Fig. 3: Testicular macrophages eliminate Leydig cell-derived mitochondria to prevent Leydig cell dysfunction.
From: An extracellular vesicle-mediated mitochondrial transfer network critical for testosterone synthesis
a, Schematic of LCs in Cyp17a1Cre; R26mitoD2 mice. b, Representative confocal image of the testes of Cyp17a1Cre; R26mitoD2 mice. Sections of the testes were immunostained with F4/80 (n = 6 mice). Arrowheads indicate LC-derived mitoD2+ mitochondria within tMacs. Scale bars, 2 μm. c, Percentage of tMacs containing mitoD2+ LC-derived mitochondria (n = 6 mice). d, Experimental design overview. FACS-sorted tdTomato+ tMacs were transplanted into the testes of Cyp17a1Cre; R26mitoD2 mice and then analysed using intravital two-photon microscopy. e, Time-lapse imaging with 3D reconstructions revealing an LC-derived mitochondria-uptake process by tMacs in vivo. Arrows indicate transferred mitochondria from LCs to tMacs. Scale bars, 10 μm (top) and 5 μm (bottom; magnified 3D reconstructions of the boxed regions). f, Representative images of testes of Cyp17a1Cre; R26mitoD2 mice immunostained with F4/80 and the lysosome marker LAMP1 (n = 6 mice). Scale bars, 2 μm. g, Percentage of LC-derived mitochondria localized inside LAMP1+ lysosomes in tMacs (n = 6 mice). h, Degradation of mitoD2+ LC-derived mitochondria by tMacs (n = 3 biological replicates). i, Representative images of sections of the testes of Cyp17a1Cre; R26tdTomato mice stained with F4/80 after tMac-depletion for seven days (n = 4 mice per group). Arrowheads indicate LC-EVs. Scale bars, 5 μm (top) and 2 μm (bottom; magnified views of the boxed regions). j, Number of extracellular LC-EVs (n = 4 mice per group, five fields of view were captured and 2,000-μm2 images were analysed for each mouse). k, Representative TEM images of mice after tMac depletion for seven days (n = 4 mice per group). Scale bars, 1 μm (top) and 200 nm (bottom; magnified views of the boxed regions). l, Ratio of cristae area to mitochondrial area (n = 80 mitochondria in LCs and LC-EVs from four mice for each group). m, Number of mitochondria within LC-EVs (n = 4 mice per group, five fields of view were captured and 2,000-μm2 images were analysed for each mouse). n, Experimental design overview. LC-EVs were isolated from Cyp17a1Cre; R26tdTomato mice and then used to treat primary LCs. o, Testosterone production of LCs co-cultured with or without LC-EVs at the indicated time points (n = 4 biological replicates per group). p, ATP levels of LCs co-cultured with or without LC-EVs (n = 3 biological replicates per group). q, Experimental design overview. Primary LCs were co-cultured with an equal volume of culture medium (control) or CCCP-treated mitochondria (defective mitochondria) for 12 h. r, Testosterone concentration in the supernatant of cultured primary LCs treated with culture medium or defective mitochondria for 12 h (n = 4 biological replicates per group). s, ATP levels of LCs co-cultured with culture medium or defective mitochondria (n = 3 biological replicates per group). c,g,h,j,m,p,s, Data are the mean ± s.e.m. o,r, Box plots: the box bounds depict the first to the third quartile, with the middle line indicating the median and the whiskers extending to the minimum and maximum values. j,l,m,o,p,r,s, Statistical significance was determined using a two-tailed Student’s t-test (j,m,o,p,r,s) or two-tailed Mann–Whitney U-test (l). mitoD2, Cyp17a1Cre; R26mitoD2. Con, control. Schematic in a,d,n,q created in BioRender. Xia, K. (2026) https://biorender.com/1l0dtjs. Source numerical data are provided.
