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: Cancer Prevention Research

Article Title: Endocrine-Immune-Paracrine Interactions in Prostate Cells as Targeted by Phytomedicines

doi: 10.1158/1940-6207.capr-08-0062

Figure Lengend Snippet: Fig. 3. Effects of DHEA, TGFβ1, and IL-6 on testosterone secretion in cocultured LAPC-4/6S cells. LAPC-4 cells were seeded in treatment media in triplicate onto 30-mm inserts coated with a film of Matrigel at a density of 5 × 105 per insert. Stromal cells (6S) were seeded in triplicate at 1 × 105 per well in 24-well plates. TGFβ1 was added to stromal cultures on the same day at 40 pmol/L to stimulate a reactive stromal phenotype. Cocultures were combined after 2 d while monocultures remained separated; hormones were added; and cells allowed to coculture for 3 d. Media containing hormones were replaced and allowed to condition for 48 h. Conditioned media were assayed for testosterone by ELISA. Each original triplicate experimental sample was assayed in duplicate. Testosterone values were normalized to cell numbers as determined by the modified MTT assay. Columns, mean from three separate experiments; bars, SE. *, P = 0.05; **, P = 0.01, within monoculture or coculture. +, P = 0.05; +++, P < 0.001, between monoculture and coculture.

Article Snippet: Total testosterone was also measured with an ELISA kit (ALPCO).

Techniques: Enzyme-linked Immunosorbent Assay, Modification, MTT Assay

Journal: Cancer Prevention Research

Article Title: Endocrine-Immune-Paracrine Interactions in Prostate Cells as Targeted by Phytomedicines

doi: 10.1158/1940-6207.capr-08-0062

Figure Lengend Snippet: Fig. 4. Red clover effects on TGFβ1 + DHEA–stimulated LAPC-4/6S cocultures. A, LAPC-4 PSA production. The same experimental procedure was used as in the PSA ELISA experiment depicted in Fig. 2A, but in addition to hormone treatments of 100 nmol/L DHEA +/−40 pmol/L TGFβ1, cells were also treated with DHEA/TGFβ1 + 100 nmol/L red clover (RC) isoflavones; DHEA/ TGFβ1 + red clover plus 100 nmol/L ICI 182,780 (ICI; estrogen receptor antagonist); DHEA/TGFβ1 + 100 nmol/L E2; TGFβ1 alone; or 10 nmol/L R1881. Columns, average from three separate experiments; bars, SE. B, LAPC-4 PSA gene expression. LAPC-4 cells were plated in triplicate in monoculture and in coculture with 6S stromal cells, as described in Fig. 2B. Stromal cells were pretreated with 40 pmol/L TGFβ1 for 3 d, then cultures were combined and treated with 100 nmol/L DHEA +/−40 pmol/L TGFβ1; DHEA/TGFβ1 + 100 nmol/L red clover isoflavones; DHEA/TGFβ1 + red clover + 1 μmol/L ICI 182,780 (estrogen receptor antagonist); DHEA/TGFβ1 + 100 nmol/L E2; or 10 nmol/L R1881 for 48 h. RNA was extracted and cDNA was reverse transcribed and probed by real-time PCR for PSA expression, standardized to ribosomal phosphoprotein PO expression. Columns, mean from three experiments; bars, SE. C, stromal testosterone secretion in cocultured LAPC-4/ 6S cells. Testosterone concentrations were determined in conditioned media from stromal cell monocultures, compared with cocultures from the same experiments illustrated in Fig. 3. Hormone treatments include 100 nmol/L DHEA +/−40 pmol/L TGFβ1; DHEA/TGFβ1 + 100 nmol/L red clover isoflavones; DHEA/TGFβ1 + red clover plus 100 nmol/L ICI 182,780; DHEA/TGFβ1 + 100 nmol/L E2; TGFβ1 alone; or 10 nmol/L R1881. Columns, mean from three experiments; bars, SE. *, P = 0.05; **, P = 0.01; +, P = 0.05; ++, P = 0.01; +++, P = 0.001; ⧫, P = 0.05, compared with DHEA + TGFβ1 alone.

Article Snippet: Total testosterone was also measured with an ELISA kit (ALPCO).

Techniques: Enzyme-linked Immunosorbent Assay, Gene Expression, Reverse Transcription, Real-time Polymerase Chain Reaction, Expressing

Journal: Cancer Prevention Research

Article Title: Endocrine-Immune-Paracrine Interactions in Prostate Cells as Targeted by Phytomedicines

doi: 10.1158/1940-6207.capr-08-0062

Figure Lengend Snippet: Fig. 5. Dose-responsive effects of red clover isoflavones to inhibit DHEA + TGFβ1–induced PSA expression in LAPC-4 cell monocultures and cocultures and testosterone metabolism in 6S stromal cell monocultures and cocultures. A, the same experimental procedure for PSA ELISA was used as in Figs. 2 and 4, but in addition to hormone treatments of 100 nmol/L DHEA +/−40 pmol/L TGFβ1 and 10 nmol/L R1881, cells were also treated with DHEA + TGFβ1 + red clover isoflavones at 10, 30, 100, or 300 nmol/L. B, testosterone concentrations were determined in conditioned media from stromal cell monocultures, compared with cocultures from the same experiments represented in A. Hormone treatments included 100 nmol/L DHEA +/−40 pmol/L TGFβ1; DHEA/TGFβ1 + red clover isoflavones at 10, 30, 100, or 300 nmol/L; and 10 nmol/L R1881. Columns, mean from three experiments; bars, SE. *, P = 0.05; **, P = 0.01; ***, P = 0.001; +, P = 0.05; ++, P = 0.01; +++, P = 0.001; ⧫, P = 0.05, compared with DHEA + TGFβ1 alone.

Article Snippet: Total testosterone was also measured with an ELISA kit (ALPCO).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay