Journal: The FASEB Journal

Article Title: Plasma membrane cholesterol trafficking in steroidogenesis

doi: 10.1096/fj.201800697rrr

Figure Lengend Snippet: Figure 1. Plasma membranes can support steroidogenesis. PMs from rat adrenals, MLTCs, Y1 adrenocortical cells, and liver were purified and added to the in vitro mitochondrial reconstitution assay, in the absence of any other sources of cholesterol or accessory proteins. Pregnenolone production was measured by ELISA. A) The relative pregnenolone production was measured with rat adrenal PMs, MLTC, Y1 adrenocortical cell, and liver PMs as the sources of cholesterol. The basal pregnenolone production for mitochondria without PMs ranged from 0.5 to 3 ng/ml. B) Comparison of rat adrenal PMs and lipid emulsion as cholesterol sources for pregnenolone production in the absence or presence of accessory proteins. LE, lipid emulsion; Mito, mitochondria; NS, not significant; SNAREs/StAR, a cocktail containing recombinant SNAP23, SNAP25, a-SNAP, STX17, and N62-StAR. Results are representative of 3 independent experiments (n = 3). **P , 0.01, ***P , 0.001.

Article Snippet: Mouse Leydig tumor cells (MLTCs) were obtained from ATCC and were cultured in RPMI 1640 plus 10% fetal bovine serum, 100 U/ml penicillin, and 100 g/ml streptomycin.

Techniques: Clinical Proteomics, In Vitro, Reconstitution Assay, Enzyme-linked Immunosorbent Assay, Comparison, Emulsion, Recombinant

Journal: PLOS Pathogens

Article Title: Interferon epsilon is produced in the testis and protects the male reproductive tract against virus infection, inflammation and damage

doi: 10.1371/journal.ppat.1012702

Figure Lengend Snippet: (A) Schematic diagram of the anatomy of the male reproductive tract. (B) Ifne and (C) Ifnb1 mRNA expression in the testis, epididymis (caput, corpus and cauda regions) and vas deferens (P. vas = proximal and D. vas = distal region of vas deferens) in 56-day-old mice (n = 8), (D) Ifne mRNA expression in the testis in mice aged 5–180 days (n = 6–8 per group). (E) Ifne and (F) type I interferon receptor Ifnar1 and (G) Ifnar2 mRNA expression in testicular cell types isolated from 44-day-old WT mice (n = 3 cell preps per cell type, Sp’cytes = spermatocytes). All mRNA expression data is relative to the reference gene Rplp0 (2^-dCt = relative copy number). (H) IFNε localisation in 56- and 25-day-old WT adult mouse male reproductive tract, compared to that of 56-day-old Ifne -/- mice using an anti-human IFNɛ antibody validated for mouse. Representative images from n = 4 mice. Inset: Isotype control. Yellow: IFNɛ. Blue: DAPI. Scale bar: 50 μm. Red arrows: Leydig cell and macrophage clusters. Arrowhead: post-meiotic spermatogenic cells. White arrows: Epithelial staining in vas deferens. Asterisk: stereocilia layer of the epididymal epithelium. E = epithelium of epididymis and vas deferens. L = lumen. The Biorender software was used to create Fig 1A.

Article Snippet: Cells were grown in Leydig Cell Medium (ScienCell Cat. #4511) at 37°C in 5% CO 2 , and passaged at 90% confluency (~ every 3 days) using mild trypsinization.

Techniques: Expressing, Isolation, Control, Staining, Software

Journal: PLOS Pathogens

Article Title: Interferon epsilon is produced in the testis and protects the male reproductive tract against virus infection, inflammation and damage

doi: 10.1371/journal.ppat.1012702

Figure Lengend Snippet: (A) IFNɛ localisation in the adult human testis, using an anti-human IFNɛ antibody at low and higher magnification; scale bars 50 and 20 μm respectively. Inset: Isotype control. Yellow: IFNɛ. Blue: DAPI. Arrows: Leydig cell and macrophage clusters. Arrowhead: post-meiotic spermatogenic cells embedded in Sertoli cell cytoplasm. Representative images from n = 3 testicular biopsy samples exhibiting normal testicular histology. (B) IFNE , IFNB1 and their receptors IFNAR1 and IFNAR2 mRNA expression measured by qRT-PCR in testicular tissue samples showing intact spermatogenesis without any signs of inflammation (Normal) compared with biopsies showing impaired spermatogenesis and focal immune cell infiltrates (I + WBC). n = 5–19 per group. Student’s t-test, ***P < 0.001. (C) Correlation analysis of IFNAR2 mRNA expression and the histological degree of immune cell infiltration in the testis biopsy site (scored from 0–4; 4 = dense, 3 = sparse, 2 = scattered, 1 = single cells, 0 = absent). n = 5–19 per group. Spearman’s coefficient method. (D) IFNɛ protein measured using a two-site ELISA assay in testicular interstitial fluid from three patient groups with different causes of infertility. MTA = Mixed testicular atrophy, SCO = Sertoli Cell Only phenotype, SA = spermatogenic arrest. LoD = Limit of detection: 3.9 pg/ml. Pooled samples, 25 μl testicular interstitial fluid per patient from 4 patients in each category.

Article Snippet: Cells were grown in Leydig Cell Medium (ScienCell Cat. #4511) at 37°C in 5% CO 2 , and passaged at 90% confluency (~ every 3 days) using mild trypsinization.

Techniques: Control, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay