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: Ecotoxicology and environmental safety

Article Title: Ameliorative effect of betulinic acid against zearalenone exposure triggers testicular dysfunction and oxidative stress in mice via p38/ERK MAPK inhibition and Nrf2-mediated antioxidant defense activation.

doi: 10.1016/j.ecoenv.2022.113561

Figure Lengend Snippet: Fig. 2. BA protected the aggravation of male reproduction injury in ZEA-induced mice. The morphology of the sperm was photographed using an optical microscope (A). The sperm motility was used to evaluate the male repro duction of mice, including sperm survival rate (B), sperm malformation rate (C), and sperm mortality rate (D). Protein and mRNA levels of ERα in testis were detected by immunoblotting and RT-PCR analysis, respectively, and the pro tein and mRNA levels were normalized to β-actin (E-G). The content of testosterone in serum was detected by ELISA kit (H). The mRNA expression of CLDN11 (I), CDH2 (J), and Vim (K) were measured by RT-PCR. Mean ± SEM, *P < 0.05 and **P < 0.01 represented a significant differ ence compared to the control group, while #P < 0.05 and ##P < 0.01 represented a signifi cant difference compared to the ZEA group.

Article Snippet: Testosterone enzyme linked immunosorbent assay (ELISA) kit (CSB-E05101m) was obtained from Cusabio Biotech Co. Ltd. (Wuhan, China).

Techniques: Microscopy, Western Blot, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Expressing, Control

Journal: Research

Article Title: CCN5 Drives Leydig Cell Aging and Testicular Dysfunction: Insights into Fibrosis, Lipid Dysregulation, and Therapeutic Potential

doi: 10.34133/research.0762

Figure Lengend Snippet: CCN5 overexpression leads to LC senescence and a decline in testicular function. (A) Western blot shows the CCN5 protein level of LCs before and after transfected with CCN5 overexpression or control plasmids. (B) SA-β-gal staining of LCs transfected with CCN5 overexpression or control plasmids. The scale bar represents 10 μm. n = 4 technical repetitions. (C) CCK-8 assay shows the proliferation of LCs transfected with CCN5 overexpression or control plasmids. n = 5 technical repetitions. (D) Flow cytometry demonstrated the influence of LCs transfected with CCN5 overexpression or control plasmids on the early (Q4) and late apoptosis (Q2) of LCs. The existence of Annexin V ( x -axis) and the nuclear staining of PI ( y -axis) by flow cytometry were shown. n = 3 technical repetitions. (E) Schematic illustration of the experimental workflow in vivo. (F) Immunofluorescence staining of CCN5 in testis after injection with CCN5-AAV9 or vector. The scale bar represents 100 μm. The right panel shows the fluorescence intensity statistics. n = 5 biological independent samples. (G) Testis after injection with CCN5-AAV9 showed a lower testis/body weight index. n = 5 biological independent samples. (H) ELISA results showing the testosterone concentration in the supernatant of cultured LC. n = 5 biological independent samples. (I) PAS staining of testis after injection with CCN5-AAV9 or vector. Asterisks indicate severely atrophied and degenerated seminiferous tubules. The scale bar represents 200 μm. (J) SA-β-gal staining of testis after injection with CCN5-AAV9 or vector. The scale bar represents 100 μm. n = 5 biological independent samples. (K) Immunofluorescence staining of CYP11A1 (LCs marker), SOX9 (SC marker), or DDX4 (germ cell marker) in testis after injection with CCN5-AAV9 or vector. The scale bar represents 200 μm. n = 5 biological independent samples. (L) The rotarod is used to assess endurance in mice. n = 5 biological independent samples. (M and N) Sexual behaviors including sniffing (M) and mating (N) in mice injection with CCN5-AAV9 or vector. n = 5 biological independent samples.

Article Snippet: Testosterone levels were measured using a Total Testosterone ELISA Kit (abclonal, RK00724) according to the manufacturer’s instructions.

