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Placental expression of estrogen receptor beta and its hormone binding variant--comparison with estrogen receptor alpha and a role for estrogen receptors in asymmetric division and differentiation of estrogen-dependent cells.

Bukovsky A, Caudle MR, Cekanova M, Fernando RI, Wimalasena J, Foster JS, Henley DC, Elder RF - Reprod. Biol. Endocrinol. (2003)

Bottom Line: During human pregnancy, the production of 17-beta-estradiol (E2) rises steadily to eighty fold at term, and placenta has been found to specifically bind estrogens.In trophoblast cultures, development of ST aggregates was associated with diminution of ER-alpha and appearance of ER-beta immunoreactivity.Our data indicate that during trophoblast differentiation, the ER-alpha is associated with a less, and ER-beta with the more differentiated state.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory for Development, Differentiation and Cancer, Department of Obstetrics and Gynecology, The University of Tennessee Graduate School of Medicine, Knoxville, Tennessee 37920, USA. rbe@utk.edu

ABSTRACT
During human pregnancy, the production of 17-beta-estradiol (E2) rises steadily to eighty fold at term, and placenta has been found to specifically bind estrogens. We have recently demonstrated the expression of estrogen receptor alpha (ER-alpha) protein in human placenta and its localization in villous cytotrophoblast (CT), vascular pericytes, and amniotic fibroblasts. In vitro, E2 stimulated development of large syncytiotrophoblast (ST) aggregates. In the present study we utilized ER-beta affinity purified polyclonal (N19:sc6820) and ER-alpha monoclonal (clone h-151) antibodies. Western blot analysis revealed a single approximately 52 kDa ER-beta band in chorionic villi (CV) protein extracts. In CV, strong cytoplasmic ER-beta immunoreactivity was confined to ST. Dual color immunohistochemistry revealed asymmetric segregation of ER-alpha in dividing villous CT cells. Prior to separation, the cell nuclei more distant from ST exhibited high ER-alpha, while cell nuclei associated with ST showed diminution of ER-alpha and appearance of ER-beta. In trophoblast cultures, development of ST aggregates was associated with diminution of ER-alpha and appearance of ER-beta immunoreactivity. ER-beta was also detected in endothelial cells, amniotic epithelial cells and fibroblasts, extravillous trophoblast (nuclear and cytoplasmic) and decidual cells (cytoplasmic only). In addition, CFK-E12 (E12) and CWK-F12 (F12) monoclonal antibodies, which recognize approximately 64 kDa ER-beta with hormone binding domain, showed nuclear-specific reactivity with villous ST, extravillous trophoblast, and amniotic epithelium and fibroblasts. Western blot analysis indicated abundant expression of a approximately 64 kDa ER-beta variant in trophoblast cultures, significantly higher when compared to the chorionic villi and freshly isolated trophoblast cell protein extracts. This is the first report on ER-beta expression in human placenta and cultured trophoblast. Our data indicate that during trophoblast differentiation, the ER-alpha is associated with a less, and ER-beta with the more differentiated state. Enhanced expression of approximately 64 kDa ER-beta variant in trophoblast cultures suggests a unique role of ER-beta hormone binding domain in the regulation of trophoblast differentiation. Our data also indicate that asymmetric segregation of ER-alpha may play a role in asymmetric division of estrogen-dependent cells.

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Immunolocalization of ERβ and ERα in the late trophoblast culture (68 h). [A] Isolated CT cells are unstained (arrowhead) but strong ERβ nuclear and cytoplasmic immunostaining accompanies young ST aggregates (y). [B] CT cell associated with ST shows ERβ immunoreactivity (arrowhead). [C] Mature ST (m) shows a diminution of ERβ nuclear staining (solid > white arrows) with persisting cytoplasmic immunoreactivity. [D] Secondary antibody control. [E] Isolated CT cells show strong ERα immunostaining (arrowhead) persisting in young ST aggregates [y, E and F] but diminishing in mature ST [m; F and G]. [H] Secondary antibody control.
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Figure 4: Immunolocalization of ERβ and ERα in the late trophoblast culture (68 h). [A] Isolated CT cells are unstained (arrowhead) but strong ERβ nuclear and cytoplasmic immunostaining accompanies young ST aggregates (y). [B] CT cell associated with ST shows ERβ immunoreactivity (arrowhead). [C] Mature ST (m) shows a diminution of ERβ nuclear staining (solid > white arrows) with persisting cytoplasmic immunoreactivity. [D] Secondary antibody control. [E] Isolated CT cells show strong ERα immunostaining (arrowhead) persisting in young ST aggregates [y, E and F] but diminishing in mature ST [m; F and G]. [H] Secondary antibody control.

Mentions: Quantitative evaluation of ERα immunoreactivity in quiescent (q), parental (p) and daughter cells (d) during asymmetric divisions is shown in Fig. 3E. The ERα immunoreactivity was lowest in quiescent cells and highest in parental cells during asymmetric division. However, daughter cells showed significant diminution of ERα when compared to parental cells. Statistical analysis indicated overall ANOVA P < 0.0001, and differences between all columns P < 0.01.


