Fig. 5: Testicular macrophages transfer extracellular vesicles with functional mitochondria.
From: An extracellular vesicle-mediated mitochondrial transfer network critical for testosterone synthesis
a, Detection (left) and quantification (right) of WGA–Alexa Fluor 594 (AF594) signal in flow cytometry-purified tMac particles (n = 3 biological replicates). b, Experimental strategy to label tMacs with GFP and membrane-targeted tdTomato in Cx3cr1GFP; mTmG mice. c, Representative confocal image of the testes of Cx3cr1GFP; mTmG mice (n = 4 mice). Scale bars, 5 μm (main image) and 0.5 μm (inset, 3D reconstruction of the boxed regions). d, Representative confocal image of LCs containing tMac-EVs. Sections of the testes of Cx3cr1GFP; mTmG mice were immunostained with the LC marker CYP17A1 and counterstained with DAPI (n = 3 mice). Scale bars, 5 μm. e, Experimental design overview. Following isolation from Cx3cr1GFP mice using FACS, tMac-EVs were subjected to proteomics analysis. f, Donut chart showing the proteome composition of tMac-EVs. CV, cytoplasmic vesicle; ER, endoplasmic reticulum; lyso, lysosome; mito, mitochondrion; PM, plasma membrane; ribo, ribosome. g, Venn diagrams of the tMac-EV proteome with proteins reported in the MitoCarta3.0 and Vesiclepedia database. h, Representative confocal image (left) and 3D reconstruction (right) of tMac-EVs. Sections of the testes of TAM-treated Cx3cr1CreER; mTmG mice were immunostained with TOMM20 and counterstained with DAPI (n = 4 mice). Scale bars, 5 μm (confocal image) and 0.5 μm (magnified 3D reconstructions of the boxed regions). i, Analysis of mitochondrial signal in flow cytometry-purified tMac-EVs (n = 3 biological replicates). j, Representative TEM image showing mitochondria in tMac-EVs (i) and tMacs (ii) (left; n = 3 mice). Scale bars, 2 μm (left) and 200 nm (right; magnified views of the boxed regions). k, Ratio of cristae area to mitochondrial area (n = 50 mitochondria in tMacs and tMac-EVs from three mice). l, Assessment of MMP in RAW264.7 cells (mouse cell line of macrophages; left) and tMac-EVs (right) via MitoNIR staining following treatment with FCCP or oligomycin (n = 3 biological replicates). m, Representative confocal images of tMac-EVs and tMacs stained with TMRE. The tMacs were sorted from TAM-injected Cx3cr1CreER; R26mitoD2 (mitoD2) mice (n = 3 biological replicates). n, TMRE intensity per mitochondrion. o, Representative confocal images of tMac-EVs and tMacs stained with ATP probe (n = 3 biological replicates). p, ATP intensity per mitochondrion. q, Representative confocal images of tMac-EVs and tMacs stained with MitoSOX (n = 3 biological replicates). r, Intensity of MitoSOX per mitochondrion. m,o,q, Scale bars, 3 μm (left) and 2 μm (right; magnified views of the boxed regions). n,p,r, n = 21 mitochondria in tMacs and tMac-EVs from three biological replicates. a,i,k,l, Data are the mean ± s.e.m. k,l,n,p,r, Statistical significance was determined using a two-tailed Student’s t-test (k,n,p), two-tailed Welch’s t-test (r) or one-way ANOVA (l). Schematics in b,e,h created in BioRender. Xia, K. (2026) https://biorender.com/u5q8ux2. Source numerical data are provided.
