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

SIRT1/MFN2-Mediated Regulation of Mitochondrial Dynamics and Mitophagy in Age-Related Decline of Leydig Cell Function | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article SIRT1/MFN2-Mediated Regulation of Mitochondrial Dynamics and Mitophagy in Age-Related Decline of Leydig Cell Function zhengmei Lv, chao Liu, zimeng Xu, Chongkang Wu, Yajing Liu, Xin Chen, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9553901/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 6 You are reading this latest preprint version Abstract 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. Mitochondrial dynamics Mitofusin 2 (MFN2) Cellular senescence Late-onset hypogonadism Oxidative stress SIRT1 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Late-onset hypogonadism (LOH) is a prevalent clinical syndrome in aging males, characterized by a significant decline in serum testosterone levels and associated with a spectrum of symptoms including diminished libido, fatigue, reduced muscle mass, mood disturbances, and metabolic alterations (Huhtaniemi and Forti 2011 ). As the global population ages, the incidence of LOH continues to rise, posing a substantial challenge to the quality of life and overall health of middle-aged and elderly men (Zhou et al. 2020 ). Testosterone is primarily synthesized and secreted by testicular Leydig cells. The age-related decline in testosterone levels is fundamentally attributed to the functional degeneration of Leydig cells, the decrease in the number of Leydig cells, along with a reduction in the number of intracellular organelles (Mularoni et al. 2020 ; Santiago et al. 2019 ). However, the precise molecular mechanisms driving this functional decline during aging remain incompletely elucidated, thereby limiting the development of targeted and effective therapeutic strategies. Among the various mechanisms implicated in cellular senescence, mitochondrial dysfunction has emerged as a central player. Mitochondria are not only the cellular "powerhouses," responsible for generating ATP, but are also crucial sites for steroid hormone biosynthesis (Selvaraj and Stocco. 2015). Mitochondria are highly dynamic organelles that maintain their network homeostasis, function, and quality control through continuous fusion and fission processes. The damaged and dysfunctional mitochondria can be removed by mitophagy (Harrington et al. 2023 ). Therefore, the balance of mitochondrial dynamics and mitophagy is critical for preserving mitochondrial health, including the normal synthesis of steroid hormones. Mitochondrial imbalance can lead to mitochondrial fragmentation, decreased membrane potential (MMP), reduced ATP production, and exacerbated oxidative stress (Chourasia et al. 2025 ; Sun et al. 2021 ). Accumulating evidence indicates that an imbalance in mitochondrial dynamics and disruption of mitophagy suppress steroid hormone biosynthesis in testicular Leydig cells (Garza et al. 2022 ). Despite this, the specific role of imbalanced mitochondrial dynamics in the functional decline of aging Leydig cells and its upstream regulatory mechanisms require in-depth exploration. Among the key proteins regulating mitochondrial fusion, Mitofusin 2 (MFN2) stands out. MFN2 not only mediates the fusion of the mitochondrial outer membrane but is also involved in mitochondria-endoplasmic reticulum tethering and the initiation of mitophagy (Gatti et al. 2025 ; Gordaliza-Alaguero et al. 2025 ; Tur et al. 2020 ). Downregulation of MFN2 has been linked to various pathological states, including muscle atrophy, vascular calcification, metabolic diseases, and reduced steroidogenesis (Cefis et al. 2024 ; Wu et al. 2025 ; Di Rienzo et al. 2024 ). Moreover, MFN2 upregulation facilitates cholesterol transport, thereby stimulating testosterone synthesis (Duarte et al. 2014 ; Lv et al. 2024 ). We therefore hypothesized that MFN2 could be a critical node connecting aging signals, mitochondrial homeostasis, and Leydig cell steroidogenic function. Sirtuin 1 (SIRT1), an NAD+-dependent deacetylase, is a key regulator of energy metabolism and stress responses, renowned for its anti-aging properties. The AMPK/SIRT1 signaling pathway plays a vital role in modulating energy homeostasis, oxidative stress, and cellular senescence (Salminen et al. 2012). Existing evidence suggests that SIRT1 can influence mitochondrial biogenesis and function (Behera et al. 2024 ). Nevertheless, the role of SIRT1 in regulating mitochondrial homeostasis in Leydig cells within the context of LOH, and its functional interplay with key mitochondrial dynamics proteins, remains poorly defined. This study aims to elucidate the central role of MFN2-mediated mitochondrial dynamics in the pathogenesis of LOH and to clarify its relationship with the SIRT1 pathway. In this study, we employed an integrated approach utilizing natural aging mouse models and H₂O₂-induced senescent TM3 Leydig cells. First, we systematically assessed the impact of aging on Leydig cell senescence markers, AMPK/SIRT1 pathway activity, the expression of key steroidogenic enzymes, and mitochondrial dynamics balance. Subsequently, using genetic (siMFN2 knockdown) and pharmacological (M1, a mitochondrial fusion promoter) interventions to modulate MFN2 expression, we investigated the causal role of MFN2 in regulating mitochondrial function, oxidative stress, mitophagy, and testosterone synthesis. Finally, through combined SIRT1 knockdown and MFN2 knockdown experiments, we meticulously dissected the complex hierarchical relationship between MFN2 and the SIRT1 signaling pathway. Our findings are expected to deepen the understanding of the molecular mechanisms underlying age-related testosterone decline and to provide a solid theoretical and experimental foundation for the development of novel, MFN2-targeted therapeutic strategies for LOH, aimed at restoring mitochondrial function and steroidogenic capacity in aging Leydig cells. Materials and Methods Antibodies and reagents The primary antibodies against AMPKα, p-AMPKα, LC3B, ATG7, ATG5, p-DRP1(Ser637), p-DRP1(Ser616), and COX4 were obtained from Cell Signaling Technology (Massachusetts, USA), and Lamin B, GAPDH, LAMP2, MFN2, MFN1, P62, FIS1, PINK1, PARK2, GPX5, Acsl4, ATGL and ADRP from Proteintech (Wuhan, China), NIX and α-Tubulin from Bioworld (Minnesota, USA). SIRT1, Acetyl-Lysine, and mt-TFA from Abcam (Cambridge, England). GPX4 and SOD2 from Boster Biological Technology (Wuhan, China). LAMP1 and RAB7 from Bioswamp (Wuhan, China). HSL from Affinity (Jiangsu, China). P21 from Beyotime (Shanghai, China). The secondary antibodies goat anti-rabbit IgG (H + L) HRP-conjugated and goat anti-mouse IgG (H + L) HRP-conjugated were obtained from Elabscience (Wuhan, China). Alexa Fluor 488 donkey anti-mouse IgG (H + L) and Alexa Fluor 594 donkey anti-rabbit IgG (H + L) from Invitrogen (Carlsbad, CA, USA). The Senescence β-Galactosidase Staining Kit (C0602), Testosterone ELISA Kit (PT872), ATP Assay Kit (S0026), Reactive Oxygen Assay (S0033S), 4′,6-diamidino2-phenylindole (DAPI) (C1006) and bicinchoninic acid (BCA) protein assay kit (P0012) were purchased from Beyotime Biotechnology (Shanghai, China), MitoTracker™ Green FM (M7514) and MitoSOX™ Red Mitochondrial Superoxide Indicator (M36008) from Invitrogen (Carlsbad, CA, USA), Mitophagy Detection Kit (MD01) from Dojindo (Kumamaoto, Japan). Animals and treatments Two-month-old and 18-month-old male C57BL/6J mice were assigned to a young group (Y, n = 6) and an old group (O, n = 6), respectively. Testicular tissues from one side were collected and processed for frozen sectioning or Western blot analysis. The contralateral testes were used for Leydig cell isolation, followed by steroidogenesis stimulation experiments. The mouse serum was collected to assess testosterone levels using a commercial ELISA kit. All animal experimental procedures were in accordance with the animal welfare management regulations formulated by the Experimental Animal Ethics Committee of Anhui Medical University. Isolation of mouse primary Leydig cells Briefly, following the protocols described in the previous studies (Lv et al. 2024 ), we isolated Leydig cells by first decapsulating the mouse testes and digesting them in 0.05% collagenase type IV under constant agitation. Digestion continued until the tissue was adequately dispersed while minimizing disruption of the seminiferous tubules. The suspension was then filtered through a 100-mesh sieve, and the collected filtrate underwent washing with M199 medium. Following Percoll density gradient centrifugation, the purified Leydig cells showed about 81% purity, which was confirmed through 3β-HSD histochemical staining. Then the isolated Leydig cells were stimulated in M199 medium with 10 ng/L human chorionic gonadotropin (hCG), or with 1mM double cyclic adrenosine monophosphate (db-cAMP) for 3h. Cell culture and intervention The TM3 mouse Leydig cell line was obtained from the cell bank of the Chinese Academy of Sciences (ATCC) and maintained in DMEM/F12 medium containing 10% fetal bovine serum (FBS). To assess their effects on senescence, TM3 cells were first pretreated with either 20 µM M1 agonist (4 h) or MFN2 siRNA (24 h) and then subjected to senescence induction by 400 µM H 2 O 2 treatment for 2 h. To examine whether the role of MFN2 in senescence depends on SIRT1 levels, SIRT1 was knocked down using siRNA in TM3 cells, after which the cells were treated with 20 µM M1 agonist and then exposed to H₂O₂ to induce senescence. To determine whether MFN2 participates in the regulation of senescence by SIRT1, MFN2 was knocked down in TM3 cells using siRNA. These cells were then treated with 1 mM nicotinamide riboside chloride (NRC) (12 h) activate SIRT1 or subjected to SIRT1 siRNA knockdown (24 h), followed by senescence induction with 400 µM H 2 O 2 for 2 h. Small inhibitory RNA (siRNA) knockdown Sirt1 siRNA and mfn2 siRNA were purchased from GenePharma (Shanghai, China). The sirt1 siRNA sequences were 5'-GCA CUA AUU CCA AGU UCU ATT-3' (sense) and 5'-UAG AAC UUG GAA UUA GUG CTT-3' (antisense). The MFN2 siRNA sequences were 5'-CUG CCA UGA ACA ACA AGA AAG UTT-3' (sense) and 5'-ACU UUC UUG UUC AUG GCA GTT-3' (antisense). The experiments were performed according to the manufacturer’s instructions. Briefly, liposomal Lipo3000 was mixed with Opti-MEM, then specific siRNA or the scramble siRNA was mixed with Opti-MEM and the concentration of siRNA was adjusted to 1 µM. Then the mixture was added to a petri dish and incubated for 24 hours to knockdown mfn2 or sirt1 respectively in TM3 cells. Western blot analysis The testicular tissues or cell pellets were homogenized in RIPA lysis buffer on ice. Following centrifugation at 12,000 ×g for 15 min at 4°C, the supernatant was collected, and its protein concentration was quantified with a BCA assay. Equal amounts of protein were separated by SDS-PAGE using 8–12% gradient gels and then transferred onto PVDF membranes. After blocking with 5% non-fat dry milk in TBST for 2 h at room temperature, the membranes were incubated with primary antibodies overnight at 4°C. Next, the membranes were incubated with corresponding horseradish peroxidase (HRP)-conjugated secondary antibodies for 2 h at room temperature. Finally, the immunoreactive bands were detected using a Tanon-5200 chemiluminescence imaging system. Immunofluorescence staining Frozen sections of mouse testicular tissue were thawed for 30 min, washed with PBS, and permeabilized with 1% Triton X-100 in PBS for 30 min. Antigen retrieval was performed using sodium citrate buffer (pH 6.0). The sections were then blocked with 5% BSA in PBS for 30 min at room temperature. After blocking, sections were incubated with specific primary antibodies overnight at 4°C, followed by incubation with fluorescent secondary antibodies for 1 h at room temperature. Following three PBS washes, nuclei were counterstained with DAPI for 10 min. Finally, the sections were mounted and imaged using a Leica THUNDER Imager (Germany). Detection of reactive oxygen species (ROS) The levels of intracellular ROS production were analyzed by the fluorescent dye dichlorodihydrofluorescein dihydrate (DCFH-DA) using a commercial assay kit according to the manufacturer’s instructions. For tissue staining, frozen testicular sections were incubated with ROS staining working solution at 37°C for 30 min in the dark. For cell staining, cells were incubated within a 5% CO 2 incubator. After incubation, all samples were washed and tissue sections were then mounted with an antifade medium containing DAPI. Images of both sections and cells were acquired immediately using a Leica THUNDER Imager (Germany). Detection of ATP content ATP assay kit was used to measure ATP content in the sample, according to the manufacturer's instructions. Briefly, Leydig cells (LCs) and TM3 cells were homogenized and centrifuged. For the ATP measurement, 20 µL of the supernatant was rapidly mixed with 100 µL of the ATP Assay Working Solution. A standard curve was generated using known concentrations of ATP, and the protein concentration in each treatment group was determined using a BCA Protein Assay Kit. Finally, the total ATP levels were calculated and expressed as micromoles per nanogram of protein (µmol/ng protein). Senescence-associated β-galactosidase (SA-β-Gal) staining Cellular senescence was evaluated by SA-β-Gal staining according to the manufacturer’s instructions. For tissue samples, frozen testicular sections were incubated overnight at 37°C with SA-β-Gal staining solution, followed by hematoxylin nuclear counterstaining and mounting with antifade medium. For cultured cells, after fixation, the SA-β-Gal working solution was added to the samples and incubated overnight at 37°C in the dark. Images were acquired using a Leica THUNDER Imager (Germany). MitoTracker™ Green FM staining TM3 Cells were incubated with 100 nM MitoTracker™ Green FM (Invitrogen, USA) diluted in serum-free DMEM/F12 medium at 37°C for 30 minutes in the dark. After staining, the solution was replaced with prewarmed fresh medium. Mitochondrial morphology was subsequently examined and imaged using a laser scanning confocal microscope (Leica TCS SP5, Germany). Mitochondrial Membrane Potential Detection