What is LongevityOne?

LongevityOne is a global platform dedicated to curing human aging. We consolidate advanced research, emerging technology, and unified data to accelerate innovation in the longevity space. Our mission is to serve scientists, bio-hackers, and advocates with trustworthy resources and collaborative tools.

Who founded LongevityOne?

LongevityOne was founded by Michael Peres, a serial entrepreneur and biomedical researcher with a background in Mathematics, Computer Science, and Jewish Studies. He is currently pursuing Biomedical Engineering to strengthen the technical foundation of LongevityOne’s work. Learn more about him at michaelperes.com.

What makes LongevityOne different?

We don’t just report on breakthroughs — we unify them. LongevityOne acts as a central repository where research, news, tools, and contributors converge. By removing silos in the aging research ecosystem, we accelerate progress toward reversing age-related decline.

Who is LongevityOne for?

Our platform is built for:Researchers in biotechnology, genetics, and agingMedical professionals and institutionsBio-hackers and health optimizersInvestors in life-extension technologiesPublic advocates for healthy aging

What are your long-term goals?

Our core objective is to contribute to the development of a foundational cure for aging. We aim to reshape the human experience of aging by supporting breakthrough research, increasing public understanding, and fostering international collaboration over the next 15 years.

How do I get involved?

Whether you’re a researcher, journalist, developer, or longevity enthusiast, we welcome your participation. Email us at contact@longevityone.org to explore partnerships, media opportunities, or to join our contributor network.

Impaired ketogenesis in Leydig Cells drives testicular aging

Introduction

The testis is fundamental to male reproductive health, playing a pivotal role in both spermatogenesis and the synthesis of male sex hormones throughout an individual’s life^1,2. As men age, a progressive decline in spermatogenesis significantly contributes to male infertility and increases the risk of genetic and other abnormalities in their progeny^3,4. Additionally, testicular aging is also characterized by a reduction in testosterone, which is linked to various male reproductive disorders and a diminished quality of life in the elderly⁵. Currently, testosterone replacement therapy (TRT) serves as the primary intervention for alleviating symptoms associated with testicular aging⁶. However, TRT is accompanied by notable adverse effects, including an elevated risk of cardiovascular diseases, exacerbation of sleep apnea syndrome, and an increased possibility of developing prostate cancer^7,8. Moreover, TRT fails to mimic the physiological secretion patterns of testosterone and can negatively impact spermatogenesis^9,10. Consequently, there is a pressing need to explore novel therapeutic strategies for addressing testicular aging.

Cellular senescence has been classically described as a stable cell cycle arrest that develops in response to stress or damage, thereby triggering multiple intracellular phenotypic changes¹¹. Senescent cells are characterized by features such as enlarged cell size, DNA damage, and the secretion of senescence-associated secretory phenotype (SASP) factors^11,12. Notably, senescent cells accumulate in vivo with aging and are associated with disrupted tissue homeostasis and diminished regenerative capacity¹³. Recent evidence has implicated accumulation of senescent cells as a contributing factor to aging and age-related diseases in fast-aging and naturally aged mice models. For instance, the transplantation of a small number of senescent cells around the knee joint was sufficient to induce osteoarthritis¹⁴. In addition, transplanting senescent ear fibroblasts or syngeneic preadipocytes into middle-aged mice can induce frailty, accelerate the onset of all age-related diseases, and lead to premature death when compared to transplanting equivalent numbers of non-senescent cells¹⁵. Conversely, recent studies have showed that alleviating cellular senescence could effectively delay organ aging and address aging-related diseases. In muscle, inhibiting 15-PGDH (15-hydroxyprostaglandin dehydrogenase) in senescent myofibers could enhance aged muscle mass, strength, and exercise performance¹⁶. In blood vessels, knockout of GIGYF2(Grb10-interacting GYF protein 2) in senescent vascular endothelial cells could protect aged mice from aging-associated vascular endothelium-dependent relaxation and arterial stiffness¹⁷. Therefore, targeting senescent cells presents a promising pathway for developing novel and effective anti-aging therapeutics.

