Journal: Redox Biology

Article Title: Methylation reader MBD2-mediated GPX4 transcriptional repression drives ovarian granulosa cell ferroptosis in PCOS

doi: 10.1016/j.redox.2026.104034

Figure Lengend Snippet: The PCOS ovary exhibits severer ferroptosis and transcriptional suppression of GPX4. PCOS mouse model was constructed by subcutaneous injection of dehydroepiandrosterone (DHEA, 60 mg/kg) for 21 consecutive days, while control (Ctrl) mice were injected with oil vehicle ( n = 10 in each group). (a) Body weight (g) and ovary weight (mg) of control (Ctrl) and DHEA-treated (DHEA) mice. ∗ P < 0.05, Student's t -test. (b) Representative H&E-stained ovarian sections from Ctrl and DHEA mice. Asterisks indicate corpora lutea; black arrows indicate preantral follicles. (c) Quantification of ( b ). Box-and-whisker plots with data points ( n = 6). ∗ P < 0.05, Student's t -test. (d) Representative photomicrographs of ovarian sections from Ctrl and DHEA mice following Masson trichrome (collagen deposition, yellow arrows), TUNEL (positive cells, white arrows), and Perl's (iron deposition, red arrows) staining, alongside a transmission electron microscopy (TEM) micrograph (ferroptotic mitochondria, orange arrows). (e) Quantification of ( d ). Box-and-whisker plots with data points ( n = 6). ∗ P < 0.05, Student's t -test. (f) Quantitative real-time PCR (qRT-PCR) analysis of ovarian Gpx4 mRNA in Ctrl and DHEA mice. The Beta-actin gene ( Actb ) was used as an internal control. Data were presented as mean ± SEM, n = 6. ∗ P < 0.05, Student's t -test. (g) Western blot analysis of ovarian GPX4, 4-HNE, α-SMA, and Collagen I (Col1α) proteins from Ctrl and DHEA mice. GAPDH served as a loading control. Blots (left panel) are representative of two samples per group. Quantitative data (right panel) were presented as mean ± SEM, n = 6. ∗ P < 0.05, Student's t -test. (h) Volcano plot of gene expression profile from the database [GEO Dataset Accession Number: GSE293353 and GSE277906 ], comprising RNA-seq data ovarian granulosa cells of 24 women diagnosed with PCOS and 24 healthy control women. The number and position of genes statistic-significantly increased (red, 262), no difference (gray, 20,075), or decreased (blue, 606) including GPX4 (Log 2 (fold-change) = −0.55294), orange point was marked. (i) Representative photomicrographs of ovarian sections from Ctrl and DHEA mice stained for GPX4 by immunohistochemistry (IHC) staining. (j) Western blot analysis of 4-HNE and GPX4 expression in primary granulosa cells (GCs) treated with or without DHEA (50 μM, 48 h). GAPDH was used as a loading control. Quantification was shown below. Data were presented as mean ± SEM, n = 4. ∗ P < 0.05, Student's t -test. (k) Representative images of C11-BODIPY and TUNEL staining in GCs treated with or without DHEA. (Left) Non-oxidized (N-) and oxidized (O-) BODIPY were in red and green respectively, and then merged. (Right) The positively-stained cells by TUNEL (TUNEL Bright Red kit) were shown.

Article Snippet: Perl's Prussian blue staining (G1029, Servicebio, China) and TUNEL (TdT-mediated dUTP Nick-End Labeling, A111-01, Vazyme, China) assays were performed according to the manufacturers' instructions.

Techniques: Construct, Injection, Control, Staining, Whisker Assay, TUNEL Assay, Transmission Assay, Electron Microscopy, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Gene Expression, RNA Sequencing, Immunohistochemistry, Expressing