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ET rescues testosterone biosynthesis from lipotoxic stress by reinstating PKA-CREB-StAR signaling. (A) Schematic illustration of the canonical steroidogenic pathway in Leydig cells, highlighting the key enzymes and intermediates from cholesterol to testosterone. (B-D) Representative immunoblotting images and densitometric quantification of phosphorylated PKA ( p -PKA), p -CREB (Ser133), total CREB, and StAR protein abundance in TM3 Leydig cells challenged with palmitate (PA) in the presence or absence of ET. GAPDH was utilized as the internal loading control. (E-F) Quantitative analysis of pregnenolone and testosterone secretion levels in the culture supernatant of TM3 cells via Enzyme-Linked Immunosorbent Assay <t>(ELISA),</t> demonstrating the functional rescue of steroidogenesis by ET. (G) ELISA-based quantification of intratesticular 5α-dihydrotestosterone <t>(DHT)</t> levels in the Control, HFD, and HFD + ET mouse cohorts. (H) Schematic workflow of the ex vivo organ culture system using human seminiferous tubules. Specimens were obtained via therapeutic testicular sperm extraction (TESE) from obese patients presenting with oligoasthenoteratozoospermia. (I) Quantification of mitochondrial ROS levels in human seminiferous tubules treated with PA and ET. (J) Functional assessment of testosterone secretion in the human ex vivo tubule culture system following PA challenge and ET intervention. Data are presented as means ± SEM ( n = 3). The normality of all datasets, including Western blot densitometry and ELISA measurements, was rigorously verified using the Shapiro-Wilk test. Statistical significance was determined by one-way ANOVA followed by Tukey's post-hoc test. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001 versus the respective stress group (PA).
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ET rescues testosterone biosynthesis from lipotoxic stress by reinstating PKA-CREB-StAR signaling. (A) Schematic illustration of the canonical steroidogenic pathway in Leydig cells, highlighting the key enzymes and intermediates from cholesterol to testosterone. (B-D) Representative immunoblotting images and densitometric quantification of phosphorylated PKA ( p -PKA), p -CREB (Ser133), total CREB, and StAR protein abundance in TM3 Leydig cells challenged with palmitate (PA) in the presence or absence of ET. GAPDH was utilized as the internal loading control. (E-F) Quantitative analysis of pregnenolone and testosterone secretion levels in the culture supernatant of TM3 cells via Enzyme-Linked Immunosorbent Assay <t>(ELISA),</t> demonstrating the functional rescue of steroidogenesis by ET. (G) ELISA-based quantification of intratesticular 5α-dihydrotestosterone <t>(DHT)</t> levels in the Control, HFD, and HFD + ET mouse cohorts. (H) Schematic workflow of the ex vivo organ culture system using human seminiferous tubules. Specimens were obtained via therapeutic testicular sperm extraction (TESE) from obese patients presenting with oligoasthenoteratozoospermia. (I) Quantification of mitochondrial ROS levels in human seminiferous tubules treated with PA and ET. (J) Functional assessment of testosterone secretion in the human ex vivo tubule culture system following PA challenge and ET intervention. Data are presented as means ± SEM ( n = 3). The normality of all datasets, including Western blot densitometry and ELISA measurements, was rigorously verified using the Shapiro-Wilk test. Statistical significance was determined by one-way ANOVA followed by Tukey's post-hoc test. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001 versus the respective stress group (PA).
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ET rescues testosterone biosynthesis from lipotoxic stress by reinstating PKA-CREB-StAR signaling. (A) Schematic illustration of the canonical steroidogenic pathway in Leydig cells, highlighting the key enzymes and intermediates from cholesterol to testosterone. (B-D) Representative immunoblotting images and densitometric quantification of phosphorylated PKA ( p -PKA), p -CREB (Ser133), total CREB, and StAR protein abundance in TM3 Leydig cells challenged with palmitate (PA) in the presence or absence of ET. GAPDH was utilized as the internal loading control. (E-F) Quantitative analysis of pregnenolone and testosterone secretion levels in the culture supernatant of TM3 cells via Enzyme-Linked Immunosorbent Assay <t>(ELISA),</t> demonstrating the functional rescue of steroidogenesis by ET. (G) ELISA-based quantification of intratesticular 5α-dihydrotestosterone <t>(DHT)</t> levels in the Control, HFD, and HFD + ET mouse cohorts. (H) Schematic workflow of the ex vivo organ culture system using human seminiferous tubules. Specimens were obtained via therapeutic testicular sperm extraction (TESE) from obese patients presenting with oligoasthenoteratozoospermia. (I) Quantification of mitochondrial ROS levels in human seminiferous tubules treated with PA and ET. (J) Functional assessment of testosterone secretion in the human ex vivo tubule culture system following PA challenge and ET intervention. Data are presented as means ± SEM ( n = 3). The normality of all datasets, including Western blot densitometry and ELISA measurements, was rigorously verified using the Shapiro-Wilk test. Statistical significance was determined by one-way ANOVA followed by Tukey's post-hoc test. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001 versus the respective stress group (PA).
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ET rescues testosterone biosynthesis from lipotoxic stress by reinstating PKA-CREB-StAR signaling. (A) Schematic illustration of the canonical steroidogenic pathway in Leydig cells, highlighting the key enzymes and intermediates from cholesterol to testosterone. (B-D) Representative immunoblotting images and densitometric quantification of phosphorylated PKA ( p -PKA), p -CREB (Ser133), total CREB, and StAR protein abundance in TM3 Leydig cells challenged with palmitate (PA) in the presence or absence of ET. GAPDH was utilized as the internal loading control. (E-F) Quantitative analysis of pregnenolone and testosterone secretion levels in the culture supernatant of TM3 cells via Enzyme-Linked Immunosorbent Assay <t>(ELISA),</t> demonstrating the functional rescue of steroidogenesis by ET. (G) ELISA-based quantification of intratesticular 5α-dihydrotestosterone <t>(DHT)</t> levels in the Control, HFD, and HFD + ET mouse cohorts. (H) Schematic workflow of the ex vivo organ culture system using human seminiferous tubules. Specimens were obtained via therapeutic testicular sperm extraction (TESE) from obese patients presenting with oligoasthenoteratozoospermia. (I) Quantification of mitochondrial ROS levels in human seminiferous tubules treated with PA and ET. (J) Functional assessment of testosterone secretion in the human ex vivo tubule culture system following PA challenge and ET intervention. Data are presented as means ± SEM ( n = 3). The normality of all datasets, including Western blot densitometry and ELISA measurements, was rigorously verified using the Shapiro-Wilk test. Statistical significance was determined by one-way ANOVA followed by Tukey's post-hoc test. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001 versus the respective stress group (PA).
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ET rescues testosterone biosynthesis from lipotoxic stress by reinstating PKA-CREB-StAR signaling. (A) Schematic illustration of the canonical steroidogenic pathway in Leydig cells, highlighting the key enzymes and intermediates from cholesterol to testosterone. (B-D) Representative immunoblotting images and densitometric quantification of phosphorylated PKA ( p -PKA), p -CREB (Ser133), total CREB, and StAR protein abundance in TM3 Leydig cells challenged with palmitate (PA) in the presence or absence of ET. GAPDH was utilized as the internal loading control. (E-F) Quantitative analysis of pregnenolone and testosterone secretion levels in the culture supernatant of TM3 cells via Enzyme-Linked Immunosorbent Assay <t>(ELISA),</t> demonstrating the functional rescue of steroidogenesis by ET. (G) ELISA-based quantification of intratesticular 5α-dihydrotestosterone <t>(DHT)</t> levels in the Control, HFD, and HFD + ET mouse cohorts. (H) Schematic workflow of the ex vivo organ culture system using human seminiferous tubules. Specimens were obtained via therapeutic testicular sperm extraction (TESE) from obese patients presenting with oligoasthenoteratozoospermia. (I) Quantification of mitochondrial ROS levels in human seminiferous tubules treated with PA and ET. (J) Functional assessment of testosterone secretion in the human ex vivo tubule culture system following PA challenge and ET intervention. Data are presented as means ± SEM ( n = 3). The normality of all datasets, including Western blot densitometry and ELISA measurements, was rigorously verified using the Shapiro-Wilk test. Statistical significance was determined by one-way ANOVA followed by Tukey's post-hoc test. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001 versus the respective stress group (PA).
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Image Search Results