Aging testes undergo profound alterations in both germ cells and somatic cells, leading to reduced functionality^18,19. Previous studies have shown that testicular aging is marked by a decline in the number of spermatogonia and spermatocytes, as well as the accumulation of DNA damage and mutations within germline cells^4,20. As the primary cells producing testosterone, LCs play a crucial role in spermatogenesis and male fertility²¹. LCs are thought to be vulnerable to age-related damage, primarily due to oxidative stress induced by reactive oxygen species (ROS). This susceptibility can contribute to the development of late-onset hypogonadism^22,23. An alternative view suggests that Sertoli cells are also susceptible to aging, since Sertoli cells expressed senescent markers in the aged testes²⁴. A recent study reported that compromised lysosomal acidity in aged Sertoli cells disrupts autophagy and phagocytic flow, leading to testicular aging²⁵. Nevertheless, a systematic profiling of senescent cells in the testes during aging has yet to be established.

In this study, we characterize testicular aging by detecting the senescence marker senescence-associated β-galactosidase (SA-β-gal), identifying that LCs are the most susceptible cells to aging in the testis. Single-cell transcriptomics reveals a significant downregulation of 3-Hydroxy-3-methylglutaryl-CoA synthase 2 (Hmgcs2), which encodes the rate-limiting enzyme in ketogenesis, in aged LCs. Moreover, silence of Hmgcs2 in young LCs impairs ketogenesis, causing premature senescence and accelerating testicular aging. Mechanistically, β-hydroxybutyric acid (BHB), a ketogenic product and inhibitor of histone deacetylase 1 (HDAC1), promotes Foxo3a expression by enhancing histone acetylation, thereby alleviating LCs senescence and improving steroidogenic function. In vivo studies further demonstrate that enhancing ketogenesis via Hmgcs2 overexpression or BHB supplementation reduces LCs senescence and improves testicular function in aged mice.

Results

LCs are susceptible to senescence during the process of aging in mice

To investigate the phenotypic changes and underlying mechanisms of testicular aging, testes were harvested from young (2-month-old) and aged (24-month-old) mice. Morphological analysis revealed a reduction in the diameter of seminiferous tubules and the thickness of the seminiferous epithelium in aged mice (Supplementary Fig. 1a–c). Consistently, sperm analysis demonstrated a significant decrease in sperm concentration and motility in the aged group (Supplementary Fig. 1d–g). Subsequently, we analyzed the levels of sex hormones in the aged group. Testosterone assays revealed that serum and intratesticular testosterone levels decreased significantly in aged mice (Supplementary Fig. 1h, i). In addition, Insulin-like peptide 3 (Insl3), indicative of LCs function, was reduced in the aged group (Supplementary Fig. 1j). Since testosterone levels are regulated by gonadotropins, we measured the concentrations of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) levels in both groups. As expected, LH and FSH levels were elevated in the aged group (Supplementary Fig. 1k, l). Collectively, these histological and functional analysis revealed significant testicular aging in aged mice.

The accumulation of senescent cells is a driving force in physical functional decline in various tissues during aging²⁶. To further quantify cellular senescence in the testis, we performed SA-β-gal staining on the testes of 2-, 8-, 16-, and 24-month-old mice. Surprisingly, we observed a notable increase in SA-β-gal⁺ cells appeared as early as 8 months of age and SA-β-gal⁺ signals were specifically located in the interstitium of testes during aging, indicating that interstitial cells are more susceptible to aging than Sertoli cells and germ cells (Supplementary Fig. 1m). The specific cell types from testes were further analyzed by co-immunostaining with SA-β-gal and previously described cell markers. The results revealed that SA-β-gal⁺ cells largely co-expressed with the LCs marker CYP17A1 (Supplementary Fig. 1n, o). However, macrophages and Sertoli cells did not show SA-β-gal staining on testes of 8-month-old mice (Supplementary Fig. 1p). These results underscore that LCs are highly vulnerable to aging.

