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. 2021 Apr 23;11:8847. doi: 10.1038/s41598-021-87492-5

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© The Author(s) 2021

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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/.

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Characterization of the ovaries and uteri of transgenic mice. (A): H&E staining on representative ovary samples of TG-LHR-frt-200 mouse (left panel) and TG-LHR-frt-100 mouse (right panel). The ovaries do not show any histological alterations: antral follicles (1), corpus luteum (2) and the oviduct (3) are indeed still visible. Bar = 100 μm. (a): Resuming table in which the mean number ± SEM of antral and atretic follicles observed in each ovary slide is reported. We evaluated 5 mice per group (n = 5), 5 slides per mouse. (B): Table summarizing the characterization of the uteri of TG and WT mice in terms of size (x and y axes) and morphometry parameters (UR, ICM and LEH). Mice 3-12 months old: UR: TG vs WT p = 0.43; ICM: TG vs WT p = 0.21; LEH: TG vs WT p = 0.96. Mice > 12 months old: UR: TG vs WT p = 0.72; ICM: TG vs WT p = 0.64; LEH: TG vs WT p = 0.87. Values are shown as mean ± SEM. (b): The uterus longitudinal axis (x) and the transversal axis (y) of TG (of both the TG lines) and WT mice are reported. In the box a detail of a representative H&E staining showing UR, ICM and LEH is reported. (C): Representative IHC pictures of the uteri of WT and transgenic mice labelled with CK-8: (C’): 13 months-old WT mouse negative to cytokeratin 8; (C’’): 9 months-old TG-LH-R-frt-100, weakly positive; (C’’’): 9 month-old TG-LH-R-frt-111 (belonging to the TG-LH-R-frt-100 line) strongly positive to CK-8. Nuclei are counterstained with hematoxylin. Bar = 200 μm (c): Histogram summarizing CK-8 scoring in the different uteri samples (WT, TG-LH-R-frt-100 and TG-LH-R-frt-111). Sample size: 6 mice per group. (D): Representative IHC pictures of the uteri of WT and transgenic mice labelled with anti Ki67 antibody. Left panel: WT mouse. Right panel: TG-LH-R-frt mouse. Nuclei are counterstained with hematoxylin. Bar = 200 μm (d): Table summarizing Ki67 scoring in the different samples. The mean percentage of labeled nuclei evaluated in three different areas is reported. Mice are divided into two groups of age: 3-12 months old and older than 12 months. Statistically significant differences are observed between WT vs TG animals in glandular cells (TG vs WT p = 0.008, Fisher’s exact test) and stromal cells (TG vs WT p = 0.015, Fisher’s exact test) of 3-12 months-old mice. Sample size: 8 TG-LH-R-frt mice, 6 WT. 4 TG and 3 WT mice for each group of age. (E): Representative IHC pictures of the uteri of WT and TG mice labelled with anti α-sma antibody. Left panel: WT mouse. Right panel: TG-LH-R-frt mouse (of both the TG lines). (E’): Uterine slice of TG-LH-R-frt mouse stained with CD31 antibody: the staining is not present in the gland. (E’’): Uterine slice of TG-LH-R-frt mouse stained with FOXA2 antibody: only the glands are positive. Nuclei are counterstained with hematoxylin. Bar = 200 μm.