Fig 2: Sema7A expression in ME tanycytes varies as a function of the oestrous cycle.(a) Western blotting for Sema7A in the ME during the oestrous cycle. Bar graph illustrates the mean ratio (±s.e.m.) of the signal intensity obtained for Sema7A to that of total actin (n=4 independent experiments per oestrous cycle phase; 3- to 4-month-old females rats, Kruskal–Wallis test. *P<0.05, values are normalized to the Diestrus group). (b) Western blot analysis for Sema7A and actin in the ME of control OVX rats and those treated with saline, 17β-estradiol 3-benzoate (OVX+E2), progesterone (OVX+P4) and 17β-estradiol 3-benzoate+progesterone (OVX+E2+P4). N=4; 3- to 4-month-old females rats, Kruskal–Wallis test *P<0.05, values are normalized to the OVX group. (c–f) Immunofluorescence for Sema7A, GnRH, BSLI and vimentin in coronal sections of the rat ME (n=4 brains for each experiment, 3- to 4-month-old females rats. Experiments were repeated five times). (c) Simultaneous triple-labelling experiments for Sema7A (green), GnRH (blue) and the endothelial-cell marker BSLI (red). (d–f) Confocal images of double immunofluorescence for Sema7A (green) and vimentin (red). Tanycytic processes (d, arrows) exhibit Sema7A immunoreactivity (c, arrows, f). 3 V, third ventricle. (g) Tanycyte isolation by FACS and real-time PCR analysis in Tomato-positive (pos; tanycytes) and -negative cells (neg) in tdTomatoloxP/+ mice infused i.c.v. with Tat-cre. (h) Tat-cre injection into the third ventricle results in Cre-Lox recombination (red) exclusively in tanycytes (vimentin immunoreactive, green), whose cell bodies line the wall of the third ventricle (asterisks). (i) Real-time PCR analysis of DARPP32 (n=5 Tat-cre; tdTomatoloxP/+) mRNA in tanycytes isolated from Tat-cre;tdTomatoloxP/+ mice, normalized to values in Tomato-negative cells. *P<0.01, Wilcoxon–Mann–Whitney test. (l) Real-time PCR analysis of Sema7A mRNA in tanycytes isolated from Tat-cre;tdTomatoloxP/+ mice, normalized to values in Tomato-negative cells isolated from Tat-cre;tdTomatoloxP/+ mice at diestrus and estrus (n=4 Diestrus Tat-cre; tdTomatoloxP/+ versus n=4 Estrus Tat-cre; tdTomatoloxP/+). *P<0.05, Kruskal–Wallis test. Values shown are means±s.e.m. Scale bars, (c) 40 μm, (d–f) 80 μm, (h) 100 μm.

Fig 3: Schematic of the Sema7A-action in the ME of the hypothalamus.Schematic representation of the bifunctional mechanism of action of Sema7A on tanycytes (yellow cells) and GnRH terminals (blue) as a function of the oestrous phase. During diestrus, progesterone binds to its specific receptors, PR-A/B in tanycytes. Tanycytes then overexpress TGF-ß1, which is responsible for the rise in Sema7A expression in tanycytes. Sema7A induces the elongation of tanycytic end feet through Itgb1 activation and retraction of GnRH nerve endings through the engagement of PlexinC1 and inactivation of Rap1 in GnRH terminals.

Fig 4: Sema7A promotes tanycytic end-feet growth through the Itgb1 signalling pathway.(a) Representative western blotting showing the expression of PlexinC1 and actin in primary tanycytes, in GnV3 cells and in median eminence (ME) lysates (positive control). Double amount of tanycytes cell lysates as compared with GnV3 protein extracts were intentionally loaded (see actin bands) to ascertain the lack of PlexinC1 expression in these cells. (b) Sema7A activates the Itgb1/MAPK pathway in primary tanycytes. Cells were treated with the indicated doses of recombinant Sema7A for 30 min and total cell lysates subjected to 3–8% SDS–PAGE and immunoblotting with the indicated antibodies. Experiments were done in triplicate with comparable results. (c–j) Representative photomicrographs of a scratch-wound-healing assay performed on primary tanycytes treated or not for 24 h with recombinant Sema7A (250 ng ml-1; n=5 independent experiments). Continuous (e,f) and dotted (g,h) lines indicate the borders of the scratch at the initial recording time. After 24 h of exposure to Sema7A, tanycytes invade a greater area of the scratch than in control cultures (e–h). (g–j) Immunolabelling of tanycytes for paxillin (green) and F-actin (red) at 1 day in vitro (div) after treatment shows that Sema7A induces cytoskeletal remodelling in tanycytes, which quickly develop end-feet-like structures (arrowheads, h) barely seen in control cells (arrowheads, g). Scale bars, (c–f) 30 µm, (g–j) 20 µm.

Fig 5: Tanycytic Sema7A expression is progesterone and TGF-ß1 dependent.(a) Western blot analysis of Sema7A-secreted protein in the conditioned media (CM) of four representative tanycytic cultures 48 h after plating. (b) Representative western blotting showing the expression of progesterone receptors, PR-A and PR-B, in primary tanycytes cell lysates (n=4). Primary tanycytes were treated with progesterone (P) with or without its antagonist Mifepristone (Mif), and TGF-ß1 (c) and Sema7A (d) expression were evaluated by immunoblotting on total cell lysates (n=3 independent experiments for each treatment condition). (e) Representative immunoblot depicting a TGF-ß1 dose–response experiment (6 h of treatment) on Sema7A protein levels in primary tanycytes. Bar graphs illustrate the mean ratio of the signal intensity obtained for Sema7A to that for total actin (n=3 independent experiments per treatment; Kruskal–Wallis test *P<0.05; values are normalized to the control groups).