The role of progesterone in regulating human granulosa cell proliferation and differentiation in vitro

To further elucidate the role of progesterone in regulating granulosa cell function, human granulosa and luteal cells were obtained from follicular aspirates of women undergoing in vitro fertilization and placed in culture. Cells plated at 5 x 10(3) cells/mL doubled after 3 days. In contrast, cells plated at 50 x 10(3) cells/mL did not proliferate, but differentiated, secreting high levels of progesterone. Cells plated at 5 x 10(3) cells/mL and cultured with spent medium from cells plated at 50 x 10(3) cells/mL did not increase in number over 3 days of culture. The growth-inhibiting action of the spent medium was removed by either RU 486 (a progesterone receptor antagonist) or charcoal extraction, but not by heat inactivation. The addition of progesterone to fresh medium also prevented cell proliferation. Progesterone's ability to inhibit cell division was attenuated by either RU 486 or aminoglutethamide, which blocked progesterone synthesis. Further, epidermal growth factor (EGF) stimulated cell proliferation, and continuous exposure to progesterone blocked EGF-induced mitosis. When progesterone was added 2 h after EGF, it did not block EGF-stimulated cell proliferation. Progesterone also increased the percentage of granulosa cells and decreased the percentage of large luteal cells present after 3 days of culture, indicating that progesterone inhibited differentiation. Progesterone's effect on differentiation was dose dependent, reversible, and could be overridden by hCG or 8-bromo-cAMP. These observations suggest that progesterone acts directly on granulosa cells through its receptor to inhibit mitosis and that progesterone mediates its antiproliferative effects within 2 h of mitotic stimulation. Progesterone also blocks differentiation, but this effect of can be overcome by hCG or cAMP analogs. These data indicate that progesterone plays a major role in controlling the number of luteal cells that ultimately develop within a corpus luteum by regulating both granulosa cell proliferation and differentiation.