Journal: International Journal of Biological Sciences

Article Title: Hypoxia Reprograms a Ciliary Scaffold into a Metabolic-Epigenetic Trafficking Hub via a Succinate-Driven DNMT3L-ASXL2 Axis

doi: 10.7150/ijbs.137355

Figure Lengend Snippet: Overexpressing CEP162 reduces sperm motility in mice. (A) Sperm viability and kinematic analysis following CEP162 overexpression under normoxia (21% O 2 ) and hypoxia (11.7% O 2 ). The sperm motility parameters, including progressive motility, VCL, VSL, VAP, STR, BCF, ALH, LIN, and WOB, were quantified using computer-assisted sperm analysis (CASA; Microptic S.L., Barcelona, Spain). (B) Ultrastructural analysis of sperm flagella by transmission electron microscopy. Representative micrographs show transverse sections of the flagellum, including the midpiece (with a mitochondrial sheath), principal piece (with a fibrous sheath), and end piece (showing axonemal termination). All scale bars, 200 nm. The lower panel shows the quantification of axonemal DMT defects (%). (C) Microtubule regrowth following nocodazole washout in CEP162-overexpressing G2 cells. The percentage of pericentrin-expressing cells was calculated by dividing the number of pericentrin-positive cells by the total number of cells in each respective group. (D) Targeted proteomic profiling of CEP162-overexpressing sperm flagella. Heatmap showing Z score-normalized expression of selected TEKT family members involved in sperm axoneme assembly and Rab GTPases across three biological replicates. t-SNE visualization of the correlations among TEKT1, TEKT2, TEKT3, TEKT4, TEKT5, EFHC1, Rab1a, RAB1b, and RAB14 expression in the sperm flagella of control and CEP162-overexpressing mice. (E) Western blot analysis of TEKT2 protein expression in whole sperm and flagella isolated from CEP162-overexpressing mice maintained under normoxia (21% O 2 ) or hypoxia (11.7% O 2 ) for 10 weeks. Note: This Western blot detected endogenous TEKT2 (~50 kDa); the RFP-TEKT2-FKBP fusion protein (~87 kDa) was used exclusively for immunofluorescence localization in Panels F-G. (F-G) Mice were injected with recombinant AAV9 vectors encoding RFP-TEKT2-FKBP (~87 kDa) and maintained under (F) 21% O₂ (normoxia) or (G) 11.7% O₂ (hypoxia) for 10 weeks. Sperm were collected from the cauda epididymis and subjected to triple-label immunofluorescence for RFP (a fusion protein), acetylated α-tubulin (an axoneme marker), and DAPI (a nuclear counterstain). Imaging confirmed the flagellar localization of each RFP-tagged construct, with spatial resolution of the sperm head and flagellum enabled by DAPI counterstaining. Scale bar: 20 μm. Quantification of the head-to-tail TEKT2 fluorescence intensity ratio in the indicated groups. All the data are presented as the means ± SDs. In (A-C), n = 12 biologically independent mice (A-B) or n = 12 fields from 3 biological replicates (C); the data were normalized to the mean of the control group where indicated; two-way ANOVA with Tukey's post hoc test; **** P < 0.0001. In (D), n = 3 biological replicates; proteomic data are shown as Z scores. Differential protein expression was determined by two-tailed Student's t test with FDR correction (thresholds: P < 0.05, |log₂FC| > 1). (E) shows a representative image from a single independent experiment. The TEKT2 signal was normalized to that of β-actin (whole sperm or flagella). No formal statistical testing was applied to this single replicate. In (F-G), n = 10 biologically independent mice; ≥100 sperm scored per mouse for immunofluorescence analysis; the head-to-tail TEKT2 fluorescence intensity ratio was quantified using ImageJ; unpaired two-tailed Student's t test; **** P < 0.0001.

Article Snippet: Finally, nuclear counterstaining was performed using DAPI (C1005, Beyotime) for 5 minutes.

Techniques: Analysis, Over Expression, Transmission Assay, Electron Microscopy, Expressing, Profiling, Control, Western Blot, Isolation, Immunofluorescence, Injection, Recombinant, Marker, Imaging, Construct, Fluorescence, Two Tailed Test