Single-cell transcriptomic profile of testicular aging reveals a senescence signature of LCs

To investigate the molecular changes associated with LCs senescence, testicular cells were isolated from 3 young mice (2-month-old) and 3 aged mice (24-month-old), followed by single-cell RNA sequencing (scRNA-seq) using the BD Rhapsody platform (Fig. 1a). Data from 2584 and 4344 cells passed quality control (Supplementary Fig. 2a, b) and were included in the subsequent analysis for young and aged samples, respectively (Fig. 1a). Using unbiased clustering and t-Distributed Stochastic Neighbor Embedding (tSNE) analysis, we identified the following 7 cell types based on the expression of specific marker genes: LCs (Cyp17a1, Star, Lhcgr), mesenchymal cells (MCs; Pdgfrα, Arx, Tcf21), macrophages (Ms; Adgre1, Mrc1, Itgam), spermatogonia (SPGs; Id4, Tspan33, Dazl), spermatocytes (SPCs; Sycp3, Zpbp2, Pgk2), round spermatids (RSs; Acrv1, Spag6, Tssk1), and elongating spermatids (ESs; Prm1, Tnp2, Prm3) (Fig. 1b, c; Supplementary Fig. 2c). Analysis of the top 30 marker genes revealed that each cell type had unique transcriptional features and enriched pathways relevant to their distinct biological functions (Fig. 1d).

**Fig. 1: Single-cell transcriptome profiling of young and aged mice testes.**

We next performed a focused analysis of age-associated changes in the LCs cluster. Not surprisingly, clustering analysis revealed that LCs from young and aged mice exhibited two distinct subpopulations (Fig. 1e). Furthermore, we examined the expression of senescence-related markers and found that p21 (known as Cyclin-dependent kinase inhibitor 1 A, Cdkn1a) and Cxcl10 exhibited transcriptional upregulation in LCs obtained from the aged testes compared to the young group (Fig. 1e). To validate the gene expression pattern, we performed quantitative RT-PCR analysis, revealing significantly increased expression of p21 and Cxcl10 in the aged group (Fig. 1f, g). Consistently, immunostaining analysis confirmed that the protein levels of p21 showed a substantial increase in LCs from the aged group compared to the young group (Fig. 1h). These findings collectively indicate that testicular aging is accompanied by the accumulation of senescent LCs, which may lead to morphological changes and functional impairment.

Senescent LCs of aged mice exhibit impaired ketogenesis

To further characterize the ageing-associated changes of gene expression in LCs during testicular aging, we performed gene set enrichment analysis (GSEA) based on gene ontology (GO) database. Our results revealed that the genes upregulated with age were mainly associated with response to inflammation-related terms, such as “cellular response to interleukin 1” and “positive regulation of alpha beta t cell activation”. By comparison, the downregulated DEGs were enriched in metabolism-related terms, including “aerobic respiration”, “generation of precursor metabolites and energy”, and “cholesterol catabolic process” (Fig. 2a). To identify critical regulators linked to LCs aging, we analyzed aging-associated differentially expressed genes (DEGs) based on the above GO terms. Among these DEGs, Hmgcs2 exhibited the most prominent changes, with notable downregulation in LCs from the aged group (Fig. 2b). Hmgcs2 is the gene encoding the rate-limiting enzyme for ketogenesis^27,28 (Fig. 2c). Therefore, we examined the expression of genes encoding other ketogenic enzymes, including acetyl-CoA acetyltransferase 1 (Acat1), Hmg-CoA lyase (Hmgcl) and 3-hydroxybutyrate dehydrogenase 1 (Bdh1)²⁷. The analysis of scRNA-seq data revealed downregulation of Hmgcl in aged LCs, however, Bdh1 and Acat showed no significant difference between young and aged LCs (Fig. 2d). To validate these findings, we isolated primary LCs from the young and aged testes and assessed genes expression using quantitative RT-PCR. The results demonstrated a substantial decrease in the expression of ketogenesis-related enzymes, including Acat, Hmgcs2, and Hmgcl in aged mice (Fig. 2e–g). Additionally, immunofluorescence staining revealed a significant reduction in HMGCS2 expression in LCs from aged mice (Fig. 2h). The primary ketone bodies include acetoacetic acid (AcAc) and its redox partner β-hydroxybutyric acid (BHB)²⁸. To assess ketogenesis levels in the testis, we measured intratesticular ketone body concentrations. As expected, BHB and AcAc concentrations showed significant decreases in the aged testes compared to the young testes (Fig. 2i, j). However, BHB and AcAc concentrations in serum were comparable between the young and the aged group (Fig. 2k, l). Remarkably, the concentration of ketone bodies in the testes were more than tenfold higher than that in serum (Fig. 2i–l), implying ketone bodies might have testis-specific functions. In summary, these results indicate that ketogenesis is impaired in the LCs of aged testis.