To evaluate mitochondrial membrane potential, TM3 cells were incubated with 10 µM JC-1 staining solution at 37°C for 20 minutes, protected from light. The cells were then washed twice with JC-1 staining buffer (1X) and prewarmed PBS. Imaging was performed using an inverted fluorescence microscope (Nikon Ts2, Japan). . Mitochondrial Superoxide Assay To detect mitochondrial superoxide production, cells were incubated with 5 µM MitoSOX™ Red mitochondrial superoxide indicator (Invitrogen, USA) in Hanks' balanced salt solution (HBSS) for 10 minutes at 37°C in the dark. After incubation, cells were gently washed three times with PBS and maintained in pre-warmed HBSS for immediate observation under an inverted fluorescence microscope (Nikon Ts2, Japan). Mitophagy Assay Mitophagy was assessed using the Mitophagy Detection Kit (MD01, Dojindo, Kumamoto, Japan). Cells were sequentially stained with a 100 nmol/L Mitophagy Dye and a 1 µmol/L Lyso Dye according to the manufacturer’s protocol. Briefly, after incubation with the Mitophagy Dye working solution at 37°C for 30 min in the dark, the dye was removed and replaced with fresh complete medium. Cells were then cultured for an additional 24 h to allow mitophagic flux to proceed. Subsequently, the Lyso Dye working solution was applied to label acidic compartments. Imaging was performed using a Leica THUNDER Imager(Germany), and fluorescence puncta were quantified with ImageJ software. Puncta per cell were determined based on at least 50 cells. Statistical analysis Western blot band gray values and fluorescence intensities were measured using ImageJ software. Data are presented as mean ± SEM. Statistical analyses were performed using GraphPad Prism software (version 8.0.2). Group comparisons were conducted using Student’s t-test or one-way ANOVA, followed by Bonferroni post hoc test where applicable. A P value < 0.05 was considered statistically significant Results Age-related changes in mouse testicular tissues and H₂O₂-induced senescence in TM3 cells To observe age-related changes in testicular tissues, SA-β-gal staining was performed on frozen sections from 2-month-old (Y group) and 18-month-old (O group) mice. The results revealed prominent SA-β-gal-positive signals in the cytoplasm of Leydig cells in the O group (Fig. 1 A, B). Western blot analysis was used to assess the protein levels of aging markers Lamin B and P21 in Leydig cells from both aged and young mice. Compared with the Y group, the O group exhibited a significant decrease in Lamin B protein expression and a marked increase in p21 protein levels in Leydig cells (Fig. 1 C-D and S1A). Moreover, the protein expressions of the AMPK/SIRT1 anti-aging pathway were analyzed in Leydig cells from both aged and young mice. Our data showed that the O group had generally downregulated expression of SIRT1, AMPKα, and p-AMPKα (Fig. 1 E-F and S1B), along with elevated protein acetylation levels compared to the Y group (Figs. 1 G and S1C). In addition, immunofluorescence staining results also demonstrated that compared to the Y group, the SIRT1 fluorescence intensity was significantly reduced, while the protein acetylation level was markedly elevated in Leydig cells in the O group (Figs. 1 H-J). Furthermore, we treated TM3 cells with different H₂O₂ concentrations to model cellular senescence. We found that a concentration of 400 µM H₂O₂ produced the most potent senescence-inducing effect (Figs. 1 K-L and S1D). Therefore, this concentration was selected for subsequent experiments to induce senescence in TM3 cells. Age-related decline in testosterone synthesis by Leydig cells To investigate whether aging leads to a decline in testosterone synthesis in mice, serum testosterone levels were measured using ELISA. As shown in Fig. 2 A, serum testosterone levels in the O group were significantly lower than those in the Y group. Moreover, the protein levels of P450scc, 3β-HSD, and StAR, three key enzymes of testosterone synthesis, were significantly downregulated in Leydig cells from O group (Fig. 2 B-C and S2A). Immunofluorescence staining revealed a significant decrease in the fluorescence intensity of 3β-HSD, which is localized to Leydig cells, in the O group compared to the Y group (Fig. 2 D, E). Furthermore, hCG) and db-cAMP were used to stimulate endogenous testosterone production. As expected, hCG and db-cAMP treatment significantly upregulated the expression of testosterone synthesis-related enzymes in Leydig cells from both aged and young mice (Fig. 2 F-G and S2B-C). However, even after stimulation, the expression levels of testosterone synthesis-related enzymes in the stimulated O group remained lower than those in the stimulated Y group (Figs. 2 F-G and S2B-C). Imbalanced mitochondrial dynamics in Leydig cells from aged mice To investigate the mechanism underlying impaired testosterone synthesis in aging Leydig cells, mitochondrial dynamics were assessed. The results showed that the mitochondrial fusion regulatory proteins MFN2 and MFN1, as well as the mitochondrial fission protein FIS1, were significantly downregulated in Leydig cells from aged mice (Figs. 3 A-D and S3A). Dynamin-related protein 1 (DRP1) promotes mitochondrial fission when phosphorylated at Ser616, but conversely inhibits it when phosphorylated at Ser637. Accordingly, we measured its phosphorylation levels at these two sites. The level of p-DRP1 (Ser637) was significantly downregulated in Leydig cells from aged mice. Conversely, the level of p-DRP1 (Ser616) was significantly upregulated (Figs. 3 C-D and S3A). Furthermore, hCG and db-cAMP were used to stimulate endogenous testosterone production. Following hCG and db-cAMP treatment, we observed a significant upregulation in MFN2, MFN1, FIS1, and p-DRP1 (Ser637) expression, alongside a downregulation of p-DRP1 (Ser616) (Fig. 3 E-F and S3B-C). However, even after stimulation, the protein levels of MFN2, MFN1, FIS1, and p-DRP1 (Ser637) in the stimulated O group remained lower than those in the stimulated Y group (Fig. 3 E-F and S3B-C). In addition, after stimulation, the protein level of p-DRP1 (Ser616) in the stimulated O group remained higher than those in the stimulated Y group (Fig. 3 E-F and S3B-C). Mitochondrial dynamics imbalance resulting from MFN2 downregulation contributes to the aging process To further verify whether mitochondrial dynamics imbalance is involved in the aging process, TM3 cells were cultured in vitro and treated with the mitochondrial fusion promoter M1 or transfected with MFN2-targeting short interfering RNA (siMFN2) to knock down MFN2. Mitochondrial morphology was labeled using the MitoTracker Green probe. We found that compared with the control group, TM3 cells in the H 2 O 2 -induced senescence group exhibited a transformation of mitochondria into numerous small fragments (Figs. 4 A, B). Interestingly, M1 treatment rescued the H₂O₂-induced mitochondrial fragmentation in TM3 cells, whereas MFN2 knockdown exacerbated it (Fig. 4 A, B). Consistent with the mitochondrial morphological findings, TM3 cells in the H₂O₂-induced senescence group exhibited significant downregulation of the mitochondrial fusion regulators MFN2, MFN1, and p-DRP1 (Ser637), along with upregulation of the fission regulator p-DRP1 (Ser616), compared to the control group (Fig. 4 C, D, and S4). M1 treatment rescued the H₂O₂-induced downregulation of MFN1, MFN2, and p-DRP1 (Ser637) proteins and upregulation of p-DRP1 (Ser616) protein in TM3 cells (Fig. 4 C-D and S4). In addition, MFN2 knockdown exacerbated the H₂O₂-induced downregulation of MFN1, MFN2, and p-DRP1 (Ser637) protein, as well as the upregulation of p-DRP1 (Ser616) protein in TM3 cells (Fig. 4 C-D and S4). Furthermore, SA-β-gal staining was used to assess the senescence level of TM3 cells. The results revealed a large number of SA-β-gal-positive cells in the H₂O₂-induced senescence group (Fig. 4 E, F). M1 treatment markedly attenuated the H₂O₂-induced senescence in TM3 cells (Fig. 4 E, F). In contrast, MFN2 knockdown exacerbated the H₂O₂-induced senescence in TM3 cells (Fig. 4 E, F). MFN2 plays a role in counteracting oxidative stress during aging Oxidative stress is an important pathophysiological mechanism in aging-related diseases. DCFH-DA was used to mark the intracellular reactive oxygen species (ROS). The results showed that the ROS level in the testicular interstitium of the aged group was significantly higher than that in the young group (Figs. 5 A, B). Western blot was further used to detect the expression of redox enzymes in Leydig cells from aged and young mice. As compare with the Y group, the proteins glutathione peroxidase 4 (Gpx4), GPX5, and superoxide dismutase 2 (SOD2) were significantly downregulated in the O group (Figs. 5 C-D and S5A). Moreover, after hCG and db-cAMP stimulation, the protein levels of Gpx4, GPX5, SOD2, and p-DRP1 (Ser637) in the stimulated O group remained lower than those in the stimulated Y group (Figs. 5 E-F and S5B-C). Furthermore, we assessed the effects of M1 treatment and MFN2 knockdown on oxidative stress in the H₂O₂-induced senescence model using TM3 cells. M1 treatment reduced the H₂O₂-induced increases in both intracellular and mitochondrial ROS levels in TM3 cells (Fig. 5 G, H). In contrast, MFN2 knockdown further heightened the ROS levels in both the cytosol and mitochondria induced by H₂O₂ in TM3 cells (Fig. 5 G, H). In addition, M1 treatment attenuated the H₂O₂-induced downregulation of GPX4, GPX5 and SOD2 protein expression (Fig. 5 I-J and S5D-E). Conversely, MFN2 knockdown further exacerbated this downregulation (Fig. 5 I-J and S5D-E). MFN2 counteracts age-related mitophagy decline Mitochondria are key sites for testosterone synthesis in Leydig cells. This study investigates the effect of mitophagy and mitochondrial function on testosterone synthesis in aging Leydig cells. The results showed that the expression levels of mitophagy-related proteins PINK1, Parkin, and NIX, as well as mitochondrial function-related proteins mitochondrial transcription factor A (mt-TFA) and cytochrome c oxidase 4 (COX4), were significantly lower in the O group compared to the Y group (Figs. 6 A-B and S6A). Stimulation of testosterone synthesis with hCG and db-cAMP significantly increased the expression of these proteins in the O group, but the levels of PINK1, Parkin, NIX, mt-TFA, and COX4 proteins remained lower than those in the stimulated Y group (Figs. 6 C-D and S6B-C). Further detection of ATP levels in Leydig cells revealed a significant decrease in ATP in the O group, while hCG or db-cAMP stimulation significantly increased ATP levels (Fig. 6 E). In TM3 cell experiments, the expression levels of PINK1, Parkin, NIX, mt-TFA, and COX4 were also significantly lower in the H₂O₂-induced senescence group compared to the control group (Figs. 6 F-G and S6D-E). M1 treatment alleviated the downregulation of these proteins in the H₂O₂-induced senescence group (Figs. 6 -G and S6D-E). Additionally, compared to the H₂O₂-induced senescence group, the protein levels of PINK1, Parkin, NIX, mt-TFA, and COX4 were further downregulated in TM3 cells treated with H₂O₂ + siMFN2 (Figs. 6 F-G S6D-E). Mitophagy staining results were consistent with the downregulations of these proteins, as shown in Figs. 6 H-J, where mitophagy staining and colocalization of mitochondrial staining with lysosomal staining were reduced in the H₂O₂-induced senescence group. M1 treatment attenuated H₂O₂-induced mitophagy decline in TM3 cells (Figs. 6 H-J). Conversely, MFN2 knockdown further exacerbated H₂O₂-induced mitophagy decline in TM3 cells (Figs. 6 H-J). Moreover, ATP levels in the H₂O₂-induced senescence group were significantly lower than those in the control group in TM3 cells (Fig. 6 K). The H₂O₂-induced decrease in ATP levels was ameliorated by M1 and, conversely, exacerbated by MFN2 knockout in TM3 cells (Fig. 6 K). Further detection of mitochondrial membrane potential (MMP), an important indicator of normal mitochondrial function, showed that MMP was significantly reduced in the H₂O₂-induced senescence group (Figs. 6 L, M). The H₂O₂-induced decrease in MMP levels was ameliorated by M1 and, conversely, exacerbated by MFN2 knockout in TM3 cells (Figs. 6 L, M). M1-induced mitochondrial fusion protects mitochondrial function and testosterone synthesis independently of SIRT1 To investigate whether the protective effects of MFN2 on testosterone synthesis and mitochondrial function are related to SIRT1 levels, SIRT1 was knocked down in TM3 cells using SIRT1-targeted short interfering RNA (siSIRT1). As expected, the protein levels of SIRT1, P450scc, 3β-HSD, StAR, MFN1, MFN2, and p-DRP1 (Ser637) were downregulated, while p-DRP1 (Ser616) was upregulated in the H₂O₂-induced senescence group compared to the control group (Figs. 7 A-C and S7). M1 treatment alleviated H₂O₂-induced downregulation of those proteins and the upregulation of p-DRP1 (Ser616) (Figs. 7 B-C and S7B-C). Moreover, SIRT1 knockdown did not significantly affect the ability of M1 treatment to rescue the H₂O₂-induced downregulation of P450scc, 3β-HSD, StAR, MFN1, MFN2, p-DRP1 (Ser637) and upregulation of p-DRP1 (Ser616) (Figs. 7 B-C and S7B-C). Furthermore, SIRT1 knockdown did not significantly affect the ability of M1 treatment to rescue the H₂O₂-induced increase the proportion of SA-β-gal positive cells and ROS levels, and decrease MMP and ATP levels (Figs. 7 D-G). These results indicate that SIRT1 may be not required for M1 to counteract aging-induced mitochondrial dysfunction and the suppression of testosterone synthesis. MFN2 is required for the SIRT1-mediated anti-aging effects SIRT1 possesses various anti-aging properties. To investigate whether the protective role of SIRT1 against aging-induced suppression of testosterone synthesis in Leydig cells requires the involvement of MFN2, MFN2 was knocked down in TM3 cells using siMFN2. Nicotinamide riboside chloride (NRC), a precursor of NAD+, was used to increase NAD levels in TM3 cells. As expected, NRC treatment significantly reversed the H₂O₂-mediated decrease in SIRT1, AMPKα, and p-AMPKα protein levels, indicating that NRC can activate SIRT1 signaling (Fig. 8 and S8C). Of interest, NRC treatment significantly rescued the H₂O₂-mediated downregulation of P450scc, 3β-HSD, StAR, MFN1, MFN2, p-DRP1 (Ser637) protein, and the upregulation of p-DRP1 (Ser616) protein in TM3 cells (Figs. 8 B-C and S8B-C). However, MFN2 knockdown abolished the protective effects of NRC against H₂O₂-induced inhibition in testosterone synthesis and imbalance in mitochondrial dynamics (Figs. 8 B-C and S8B-C). Next, we measured mitochondrial morphology. We found that NRC treatment significantly rescued the H₂O₂-mediated mitochondrial fragmentation, increase of ROS levels, and decrease of MMP and ATP levels in TM3 cells (Figs. 8 D-E, G). However, MFN2 knockdown abolished the protective effects of NRC against H₂O₂-induced mitochondrial dysfunction (Figs. 8 D-F). Furthermore, NRC treatment significantly rescued the H₂O₂-mediated increase in the proportion of SA-β-gal positive cells in TM3 cells (Figs. 8 D, F). However, MFN2 knockdown abolished the protective effects of NRC against H₂O₂-induced senescence (Figs. 8 D, F). Discussion The age-related decline in testosterone production, a hallmark of late-onset hypogonadism (LOH), poses a significant challenge to the health and well-being of aging males. While the central role of Leydig cell dysfunction in this process is recognized, the underlying molecular drivers have remained incompletely defined. In this study, we present a comprehensive investigation that positions an imbalance in mitochondrial dynamics, orchestrated by the downregulation of Mitofusin 2 (MFN2), as a pivotal mechanism in aging-related Leydig cell failure. Our data, derived from both in vivo aged mouse models and in vitro H₂O₂-induced senescent TM3 Leydig cells, delineate a clear pathogenic cascade beginning with aging-induced MFN2 downregulation, which leads to mitochondrial fragmentation, and in turn promotes oxidative stress and impaired mitophagy, ultimately resulting in mitochondrial dysfunction and suppressed steroidogenesis. Furthermore, we unravel a novel and hierarchically structured relationship between the nutrient-sensor SIRT1 and MFN2, revealing that MFN2 operates independently of SIRT1 to exert its protective effects, yet is absolutely required for SIRT1-mediated anti-aging benefits. This establishes MFN2 not merely as a participant but as a critical executioner of mitochondrial and cellular homeostasis in aging Leydig cells. Our initial findings confirm and extend the established hallmarks of aging in Leydig cells. The increased SA-β-gal activity, reduced Lamin B, elevated p21, and dampened AMPK/SIRT1 pathway activity in aged mice collectively paint a picture of progressive cellular senescence (Salminen et al. 2012; López-Otín et al. 2023 ). Crucially, this senescent state is intrinsically linked to a profound deficit in steroidogenic capacity, as evidenced by significantly lower serum testosterone and the downregulation of key synthesizing enzymes P450scc, 3β-HSD, and StAR. The failure of hCG and db-cAMP stimulation to fully restore enzyme levels in aged cells suggests a fundamental defect lies beyond cell surface receptors, within the steroidogenic machinery itself. This indicates an insufficiency in the core cellular energetic and biosynthetic platforms, namely, the mitochondria. We therefore focused on mitochondrial dynamics, a key determinant of mitochondrial health. The mitochondrion is a dynamic organelle that frequently undergoes fission and fusion to maintain its integrity in response to various physiological and pathological processes (Pernas et al. 2016 ; Chen et al. 2026 ). Mitochondrial fusion facilitates content exchange between mitochondria, leading to larger, more interconnected networks, whereas fission promotes their fragmentation and generates isolated and shortened mitochondria (Jiang et al. 2022 ; Ng et al. 2021 ). Mitochondrial outer membrane fusion is mediated by two proteins, mitofusin 1 (MFN1) and MFN2. Conversely, mitochondrial fission is mainly driven by the proteins dynamin-related protein 1 (DRP1) (Tábara et al. 2025 ). When phosphorylated at Ser616, DRP1 stimulates mitochondrial fission during mitosis. Conversely, fission is inhibited when DRP1 is phosphorylated at Ser637 (Xie et al. 2020 ; Shi et al. 2022 ). Our results demonstrate a pronounced shift towards fission in aged Leydig cells. The downregulation of fusion proteins MFN2 and MFN1, coupled with the inhibitory phosphorylation of DRP1 at Ser637 and its activating phosphorylation at Ser616, creates a molecular environment permissive for mitochondrial fragmentation. The resulting fragmented mitochondrial network is associated with a loss of membrane potential (MMP), reduced ATP production, and elevated oxidative stress creating an internal milieu that is fundamentally incompatible with the high-energy demands of steroid hormone biosynthesis (Selvaraj et al. 2015 ). Moreover, the observation that hCG/db-cAMP stimulation partially improves this dynamic imbalance, yet fails to normalize it in aged cells. These findings suggests that restoring mitochondrial dynamics could be a more upstream and effective therapeutic strategy than merely providing hormonal stimulation. To establish the role for MFN2 in this process, we employed gain-of-function and loss-of-function approaches in our cellular model. The finding demonstrated that the mitochondrial fusion promoter M1 could robustly reverse H 2 O 2 -induced mitochondrial fragmentation, senescence, and steroidogenic decline in TM3 cells. Conversely, siRNA-mediated knockdown of MFN2 exacerbated all these deficits, confirming its non-redundant, central role. These results provide compelling evidence that promoting fusion is a viable strategy to rescue aged Leydig cell function. Sirtuin 1 (SIRT1), an NAD+-dependent deacetylase, is a key regulator of energy metabolism and stress responses, renowned for its anti-aging properties (Hekmatimoghaddam et al. 2017 ). In this study, we investigate the functional interplay between the well-known anti-aging protein SIRT1 and MFN2. The observed downregulation of SIRT1 in aged Leydig cells is consistent with its established role as a guardian against cellular aging (Chen 2018 ). However, the mechanistic link between SIRT1 and mitochondrial function in this specific context was unclear. We initially hypothesized that SIRT1 might act upstream of MFN2, given its role as a deacetylase that can modify numerous targets. Surprisingly, our data compellingly demonstrate that the protective effects of pharmacological MFN2 upregulation with M1 against aging and mitochondrial damage are completely independent of SIRT1. Furthermore, SIRT1 knockdown did not attenuate M1's ability to restore steroidogenic enzymes, rebalance mitochondrial dynamics, improve ATP/MMP, reduce ROS, or suppress senescence. This indicates that MFN2 functions in a pathway parallel to or downstream of SIRT1, and its activation is sufficient to counteract aging phenotypes even when SIRT1 is deficient. In stark contrast, the converse was not true. The protective effects of SIRT1 activation with nicotinamide riboside chloride (NRC), a precursor of NAD+, were completely abrogated upon MFN2 knockdown. NRC successfully activated the SIRT1/AMPK pathway, thereby rescuing the H₂O₂-induced imbalance in mitochondrial dynamics, mitochondrial dysfunction, and the decline in steroidogenesis in TM3 cells. However, in the absence of MFN2, NRC was rendered completely ineffective. This reveals a critical, non-redundant dependency of SIRT1 on MFN2 to execute its anti-aging program in Leydig cells. Therefore, the concurrent decline of both SIRT1 and MFN2 may create a doubly detrimental scenario in aging. However, our findings suggest that therapeutic strategies aimed solely at activating SIRT1 may fail if MFN2 levels are not concurrently supported. Mitophagy serves to eliminate damaged and dysfunctional mitochondria (Zhang et al. 2025 ). Our findings further illuminate the critical role of impaired mitophagy, a selective form of autophagy for damaged mitochondria, in the vicious cycle of mitochondrial dysfunction and steroidogenic decline in aging Leydig cells. The age-related downregulation of key mitophagy initiators, PINK1 and Parkin, along with the receptor NIX, indicates a significant failure in the quality control system tasked with eliminating fragmented and dysfunctional mitochondria. This deficiency is not merely a consequence of aging but an active contributor to the pathology. As our data show, the imbalance in mitochondrial dynamics, characterized by MFN2 downregulation and excessive DRP1-mediated fission, generates a surplus of damaged, ROS-producing mitochondria. Under normal conditions, these damaged organelles would be promptly recognized and cleared by mitophagy. However, in the aging Leydig cell, this clearance mechanism is blunted. The resulting accumulation of defective mitochondria creates a state of chronic oxidative stress and bioenergetic crisis, as evidenced by elevated ROS and reduced ATP, which directly poisons the steroidogenic machinery and starves it of the necessary energy and cholesterol precursors for testosterone synthesis. The interconnection between mitochondrial dynamics and mitophagy is particularly underscored by the role of MFN2. Beyond its canonical function in fusion, MFN2 is increasingly recognized as a regulatory node for mitophagy, serving as a receptor for Parkin recruitment on the outer mitochondrial membrane (Chen and Dorn 2013 ). Our rescue experiments demonstrated that the mitochondrial fusion promoter M1 rescued H₂O₂-induced suppression of mitophagy. Conversely, MFN2 knockdown exacerbated the suppression of mitophagy. This creates a self-reinforcing loop: mitochondrial damage promotes fission, and the resulting fragments, if not cleared, exacerbate the damage. While our study provides strong evidence for the central role of MFN2, several questions remain for future investigation. First, the precise upstream mechanism responsible for the age-related downregulation of MFN2 in Leydig cells is still unknown. It could involve transcriptional repression, post-translational modifications, or enhanced degradation. Identifying these regulators could unveil new targets for intervention. Second, our in vitro model utilizes H 2 O 2 to induce senescence, which, while robust, may not capture all aspects of the complex, low-grade chronic inflammation and metabolic dysregulation seen in physiological aging in vivo. Future studies could benefit from using genetic models of accelerated aging or conducting long-term longitudinal analyses. Third, the specific molecular link whereby MFN2 loss impairs the expression of steroidogenic enzymes like P450scc and StAR warrants deeper exploration. It may involve disrupted cholesterol transport due to impaired mitochondria-ER contact sites, a known function of MFN2, or a more general failure in providing adequate ATP for the steroidogenic process. From a translational perspective, our findings position MFN2 as a highly promising therapeutic target for LOH. The demonstration that a pharmacological promoter of mitochondrial fusion (M1) can reverse key aging phenotypes in Leydig cells opens up a novel avenue for drug development. Rather than merely supplementing testosterone, a strategy with known limitations and side effects, enhancing mitochondrial health via MFN2 could address the root cause of the hormonal deficit in a subset of aging men. Future research should focus on developing more specific and potent MFN2-stabilizing compounds and testing their efficacy and safety in pre-clinical models of LOH. In summary, our work delineates a detailed pathway through which aging impairs Leydig cell function, centering on the downregulation of MFN2 and the consequent disintegration of mitochondrial homeostasis. We establish that MFN2 is a critical regulator that governs mitochondrial dynamics, oxidative stress, and mitophagy, and its decline is a principal driver of steroidogenic failure. Moreover, we redefine the functional hierarchy between SIRT1 and MFN2, revealing that MFN2 is the indispensable effector through which SIRT1 must act to confer its anti-aging benefits. By identifying MFN2 as a central executioner of mitochondrial and cellular health, this study not only deepens our understanding of the molecular etiology of LOH but also provides a compelling rationale for targeting mitochondrial fusion as a novel therapeutic strategy for age-related hypogonadism. Declarations Funding This study was supported by the National Natural Science Foundation of China (82173559), Major Project of Natural Science Research in Universities of Anhui Province (2025AHGXZK20014), Key Research and Development Projects of Anhui Province (202104j07020035), Key Research Program of Anhui Science and Technology Innovation Platform (202305a12020016), Key Project of Natural Science Research of Anhui Provincial Department of Education (2024AH050752), Research Fund of Anhui Institute of translational medicine (2022zhyx-C45). Author contributions Zheng-Mei Lv: Conceptualization, Funding acquisition, Investigation, data curation and formal analysis. Chao Liu: Investigation. Chong-Kang Wu: Investigation. Zi-meng Xu: Investigation. Ya-jing Liu: Formal analysis, Xin Chen: Formal analysis, Yuan-Hua Chen: Conceptualization, Supervision, data curation and formal analysis, Funding acquisition, Writing-Original Draft, Writing-review & editing. All authors reviewed the manuscript. Disclosures: the authors declare no conflicts of interest. Clinical trial number: not applicable. Data Availability Statement: the data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions. References Behera BP, Mishra SR, Mahapatra KK, Patil S, Efferth T, Bhutia SK. SIRT1-activating butein inhibits arecoline-induced mitochondrial dysfunction through PGC1α and MTP18 in oral cancer. Phytomedicine. 2024;129:155511. Cefis M, Dargegen M, Marcangeli V, Taherkhani S, Dulac M, Leduc-Gaudet JP, et al. MFN2 overexpression in skeletal muscles of young and old mice causes a mild hypertrophy without altering mitochondrial respiration and H2O2 emission. Acta Physiol (Oxf). 2024;240(5):e14119. Chen K, Sun Z. Activation of DNA demethylases attenuates aging-associated arterial stiffening and hypertension. Aging Cell. 2018;17(4):e12762. 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Zhou SJ, Zhao MJ, Yang YH, Guan D, Li ZG, Ji YD, et al. The Epidemiological Characteristics of Late-Onset Hypogonadism in Chinese Middle-Aged and Elderly Men: Two Cross-Sectional Studies in the Same Community. Am J Mens Health. 