Binding of environmental estrogens to estrogen receptor α and β of japanese quail (Coturnix japonica)‎

Abstract EN

The adverse effects of estrogenic compounds have recently received much attention in particular their activity on the impaired sexual differentiation and reproductive dysfunction.

Estrogenic chemicals must bind to estrogen receptors (ER) and modulate estrogen-inducible gene expression.

In order to examine the binding affinity of various environmental estrogens to bacterially-expressed ER α and ER β of quail, first-strand cDNA was synthesized from total RNA isolated from mature female quail liver with oligo-dT primed reverse transcription.

The cDNA included the hinge domain, the ligand-binding domain and the C-terminal domain of quail ER a and ER β.

The def domains of quail ERa (qERa-def) and ER a (qER β-def) were amplified by PCR and the PCR product was ligated into GST fusion protein expression vector, pGEX-6P-3, and transfected to E.

coli DH5a strain.

The binding assay using clear supernatant of cell lysates was performed by incubation with [3H]estradiol-17 β and increasing concentrations of competitor at 4°C for 24 h.

After removing unbound steroids by addition of dextran-coated charcoal, radioactivity of the supernatant was counted by the liquid scintillation counter.

Quail ER expressed bacterially showed one single class of binding site for estradiol-17 β with a dissociation constant of 1.74 ± 0.34 × 10-10 M for ERa and 4.90 ± 0.16 × 10-9 M for ER β.

The competition studies indicated that relative binding affinities for synthetic estrogens, diethylstilbestrol and ethinyl estradiol, were very high, while those of xenoestrogens, bisphenol A and nonylphenol, were very low.

Phytoestrogens, coumestrol and genistein can compete with estradiol-17b (E2) with higher binding affinity for ER β than Era.