**Fig. 2: LCs in the aged testis show impaired ketogenesis, which leads to a decrease in testicular ketone body concentration.**

Suppression of ketogenesis induces senescence of LCs in vitro

Next, we investigated whether impaired ketogenesis induces premature senescence of LCs. For this purpose, MLTC-1 cells (cell line of LCs) were treated with an HMGCS2 inhibitor Hymeglusin (3 μM) for 4 days in culture (Fig. 3a). As evidenced by the reduced levels of ketone bodies in MLTC-1 cells, Hymeglusin treatment effectively inhibited ketogenesis (Fig. 3b, c). After Hymeglusin treatment, an increase in SA-β-gal⁺ cells was observed (Fig. 3d), along with increased immunofluorescent staining for the senescence marker γ-H2AX and H2AY, as well as reduced Lamin B expression (Fig. 3e–g). Quantitative RT-PCR analysis revealed a significant upregulation of senescence markers p21 and Plasminogen activator inhibitor-1 (Pai1) in Hymeglusin treated MLTC-1 cells (Fig. 3h, i). Additionally, Hymeglusin treatment resulted in a significant decrease in progesterone level in the culture medium of MLTC-1 cells, indicating suppression of ketogenesis impairs steroidogenic function of LCs (Fig. 3j). To confirm these findings, we performed bulk RNA-seq on MLTC-1 cells treated with Hymeglusin or vehicle. The results revealed marked alterations in global gene expression following Hymeglusin treatment (Fig. 3k). Notably, these changes closely resembled those observed during natural aging. Specifically, DEGs upregulated by Hymeglusin were primarily associated with inflammation and cell cycle arrest, such as terms involved in “negative regulation of cell cycle phase transition” and “innate immune response”. In contrast, downregulated DEGs were enriched in metabolism-related pathways, including “generation of precursor metabolites and energy”, “fatty acid metabolic process”, and “cholesterol metabolic process” (Fig. 3l). These findings suggest that impaired ketogenesis leads to the appearance of aging phenotype in LCs.

**Fig. 3: Inhibition of Hmgcs2 accelerates senescence of Leydig cells.**

To specifically investigate the effects of Hmgcs2 on senescence, we further used a genetic approach to reduce enzyme levels in MLTC-1 cells by introducing either shRNA to Hmgcs2 or a scrambled (scr) shRNA under the control of a ubiquitous promoter CAG through lentivirus. The knockdown efficiency of Hmgcs2 was confirmed by Western blot analysis, and a reduction in ketone bodies levels was observed in MLTC-1 cells 7 days after transfection (Supplementary Fig. 3a–c). Similarly, knockdown of Hmgcs2 increased the number of SA-β-gal⁺ cells and enhanced expression of γ-H2AX, H2AY, as well as reduced expression of Lamin B in MLTC-1 cells (Supplementary Fig. 3d–g). Quantitative RT-PCR also revealed significant upregulation of p21 and Pai1 expression in the Hmgcs2-knockdown cells (Supplementary Fig. 3h, i). Furthermore, knockdown of Hmgcs2 significantly suppressed progesterone production compared to the control group (Supplementary Fig. 3j). Collectively, these findings suggest that impaired ketogenesis drives the senescence of LCs.

Knockout of Hmgcs2 drives LCs senescence and testicular aging in young mice

To further elucidate the effect of ketogenesis on LCs senescence in vivo, we generated a Hmgcs2-flox (Hmgcs2^fl/fl) mouse line on C57BL/6 background. Recent studies have indicated that adeno-associated virus (AAV) vectors exhibit tropism for LCs when injected into testes^29,30. We therefore interstitially injected AAV serotype DJ encoding GFP under the control of the CAG promoter (AAV-GFP) at titers of 2×10⁹ genome copies (gc) into each testis of 8-week-old wild type mice. Seven days post-injection, immunofluorescence analysis revealed that GFP was expressed exclusively in the interstitium of testis, with no off-target transduction detected in the liver, kidney, heart, brain, lung, intestine, or muscle (Supplementary Fig. 4a). Moreover, GFP expression was specific to CYP17A1⁺ cells in the injected testes, confirming that AAV specifically target LCs when delivered into the interstitial compartment (Supplementary Fig. 4b). Next, 8-week-old Hmgcs2^fl/fl mice were subjected to intratesticular injections of AAV-GFP or AAV-Cre at titers of 2×10^9 genome copies (gc) into each testis (Fig. 4a). Four weeks after injection, quantitative RT-PCR analysis of testicular tissue showed a significant decline in Hmgcs2 transcripts (Supplementary Fig. 4c), and Western blot assays demonstrated a corresponding decrease in HMGCS2 protein levels in AAV-Cre group compared with AAV-GFP group (Supplementary Fig. 4d). Immunofluorescence staining also confirmed a reduction of HMGCS2 in LCs from AAV-Cre group (Fig. 4b). Moreover, the concentrations of testicular BHB and AcAc showed significant decline in AAV-Cre treated mice (Fig. 4c, d; Supplementary Fig. 4e, f).