Techniques: Over Expression, Western Blot, Transfection, Control, Staining, CCK-8 Assay, Flow Cytometry, In Vivo, Immunofluorescence, Injection, Plasmid Preparation, Fluorescence, Enzyme-linked Immunosorbent Assay, Concentration Assay, Cell Culture, Marker

Journal: Research

Article Title: CCN5 Drives Leydig Cell Aging and Testicular Dysfunction: Insights into Fibrosis, Lipid Dysregulation, and Therapeutic Potential

doi: 10.34133/research.0762

Figure Lengend Snippet: CCN5 promotes LCs senescence by down-regulating RNF213. (A) This table shows the top 10 proteins that combined with CCN5 identified by LC-MS/MS. (B) Bar plot shows the enrichment analysis of proteins that combined with CCN5. (C) The LCs (top panel) and 293t (bottom panel) cells lysates were immunoprecipitated by anti-FLAG antibody (fused with CCN5) or IgG with protein A/G magnetic beads. The precipitated proteins were analyzed by Western blot using anti-DOCK7 and anti-RNF213 antibody, respectively. (D) Immunofluorescence co-staining of RNF213 (red) and CCN5 (green) in young and old mice testis. The scale bar represents 100 μm. n = 10 different regions. (E) Scatter plot showing the correlation between CCN5 and RNF213 expression levels in the testicular interstitial region. (F) Western blot shows the level of RNF213 in LCs after recombinant CCN5 protein with CCN5(ΔSP) or CCN5(wt) overexpression. n = 3 technical repeats. (G) Western blot analysis to validate the knockdown efficiency of RNF213 in LCs. n = 3 technical repeats. (H) Western blot shows the fold change of FOXO1, FOXO3, p16, and p21 in LCs after knockdown of RNF213. n = 3 technical repeats. (I) ELISA was used to measure the change of testosterone concentration in the culture supernatant of LCs after knockdown of RNF213. n = 3 technical repeats. (J) SA-β-gal staining of LCs with RNF213 knockdown. The scale bar represents 20 μm. n = 5 technical repeats. (K) Crystal violet staining marks colony formation in LCs with RNF213 knockdown. n = 2 technical repeats. (L) Oil red staining of LCs with RNF213 knockdown. The scale bar represents 10 μm. n = 10 cells.

Article Snippet: Testosterone levels were measured using a Total Testosterone ELISA Kit (abclonal, RK00724) according to the manufacturer’s instructions.

Techniques: Liquid Chromatography with Mass Spectroscopy, Immunoprecipitation, Magnetic Beads, Western Blot, Immunofluorescence, Staining, Expressing, Recombinant, Over Expression, Knockdown, Enzyme-linked Immunosorbent Assay, Concentration Assay

Journal: Research

Article Title: CCN5 Drives Leydig Cell Aging and Testicular Dysfunction: Insights into Fibrosis, Lipid Dysregulation, and Therapeutic Potential

doi: 10.34133/research.0762

Figure Lengend Snippet: Knockdown of CCN5 in vivo alleviates age-related testicular degeneration in aged mice. (A) Schematic illustration of the experimental workflow in vivo. (B) Immunofluorescence staining of CCN5 in testis after injection with shRNA of CCN5 or scramble. The scale bar represents 100 μm (bottom panel) or 200 μm (top panel). n = 10 regions of 5 biological independent samples. (C) Photograph of aged mice after testicular injection of shRNA-AAV. (D) The body weight of aged mice after testicular injection of shRNA-AAV. n = 5 biological independent samples. (E) Photograph of testis from aged mice after testicular injection of shRNA-AAV. Testis/body weight index data from 5 biological independent samples. (F) PAS staining of testis from aged mice after testicular injection of shRNA-AAV. The scale bar represents 200 μm. (G) SA-β-gal staining of testis from aged mice after testicular injection of shRNA-AAV. The scale bar represents 100 μm. n = 5 biological independent samples. (H) Nile red staining of mice testis tissue after injection with shRNA-AAV of CCN5 or scramble. The scale bar represents 50 μm. n = 5 biological independent samples. (I and J) Immunofluorescence staining of (I) CYP11A1 (LCs marker) or (J) DDX4 (germ cell marker) in testis after injection with shRNA-AAV of CCN5 or scramble. The scale bar represents (I) 50 μm or (J) 100 μm. n = 5 biological independent samples. (K) ELISA results showing the testosterone concentration in the testicular tissue homogenate from aged mice after testicular injection of shRNA-AAV. n = 5 biological independent samples. (L) The rotarod is used to assess endurance in mice. n = 5 biological independent samples. (M and N) Sexual behaviors including sniffing (M) and mating (N) in mice after injection with shRNA-AAV or scramble. n = 5 biological independent samples.

