1. Levels of liver X receptors in testicular biopsies of patients with azoospermia 
Christine Rondanino, Ph.D., Lemlih Ouchchane, M.D., Ph.D., Candice Chauffour, M.Sc., Geoffroy Marceau, Pharm.D., Ph.D., Pierre Déchelotte, M.D., Ph.D., Benoît Sion, Pharm.D., Ph.D., Hanae Pons-Rejraji, Ph.D., Laurent Janny, M.D., David H. Volle, Ph.D., Jean- Marc A. Lobaccaro, Ph.D., Florence Brugnon, M.D., Ph.D. 
Fertility and Sterility 
Volume 102, Issue 2, Pages e5 (August 2014)
DOI: /j.fertnstert
Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions

2. Figure 1
Accumulation of the various liver X receptor (LXR) transcripts in the testes of azoospermic patients. Relative expression of genes encoding (A) LXRα1–3 and (B) LXRβ in the testes of patients with either obstructive azoospermia (OA) or nonobstructive azoospermia (NOA) with different histologic alteration (HS = hypospermatogenesis; MA = maturation arrest; SCO = Sertoli cell–only syndrome). (C) Scatter plots showing LXRα1–3 and LXRβ mRNA levels in the testes of azoospermic patients. Each patient is represented by a dot and the means are represented by a bold line. (D) Representative Western blot of LXRβ and of GAPDH loading control in testes from OA, HS, MA, and SCO patients. (E) Densitometric quantification of relative LXRβ protein levels normalized to GAPDH (n = 3). (F) Relative expression of genes encoding RXRα and RXRβ in OA and NOA testes. Histograms are expressed as mean ± SEM. In quantitative polymerase chain reaction (qPCR) experiments, β-ACTIN was used as internal control. *Analysis of variance: P<.05.
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3. Figure 2
Expression of LXR downstream genes involved in lipid homeostasis. (A) Schematic diagram representing the regulation of lipid homeostasis by LXRα and LXRβ. LXR activity limits low-density lipoprotein (LDL) cholesterol uptake by stimulating the expression of IDOL, an E3 ubiquitin ligase which induces the degradation of LDL receptor (LDLR). LXR controls the efflux of intracellular cholesterol by inducing ABCA1 and ABCG1 transporters. LXR also modulates fatty acid synthesis by activating the transcription of SREBP1c, which in turn regulates the expression of lipogenic enzymes, including FASN, SCD1, and ELOVL6. (B–D) Relative expression of the (B) IDOL and LDLR genes involved in cholesterol capture, (C) ABCA1 and ABCG1 genes involved in cholesterol efflux, and (D) SREBP1c, FASN, SCD1, and ELOVL6 genes involved in fatty acid synthesis in the testes of patients with either OA or NOA. β-ACTIN was used as internal control. (E) Representative Western blot of mature SREBP1c and GAPDH loading control in OA and NOA testes. (F) Quantification of intratesticular cholesterol, cholesterol esters, triglycerides, and free fatty acids levels (μg/mg of tissue) in OA and NOA testes. Histograms are expressed as mean ± SEM. Abbreviations as in Figure 1. *Analysis of variance: P<.05.
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4. Figure 3
Correlation between LXR transcript levels and the presence of germ cells. (A) Quantification of germ cells in testicular biopsies from azoospermic patients. At least 20 random seminiferous tubules were scored for each patient. (B–D) Relative expression of genes encoding specific markers of (B) spermatogonial stem cells (PLZF), (C) spermatocytes (SCP3), and (D) spermatids/spermatozoa (PROTAMINE 2) in OA and NOA (HS, MA, and SCO) testes. (E) Quantification of proliferating cell nuclear antigen (PCNA)–positive cells in immunostained testicular biopsies from azoospermic patients. At least 20 random seminiferous tubules were scored for each patient. (F–H) Relative expression of genes encoding specific markers of (F) proliferation (PCNA), (G) mitosis (CYCLIN A2), and (H) meiosis (DMC1) in testes. (I) Correlation between LXRα2 mRNA expression and the number of germ cells per seminiferous tubule. Each point represents the value obtained for a patient. The 95% confidence interval is represented in grey. Dotted lines indicate the 95% prediction limits. (J) Spearman correlation coefficients obtained from linear regression analyses. (K) Percentage of apoptotic (TUNEL-positive) germ cells per seminiferous tubule in each group of patients. (L) Relative expression of genes encoding proteins involved in apoptosis (BAD, BAX, BCL-2, BCL-XL, FAS, TNFα, and PRO-CASPASE 3). In qPCR experiments, β-ACTIN was used as internal control. Histograms are expressed as mean ± SEM. Abbreviations as in Figure 1. *Analysis of variance: P<.05.
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5. Figure 4
Cellular localization of LXRα and accumulation of LXR downstream genes involved in steroidogenesis. (A–D) Immunolocalization of LXRα in testes from OA patients. The cellular localization of LXRα was assessed with the use of immunofluorescence. Representative microscopy images show the distribution of (A) LXRα (green) and (B) the nuclei (red) in OA biopsies. A merged image is presented in (C). (D) An adjacent tissue section was stained with anti–androgen receptor (AR) antibody. Antibody binding was visualized with the use of Novared horseradish peroxidase substrate. The image shows the localization of AR in the nuclei of Leydig cells (within the interstitial compartment) and of Sertoli cells (within the seminiferous tubule). L = Leydig cell; S = Sertoli cell; T = seminiferous tubule; Go = spermatogonia; Cy = spermatocytes; Ti = spermatids; Spz = spermatozoa; BV = blood vessel. Magnification = ×400; bar = 50 μm. (E–G) Immunolocalization of LXRα in testes of NOA patients with (E) HS, (F) MA with arrest at the spermatogonia stage, and (G) SCO. Shown are merged images of the distribution of LXRα (green) and the nuclei (red). Dashed lines mark the basal membrane of the seminiferous tubules. (H) Schematic diagram depicting the regulation of testosterone synthesis by LXRα in the mouse testis. In Leydig cells, LXRα modulates the accumulation of the steroidogenic proteins StAR and 3βHSD (7). StAR is crucial for the transport of cholesterol to mitochondria where the biosynthesis of steroids is initiated, and 3βHSD is an enzyme involved in androgen synthesis. (I) Relative expression of LXR downstream genes encoding the steroidogenic enzymes StAR and 3βHSD2. β-ACTIN was used as internal control. (J) Intratesticular testosterone levels (nmol/mg of tissue) in OA and NOA biopsies. Histograms are expressed as mean ± SEM. Abbreviations as in Figure 1. *Analysis of variance: P<.05.
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6. Supplemental Figure 1
Immunostaining of proliferating cell nuclear antigen (PCNA) in testicular biopsies of azoospermic patients. Representative microscopy images show the localization of PCNA in the nuclei of spermatogonia and spermatocytes in the seminiferous tubules of testicular biopsies obtained from patients with (A) obstructive azoospermia, (B) hypospermatogenesis, (C) maturation arrest with arrest at the spermatocyte stage, (D) maturation arrest with arrest at the spermatogonia stage, or (E) Sertoli cell–only syndrome (SCO). Testicular biposies from HS patients were heterogeneous, with foci of full spermatogenesis represented in (B) and SCO-like areas (not shown). Antibody binding was visualized with the use of Novared horseradish peroxidase substrate, and sections were counterstained with Mayer hematoxylin. L = Leydig cell; S = Sertoli cell; T = seminiferous tubule; Go = spermatogonia; Cy = spermatocytes; Ti = spermatids; Spz = spermatozoa; BV = blood vessel. Dashed lines mark the basal membrane of the seminiferous tubules. Magnification = ×400; bar = 50 μm.
Fertility and Sterility  , e5DOI: ( /j.fertnstert )
Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions

7. Supplemental Figure 2
Expression of Sertoli cell– and Leydig cell–specific genes. Relative accumulation of genes specifically expressed in (A) Sertoli cells (FSHR, INHBB) or (B) Leydig cells (LHCGR) in the testes of OA and nonobstructive azoospermia patients. β-ACTIN was used as internal control. Histograms are expressed as mean ± SEM. HS = hypospermatogenesis; MA = maturation arrest; OA = obstructive azoospermia; SCO = Sertoli cell–only syndrome. *P<.05.
Fertility and Sterility  , e5DOI: ( /j.fertnstert )
Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions