Canine decidualization <i>in vitro</i>: extracellular matrix modification, progesterone mediated effects and selective blocking of prostaglandin E2 receptors

Abstract

Recently, we established an in vitro model with immortalized dog uterine stromal (DUS) cells for investigations into canine-specific decidualization. Their capability to decidualize was assessed with cAMP and prostaglandin (PG) E2. Here, we show that the effects of PGE2 are mediated through both of the cAMP-mediating PGE2 receptors (PTGER2/4). Their functional inhibition suppressed gene expression of PRLR and PGR in DUS cells. We also assessed the effects of cAMP and PGE2 on selected extracellular matrix components and CX43, and showed that cAMP, but not PGE2, increases COL4, extracellular matrix protein 1 (ECM1) and CX43 protein levels during in vitro decidualization, indicating a mesenchymal-epithelial decidual transformation in these cells. Thus, although PGE2 is involved in decidualization, it does not appear to regulate extracellular matrix. Further, the role of progesterone (P4) during in vitro decidualization was addressed. P4 upregulated PRLR and PGR in DUS cells, but these effects were not influenced by PGE2; both P4 and PGE2 hormones appeared to act independently. P4 did not affect IGF1 expression, which was upregulated by PGE2, however, it suppressed expression of IGF2 , also in the presence of PGE2. Similarly, P4 did not affect PGE2 synthase ( PTGES ), but in the presence of PGE2 it increased PTGER2 levels and, regardless of the presence of PGE2, suppressed expression of PTGER4 . Our results indicate a reciprocal regulatory loop between PGE2 and P4 during canine in vitro decidualization: whereas P4 may be involved in regulating PGE2-mediated decidualization by regulating the availability of its receptors, PGE2 regulates PGR levels in a manner dependent on PTGER2 and -4.

Antigestagens Mediate the Expression of Decidualization Markers, Extracellular Matrix Factors and Connexin 43 in Decidualized Dog Uterine Stromal (DUS) Cells

“…The morphological and biological properties of DUS cells were monitored throughout the experiments, and the cells were found to retain their mesenchymal character and vimentin expression, as well as the nuclear expression of the immortalization factor pSV40 [ 3 ] ( Figure 1 B). Their morphology changed during decidualization, becoming more rounded and larger in size, indicating the morphological mesenchymal-epithelial transition observed previously [ 3 , 25 ] ( Figure 1 C). No changes in morphology or confluence of cells were noted in response to antigestagens ( Figure 1 C).…”

“…Here, to gain initial insights into PGR-mediated effects in decidual cells, a well-characterized model of canine decidualization with DUS cells was employed [ 3 , 9 , 25 ]. Type II antigestagens were used to interfere with PGR-signaling in decidualized DUS cells, and then the effects on the cells’ decidualization features were assessed.…”

“…The immortalized dog uterine stromal (DUS) cell line was used as the cell culture model in this study [ 3 , 25 ], following previously described procedures [ 3 , 9 , 25 ]. Briefly, cells were grown in 150 cm 2 cell culture flasks (Corning, New York, NY, USA) in maintenance medium consisting of DMEM-High Glucose (4.5 g/L; Bio Concept, Allschwil, Switzerland; pH = 7.2–p7.4) with 10% heat inactivated fetal bovine serum (FBS; Thermo Fisher Scientific AG, Reinach, Switzerland), 100 U/mL penicillin and 100 μg/mL streptomycin (PAN Biotech, Aidenbach, Germany), and 1% insulin-transferrin-selenium (ITS; Corning from Thermo Fisher Scientific AG, Reinach, Switzerland), under standard culture conditions (i.e., 37 °C, 5% CO 2 in air, in a humidified incubator).…”

“…Negative controls were created by staining slides without primary or secondary antibodies. The quantification of the intensity of positive signals was performed as previously described [ 25 ]. Pictures of six randomly selected areas were taken with 63× dry magnification lens.…”

“…The in vitro decidualization of these cells also leads to upregulation of components of the extracellular matrix, e.g., ECM1, collagen (COL) 4, and connexin (CX) 43. While decidualized DUS cells retain the expression of mesenchymal markers like vimentin (VIM) [ 9 , 25 ], their upregulated expression of COL4, which marks the mesenchymal-epithelial transition of stromal cells, is of particular importance [ 25 ]. Also, this phenomenon is similar between the dog and other decidualization models [ 26 , 27 ].…”