Identification of a Crosstalk among TGR5, GLIS2, and TP53 Signaling Pathways in the Control of Undifferentiated Germ Cell Homeostasis and Chemoresistance

Abstract

Spermatogonial stem cells regenerate and maintain spermatogenesis throughout life, making testis a good model for studying stem cell biology. The effects of chemotherapy on fertility have been well-documented previously. This study investigates how busulfan, an alkylating agent that is often used for chemotherapeutic purposes, affects male fertility. Specifically, the role of the TGR5 pathway is investigated on spermatogonia homeostasis using in vivo, in vitro, and pharmacological methods. In vivo studies are performed using wild-type and Tgr5-deficient mouse models. The results clearly show that Tgr5 deficiency can facilitate restoration of the spermatogonia homeostasis and allow faster resurgence of germ cell lineage after exposure to busulfan. TGR5 modulates the expression of key genes of undifferentiated spermatogonia such as Gfra1 and Fgfr2. At the molecular level, the present data highlight molecular mechanisms underlying the interactions among the TGR5, GLIS2, and TP53 pathways in spermatogonia associated with germ cell apoptosis following busulfan exposure. This study makes a significant contribution to the literature because it shows that TGR5 plays key role on undifferentiated germ cell homeostasis and that modulating the TGR5 signaling pathway could be used as a potential therapeutic tool for fertility disorders.

Keywords: GLIS2; TGR5; TP53; chemodrugs; germ cells; male fertility; stem cell regeneration.

Figure 1

A) Percentage of fertile Wt or Tgr5^–/– males 6–8, 10–12, or 18–20 weeks after busulfan or vehicle treatments. n = 10 to 15 males from 3 to 5 independent experiments. Data are expressed as the means ± SEM. ANOVA2 followed by Holm‐Sidak's test for multiple comparisons. ***, p < 0.001 versus respective vehicle group for each genotype. #, p < 0.05 between genotypes exposed to same treatments. The horizontal square brackets underline the groups statistically compared between two conditions of different genotypes. Veh: vehicle and Bu: Busulfan. B) Number of pups per litter obtained from fertile Wt or Tgr5^–/– males 10–12 or 18–20 weeks after busulfan or vehicle treatments. n = 12–15 litters from 3 to 5 independent experiments. Data are expressed as the means ± SEM. ANOVA2 followed by Holm‐Sidak's test for multiple comparisons. *, p < 0.05 versus respective vehicle group for each genotype. ## p < 0.01 between genotypes exposed to same treatments. The horizontal square brackets underline the groups statistically compared between two conditions of different genotypes. Veh: vehicle and Bu: Busulfan. C) Number of sperm count in the epididymis head of Wt or Tgr5^–/– males 8, 12 and 20 weeks after busulfan or vehicle treatments. n = 12–40 from 3 to 6 independent experiments. Data are expressed as the means ± SEM. ANOVA2 followed by Holm‐Sidak's test for multiple comparisons. ***, p < 0.001 versus respective vehicle group for each genotype. #, p < 0.05; ##; p < 0.01; between genotypes exposed to same treatments. The horizontal square brackets underline the groups statistically compared between two conditions of different genotypes. Veh: vehicle and Bu: Busulfan. D) Representative micrographs of hematoxylin/eosin‐stained testes of Wt or Tgr5^–/‐ males treated with the vehicle or 4, 8, 12 and 20 weeks after busulfan treatment. The arrowhead indicates tubules with germ cell loss. E) Representative micrographs of testis of vehicle or Bu treated Wt or Tgr5^–/‐ males stained for TUNEL. F) (Left panel) Quantification of the raw number of TUNEL positive cells for 100 seminiferous tubules of Wt or Tgr5^–/– males treated with the vehicle treatment. (Right panel) Quantification of the relative number of TUNEL positive cells in Wt or Tgr5^–/– males treated with the vehicle or busulfan (4 weeks after treatment). n = 10–24 from 3 to 6 independent experiments. Vehicle groups of each genotype were arbitrarily set at 1. Data are expressed as the means ± SEM. ANOVA2 followed by Holm‐Sidak's test for multiple comparisons. ***, p < 0.001 versus respective vehicle group for each genotype. ##; p < 0.01 between genotypes exposed to same treatments. The horizontal square brackets underline the groups statistically compared between two conditions of different genotypes. Veh: vehicle and Bu: Busulfan.

