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-17␤ (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)␣ and progesterone receptor (PR), but not ER␤, appeared to be abundant, presumably indicating that specific ER or PR coregulator expression might be responsible for this difference. Consistently, an agonist of ER␣, but not ER␤, 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 ER␣ 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. (Endocrinology 152: 3246 -3258, 2011) T he uterus is composed of heterogeneous cell types that respond uniquely to estradiol-17␤ (E2) and progesterone (P4). In the adult ovariectomized mouse uterus, E2 stimulates proliferation of luminal and glandular epithelia, whereas in the stroma, this process requires P4 and is potentiated by E2 (1, 2). P4 also plays a major role in the inhibition of E2-induced epithelial cell proliferation (1, 2). A similar hormonal action is also revealed in the early pregnancy of mice. For example, preovulatory ovarian estrogen directs epithelial cell proliferation on d 1 and 2 of pregnancy, whereas on d 3, P4 from newly formed corpora lutea initiates proliferation of stromal cells, but inhibition of epithelial cell proliferation; this is further potentiated by the preimplantation estrogen secretion on d 4 (2, 3). It has been widely viewed that ovarian steroid hormones control uterine cell proliferation and differentiation via alteration of cell-cell communication signaling and gene regulation primarily to restore uterine receptivity for the onset of embryo implantation ...

Entosis Allows Timely Elimination of the Luminal Epithelial Barrier for Embryo Implantation

“…Epithelial cells were isolated by enzymatic digestion of uterine tissues on day 4 of pseudopregnancy as previously described (Chung and Das, 2011; Daikoku et al, 2011). Sheets of epithelial cells were grown on matrigel-coated cell culture dishes with complete media, consisting of DMEM/F12 supplemented with charcoal-stripped 10% fetal bovine serum (FBS).…”

Suppression of autophagic activation in the mouse uterus by estrogen and progesterone

“…We examined whether a similar response is observed in primary cells isolated from the uterine stroma and epithelium (Chung & Das 2011). Uterine epithelial cells and stromal cells were isolated from random cycling mice and cultured separately.…”

“…Cells were isolated from the uteri of random cycling mice (nZ6) (Chung & Das 2011). Uterine horns were dissected, slit longitudinally, and cut into 3-4 mm pieces.…”

“…Uterine horns were dissected, slit longitudinally, and cut into 3-4 mm pieces. The isolation of uterine cells was carried out with pancreatin (Sigma-Aldrich), dispase (Invitrogen), and collagenase, following the procedure of Chung & Das (2011). Uterine epithelial cells and stromal cells were cultured separately in DMEM:F-12 (1:1) media with antibiotics and 10% charcoal-stripped fetal bovine serum (FBS) (Sigma-Aldrich).…”

Effects of steroid hormones on differentiated glandular epithelial and stromal cells in a three dimensional cell culture model of the canine endometrium

“…In a human endometrial co-culture system, addition of E induced increased PR-positive immunoreactivity of endometrial epithelial cells [47]. Comparable results were observed in the murine endometrium model of Chung et al [48], demonstrating an increase of PR expression in co-cultured endometrial epithelial cells due to E supplementation. In both studies, endometrial epithelial cells were separated by membranes (inserts) from the stromal cells.…”