Fig. 5.

Expression of AMH and AMHR2 in ME tanycytes. (a) Tanycyte isolation by FACS and real-time PCR analysis in Tomato-positive (pos; tanycytes) and -negative cells (neg) in tdTomato^loxP/+ mice infused i.c.v. with Tat-cre. Tat-cre injection into the third ventricle results in Cre-Lox recombination (red) exclusively in tanycytes. (b) Real-time PCR analysis of indicated genes in tanycytes isolated from Tat-cre;tdTomato^loxP/+ mice, normalized to values in Tomato-negative cells (n = 6 Tomato Pos and n = 6 Tomato Neg; ∗P < 0.05, ∗∗P < 0.005, Wilcoxon–Mann–Whitney test). The horizontal line in each plot corresponds to the median value. The vertical line represents the 25th–75th percentile range. (c and d) Representative immunofluorescence experiment for AMH_C in coronal sections of the mouse ME depicting AMH expression in ME tanycytes and along their processes (n = 3 brains for each experiment, 3- to 4-month-old female mice. Experiments were repeated three times). (e and f) AMHR2 expression was analysed in coronal sections of ME of AMHR2-Cre;Z/E^{loxP/-,βgeo/+} (AMHR2-EGFP) knock-in reporter line (n = 3 brains for each experiment, 3- to 4-month-old female mice. Experiments were done in triplicates). EGFP is expressed by tanycytes (Tan), endothelial cells (ec) and by cells of the arcuate nucleus (ARN). Scale bars, (c and e) 50 μm; (d and f) 20 μm. (g) Schematic representing the generation of primary tanycytic cultures. (h) Western blotting for AMH and AMHR2 in rat tanycytic primary cultures. (i and j) Representative photomicrographs of a scratch-wound-healing assay performed on primary tanycytes treated or not for 48 h with recombinant AMH (100 ng/ml; n = 5 independent experiments). Dotted lines indicate the borders of the scratch. After 48 h of exposure to AMH, tanycytes invade a greater area of the scratch in control cultures (PBS-treated; i) than in AMH-treated cultures (j). Tanycytes were stained using F-actin (red). Scale bars, (i and j) 100 μm. (k) Quantitative analysis of the % area covered by tanycytes in control and AMH-treated conditions (n = 13 Control and n = 17 AMH-treated; ∗∗∗P < 0.0005, Wilcoxon–Mann–Whitney test). The horizontal line in each plot corresponds to the mean value. The vertical line represents the S.E.M.