Fig. 6: Leydig cells integrate testicular macrophage-derived mitochondria to support testosterone production.
From: An extracellular vesicle-mediated mitochondrial transfer network critical for testosterone synthesis
a, Schematic of tMacs in TAM-induced Cx3cr1CreER; R26mitoD2 mice. b, Representative confocal image of the testes of TAM-injected Cx3cr1CreER; R26mitoD2 mice. Sections of the testes were immunostained with CYP17A1 and counterstained with TWO-PRO-3 (n = 5 mice). Arrows indicate tMac-derived mitoD2+ mitochondria within LCs. Scale bar, 3 μm. c, Percentage of LCs containing mitoD2+ tMac-derived mitochondria (n = 5 mice). d, Experimental design overview. FACS-sorted mitoD2+ tMacs were transplanted into the testes of Cyp17a1Cre; R26tdTomato mice and then analysed using intravital two-photon microscopy. e, Time-lapse imaging (top) with 3D reconstructions (bottom) revealing a tMac-derived mitochondrial transfer process into LCs in vivo. Arrows indicate transferred mitochondria from tMacs to LCs. Scale bars, 5 μm. f, Experimental strategy to label LC mitochondria by intratesticular injection of AAV-mito–mCherry into the testes of TAM-induced Cx3cr1CreER; R26mitoD2 mice. g, Representative confocal images of the testes of AAV-mito–mCherry-injected Cx3cr1CreER; R26mitoD2 mice. Sections of the testes were immunostained with CYP17A1 (n = 5 mice). Arrows indicate tMac-derived mitoD2+ mitochondria fused (right) or not fused (left) with LC-derived mCherry+ mitochondria within LCs. Scale bars, 2 μm. h, Percentage of tMac-derived mitochondria fused with mCherry+ mitochondria in LCs (n = 5 mice). i, Experimental design overview. FACS was used to isolate tMac-EVs from Cx3cr1GFP mice. Primary LCs were treated with culture medium (control), culture medium containing tMac-EVs (tMac-EVs) or culture medium containing sonicated tMac-EVs (sonicated tMac-EVs). j, Testosterone production of LCs from the three groups at the indicated time points. k, ATP levels of LCs from the three groups. l, Experimental design overview. FACS was used to isolate tMac-EVs from Cx3cr1GFP mice. Primary LCs were treated with culture medium (control), culture medium containing tMac-EVs (tMac-EVs) or culture medium containing tMac-EVs pretreated with antimycin A (mito-inhibited tMac-EVs). m, Testosterone production of LCs from the three groups at 6 h. n, ATP levels of LCs from the three groups. o, Experimental design overview. Mitochondria were isolated from tMacs (tMac-mito) sorted from Cx3cr1GFP mice using FACS. Primary LCs were treated with culture medium (control), culture medium containing tMac-mito or culture medium containing sonicated tMac-mito (sonicated tMac-mito). p, Testosterone production of LCs from the three groups at 6 h. q, ATP levels of LCs from the three groups. c,h,j,k,m,n,p,q, Data are the mean ± s.e.m. j,k,m,n,p,q, Statistical significance was determined using a one-way ANOVA; n = 5 biological replicates per group. Con, control. Schematic in a,d,f,i,l,o created in BioRender. Xia, K. (2026) https://biorender.com/71c5gkk. Source numerical data are provided.
