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. 2020 Sep 21;11:4752. doi: 10.1038/s41467-020-18500-x

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© The Author(s) 2020

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 3 — a Upper panel: Macroscopic analysis of the pituitary gland from 27-week-old WT (+/+) and Ghrhr^Gpr101 (+/T) mice. Lower panel: microscopic visualization of pituitary sections after H&E staining. Scale bar: 150 µm. b High magnification of anterior and posterior pituitaries stained with H&E. Scale bar: 15 µm. c Left panel: Immunohistochemical staining of the anterior pituitary sections with the cell proliferation marker Ki-67 (scale bar: 15 µm). Blue arrows indicate Ki-67-positive nuclei staining. Right panel: quantification of the Ki-67 labeling index in pituitary sections of 27-week-old WT (+/+) and Ghrhr^Gpr101 (+/T) mice. The Ki-67 labeling index represents the percentage of positive nuclei stained by anti-Ki-67 antibody. n = 11 independent areas from staining section of WT (+/+) and Ghrhr^Gpr101 (+/T) (n = 4 mice per group, p = 0.9487). d Immunofluorescent staining of GH (green) and Ki-67 (Red). Scale bar: 10 µm. e Reticulin staining of the anterior and posterior pituitaries of WT (+/+) and Ghrhr^Gpr101 (+/T) mice (scale bar: 15 µm). f The expression of GH in the pituitary of WT and Ghrhr^Gpr101 mice (aged 27 weeks n = 5, p = 0.0079) was quantified by RT-qPCR. GAPDH was used as a control housekeeping gene. g The content of the GH protein was quantified by ELISA and normalized to total protein in pituitary lysates of both males and females of the WT (+/+) and Ghrhr^Gpr101 (+/T) genotypes (aged 29 weeks, n = 4, p = 0.0286). h Ex vivo pituitary superfusion analysis. Pituitary glands of WT (+/+) and Ghrhr^Gpr101 (+/T) (aged 29 weeks, n = 3 mice) were superfused at 0.1 ml min⁻¹ in superfusion chambers. Effluents were collected every 5 min for GH measurement. GHRH (100 nM) was added to the medium for 15 min and KCl (0.03 M) for 20 min (as it is indicated with arrows). GH secretion was quantified by ELISA at indicated time points. All the experiments were independently repeated three times unless stated otherwise. F Female, M Male. For statistical analysis of all data, a two-sided Mann–Whitney test was used unless stated otherwise. ns not significantly different; *p < 0.05; **p < 0.01; ***p < 0.001.