Prostaglandin F2alpha Receptors (FP) in Human Granulosa-Lutein Cells: Acquisition of Function During Transition from Granulosa to Luteal Cell Phenotype.

Abstract

Prostaglandin F2alpha (PGF2alpha) is an important physiological regulator of the corpus luteum. Data from our laboratory suggest that monkey granulosa cells expressed FP receptor protein, but granulosa cell response to the FP receptor agonist fluprostenol was not detected. In contrast, monkey luteal cells expressed functional FP receptors at mid luteal phase. The present study was performed to examine the acquisition of FP receptor function as granulosa cells differentiate into the granulosa-lutein cells of the corpus luteum. Human luteinizing granulosa cells were obtained from healthy women donating oocytes for in vitro fertilization. These cells were cultured for a total of 6 days under conditions which promote transition to the granulosa-lutein cell phenotype. Western blot and immunostaining were performed to determine FP protein levels and cellular location, respectively. Real time RT-PCR was used to quantify mRNAs, and media progesterone was measured by ELISA. FP protein levels did not change during the culture period. FP protein was distributed throughout the cytoplasm and nucleus of luteinizing granulosa cells on day 0. However, FP protein was located primarily in the nucleus of cells on day 2-6 of culture, comparable to the nuclear location of FP receptors in large, granulosa-derived cells of the monkey corpus luteum. To further examine the function of nuclear FP receptors, additional studies were conducted using human cells after 2 days of culture. Stimulation of FP by fluprostenol reduced progesterone production and early growth response factor (EGR)-1 mRNA levels. Furthermore, fluprostenol inhibited the hCG-stimulated increase in EGR-1 mRNA. These data show that FP receptor stimulation regulates progesterone and EGR-1 expression in cultured human granulosa cells in a manner similar to previous studies in luteal cells. The FP receptor is a member of the G protein-coupled receptor superfamily and interacts with Gq to activate phopholipase C (PLC)-dependent protein kinase C (PKC) in the corpus luteum. In human luteinizing granulosa cells cultured for 2 days, fluprostenol did not alter basal or hCG-stimulated cAMP. FP-mediated decrease of progesterone and EGR-1 mRNA levels was blocked by the PLC inhibitor U73122 and the selective PKC inhibitor Ro-31-8220. Treatment with the mTOR inhibitor rapamycin also prevented the FP-mediated decrease in EGR-1 mRNA. Therefore, FP receptor movement into the nucleus correlates with acquisition of FP receptor function. FP receptor likely regulates luteal functions (progesterone production and EGR-1 mRNA) via a PLC and PKC-dependent pathway in primate granulosa-lutein cells. Supported by NIH HD054691 and Schering-Plough, now Merck & Co. (poster)