2020;14(6):1557988320977991. Additional Declarations No competing interests reported. Supplementary Files SupportingInformation.docx Cite Share Download PDF Status: Under Review Version 1 posted Reviewers agreed at journal 13 May, 2026 Reviewers agreed at journal 07 May, 2026 Reviewers invited by journal 05 May, 2026 Editor assigned by journal 28 Apr, 2026 Submission checks completed at journal 28 Apr, 2026 First submitted to journal 28 Apr, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9553901","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":639305697,"identity":"dc6d5ea8-c79f-4118-8aaa-854d32219b25","order_by":0,"name":"zhengmei Lv","email":"","orcid":"","institution":"Anhui Medical University","correspondingAuthor":false,"prefix":"","firstName":"zhengmei","middleName":"","lastName":"Lv","suffix":""},{"id":639305702,"identity":"37e9dadf-ccb2-4b2f-b0ab-6b478bb65a74","order_by":1,"name":"chao Liu","email":"","orcid":"","institution":"Anhui Medical University","correspondingAuthor":false,"prefix":"","firstName":"chao","middleName":"","lastName":"Liu","suffix":""},{"id":639305706,"identity":"a2ff3ec4-123b-4868-97d3-4bc72ec7196f","order_by":2,"name":"zimeng Xu","email":"","orcid":"","institution":"Anhui Medical University","correspondingAuthor":false,"prefix":"","firstName":"zimeng","middleName":"","lastName":"Xu","suffix":""},{"id":639305708,"identity":"1b99d942-301a-404f-bb82-b8161a96f726","order_by":3,"name":"Chongkang Wu","email":"","orcid":"","institution":"Anhui Medical University","correspondingAuthor":false,"prefix":"","firstName":"Chongkang","middleName":"","lastName":"Wu","suffix":""},{"id":639305710,"identity":"71d5f182-63da-4ce5-bd7f-00e700a96027","order_by":4,"name":"Yajing Liu","email":"","orcid":"","institution":"Anhui Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yajing","middleName":"","lastName":"Liu","suffix":""},{"id":639305711,"identity":"8bc8ab48-0b96-42e9-a628-8e470226c556","order_by":5,"name":"Xin Chen","email":"","orcid":"","institution":"Second Hospital of Anhui Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xin","middleName":"","lastName":"Chen","suffix":""},{"id":639305712,"identity":"43fe5393-e5b6-4e57-bc0b-59909923b89d","order_by":6,"name":"Yuanhua Chen","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7UlEQVRIiWNgGAWjYJACZhBhwMDA+ICxgQHKJlILswHJWtgkiNJicCP52ePCNjt5c/beYxU/d2xLbGBv3ibBUHMHj5Y0c+OZbcmGO3vOpd3sPXM7sYHnWJkEw7FneLQkmEnztjEnGNzIMbvB2wbUIpFjBnThYTxa0r8BtdQnGNx/Y1b4F6RF/g0hLTkgWw4DbeExY4bYwoNfi+SZN2XSPOeOG244k2MsLdt227iNJ63YIuEYbi18x9O3SfOUVcsbHD9j+PFt223ZfvbDG298qMGtReEAuggbiEjAqYGBQb4Bj+QoGAWjYBSMAjAAAM6zV09FO2JnAAAAAElFTkSuQmCC","orcid":"","institution":"Anhui Medical University","correspondingAuthor":true,"prefix":"","firstName":"Yuanhua","middleName":"","lastName":"Chen","suffix":""}],"badges":[],"createdAt":"2026-04-28 12:08:27","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9553901/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9553901/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":109170260,"identity":"f2ea4cc6-bef8-472c-a0d1-9c24112e51be","added_by":"auto","created_at":"2026-05-13 08:46:50","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":692603,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAge-related changes in mouse testicular tissues and H₂O₂-induced senescence in TM3 cells. \u0026nbsp;\u003c/strong\u003e(A-J)\u003cstrong\u003e \u003c/strong\u003eTwo-month-old and 18-month-old male mice were divided into young (group Y) and old (group O) groups. For SA-β-gal and immunofluorescence staining, Cryosections were prepared from testicular tissue samples. Leydig cells were isolated from the other testicular tissue and used for Western Blot analysis. α-Tubulin was used as a loading control for western blots. (A) Representative images showing senescence (SA-β-Gal staining). (B) Quantitative analysis of SA-β-Gal staining per field. (C) Lamin B and P21 proteins in the testicular tissues were measured using Western blot. Representative images were shown. (D) Quantitative analysis of the protein expression levels shown in (C). (E) SIRT1 and AMPKα/p-AMPKα proteins in the testicular tissues were measured using Western blot. Representative images were shown. (F) Quantitative analysis of the protein expression levels shown in (E). (G) Protein acetylation levels in the testicular tissues were measured. Representative images were shown. (H) SIRT1 and protein acetylation were measured using immunofluorescence staining. Representative images were shown. (J) Quantitative analysis of the fluorescence intensity shown in (H). (K-L) TM3 cells were treated with different concentrations of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. (K) Representative images showing senescence (SA-β-Gal staining). (L) Lamin B and P21 proteins were measured using Western blot. Representative images were shown. Data are presented as mean ± SEM (n = 6). **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"image1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9553901/v1/376f0aeb121b056416d2933e.jpeg"},{"id":109170293,"identity":"75cce5e4-618e-4c77-a868-1a6ac30f67f4","added_by":"auto","created_at":"2026-05-13 08:47:06","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":353997,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAge-related decline in testosterone synthesis by Leydig cells. \u003c/strong\u003eTwo-month-old and 18-month-old male mice were divided into young (group Y) and old (group O) groups. Cryosections were prepared from testicular tissue samples. Leydig cells were isolated from the other testicular tissue and used for Western Blot analysis. α-Tubulin was used as a loading control for western blots. (A) Plasma testosterone levels measured by ELISA in young (Y) and old (O) mice. (B) P450scc, 3β-HSD, and StAR proteins in the testicular tissues were measured using Western blot. Representative images were shown. (C) Quantitative analysis of the protein expression levels shown in (B). (D) Representative immunofluorescence staining of 3β-HSD (green) in testicular sections from Y and O mice. Nuclei were counterstained with DAPI (blue). Scale bar: 50 µm. (E) Quantitative analysis of the fluorescence intensity shown in (D). (F) Representative images of P450scc, 3β-HSD, and StAR proteins in Leydig cells from Y and O mice after stimulation with hCG. (G) Representative images of P450scc, 3β-HSD, and StAR proteins in Leydig cells from Y and O mice after stimulation with db-cAMP. Data are presented as mean ± SEM (n = 6). **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"image2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9553901/v1/e3dbfa47e479a10645f6beac.jpeg"},{"id":109170272,"identity":"ad1aca7e-c349-44c6-928a-675a3edaf6f0","added_by":"auto","created_at":"2026-05-13 08:46:56","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":615130,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImbalanced mitochondrial dynamics in Leydig cells from aged mice. \u003c/strong\u003eTwo-month-old and 18-month-old male mice were divided into young (group Y) and old (group O) groups. Cryosections were prepared from testicular tissue samples. Leydig cells were isolated from the other testicular tissue and used for Western Blot analysis. α-Tubulin was used as a loading control for western blots. (A, B) Representative immunofluorescence staining of MFN2 and MFN1 (red) in testicular sections from Y and O mice. Nuclei were counterstained with DAPI (blue). Scale bar: 50 µm. (C) MFN2, MFN1, p-DRP1 (Ser637), p-DRP1 (Ser616), and Fis1 proteins in the testicular tissues were measured using Western blot. Representative images were shown. (D) Quantitative analysis of the protein expression levels shown in (C). (E) Representative images of MFN2, MFN1, p-DRP1 (Ser637), p-DRP1 (Ser616), and Fis1 proteins in Leydig cells from Y and O mice after stimulation with hCG. (F) Representative images of MFN2, MFN1, p-DRP1 (Ser637), p-DRP1 (Ser616), and Fis1 proteins in Leydig cells from Y and O mice after stimulation with db-cAMP. Data are presented as mean ± SEM (n = 6). *\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05; **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"image3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9553901/v1/bbf84cbfe6e45594b1da02d0.jpeg"},{"id":109170254,"identity":"7ff8cd64-6f20-47c1-8bfe-b8dc3d633d64","added_by":"auto","created_at":"2026-05-13 08:46:49","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":764311,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMitochondrial dynamics imbalance resulting from MFN2 downregulation contributes to the aging process. \u003c/strong\u003eTM3 cells were treated with or without H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2 \u003c/sub\u003e(400 μM). siRNA targeting MFN2 (siMFN2) was used to knockdown MFN2. Mitochondrial fusion promoter M1 was used to induce MFN2. (A) Mitochondrial network morphology was labeled with MitoTracker Green probes. Representative photomicrographs were shown. (B) Mitochondrial morphology was scored by counting individual cell that has either elongated (\u0026gt;50% tubular) or fragmented (\u0026gt;50% fragmented) mitochondria. (C-D) MFN2, MFN1, p-DRP1 (Ser637), p-DRP1 (Ser616), and Fis1 proteins in response to M1 treatment and MFN2 knockdown were measured using Western blot. Representative images were shown. (E) Representative images showing senescence (SA-β-Gal staining). (F) Quantitative analysis of SA-β-Gal staining per field. All data were expressed as means ± SEM. **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01 as compare with the Ctrl group; ##\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01 as compare with the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2 \u003c/sub\u003egroup.\u003c/p\u003e","description":"","filename":"image4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9553901/v1/a03da184c694ab9ac91bff1c.jpeg"},{"id":109170297,"identity":"4e42c15e-3904-495a-93fe-2e5468018d21","added_by":"auto","created_at":"2026-05-13 08:47:07","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":586920,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMFN2 plays a role in counteracting oxidative stress during aging. \u003c/strong\u003e(A-F)\u003cstrong\u003e \u003c/strong\u003eTwo-month-old and 18-month-old male mice were divided into young (group Y) and old (group O) groups. Cryosections were prepared from testicular tissue samples. Leydig cells were isolated from the other testicular tissue and used for Western Blot analysis. α-Tubulin was used as a loading control for western blots.\u003cstrong\u003e (A)\u003c/strong\u003e\u0026nbsp;Representative images of ROS staining (Green) in testicular sections from young (Y) and old (O) mice. Nuclei were counterstained with DAPI (Blue). Scale bar: 50 µm.\u003cstrong\u003e (B)\u003c/strong\u003e\u0026nbsp;Quantitative analysis of ROS intensity normalized to DAPI from (A).\u003cstrong\u003e (C)\u003c/strong\u003e\u0026nbsp;Representative images of antioxidant enzymes GPX4, GPX5, and SOD2 in Leydig cells from Y and O mice. \u003cstrong\u003e(D)\u003c/strong\u003e\u0026nbsp;Quantitative analysis of the protein expression levels shown in (C).\u003cstrong\u003e (E)\u003c/strong\u003e\u0026nbsp;Representative Western blot images of antioxidant enzymes GPX4, GPX5, and SOD2 proteins in Leydig cells from Y and O mice after stimulation with hCG.\u003cstrong\u003e (F)\u003c/strong\u003e\u0026nbsp;Representative images of antioxidant enzymes GPX4, GPX5, and SOD2 proteins in Leydig cells from Y and O mice after stimulation with db-cAMP. (G-K) TM3 cells were treated with or without H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (400 μM). siRNA targeting MFN2 (siMFN2) was used to knock down MFN2. Mitochondrial fusion promoter M1 was used to induce MFN2. α-Tubulin was used as a loading control for western blots. (G) Representative images of intracellular ROS (Green) staining and mitochondrial ROS (mt-SOX Red) staining in TM3 cells under different treatments.\u003cstrong\u003e (H)\u003c/strong\u003e\u0026nbsp;Quantitative analysis of ROS intensity and mt-SOX Red intensity per cell from (G). \u003cstrong\u003e(I-J)\u003c/strong\u003e\u0026nbsp;Representative images of antioxidant enzymes GPX4, GPX5, and SOD2 proteins in TM3 cells under different treatments. Data are presented as mean ± SEM (n = 6). **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01 as compare with the Ctrl group; ##\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01 as compare with the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2 \u003c/sub\u003egroup.\u003c/p\u003e","description":"","filename":"image5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9553901/v1/01d621a09ff25f485688c135.jpeg"},{"id":109170341,"identity":"8910004e-d49e-4f49-9736-a16d1b8627d0","added_by":"auto","created_at":"2026-05-13 08:47:10","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":699559,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMFN2 counteracts age-related mitophagy decline.\u003c/strong\u003e (A-E) Two-month-old and 18-month-old male mice were divided into young (group Y) and old (group O) groups. Leydig cells were isolated from the testicular tissue and used for Western Blot analysis. GAPDH was used as a loading control for western blots. (A) Representative images of mitophagy-related proteins (PINK1, Parkin, NIX) and mitochondrial function-related proteins (mt-TFA, COX4) in Leydig cells from young (Y) and old (O) groups. (B) Quantitative analysis of the protein expression levels shown in (A). (C) Representative images of PINK1, Parkin, NIX, mt-TFA, and COX4 proteins in Leydig cells from Y and O groups following testosterone synthesis stimulation. (D) Quantitative analysis of the protein expression levels shown in (C). (E) ATP levels in Leydig cells from Y and O groups, with or without hCG or db-cAMP stimulation. (F-M) TM3 cells were treated with or without H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (400 μM). siRNA targeting MFN2 (siMFN2) was used to knock down MFN2. Mitochondrial fusion promoter M1 was used to induce MFN2. GAPDH was used as a loading control for western blots. (F) Representative images of PINK1, Parkin, NIX, mt-TFA, and COX4 proteins in TM3 cells. (G) Quantitative analysis of the protein expression levels shown in (F). (H) Representative images of mitophagy staining in TM3 cells. Red puncta indicate mitophagosomes, green puncta indicate lysosomes, and yellow puncta in the merged images indicate mitophagic vesicles. Scale bar: H, 10 μm; L, 50 μm. (I) Quantitative analysis of relative yellow puncta (mitophagic vesicles) per cell from (H). (J) Quantitative analysis of relative red puncta (mitophagosomes) per cell from (H). (K) ATP levels in TM3 cells among different groups. (L) Representative images of MMP detection in TM3 cells. Scale bar: H, 10 μm; L, 50 μm. (M) Quantitative analysis of MMP shown in (L). GAPDH was used as a loading control for Western blot analyses. Data are presented as mean ± SEM (n = 6). **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01 as compare with the Ctrl group; ##\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01 as compare with the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2 \u003c/sub\u003egroup.