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Cloning of Guinea Pig Cyclooxygenase-2 and 15-Hydroxyprostaglandin Dehydrogenase Complementary Deoxyribonucleic Acids: Steroid-Modulated Gene Expression Correlates to Prostaglandin F₂ Secretion in Cultured Endometrial Cells¹

Institute for Hormone and Fertility Research, Division of Reproductive Sciences, University of Hamburg, 22529 Hamburg, Germany and Entec GmbH (W.E.), 07745 Jena, Germany

Address all correspondence and requests for reprints to: Birgit Gellersen, Institute for Hormone and Fertility Research, Division of Reproductive Sciences, Grandweg 64, 22529 Hamburg, Germany. E-mail: 100607.1557{at}compuserve.com

Prostaglandin F₂ (PGF₂) secretion is lowest at midcycle and highest on day 15 at luteolysis in the cycling guinea pig uterus and is inversely related to serum progesterone levels. An increase in 17-ß estradiol (E₂) occurs only towards the end of the cycle. To investigate the effect of steroids on the control of uterine PGF₂ metabolism at the level of gene expression we established a primary cell culture model of day 15 cycling guinea pig endometrial cells. We cloned guinea pig cDNAs for cyclooxygenase 2 (COX-2), 15-hydroxyprostaglandin dehydrogenase (PGDH) that converts PGF₂ to biologically inactive 13,14-dihydro-15-keto PGF₂ (PGFM) and a fragment of cyclooxygenase-1 (COX-1). They were found to bear 87% and 90% homology at the amino acid level to their human counterparts for COX-2 and PGDH, respectively, retaining all functional sites. Purified epithelial and stromal cell subcultures were primed with medium containing either E₂ or medroxyprogesterone acetate (MPA) for 24 h. They were then treated for a further 4 or 24 h either withdrawing the steroid, maintaining the priming steroid, or supplementing with both steroids, before harvesting conditioned media and RNA. Epithelial cells secreted 30-fold more PGF₂ compared with stromal cells (e.g. 7.8 ± 0.7 vs. 0.26 ± 0.09 pg/ng DNA•24 h), and PGF₂ secretion levels were approximately 15-fold higher than those of PGFM (e.g. 7.8 ± 0.7 vs. 0.45 ± 0.16 pg/ng DNA·24 h, for epithelial cells). COX-1 transcripts were low and unaffected by treatment in both cell types. COX-2 transcripts were more abundant in epithelial than stromal cells. Steroid-modulated, COX-2 dependent changes in PGF₂ secretion were observed. The addition of MPA to E₂ primed cells caused a decrease in PGF₂ secretion and COX-2 messenger RNA levels after 4 h. Conversely, the addition of E₂ to MPA primed epithelial cells led to an increase in PGF₂ secretion and COX-2 messenger RNA levels after 4 and 24 h. The withdrawal of E₂ caused a fall in PGF₂ secretion and COX-2 transcripts after 24 h. In contrast, PGDH transcripts were more abundant in stromal than epithelial cells and were up-regulated by the addition of MPA to E₂ primed cells. These in vitro observations are in keeping with the secretory profile seen in vivo in the cycling guinea pig uterus suggesting that 1) the fall of E₂ and the coinciding rise in progesterone seen in the early cycle lead to a reduction in PGF₂ levels; and 2) the rise of E₂ in the late cycle on a progesterone primed uterus is the stimulus for an increase in uterine PGF₂ production. Our findings suggest a differential role for uterine stroma and epithelium in vivo whereby the former acts to remove (via PGDH), and the latter to produce (via COX-2) biologically active prostaglandin.

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