Intracellular regulation of estradiol and progesterone production by cultured bovine granulosa cells

The objective of the present study was to investigate the implication of pr otein kinase A (PKA), protein kinase C (PKC), and receptor protein tyrosine kinase (R-PTK) pathways in the regulation of estradiol (E2) and progestero ne (P4) production by bovine granulosa cells. Cells were harvested from bov ine follicles (8-15 mm diameter) and cultured without serum for an initial 3 days (37 degrees C; 5% CO2 in air; D1-D3). On the fourth day of culture ( D4), E2 and P4 production were stimulated with FSH (1-6 ng/ml) or forskolin (FSK) in the presence or absence of intracellular effecters of PKA, PKC, a nd R-PTK. Culture medium was collected and replaced each day. Stimulation o f granulosa cell adenylate cyclase activity with FSK (0.06-3.75 mu M) mimic ked FSH, inducing a quadratic increase (P < 0.001) of E2 production and a c ontinuous elevation of P4 (P < 0.01). Inhibition of R-PTK activity with gen istein (25-50 mu M) increased the sensitivity of cells to FSH as demonstrat ed by a leftward shift in the dose response curve (P < 0.001). Treatment wi th transforming growth factor-alpha (TGF alpha; 0.1 ng/ml) abolished the FS H-induced E2 production (P < 0.001) and this effect was not reversed (P < 0 .001) by FSK or by genistein. Furthermore, the inhibitory effect of TGF alp ha on FSH-induced E2 production was reproduced by phorbol 12-myristate 13-a cetate (PMA; 1.25-2.5 mu M), a PKC activator (P < 0.001). Interestingly, ge nistein inhibited P4 production (P < 0.05). From these results, we conclude that E2 production by bovine granulosa cells is mediated by intracellular factors and can be stimulated downstream from the FSH receptor. The results also suggest that stimulation of R-PTK and/or PKC activities, as probably occurs with TGF alpha, negatively affects the PKA pathway, thus decreasing E2 production. Furthermore, inhibition of R-PTK leads to an increase produc tion of E2 and may limit luteinization of bovine granulosa cells. (C) 1999 Wiley-Liss, Inc.