GPER triggers JKT-1 cell proliferation in vitro.

<p>JKT-1 cells were seeded in six-well plates (0,6×10⁶ cells/well). After 48 h, the JKT-1 cells were washed and oestrogen starved overnight in phenol red-free DMEM supplemented with 1% charcoal-stripped FBS. Serum-deprived JKT-1 cells were then incubated for 24 h with E2-BSA (1 nM), an impermeable E2-conjugate, which cannot thus interact with classical nuclear or cytoplasmic estrogen receptors, after a pre-treatment with G15 (1 nM), a GPER antagonist, or ICI-182,780 (1 µM), a pure ER antagonist, or <i>pertussis</i> toxin (100 ng/mL), a GPCR protein inhibitor. G1 (1 nM), a GPER-specific agonist, was used as a positive control. E2 at the same concentration (1 nM), which induces an inhibitory effect on cell proliferation neutralized by ER antagonist ICI (1 µM), was used for comparison. Histograms represent percentages of variation in the JKT-1 cell number compared with the control (0%), which was measured at 1,2×10⁶ cells/well after 24 hours of incubation in the steroid-free medium with ethanol (10% of variation represent an increase or a decrease of 60 000 cells). All results are expressed as means ± SEM of three independents experiments. * P<0.01.</p

Effects of GPER knockdown on E2 and E2-BSA-induced JKT-1 cell proliferation.

<p>(A) JKT-1 cells were transfected with 50 nM of siRNA designed to knockdown GPER or with control siRNA. After 72-h incubation, proteins were extracted and subjected to Western blotting to confirm the specific inhibitory activity of GPER siRNA after normalization with β-actin, which was evaluated as a house-keeping gene. (B) After 72 h transfection with 50 nM of GPER siRNA or control siRNA, JKT-1 cells were incubated for 24 h with E2 (1 nM) or E2-BSA (1 nM). Histograms represent the percentages of variation of the JKT-1 cell number compared to control without estrogens. Cell number was measured at 1,5×10⁶ cells/well after 24 hours of incubation in the steroid-free medium with ethanol (10% of variation representing an increase or a decrease of 90 000 cells). All results are expressed as means ± SEM of three independents experiments (*P<0.01).</p

Immunolocalization of GPER in JKT-1 seminoma cells.

<p>In JKT-1 cells, GPER (red fluorescence) had both a membranous and intracytoplasmic perinuclear localization (A), whereas the classical oestrogen receptor ERβ had an intracytoplasmic perinuclear and nuclear localization (B, blue fluorescence). E2-BSA, an impermeable E2 conjugate that does not cross the membrane, stained the cell membrane when coupled to FITC (C, green fluorescence). By double staining (D), E2-BSA (green) and GPER (red) co-localized at the membrane (yellow [merge]); GPER was also expressed in the cytoplasm (red). (A, B, C, D: magnification, ×63).</p

Bile Acid Alters Male Mouse Fertility in Metabolic Syndrome Context

<div><p>Bile acids have recently been demonstrated as molecules with endocrine activities controlling several physiological functions such as immunity and glucose homeostases. They act mainly through two receptors, the nuclear receptor Farnesol-X-Receptor alpha (FXRα) and the G-protein coupled receptor (TGR5). These recent studies have led to the idea that molecules derived from bile acids (BAs) and targeting their receptors must be good targets for treatment of metabolic diseases such as obesity or diabetes. Thus it might be important to decipher the potential long term impact of such treatment on different physiological functions. Indeed, BAs have recently been demonstrated to alter male fertility. Here we demonstrate that in mice with overweight induced by high fat diet, BA exposure leads to increased rate of male infertility. This is associated with the altered germ cell proliferation, default of testicular endocrine function and abnormalities in cell-cell interaction within the seminiferous epithelium. Even if the identification of the exact molecular mechanisms will need more studies, the present results suggest that both FXRα and TGR5 might be involved. We believed that this work is of particular interest regarding the potential consequences on future approaches for the treatment of metabolic diseases.</p></div

CA-supplementation reverse HFD induced overweight.

<p><b>(A)</b> Weight gain of C57BL/6J mice along the experiments. After 90 days of high fat diet (HFD) (red arrow), half of the mice on the HFD (triangles) were switched to HFD supplemented with CA (HF-CA) (squares) (n = 12–35 per group). Black arrows indicated the timing of fertility test. <b>(B)</b> Relative body weight 4 months after the switch to HF-CA diet. <b>(C)</b> Relative liver weight normalized to body weight in C57Bl/6 mice fed HFD and HF-CA diet 4 months after the switch to HF-CA diet. (n = 18–25 per group). <b>(D)</b> Plasma bile acid levels and pool composition in mice under HFD or HF-CA diet 4 months after the switch to HF-CA diet. (<b>D)</b> Plasma cholesterol, cholesterol ester, triglycerides and glucose levels in mice fed to HFD or HF-CA diets 4 months after the switch to HF-CA diet. (n = 19–25 per group). Data represent mean ± SEM; Statistical analyses: * p<0,05; ** p<0,01 and *** p<0,001.</p

Potential intratesticular action of BAs.

