Altered Gene Expression Profile in Ovarian Follicle in Rats Treated with Indomethacin and RU486

It is well-known that indomethacin (the cyclooxygenase 1 & 2 inhibitor) and RU486 (or mifepristone, the progesterone receptor antagonist) block follicular rupture in rats. To characterize genetic alterations in unruptured follicles, gene expression profiles in ovarian follicle were analyzed in indomethacin- and RU486-treated female Sprague-Dawley rats. Ovaries are collected at 22:00 on the proestrus day and 10:00 on the following estrus day after a single dose of indomethacin and RU486. Histopathologically, changes depicting responses to LH surge were observed in ovaries, uteri and vagina. Total RNA was extracted from pre-ovulatory follicles or unruptured follicles collected by laser microdissection and analyzed by GeneChip. Among genes showing statistically significant changes compared to control groups, following changes were considered relevant to induction of unruptured follicles. In indomethacin-treated rats, Wnt4 was down-regulated, suggesting effect on tissue integrity and steroid genesis. In RU486-treated rats, Adamts1, Adamts9, Edn2, Ednra, Lyve1, Plat, and Pparg were down-regulated. These changes suggest effects on proteolysis for extracellular matrix or surrounding tissue (Adamts1 & 9, and Plat), constriction of smooth muscle surrounding follicles (Edn2, Ednra, and Pparg), follicular fluid (Lyve1), and angiogenesis (Pparg). Down-regulation of angiogenesis related genes (Angpt2, Hmox1, and Vegfa) was observed in both treatment groups. Here, we clarify genetic alterations induced by the inhibition of cyclooxygenase or progesterone receptor. Animals were singly administered orally 4mg/kg of Indomethacin (IM) at 16:00 or 100 mg/kg of RU486 (RU) at 10:00 on the proestrus day, and sacrificed by exsanguination under anesthesia at 22:00 on the proestrus day, and 10:00 on the following estrus day. Three animals were contained per each treatment group at each timing. Pre-ovulatory follicle (PeF) and unruptured follicles (UF) were laser-microdissected from ovaries collected on the proestrus day, and the estrus day, respectively. From untreated animals, PeF and post-ovulatory follicle (PoF) were also laser-microdissected from the ovaries collected on the proestrus day, and the estrus day, respectively, as the control group. The ovaries were removed and embedded in OCT (Tissue-Tek, Sakura Finetek, Torrance, CA), frozen in dry-ice-cold isopentan, and stored at -70 °C until used. Frozen sections (8 micro meter thick) were mounted on the membrane slides (MMI, Glatteburg, Zurich, Switzerland), and stained with HistogeneTM LCM frozen section staining kit (Arcturus Engineering, Mountain View, CA). Then, 15 follicles in each phase were retrieved by Laser Micro Dissection system (LMD, CellCut Plus®, MMI). The collected follicle sections were lysed by the buffer RLT in the collection tube supplied in RNeasy Micro Kit (Qiagen, Hilden, Germany), and total RNA was extracted according to manufacturer's instructions.