**Fig. 4: Conditional knockout of *Hmgcs2* in LCs leads to testicular aging in young mice.**

To determine whether the conditional knockout of Hmgcs2 induces LCs senescence, we performed SA-β-gal staining on the testes 2 months post-injection. The number of SA-β-gal⁺ LCs increased significantly in the testes from AAV-Cre treated group (Fig. 4e). Immunofluorescence analysis also revealed a significant increase in the senescence marker p21 in LCs from AAV-Cre treated group (Fig. 4f). Quantitative RT-PCR of isolated LCs showed significant upregulation of senescence markers p21 and Cxcl10 in AAV-Cre treated group compared to AAV-GFP treated group (Fig. 4g, h). Additionally, serum and intratesticular testosterone concentrations decreased significantly in AAV-Cre treated mice compared to AAV-GFP treated mice (Fig. 4i, j). Similarly, serum Insl3 concentrations were significantly reduced (Fig. 4k), while serum LH concentrations increased in AAV-Cre treated mice (Fig. 4l). Furthermore, histological analysis showed thinning of the seminiferous epithelium in AAV-Cre treated mice (Fig. 4m–o). Sperm analysis indicated a decrease in sperm concentration in AAV-Cre treated mice compared to AAV-GFP treated mice, however, no significant difference was detected in sperm motility or progressive motility between the two groups (Fig. 4p–s). These findings indicate that ketogenesis exerts protective effects on LCs, and loss of Hmgcs2 leads to premature senescence of LCs and accelerates testicular aging.

The protective effects of ketogenesis on LCs are mediated by BHB but not AcAc

To determine which ketogenic products contribute to the protective effects of ketogenesis on LCs, we examined their ability to mitigate cellular senescence caused by HMGCS2 inhibition or silence. Our results demonstrated that BHB (5 mM) alleviated Hymeglusin-induced cellular senescence, as evidenced by the reduced proportion of SA-β-gal⁺ cells (Supplementary Fig. 5a) and the downregulation of senescence markers p16 and p53 (Supplementary Fig. 5b). Moreover, quantitative RT-PCR analysis showed that BHB decreased the expression of p21 and Pai1 (Supplementary Fig. 5c, d). However, AcAc (5 mM) had no effect on the senescence of Hymeglusin-treated MLTC-1 cells and, in some cases, even appeared to exacerbate senescence (Supplementary Fig. 5a–d). Consistent with these findings, BHB restored the steroidogenic function impaired by Hymeglusin, as demonstrated by increased progesterone levels in the culture medium supernatant, whereas AcAc offered no such protective effect (Supplementary Fig. 5e). Similarly, BHB attenuated Hmgcs2-knockdown-induced cellular senescence, as indicated by the reduced proportion of SA-β-gal⁺ cells (Supplementary Fig. 5f, g) and decreased p21 and Pai1 expression (Supplementary Fig. 5h–k). Collectively, these findings suggest that BHB mediates the protective effects of ketogenesis on LCs.

