Journal: Endocrinology and Metabolism

Article Title: Fancd2os Reduces Testosterone Production by Inhibiting Steroidogenic Enzymes and Promoting Cellular Apoptosis in Murine Testicular Leydig Cells

doi: 10.3803/enm.2022.1431

Figure Lengend Snippet: Fig. 4. Fancd2 opposite-strand (Fancd2os) reduces testosterone production and steroidogenic enzyme expression in TM3 cells. The testoster one levels in Fancd2os-overexpressing (A) or knockdown TM3 cells (B) were detected by enzyme-linked immunosorbent assays (n=3 per group). (C, D, E) Relative quantities of mRNA expression of steroidogenic acute regulatory protein (StAR), P450 cholesterol side-chain cleavage (P450scc), and 3β-hydroxysteroid dehydrogenase (3β-HSD) in Fancd2os-overexpressing TM3 cells or Fancd2os knockdown TM3 cells (F, G, H) were determined real-time polymerase chain reaction using β-actin as a housekeeping gene. Each bar represents the mean± standard deviation from three separate experiments. Significant difference compared to TM3, vector/TM3 or NC/TM3. aP<0.05; bP<0.01.

Article Snippet: The testosterone concentration of both serum and cell supernatants was measured using a testosterone ELISA Kit (Elabscience, Houston, TX, USA) according to the manufacturer’s protocol.

Techniques: Expressing, Knockdown, Real-time Polymerase Chain Reaction, Standard Deviation, Plasmid Preparation

Journal: Endocrinology and Metabolism

Article Title: Fancd2os Reduces Testosterone Production by Inhibiting Steroidogenic Enzymes and Promoting Cellular Apoptosis in Murine Testicular Leydig Cells

doi: 10.3803/enm.2022.1431

Figure Lengend Snippet: Fig. 6. Higher Fancd2 opposite-strand (Fancd2os) levels in older mouse Leydig cells result in cellular apoptosis and lower serum testosterone production. (A) The serum testosterone levels from mice of different ages were measured using enzyme-linked immunosorbent assays. (B, C) The testis tissues from different aged mice were sliced and then Fancd2os protein expression and apoptosis were analyzed using immuno chemistry and the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assay, respectively. ST represents seminiferous tubule. Black arrows and red arrows indicate Fancd2os-positive cells and TUNEL-positive cells, respectively. Scale bar repre sents 25 μm. The results are given as the mean±standard deviation (n=3). (D) The correlations between Fancd2os expression (B) and the TUNEL-positive staining rate (C) were analyzed using Pearson correlation coefficients. An r≥0.5 was considered to indicate a strong correla tion, and P<0.05 was considered statistically significant. aP<0.05 compared with juvenile mice; bP<0.05 compared with young mice; cP<0.05 compared with middle-aged mice.

Article Snippet: The testosterone concentration of both serum and cell supernatants was measured using a testosterone ELISA Kit (Elabscience, Houston, TX, USA) according to the manufacturer’s protocol.

Techniques: Expressing, End Labeling, TUNEL Assay, Standard Deviation, Staining

Journal: Science immunology

Article Title: Aire drives steroid hormone biosynthesis by medullary thymic epithelial cells

