Mitochondrial Ca2+ uptake plays a vital role in regulating cellular functions, involving stimulation of ATP production, inhibition of autophagy, correction of intracytoplasmic Ca2+ signaling, and regulation of cell death. Another essential function of mitochondria is to regulate intracellular calcium (Ca2+) homeostasis. Mitochondrial fission separates the damaged part from the healthy mitochondrial network or occurs during cell mitosis, producing two new mitochondria to meet cell division needs (33, 37). Just as mitochondrial biogenesis and mitochondrial autophagy dynamically regulate mitochondrial quantity and quality, mitochondrial fusion and fission serve as another set of opposing processes to regulate mitochondrial production and morphology.
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).
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.
28-day oxidative stress induced by the reduced activity of complex I in GDX rats might not be enough to affect complexes II, III, and IV. Immunocytochemical 59, 62 and in situ hybridization 60, 61 studies identify the subpopulations of intracellular gonadal hormone receptor-bearing neurons in the SN, which suggested that specific subsets of midbrain dopaminergic neurons might be direct targets of gonadal hormones. Another reason might be that testosterone specifically regulates the subunits of complex I either via androgen receptor 59, 60 or via estrogen receptor 61, 62 when testosterone is aromatized to estrogen. Complex I seem more vulnerable than complexes II, III, and IV to oxidative damage caused by testosterone deficiency. The following reasons might explain the reduced activity of complex I in the SN of GDX rats. The reduced activity of complex I was found in the SN of GDX rats. Supplement of TP improves the decreased behavioral parameters of open-field activity in GDX rats .
Use red light therapy, which has been shown to support mitochondrial function and enhance testosterone production by stimulating ATP production in cells. Supplements of testosterone propionate to castrated male rats ameliorated the activity of mitochondrial complex I and upregulated the expression of mitochondrial ND1 and ND4. In conclusion, our study revealed that testosterone supplementation improved exploratory behavior, attenuated neuronal dysfunction and neuronal loss, and ameliorated mitochondrial dysfunction by enhancing both mitochondrial antioxidative capacity and mitochondrial biogenesis of aged male rats. Increased PINK1/Parkin and decreased P62 expression in the SN and HIPP of testosterone-supplemented rats further suggested that enhanced mitophagy likely contributes to the beneficial effect of testosterone supplementation against mitochondrial dysfunction in the aged rat brain. These findings, along with those of the present study, demonstrated that cognitive/behavioral deficits and mitochondrial dysfunction in the aged male brain are, to some extent, related to decreased serum testosterone levels. In turn, orchiectomy was shown to disturb mitochondrial function, evidenced by increased mitochondrial H2O2 production and decreased MMP in the SN of adult male rats . These findings strongly suggest that testosterone supplementation ameliorates age-related brain mitochondria dysfunction in male rats by enhancing both mitochondrial antioxidative capacity and mitochondrial biogenesis.
Age-related mitochondrial alterations are demonstrated in the human skeletal muscle beginning at 40 ~ 50 years of age 30, 31. Normal neuronal activities are critically dependent on mitochondrial function . Mitochondrial DNA (mtDNA) copy number was determined by quantifying mitochondrial ND1 (mtND1) and nuclear-encoded beta-2-microglobulin (β2MG) gene expression via qPCR. Therefore, further analysis is required to discriminate testosterone-specific effects on age-related mitochondria dysfunction in the brain. Indeed, consistent with our preclinical findings and based on robust clinical evidence, it was hypothesized that the age-related decline in testosterone levels in men may act as a "second hit" to impair neurocognitive function and precipitate neurodegenerative disease . Accumulation of amyloid β peptide, a hallmark of AD, impairs the activity of mitochondrial complex IV, leading to increased ROS levels and ATP depletion in AD brains . Normal mitochondrial function is inextricably linked with proper and sustained activity of oxidative phosphorylation complexes.
Adult Cd11bDTR mice were weekly injected with vehicle (vehicle) or Diphtheria Toxin (DT) for a total of 3 consecutive injections, and testes were collected for analysis 7 days after the last injection. L, Representative confocal images of the testes from Cyp17a1Cre; R26tdTomato mice. K, Representative confocal images of the testes from Cyp17a1Cre; R26tdTomato mice. E, Representative confocal images of the testes from AAV-DIO-Lck–EGFP-injected Cyp17a1Cre; R26tdTomato mice. D, Experimental strategy to label LCs membranes by intratesticular injection of AAV-DIO-Lck–EGFP into the testes of Cyp17a1Cre; R26tdTomato mice.