\u003c/p\u003e","description":"","filename":"image6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9553901/v1/1876de1ea0bce87cf2e7f8aa.jpeg"},{"id":109205216,"identity":"5dfd98f8-4494-4a99-95a0-08ba8478a986","added_by":"auto","created_at":"2026-05-13 15:03:47","extension":"jpeg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":722849,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eM1-induced mitochondrial fusion protects mitochondrial function and testosterone synthesis independently of SIRT1. \u003c/strong\u003eTM3 cells were treated with or without H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (400 μM). siRNA targeting SIRT2 (siSIRT) was used to knock down SIRT. Mitochondrial fusion promoter M1 was used to induce MFN2. α-Tubulin was used as a loading control for western blots. (A) SIRT1 and AMPKα/p-AMPKα proteins were measured using Western blot. Representative images were shown. (B) P450scc, 3β-HSD, and StAR proteins were measured using Western blot. Representative images were shown. (C) MFN2, MFN1, p-DRP1 (Ser637), p-DRP1 (Ser616), and Fis1 proteins were measured using Western blot. Representative images were shown. (D) Representative images (from up to down) showing senescence (SA-β-Gal staining), intracellular ROS levels (detected by a DCFH-DA probe), mitochondrial ROS levels (the pH-insensitive indicator MitoSOX Red), and mitochondrial membrane potential (JC-1 staining). (E) Quantitative analysis of SA-β-Gal staining per field. (F) Quantitative analysis of intracellular ROS per field. (G) ATP contents were measured. All data were expressed as means ± SEM. **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"image7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9553901/v1/9244e89c92be7eeb48fba166.jpeg"},{"id":109170259,"identity":"c9788940-59ad-4371-b5d7-38d83e9f53ce","added_by":"auto","created_at":"2026-05-13 08:46:50","extension":"jpeg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":974297,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMFN2 is required for the SIRT1-mediated anti-aging effects. \u003c/strong\u003eTM3 cells were treated with or without H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (400 μM). siRNA targeting MFN2 (siMFN2) was used to knock down MFN2. Nicotinamide riboside chloride (NRC), a precursor of NAD+, was used to activate SIRT1. α-Tubulin was used as a loading control for western blots. (A) SIRT1 and AMPKα/p-AMPKα proteins were measured using Western blot. Representative images were shown. (B) P450scc, 3β-HSD, and StAR proteins were measured using Western blot. Representative images were shown. (C) MFN2, MFN1, p-DRP1 (Ser637), p-DRP1 (Ser616), and Fis1 proteins were measured using Western blot. Representative images were shown. (D) Representative images (from up to down) showing mitochondrial morphology (stained with MitoTracker Red), intracellular ROS levels (detected by a DCFH-DA probe), mitochondrial ROS levels (the pH-insensitive indicator MitoSOX Red), Mitochondrial membrane potential (JC-1 staining), and senescence (SA-β-Gal staining). (E) Quantitative analysis of intracellular ROS per field. (F) Quantitative analysis of SA-β-Gal staining per field. (G) ATP contents were measured. All data were expressed as means ± SEM. **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"image8.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9553901/v1/e96bdb2b04cfe633d0cff08b.jpeg"},{"id":109207910,"identity":"a60bada0-3c55-421f-a78f-3e98161d8b19","added_by":"auto","created_at":"2026-05-13 15:22:25","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5660925,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9553901/v1/dfbccb3d-551c-45a0-8ee3-b38b4e4caa4d.pdf"},{"id":109170257,"identity":"2dc83f25-5a90-4353-9820-d6c261071623","added_by":"auto","created_at":"2026-05-13 08:46:50","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":4241120,"visible":true,"origin":"","legend":"","description":"","filename":"SupportingInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-9553901/v1/b6d1f409d5939ce986314e40.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"SIRT1/MFN2-Mediated Regulation of Mitochondrial Dynamics and Mitophagy in Age-Related Decline of Leydig Cell Function","fulltext":[{"header":"Introduction","content":"\u003cp\u003eLate-onset hypogonadism (LOH) is a prevalent clinical syndrome in aging males, characterized by a significant decline in serum testosterone levels and associated with a spectrum of symptoms including diminished libido, fatigue, reduced muscle mass, mood disturbances, and metabolic alterations (Huhtaniemi and Forti \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). As the global population ages, the incidence of LOH continues to rise, posing a substantial challenge to the quality of life and overall health of middle-aged and elderly men (Zhou et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Testosterone is primarily synthesized and secreted by testicular Leydig cells. The age-related decline in testosterone levels is fundamentally attributed to the functional degeneration of Leydig cells, the decrease in the number of Leydig cells, along with a reduction in the number of intracellular organelles (Mularoni et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Santiago et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). However, the precise molecular mechanisms driving this functional decline during aging remain incompletely elucidated, thereby limiting the development of targeted and effective therapeutic strategies.\u003c/p\u003e \u003cp\u003eAmong the various mechanisms implicated in cellular senescence, mitochondrial dysfunction has emerged as a central player. Mitochondria are not only the cellular \"powerhouses,\" responsible for generating ATP, but are also crucial sites for steroid hormone biosynthesis (Selvaraj and Stocco. 2015). Mitochondria are highly dynamic organelles that maintain their network homeostasis, function, and quality control through continuous fusion and fission processes. The damaged and dysfunctional mitochondria can be removed by mitophagy (Harrington et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Therefore, the balance of mitochondrial dynamics and mitophagy is critical for preserving mitochondrial health, including the normal synthesis of steroid hormones. Mitochondrial imbalance can lead to mitochondrial fragmentation, decreased membrane potential (MMP), reduced ATP production, and exacerbated oxidative stress (Chourasia et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Sun et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Accumulating evidence indicates that an imbalance in mitochondrial dynamics and disruption of mitophagy suppress steroid hormone biosynthesis in testicular Leydig cells (Garza et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Despite this, the specific role of imbalanced mitochondrial dynamics in the functional decline of aging Leydig cells and its upstream regulatory mechanisms require in-depth exploration.\u003c/p\u003e \u003cp\u003eAmong the key proteins regulating mitochondrial fusion, Mitofusin 2 (MFN2) stands out. MFN2 not only mediates the fusion of the mitochondrial outer membrane but is also involved in mitochondria-endoplasmic reticulum tethering and the initiation of mitophagy (Gatti et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Gordaliza-Alaguero et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Tur et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Downregulation of MFN2 has been linked to various pathological states, including muscle atrophy, vascular calcification, metabolic diseases, and reduced steroidogenesis (Cefis et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Wu et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Di Rienzo et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Moreover, MFN2 upregulation facilitates cholesterol transport, thereby stimulating testosterone synthesis (Duarte et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Lv et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). We therefore hypothesized that MFN2 could be a critical node connecting aging signals, mitochondrial homeostasis, and Leydig cell steroidogenic function.\u003c/p\u003e \u003cp\u003eSirtuin 1 (SIRT1), an NAD+-dependent deacetylase, is a key regulator of energy metabolism and stress responses, renowned for its anti-aging properties. The AMPK/SIRT1 signaling pathway plays a vital role in modulating energy homeostasis, oxidative stress, and cellular senescence (Salminen et al. 2012). Existing evidence suggests that SIRT1 can influence mitochondrial biogenesis and function (Behera et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Nevertheless, the role of SIRT1 in regulating mitochondrial homeostasis in Leydig cells within the context of LOH, and its functional interplay with key mitochondrial dynamics proteins, remains poorly defined.\u003c/p\u003e \u003cp\u003eThis study aims to elucidate the central role of MFN2-mediated mitochondrial dynamics in the pathogenesis of LOH and to clarify its relationship with the SIRT1 pathway. In this study, we employed an integrated approach utilizing natural aging mouse models and H₂O₂-induced senescent TM3 Leydig cells. First, we systematically assessed the impact of aging on Leydig cell senescence markers, AMPK/SIRT1 pathway activity, the expression of key steroidogenic enzymes, and mitochondrial dynamics balance. Subsequently, using genetic (siMFN2 knockdown) and pharmacological (M1, a mitochondrial fusion promoter) interventions to modulate MFN2 expression, we investigated the causal role of MFN2 in regulating mitochondrial function, oxidative stress, mitophagy, and testosterone synthesis. Finally, through combined SIRT1 knockdown and MFN2 knockdown experiments, we meticulously dissected the complex hierarchical relationship between MFN2 and the SIRT1 signaling pathway. Our findings are expected to deepen the understanding of the molecular mechanisms underlying age-related testosterone decline and to provide a solid theoretical and experimental foundation for the development of novel, MFN2-targeted therapeutic strategies for LOH, aimed at restoring mitochondrial function and steroidogenic capacity in aging Leydig cells.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAntibodies and reagents\u003c/h2\u003e \u003cp\u003eThe primary antibodies against AMPKα, p-AMPKα, LC3B, ATG7, ATG5, p-DRP1(Ser637), p-DRP1(Ser616), and COX4 were obtained from Cell Signaling Technology (Massachusetts, USA), and Lamin B, GAPDH, LAMP2, MFN2, MFN1, P62, FIS1, PINK1, PARK2, GPX5, Acsl4, ATGL and ADRP from Proteintech (Wuhan, China), NIX and α-Tubulin from Bioworld (Minnesota, USA). SIRT1, Acetyl-Lysine, and mt-TFA from Abcam (Cambridge, England). GPX4 and SOD2 from Boster Biological Technology (Wuhan, China). LAMP1 and RAB7 from Bioswamp (Wuhan, China). HSL from Affinity (Jiangsu, China). P21 from Beyotime (Shanghai, China). The secondary antibodies goat anti-rabbit IgG (H\u0026thinsp;+\u0026thinsp;L) HRP-conjugated and goat anti-mouse IgG (H\u0026thinsp;+\u0026thinsp;L) HRP-conjugated were obtained from Elabscience (Wuhan, China). Alexa Fluor 488 donkey anti-mouse IgG (H\u0026thinsp;+\u0026thinsp;L) and Alexa Fluor 594 donkey anti-rabbit IgG (H\u0026thinsp;+\u0026thinsp;L) from Invitrogen (Carlsbad, CA, USA). The Senescence β-Galactosidase Staining Kit (C0602), Testosterone ELISA Kit (PT872), ATP Assay Kit (S0026), Reactive Oxygen Assay (S0033S), 4\u0026prime;,6-diamidino2-phenylindole (DAPI) (C1006) and bicinchoninic acid (BCA) protein assay kit (P0012) were purchased from Beyotime Biotechnology (Shanghai, China), MitoTracker\u0026trade; Green FM (M7514) and MitoSOX\u0026trade; Red Mitochondrial Superoxide Indicator (M36008) from Invitrogen (Carlsbad, CA, USA), Mitophagy Detection Kit (MD01) from Dojindo (Kumamaoto, Japan).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eAnimals and treatments\u003c/h3\u003e\n\u003cp\u003eTwo-month-old and 18-month-old male C57BL/6J mice were assigned to a young group (Y, n\u0026thinsp;=\u0026thinsp;6) and an old group (O, n\u0026thinsp;=\u0026thinsp;6), respectively. Testicular tissues from one side were collected and processed for frozen sectioning or Western blot analysis. The contralateral testes were used for Leydig cell isolation, followed by steroidogenesis stimulation experiments. The mouse serum was collected to assess testosterone levels using a commercial ELISA kit. All animal experimental procedures were in accordance with the animal welfare management regulations formulated by the Experimental Animal Ethics Committee of Anhui Medical University.\u003c/p\u003e\n\u003ch3\u003eIsolation of mouse primary Leydig cells\u003c/h3\u003e\n\u003cp\u003eBriefly, following the protocols described in the previous studies (Lv et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), we isolated Leydig cells by first decapsulating the mouse testes and digesting them in 0.05% collagenase type IV under constant agitation. Digestion continued until the tissue was adequately dispersed while minimizing disruption of the seminiferous tubules. The suspension was then filtered through a 100-mesh sieve, and the collected filtrate underwent washing with M199 medium. Following Percoll density gradient centrifugation, the purified Leydig cells showed about 81% purity, which was confirmed through 3β-HSD histochemical staining. Then the isolated Leydig cells were stimulated in M199 medium with 10 ng/L human chorionic gonadotropin (hCG), or with 1mM double cyclic adrenosine monophosphate (db-cAMP) for 3h.