Figure 2

A) Representative micrographs of vehicle or Bu treated Wt or Tgr5^–/‐ testis stained for acetylated histone H4 (acetylated H4). B) Quantification of the number of acetylated H4 positive seminiferous tubules in testis of Wt or Tgr5^–/‐ males treated with the vehicle or busulfan (1, 2, 4, and 8 weeks after treatment). The arrowhead indicates acetylated H4 positive seminiferous tubules, and the star indicates acetylated H4 negative tubules. C) Representative micrographs of vehicle or Bu treated Wt or Tgr5^–/‐ testis stained for PLZF green) and PCNA (red). The arrowhead indicates PLZF‐PCNA positive cells, and the star indicates PCNA negative and PLZF positive cells. D) Quantification of the relative number of PLZF positive cells per seminiferous tubule in Wt or Tgr5^–/– testis treated with busulfan (1 week and 2 weeks after treatment). The mean number of PLZF+ cells observed in Wt males treated with Bu was arbitrarily set at 1 for comparison with the number of PLZF+ cells observed one and two weeks after treatment. E) Quantification of the relative number of PLZF positive cells per seminiferous tubule in Wt or Tgr5^–/– males treated with the vehicle or busulfan (4 weeks or 8 weeks after treatment). Vehicle groups of each genotype were arbitrarily set at 1. F) Quantification of the relative number of double stained PLZF‐PCNA cells per seminiferous tubule in Wt or Tgr5^–/‐ males treated with the vehicle or busulfan (2 weeks after treatment). Vehicle groups of each genotype were arbitrarily set at 1. In all panels, n = 11–23 from 3 to 6 independent experiments. Data are expressed as the means ± SEM. ANOVA2 followed by Holm‐Sidak's test for multiple comparisons. **, p < 0.01; ***, p < 0.001 versus respective vehicle group for each genotype. #, p < 0.05; ##, p < 0.01 between genotypes exposed to same treatments. The horizontal square brackets underline the groups statistically compared between two conditions of different genotypes. Veh: vehicle and Bu: Busulfan.

Figure 3

A) (Left panel) Representative micrographs of GFP immuno‐staining in GFP‐negative mouse and in hTGR5‐T2A‐GFP mouse model. (Right panel) Representative micrographs of hTGR5‐T2A‐GFP mouse model testis stained for GFP (green) and PLZF (red), or LIN28 (red) or SYCP3 (red). The white arrowheads indicate co‐stained cells. Experiment was performed on 4 different hTGR5‐T2A‐GFP males. The dotted lines delineate the seminiferous tubules. B) Cyp11a1, Fshr, Gfra1, Plzf, and Tgr5 mRNA accumulations normalized to β‐actin in THY1+ cells isolated from 6 to 7 days old males. n = 5. The THY1‐ unsorted cell group was arbitrary set at 1 (Dotted line). Data are expressed as the means ± SEM. T‐test statistical analysis; *, p < 0.05. C) (Left panel) Percentage of fertile Tgr5^{f/‐ ;} Cre^–/‐ , and Tgr5^GC‐KO males 6–8, 8–10, and 10–13 weeks after vehicle or busulfan treatment following breeding with C57Bl6J females for 10 days. (Right panel) Number of pups per litter obtained in breeding with C57Bl6J females with Tgr5^{f/‐ ;} Cre^–/ , ^‐ or Tgr5^GC‐KO males 8–10 and 10–13 weeks after busulfan treatment. n = 12–20 from 3 independent experiments. Data are expressed as the means ± SEM. ANOVA2 followed by Holm‐Sidak's test for multiple comparisons. #, p < 0.05; ##; p < 0.01; ###, p < 0.001 between genotypes exposed to same treatments. The horizontal square brackets underline the groups statistically compared between two conditions of different genotypes. Veh: vehicle and Bu: Busulfan.

