Inhibition of hormone-induced steroidogenesis during cell proliferation in serum-free cultures of rat granulosa cells.

Long term cultures of rat granulosa cells were grown in serum-free medium, consisting of Dulbecco's modified Eagle's medium mixed 1:1 with Ham's nutrient F-12 medium and supplemented with insulin, transferrin, hydrocortisone, and fibronectin (4F medium). In sparse cultures (10(4) cells/cm2), the granulosa cells were steroidogenically responsive to ovine FSH (NIADDK-oFSH-15) during days 1-2 and 10-14 (responsive periods). The major steroids produced were 20 alpha-hydroxyprogesterone (20 alpha-OH-P) and 5 alpha-pregnane, 3 alpha,20 alpha-diol (pregnanediol). However, as of day 3, the cells gradually lost their steroidogenic responsiveness which was inhibited by 88% at day 7 (refractory period). Nevertheless, from day 8 onward, the cells regained their responsiveness which was fully restored at day 12. The transient loss of responsiveness was uniquely associated with progestin biosynthesis, since FSH-induced aromatase activity declined to background levels within 12 days and was never restored again. The loss of progestin responsiveness was not due to lack of cAMP because FSH induced increasing levels of cAMP accumulation, reaching maximal values on day 7 in culture. On the other hand, the onset of the refractory period occurred concomitantly with the entry of the cultured cells into a synchronous proliferation phase, during which the cell population doubled. Thereafter, as DNA synthesis ceased, the cells regained their steroidogenic responsiveness. A deliberate arrest of cell replication, in the presence of excess thymidine or in high density cultures, prevented the temporal loss of activity. The data presented favor the notion that cell proliferation and expression of differentiated functions are inversely related. It is suggested that growth-related processes suppress steroidogenesis by an as yet unknown mechanism.

Low molecular weight substance from rat ovary induces steroidogenesis in cultured granulosa cells.

Ovaries from immature intact rats contain an apparently low molecular weight substance which mimics the action of follitropin (FSH) on ovarian granulosa cells in culture. Similar to FSH action, the ovarian substance (OS) induced cell-shape changes followed by intensive progestin production. Like FSH action, OS-induced steroidogenesis reversibly ceased upon washing the factor from the cultured cells, and could be blocked in the presence of cycloheximide or alpha-amanitin. Although OS stimulated aromatase activity in granulosa cells, it failed to elicit LH responsiveness in the cultured cells. Androstenedione synergistically augmented OS-induced progestin production and aromatase activity. OS itself synergistically augmented FSH-induced progestin but did not have any effect on FSH-induced aromatase activity. In contrast to FSH action which is mediated via cAMP formation, OS doses which evoked extensive synthesis of progestin products failed to stimulate significant increases in intracellular cAMP accumulation. These results suggest the existence of a putative intraovarian hormone-like substance which can mimic some effects of the gonadotropins on the follicular granulosa cell differentiation and may facilitate FSH action at yet unknown stages of the follicular development.