynthesis. In the present study we have compared BGC-1 cells and primary cultures of bovine granulosa cells in terms of protein secretion, steroid metabolism, and mitogenic responses to growth factors. The pattern of protein secretion in BGC-1 cells was qualitatively similar to that of primary cultures. The main differences were a higher proportion of fibronectin and the relative amounts of several other unidentified proteins. Progesterone levels in BGC-1 cultures were undetectable. When BGC-1 cells and primary cultures were incubated with [3H]-pregnenolone, the former showed a lower conversion rate to progesterone. In contrast, the conversion rate of [3H]-progesterone to 5 alpha-reduced metabolites was markedly increased in BGC-1 cells. We also examined the effects of epidermal growth factor (EGF), insulin like growth factor-I (IGF-I), and transforming growth factor-beta (TGF-beta) on DNA synthesis under serum-free conditions. Both primary cultures and BGC-1 cells exhibited a stimulatory response to EGF and IGF-I on [3H]-thymidine incorporation. Neither BGC-1 cells nor primary cultures showed a significant response to TGF-beta when added alone. However, in the presence of a combination of EGF and IGF-I, TGF-beta displayed an inhibitory effect on primary cultures while it stimulated DNA synthesis in BGC-1 cells even further. The addition of conditioned medium from BGC-1 cells (BGC-1-CM) stimulated DNA synthesis on primary cultures to a greater extent than the addition of conditioned medium from primary cultures. These results suggest that BGC-1 cells may be a useful model to study the regulation of granulosa cell function during the period previous to the preovulatory stage of follicular development. The differential responses of the immortalized cells to growth regulators may offer some clues on the mechanisms that control cell proliferation in normal tissues.