Next, we investigated whether ketogenic metabolites confer protection against senescence induced by common stressors³¹. Hydrogen peroxide (H₂O₂) triggers premature senescence by increasing oxidative stress³². As expected, H₂O₂ (100 μM) exposure increased the number of SA-β-gal⁺ cells and led to a significant decline in Hmgcs2 transcripts in MLTC-1 cells (Fig. 5a, b; Supplementary Fig. 6a). Treatment with BHB (5 mM) decreased SA-β-gal⁺ cells and lowered expression of senescence markers p21 and Cxcl10 (Fig. 5b–e). Additionally, BHB restored progesterone levels impaired by H₂O₂, whereas AcAc (5 mM) had no protective effect (Fig. 5f). Similarly, doxorubicin (DXR, 150 nM), a chemotherapeutic agent known to induce senescence³³, increased SA-β-gal⁺ cells and significantly reduced Hmgcs2 expression (Supplementary Fig. 6b,e). BHB alleviated DXR-induced senescence by decreasing SA-β-gal⁺ cells and suppressing the expression of p21 and Pai1, while AcAc showed no effect (Supplementary Fig. 6d–g). Lastly, ionizing radiation (IR, 8 Gy), which induces senescence through DNA damage³⁴, also resulted in downregulation of Hmgcs2 expression and an increase in SA-β-gal⁺ cells in MLTC-1 cells (Supplementary Fig. 6c). Treatment with BHB significantly reduced SA-β-gal⁺ cells and downregulated senescence markers p21 and Cxcl10, whereas AcAc remained ineffective (Supplementary Fig. 6h–k). Collectively, these results demonstrate that BHB consistently mitigates senescence induced by oxidative stress, chemotherapy, and radiation, underscoring its protective role in LCs.

**Fig. 5: BHB alleviates cellular senescence by promoting histone acetylation via HDAC inhibition.**

BHB alleviates testicular aging by enhancing histone acetylation

We next elucidate the molecular mechanism by which BHB alleviates LCs senescence. Recent studies have suggested that BHB acts as an epigenetic modifier, particularly by altering histone H3 at lysine 9 (H3K9) through β-hydroxybutyrylation (Kbhb)³⁵. However, our findings demonstrate that BHB treatment did not increase the Kbhb of H3K9 in MLTC-1 cells, thereby excluding the possibility that BHB regulates senescence via Kbhb (Fig. 5g). In addition to Kbhb, BHB serves as an endogenous inhibitor of histone deacetylase (HDAC), thereby modulating H3K9 acetylation (H3K9ac)³⁶. Western blot and immunofluorescence staining analyses revealed that H3K9ac was dose-dependently upregulated by BHB in MLTC-1 cells (Fig. 5g, h), consistent with its role as a potential HDAC inhibitor. To verify that BHB alleviates LCs senescence by inhibiting HDAC, we first demonstrated that BHB treatment reduced HDAC activity in MLTC-1 cells, similar to the pharmacological inhibitor vorinostat (Fig. 5i). Then, senescent MLTC-1 cells were treated with BHB (5 mM) or vorinostat (1 μM). Both treatments reduced the number of SA-β-gal⁺ cells (Fig. 5j) and decreased the expression of senescence markers p16, p21, and Cxcl10 (Fig. 5k–m). Additionally, BHB and vorinostat individually increased progesterone concentration in the supernatant of the culture medium (Fig. 5n). However, co-treatment with vorinostat and BHB did not further increase progesterone concentrations or decrease the expression of senescence markers, indicating redundancy between BHB signaling and HDAC inhibition (Fig. 5j–n). These data support that BHB mitigates cell senescence by enhancing histone acetylation via HDAC inhibition.

BHB-mediated HDAC1 inhibition enhances Foxo3a expression

BHB is as an endogenous inhibitor of HDAC1, HDAC2, and HDAC3^27,37. To identify which HDAC subtype mediates the alleviation of cell senescence by BHB, we conducted a focused analysis of the LCs cluster from scRNA-seq data. This analysis revealed that the expression of Hdac1 in LCs from aged mice is higher than that in young mice (Supplementary Fig. 7a). Then, we performed quantitative RT-PCR and found that Hdac1 expression increased significantly in the aged group, while Hdac2 and Hdac3 expressions were comparatively lower, with no significant differences between the groups (Supplementary Fig. 7b).

To determine whether the protective effect of BHB required Hdac1 expression, we applied shRNA to knockdown Hdac1 in MLTC-1 cells (Supplementary Fig. 7c). As expected, silencing of Hdac1 in MLTC-1 cells abrogated the ability BHB to alleviate senescence. Specifically, BHB treatment did not reduce the proportion of SA-β-gal⁺ cells in the Hdac1-silenced group (Supplementary Fig. 7d, e). Similarly, quantitative RT-PCR analysis showed that BHB treatment failed to decrease the expression of senescence markers p21 and Cxcl10 in Hdac1-silenced cells (Supplementary Fig. 7f, g). Furthermore, gene silencing of Hdac1 prevented BHB from enhancing steroidogenic function of MLTC-1 cells (Supplementary Fig. 7h). These results suggest that BHB mitigates senescence primarily by inhibiting HDAC1.

