This project was designed to test the hypothesis that antisense oligonucleotides generated against progesterone receptor (PR) mRNA can modulate PR expression and hence P action (e.g., P production) in monkey luteinized granulosa cells during culture. Granulosa cells isolated from gonadotropin-treated rhesus monkeys receiving an ovulatory stimulus of human chorionic gonadotropin (hCG, 1000 IU) were cultured (8 x 104 cells/well) on fibronectin-coated 8-well glass chamber slides in chemically-defined medium containing low-density lipoprotein ( hLDL 25 fg/ml) and hCG (100 ng/ml) for 18 hr. Medium was changed and one of six 18-mer antisense PR phosphorothioate-oligonucleotides (PR/ODN) added to each well (8 fg/ml) with or without a liposome formulation, Lipofectin (8 fg/ml). Cultures were continued for an additional 2 days with daily medium changes. Spent medium was assayed for progesterone using RIA. On days 0 and 3, granulosa cells were fixed for PR analysis by immunocytochemistry. Preliminary data indicate that one PR/ODN, 1440, which hybridizes to the mRNA start site for the B form of PR, decreased the % PR-positive cells by day 3 of culture compared to controls. Likewise, progesterone production was greater by day 3 in cultures incubated with 1440. Experiments are being repeated with elimination of the 18 hr incubation prior to the addition of PR/ODN and lipofectin.
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