Fig. 2: Leydig cells release extracellular vesicles with defective mitochondria.
From: An extracellular vesicle-mediated mitochondrial transfer network critical for testosterone synthesis
a, Detection (left) and quantification (right) of WGA–Alexa Fluor 488 (AF488) signal in purified LC particles by flow cytometry (n = 3 biological replicates). b, Experimental strategy to label LC membranes by intratesticular injection of AAV-DIO-Lck–EGFP into the testes of Cyp17a1Cre; R26tdTomato mice. c, Representative confocal image of the testes of Cyp17a1Cre; R26tdTomato mice injected with AAV-DIO-Lck–EGFP (n = 3 mice). The LC particles were labelled with tdTomato and membrane EGFP. d, Representative confocal image of tMacs containing LC-EVs. Sections of the testes of Cyp17a1Cre; R26tdTomato mice injected with AAV-DIO-Lck–EGFP were immunostained with the tMacs marker F4/80 (n = 3 mice). c,d, Inset: 3D reconstruction showing LC-derived particles. Scale bars, 5 μm (main images) and 2 μm (insets). e, Experimental design overview. LC-EVs were isolated from Cyp17a1Cre; R26tdTomato mice using FACS and then subjected to proteomics analysis. f, Donut chart showing the proteome composition of LC-EVs. CV, cytoplasmic vesicle; ER, endoplasmic reticulum; lyso, lysosome; mito, mitochondrion; PM, plasma membrane; ribo, ribosome. g, Venn diagrams of the LC-EV proteome with proteins reported in the MitoCarta3.0 and Vesiclepedia databases. h, Representative confocal image (left) and 3D reconstruction of LC-EVs (right). Sections of the testes of Cyp17a1Cre mice injected with AAV-DIO-Lck–EGFP were immunostained with mitochondrial marker (TOMM20) and counterstained with DAPI (n = 3 mice). Scale bars, 3 μm (confocal image) and 0.5 μm (3D reconstruction). i, Analysis of mitochondrial signal in purified LC-EVs by flow cytometry (n = 3 biological replicates). MTG, MitoTracker Green. j, Representative TEM micrographs showing mitochondria (arrowhead) in LCs (i) and LC-EVs (ii) (n = 3 mice). Scale bars, 500 nm (main image; left) and 200 nm (magnified view of the boxed regions; right). k, Ratio of cristae area to mitochondrial area (n = 50 mitochondria in LCs and LC-EVs from three mice). l, Assessment of MMP in mouse MA-10 cells and LC-EVs via MitoNIR staining following treatment with FCCP or oligomycin (n = 3 biological replicates). MFI, mean fluorescence intensity. m, Representative confocal images of LCs and LC-EVs stained with TMRE. Primary LCs were isolated from Cyp17a1Cre; R26mitoD2 (mitoD2) mice. n, TMRE intensity per mitochondrion in LCs and LC-EVs. o, Representative confocal images of LCs and LC-EVs labelled with ATP probe. p, ATP intensity per mitochondrion. q, Representative confocal images of LCs and LC-EVs labelled with the dye MitoSOX. r, MitoSOX intensity per mitochondrion. m,o,q, Scale bars, 10 μm (main image; left) and 1 μm (magnified view of the boxed regions; right); n = 3 biological replicates. a,i,l, Data are the mean ± s.e.m. n,p,r, n = 21 mitochondria in LCs and LC-EVs from three biological replicates. k,l,n,p,r, Statistical significance was determined using a two-tailed Student’s t-test (k,n,r), two-tailed Welch’s t-test (p), one-way ANOVA (l(left)) or Kruskal–Wallis test (l(right)). Schematic in b,e created in BioRender. Xia, K. (2026) https://biorender.com/jflr23k. Source numerical data are provided.