Article Snippet: Testosterone levels were measured using a Total Testosterone ELISA Kit (abclonal, RK00724) according to the manufacturer’s instructions.

Techniques: Knockdown, In Vivo, Immunofluorescence, Staining, Injection, shRNA, Marker, Enzyme-linked Immunosorbent Assay, Concentration Assay

Journal: Molecular Medicine

Article Title: MicroRNA regulation of the proliferation and apoptosis of Leydig cells in diabetes

doi: 10.1186/s10020-021-00370-8

Figure Lengend Snippet: Effects of diabetes on testicular function and apoptosis. Eight weeks after diabetes was established, the right testis of each rat was removed and separately photographed ( A ) and the testis index {(testis weight/body weight) × 100%} was calculated ( B ). Concentrations of serum ( C ) and testicular ( D ) testosterone detected by ELISA in each group. Representative hematoxylin & eosin (H&E) and TUNEL staining of rat testicular tissues from ND (first 2 panels) and DM (last 2 panels) groups. For a better comparison, the second panel in each group is a partially enlarged panel (black box) of the first panel. Scale bar = 100 μm (first panel) and 40 μm (second panel) ( E ). Data are presented as mean ± SD.*p < 0.05 **p < 0.01 compared with the ND group

Article Snippet: Total testosterone was measured using the Rat or Human Testosterone ELISA kit (Cusabio, Wuhan, China) according to the manufacturer’s instructions.

Techniques: Enzyme-linked Immunosorbent Assay, TUNEL Assay, Staining, Comparison

Journal: Molecular Medicine

Article Title: MicroRNA regulation of the proliferation and apoptosis of Leydig cells in diabetes

doi: 10.1186/s10020-021-00370-8

Figure Lengend Snippet: Effect of glucose concentration on miRNAs and apoptosis. Expression of miR-504 ( A ) and miR-935 ( B ) in R2C cells at 24 h after culturing in a glucose concentration gradient (basal glucose for R2C cell was 5 mM and stimulated concentrations were 15 mM and 30 mM). Data were normalised to U6 RNA, used as an internal control. Expression of MEK5 ( C ) and MEF2C ( D ) determined using RT-qPCR analysis. β-actin was used as an internal control. Representative immunoblotting ( E ) and cumulative quantification of the protein levels of MEK5 ( F ) and MEF2C ( G ) in R2C cells. Media were collected and assayed for concentration of testosterone using ELISA ( H ). Detection of apoptotic cells using FACS analysis with FITC-labelled annexin V and PI staining ( I ). Bar graphs represent the percentage of apoptotic cells in each group ( J ). *p < 0.05, **p < 0.01, ***p < 0.001. n = 3

Article Snippet: Total testosterone was measured using the Rat or Human Testosterone ELISA kit (Cusabio, Wuhan, China) according to the manufacturer’s instructions.

Techniques: Concentration Assay, Expressing, Control, Quantitative RT-PCR, Western Blot, Enzyme-linked Immunosorbent Assay, Staining

Journal: Molecular Medicine

Article Title: MicroRNA regulation of the proliferation and apoptosis of Leydig cells in diabetes

doi: 10.1186/s10020-021-00370-8

Figure Lengend Snippet: Modulation of proliferation and apoptosis of Leydig cells by mRNA targets of miR-504. Expression of miR-504 in miR-504 mimic-or miR-504 inhibitor-infected R2C cells at 24 h after culturing in normal or high glucose (HG). Data were normalised to U6 RNA, used as an internal control ( A ). Expression of MEK5 and MEF2C determined by RT-qPCR analysis. β-actin was used as an internal control ( B , C ). Representative immunoblotting ( D ) and cumulative quantification ( E , F ) of the protein levels of MEK5 and MEF2C in R2C cells transfected with miR-504 mimic, miR-504 inhibitor, mimic NC, or inhibitor NC. Media were collected and assayed for concentration of testosterone using ELISA ( G ). Cell proliferation was assayed using CCK8 ( H ). Detection of apoptotic cells by FACS analysis with FITC-labelled annexin V and PI staining ( I ). Bar graphs represent the percentage of apoptotic cells in each group (J). *p < 0.05, **p < 0.01, ***p < 0.001. n = 3

Article Snippet: Total testosterone was measured using the Rat or Human Testosterone ELISA kit (Cusabio, Wuhan, China) according to the manufacturer’s instructions.

