Journal: bioRxiv
Article Title: Testosterone-Induced Metabolic Changes in Seminal Vesicle Epithelial cells Alter Plasma Components to Enhance Sperm Fertility
doi: 10.1101/2024.01.16.575926
Figure Lengend Snippet: (A) Tracing of oxygen consumption rate (OCR) basal and injection with 10 nM oleic acid (OA) or OA + 40 nM Etomoxir (Eto). (B) The sperm, after 1 hour of incubation with or without seminal vesicle secretions (SV) from nontreated (Ctrl) or flutamide-treated mice(Flu), were used for IVF, and the cleavaged oocytes were observed. The absolute number of oocytes collected from the oviduct and the cleavage rate are shown. (C) Western blot analysis for phosphotyrosine (P-Tyr) in capacitated spermatozoa (Ctrl; 1-hour incubation in HTF medium) and treated with SV from healthy mice or 10 nM oleic acid (OA). (D) Quantitative analysis of GLUT4 relative to α-tubulin obtained from Western blot. (E) Flow cytometric analysis of the sperm after 1 hour incubation in control medium (HTF) and medium containing SV or 10 nM OA, using fluorescein isothiocyanate-conjugated peanut agglutinin (PNA-FITC; to distinguish between acrosome-reacted and non-reacted cells) and propidium iodide (PI; to distinguish between dead and viable cells). (F) The percentage of viable sperm with an acrosome reaction (PI-, PNA-FITC+) was evaluated by flow cytometry in the 4th quadrant (4Q; red square). Data are mean ± SEM. At least three independent replicates. Percentage data were subjected to arcsine transformation before statistical analysis. (B) Dunnett’s test was used to analyze the cleavage rate. Different letters represent significantly different groups. (D,F) Differences between groups were assessed by one-way analysis of variance (ANOVA). When ANOVA was significant, differences among values were analyzed by Tukey’s Honest Significant Difference test for multiple comparisons.
Article Snippet: The membranes were incubated overnight at 4°C with primary antibodies: anti-phosphotyrosine antibody (1:10,000; 8959; Cell Signaling), anti-GLUT4 antibody (1:100; ab33780; Abcam), anti-ACLY antibody (1:10,000; ab40793; Abcam) or anti-α/β-Tubulin antibody (1:1,000; 2148S; Cell Signaling).
Techniques: Injection, Incubation, Western Blot, Control, Flow Cytometry, Transformation Assay