Indexed on: 01 Jan '88Published on: 01 Jan '88Published in: Endocrinology
Epidermal growth factor (EGF) stimulates granulosa cell (GC) proliferation of certain species and modulates FSH-induced GC differentiation. The present study was undertaken to characterize the binding properties of the EGF receptor in porcine GCs to determine if the EGF responsiveness of mitotically active porcine GCs was related to their differentiated state and was regulated by reproductive hormones in vitro. Characterization of the EGF receptor-binding properties of porcine GCs revealed that saturation binding was achieved with 10 ng/ml [125I]iodo-EGF after 1 h at 37 C. In all states of differentiation, porcine GCs expressed few (less than 20,000/cell), specific, high affinity EGF receptors with apparent Kd values of 5.5 +/- 0.7 X 10(-10) M (mean +/- SEM; n = 6). Freshly harvested GCs obtained from small follicles were considered slightly differentiated (SDs) and bound, on the average, 2.6-fold more [125I]iodo-EGF than highly differentiated cells (HDs) obtained from large follicles which had further differentiated in vivo. The difference in binding was due to a decrease in receptor number and not to a change in receptor affinity. This relationship observed in freshly harvested cells was maintained in culture for a limited period. At 48 h of culture, the [125I]iodo-EGF-binding capacity of SDs was higher than that of HDs and was inversely related to the state of differentiation, as measured by [125I]iodo-LH/hCG-binding capacity. After 96 h, however, the EGF-binding capacity of HDs increased 3.7-fold from the level of binding at 48 h, while the LH/hCG-binding capacity decreased 10-fold. Conversely, the EGF-binding capacity of SDs decreased 28%, while the LH/hCG-binding capacity remained low. These experiments indicated that the state of GC differentiation was inversely correlated with EGF receptor number and that this relationship was not maintained in culture beyond 48 h. FSH treatment within the first 48 h of culture decreased the EGF-binding capacity of SDs 35% relative to the control value, but estradiol and dihydrotestosterone had no effect. FSH also regulated the mitotic responsiveness to EGF. EGF treatment of cultured SDs stimulated an 84% increase in cell number and a 178% increase in [3H]thymidine incorporation. These effects were suppressed by a high concentration of FSH. Thus, the ability of porcine GCs to bind EGF was changed with differentiation in vivo, while both EGF-binding capacity and mitotic responsiveness were regulated by exposure to FSH in vitro.