F R Tekpetey1, S A Daniel, A Yuzpe. 1. Department of Obstetrics & Gynaecology, University of Western Ontario, London, Canada.
Abstract
PURPOSE: The purpose of this study was to investigate the expression of EGF receptor (EGF-R) in human granulosa cells undergoing luteinization and progesterone production by these cells in response to EGF an TGF-alpha alone or in combination with luteinizing hormone (LH). Granulosa cells were obtained from IF patients following oocyte retrieval 34 to 36 hr post-hCG injection. EGF receptor was localized in cells by means of immunoperoxidase staining using a polyclonal primary antibody directed against the human EGF-R. To assess progesterone production, cells were seeded overnight, washed, and cultured with the growth factors +/- LH. Medium and treatments were changed every 24 hr for 3 days. RESULTS: Specific EGF-R staining was observed in the cultured cells compared to those incubated with antibody that was preabsorbed with a 10-fold excess of EGF. Basal progesterone accumulation per 24-hr period was stimulated dose dependently on each day of culture, by both EGF (up to 3.5-fold at 5 or 50 ng/ml) and TGF-alpha (up to 4-fold at 50 ng/ml). The addition of LH alone also stimulated progesterone accumulation daily, and this effect was further enhanced dose dependently by cotreatment with EGF or TGF-alpha. Furthermore, tyrphostin, an EGF-R-specific tyrosine kinase inhibitor, inhibited both basal and growth factor-stimulated progesterone production. CONCLUSION: These data suggest an EGF receptor-mediated physiological role for EGF and TGF-alpha in human luteal function involving an autocrine and/or a paracrine stimulation of progesterone production.
PURPOSE: The purpose of this study was to investigate the expression of EGF receptor (EGF-R) in human granulosa cells undergoing luteinization and progesterone production by these cells in response to EGF an TGF-alpha alone or in combination with luteinizing hormone (LH). Granulosa cells were obtained from IF patients following oocyte retrieval 34 to 36 hr post-hCG injection. EGF receptor was localized in cells by means of immunoperoxidase staining using a polyclonal primary antibody directed against the humanEGF-R. To assess progesterone production, cells were seeded overnight, washed, and cultured with the growth factors +/- LH. Medium and treatments were changed every 24 hr for 3 days. RESULTS: Specific EGF-R staining was observed in the cultured cells compared to those incubated with antibody that was preabsorbed with a 10-fold excess of EGF. Basal progesterone accumulation per 24-hr period was stimulated dose dependently on each day of culture, by both EGF (up to 3.5-fold at 5 or 50 ng/ml) and TGF-alpha (up to 4-fold at 50 ng/ml). The addition of LH alone also stimulated progesterone accumulation daily, and this effect was further enhanced dose dependently by cotreatment with EGF or TGF-alpha. Furthermore, tyrphostin, an EGF-R-specific tyrosine kinase inhibitor, inhibited both basal and growth factor-stimulated progesterone production. CONCLUSION: These data suggest an EGF receptor-mediated physiological role for EGF and TGF-alpha in human luteal function involving an autocrine and/or a paracrine stimulation of progesterone production.