doi: 10.1126/sciimmunol.abo7975

Figure Lengend Snippet: (A) Gene expression of glucocorticoid biosynthetic enzyme genes in mTEC cells of WT and Aire−/− mice. Bulk RNA-seq data from Aire−/− mTEC are shown for each gene relative to WT expression of the same gene. (B) Gene expression of glucocorticoid biosynthetic enzyme genes in mTEC cells of WT and Fezf2−/− mice. Bulk RNA-seq data from Fezf2−/− mTEC are shown for each gene relative to WT expression of the same gene. (C) Expression of glucocorticoid biosynthetic enzyme genes in bulk mTEC subset samples of WT and Aire−/− mice. The expression level of each gene is presented as a proportion of its maximal expression (1.0) across analyzed cell types as indicated by the color scale. (D) Single-cell transcriptomes obtained from deep sequencing of 174 mTECs were analyzed for the coincident expression of glucocorticoid synthesis genes (Star, Cyp11a1, Hsd3b6, Cyp21a1, and Cyp11b1) with each other or with Aire- and Fezf2-dependent TRA genes (Ins1, Spt1, Mup4, S100a8, Resp18, Rabp9, Krt10, and Maoa) in the same cell (see Materials and Methods for calculation details). Data are presented as means ± SEM. (E) Thymi from 2-week-old WT, KI, and KI Aire−/− mice were digested and cells analyzed by flow cytometry. Representative contour plots show each subset, and numbers indicate the percentage of cells within the Cyp11b1mScarlet+ gate. (F) Data collected and analyzed as in (E), pooled from two experiments with 2- and 3-week-old mice, presented as means ± SEM. Data in (E) and (F) are representative or pooled from two independent experiments with N = 7 WT, 9 KI, and 7 KI Aire−/− mice. (G) WT, KI, and KI Aire−/− thymi from 1-week-old mice were fixed, sectioned, and stained for UEA-1 (mTEC, FITC) and Cyp11b1mScarlet (Atto 565). Confocal images were taken at ×60 oil immersion magnification and are representative of two independent experiments. Scale bars, 100 μm. (H) Total RNAwas extracted from whole thymus lobes of WT and Aire−/− mice, and RT-qPCR was used to quantify Cyp11b1 gene expression. Expression was normalized to 18S RNA and is shown relative to WT expression. Data are pooled from four independent experiments, shown as means ± SEM with N = 17 WT and 9 Aire−/− thymi. (I) Thymus lobes were dissected, minced, and cultured for 72 hours in steroid-free medium. Supernatants were collected, and corticosterone was quantified by a commercial ELISA kit. Data are pooled from four independent experiments, with N = 19 WT and 11 Aire−/− thymi. Statistical significance in (D), (H), and (I) was determined by one-tailed t tests and that in (F) was determined by a mixed linear model with post hoc unpaired t tests applying the Holm-Šidák correction for multiple comparisons. *P < 0.05, **P < 0.01, and ***P < 0.001. n.s., not significant.

Article Snippet: Steroids were quantified using commercial enzyme-linked immunosorbent assay (ELISA) kits (corticosterone, Arbor Assays, K014; progesterone, Cayman Chemical, 582601; testosterone, Cayman Chemical, 582701; estradiol, Cayman Chemical, 501890) according to the manufacturer’s protocol, with standards diluted in steroid-free culture medium.

Techniques: Gene Expression, RNA Sequencing, Expressing, Sequencing, Flow Cytometry, Staining, Quantitative RT-PCR, Cell Culture, Enzyme-linked Immunosorbent Assay, One-tailed Test

Journal: Science immunology

Article Title: Aire drives steroid hormone biosynthesis by medullary thymic epithelial cells

doi: 10.1126/sciimmunol.abo7975

Figure Lengend Snippet: (A) Single-cell RNA-seq analysis of mTEC expression of androgen (Hsd17b3) and estrogen (Cyp19a1) biosynthetic enzymes, with relative expression, as indicated by the color scale, mapped onto a UMAP plot. Numbered populations indicate different cTEC and mTEC cell populations as defined in fig. S4. (B) mTEC specificity and Aire dependence of gene expression in biosynthetic pathways of progestins, corticosteroids (glucocorticoids and mineralocorticoids), androgens, and estrogens. Relative gene expression in bulk Aire+ versus Aire−/− mTEC samples is presented to show Aire-dependent expression, and relative expression in mTECs versus cTECs is presented to show mTEC specificity. Each point represents one gene in the biosynthetic pathway, and the gray box highlights genes with at least twofold higher expression in mTECs versus cTECs and twofold higher expression in Aire+ vs Aire−/− mTEC. A large pink point at the top right represents two or more genes. (C) Total RNAwas extracted from whole thymus lobes of WT and Aire−/− mice, and RT-qPCR was used to quantify gene expression. Ins2 and Spt1 are Aire-dependent TRA genes, and others encode steroidogenic enzymes. Expression was normalized to 18S RNA and is shown relative to WT expression. Data are pooled from four independent experiments, shown as means ± SEM with N = 17 WT and 9 Aire−/− thymi. (D) Thymus lobes were dissected, minced, and cultured for 72 hours in steroid-free medium. Supernatants were collected, and steroids were quantified by commercial ELISA kits. Data were pooled from four independent experiments, with N = 19 WT and 11 Aire−/− thymi. (E) mTEC specificity and Aire dependence of enzyme gene expression in biosynthetic pathways of lipid signaling molecules (prostaglandins, retinoic acid, and thyroid hormone) and amino acid–derived molecules (catecholamines). Relative expression in bulk Aire+ versus Aire−/− mTEC samples is presented to show Aire-dependent expression, and relative expression in mTECs versus cTECs is presented to show mTEC specificity. The gray box highlights genes with at least a twofold expression difference on both axes. Statistical significance in (C) was determined using unpaired t tests with the family-wise error rate set to 0.05 and determined using the correction of Benjamini, Krieger, and Yekutieli and in (D) using one-tailed t tests. *P < 0.05, **P < 0.01, and ***P < 0.001. n.s., not significant.