ET rescues testosterone biosynthesis from lipotoxic stress by reinstating PKA-CREB-StAR signaling. (A) Schematic illustration of the canonical steroidogenic pathway in Leydig cells, highlighting the key enzymes and intermediates from cholesterol to testosterone. (B-D) Representative immunoblotting images and densitometric quantification of phosphorylated PKA ( p -PKA), p -CREB (Ser133), total CREB, and StAR protein abundance in TM3 Leydig cells challenged with palmitate (PA) in the presence or absence of ET. GAPDH was utilized as the internal loading control. (E-F) Quantitative analysis of pregnenolone and testosterone secretion levels in the culture supernatant of TM3 cells via Enzyme-Linked Immunosorbent Assay (ELISA), demonstrating the functional rescue of steroidogenesis by ET. (G) ELISA-based quantification of intratesticular 5α-dihydrotestosterone (DHT) levels in the Control, HFD, and HFD + ET mouse cohorts. (H) Schematic workflow of the ex vivo organ culture system using human seminiferous tubules. Specimens were obtained via therapeutic testicular sperm extraction (TESE) from obese patients presenting with oligoasthenoteratozoospermia. (I) Quantification of mitochondrial ROS levels in human seminiferous tubules treated with PA and ET. (J) Functional assessment of testosterone secretion in the human ex vivo tubule culture system following PA challenge and ET intervention. Data are presented as means ± SEM ( n = 3). The normality of all datasets, including Western blot densitometry and ELISA measurements, was rigorously verified using the Shapiro-Wilk test. Statistical significance was determined by one-way ANOVA followed by Tukey's post-hoc test. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001 versus the respective stress group (PA).

