Estrogenic in vitro assay on mouse embryonic Leydig cells.
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
Gina
G
Department of Public Health and Cell Biology, University of Rome Tor Vergata, Rome, Italy.
Farini
Donatella
D
De Felici
Massimo
M
D000818:Animals; D001681:Biological Assay; D002454:Cell Differentiation / Q000187:drug effects / Q000235:genetics; D002478:Cells, Cultured; D004958:Estradiol / Q000031:analogs & derivatives; D004965:Estrogen Antagonists / Q000494:pharmacology*; D047628:Estrogen Receptor alpha / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D004967:Estrogens / Q000235:genetics / Q000494:pharmacology*; D004970:Estrone / Q000235:genetics; D005260:Female; D000077267:Fulvestrant; D017930:Genes, Reporter / Q000187:drug effects; D006801:Humans; D007985:Leydig Cells / Q000378:metabolism*; D008156:Luciferases / Q000235:genetics; D008297:Male; D051379:Mice; D048789:Phytoestrogens / Q000494:pharmacology; D020218:Response Elements / Q000235:genetics; D013737:Testis / Q000196:embryology*; D018808:Transcription Factor AP-1 / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D004965:Estrogen Antagonists; D047628:Estrogen Receptor alpha; D004967:Estrogens; D048789:Phytoestrogens; D018808:Transcription Factor AP-1; D000077267:Fulvestrant; D004970:Estrone; D004958:Estradiol; D008156:Luciferases