Characterization of progesterone biosynthesis in a transformed granulosa cell line

The study of regulation of steroidogenesis in primary cultures of rat granulosa cells is difficult because the cells do not undergo more than one cell doubling in culture. Furthermore, there is size and steroidogenic heterogeneity in granulosa cells and it is difficult to obtain pure, functionally defined populations. Hence, it is advantageous to develop a homogeneous population of granulosa cells. In this report we describe the characterization of one such cell line (Rao-gcl-29) developed from diethylstilbestrol treated immature rat granulosa cells by transformation with SV40 T antigen. In this cell line cyclic AMP analogs induce high levels of progesterone biosynthesis, though there was no effect on estradiol biosynthesis. Also, FSH and hCG have no effect on progesterone biosynthesis. In the presence of FBS medium (20% fetal bovine serum in DMEM/F-12) and enriched medium (10% fetal bovine serum, 10% horse serum and 2% UltraSer G in DMEM/F-12 medium), 1 mM cAMP analogs induce high levels of progesterone biosynthesis up to 96 h. Ultrastructural features of the cell line resemble those of primary granulosa cells, in addition to forming gap junctions. Cyclic AMP analogs also induced cytochrome P450scc mRNA in this cell line by 48 h, and this effect is apparent by 24 h. Thus, this cell line could be useful in understanding the molecular mechanisms of regulation of cytochrome P450scc gene regulation.