Correlation of Cell Surface Features with Progesterone Secretion in Rat Granulosa Cell Cultures1, 2

The surface ultrastructure of rat granulosa cells in culture was studied and correlated with progesterone secretion. Granulosa cells were harvested from pregnant mares’ serum-luteinizing hormone (PMS-LH) treated immature female rats and cultured on glass coverslips in Leighton tubes. Progesterone secretion as measured by specific radioimmunoassay and cell surface morphology utilizing routine procedures were studied in rat granulosa cell cultures under varying stimulatory and inhibitory conditions. Cultures maintained without exogenous hormones added to the culture medium (controls) secreted relatively low levels of progesterone throughout a 2–12 day culture period. The surface morphology of these cells was characterized by numerous smooth surfaced blebs. All cells regardless of hormone stimulation exhibited a centric clump of smooth surfaced blebs. The cell borders of control granulosa cells exhibited numerous slender filopodia and occasional ruffles. Granulosa cells cultured in the presence of rat prolactin (rPRL) or human placental lactogen (hPL) secreted enhanced levels of progesterone. The cell surface morphology of this group was characterized by smooth surfaced blebs which were fewer in number than control cultures. Stimulated granulosa cells had many more microvilli than control cells did. Filopodia were few in number, or in most cases entirely absent. Granulosa cells cultured in the presence of prolactin plus cyanoketone (CK, an enzyme inhibitor of progesterone biosynthesis) secreted very low levels of progesterone. Three methods chosen to test cell viability from the CK treated group a) trypan blue exclusion, b) [14C]-glucose uptake and metabolism to 14CO2 and c) restimulation of inhibted cultures following removal of cyanoketone treated granulosa cells possessed a few smooth surfaced blebs. Filopodia were fewer in number than they were in controls. The appearance of microvilli on the surface of prolactin stimulated rat granulosa cells in culture was the single most striking surface feature which could be correlated with increased progesterone secretion.