Granulosa cell differentiation in vitro: induction and maintenance of follicle-stimulating hormone receptors by adenosine 3',5'-monophosphate

Granulosa cell differentiation is stimulated in vitro by FSH and other agents that increase cAMP production. To determine if alterations in FSH receptors are associated with cAMP-mediated granulosa cell maturation, FSH-binding sites were measured during culture of undifferentiated granulosa cells from hypophysectomized diethylstilbestrol-implanted rats. FSH receptors decreased rapidly in the absence of stimulatory ligands, with loss of 75% and 90% of the FSH-binding activity from freshly prepared cells after 24 and 48 h, respectively. The decline in FSH receptors during culture was accompanied by a corresponding decrease in cAMP responses to added FSH. In cells cultured with FSH, the available FSH receptor content fell to 15% after 8 h, then rose to 25% of the initial receptor levels from 24-48 h of culture. Cells treated with 8-bromo-cAMP or choleragen for 48 h retained about 40% and 90%, respectively, of their initial FSH-binding activity. Although choleragen did not prevent the 60-70% fall in FSH binding during the first 6 h of culture, it increased receptors 2.5-fold from 12-48 h. Also, the addition of choleragen after 3, 6, or 12 h of culture elevated FSH receptors at 48 h in proportion to the amount of cAMP produced. The FSH receptors induced by choleragent treatment were functionally active, as shown by their ability to mediate FSH-stimulated cAMP production. A GnRH agonist prevented the choleragen-induced rise in FSH receptors and cAMP accumulation from 24-48 h of culture. Scatchard analysis revealed a single population of FSH receptors after all treatments, with association constant of 5 X 10(9) M-1. Freshly prepared cells contained approximately 1600 FSH binding sites/cell, while treatment with choleragen or FSH for 48 h restored receptor levels to 1150 and 500 sites/cell, respectively. These results indicate that the expression of functional FSH receptors during granulosa cell maturation is mediated by cAMP.