Identification of estrogen-responsive cells involved in cell proliferation in response to estrogen

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Research project 11)Using rat anterior pituitary cells in culture, we established triple immunostaining for Ki67, estrogen receptor (ER), and prolactin (PRL).2)When estrogen stimulated proliferation of lactotrophs, the proportion of ER-imnumoreadive cells in proliferating lactctrophs was decreased. The proportion of the ER-immunoreactive cells was not changed when estrogen inhibited IGF-1-induced proliferation.Research project 21)We produced recombinant adenovirus vectors that carry PRL promoter-driven dominant negative ER mutants.2)Using an adenovirus vector that carries PRL promoter-driven luciferase reporter gene, infection conditions in pituitary cultures were addressed. A maximal infection efficiency was obtained by infection at 5 MOI during the early culture period in the presence of insulin. Even at this infection efficiency, the proportion of reporter gene-expressing cells in lactotrophs was no more than 30%.Research project 31)We produced several lines of transgenic rats carrying PRL promoter-driven GFP gene.2)Among the transgenic rat lines, we chose MI-11-90 line on the basis of the fluorescence intensity, the proportion of GFP-expressing cells in lactotraphs, and the lactotroph-specificity for GFP expression.3)Using FACS, we succeeded in selectively collect ting GFP-expressing pituitary cells, 90% of which were lactoarophs.4)The stimulatory action of estrogen on lactotroph proliferation was enhanced in this lactotroph-enriched population while its inhibitory action of proliferation in the presence of IGF-1 was completely eliminated.