Implication of PGC-1α in GR transactivation.

<p>A/ Expression of PGC-1α. Total RNA was prepared from 158N cells. RT-PCR experiments were performed using primers recognizing specifically PGC-1α. PCR products were analyzed on agarose gel (2%) and visualized under UV. 18S RNA was detected by specific primers and used to normalize PGC-1α expression levels. B/ Implication of PGC-1α in the glucocorticoid pathway. 158N cells were transiently cotransfected with either 0.2 µg of (GRE)2-TATA-Luc, 0.1 µg of pRSV-βGal plasmids and increasing amounts of PGC-1α expression vector, as indicated. Eighteen hours after transfection, cells were incubated with Dex (10⁻⁶ M) for 24 h, and then luciferase and β-galactosidase activities were analyzed. Results are expressed as percentage of the basal activity and represent the mean +/− SEM of at least four independent experiments performed in duplicate. **p<0.01 when compared between cells transfected with PGC-1α using Bonferroni's test after ANOVA.</p

Cross-talk between oxysterols and glucocorticoids in oligodendrocyte death.

<p>A/ Histograms showing cells treated with or without 25-OH in presence or absence of dexamethasone. As described here, the mitochondrial membrane potential was determined by DiOC6(3) and propidium iodide staining of the cells, followed by flow cytometry analysis. The different categories of cell populations as described in (A) have been simultaneously plotted. In all cases, 10.000 cells have been counted. The standard deviations have been extracted from five different experiments. B/ Biparametric flow cytometric analysis of oligodendrocytes treated with or without 25-OH in presence or absence of dexamethasone. Apoptotic cells were identified as Annexin V-FITC positive/PI negatives, whereas necrotic cells or cells exhibiting secondary necrosis were considered as Annexin V-FITC positive and PI positive. The addition of the two populations gives the total cell death in the present conditions. A typical experiment with cells treated for 24 h or 48 h is presented. 10.000 cells have been counted for each representation (the cell debris and aggregated cells have been excluded from the analysis by selection based on their light scattering properties). C/ Histograms of the cells treated with or without 25-OH in the presence or absence of dexamethasone. The different categories of cell populations as described in (B) have been simultaneously plotted. In all cases 10.000 cells have been counted. The standard deviations have been extracted from five independent experiments.</p

Effect of Dex and implication of the GR in sPLA2-IIA promoter activity.

<p>A/ 158N cells were transiently transfected with 0.2 µg of sPLA2-IIA(1 kb) and 0,1 µg of pRSV-βGal plasmids in the presence of 0.2 µg of mock vector (NT) or siRNA directed against the GR (siGR). Eighteen hours after transfection, cells were incubated with 25-OH (10⁻⁵ M) and/or Dex (10⁻⁶ M) for 24 h, and then luciferase and β-galactosidase activities were analyzed. Results are expressed as percentage of the basal activity; they represent the mean +/− SEM of 10 independent experiments performed in duplicate. In all experiments *p<0.05, **p<0.01, ***p<0.001 by using Bonferroni's test after ANOVA. B/ Test of the efficacy of the siRNA: Total RNA from 158N cells transfected with either non targeting siRNA or siRNA against GR was prepared. Real time RT-PCR was performed. 26S RNA was detected by specific primers and used to normalize GR expression levels.</p

PGC-1α implication in sPLA2-IIA promoter regulation.

<p>158N cells were transiently transfected with 0.2 µg of sPLA2-IIA(1 kb), 0.1 µg of pRSV-βGal plasmids and with 0.1 µg of PGC-1α expression vector or mock vector, as indicated. Eighteen hours after transfection, cells were incubated with 25-OH (10⁻⁵ M) and/or Dex (10⁻⁶ M) for 24 h, and then luciferase and β-galactosidase activities were analyzed. Results are expressed as percentage of the basal activity; they represent the mean +/− SEM of at least four independent experiments performed in duplicate. **p<0.01, when comparing between 25-OH and Dex+25-OH using Bonferroni's test after ANOVA.</p

Effect of dexamethasone on the expression of LXRβ and PXR.

<p>158N cells were incubated with Dex (1 µM) during 24 h. Total RNA was extracted and real-time PCR experiments were performed using primers recognizing specifically LXRβ or PXR. Results are expressed as the ratio of LXR or PXR expression over 26S. 100% is the level in control cells. They represent the mean +/− SEM of three independent experiments. *p<0.05 when compared to control by using Student's t test.</p

The density of oligodendroglial cells becomes sexually differentiated between P5 and P10.

<p>(A) Sagittal section showing EGFP expression in the adult PLP-EGFP mouse brain. White matter tracts display intense green fluorescence, including the three subregions of the corpus callosum: genu, center and splenium. Crb = cerebellum; OB = olfactory bulb; Str = striatum.(B) Comparison of the densities of EGFP⁺ oligodendroglial cells in corpus callosum (CC) between adult male and female PLP-EGFP mice (n = 5). (C) Immunostaining of Olig2⁺ gliogenic progenitors (red) and EGFP⁺ oligodendroglial cells (green) on a sagittal brain section of a PLP-EGFP male mouse at P5. CC = corpus callosum; Str = striatum; V = third ventricle. (D) Comparison of the densities of Olig2⁺, EGFP⁺ and Olig2⁺/EGFP⁺ double-positive cells in corpus callosum between P5 male and female PLP-EGFP mice (n = 5). Immunostaining of Olig2⁺ cells (red) and EGFP⁺ cells (green) on sagittal brain sections at P10 of (E and F) a male and (G) a female. CC = corpus callosum; Str = striatum; V = third ventricle. (H) Comparison of the densities of Olig2⁺, EGFP⁺ and Olig2⁺/EGFP⁺ double-positive cells in corpus callosum between P10 males and females PLP-EGFP mice (n = 9). (I and J) Immunostaining of Olig2⁺ cells (red) and CC1⁺ mature oligodendrocytes (green) on sagittal corpus callosum sections at P10 of (I) a male and (J) a female C57Bl/6 mouse. (K) Comparison of the densities of CC1⁺ oligodendrocytes and CC1⁺/Olig2⁺ double-positive cells in corpus callosum at P10 between males and females (n = 5). Cell densities are presented as means ± SEM. Significance was calculated using two-tailed Student’s t test (**p < 0.01; *p < 0.05 when compared to the corresponding male group). Scale bars: (A) 1 mm; (C and E) 50 μm; (F-J) 20 μm.</p

Androgen-dependent sex differences in myelin are maintained over time: <i>In vivo</i> and <i>in vitro</i> studies.