Figure 4

A) (Left panel) Raw number of adherent cells after 24 or 48 h of treatment with vehicle in siCtrl and siTgr5 GC1spg transfected cells; and (Right panel) relative number of adherent cells after 24 or 48 h of treatment with vehicle or busulfan in siCtrl and siTgr5 GC1spg transfected cells. Vehicle groups of each genotype were arbitrarily set at 1. In panel, n = 20–30 from 6 independent experiments. Data are expressed as the means ± SEM. ANOVA2 followed by Holm‐Sidak's test for multiple comparisons. *, p < 0.05; **, p < 0.01; ***, p < 0.001 versus respective vehicle group for each genotype. ##, p < 0.01 between genotypes exposed to same treatments. The horizontal square brackets underline the groups statistically compared between two conditions of different genotypes. Veh: vehicle and Bu: Busulfan. B) (Upper panel) Representative micrographs of GC1spg cells transfected with siCtrl or siTgr5 and treated with vehicle or Bu for 24 h and stained for TUNEL. (Lower left panel) Quantification of the raw number of TUNEL positive GC1spg cells transfected with siCtrl and siTgr5 and treated with vehicle. (Lower right panel) Quantification of the relative number of TUNEL positive GC1spg cells transfected with siCtrl and siTgr5 and treated with vehicle or Bu for 24 h. n = 5–15 from 3 to 5 independent experiments. Vehicle groups of each genotype were arbitrarily set at 1. Data are expressed as the means ± SEM. ANOVA2 followed by Holm‐Sidak's test for multiple comparisons. ***, p < 0.001 versus respective vehicle group for each genotype. ##, p < 0.01 between genotypes exposed to same treatments. The horizontal square brackets underline the groups statistically compared between two conditions of different genotypes. Veh: vehicle and Bu: Busulfan. C) Relative Gfra1 mRNA accumulation normalized to β‐actin on GC1spg cells transfected with siCtrl or siTgr5 and treated 24 h with vehicle or with 200 × 10⁻⁶ m of Bu. In panel, n = 25–27 from 6 independent experiments. Data are expressed as the means ± SEM. ANOVA2 followed by Holm‐Sidak's test for multiple comparisons. ***, p < 0.001 versus respective vehicle group for each genotype. ##, p < 0.01between genotypes exposed to same treatments. The horizontal square brackets underline the groups statistically compared between two conditions of different genotypes. Veh: vehicle and Bu: Busulfan. D) Representation of the RNAseq groups: GC1spg cells transfected with siCtrl or siRNA directed against Tgr5 (siTgr5) and treated 24 h with vehicle or with 200 × 10⁻⁶ m of Bu. E) Venn diagram for differentially expressed genes in GC1spg cells transfected with siCtrl or siTgr5 and treated 24 h with vehicle or with 200 × 10⁻⁶ m of Bu. F) Heatmap of differentially downregulated genes following 24 h exposure to Bu specifically in GC1spg cells transfected with siCtrl based on Table S1c1 in the Supporting Information. G) Heatmap of differentially increased genes following 24 h exposure to Bu specifically in GC1spg cells transfected with siCtrl based on Table S1c2 in the Supporting Information. H) Gene ontology analysis based on the list of differentially expressed genes specifically in GC1spg cells transfected with siCtrl following Bu exposure compared to siTgr5‐transfected cells treated with Bu. Based on gene lists Table S1c1,c2 in the Supporting Information. In panel D to H, RNAseq was performed from one experiment with n = 5 per group. Statistical analyses were performed as mentioned in Materials and Methods section. P value was set to p < 0.01 between genotypes and/or treatment.

