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. 2023 Mar 29;90:104535. doi: 10.1016/j.ebiom.2023.104535

Fig. 3.

Fig. 3

AMH levels and ultrastructural organization of the ME in control and PCOS-animals. (a) Experimental design employed to generate PAMH offspring. (b) Blood samples were derived from control and PAMH female mice at postnatal day 25 (P25; n = 6 controls, n = 6 PAMH) and in adult diestrous animals (2 months; n = 9 controls, n = 11 PAMH) and AMH concentration was measured by ELISA. Mean AMH levels are significantly higher in PAMH females as compared to control mice (unpaired two-tailed Student's t test, ∗P < 0.05). Experiments were replicated three times with comparable results. (c and d) Representative electron micrographs of GnRH-immunoreactive axon terminals (immunogold) from hypothalamic ME dissected from diestrous control (CNTR) or PAMH mice (P90–P120). GnRH axonal endings are highlighted in blue, other neuroendocrine nerve terminals contained in the external layer of the ME are pseudo-coloured in yellow, tanycytic end-feet are pseudo-coloured in green and the pericapillary space (p.s.) in pink. (e) Quantitative analysis of the percentage of area occupied by tanycytes in the external zone of the ME at 2 μm from the pericapillary space, in ME explants from CNTR diestrus mice and PAMH mice (∗P < 0.05; Wilcoxon–Mann–Whitney test; n = 5 controls, N = 58 number of analysed EM photomicrographs in controls; n = 3 PAMH mice, N = 35 number of analysed EM photomicrographs in PAMH mice). (f) Quantitative analysis of the percentage of GnRH nerve terminals located at less than 1 μm from the pericapillary space in the external zone of the ME, in explants from control and PAMH diestrous mice (∗P < 0.05; Wilcoxon–Mann–Whitney test; n = 5 controls, N = 100 number of GnRH-immunoreactive axon terminals measured per explant in controls; n = 3 PAMH mice, N = 100 number of GnRH-immunoreactive axon terminals measured per explant in PAMH mice). The horizontal line in each plot corresponds to the median value. The vertical line represents the 25th–75th percentile range. (g) Correlation of % of GnRH terminals located at less than 1 μm from the pericapillary space versus the % of area occupied by tanycytes in the external zone of the ME at 2 μm from the pericapillary space (r = −0.8, P = 0.015, Spearman's correlation). (h) Electron microscopic image of GnRH-positive terminals (arrows) in the median eminence located in proximity of the portal capillary basal lamina. (i) Electron microscopic image at the same magnification as h showing the lack of gold particles in an ultra-thin section of the median eminence after omission of the primary antibody. (j and k) Representative electron micrographs of GnRH-immunoreactive axon terminals from hypothalamic ME of diestrous control (CNTR) and PAMH mice (P90–P120). Dotted lines depict the surface of a GnRH terminal in a CNTR mouse (blue dotted lines, j) and in a PAMH mouse (red dotted lines, k). Arrows point to individual 18 nm gold particles showing immunogold labelling in large dense-core vesicles. (l) Quantitative analysis of the number of gold particles contained in the GnRH terminals (n = 5 controls, N = 877 number of GnRH terminals analysed in control mice; n = 3 PAMH, N = 682 number of GnRH terminals analysed in PAMH mice). Wilcoxon–Mann–Whitney test, ∗∗∗P < 0.0001. The horizontal line in each plot corresponds to the median value. The vertical line represents the 25th–75th percentile range. Abbreviations: b.l., basal lamina; cap, capillary; ME, median eminence; n, neurons; p.s., pericapillary space; tan, tanycytes; EM, electron microscopy.