\u003c/p\u003e\n\u003ch3\u003eCell culture and intervention\u003c/h3\u003e\n\u003cp\u003eThe TM3 mouse Leydig cell line was obtained from the cell bank of the Chinese Academy of Sciences (ATCC) and maintained in DMEM/F12 medium containing 10% fetal bovine serum (FBS). To assess their effects on senescence, TM3 cells were first pretreated with either 20 \u0026micro;M M1 agonist (4 h) or MFN2 siRNA (24 h) and then subjected to senescence induction by 400 \u0026micro;M H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e treatment for 2 h. To examine whether the role of MFN2 in senescence depends on SIRT1 levels, SIRT1 was knocked down using siRNA in TM3 cells, after which the cells were treated with 20 \u0026micro;M M1 agonist and then exposed to H₂O₂ to induce senescence. To determine whether MFN2 participates in the regulation of senescence by SIRT1, MFN2 was knocked down in TM3 cells using siRNA. These cells were then treated with 1 mM nicotinamide riboside chloride (NRC) (12 h) activate SIRT1 or subjected to SIRT1 siRNA knockdown (24 h), followed by senescence induction with 400 \u0026micro;M H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e for 2 h.\u003c/p\u003e\n\u003ch3\u003eSmall inhibitory RNA (siRNA) knockdown\u003c/h3\u003e\n\u003cp\u003eSirt1 siRNA and mfn2 siRNA were purchased from GenePharma (Shanghai, China). The sirt1 siRNA sequences were 5'-GCA CUA AUU CCA AGU UCU ATT-3' (sense) and 5'-UAG AAC UUG GAA UUA GUG CTT-3' (antisense). The MFN2 siRNA sequences were 5'-CUG CCA UGA ACA ACA AGA AAG UTT-3' (sense) and 5'-ACU UUC UUG UUC AUG GCA GTT-3' (antisense). The experiments were performed according to the manufacturer\u0026rsquo;s instructions. Briefly, liposomal Lipo3000 was mixed with Opti-MEM, then specific siRNA or the scramble siRNA was mixed with Opti-MEM and the concentration of siRNA was adjusted to 1 \u0026micro;M. Then the mixture was added to a petri dish and incubated for 24 hours to knockdown mfn2 or sirt1 respectively in TM3 cells.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eWestern blot analysis\u003c/h2\u003e \u003cp\u003eThe testicular tissues or cell pellets were homogenized in RIPA lysis buffer on ice. Following centrifugation at 12,000 \u0026times;g for 15 min at 4\u0026deg;C, the supernatant was collected, and its protein concentration was quantified with a BCA assay. Equal amounts of protein were separated by SDS-PAGE using 8\u0026ndash;12% gradient gels and then transferred onto PVDF membranes. After blocking with 5% non-fat dry milk in TBST for 2 h at room temperature, the membranes were incubated with primary antibodies overnight at 4\u0026deg;C. Next, the membranes were incubated with corresponding horseradish peroxidase (HRP)-conjugated secondary antibodies for 2 h at room temperature. Finally, the immunoreactive bands were detected using a Tanon-5200 chemiluminescence imaging system.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eImmunofluorescence staining\u003c/h3\u003e\n\u003cp\u003eFrozen sections of mouse testicular tissue were thawed for 30 min, washed with PBS, and permeabilized with 1% Triton X-100 in PBS for 30 min. Antigen retrieval was performed using sodium citrate buffer (pH 6.0). The sections were then blocked with 5% BSA in PBS for 30 min at room temperature. After blocking, sections were incubated with specific primary antibodies overnight at 4\u0026deg;C, followed by incubation with fluorescent secondary antibodies for 1 h at room temperature. Following three PBS washes, nuclei were counterstained with DAPI for 10 min. Finally, the sections were mounted and imaged using a Leica THUNDER Imager (Germany).\u003c/p\u003e\n\u003ch3\u003eDetection of reactive oxygen species (ROS)\u003c/h3\u003e\n\u003cp\u003eThe levels of intracellular ROS production were analyzed by the fluorescent dye dichlorodihydrofluorescein dihydrate (DCFH-DA) using a commercial assay kit according to the manufacturer\u0026rsquo;s instructions. For tissue staining, frozen testicular sections were incubated with ROS staining working solution at 37\u0026deg;C for 30 min in the dark. For cell staining, cells were incubated within a 5% CO\u003csub\u003e2\u003c/sub\u003e incubator. After incubation, all samples were washed and tissue sections were then mounted with an antifade medium containing DAPI. Images of both sections and cells were acquired immediately using a Leica THUNDER Imager (Germany).\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eDetection of ATP content\u003c/h2\u003e \u003cp\u003eATP assay kit was used to measure ATP content in the sample, according to the manufacturer's instructions. Briefly, Leydig cells (LCs) and TM3 cells were homogenized and centrifuged. For the ATP measurement, 20 \u0026micro;L of the supernatant was rapidly mixed with 100 \u0026micro;L of the ATP Assay Working Solution. A standard curve was generated using known concentrations of ATP, and the protein concentration in each treatment group was determined using a BCA Protein Assay Kit. Finally, the total ATP levels were calculated and expressed as micromoles per nanogram of protein (\u0026micro;mol/ng protein).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eSenescence-associated β-galactosidase (SA-β-Gal) staining\u003c/h2\u003e \u003cp\u003eCellular senescence was evaluated by SA-β-Gal staining according to the manufacturer\u0026rsquo;s instructions. For tissue samples, frozen testicular sections were incubated overnight at 37\u0026deg;C with SA-β-Gal staining solution, followed by hematoxylin nuclear counterstaining and mounting with antifade medium. For cultured cells, after fixation, the SA-β-Gal working solution was added to the samples and incubated overnight at 37\u0026deg;C in the dark. Images were acquired using a Leica THUNDER Imager (Germany).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eMitoTracker\u0026trade; Green FM staining\u003c/h2\u003e \u003cp\u003eTM3 Cells were incubated with 100 nM MitoTracker\u0026trade; Green FM (Invitrogen, USA) diluted in serum-free DMEM/F12 medium at 37\u0026deg;C for 30 minutes in the dark. After staining, the solution was replaced with prewarmed fresh medium. Mitochondrial morphology was subsequently examined and imaged using a laser scanning confocal microscope (Leica TCS SP5, Germany).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eMitochondrial Membrane Potential Detection\u003c/h2\u003e \u003cp\u003eTo evaluate mitochondrial membrane potential, TM3 cells were incubated with 10 \u0026micro;M JC-1 staining solution at 37\u0026deg;C for 20 minutes, protected from light. The cells were then washed twice with JC-1 staining buffer (1X) and prewarmed PBS. Imaging was performed using an inverted fluorescence microscope (Nikon Ts2, Japan). .\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eMitochondrial Superoxide Assay\u003c/h2\u003e \u003cp\u003eTo detect mitochondrial superoxide production, cells were incubated with 5 \u0026micro;M MitoSOX\u0026trade; Red mitochondrial superoxide indicator (Invitrogen, USA) in Hanks' balanced salt solution (HBSS) for 10 minutes at 37\u0026deg;C in the dark. After incubation, cells were gently washed three times with PBS and maintained in pre-warmed HBSS for immediate observation under an inverted fluorescence microscope (Nikon Ts2, Japan).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eMitophagy Assay\u003c/h2\u003e \u003cp\u003eMitophagy was assessed using the Mitophagy Detection Kit (MD01, Dojindo, Kumamoto, Japan). Cells were sequentially stained with a 100 nmol/L Mitophagy Dye and a 1 \u0026micro;mol/L Lyso Dye according to the manufacturer\u0026rsquo;s protocol. Briefly, after incubation with the Mitophagy Dye working solution at 37\u0026deg;C for 30 min in the dark, the dye was removed and replaced with fresh complete medium. Cells were then cultured for an additional 24 h to allow mitophagic flux to proceed. Subsequently, the Lyso Dye working solution was applied to label acidic compartments. Imaging was performed using a Leica THUNDER Imager(Germany), and fluorescence puncta were quantified with ImageJ software. Puncta per cell were determined based on at least 50 cells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eWestern blot band gray values and fluorescence intensities were measured using ImageJ software. Data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. Statistical analyses were performed using GraphPad Prism software (version 8.0.2). Group comparisons were conducted using Student\u0026rsquo;s t-test or one-way ANOVA, followed by Bonferroni post hoc test where applicable. A \u003cem\u003eP\u003c/em\u003e value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eAge-related changes in mouse testicular tissues and H₂O₂-induced senescence in TM3 cells\u003c/h2\u003e \u003cp\u003eTo observe age-related changes in testicular tissues, SA-β-gal staining was performed on frozen sections from 2-month-old (Y group) and 18-month-old (O group) mice. The results revealed prominent SA-β-gal-positive signals in the cytoplasm of Leydig cells in the O group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, B). Western blot analysis was used to assess the protein levels of aging markers Lamin B and P21 in Leydig cells from both aged and young mice. Compared with the Y group, the O group exhibited a significant decrease in Lamin B protein expression and a marked increase in p21 protein levels in Leydig cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC-D and S1A). Moreover, the protein expressions of the AMPK/SIRT1 anti-aging pathway were analyzed in Leydig cells from both aged and young mice. Our data showed that the O group had generally downregulated expression of SIRT1, AMPKα, and p-AMPKα (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE-F and S1B), along with elevated protein acetylation levels compared to the Y group (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG and S1C). In addition, immunofluorescence staining results also demonstrated that compared to the Y group, the SIRT1 fluorescence intensity was significantly reduced, while the protein acetylation level was markedly elevated in Leydig cells in the O group (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH-J). Furthermore, we treated TM3 cells with different H₂O₂ concentrations to model cellular senescence. We found that a concentration of 400 \u0026micro;M H₂O₂ produced the most potent senescence-inducing effect (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eK-L and S1D). Therefore, this concentration was selected for subsequent experiments to induce senescence in TM3 cells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eAge-related decline in testosterone synthesis by Leydig cells\u003c/h2\u003e \u003cp\u003eTo investigate whether aging leads to a decline in testosterone synthesis in mice, serum testosterone levels were measured using ELISA. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, serum testosterone levels in the O group were significantly lower than those in the Y group. Moreover, the protein levels of P450scc, 3β-HSD, and StAR, three key enzymes of testosterone synthesis, were significantly downregulated in Leydig cells from O group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB-C and S2A). Immunofluorescence staining revealed a significant decrease in the fluorescence intensity of 3β-HSD, which is localized to Leydig cells, in the O group compared to the Y group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD, E). Furthermore, hCG) and db-cAMP were used to stimulate endogenous testosterone production. As expected, hCG and db-cAMP treatment significantly upregulated the expression of testosterone synthesis-related enzymes in Leydig cells from both aged and young mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF-G and S2B-C). However, even after stimulation, the expression levels of testosterone synthesis-related enzymes in the stimulated O group remained lower than those in the stimulated Y group (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF-G and S2B-C).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eImbalanced mitochondrial dynamics in Leydig cells from aged mice\u003c/h2\u003e \u003cp\u003eTo investigate the mechanism underlying impaired testosterone synthesis in aging Leydig cells, mitochondrial dynamics were assessed. The results showed that the mitochondrial fusion regulatory proteins MFN2 and MFN1, as well as the mitochondrial fission protein FIS1, were significantly downregulated in Leydig cells from aged mice (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-D and S3A). Dynamin-related protein 1 (DRP1) promotes mitochondrial fission when phosphorylated at Ser616, but conversely inhibits it when phosphorylated at Ser637. Accordingly, we measured its phosphorylation levels at these two sites. The level of p-DRP1 (Ser637) was significantly downregulated in Leydig cells from aged mice. Conversely, the level of p-DRP1 (Ser616) was significantly upregulated (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC-D and S3A). Furthermore, hCG and db-cAMP were used to stimulate endogenous testosterone production. Following hCG and db-cAMP treatment, we observed a significant upregulation in MFN2, MFN1, FIS1, and p-DRP1 (Ser637) expression, alongside a downregulation of p-DRP1 (Ser616) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE-F and S3B-C). However, even after stimulation, the protein levels of MFN2, MFN1, FIS1, and p-DRP1 (Ser637) in the stimulated O group remained lower than those in the stimulated Y group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE-F and S3B-C). In addition, after stimulation, the protein level of p-DRP1 (Ser616) in the stimulated O group remained higher than those in the stimulated Y group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE-F and S3B-C).