Figure 5

A) Perp, Phlda3, and Mmp24 mRNA accumulations normalized to β‐actin on GC1spg cells transfected with siCtrl or siTgr5 and treated 24 h with vehicle or with 200 × 10⁻⁶ m of Bu. Vehicle groups of each genotype were arbitrarily set at 1. B) Representative western blots of GAPDH, TP53 and Phospho‐TP53 (P‐TP53), and quantification of ratios in GC1spg cells transfected with siCtrl and siTgr5 and treated with vehicle or Bu for 24 h. Vehicle groups of each genotype were arbitrarily set at 1. C) Number of adherent GC1spg and GC1spg^KO‐Tgr5 cells exposed to veh. or Bu 200µM for 24 or 48 h. Vehicle groups of each genotype were arbitrarily set at 1. D) (Left) Gfra1 mRNA accumulation normalized to β‐actin in GC1spg and GC1spg^KO‐Tgr5 cells; GC1spg were arbitrarily set at 1. (Right) Gfra1 mRNA accumulation normalized to β‐actin in GC1spg and GC1spg^KO‐Tgr5 cells exposed to vehicle or Bu 200µM for 24 h. Vehicle groups of each genotype were arbitrarily set at 1. E) (Left) Representative micrographs of GC1spg and GC1spg^KO‐Tgr5cells exposed to vehicle or Bu 200µM for 24 h and stained for TUNEL. (Right) Quantification of the relative number of TUNEL positive GC1spg cells and GC1spg^KO‐Tgr5 cells exposed to vehicle or Bu 200µM for 24 h. Vehicle groups of each genotype were arbitrarily set at 1. F) Representative western blots of GAPDH, TP53 and Phospho‐TP53 (P‐TP53), and quantification of ratios in GC1spg cells and GC1spg^KO‐Tgr5 cells treated with vehicle or Bu for 24 h. Vehicle groups of each genotype were arbitrarily set at 1. G) Perp mRNA accumulation normalized to β‐actin in GC1spg cells and GC1spg^KO‐Tgr5 cells treated with vehicle or with 200 × 10⁻⁶ m of Bu for 24 h. Vehicle groups of each genotype were arbitrarily set at 1. In all panels, n = 6–18 from 3 to 6 independent experiments. Data are expressed as the means ± SEM. ANOVA2 followed by Holm‐Sidak's test for multiple comparisons. **, p < 0.01; ***, p < 0.001 versus respective vehicle group for each genotype. #, p < 0.05; ##, p < 0.01; ###, p < 0.001 between genotypes exposed to same treatments. The horizontal square brackets underline the groups statistically compared between two conditions of different genotypes. Veh: vehicle and Bu: Busulfan.

Figure 6

A) Glis2 mRNA accumulation normalized to β‐actin on GC1spg cells transfected with siCtrl or siTgr5 and treated 6 h with vehicle or with 200 × 10⁻⁶ m of Bu. Vehicle‐treated siCtrlt‐transfected cells were arbitrarily set at 1. B) Fgfr2, Izumo4, Adgrg1 and Wfdc1 mRNA accumulation normalized to β‐actin on GC1spg cells transfected with siCtrl or siTgr5 and treated 24 h with vehicle or with 200 × 10⁻⁶ m of Bu. For quantification, vehicle‐treated siCtrlt‐transfected cells were arbitrarily set at 1. C) Glis2 and Fgfr2 mRNA accumulations normalized to β‐actin in vehicle treated GC1spg cells and GC1spg^KO‐Tgr5. For quantification, GC1spg cells were arbitrarily set at 1. D) Representative western blots of GLIS2 in GC1spg cells and GC1spg^KO‐Tgr5 cells treated with vehicle. Normalization was performed against total protein using stain‐free gels. For quantification, GC1spg cells were arbitrarily set at 1. E) Representative western blots of GLIS2 and quantification of ratios in GC1spg cells and GC1spg^KO‐Tgr5 cells treated with vehicle or Bu for 24 h. Normalization was performed against total protein using stain‐free gels. Vehicle groups of each genotype were arbitrarily set at 1. F) Izumo4 and Fgfr2 mRNA accumulations normalized to β‐actin in GC1spg cells and GC1spg^KO‐Tgr5 treated for 24 h with vehicle treated or Bu 200 × 10⁻⁶ m. Vehicle groups of each genotype were arbitrarily set at 1. In all panels, n = 9–26 from 3 to 5 independent experiments. Data are expressed as the means ± SEM. ANOVA2 followed by Holm‐Sidak's test for multiple comparisons. *, p < 0.05; **, p < 0.01 versus respective vehicle group for each genotype. #, p < 0.05; ##, p < 0.01; ###, p < 0.001 between genotypes exposed to same treatments. The horizontal square brackets underline the groups statistically compared between two conditions of different genotypes. Veh: vehicle and Bu: Busulfan.

