Mouse primary uterine cell coculture system revisited: ovarian hormones mimic the aspects of in vivo uterine cell proliferation

Abstract

Previously, the uterine epithelial-stromal coculture system had limited success mimicking in vivo ovarian hormone-dependent cell-specific proliferation. Here, we established a mouse primary uterine coculture system, in which cells collected in pseudopregnancy specifically on d 4 are conducive to supporting hormone-induced cell-specific proliferation. When two cell types are placed in coculture without direct contact via cell culture inserts (nonadjacent), as opposed to with contact (adjacent), epithelial cells exhibit significant proliferation by estradiol-17β (E2), whereas progesterone in combination with E2 caused inhibition of epithelial cell proliferation and a major shift in proliferation from epithelial to stromal cells. Epithelial cell integrity, with respect to E-cadherin expression, persisted in nonadjacent, but not adjacent, conditions. In subsequent studies of nonadjacent cocultures, localization of estrogen receptor (ER)α and progesterone receptor (PR), but not ERβ, appeared to be abundant, presumably indicating that specific ER or PR coregulator expression might be responsible for this difference. Consistently, an agonist of ERα, but not ERβ, was supportive of proliferation, and antagonists of ER or PR totally eliminated cell-specific proliferation by hormones. RT-PCR analyses also revealed that hormone-responsive genes primarily exhibit appropriate regulation. Finally, suppression of immunoglobulin heavy chain binding protein, a critical regulator of ERα signaling, in epithelial and/or stromal cells caused dramatic inhibition of E2-dependent epithelial cell proliferation, suggesting that a molecular perturbation approach is applicable to mimic in vivo uterine control. In conclusion, our established coculture system may serve as a useful alternative model to explore in vivo aspects of cell proliferation via communication between the epithelial and stromal compartments under the direction of ovarian hormones.

Fig. 1.

A, A diagram of both the adjacent and nonadjacent cocultures. B, Analysis of cell proliferation using the adjacent coculture system in the presence or absence of ovarian hormones. Both epithelial and stromal cells collected on d 4 of pseudopregnancy were cultured for 2 d in the presence of vehicle (control), E2 (10 nm), or E2 (10 nm) plus P4 (1 μm). Ethanol-fixed cells were analyzed by confocal microscopy with primary antibodies for BrdU (red), cytokeratin (green), or desmin (yellow). 4′,6-diamidino-2-phenylindole (DAPI) (blue) was used to stain all nuclei. Des, Desmin; Cyto, cytokeratin.

Fig. 2.

Analysis of cell proliferation using the nonadjacent coculture system in the presence or absence of E2. Both epithelial and stromal cells collected on d 4 of pseudopregnancy were subjected to nonadjacent cocultures, as described in Materials and Methods, and treated with vehicle (control) or E2 (1, 10, or 25 nm) for 1, 2, or 3 d. A, Quantitation of E2-dependent response to epithelial and stromal cell proliferation. The data presented here are after the analysis of at least 1000 cells from each group. The error bars represent se. Asterisk, Values are statistically different (P < 0.05) against control. B, Representative confocal images for E2 (10 nm)-dependent cell proliferative response on d 2 in the culture are shown by BrdU (red) staining. Epithelial or stromal cells are indicated by the staining for cytokeratin (green) or desmin (green), respectively. 4′,6-diamidino-2-phenylindole (DAPI) (blue) was used to stain all nuclei. These experiments were repeated at least five times with similar results.

Fig. 3.

Analysis of cell proliferation using the nonadjacent coculture system in the presence of vehicle, P4, or E2 plus P4. Day-four uterine epithelial and stromal cells in the nonadjacent cocultures were subjected to vehicle (control), P4 (1 μm), or E2 (10 nm) plus P4 (1 μm or 10 μm) for 2 d. A, Immunofluorescence analysis of proliferation by BrdU (red) incorporation. Epithelial or stromal cells are indicated by the staining for cytokeratin (green) or desmin (green), respectively. 4′,6-diamidino-2-phenylindole (DAPI) (blue) was used to stain all nuclei. B, Quantitative analysis of cellular proliferation. The data presented here are after the analysis of at least 1000 cells from each group. The error bars represent se. Asterisk, Values are statistically different (P < 0.05) against control. These experiments were repeated at least five times with similar results.