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eMitochondrial dynamics imbalance resulting from MFN2 downregulation contributes to the aging process\u003c/h2\u003e \u003cp\u003eTo further verify whether mitochondrial dynamics imbalance is involved in the aging process, TM3 cells were cultured \u003cem\u003ein vitro\u003c/em\u003e and treated with the mitochondrial fusion promoter M1 or transfected with MFN2-targeting short interfering RNA (siMFN2) to knock down MFN2. Mitochondrial morphology was labeled using the MitoTracker Green probe. We found that compared with the control group, TM3 cells in the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced senescence group exhibited a transformation of mitochondria into numerous small fragments (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, B). Interestingly, M1 treatment rescued the H₂O₂-induced mitochondrial fragmentation in TM3 cells, whereas MFN2 knockdown exacerbated it (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, B). Consistent with the mitochondrial morphological findings, TM3 cells in the H₂O₂-induced senescence group exhibited significant downregulation of the mitochondrial fusion regulators MFN2, MFN1, and p-DRP1 (Ser637), along with upregulation of the fission regulator p-DRP1 (Ser616), compared to the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, D, and S4). M1 treatment rescued the H₂O₂-induced downregulation of MFN1, MFN2, and p-DRP1 (Ser637) proteins and upregulation of p-DRP1 (Ser616) protein in TM3 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC-D and S4). In addition, MFN2 knockdown exacerbated the H₂O₂-induced downregulation of MFN1, MFN2, and p-DRP1 (Ser637) protein, as well as the upregulation of p-DRP1 (Ser616) protein in TM3 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC-D and S4). Furthermore, SA-β-gal staining was used to assess the senescence level of TM3 cells. The results revealed a large number of SA-β-gal-positive cells in the H₂O₂-induced senescence group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE, F). M1 treatment markedly attenuated the H₂O₂-induced senescence in TM3 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE, F). In contrast, MFN2 knockdown exacerbated the H₂O₂-induced senescence in TM3 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE, F).\u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eMFN2 plays a role in counteracting oxidative stress during aging\u003c/h2\u003e \u003cp\u003eOxidative stress is an important pathophysiological mechanism in aging-related diseases. DCFH-DA was used to mark the intracellular reactive oxygen species (ROS). The results showed that the ROS level in the testicular interstitium of the aged group was significantly higher than that in the young group (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, B). Western blot was further used to detect the expression of redox enzymes in Leydig cells from aged and young mice. As compare with the Y group, the proteins glutathione peroxidase 4 (Gpx4), GPX5, and superoxide dismutase 2 (SOD2) were significantly downregulated in the O group (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC-D and S5A). Moreover, after hCG and db-cAMP stimulation, the protein levels of Gpx4, GPX5, SOD2, and p-DRP1 (Ser637) in the stimulated O group remained lower than those in the stimulated Y group (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE-F and S5B-C). Furthermore, we assessed the effects of M1 treatment and MFN2 knockdown on oxidative stress in the H₂O₂-induced senescence model using TM3 cells. M1 treatment reduced the H₂O₂-induced increases in both intracellular and mitochondrial ROS levels in TM3 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG, H). In contrast, MFN2 knockdown further heightened the ROS levels in both the cytosol and mitochondria induced by H₂O₂ in TM3 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG, H). In addition, M1 treatment attenuated the H₂O₂-induced downregulation of GPX4, GPX5 and SOD2 protein expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eI-J and S5D-E). Conversely, MFN2 knockdown further exacerbated this downregulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eI-J and S5D-E).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eMFN2 counteracts age-related mitophagy decline\u003c/h2\u003e \u003cp\u003eMitochondria are key sites for testosterone synthesis in Leydig cells. This study investigates the effect of mitophagy and mitochondrial function on testosterone synthesis in aging Leydig cells. The results showed that the expression levels of mitophagy-related proteins PINK1, Parkin, and NIX, as well as mitochondrial function-related proteins mitochondrial transcription factor A (mt-TFA) and cytochrome c oxidase 4 (COX4), were significantly lower in the O group compared to the Y group (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA-B and S6A). Stimulation of testosterone synthesis with hCG and db-cAMP significantly increased the expression of these proteins in the O group, but the levels of PINK1, Parkin, NIX, mt-TFA, and COX4 proteins remained lower than those in the stimulated Y group (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC-D and S6B-C). Further detection of ATP levels in Leydig cells revealed a significant decrease in ATP in the O group, while hCG or db-cAMP stimulation significantly increased ATP levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE). In TM3 cell experiments, the expression levels of PINK1, Parkin, NIX, mt-TFA, and COX4 were also significantly lower in the H₂O₂-induced senescence group compared to the control group (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF-G and S6D-E). M1 treatment alleviated the downregulation of these proteins in the H₂O₂-induced senescence group (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e-G and S6D-E). Additionally, compared to the H₂O₂-induced senescence group, the protein levels of PINK1, Parkin, NIX, mt-TFA, and COX4 were further downregulated in TM3 cells treated with H₂O₂ + siMFN2 (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF-G S6D-E). Mitophagy staining results were consistent with the downregulations of these proteins, as shown in Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eH-J, where mitophagy staining and colocalization of mitochondrial staining with lysosomal staining were reduced in the H₂O₂-induced senescence group. M1 treatment attenuated H₂O₂-induced mitophagy decline in TM3 cells (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eH-J). Conversely, MFN2 knockdown further exacerbated H₂O₂-induced mitophagy decline in TM3 cells (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eH-J). Moreover, ATP levels in the H₂O₂-induced senescence group were significantly lower than those in the control group in TM3 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eK). The H₂O₂-induced decrease in ATP levels was ameliorated by M1 and, conversely, exacerbated by MFN2 knockout in TM3 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eK). Further detection of mitochondrial membrane potential (MMP), an important indicator of normal mitochondrial function, showed that MMP was significantly reduced in the H₂O₂-induced senescence group (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eL, M). The H₂O₂-induced decrease in MMP levels was ameliorated by M1 and, conversely, exacerbated by MFN2 knockout in TM3 cells (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eL, M).\u003c/p\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003eM1-induced mitochondrial fusion protects mitochondrial function and testosterone synthesis independently of SIRT1\u003c/h2\u003e \u003cp\u003eTo investigate whether the protective effects of MFN2 on testosterone synthesis and mitochondrial function are related to SIRT1 levels, SIRT1 was knocked down in TM3 cells using SIRT1-targeted short interfering RNA (siSIRT1). As expected, the protein levels of SIRT1, P450scc, 3β-HSD, StAR, MFN1, MFN2, and p-DRP1 (Ser637) were downregulated, while p-DRP1 (Ser616) was upregulated in the H₂O₂-induced senescence group compared to the control group (Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA-C and S7). M1 treatment alleviated H₂O₂-induced downregulation of those proteins and the upregulation of p-DRP1 (Ser616) (Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB-C and S7B-C). Moreover, SIRT1 knockdown did not significantly affect the ability of M1 treatment to rescue the H₂O₂-induced downregulation of P450scc, 3β-HSD, StAR, MFN1, MFN2, p-DRP1 (Ser637) and upregulation of p-DRP1 (Ser616) (Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB-C and S7B-C). Furthermore, SIRT1 knockdown did not significantly affect the ability of M1 treatment to rescue the H₂O₂-induced increase the proportion of SA-β-gal positive cells and ROS levels, and decrease MMP and ATP levels (Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD-G). These results indicate that SIRT1 may be not required for M1 to counteract aging-induced mitochondrial dysfunction and the suppression of testosterone synthesis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e \u003ch2\u003eMFN2 is required for the SIRT1-mediated anti-aging effects\u003c/h2\u003e \u003cp\u003eSIRT1 possesses various anti-aging properties. To investigate whether the protective role of SIRT1 against aging-induced suppression of testosterone synthesis in Leydig cells requires the involvement of MFN2, MFN2 was knocked down in TM3 cells using siMFN2. Nicotinamide riboside chloride (NRC), a precursor of NAD+, was used to increase NAD levels in TM3 cells. As expected, NRC treatment significantly reversed the H₂O₂-mediated decrease in SIRT1, AMPKα, and p-AMPKα protein levels, indicating that NRC can activate SIRT1 signaling (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e and S8C). Of interest, NRC treatment significantly rescued the H₂O₂-mediated downregulation of P450scc, 3β-HSD, StAR, MFN1, MFN2, p-DRP1 (Ser637) protein, and the upregulation of p-DRP1 (Ser616) protein in TM3 cells (Figs.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB-C and S8B-C). However, MFN2 knockdown abolished the protective effects of NRC against H₂O₂-induced inhibition in testosterone synthesis and imbalance in mitochondrial dynamics (Figs.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB-C and S8B-C). Next, we measured mitochondrial morphology. We found that NRC treatment significantly rescued the H₂O₂-mediated mitochondrial fragmentation, increase of ROS levels, and decrease of MMP and ATP levels in TM3 cells (Figs.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eD-E, G). However, MFN2 knockdown abolished the protective effects of NRC against H₂O₂-induced mitochondrial dysfunction (Figs.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eD-F). Furthermore, NRC treatment significantly rescued the H₂O₂-mediated increase in the proportion of SA-β-gal positive cells in TM3 cells (Figs.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eD, F). However, MFN2 knockdown abolished the protective effects of NRC against H₂O₂-induced senescence (Figs.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eD, F).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe age-related decline in testosterone production, a hallmark of late-onset hypogonadism (LOH), poses a significant challenge to the health and well-being of aging males. While the central role of Leydig cell dysfunction in this process is recognized, the underlying molecular drivers have remained incompletely defined. In this study, we present a comprehensive investigation that positions an imbalance in mitochondrial dynamics, orchestrated by the downregulation of Mitofusin 2 (MFN2), as a pivotal mechanism in aging-related Leydig cell failure. Our data, derived from both in vivo aged mouse models and in vitro H₂O₂-induced senescent TM3 Leydig cells, delineate a clear pathogenic cascade beginning with aging-induced MFN2 downregulation, which leads to mitochondrial fragmentation, and in turn promotes oxidative stress and impaired mitophagy, ultimately resulting in mitochondrial dysfunction and suppressed steroidogenesis. Furthermore, we unravel a novel and hierarchically structured relationship between the nutrient-sensor SIRT1 and MFN2, revealing that MFN2 operates independently of SIRT1 to exert its protective effects, yet is absolutely required for SIRT1-mediated anti-aging benefits. This establishes MFN2 not merely as a participant but as a critical executioner of mitochondrial and cellular homeostasis in aging Leydig cells.\u003c/p\u003e \u003cp\u003eOur initial findings confirm and extend the established hallmarks of aging in Leydig cells. The increased SA-β-gal activity, reduced Lamin B, elevated p21, and dampened AMPK/SIRT1 pathway activity in aged mice collectively paint a picture of progressive cellular senescence (Salminen et al. 2012; L\u0026oacute;pez-Ot\u0026iacute;n et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Crucially, this senescent state is intrinsically linked to a profound deficit in steroidogenic capacity, as evidenced by significantly lower serum testosterone and the downregulation of key synthesizing enzymes P450scc, 3β-HSD, and StAR. The failure of hCG and db-cAMP stimulation to fully restore enzyme levels in aged cells suggests a fundamental defect lies beyond cell surface receptors, within the steroidogenic machinery itself. This indicates an insufficiency in the core cellular energetic and biosynthetic platforms, namely, the mitochondria.