Article Snippet: Steroids were quantified using commercial enzyme-linked immunosorbent assay (ELISA) kits (corticosterone, Arbor Assays, K014; progesterone, Cayman Chemical, 582601; testosterone, Cayman Chemical, 582701; estradiol, Cayman Chemical, 501890) according to the manufacturer’s protocol, with standards diluted in steroid-free culture medium.

Techniques: RNA Sequencing, Expressing, Gene Expression, Quantitative RT-PCR, Cell Culture, Enzyme-linked Immunosorbent Assay, Derivative Assay, One-tailed Test

Journal: Vaccines

Article Title: Oral Vaccine Formulation for Immunocastration Using a Live-Attenuated Salmonella ΔSPI2 Strain as an Antigenic Vector

doi: 10.3390/vaccines12121400

Figure Lengend Snippet: IgG anti-GnRXG/Q antibody levels expressed as arithmetic means of absorbance values measured at 450 nm on days 0, 15, 30, 45, and 60 post immunization. Male BALB/c mice ( n = 3) were orally immunized on days 0 and 30 with 10 9 CFU of S. Typhimurium 14028s ΔSPI2/pDNAX3::GnRXG/Q (pDNAX3) with 10 9 CFU of S. Typhimurium 14028s ΔSPI2/pJEX::GnRXG/Q (pJEX), subcutaneously with 100 µg of GnRXG/Q purified by affinity chromatography using aluminum as an adjuvant (Control +), and orally with 200 µL of PBS (Control −). IgG anti-GnRXG/Q levels were measured by ELISA on days 0, 15, 30, 45, and 60 post immunization. (*) Indicates a significant difference with p < 0.05 for any of the treated groups compared with the negative control.

Article Snippet: The quantitative determination of testosterone in the serum samples obtained from the study animals was performed using the commercial ELISA kit for testosterone (IBL-America, Inc., Minneapolis, MN, USA).

Techniques: Purification, Affinity Chromatography, Adjuvant, Control, Enzyme-linked Immunosorbent Assay, Negative Control

Journal: Vaccines

Article Title: Oral Vaccine Formulation for Immunocastration Using a Live-Attenuated Salmonella ΔSPI2 Strain as an Antigenic Vector

doi: 10.3390/vaccines12121400

Figure Lengend Snippet: Serum testosterone concentration of the study animals, based on a standard curve. ( A ) Day 15 post immunization. ( B ) Day 30 post immunization. ( C ) Day 45 post immunization. Male BALB/c mice ( n = 3) were orally immunized on days 0 and 30 with 10 9 CFU of S. Typhimurium 14028s ΔSPI2/pDNAX3::GnRXG/Q (pDNAX3) with 10 9 CFU of S. Typhimurium 14028s ΔSPI2/pJEX::GnRXG/Q (pJEX), subcutaneously with 100 µg of GnRXG/Q purified by affinity chromatography using aluminum as an adjuvant (Control +), and orally with 200 µL of PBS (Control −). Serum testosterone levels were measured by commercial ELISA (IBL-AMERICA kit) on days 15, 30, and 45 post immunization. (***) Indicates a difference of p < 0.001 compared to the negative control. (**) Indicates a difference of p < 0.01 compared to the negative control. (*) Indicates a difference of p < 0.05 compared to the negative control.

Article Snippet: The quantitative determination of testosterone in the serum samples obtained from the study animals was performed using the commercial ELISA kit for testosterone (IBL-America, Inc., Minneapolis, MN, USA).

Techniques: Concentration Assay, Purification, Affinity Chromatography, Adjuvant, Control, Enzyme-linked Immunosorbent Assay, Negative Control