Fig. 7: The adhesive molecule VCAM1 in Leydig cells mediates the uptake of testicular macrophage-derived mitochondria.

a, Representative images of FACS-sorted tdTomato⁺ LCs co-cultured with CD206^hi or MHCII^hi tMacs isolated from Cx3cr1^CreER; R26^mitoD2 (mitoD2) mice (n = 4 biological replicates per group). Arrows indicate tMac-derived mitoD2⁺ mitochondria within tdTomato⁺ LCs. Scale bars, 10 μm (top) and 5 μm (bottom; magnified views of the boxed regions). b, Levels of mitoD2⁺ signal in tdTomato⁺ LCs (n = 4 biological replicates per group). c, Representative images of tdTomato⁺ LCs containing CD206^hi or MHCII^hi tMac-derived mitochondria (mitoD2⁺). CD206^hi or MHCII^hi tMacs isolated from TAM-induced Cx3cr1^CreER; R26^mitoD2 mice were transplanted into the testes of tMacs-depleted Cyp17a1^Cre; R26^tdTomato mice (n = 3 mice per group). Arrows indicate tMac-derived mitoD2⁺ mitochondria within tdTomato⁺ LCs. Scale bars, 5 μm (top) and 2 μm (bottom; magnified views of the boxed regions). d, Levels of mitoD2⁺ signal in tdTomato⁺ LCs (n = 3 mice per group). e, Illustration of intercellular communication between LCs and tMacs via ligand–receptor pairs. f, Circle plots showing cell–cell communication between tMacs and LCs via ligand–receptor pair signalling. The width of the arrows indicates the communication probability. g, Sections of testes of Cyp17a1^Cre; R26^tdTomato mice were immunostained with VCAM1 and counterstained with DAPI (n = 3 biological replicates). Scale bar, 20 μm (left) and 10 μm (right; magnified view of the boxed region). h, Flow cytometry analysis of tMac-EVs labelled with ITGβ1. i, Experimental design overview. Cyp17a1^Cre; R26^{Cas9-tdTomato} mice were injected with sgScr or sgVcam1. j, The levels of Vcam1 messenger RNA expression in the testes of Cyp17a1^Cre; R26^{Cas9-tdTomato} mice were determined by quantitative PCR with reverse transcription 14 days post AAV treatment (n = 3 mice per group). k, Representative images of testes of Cyp17a1^Cre; R26^{Cas9-tdTomato} mice stained with VCAM1 and DAPI 14 days after treatment with sgScr or sgVcam1 (n = 6 mice per group). Scale bars, 20 μm. l, Levels of VCAM1 expression on LCs (n = 6 mice per group). m, Experimental design overview. FACS was used to sort tMac-EVs containing mitochondria (WGA⁺ mtD2⁺ tMac-EVs), which were used to treat primary LCs from the sgScr and sgVcam1 groups. n, Representative images of primary LCs from the sgScr and sgVcam1 groups after treatment with tMac-EVs containing mitochondria. Arrows indicate tMac-derived mitoD2⁺ mitochondria within tdTomato⁺ LCs. Scale bars, 10 μm (top) and 4 μm (bottom; magnified views of the boxed regions). o, Percentage of LCs containing tMac-derived mitochondria. p, ATP production of primary LCs from the sgScr and sgVcam1 groups treated with tMac-EVs containing mitochondria. q, Testosterone levels of Cyp17a1^Cre; R26^{Cas9-tdTomato} mice 14 days following injection with sgScr or sgVcam1. r, Sperm counts in the sgScr and sgVcam1 groups (n = 6 mice per group). s, Number of litters obtained from the two groups. t, Number of pups per litter. u, Grip strength of the mice in the two groups. v, Muscle endurance of the two groups. h,o–q,s–v, n = 5 biological replicates (h,o–q) or mice (s–v) per group. b,d,h,j,u,v, Data are the mean ± s.e.m. l,o–t, 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. b,d,j,l,o–v, Statistical significance was determined using a two-tailed Student’s t-test (b,d,j,o–r,t,u), two-tailed Mann–Whitney U-test (l,s) or two-tailed Welch’s t-test (v). Schematic in e,i,m,u,v created using BioRender. Xia, K. (2026) https://biorender.com/cv4gh8q. Source numerical data are provided.

Source Data