Techniques: Expressing, Infection, Control, Quantitative RT-PCR, Western Blot, Transfection, Concentration Assay, Enzyme-linked Immunosorbent Assay, Staining

Journal: Molecular Medicine

Article Title: MicroRNA regulation of the proliferation and apoptosis of Leydig cells in diabetes

doi: 10.1186/s10020-021-00370-8

Figure Lengend Snippet: Modulation of proliferation and apoptosis of Leydig cells by mRNA targets of miR-935. Expression of miR-935 in miR-935 mimic-or miR-935 inhibitor-infected R2C cells at 24 h after culturing in normal or high glucose (HG). Data were normalised to U6 RNA used as an internal control ( A ). Expression of MEF2C determined by RT-qPCR analysis. β-actin was used as an internal control ( B ). Representative immunoblotting ( C ) and cumulative quantification ( D ) of the protein levels of MEF2C in R2C cells transfected with miR-935 mimic, miR-935 inhibitor, mimic NC, or inhibitor NC. Media were collected and assayed for concentration of testosterone using ELISA ( E ). Cell proliferation was assayed using CCK8 ( F ). Detection of apoptotic cells by FACS analysis with FITC-labelled annexin V and PI staining ( G ). Bar graphs represent the percentage of apoptotic cells in each group (H). *p < 0.05, **p < 0.01, ***p < 0.001. n = 3

Article Snippet: Total testosterone was measured using the Rat or Human Testosterone ELISA kit (Cusabio, Wuhan, China) according to the manufacturer’s instructions.

Techniques: Expressing, Infection, Control, Quantitative RT-PCR, Western Blot, Transfection, Concentration Assay, Enzyme-linked Immunosorbent Assay, Staining

Journal: Sexual Medicine

Article Title: Icariin inhibits hyperglycemia-induced cell death in penile cavernous tissue and improves erectile function in type 1 diabetic rats

doi: 10.1093/sexmed/qfaf017

Figure Lengend Snippet: Effect of icariin on oxidative stress in the penile cavernous tissue of DM rats. (A) MDA levels, (B) GSH levels, (C) the GSH/GSSG ratio, and (D) SOD activity in each group were measured via ELISA. (E) Semiquantitative analysis and (G) representative images (40×) of ROS in each group. (F) Semiquantitative analysis and (H) representative images (15×) of Prussian blue staining showing nucleus (red areas) and iron deposition (blue areas). And P < .05 vs the control group. * P < .05 vs the control + ICA group. # P < .05 vs the DM group.

Article Snippet: Serum testosterone was detected according to the instructions of the rat serum testosterone ELISA kit (Elabscience Biotechnology, Wuhan, China; E-OSEL-R0003).

Techniques: Activity Assay, Enzyme-linked Immunosorbent Assay, Staining, Control

Journal: Sexual Medicine

Article Title: Icariin inhibits hyperglycemia-induced cell death in penile cavernous tissue and improves erectile function in type 1 diabetic rats

doi: 10.1093/sexmed/qfaf017

Figure Lengend Snippet: Effect of icariin on the fibrosis of penile cavernous tissue in DM rats. (A) Representative images (15×) of Masson's trichrome staining showing smooth muscle cells (SMCs) and collagen fibers. (B) Semiquantitative analysis of the SM/C ratio of each group. (C) NO levels in each group were measured using ELISA. (D) Western blot analysis of the expression of eNOS and peNOS in penile cavernous tissue from each group. (E, F) Semiquantitative analysis of eNOS and p-eNOS levels and the p-eNOS/eNOS ratio in penile cavernous tissue from each group. And P < .05 vs the control group. * P < .05 vs the control + ICA group. # P < .05 vs the DM group.

Article Snippet: Serum testosterone was detected according to the instructions of the rat serum testosterone ELISA kit (Elabscience Biotechnology, Wuhan, China; E-OSEL-R0003).

Techniques: Staining, Enzyme-linked Immunosorbent Assay, Western Blot, Expressing, Control