Journal: Frontiers in Cell and Developmental Biology

Article Title: Dialogue between nutrition and reproduction: preliminary exploration of sperm quality response to high-fat diet in mice

doi: 10.3389/fcell.2025.1640714

Figure Lengend Snippet: Differential mRNA expression of autophagy- and apoptosis-related genes in the epididymal caput, cauda, and testis of mice fed a control diet (CD) (n = 3) or high-fat diet (HFD) (n = 3). (A) Relative mRNA expression levels of Beclin-1, LC3B, and p62 in the epididymal caput. HFD mice showed mild upregulation of autophagy markers (Beclin-1, LC3B) and downregulation of p62, suggesting activation of autophagic flux in the early epididymal region. (B) Relative mRNA expression levels of Caspase-3, Cyt-c, Bax, and Bcl-2 in the cauda epididymis. Marked elevation of pro-apoptotic genes (Caspase-3, Cyt-c, Bax) and reduced Bcl-2 expression indicated enhanced apoptosis associated with sperm storage and motility impairment. (C) Relative mRNA expression levels of Beclin-1, ATG5, Caspase-3, and Apaf-1 in the testis. HFD mice exhibited significant activation of both autophagy and apoptosis pathways, consistent with histological evidence of germ cell loss. Data are expressed as mean ± standard deviation (x̄ ± s). Statistical comparisons between CD and HFD groups were performed using independent-samples t-tests. P < 0.05 was considered statistically significant ( P < 0.05, * P < 0.01 vs. CD group).

Article Snippet: Equal amounts (20 μg) of protein were boiled in 4× SDS sample buffer at 100 °C for 10 min, separated by 12% SDS-PAGE, and transferred to PVDF membranes (Millipore, IPVH00010) at 350 mA for 90 min. Membranes were blocked with 5% BSA in TBST (0.1% Tween-20) for 1 h at room temperature and then incubated overnight at 4 °C with the following primary antibodies:Bax (1:1,000, Cell Signaling Technology, CST#2772), Bcl-2 (1:1,000, CST#3498), Caspase-3 (1:1,000, CST#9662), Caspase-8 (1:1,000, CST#9746), Caspase-9 (1:1,000, CST#9508), Mn SOD (1:1,000, Abcam, ab13533), Beclin-1 (1:1,000, CST#3495), LC3B (1:1,000, CST#3868), β-Actin (1:2,000, CST#3700) as the internal control.

Techniques: Expressing, Control, Activation Assay, Standard Deviation

Journal: Frontiers in Cell and Developmental Biology

Article Title: Dialogue between nutrition and reproduction: preliminary exploration of sperm quality response to high-fat diet in mice

doi: 10.3389/fcell.2025.1640714

Figure Lengend Snippet: Western blot analysis and quantification of apoptosis, autophagy, and oxidative stress markers. Proteins were extracted from sperm samples of the control diet (CD, n = 3) and high-fat diet (HFD, n = 3) groups using RIPA buffer. Equal amounts of protein (20 μg) were separated via 12% SDS–PAGE and transferred to PVDF membranes (350 mA, 90 min). Membranes were blocked with 5% BSA and incubated overnight at 4 °C with primary antibodies against Bax, Bcl-2, Beclin-1, Caspase-8, Caspase-9, LC3A/B, Mn SOD (all 1:1,000, CST), and β-Actin (1:2,000). After incubation with HRP-conjugated secondary antibodies (1:5,000) and ECL detection, protein bands were visualized and quantified using ImageJ. Densitometric values were normalized to β-Actin. Quantitative results are shown as bar graphs for each marker, illustrating the differential expression between groups. Note: *: P < 0.05, **: P < 0.01, ***: P < 0.001 vs. CD.

Article Snippet: Equal amounts (20 μg) of protein were boiled in 4× SDS sample buffer at 100 °C for 10 min, separated by 12% SDS-PAGE, and transferred to PVDF membranes (Millipore, IPVH00010) at 350 mA for 90 min. Membranes were blocked with 5% BSA in TBST (0.1% Tween-20) for 1 h at room temperature and then incubated overnight at 4 °C with the following primary antibodies:Bax (1:1,000, Cell Signaling Technology, CST#2772), Bcl-2 (1:1,000, CST#3498), Caspase-3 (1:1,000, CST#9662), Caspase-8 (1:1,000, CST#9746), Caspase-9 (1:1,000, CST#9508), Mn SOD (1:1,000, Abcam, ab13533), Beclin-1 (1:1,000, CST#3495), LC3B (1:1,000, CST#3868), β-Actin (1:2,000, CST#3700) as the internal control.

Techniques: Western Blot, Control, SDS Page, Incubation, Marker, Quantitative Proteomics