Fig 2: ERa and LCN2 expression in liver tissue. (A) Liver weights of wild-type (WT, male: n = 8, female: n = 9) and Esr1 null mice (male: n = 8, female: n = 6) mice. Statistical analysis was conducted by performing a two-way ANOVA. Significant differences between groups are marked with asterisks: ** p < 0.01, *** p < 0.001. Data are displayed as means ± SD. (B) Paraffin-embedded WT (female n = 9, male n = 8) and Esr1 knockout mice (female n = 6, male n = 8) livers were subjected to staining and protein analysis. Immunohistochemical detection of LCN2 was performed in liver tissues using 3,3'-diaminobenzidine (DAB) as a chromogen. The sections were counterstained with hematoxylin. (C) Male liver protein extracts were subjected to Western blot analysis for detection of ERa and LCN2. GAPDH was used to ensure equal protein loading. Unspecific bands are marked with asterisks (*) and are separated from specific bands by dashed lines. Protein expression of LCN2 was quantified densitometrically and plotted relative to GAPDH expression. The Student’s t-test was used for statistical analysis. Data are displayed as means ± SD and non-significant results are marked with ns. (D) Nuclear expression of ERa was analyzed in liver tissues using a NovaRed-HRP substrate. The sections were counterstained with methyl green. All magnifications in (B,D): 100×; scale bars: 100 µm.

Fig 3: Depletion of estrogen receptor 1 (Esr1) in mice. (A) Traditional genotyping PCR was performed in wild-type (WT, +/+), Esr1 null (-/-), and heterozygous Esr1 mice (+/-). Top bands (550 bp) represent the depleted and bottom bands (280 bp) of the WT alleles. Heterozygous animals displayed both alleles. (B) Representative reproductive tracts from adult WT and knockout females were placed on scale paper to examine phenotypical changes. (C) Total body weights of WT (female n = 9, male n = 8) and Esr1-/- mice (female n = 6, male = 8) were measured and are shown as means ± SD. For statistical analysis, a two-way ANOVA was performed. Significant differences between groups are indicated by asterisks: ** p < 0.01, *** p < 0.001. (D) Hematoxylin and eosin staining was performed for histological analysis of female ovaries taken from WT and Esr1 null mice. Scale bars: 500 µm.

Fig 4: Esr1-deficient testes show increased LCN2 and STAR expression. Testes from wild-type (WT, n = 8) and Esr1-/- null mice (n = 8) were either embedded in paraffin for immunostaining or processed for protein and RNA analyses. (A) Hematoxylin and eosin (H & E) staining was performed to acquire a histological overview. (B) Testes sections were subjected to LCN2 or ERa immunohistochemistry and counterstained with either hematoxylin (LCN2) or methyl green (ERa). Magnifications: 100×; scale bars: 100 µm. (C) LCN2, ERa, and STAR protein expression was analyzed by Western blot analysis. Probing with a GAPDH-specific antibody was used to demonstrate equal protein loading. Unspecific bands are marked with asterisks (*) and are separated from specific bands by dashed lines. Protein expression of LCN2 and STAR was quantified densitometrically and plotted relative to GAPDH expression. Mann–Whitney tests (LCN2) or Student’s t-tests (STAR) were used for statistical analysis. Significant differences between groups are indicated with asterisks: *** p < 0.001. Data are displayed as means ± SD. (D) Results were validated by RT-qPCR. Data are displayed as mean ± SD. For statistical analysis, a Student’s t-test was conducted. Significant differences between groups are marked with asterisks: * p < 0.05, ** p < 0.01.

Fig 5: Increased LCN2 expression in Esr1-deficient ovaries. Ovaries from wild-type (WT, n = 8) and Esr1 null mice (n = 6) were dissected and either embedded in paraffin or prepared for RNA analysis. (A) Hematoxylin and eosin (H & E) staining showed characteristic histological changes in Esr1-deficient compared to WT ovaries. (B) Immunohistochemical staining for LCN2 and ERa was performed. Sections were counterstained with hematoxylin (LCN2) or methyl green (ERa). Magnifications: 100×, 400×; scale bars: 100 µm, 50 µm. (C) Tissue samples were subjected to immunofluorescence double staining for LCN2 and STAR. Magnifications: 100×; scale bars: 100 µm. (D) Lcn2 and Star expression was analyzed in ovarian tissues using RT-qPCR. Data are displayed as means ± SD. In case of normal distribution, Student’s t-tests were chosen for statistical analysis, otherwise a Mann–Whitney test was performed. Significant differences between groups are marked with asterisks: *** p < 0.001.