Journal: Redox Biology

Article Title: Ergothioneine rescues obesity-induced testicular dysfunction via dual restoration of steroidogenesis and mitochondrial redox homeostasis

doi: 10.1016/j.redox.2026.104090

Figure Lengend Snippet: ET rescues testosterone biosynthesis from lipotoxic stress by reinstating PKA-CREB-StAR signaling. (A) Schematic illustration of the canonical steroidogenic pathway in Leydig cells, highlighting the key enzymes and intermediates from cholesterol to testosterone. (B-D) Representative immunoblotting images and densitometric quantification of phosphorylated PKA ( p -PKA), p -CREB (Ser133), total CREB, and StAR protein abundance in TM3 Leydig cells challenged with palmitate (PA) in the presence or absence of ET. GAPDH was utilized as the internal loading control. (E-F) Quantitative analysis of pregnenolone and testosterone secretion levels in the culture supernatant of TM3 cells via Enzyme-Linked Immunosorbent Assay (ELISA), demonstrating the functional rescue of steroidogenesis by ET. (G) ELISA-based quantification of intratesticular 5α-dihydrotestosterone (DHT) levels in the Control, HFD, and HFD + ET mouse cohorts. (H) Schematic workflow of the ex vivo organ culture system using human seminiferous tubules. Specimens were obtained via therapeutic testicular sperm extraction (TESE) from obese patients presenting with oligoasthenoteratozoospermia. (I) Quantification of mitochondrial ROS levels in human seminiferous tubules treated with PA and ET. (J) Functional assessment of testosterone secretion in the human ex vivo tubule culture system following PA challenge and ET intervention. Data are presented as means ± SEM ( n = 3). The normality of all datasets, including Western blot densitometry and ELISA measurements, was rigorously verified using the Shapiro-Wilk test. Statistical significance was determined by one-way ANOVA followed by Tukey's post-hoc test. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001 versus the respective stress group (PA).

Article Snippet: The concentration of dihydrotestosterone (DHT) in testicular tissues was quantified using a commercial DHT ELISA Kit (D751011; Sangon Biotech, Shanghai, China) according to the manufacturer's instructions.

Techniques: Western Blot, Quantitative Proteomics, Control, Enzyme-linked Immunosorbent Assay, Functional Assay, Ex Vivo, Organ Culture, Extraction