<p>(A) Immunostaining of MBP⁺ myelin (blue) and EGFP⁺ oligodendroglial cells (green) in cerebellar slices taken from PLP-EGFP male and female mice at P10 and maintained in culture for two weeks in the absence of androgens. The Purkinje neuron marker Calbindin (CaBP) (red) was used to track nerve cells. The merged triple immunostaining (bottom) documented the higher myelination of the male cerebellar slices. (B and C) Analysis of the density of EGFP⁺ oligodendroglial cells (B) and of the MBP⁺ area (C) within the male and female cerebellar slices. (n = 5 animals, three slices and three regions from each slice were analyzed per animal). (D) Injecting male PLP-EGFP mice every two days between P0 and P10 with the AR antagonist flutamide (Flut, 1 mg/kg) and control mice with sesame oil vehicle. Analysis of EGFP⁺ oligodendroglial cells was performed at 3 months of age (n = 5). (E) Injecting female PLP-EGFP mice every two days between P0 and P10 with 5α-dihydrotestosterone (DHT, 1 mg/kg) and control mice with sesame oil vehicle. Analysis of EGFP⁺ oligodendroglial cells was performed at 3 months of age (n = 5). Results are presented as means ± SEM. Significance was calculated using two-tailed Student’s t test (***p < 0.001; **p < 0.01 *p < 0.05 when compared to the corresponding control).</p

Corpus callosum myelin is sexually dimorphic at P10.

<p>(A and B) Immunostaining of myelin basic protein (MBP) on sagittal corpus callosum sections at P10 of (A) a male and (B) a female C57Bl/6 mouse. CC = corpus callosum. The dotted lines delimit the corpus callosum. (C) Quantification of the MBP immunostaining in P10 corpus callosum of male and female mice. For each animal, the MBP⁺ area was determined within 0.26 mm² fields of splenium, center and genu using NIH image software and the mean area was calculated (n = 8). (D) MBP mRNA expression within the brain of males and female mice analyzed by qRT-PCR. The <i>cyclophilin A</i> gene was used for normalization (n = 3–4). (E and F) Electron micrographs of P10 corpus callosum of a (E) male and a (F) female WT mouse. Pictures show unmyelinated (u) and myelinated axons (m). (G-H) Analysis by electron microscopy of the total number of axons (G) and the percentage of myelinated axons (H) in corpus callosum at P10 (n = 3). Results are presented as means ± SEM. Significance was calculated using two-tailed Student’s t test (*p < 0.05 when compared to the corresponding male group). Scale bars: A and B = 20 μm; E and F = 1 μm.</p

Brain levels of testosterone and 5α-dihydrotestosterone in males and females between P0 and P10 and androgen receptor expression.

<p>Comparison of brain levels of (A) testosterone, (B) 5α-dihydrotestosterone (5α-DHT) and (C) testosterone + 5α-DHT between male and female mice. Androgen levels were analyzed by GC-MS/MS at P0 (n = 16), P5 (n = 7–9) and P10 (n = 17–19). (D) Androgen receptor (AR) mRNA expression was analyzed within the brains of male and female mice by qRT-PCR. The GAPDH gene was used for normalization (n = 5). Results are presented as means ± SEM. Significance was calculated by two-way ANOVA with sex and age as factors. A significant effect of sex is indicated in the figures. (D) Comparison by two-tailed Student’s test (***p < 0.001; **p < 0.01; *p < 0.05 when compared to the corresponding male group).</p

The role of brain androgen receptors in determining sex differences in myelin at P10.

<p>(A and B) Analysis by qRT-PCR of AR (A) and MBP (B) mRNA expressions in the brain at P10 of AR^NesCre male mice. Littermates carrying a floxed exon 1 of the <i>AR</i> gene (AR^Lox) were used as controls (n = 4). GAPDH and <i>cyclophilin A</i> were used as normalization genes. (C and D) MBP protein levels, analyzed by Western blot and normalized to the endogenous β-actin protein, in the brain of AR^NesCre mice when compared to AR^Lox controls at P10 (n = 4). (E) Immunostaining of MBP (green), Olig2 (red), CC1 (mature oligodendrocytes, blue) and the merged triple immunostaining (yellow). Representative photomicrographs were taken at the level of the genu of the corpus callosum (str = striatum). Scale bar = 100 μm. (F-I) Within the P10 corpus callosum, (F) quantification of the MBP⁺ area in a 0.26 mm² field and counting of (G) Olig2⁺ oligodendroglial cells, (H) CC1⁺ mature oligodendrocytes and (I) Olig2/CC1 double positive cells in AR^NesCre mice compared with AR^Lox littermates (n = 6). Results are presented as means ± SEM. For each animal, the mean value of the corpus callosum was calculated from the splenium, the center and the genu. Significance was calculated using two-tailed Student’s t test (***p < 0.001; *p < 0.05 when compared to the corresponding control AR^Lox males).</p