We and others have reported that mouse embryonic testes contain a subpopulation of somatic cells expressing estrogen receptor alpha (ERalpha). In order to provide evidence for a possible direct estrogen effect on mammalian testes from the early stage of their differentiation, here we devised a method for the in vitro culture of the ERalpha-expressing cells from 12.5 days post coitum mouse testes and their transfection with plasmids containing the classical estrogen responsive element (ERE) or the alternative estrogen AP-1 responsive element upstream of the luciferase reporter gene (ERE-Luc and AP-1-Luc). StAR immunopositivity of the most part of the ERalpha+ cells grown in culture and subjected to the estrogenic assay, allowed their identification as embryonic Leydig cells. Maximum induction of the ERE-Luc activity was achieved with 10 nM 17-beta estradiol (E2), from 1.7 to 3-fold in such cells and from 2.3 to 5.7-fold in MCF-7 cells used for comparison; the anti-estrogen ICI 182.780 abolished such effects. AP-1-Luc was less sensitive to E2 in both cell types (10 nM E2, 1.2 to 2.7-fold increase in embryonic Leydig cells; about 3-fold in MCF-7 cells) and the effect was not ICI-dependent. Eventually, we stimulated the transfected cells with various xenoestrogens such as lindane, bisphenol A or mono-(2-ethylhexyl) pthalate and with the phytoestrogen zeralenone obtaining evidence for ERE-Luc, but not AP-1-Luc stimulation in embryonic Leydig cells. These results represent evidence of functional ERalpha-dependent genomic pathways in embryonic Leydig cells and describe an in vitro assay suitable for evaluating the activity of putative estrogenic compounds on such cells.

La Sala, G., Farini, D., de Felici, M. (2010). Estrogenic in vitro assay on mouse embryonic Leydig cells. THE INTERNATIONAL JOURNAL OF DEVELOPMENTAL BIOLOGY, 54(4), 717-722 [10.1387/ijdb.092883gs].

Estrogenic in vitro assay on mouse embryonic Leydig cells

La Sala G.;Farini D.;de Felici M.
2010-01-01

Abstract

We and others have reported that mouse embryonic testes contain a subpopulation of somatic cells expressing estrogen receptor alpha (ERalpha). In order to provide evidence for a possible direct estrogen effect on mammalian testes from the early stage of their differentiation, here we devised a method for the in vitro culture of the ERalpha-expressing cells from 12.5 days post coitum mouse testes and their transfection with plasmids containing the classical estrogen responsive element (ERE) or the alternative estrogen AP-1 responsive element upstream of the luciferase reporter gene (ERE-Luc and AP-1-Luc). StAR immunopositivity of the most part of the ERalpha+ cells grown in culture and subjected to the estrogenic assay, allowed their identification as embryonic Leydig cells. Maximum induction of the ERE-Luc activity was achieved with 10 nM 17-beta estradiol (E2), from 1.7 to 3-fold in such cells and from 2.3 to 5.7-fold in MCF-7 cells used for comparison; the anti-estrogen ICI 182.780 abolished such effects. AP-1-Luc was less sensitive to E2 in both cell types (10 nM E2, 1.2 to 2.7-fold increase in embryonic Leydig cells; about 3-fold in MCF-7 cells) and the effect was not ICI-dependent. Eventually, we stimulated the transfected cells with various xenoestrogens such as lindane, bisphenol A or mono-(2-ethylhexyl) pthalate and with the phytoestrogen zeralenone obtaining evidence for ERE-Luc, but not AP-1-Luc stimulation in embryonic Leydig cells. These results represent evidence of functional ERalpha-dependent genomic pathways in embryonic Leydig cells and describe an in vitro assay suitable for evaluating the activity of putative estrogenic compounds on such cells.
2010
Pubblicato
Rilevanza internazionale
Articolo
Esperti anonimi
Settore MED/50 - SCIENZE TECNICHE MEDICHE APPLICATE
English
Con Impact Factor ISI
Animals; Cell Differentiation; Cells, Cultured; Estradiol; Estrogen Antagonists; Estrogen Receptor alpha; Estrogens; Estrone; Female; Genes, Reporter; Humans; Leydig Cells; Luciferases; Male; Mice; Phytoestrogens; Response Elements; Testis; Transcription Factor AP-1; Transfection; Biological Assay
La Sala, G., Farini, D., de Felici, M. (2010). Estrogenic in vitro assay on mouse embryonic Leydig cells. THE INTERNATIONAL JOURNAL OF DEVELOPMENTAL BIOLOGY, 54(4), 717-722 [10.1387/ijdb.092883gs].
La Sala, G; Farini, D; de Felici, M
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2108/191010
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