\u003c/p\u003e \u003cp\u003eWe therefore focused on mitochondrial dynamics, a key determinant of mitochondrial health. The mitochondrion is a dynamic organelle that frequently undergoes fission and fusion to maintain its integrity in response to various physiological and pathological processes (Pernas et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Chen et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2026\u003c/span\u003e). Mitochondrial fusion facilitates content exchange between mitochondria, leading to larger, more interconnected networks, whereas fission promotes their fragmentation and generates isolated and shortened mitochondria (Jiang et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Ng et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Mitochondrial outer membrane fusion is mediated by two proteins, mitofusin 1 (MFN1) and MFN2. Conversely, mitochondrial fission is mainly driven by the proteins dynamin-related protein 1 (DRP1) (T\u0026aacute;bara et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). When phosphorylated at Ser616, DRP1 stimulates mitochondrial fission during mitosis. Conversely, fission is inhibited when DRP1 is phosphorylated at Ser637 (Xie et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Shi et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Our results demonstrate a pronounced shift towards fission in aged Leydig cells. The downregulation of fusion proteins MFN2 and MFN1, coupled with the inhibitory phosphorylation of DRP1 at Ser637 and its activating phosphorylation at Ser616, creates a molecular environment permissive for mitochondrial fragmentation. The resulting fragmented mitochondrial network is associated with a loss of membrane potential (MMP), reduced ATP production, and elevated oxidative stress creating an internal milieu that is fundamentally incompatible with the high-energy demands of steroid hormone biosynthesis (Selvaraj et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Moreover, the observation that hCG/db-cAMP stimulation partially improves this dynamic imbalance, yet fails to normalize it in aged cells. These findings suggests that restoring mitochondrial dynamics could be a more upstream and effective therapeutic strategy than merely providing hormonal stimulation.\u003c/p\u003e \u003cp\u003eTo establish the role for MFN2 in this process, we employed gain-of-function and loss-of-function approaches in our cellular model. The finding demonstrated that the mitochondrial fusion promoter M1 could robustly reverse H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced mitochondrial fragmentation, senescence, and steroidogenic decline in TM3 cells. Conversely, siRNA-mediated knockdown of MFN2 exacerbated all these deficits, confirming its non-redundant, central role. These results provide compelling evidence that promoting fusion is a viable strategy to rescue aged Leydig cell function.\u003c/p\u003e \u003cp\u003eSirtuin 1 (SIRT1), an NAD+-dependent deacetylase, is a key regulator of energy metabolism and stress responses, renowned for its anti-aging properties (Hekmatimoghaddam et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). In this study, we investigate the functional interplay between the well-known anti-aging protein SIRT1 and MFN2. The observed downregulation of SIRT1 in aged Leydig cells is consistent with its established role as a guardian against cellular aging (Chen \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). However, the mechanistic link between SIRT1 and mitochondrial function in this specific context was unclear. We initially hypothesized that SIRT1 might act upstream of MFN2, given its role as a deacetylase that can modify numerous targets. Surprisingly, our data compellingly demonstrate that the protective effects of pharmacological MFN2 upregulation with M1 against aging and mitochondrial damage are completely independent of SIRT1. Furthermore, SIRT1 knockdown did not attenuate M1's ability to restore steroidogenic enzymes, rebalance mitochondrial dynamics, improve ATP/MMP, reduce ROS, or suppress senescence. This indicates that MFN2 functions in a pathway parallel to or downstream of SIRT1, and its activation is sufficient to counteract aging phenotypes even when SIRT1 is deficient. In stark contrast, the converse was not true. The protective effects of SIRT1 activation with nicotinamide riboside chloride (NRC), a precursor of NAD+, were completely abrogated upon MFN2 knockdown. NRC successfully activated the SIRT1/AMPK pathway, thereby rescuing the H₂O₂-induced imbalance in mitochondrial dynamics, mitochondrial dysfunction, and the decline in steroidogenesis in TM3 cells. However, in the absence of MFN2, NRC was rendered completely ineffective. This reveals a critical, non-redundant dependency of SIRT1 on MFN2 to execute its anti-aging program in Leydig cells. Therefore, the concurrent decline of both SIRT1 and MFN2 may create a doubly detrimental scenario in aging. However, our findings suggest that therapeutic strategies aimed solely at activating SIRT1 may fail if MFN2 levels are not concurrently supported.\u003c/p\u003e \u003cp\u003eMitophagy serves to eliminate damaged and dysfunctional mitochondria (Zhang et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Our findings further illuminate the critical role of impaired mitophagy, a selective form of autophagy for damaged mitochondria, in the vicious cycle of mitochondrial dysfunction and steroidogenic decline in aging Leydig cells. The age-related downregulation of key mitophagy initiators, PINK1 and Parkin, along with the receptor NIX, indicates a significant failure in the quality control system tasked with eliminating fragmented and dysfunctional mitochondria. This deficiency is not merely a consequence of aging but an active contributor to the pathology. As our data show, the imbalance in mitochondrial dynamics, characterized by MFN2 downregulation and excessive DRP1-mediated fission, generates a surplus of damaged, ROS-producing mitochondria. Under normal conditions, these damaged organelles would be promptly recognized and cleared by mitophagy. However, in the aging Leydig cell, this clearance mechanism is blunted. The resulting accumulation of defective mitochondria creates a state of chronic oxidative stress and bioenergetic crisis, as evidenced by elevated ROS and reduced ATP, which directly poisons the steroidogenic machinery and starves it of the necessary energy and cholesterol precursors for testosterone synthesis. The interconnection between mitochondrial dynamics and mitophagy is particularly underscored by the role of MFN2. Beyond its canonical function in fusion, MFN2 is increasingly recognized as a regulatory node for mitophagy, serving as a receptor for Parkin recruitment on the outer mitochondrial membrane (Chen and Dorn \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Our rescue experiments demonstrated that the mitochondrial fusion promoter M1 rescued H₂O₂-induced suppression of mitophagy. Conversely, MFN2 knockdown exacerbated the suppression of mitophagy. This creates a self-reinforcing loop: mitochondrial damage promotes fission, and the resulting fragments, if not cleared, exacerbate the damage.\u003c/p\u003e \u003cp\u003eWhile our study provides strong evidence for the central role of MFN2, several questions remain for future investigation. First, the precise upstream mechanism responsible for the age-related downregulation of MFN2 in Leydig cells is still unknown. It could involve transcriptional repression, post-translational modifications, or enhanced degradation. Identifying these regulators could unveil new targets for intervention. Second, our in vitro model utilizes H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e to induce senescence, which, while robust, may not capture all aspects of the complex, low-grade chronic inflammation and metabolic dysregulation seen in physiological aging in vivo. Future studies could benefit from using genetic models of accelerated aging or conducting long-term longitudinal analyses. Third, the specific molecular link whereby MFN2 loss impairs the expression of steroidogenic enzymes like P450scc and StAR warrants deeper exploration. It may involve disrupted cholesterol transport due to impaired mitochondria-ER contact sites, a known function of MFN2, or a more general failure in providing adequate ATP for the steroidogenic process.\u003c/p\u003e \u003cp\u003eFrom a translational perspective, our findings position MFN2 as a highly promising therapeutic target for LOH. The demonstration that a pharmacological promoter of mitochondrial fusion (M1) can reverse key aging phenotypes in Leydig cells opens up a novel avenue for drug development. Rather than merely supplementing testosterone, a strategy with known limitations and side effects, enhancing mitochondrial health via MFN2 could address the root cause of the hormonal deficit in a subset of aging men. Future research should focus on developing more specific and potent MFN2-stabilizing compounds and testing their efficacy and safety in pre-clinical models of LOH.\u003c/p\u003e \u003cp\u003eIn summary, our work delineates a detailed pathway through which aging impairs Leydig cell function, centering on the downregulation of MFN2 and the consequent disintegration of mitochondrial homeostasis. We establish that MFN2 is a critical regulator that governs mitochondrial dynamics, oxidative stress, and mitophagy, and its decline is a principal driver of steroidogenic failure. Moreover, we redefine the functional hierarchy between SIRT1 and MFN2, revealing that MFN2 is the indispensable effector through which SIRT1 must act to confer its anti-aging benefits. By identifying MFN2 as a central executioner of mitochondrial and cellular health, this study not only deepens our understanding of the molecular etiology of LOH but also provides a compelling rationale for targeting mitochondrial fusion as a novel therapeutic strategy for age-related hypogonadism.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the National Natural Science Foundation of China (82173559), Major Project of Natural Science Research in Universities of Anhui Province (2025AHGXZK20014), Key Research and Development Projects of Anhui Province (202104j07020035), Key Research Program of Anhui Science and Technology Innovation Platform (202305a12020016), Key Project of Natural Science Research of Anhui Provincial Department of Education (2024AH050752), Research Fund of Anhui Institute of translational medicine (2022zhyx-C45).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eZheng-Mei Lv:\u003c/strong\u003e\u0026nbsp;Conceptualization, Funding acquisition, Investigation, data curation and formal analysis. \u003cstrong\u003eChao Liu:\u003c/strong\u003e Investigation. \u003cstrong\u003eChong-Kang Wu:\u003c/strong\u003e Investigation. \u003cstrong\u003eZi-meng Xu:\u0026nbsp;\u003c/strong\u003eInvestigation. \u003cstrong\u003eYa-jing Liu:\u003c/strong\u003e Formal analysis, \u003cstrong\u003eXin Chen:\u003c/strong\u003e Formal analysis, \u003cstrong\u003eYuan-Hua Chen:\u003c/strong\u003e Conceptualization, Supervision, data curation and formal analysis, Funding acquisition, Writing-Original Draft, Writing-review \u0026amp; editing. All authors reviewed the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDisclosures: \u003c/strong\u003ethe authors declare no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical trial number:\u003c/strong\u003e\u0026nbsp;not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement: \u003c/strong\u003ethe data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBehera BP, Mishra SR, Mahapatra KK, Patil S, Efferth T, Bhutia SK. SIRT1-activating butein inhibits arecoline-induced mitochondrial dysfunction through PGC1α and MTP18 in oral cancer. 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Cell Signal. 2025;138:112221.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhou SJ, Zhao MJ, Yang YH, Guan D, Li ZG, Ji YD, et al. The Epidemiological Characteristics of Late-Onset Hypogonadism in Chinese Middle-Aged and Elderly Men: Two Cross-Sectional Studies in the Same Community. Am J Mens Health. 2020;14(6):1557988320977991.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"reproductive-biology-and-endocrinology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"rbej","sideBox":"Learn more about [Reproductive Biology and Endocrinology](http://rbej.biomedcentral.com)","snPcode":"12958","submissionUrl":"https://submission.nature.com/new-submission/12958/3","title":"Reproductive Biology and Endocrinology","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Mitochondrial dynamics, Mitofusin 2 (MFN2), Cellular senescence, Late-onset hypogonadism, Oxidative stress, SIRT1","lastPublishedDoi":"10.21203/rs.3.rs-9553901/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9553901/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAge-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.\u003c/p\u003e","manuscriptTitle":"SIRT1/MFN2-Mediated Regulation of Mitochondrial Dynamics and Mitophagy in Age-Related Decline of Leydig Cell Function","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-13 08:46:06","doi":"10.21203/rs.3.rs-9553901/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"282327298030180006747325641271562401487","date":"2026-05-13T17:59:24+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"2229822647872395408898999018996177155","date":"2026-05-07T08:04:32+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-05-05T04:44:34+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-28T14:28:30+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-28T14:27:37+00:00","index":"","fulltext":""},{"type":"submitted","content":"Reproductive Biology and Endocrinology","date":"2026-04-28T11:55:29+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"reproductive-biology-and-endocrinology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"rbej","sideBox":"Learn more about [Reproductive Biology and Endocrinology](http://rbej.biomedcentral.com)","snPcode":"12958","submissionUrl":"https://submission.nature.com/new-submission/12958/3","title":"Reproductive Biology and Endocrinology","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"289691f7-7173-44ad-b1c1-524f770ba4e5","owner":[],"postedDate":"May 13th, 2026","published":true,"recentEditorialEvents":[{"type":"reviewerAgreed","content":"282327298030180006747325641271562401487","date":"2026-05-13T17:59:24+00:00","index":16,"fulltext":""},{"type":"reviewerAgreed","content":"2229822647872395408898999018996177155","date":"2026-05-07T08:04:32+00:00","index":10,"fulltext":""},{"type":"reviewersInvited","content":"9","date":"2026-05-05T04:44:34+00:00","index":"","fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-13T08:46:07+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-13 08:46:06","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9553901","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9553901","identity":"rs-9553901","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}