Figure 7

A) (Left) Glis2 mRNA accumulation normalized to β‐actin on GC1spg cells transfected with an empty vector or a vector for overexpression of GLIS2. Cells transfected with empty vector were arbitrarily set at 1. (Right) Representative western blots of GLIS2 in GC1spg cells transfected with an empty vector or a vector for overexpression of GLIS2. B) (Left panel) Raw number of adherent cells transfected with an empty vector or a vector for overexpression of GLIS2 and treated with vehicle for 24 or 48 h. (Right panel) Relative number of adherent cells transfected with an empty vector or a vector for overexpression of GLIS2 and treated with vehicle or Bu for 24 or 48 h. Vehicle groups of each genotype were arbitrarily set at 1. C) Representative western blots of Phospho‐TP53 (P‐TP53), and quantification of ratios in GC1spg cells transfected with an empty vector or a vector for overexpression of GLIS2 and treated with vehicle or Bu for 24 h. Vehicle groups of each genotype were arbitrarily set at 1. D) Perp and Mmp24 mRNA accumulation normalized to β‐actin in GC1spg cells transfected with an empty vector or a vector for overexpression of GLIS2 and treated with vehicle or Bu for 24 h. Vehicle groups of each genotype were arbitrarily set at 1. E) (Left) Representative micrographs of GC1spg cells transfected with an empty vector or a vector for overexpression of GLIS2 and treated with vehicle or Bu for 24 h and stained for TUNEL. (Right) Quantification of the raw number of the number of TUNEL positive GC1spg cells transfected with an empty vector or a vector for overexpression of GLIS2 and treated with vehicle 24 h. Vehicle groups of each genotype were arbitrarily set at 1 Quantification of the relative number of TUNEL positive GC1spg cells transfected with an empty vector or a vector for overexpression of GLIS2 and treated with vehicle or Bu for 24 h. Vehicle groups of each genotype were arbitrarily set at 1. In all panels, n = 5–26 from 3 to 5 independent experiments. Data are expressed as the means ± SEM. ANOVA2 followed by Holm‐Sidak's test for multiple comparisons. *,**, p < 0.01; ***, p < 0.001 versus respective vehicle group for each genotype. #, p < 0.05; ##, p < 0.01between genotypes exposed to same treatments. The horizontal square brackets underline the groups statistically compared between two conditions of different genotypes. Veh: vehicle and Bu: Busulfan.