Fig. 4.

Expression of E-cadherin. A, Analysis of epithelial expression in the adjacent (A) and nonadjacent (B and C) cocultures. Both epithelial and stromal cells collected on d 4 (A and B) or d 6 (C) were cultured for 2 d in the presence of vehicle (control), E2 (10 nm), or E2 (10 nm) plus P4 (1 μm). Expression of E-cadherin in basolateral and apical locations is shown in red. Nuclei are shown by 4′,6-diamidino-2-phenylindole (DAPI) (blue). In A, epithelial cells are stained with cytokeratin (green), and the locations of epithelial (Epi) and stromal (St) cells are shown in the picture. Note: Epithelial E-cadherin expression is not detected in the adjacent coculture system, whereas the basolateral E-cadherin expression is detected in the nonadjacent cultures. These experiments were repeated at least two times with similar results.

Fig. 5.

Expression of ER and PR in the nonadjacent coculture system. A and B, Confocal analysis of ERα and PR staining. Staining for cytokeratin in epithelial or desmin in stromal cells is shown in green, whereas ERα or PR is in red. Nuclei are shown by 4′,6-diamidino-2-phenylindole (DAPI) (blue). C, Western blot analysis of expression for ERα, PR-A, and PR-B. D, The quantitation of protein levels from the Western blottings was determined by densitometric scanning. Fold changes in protein levels were calculated against vehicle (control) and normalized by α-tubulin levels. The error bars represent se. Asterisk, Values are statistically different (P < 0.05). These experiments were repeated at least three times with similar results. C, Vehicle (control); E, E2; E+P; E2 plus P4.

Fig. 6.

Effects of selective agonists and antagonists for ER or PR during analysis of cell proliferation in the nonadjacent coculture system. Cells were treated with agents as indicated for 48 h and analyzed by confocal analysis for BrdU (red) incorporation. Epithelial and stromal cells are stained with cytokeratin (green) and desmin (green), respectively. These experiments were repeated at least three times with similar results. DPN, Diarylpropionitrile; ICI, ICI 182,780.

Fig. 7.

Analysis of gene expression in RNA samples of epithelial and stromal cells, after treatments of vehicle (control) (C), E2 (E), P4 (P), or E2 plus P4 (EP) in the nonadjacent coculture system. RNA samples were collected 6 and 48 h after treatments. A, Comparative RT-PCR. Total RNA was extracted and subjected to RT-PCR to achieve linear amplification with appropriate PCR cycle numbers, as indicated in Materials and Methods. Amplified DNA bands were visualized with ethidium bromide staining. B, Relative levels of gene expression. The band intensities were measured by densitometric analysis (ImageJ), and the relative levels of mRNA for gene-specific expression were determined after normalization with Rpl7 as a constitutive gene. These experiments were repeated at least three times with similar results.

Fig. 8.

The role of Bip on E2-induced epithelial cell proliferation in the nonadjacent coculture system. A, Analysis of Bip expression in epithelial and stromal cell layers, after treatments of vehicle (control) or E2 (10 nm) for 48 h. B, Western blot analyses of expression for Bip, GFP, and α-tubulin in epithelial and stromal cells after infections with rAdGFP or rAdBip(As) virus particles, in conjunction with E2 for 48 h. C, Confocal analysis of epithelial cell proliferation by incorporation of BrdU (red). 4′,6-diamidino-2-phenylindole (DAPI) (blue) was used to stain all nuclei. D, Quantitative analysis of cellular proliferation as shown in C. The data presented here represent the analysis of at least 1000 cells from each group. The error bars represent se. These experiments were repeated at least three times with similar results.