<p>In normal condition production of testosterone is involved in germ cell survival. The expression of genes involved in steroidogenic pathway is in part supported by transcriptional activity of LRH–1 and SF1. In parallel, the integrity of the seminiferous epithelium is ensured by the establishment of cell-cell interactions involving protein such as Cx43. In the context of BA exposure, lower production of testosterone is observed. This is consistent with the potential activation of the FXRαwhich in turn leads to activation of SHP a known repressor of steroidogenic pathways via the inhibition of transcriptional activity of LRH–1 and SF–1 on promoter sequences of genes such as Star or Cyp11a1. In parallel, in BA context, the integrity of blood testis barrier is altered. This is consistent with the activation of the TGR5-Tbx2 signaling pathways leading to lower accumulation of protein involved in cell-cell interactions that destabilized the structure of the seminiferous epithelium. These alterations might participate to the increase rate of germ cell apoptosis within the testis. In regards to the major role of testicular physiology, even though post-testicular impact cannot be exclude; the present work suggests that this alterations of testicular physiology induced in a HF-diet context might participate to the appearance of male infertility.</p

CA-supplementation alters testicular endocrine function.

<p><b>(A)</b> Testicular testosterone levels in mice fed HFD and HF-CA diet 4 months after the switch to HF-CA diet. (n = 7–13 per group). (<b>B)</b> Plasma testosterone levels in mice fed HFD and HF-CA diet 4 months after the switch to HF-CA diet. (n = 7–13 per group).<b>(C)</b> Testicular mRNA accumulation of <i>Star</i>, <i>Cyp11a1</i>, <i>3βHDS</i> and <i>Cyp17a1</i> normalized to <i>β-actin</i> mRNA levels in the whole testes of mice fed HFD and HF-CA diet 4 months after the switch to HF-CA diet (n = 12–22 per group). <b>(D)</b> Testicular mRNA expression of <i>Tubb3</i>, <i>Atp1a2</i> and <i>Pem</i> normalized to <i>βactin</i> m in the whole testes of mice fed HFD and HF-CA diet 4 months after the switch to HF-CA diet (n = 7–22 per group). <b>(E)</b> Testicular mRNA expression of <i>Sf–1</i>, <i>Lrh–1</i>, <i>Shp</i>, <i>Fxrα</i> and <i>Ar</i> normalized to <i>β-actin</i> m in the whole testes of mice fed HFD and HF-CA diet 4 months after the switch to HF-CA diet (n = 7–22 per group). <b>(F)</b> Immunoblot of AR and ACTIN on testicular protein extracts of HFD or HF-CA diet 4 months after the switch to HF-CA diet (n = 5–8 per groups). Quantification of AR/ACTIN ratio. HF-diet group was arbitrarily fixed at 100%.HF-diet group was arbitrarily fixed at 100%. <b>(G)</b>. Testicular mRNA expression of <i>Shp</i>, <i>Bsep</i>, <i>Fxrα</i>, <i>Cyp11a1</i>, <i>Lrh–1</i>, Sf–1 and normalize to β-actin levels in whole testis of C57BL/6 mice fed HFD or HF-CA diet 2 months after the switch to HF-CA diet (n = 16–22 per groups. Data represent mean ± SEM; Statistical analyses: * p<0,05; ** p<0,01 and *** p<0,005.</p

CA-supplementation diet induces increase of germ cell proliferation.

<p><b>(A)</b> Apoptosis in mice exposed to HFD or HF-CA diets 4 months after the switch to HF-CA diet (n = 13 to 25 per group) analyzed by TUNEL staining. The arrow indicates apoptotic spermatocytes. The original magnification was X200. The number of TUNEL-positive cells per 100 seminiferous tubes. <b>(B)</b> Proliferation in mice exposed to HFD or HF-CA diets (n = per groups) analyzed by Ki–67 staining. Representative micrographs of the testis exposed to HF and HF-CA diets. The original magnification was X100.Quantification of the number of Ki-67-positive cells per 100 seminiferous tubules after 2 and 4 months of HF and HF-CA diets (n = 4–5 per groups). <b>(C)</b> Immunoblot of PCNA and ACTIN on testicular protein extracts of HFD or HF-CA diet 4 months after the switch to HF-CA diet (n = 5–8 per groups). Quantification of PCNA/ACTIN ratio. HFD group was arbitrarily fixed at 100%. <b>(D)</b> Testicular mRNA expression of <i>G9a</i>, <i>Cyclina1 (Ccna1)</i>, <i>Smad6</i> and <i>Prm1</i> normalize to β-actin levels in whole testis of C57BL/6 mice fed HFD or HF-CA diets 4 months after the switch to HF-CA diet (n = 16–22 per groups).). <b>(E)</b> Immunoblot of EZH2 and ACTIN on testicular protein extracts of HF diets or HF-CA diets 4 months after the switch to HF-CA diet (n = 5–8 per groups). Quantification of EZH2/ACTIN ratio. HFD group was arbitrarily fixed at 100%. <b>(F)</b> Evaluation of post-meiotic elongated spermatids in mice exposed to HFD or HF-CA diets analyzed by acetylated histone H4 (H4ac) staining 4 months after the switch to HF-CA (Representative micrographs of the testis exposed to HF diet). The original magnification was X100.Quantification of the number of H4ac-positive cells (n = 4–5 per groups). Data represent mean ± SEM; Statistical analyses: * p<0,05; ** p<0,01 and *** p<0,005.</p