Parity leads to differentiation and decreases the Wnt/Notch signaling ratio in basal stem/progenitor cells. (A) Bar graph depicting the number of gene-expression changes in FACS-sorted mammary stromal and epithelial cell subpopulations from parous mice compared with age-matched virgin control mice by using a cut-off of fold change > 1.5 and an adjusted P value < 0.05. By far the most gene-expression changes were observed in basal stem/progenitor cells. Three independent experiments were performed with 10 mice (five virgins; five parous) per experiment. (B) Schematic illustration of prominent gene-expression changes in FACS-sorted basal stem/progenitor cells from parous as compared with age-matched virgin control mice. Fold changes are shown in parentheses with upregulated genes denoted as positive (+), and downregulated genes, as negative (-). Differentiation genes were upregulated (blue), Wnt target genes were downregulated (green), Wnt inhibitor Sfrp1 was upregulated (green) and overall Notch signaling (orange) was increased in basal stem/progenitor cells from parous mice. (C) qPCR validation of the changes in gene expression in basal stem/progenitor cells of parous mice. All classic Wnt target genes were downregulated, including Lgr5, Axin2, and versican (Vcan), whereas the more ubiquitously regulated target Myc was unchanged. In all cases, fold changes are shown relative to cells from age-matched virgin control mice. Ct values were normalized to the reference genes Hprt and Ubc . Data represent the mean ± SEM of three independent experiments with 10 mice (five virgins; five parous) per experiment. (D) Representative images and quantification of immunostaining for the Wnt target gene versican in mammary gland sections from age-matched virgin and parous mice in estrus. Scale bar, 50 μm. Quantitative data represent the mean ± SEM from 60 randomly selected images from three virgin and three parous mice.