The role of epidermal growth factor in follicular fluid inhibition of progesterone secretion

Bibliography : pages 34-36.Epidermal Growth Factor (EGF), a 6045 dalton polypeptide, has been identified as a component of both serum and follicular fluid. EGF has potent mitogenic effects in many cell types including porcine granulosa cells. EGF also suppresses progesterone secretion by granulosa cells from large (6-12 mm) antral follicles. Fluid from small (l-2 mm) porcine follicles also has an inhibitory effect on progesterone secretion by granulosa cells from large follicles. This study was undertaken to determine if EGF is the molecule which is primarily responsible for the inhibitory effects of follicular fluid upon progesterone secretion by granulosa cells from large follicles. Granulosa cells were aspirated from large porcine follicles. Follicular fluid was collected from large and small porcine follicles. Follicular fluids and porcine serum were charcoal-extracted and filtered to remove 99% of endogenous steroids. To determine the effects of EGF on basal progesterone secretion, cells were grown in various doses of EGF ranging from 0 to 200 ng/ml in 10% adult porcine serum for six days. No significant changes in progesterone secretion were observed. To determine the effects of EGF on luteinizing hormone (LH) stimulated progesterone secretion, cells were grown in 10% serum plus 0, 5-0, or 50 ng EGF per ml with or without LH (0.5 μg/ml) present. LH-stimulated progesterone secretion per mean microgram of DNA was decreased 86% by 50 ng/ml EGF as compared to control cultures without EGF present. To determine the antibody titer needed to counteract a known (30 ng/ml) concentration of EGF, cells were grown in 10% serum plus 0.5 μg LH/ml for six days with various anti-EGF serum concentrations. There was a great deal of variation in effective anti-EGF titers, but restoration of LH-stimulated progesterone secretion to control levels was seen in several cases. To determine the effective anti-EGF dose needed against inhibitory follicular fluid, cells were grown in media containing fluid from small follicles (SFFL) for six days. On days 4-6, 1:200, 1:100, and 1:50 anti-EGF dilutions enhanced LH-stimulated progesterone secretion in the presence of the inhibitory follicular fluid. To determine the importance of EGF in inhibitory follicular fluids, cells were grown in 50% serum or fluid from small or large follicles, with or without LH for six days. Antibody dilutions of 0, 1:1000, 1:500, or 1:200 were added to the cultures. The presence of anti-EGF increased LH-stimulated progesterone secretion in the presence of the inhibitory follicular fluid. In one experiment, basal progesterone was also increased above control levels. Moreover, progesterone levels in the serum containing cultures were also increased over controls in several cases with the addition of anti-EGF. These experiments indicate a possible role of EGF in inhibitory follicular fluid suppression of LH-stimulated progesterone secretion in mature cells. While variations in results preclude any conclusive explanations, these results as well as the observations of others implicate EGF as one of the follicular fluid factors which may act to increase the chances of follicular atresia within the ovary.M.S. (Master of Science