Expression and localization of estrogen receptor-alpha protein in normal and abnormal term placentae and stimulation of trophoblast differentiation by estradiol

Abstract

Estrogens play an important role in the regulation of placental function, and 17-beta-estradiol (E2) production rises eighty fold during human pregnancy. Although term placenta has been found to specifically bind estrogens, cellular localization of estrogen receptor alpha (ER-alpha) in trophoblast remains unclear. We used western blot analysis and immunohistochemistry with h-151 and ID5 monoclonal antibodies to determine the expression and cellular localization of ER-alpha protein in human placentae and cultured trophoblast cells. Western blot analysis revealed a ~65 kDa ER-alpha band in MCF-7 breast carcinoma cells (positive control). A similar band was detected in five normal term placentae exhibiting strong expression of Thy-1 differentiation protein in the villous core. However, five other term placentae, which exhibited low or no Thy-1 expression (abnormal placentae), exhibited virtually no ER-alpha expression. In normal placentae, nuclear ER-alpha expression was confined to villous cytotrophoblast cells (CT), but syncytiotrophoblast (ST) and extravillous trophoblast cells were unstained. In abnormal placentae no CT expressing ER-alpha were detected. Normal and abnormal placentae also showed ER-alpha expression in villous vascular pericytes and amniotic (but not villous) fibroblasts; no staining was detected in amniotic epithelial cells or decidual cells. All cultured trophoblast cells derived from the same normal and abnormal placentae showed distinct ER-alpha expression in western blots, and the ER-alpha expression was confined to the differentiating CT, but not to the mature ST. Trophoblast cells from six additional placentae were cultured in normal medium with phenol red (a weak estrogen) as above (PhR+), or plated in phenol red-free medium (PhR-) without or with mid-pregnancy levels of E2 (20 nM). Culture in PhR- medium without E2 caused retardation of syncytium formation and PhR-medium with E2 caused acceleration of syncytium formation compared to cultures in PhR+ medium. These data indicate that the considerable increase in estrogen production during pregnancy may play a role, via the ER-alpha, in the stimulation of CT differentiation and promote function in normal placentae. This mechanism, however, may not operate in abnormal placentae, which show a lack of ER-alpha expression.

Figure 1

Reactivity of h-151 and ID5 monoclonal antibodies directed against human ERα in a western blot with lysates from fibroblast culture (FBC), MCF-7 breast carcinoma cells (MCF), trophoblast culture (TRC) and ovarian adenocarcinoma lysates (AOvCa). The h-151 shows a stronger and ID5 a weaker band at ~65 kDa.

Figure 2

Reactivity of h-151 antibody [ERα (h-151), top row] with trophoblast culture (TRC) and ovarian endometrioid carcinoma (EOvCa) lysates in western blot (WB) and with rabbit anti-Cdk2 (lanes 3 and 5) immunoprecipitates (IP). Bottom rows show positive control with cyclin E antibody in WB (lanes 1 and 2) and Cdk2 IP (lanes 3 and 4). Lane 4 is a negative control for both antibodies.

Figure 3

Immunohistochemical detection of ERα in MCF-7 cells and human placenta at term. A proportion of MCF-7 breast carcinoma cells show strong nuclear staining with h-151 [A] and ID5 antibodies [B]. [C] No staining of MCF-7 is apparent with control (CD3) antibody recognizing human T lymphocytes. [D] In normal placentae, cytotrophoblast cells (c) associated with trophoblastic syncytium (s) show strong nuclear staining with ID5 antibody (used for all placental samples stained for the ERα), which decreases in cytotrophoblast cells entering the syncytium [E]. [F] CD3 control shows an isolated T cell but no staining of other villous structures. Vascular ERα immunoreactivity is not associated with the nuclei of endothelial cells (black arrows, G and H). However vascular pericytes (PC and white arrow, H) are strongly stained. [I] In abnormal placentae, the ERα immunoreactivity was associated with villous vasculature only. [J] Staining of villous fibroblasts persisting after vascular regression. Fibroblasts beneath the amniotic epithelium show ERα staining [K], but no ERα immunoreactivity is apparent in the amniotic epithelium [L], extravillous trophoblast (EVT) [M] and decidual cells [N]. [O] Strong Thy-1 staining of decidual cells (right) and no staining of EVT (left).

Figure 4

ERα immunoreactivity (h-151 antibody) in early (17 h) and late trophoblast cultures (93 h). Dividing cells in [A] exhibit distinct surface staining (arrow). Cell in [B] shows moderate cytoplasmic and perinuclear immunoreactivity (arrowhead), and cell in [C] exhibits strong nuclear staining without surface expression. [D] Early syncytium shows traces of cytoplasmic ERα staining (compare with E). [E] No ERα staining of mature syncytium.

Figure 5

A comparison of placental Thy-1 expression (PL in vivo: Thy-1) in normal (PT1, A) and abnormal placentae (PT4, D) and the extent of trophoblast differentiation (dashed lines) in early (EC) and late PT1 and PT4 trophoblast cultures (LC). Note that late culture from normal placenta shows a large syncytium aggregates (~300 μm in diameter). Abortive formation of syncytium clusters (~50 μm) is apparent in late cultures from abnormal placenta. Inserts show ERα (top) and actin bands (bottom) from western blots performed from the same cultures. [G] Quantitative evaluation of ERα expression in immunoblots shown in top inserts [A-F]. Different column superscripts indicate significant difference. Each column represents 6 measurements. Error bars = SD. Details in text.

Figure 6

Estrogen receptor expression analyzed by western blotting in placental types PT1–PT4 [PL in vivo; A] and corresponding early (TRC EC) and late trophoblast cultures (TRC LC) [B and C]. Samples are presented in the order they have been collected. [D] Cumulative data on ERα expression in normal (NP in vivo; n = 5) and abnormal placentae (AP in vivo; n = 5) and derived early (EC) and late cultures (LC). Different column superscripts indicate significant difference. Each column represents 30 measurements. Error bars = SD. Details in text.

Figure 7

Trophoblast differentiation in late cultures from normal (NP; PT1) and abnormal placentae (AP; PT3). Cells were grown in complete DMEM-HG, which contains 45 μM phenol red (PhR+), or phenol red-free DMEM-HG without estrogens (PhR-/E2-) of with 20 nM E2 (PhR-/E2+). Each medium was supplemented with 5% heat-inactivated FBS. *indicates vesicles and vacuoles characteristic of multinucleated syncytiotrophoblast [61]; s, small; m, mid-sized; l, large; xl, extra large syncytial aggregates (dashed line). Short arrows = dividing/post-mitotic trophoblasts; long arrow, adjacent trophoblast cells with preservation of cell membranes; arrowhead, isolated trophoblast cells. Details in Materials and Methods and in the text.

Figure 8

Mean area (square μm + SD) of syncytial aggregates in late cultures of trophoblast cells derived from normal (NP) and abnormal placentae (AP). Cells were grown as indicated in Materials and Methods – see also Fig. 7 for details and abbreviations. Each column represents eighteen measurements. Different column superscripts indicate significant difference.