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.