U, Grip strength of the mice in the two groups. O, Percentage of LCs containing tMac-derived mitochondria. Scale bars, 10 μm (top) and 4 μm (bottom; magnified views of the boxed regions). N, Representative images of primary LCs from the sgScr and sgVcam1 groups after treatment with tMac-EVs containing mitochondria. In the transplantation experiment of tMacs, tMacs were enriched from the indicated mice and then transplanted into hosts at a ratio of one donor to one recipient through intratesticular injection. For adoptive transfer of tMacs or LCs, host mice were anaesthetized with Avertin (250 mg kg−1 body weight) by intraperitoneal injection. The mice were injected with three consecutive doses once every seven days. Intraperitoneal injection of either vehicle or diphtheria toxin (Sigma) was used to deplete tMacs in Cd11bDTR transgenic mice. The Imaris software was used to reconstruct 3D images, determine cellular localization with 3D positional mapping and generate movies derived from time-lapsed imaging. Three-dimensional stacks consisting of multiple planes (0.5-μm step size) were captured every 2 min. The testes were pulled out and attached to a specialized mould, which was kept moist with PBS, and carefully positioned for imaging. In one study, men who incorporated ice baths into their routine saw a boost in testosterone levels, along with improved insulin sensitivity and reduced inflammation. If you’re insulin-resistant, inflamed, or living off processed foods, your mitochondria are likely in trouble—and so is your testosterone. The enzyme responsible for this conversion, P450scc, is found within the inner mitochondrial membrane. Mitochondria are responsible for energy production, metabolism, cellular repair, and yes—hormone synthesis. Most people know mitochondria as the "powerhouses of the cell." That’s a solid high school biology definition, but it barely scratches the surface. FACS-sorted mitoD2+ tMacs were transplanted into the testes of Cyp17a1Cre; R26tdTomato mice and then analysed using intravital two-photon microscopy. B, Representative confocal image of the testes of TAM-injected Cx3cr1CreER; R26mitoD2 mice. Time-lapse videography and 3D reconstructions captured mitochondrial uptake events, with mitoD2+ mitochondria trafficking from tMacs into tdTomato+ host LCs (Fig. 6e and Supplementary Video 2). To test this, we generated Cx3cr1CreER; R26mitoD2 mice, enabling the specific labelling of tMac mitochondria after TAM treatment (Fig. 6a). Super-resolution imaging of the testes of TAM-induced Cx3cr1CreER; mTmG mice revealed GFP+ membrane-encapsulated tMac-EVs within the cytoplasm of 3βHSD+ LCs (Extended Data Fig. 8d,e). Together, these data indicate that tMac-EVs are enriched for healthy mitochondria. The TMRE fluorescence was not reduced relative to tMac mitochondria (Fig. 5m,n) and the levels of ATP staining were similarly comparable (Fig. 5o,p). Through TEM analysis of testes, we identified mitochondria within the EVs with cristae area fractions comparable to mitochondria in tMacs (Fig. 5j,k). A comparison of the tMac-EV proteome with the MitoCarta3.0 and Vesiclepedia databases24,25 revealed 183 and 864 overlapping proteins, respectively (Fig. 5g). G, Venn diagrams of the tMac-EV proteome with proteins reported in the MitoCarta3.0 and Vesiclepedia database. We purified GFP+ particles devoid of nuclei from the testes of Cx3cr1GFP mice (Extended Data Fig. 8a–c). Following the administration of hCG to induce elevated testosterone levels, we observed a noticeable increase in the number of tdTomato+ LCs with GFP+ particles (Extended Data Fig. 7c–g). To confirm the transfer of tMac-derived particles to LCs, we collected testes from Cyp17a1Cre; R26tdTomato; Cx3cr1GFP mice for analysis. The signals of tetramethylrhodamine ethyl ester, perchlorate (TMRE) indicated that the MMP of mitochondria inside LC-EVs was reduced compared with those in LCs (Fig. 2m,n). Transmission electron microscopy (TEM) of testicular sections revealed mitochondria within LC-EVs that exhibited a significantly reduced cristae area compared with those in LCs (Fig. 2j,k). Primary LCs were isolated from Cyp17a1Cre; R26mitoD2 (mitoD2) mice. Moreover, through the induction of nuclear-encoded mitochondrial transcription factors, testosterone could exert a similar effect. In order to experimentally validate the findings derived from our prior computational analysis, we recently performed the co-immunoprecipitation of the mtDNA from C2C12 cells using an antibody against the AR, followed by sequencing of the precipitated fragments (ChIP-Seq) (unpublished data). So, these results show the presence of sequence inside the mitochondrial genome, that the AR could recognize, bind, and exert its function as transcription factor by modulating mitochondrial gene transcription. Indeed, evidence suggests that androgens, mediated by the AR, not only regulate the expression of nuclear genes encoding certain subunits of the MRC, but also modulate the expression of mitochondrial genes encoding subunits of this chain. The possibility that testosterone could directly influence mitochondrial oxidative phosphorylation gene transcription by way of cognate receptors present in mitochondria, has also been proposed. Concurrently, the loss of mitochondrial membrane potential, opening of the mitochondrial permeability transition pore (mPTP), and release of cytochrome c occur, and it is during this phase that the protective influence of testosterone against apoptosis becomes evident 9,10.
