Regulation of mRNA for Chemokines, Prostaglandin-Endoperoxide Synthase 2 (PTGS2), and Major Histocompatibility Complex (MHC) II Molecules in Luteinized Human Granulosa Cells. (2011)

Luteolysis is a complex process involving interaction among multiple types of cells, including immune cells, within the corpus luteum (CL). In bovine, luteolysis is induced by uterine prostaglandin F2α (PGF), is preceded by induction of chemokines and activation of immune cells, and is mimicked, in certain molecular aspects, in luteinized bovine granulosa cells (LubGC). In this study, we investigated luteinizing human granulosa cells (LuhGC) as a model for certain aspects of human luteolysis. After receiving written patient consent, human GC that were left after collection of oocytes for in vitro fertilization, were isolated by Percoll gradient, plated overnight onto 24 well plates by incubation with 10% fetal calf serum (FCS), and subsequently cultured in 2% FCS for up to 8 days. Cells were luteinized with forskolin (10 µM/ml) or human chorionic gonadotropin (hCG; 1 IU/ml). Progesterone (P4) was measured with ELISA and mRNA concentration was measured with real-time PCR. Expt 1: Cells that were luteinized with forskolin had increasing P4 production. We evaluated regulation of gene expression by PGF at early (Day 2) and later (Day 8) stages of luteinization, as previously done in LubGC. On Day 2 of luteinization, PGF did not significantly stimulate expression of the chemokine genes, CXCL2 and IL8, but did stimulate PTGS2 mRNA (243%). In contrast, the two chemokine genes and PTGS2 were dramatically induced by PGF in LuhGC on Day 8 (600% for IL8, 517% for CXCL2, 695% for PTGS2 after 2h treatment). Moreover, it was found that LuhGC on Day 8 expressed much more mRNA for MHC II DQB1 (710%) and DRB3 (703%) compared with LuhGC on Day 2; although, their expression was not regulated by PGF. Thus similar to LubGC, LuhGC at an early stage (Day 2) have FP receptors and PGF responsiveness but require prolonged luteinization to acquire capacity for PGF-induced chemokine expression and expression of MHC II, suggesting a delayed acquisition of full capacity for luteolysis, particularly immune responses. Expt 2: Experiments were similar to Expt 1, however luteinization of LuhGC was induced with hCG. Similar to Expt. 1, P4 production increased during luteinization and responsiveness to PGF was much greater in Day 8 than Day 2 LuhGC with particularly dramatic effects of PGF on PTGS2 in Day 8 LuhGC (3,851%). There was a similar dramatic increase in MHC II mRNAs after 8 d of luteinization with hCG. Thus, either forskolin or hCG can induce hGC luteinization with similar differentiation of LuhGC to PGF responsiveness. Expt 3: Human luteolysis is marked by inadequate stimulation of LH/cAMP pathways. In this experiment forskolin or hCG were withdrawn after 8 d of luteinization and responses on Days 1, 2, and 3 after withdrawal were evaluated. Withdrawal of forskolin for only 1 d dramatically increased mRNA for PTGS2 (1,223%; P=0.02), IL8 (2,472%; P=0.03), and CXCL2 (4,530%; P=0.07). Withdrawal of hCG had the most dramatic effects at 2 d after withdrawal with decreased P4 in culture media (from 998 ng/mL to 145 ng/mL; P=0.001) and induction of mRNA for IL8 (1,003%; P=0.03). Thus, similar to LubGC, we observed acquisition of immune-related pathways and PGF responsiveness after 8 d of luteinization indicating conservation of these pathways across species. The observation that withdrawal of hCG or forskolin from LuhGC induces some luteolytic pathways indicates that these cells may be useful for understanding the molecular pathways involved in human CL regression. Supported by NIH R01 HD050616. (platform)