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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 ERs in placental membranes and ERβ peptide-absorbed antibody control. [A] Amniotic epithelium with ERβ+ (solid arrow) and ERβ- nuclei (white arrow). [B] Basal plate EVT with high (solid arrow) and diminishing ERβ staining (white arrow). [C] Immunolocalization of ERβ in vascular endothelial cells (ec) of a stem villus. [D] Cytotrophoblast cell merging with ST (st) shows nuclear ERβ immunoreactivity (solid arrow) but ST nuclei are unstained (white arrow). [E] Rarely seen nuclear ERα immunoreactivity in the basal plate decidual cells. [F] Control. [G] EVT in lower magnification, representing ERβ positive control for [H], which is a parallel section incubated with peptide-absorbed ERβ antibody (PA/ERβ). Bar in C = 50 μm for [A–C], 20 μm for [D], and 100 μm for [E-H]. No nuclear counterstain except in panel [D]. Details in text.
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Figure 2: Immunolocalization of ERs in placental membranes and ERβ peptide-absorbed antibody control. [A] Amniotic epithelium with ERβ+ (solid arrow) and ERβ- nuclei (white arrow). [B] Basal plate EVT with high (solid arrow) and diminishing ERβ staining (white arrow). [C] Immunolocalization of ERβ in vascular endothelial cells (ec) of a stem villus. [D] Cytotrophoblast cell merging with ST (st) shows nuclear ERβ immunoreactivity (solid arrow) but ST nuclei are unstained (white arrow). [E] Rarely seen nuclear ERα immunoreactivity in the basal plate decidual cells. [F] Control. [G] EVT in lower magnification, representing ERβ positive control for [H], which is a parallel section incubated with peptide-absorbed ERβ antibody (PA/ERβ). Bar in C = 50 μm for [A–C], 20 μm for [D], and 100 μm for [E-H]. No nuclear counterstain except in panel [D]. Details in text.

Mentions: Specificity of ERβ polyclonal antibody was confirmed by absorption with blocking peptide. The blocking peptide sc-6820 P (Santa Cruz Biotechnology) for N-19 ERβ antibody was mixed with the ERβ antibody at a 10:1 weight ratio, and incubated overnight at 4°C then 2 h at room temperature before the peptide-absorbed antibody was utilized for immunohistochemistry (PA/ERβ, Figure 2H) and western blot controls (PA/ERβ, Figure 6B). As an additional negative control, we tested reactivity of ERα and ERβ antibodies with cdk2 immunoprecipitates (see below).


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 ERs in placental membranes and ERβ peptide-absorbed antibody control. [A] Amniotic epithelium with ERβ+ (solid arrow) and ERβ- nuclei (white arrow). [B] Basal plate EVT with high (solid arrow) and diminishing ERβ staining (white arrow). [C] Immunolocalization of ERβ in vascular endothelial cells (ec) of a stem villus. [D] Cytotrophoblast cell merging with ST (st) shows nuclear ERβ immunoreactivity (solid arrow) but ST nuclei are unstained (white arrow). [E] Rarely seen nuclear ERα immunoreactivity in the basal plate decidual cells. [F] Control. [G] EVT in lower magnification, representing ERβ positive control for [H], which is a parallel section incubated with peptide-absorbed ERβ antibody (PA/ERβ). Bar in C = 50 μm for [A–C], 20 μm for [D], and 100 μm for [E-H]. No nuclear counterstain except in panel [D]. Details in text.
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Related In: Results  -  Collection

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Figure 2: Immunolocalization of ERs in placental membranes and ERβ peptide-absorbed antibody control. [A] Amniotic epithelium with ERβ+ (solid arrow) and ERβ- nuclei (white arrow). [B] Basal plate EVT with high (solid arrow) and diminishing ERβ staining (white arrow). [C] Immunolocalization of ERβ in vascular endothelial cells (ec) of a stem villus. [D] Cytotrophoblast cell merging with ST (st) shows nuclear ERβ immunoreactivity (solid arrow) but ST nuclei are unstained (white arrow). [E] Rarely seen nuclear ERα immunoreactivity in the basal plate decidual cells. [F] Control. [G] EVT in lower magnification, representing ERβ positive control for [H], which is a parallel section incubated with peptide-absorbed ERβ antibody (PA/ERβ). Bar in C = 50 μm for [A–C], 20 μm for [D], and 100 μm for [E-H]. No nuclear counterstain except in panel [D]. Details in text.
Mentions: Specificity of ERβ polyclonal antibody was confirmed by absorption with blocking peptide. The blocking peptide sc-6820 P (Santa Cruz Biotechnology) for N-19 ERβ antibody was mixed with the ERβ antibody at a 10:1 weight ratio, and incubated overnight at 4°C then 2 h at room temperature before the peptide-absorbed antibody was utilized for immunohistochemistry (PA/ERβ, Figure 2H) and western blot controls (PA/ERβ, Figure 6B). As an additional negative control, we tested reactivity of ERα and ERβ antibodies with cdk2 immunoprecipitates (see below).

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