Dhillon JK et al. (2010), Effects of RU486 on cyclooxygenase-2 gene expre...

XB-ART-39913

Gen Comp Endocrinol 2010 Jan 01;1651:78-82. doi: 10.1016/j.ygcen.2009.06.005.

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Effects of RU486 on cyclooxygenase-2 gene expression, prostaglandin F2alpha synthesis and ovulation in Xenopus laevis.

Dhillon JK , Liu Z .

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RU486 is a synthetic analog of progesterone and functions as a progesterone receptor antagonist. It binds to the progesterone receptor to prevent progesterone from occupying its receptor in many cellular systems. Early studies from our laboratory have shown that in Xenopus laevis ovarian follicles progesterone stimulates the expression of cyclooxygenase-2 (COX-2) gene which leads to a rapid increase in the production of prostaglandin F2alpha (PGF2alpha) and subsequent ovulation. In this study, we examined the effect of RU486 on the synthesis of COX-2 mRNA, production of PGF2alpha and ovulation in X. laevis. Ovarian tissue fragments were primed with human chorionic gonadotropin (hCG) and then incubated with progesterone (P4) alone or in the presence of varying concentrations of RU486 over a period of 12h. After the incubation ovulated oocytes were counted, COX-2 expression and synthesis of PGF2alpha were measured. Results demonstrated that RU486 attenuated the expression of COX-2 gene, reduced the synthesis of PGF2alpha, and inhibited ovulation in a dose-dependent manner. This finding suggests that progesterone receptor is an important regulator in the progesterone-cyclooxygenase-prostaglandin-mediated ovulation in amphibians.

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Species referenced: Xenopus laevis
Genes referenced: kit ptgs2 Ptgs2

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	Fig. 1. Effect of increasing concentration of RU486 on ovulation in X. laevis. Pieces of ovarian tissue (100 mg) containing approximately 20 full-grown oocytes were incubated in MBS (negative control), MBS with P4 (positive control), MBS and P4 with RU486 10, 50 and 100 μM at 22 °C. Ovarian fragments were observed for ovulation by light microscopy at 0, 6 and 12 h of incubation. RU486 significantly inhibited ovulation in a dose-dependent manner. Bars represent the mean values (±SEM).
	Fig. 2. Effect of increasing concentration of RU486 on expression of COX-2 in X. laevis. Pieces of ovarian tissue containing approximately 20 full-grown oocytes were incubated in MBS (negative control), MBS with P4 (positive control), MBS with P4 and RU486 10, 50 and 100 μM for 0, 6 and 12 h at 22 °C. Total RNA was extracted from the ovarian tissue, used to synthesize cDNA by RT-PCR and observe the expression of COX-2 by real-time PCR for each sample. Bars represent the mean values (±SEM). Expression levels of COX-2 mRNA were normalized by β-actin mRNA levels.
	Fig. 3. Effect of increasing concentration of RU486 on synthesis of PGF2α in P4 and RU486 treated X. laevis follicles during ovulation in vitro. Pieces of ovarian tissue (100 mg) containing approximately 20 full-grown oocytes were incubated in MBS (negative control), MBS with P4 (positive control), MBS and P4 with RU486 10, 50 and 100 μM at 22 °C. PGF2α concentrations in the culture media from the sample wells were measured using EIA kit in all samples. Results were obtained for 0, 6 and 12 h samples. Bars represent the mean values (±SEM).