Figure 8

A) (Left panel) Relative Glis2 mRNA accumulation normalized to β‐actin on GC1spg^KO‐TGR5 cells transfected with siCtrl or siGlis2. siCtrl‐transfected cells were arbitrarily set at 1. (Right panel) Representative western blots of GLIS2 in GC1spg^KO‐TGR5 cells transfected with siCtrl or siGlis2. Normalization was performed against total protein using stain‐free gels. B) Number of adherent GC1spg^KO‐TGR5 cells transfected with siCtrl or siGlis2 and treated with vehicle or Bu for 24 h. Vehicle treated cells of each genotype were arbitrarily set at 1. C) Representative western blots of GLIS2 and Phospho‐TP53 (P‐TP53), and quantification of ratios in GC1spg^KO‐Tgr5 cells transfected with siCtrl or siGlis2 and treated with vehicle or Bu for 24 h. Normalization was performed against total protein using stain‐free gels. Vehicle groups of each genotype were arbitrarily set at 1. D) Fgfr2, Adgrg1 and Phlda3 mRNA accumulations normalized to β‐actin on GC1spg^KO‐TGR5 cells transfected with siCtrl or siGlis2 and treated 24h with vehicle or with 200 × 10⁻⁶ m of Bu. Vehicle groups of each genotype were arbitrarily set at 1. In all panels, n = 10–15 from 3 to 5 independent experiments. Data are expressed as the means ± SEM. ANOVA2 followed by Holm‐Sidak's test for multiple comparisons. *, p < 0.05; **, p < 0.01 versus respective vehicle group for each genotype. #, p < 0.05 between genotypes exposed to same treatments. The horizontal square brackets underline the groups statistically compared between two conditions of different genotypes. Veh: vehicle and Bu: Busulfan.

Figure 9

A) Plzf, Glis2, Mmp24, Miwi2, Ngn3, Gfra1, Fgfr2 and Tgr5 mRNA accumulations normalized to β‐actin in testis of Wt mice 5 days after the treatment with vehicle or Bu. In panel, n = 10 to 15 from 3 independent experiments. Vehicle groups of each genotype were arbitrarily set at 1. Data are expressed as the means ± SEM. Statistical analysis: *, p < 0.05 versus respective vehicle group for each genotype. Veh: vehicle and Bu: Busulfan. B) Mpm24 mRNA accumulations normalized to β‐actin in testis of Wt and Tgr5^–/– mice 5 days after the treatment with vehicle or Bu. In panel, n = 10 to 15 from 3 independent experiments. Vehicle groups of each genotype were arbitrarily set at 1. Data are expressed as the means ± SEM. ANOVA2 followed by Holm‐Sidak's test for multiple comparisons. *, p < 0.05 versus respective vehicle group for each genotype. #, p < 0.05 between genotypes exposed to same treatments. The horizontal square brackets underline the groups statistically compared between two conditions of different genotypes. Veh: vehicle and Bu: Busulfan. C) Correlation analyses of the mRNA levels of Tgr5 with Mmp24 or Perp normalized to β‐actin in testis of Wt mice 5 days after the treatment with vehicle or Bu. In panel, n = at least 15 from 3 independent experiments. Spearman Statistical analysis: *, p < 0.05. D) Glis2 mRNA accumulations normalized to β‐actin in testis of Wt and Tgr5^–/– mice 5 days after the treatment with vehicle or Bu. In panel, n = 15 from 3 independent experiments. Vehicle groups of each genotype were arbitrarily set at 1. Data are expressed as the means ± SEM. ANOVA2 followed by Holm‐Sidak's test for multiple comparisons. *, p < 0.05 versus respective vehicle group for each genotype. The horizontal square brackets underline statistical analysis between two conditions of different genotypes. Veh: vehicle and Bu: Busulfan. E) Correlation analyses of the mRNA levels of Glis2 with the % of PLZF + cells in testis of Wt mice 5 days after the treatment with vehicle or Bu. In panel, n = at least 15 from 3 independent experiments. Spearman Statistical analysis: *, p < 0.05. F) Correlation analyses of the mRNA levels of Glis2 with Plzf mRNA accumulations normalized to β‐actin in testis of Wt mice 5 days after the treatment with vehicle or Bu. In panel, n = at least 15 from 3 independent experiments. Spearman Statistical analysis: *, p < 0.05. G) Correlation analyses of the mRNA levels of Glis2 with Ngn3 mRNA accumulations normalized to β‐actin in testis of Wt mice 5 days after the treatment with vehicle or Bu. In panel, n = at least 15 from 3 independent experiments. Spearman Statistical analysis: *, p < 0.05. H) Correlation analyses of the mRNA levels of Glis2 with Miwi2 mRNA accumulations normalized to β‐actin in testis of Wt mice 5 days after the treatment with vehicle or Bu. In panel, n = at least 15 from 3 independent experiments. Spearman Statistical analysis: *, p < 0.05.