Placental expression of estrogen receptor beta and its hormone binding variant--comparison with estrogen receptor alpha and a role for estrogen receptors in asymmetric division and differentiation of estrogen-dependent cells.

Bukovsky A, Caudle MR, Cekanova M, Fernando RI, Wimalasena J, Foster JS, Henley DC, Elder RF - Reprod. Biol. Endocrinol. (2003)

Immunolocalization of ERβ and ERα in the late trophoblast culture (68 h). [A] Isolated CT cells are unstained (arrowhead) but strong ERβ nuclear and cytoplasmic immunostaining accompanies young ST aggregates (y). [B] CT cell associated with ST shows ERβ immunoreactivity (arrowhead). [C] Mature ST (m) shows a diminution of ERβ nuclear staining (solid > white arrows) with persisting cytoplasmic immunoreactivity. [D] Secondary antibody control. [E] Isolated CT cells show strong ERα immunostaining (arrowhead) persisting in young ST aggregates [y, E and F] but diminishing in mature ST [m; F and G]. [H] Secondary antibody control.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC155643&req=5

Figure 4: Immunolocalization of ERβ and ERα in the late trophoblast culture (68 h). [A] Isolated CT cells are unstained (arrowhead) but strong ERβ nuclear and cytoplasmic immunostaining accompanies young ST aggregates (y). [B] CT cell associated with ST shows ERβ immunoreactivity (arrowhead). [C] Mature ST (m) shows a diminution of ERβ nuclear staining (solid > white arrows) with persisting cytoplasmic immunoreactivity. [D] Secondary antibody control. [E] Isolated CT cells show strong ERα immunostaining (arrowhead) persisting in young ST aggregates [y, E and F] but diminishing in mature ST [m; F and G]. [H] Secondary antibody control.
Mentions: Quantitative evaluation of ERα immunoreactivity in quiescent (q), parental (p) and daughter cells (d) during asymmetric divisions is shown in Fig. 3E. The ERα immunoreactivity was lowest in quiescent cells and highest in parental cells during asymmetric division. However, daughter cells showed significant diminution of ERα when compared to parental cells. Statistical analysis indicated overall ANOVA P < 0.0001, and differences between all columns P < 0.01.

Bottom Line: During human pregnancy, the production of 17-beta-estradiol (E2) rises steadily to eighty fold at term, and placenta has been found to specifically bind estrogens.In trophoblast cultures, development of ST aggregates was associated with diminution of ER-alpha and appearance of ER-beta immunoreactivity.Our data indicate that during trophoblast differentiation, the ER-alpha is associated with a less, and ER-beta with the more differentiated state.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory for Development, Differentiation and Cancer, Department of Obstetrics and Gynecology, The University of Tennessee Graduate School of Medicine, Knoxville, Tennessee 37920, USA. rbe@utk.edu

ABSTRACT
During human pregnancy, the production of 17-beta-estradiol (E2) rises steadily to eighty fold at term, and placenta has been found to specifically bind estrogens. We have recently demonstrated the expression of estrogen receptor alpha (ER-alpha) protein in human placenta and its localization in villous cytotrophoblast (CT), vascular pericytes, and amniotic fibroblasts. In vitro, E2 stimulated development of large syncytiotrophoblast (ST) aggregates. In the present study we utilized ER-beta affinity purified polyclonal (N19:sc6820) and ER-alpha monoclonal (clone h-151) antibodies. Western blot analysis revealed a single approximately 52 kDa ER-beta band in chorionic villi (CV) protein extracts. In CV, strong cytoplasmic ER-beta immunoreactivity was confined to ST. Dual color immunohistochemistry revealed asymmetric segregation of ER-alpha in dividing villous CT cells. Prior to separation, the cell nuclei more distant from ST exhibited high ER-alpha, while cell nuclei associated with ST showed diminution of ER-alpha and appearance of ER-beta. In trophoblast cultures, development of ST aggregates was associated with diminution of ER-alpha and appearance of ER-beta immunoreactivity. ER-beta was also detected in endothelial cells, amniotic epithelial cells and fibroblasts, extravillous trophoblast (nuclear and cytoplasmic) and decidual cells (cytoplasmic only). In addition, CFK-E12 (E12) and CWK-F12 (F12) monoclonal antibodies, which recognize approximately 64 kDa ER-beta with hormone binding domain, showed nuclear-specific reactivity with villous ST, extravillous trophoblast, and amniotic epithelium and fibroblasts. Western blot analysis indicated abundant expression of a approximately 64 kDa ER-beta variant in trophoblast cultures, significantly higher when compared to the chorionic villi and freshly isolated trophoblast cell protein extracts. This is the first report on ER-beta expression in human placenta and cultured trophoblast. Our data indicate that during trophoblast differentiation, the ER-alpha is associated with a less, and ER-beta with the more differentiated state. Enhanced expression of approximately 64 kDa ER-beta variant in trophoblast cultures suggests a unique role of ER-beta hormone binding domain in the regulation of trophoblast differentiation. Our data also indicate that asymmetric segregation of ER-alpha may play a role in asymmetric division of estrogen-dependent cells.

Show MeSH
Related in: MedlinePlus