Changes in global gene expression during in vitro decidualization of rat endometrial stromal cells

Abstract

During the preimplantation phase of pregnancy the endometrial stroma differentiates into decidua, a process that implies numerous morphological changes and is an example of physiological transdifferentiation. Here we show that UIII rat endometrial stromal cells cultured in the presence of calf serum acquired morphological features of decidual cells and expressed decidual markers. To identify genes involved in decidualization we compared gene expression patterns of control and decidualized UIII cells using cDNA microarray. We found 322 annotated genes exhibiting significant differences in expression (>3-fold, fold discovery rate (FDR) >0.005), of which 312 have not been previously related to decidualization. Analysis of overrepresented functions revealed that protein synthesis, gene expression, and chromatin architecture and remodeling are the most relevant modified functions during decidualization. Relevant genes are also found in the functional terms differentiation, cell proliferation, signal transduction, and matrix/structural proteins. Several of these new genes involved in decidualization (Csdc2, Trim27, Eef1a1, Bmp1, Wt1, Aes, Gna12, and Men1) are shown to be also regulated in uterine decidua during normal pregnancy. Thus, the UIII cell culture model will allow future mechanistic studies to define the transcriptional network regulating reprogramming of stromal cells into decidual cells.

Figure 1. UIII cells in vitro decidualization

UIII cells were plated in M199 supplemented with 10% DCC-FCS, and 24 hours later, media was replaced by fresh M199 without serum. After two days in serum-free conditions (day 0), cells were cultured in presence of 10% DCC-FCS for 7 days (day 7). a) UIII cells were fixed and stained with Giemsa reagent and observed with a light microscope using a 10× objective. b) Number of total cells/well (line) and percentage of cells with decidual morphology (bars) during 7 days of cultures show the kinetic of cell growth and cell differentiation respectively. Data represent the media ± SEM of 3 independent experiments. NS P>0.05, * P<0.05, ***P<0.001 vs. control (day 0). c) Quantification of desmin mRNA by real-time PCR. Day 4 column shows fold up of desmin mRNA after 4 days in 10% DCC-FCS relative to β-actin mRNA levels. Data represent the media ± SD of three independent experiments with four independent measurements. ** P<0.01 vs. control (day 0). d) Immunofluorescence of desmin protein expression. Day 0 and day 4 cells were fixed, permeabilized and incubated with polyclonal Mouse anti-Desmin and 488 goat anti-Mouse (green). Nucleus was stained with propidium iodide (red).

Figure 2. Experimental Microarrays validation of up-regulated genes

UIII cells were cultured as it was described in Figure 1. After two days in serum-free conditions (day 0, non-decidualized cells), cells were cultured in presence of 10% DCC-FCS for 4 days (day 4, decidualized cells). Day 0 and day 4 cells were resuspended and RNA was extracted for microarray and RT-PCR experiments. a) Table with microarray data of selected genes. Genes up-regulated by at least a factor of 3 and FDR of 0.005 in decidualized cells as compared to control cells were selected. b) Membrane scanning of microarrays of selected genes. c) RT-PCR of selected genes at day 0 (non-decidualized cells) and day 4 (decidualized cells).

Figure 3. Experimental Microarrays validation of down-regulated genes

UIII cells were cultured as it was described in Figure 1. After two days in serum-free conditions (day 0, non-decidualized cells), cells were cultured in presence of 10% DCC-FCS for 4 days (day 4, decidualized cells). Day 0 and day 4 cells were resuspended and RNA was extracted to microarray and RT-PCR experiments. a) Table with microarray data of selected genes. Genes down-regulated by at least a factor of 3 and FDR of 0.005 in decidualized cells as compared to control cells were selected. b) Membrane scanning of microarrays of selected genes. c) RT-PCR of selected genes at day 0 (non-decidualized cells) and day 4 (decidualized cells). d) Gja1 RT-PCR six days kinetic expression.

Figure 4. In vivo gene expression of genes found during in vitro UIII differentiation

a) Total RNA from non-pregnant uterus (np) and implantation sites from 8 d.p,c uterus (8 pdc) were extracted for RT-PCR. The figure shows ethidium bromide-stained gels of RT-PCR products for: Cdsdc2, Trim27, Eef1a1, Fkbp4, Fstl1, Bmp1, Wt1, Aes, Gna12, Myst3, Suv39h1, Epim, Ovpg1, β-HSD, desmin, Prl and GAPDH. Fold changes from microarray data of selected genes are shown. ND: not determined. b) Quantitation of Men1 mRNA in non-pregnant (np) and 8 d.p.c. in uterus. The figure shows fluorescence intensity pattern of real time PCR products for Men1 from four independent samples of non-pregnant mRNA (np), four independent samples of 8 d.p.c mRNA (8dpc) and two samples without template (H2O). c) Table shows fold up of Men1mRNA from 8 d.p.c. over non-pregnant relative to GAPDH mRNA levels. Data represent the media of four independent experiments.