Figure 10

A) Thy1, Tgr5, Id4, Plzf, Gfra1, Glis2, Fgfr2, Ngn3 and Miwi2 mRNA accumulations normalized to β‐actin in THY1+ isolated spermatogonia of adult Wt mice 1 week after the treatment with vehicle or Bu. In panel, n = 10 to 15 from 3 independent experiments. Vehicle groups of each genotype were arbitrarily set at 1. Data are expressed as the means ± SEM. statistical analysis: *, p < 0.05 versus respective vehicle group for each genotype. Veh: vehicle and Bu: Busulfan. B) Thy1, Id4, Plzf, Gfra1, Glis2, Fgfr2, Ngn3, and Miwi2, mRNA accumulations normalized to β‐actin in THY1+ isolated spermatogonia of adult Wt and Tgr5 ^–/‐ mice 1 week after the treatment with vehicle or Bu. In panel, n = 10 to 15 from 3 independent experiments. Vehicle groups of each genotype were arbitrarily set at 1. Data are expressed as the means ± SEM. ANOVA2 followed by Holm‐Sidak's test for multiple comparisons. *, p < 0.05 versus respective vehicle group for each genotype. Veh: vehicle and Bu: Busulfan. C) Tp53, Rspo3, Phlda3, Perp and Mmp24 mRNA accumulations normalized to β‐actin in THY1+ isolated spermatogonia of adult Wt and Tgr5 ^–/‐ mice 3 days after the treatment with vehicle or Bu. In panel, n = 5. Vehicle groups of each genotype were arbitrarily set at 1. Data are expressed as the means ± SEM. ANOVA2 followed by Holm‐Sidak's test for multiple comparisons. *, p < 0.05 versus respective vehicle group for each genotype. Veh: vehicle and Bu: Busulfan. D) Glis2 mRNA accumulations normalized to β‐actin in isolated spermatogonia of adult Wt and Tgr5 ^–/‐ mice 3 days after the treatment with vehicle or Bu. In panel, n = 5. Vehicle groups of each genotype were arbitrarily set at 1. Data are expressed as the means ± SEM. ANOVA2 followed by Holm‐Sidak's test for multiple comparisons. *, p < 0.05 versus respective vehicle group for each genotype. Veh: vehicle and Bu: Busulfan.

