SIRT1/MFN2-Mediated Regulation of Mitochondrial Dynamics and Mitophagy in Age-Related Decline of Leydig Cell Function

Age-related decline in testosterone production, known as late-onset hypogonadism (LOH), is a common condition in aging males, but its underlying mechanisms remain incompletely understood. Here, we investigated whether disruption of mitochondrial fusion-fission balance contributes to age-related steroidogenic failure. Using aged mouse models and H₂O₂-induced senescent TM3 Leydig cells, we demonstrated that aging is associated with impaired mitochondrial function, characterized by fragmentation, decreased membrane potential (MMP), and reduced ATP production. This mitochondrial dysfunction was driven by an imbalance in mitochondrial dynamics, specifically a downregulation of the fusion protein Mitofusin 2 (MFN2) and a shift towards fission. Consequently, aged Leydig cells exhibited elevated oxidative stress, impaired mitophagy, and a significant decline in the expression of key steroidogenic enzymes, leading to reduced testosterone synthesis. Crucially, M1 treatment, a mitochondrial fusion promoter, reversed these aging phenotypes, restoring mitochondrial integrity and testosterone production. In contrast, MFN2 knockdown exacerbated them. Mechanistically, we found that MFN2's protective effects were independent of the SIRT1 pathway. However, the anti-aging benefits of SIRT1 activation were entirely dependent on MFN2. Our findings identify MFN2 as a central regulator of mitochondrial homeostasis in Leydig cells and establish mitochondrial dynamics imbalance as a key mechanism in age-related testosterone decline. Targeting mitochondrial fusion may represent a novel therapeutic strategy for LOH.