Figure 11

Representative western blots of Phospho‐CREB (P‐CREB) and quantification of ratios in GC1spg and GC1spg^KO‐Tgr5. GC1spg cells were arbitrarily set at 1. Adgrg1, Izumo4, Wfdc1, Fgfr2 and Glis2 mRNA accumulations normalized to β‐actin in GC1spg cells treated for 45 min with vehicle or H89 and harvested 24 h later. Vehicle groups were arbitrarily set at 1. Representative western blots of GLIS2 and quantification ratios in GC1spg cells treated with vehicle or H89 for 45 min and harvested 24 h later. Normalization was performed against total protein using stain‐free gels. Glis2 mRNA accumulations normalized to β‐actin in GC1spg and GC1spg^KO‐Tgr5 cells treated for 45 min with vehicle or H89 and harvested 24 h later. Vehicle treated GC1spg cells were arbitrarily set at 1 Representative western blots of Phospho‐TP53 (P‐TP53), and quantification of ratios in GC1spg and GC1spg^KO‐Tgr5 cells treated with vehicle or INT‐777 for 24h. Vehicle treated cells were arbitrarily set at 1. Representative western blots of Phospho‐TP53 (P‐TP53), and quantification ratios in GC1spg cells treated with vehicle or H89 for 45 min and then with vehicle or INT‐777 for 24 h. Vehicle treated cells were arbitrarily set at 1. Normalization was performed against total protein using stain‐free gels. Representative western blots of Phospho‐TP53 (P‐TP53), and quantification of ratios in GC1spg cells transfected with an empty vector or a vector for overexpression of GLIS2 and treated with vehicle or INT‐777 (25 × 10⁻⁶ m) for 24 h as well as GC1spg^KO‐Tgr5 cells transfected with an empty vector and treated with vehicle for 24 h. Normalization was performed against total protein using stain‐free gels. Vehicle treated cells were arbitrarily set at 1. Representative western blots of GLIS2, and quantification of ratios in GC1spg cells transfected with an empty vector or a vector for overexpression of GLIS2 and treated with vehicle or INT‐777 (25 × 10⁻⁶ m) for 24 h as well as GC1spg^KO‐Tgr5 cells transfected with an empty vector and treated with vehicle for 24 h. Normalization was performed against total protein using stain‐free gels. Vehicle treated cells were arbitrarily set at 1. In all panels, n = 15 from 3 independent experiments. Data are expressed as the means ± SEM. ANOVA2 followed by Holm‐Sidak's test for multiple comparisons. *, p < 0.05 versus respective vehicle group for each genotype. Veh: vehicle and Bu: Busulfan.

Figure 12

A) Relative number of adherent cells in GC1spg and GC1spg^KO‐Tgr5 cells pre‐exposed 24h with INT‐777 and then to Bu (200µM) for 24 or 48 h. Vehicle treated cells were arbitrarily set at 1. B) (Left) Representative micrographs of GC1spg and GC1spg^KO‐Tgr5 cells pre‐exposed 24h with INT‐777 and then to Bu 200µM for 48 h and stained for TUNEL. (Right) Quantification of the relative number of TUNEL positive GC1spg and GC1spg^KO‐Tgr5 cells pre‐exposed 24h with INT‐777 and then with Bu (200µM) for 48 h. Vehicle treated cells were arbitrarily set at 1. C) Perp, Phld3a and Mmp24 mRNA accumulations normalized to β‐actin in GC1spg cells and GC1spg^KO‐Tgr5 cells pre‐exposed 24h with INT‐777 and then to Bu 200µM for 24 h. Vehicle treated cells were arbitrarily set at 1. In A to C panels, n = 6–24 from 3 independent experiments. Data are expressed as the means ± SEM. ANOVA2 followed by Holm‐Sidak's test for multiple comparisons. **, p < 0.01, ***, p < 0.001 versus respective vehicle group for each genotype. #, p < 0.05; ##, p < 0.01; ###, p < 0.001 between genotypes exposed to same treatments. The horizontal square brackets underline the groups statistically compared between two conditions of different genotypes. Veh: vehicle and Bu: Busulfan. (D) (Left) Representative micrographs of testis of Bu or Bu+CA treated Wt males stained for PLZF. (Right) Quantification of the number of positive PLZF cells per seminiferous tubule of males treated with the Bu or Bu+CA (4 weeks after treatment). E) (Left) Representative micrographs of testis of Bu or Bu+CA treated Wt males stained for acetylated H4. (Right) Quantification of the number of acetylated H4 positive seminiferous tubules of males treated with the Bu or Bu+CA (8 weeks after treatment). F) (Left) Representative micrographs of testis of Bu or Bu+CA treated Wt males stained for TUNEL. (Right) Quantification of the number of TUNEL positive cells per seminiferous tubule of testes from Wt males treated with Bu or Bu+CA (8 weeks after treatment). In D to F panels, n = 10–24 from 3 independent experiments. Data are expressed as the means ± SEM. Statistical analysis: *, p < 0.05; ** p < 0.01; *** p < 0.001 versus Bu treated group. Bu: Busulfan and Bu+CA: Busulfan+ cholic acid.

Figure 13

Proposed model for the crosstalk between TGR5 and Bu signaling pathways.