Histone acetylation induced by BHB is associated with the transcriptional activation of genes³⁶. FOXO3a, a longevity-associated transcription factor, has been reported to be upregulated by BHB-mediated histone acetylation^36,38. To investigate the role of Foxo3a in LCs senescence, we performed Western blot analysis, which demonstrated that FOXO3a expression is downregulated in H₂O₂-induced senescent MLTC-1 cells. As expected, BHB treatment upregulated the expression of FOXO3a (Supplementary Fig. 8a). To assess whether BHB alleviates cell senescence through Foxo3a pathway, we applied shRNA to knockdown Foxo3a in MLTC-1 cells (Supplementary Fig. 8b). SA-β-gal staining showed that Foxo3a silencing invalidated the BHB’s ability to alleviate senescence (Supplementary Fig. 8c, d). Similarly, quantitative RT-PCR analysis showed that BHB treatment did not decrease the expression of senescence markers p21 and Cxcl10 in Foxo3a-silenced cells (Supplementary Fig. 8e, f). To demonstrate that Foxo3a expression is regulated by HDAC1, we conducted chromatin immunoprecipitation-qPCR (ChIP-qPCR) analysis, which showed that BHB restored histone H3K9 acetylation at the Foxo3a promoter in MLTC-1 cells (Supplementary Fig. 8g). Furthermore, Hdac1 knockdown significantly increased Foxo3a expression, indicating that BHB upregulates Foxo3a expression by inhibiting HDAC1 (Supplementary Fig. 8h). These results demonstrate that BHB promotes Foxo3a expression by inhibiting HDAC1 activity, thereby mitigating cell senescence.

Previous reports have shown that FOXO3a can exert anti-aging effects by inhibiting inflammation or promote DNA repair^36,39,40. To further investigate these possibilities, we sorted LCs from young (2-month-old) and aged (24-month-old) mice and performed bulk RNA sequencing. The analysis revealed that Foxo3a expression was significantly downregulated in LCs from aged mice, accompanied by a downregulation of DNA repair genes (Supplementary Fig. 9a). In contrast, inflammation-related genes were significantly upregulated in LCs from aged mice (Supplementary Fig. 9a).

Enhancement of ketogenesis alleviates LCs senescence and testicular aging in aged mice

Given that BHB exhibits protective effects on LCs in vitro, we asked whether overexpression of Hmgcs2 in LCs of aged mice could exert an anti-aging effect on testicular function. To test this hypothesis, we used AAV vectors to deliver and overexpress the Hmgcs2 gene (AAV-Hmgcs2, O.E.) or mCherry (AAV-mCherry, Con) under the control of the CAG promoter in the testes of aged mice (15-month-old) (Fig. 6a). Immunofluorescence staining confirmed a significant increase in HMGCS2 expression 2 weeks after testicular injection of AAV-Hmgcs2 (Fig. 6b). Additionally, analysis of ketogenic products revealed a marked increase of BHB in AAV-Hmgcs2 treated testes, approaching the BHB levels observed in young testes (Fig. 6c). Consistent with in vitro results, Western blot analysis demonstrated that Hmgcs2 overexpression increased the levels of H3K9ac and FOXO3a in aged testicular tissue, similar to the levels in young testes (Fig. 6d, e).

**Fig. 6: Enhancing ketogenesis in LCs of aged mice alleviates testicular aging.**

Next, we determined whether Hmgcs2 overexpression alleviates LCs senescence. Overexpression of Hmgcs2 resulted in a significant decrease in the number of SA-β-gal⁺ LCs in aged mice (Fig. 6f). Immunofluorescence analysis revealed a significant decrease of the senescence marker p21 in LCs from AAV-Hmgcs2-treated mice (Fig. 6g). Quantitative RT-PCR analysis showed that Hmgcs2 overexpression significantly downregulated senescence markers p21 and Cxcl10 (Fig. 6h, i). Additionally, serum and intratesticular testosterone concentrations increased significantly in AAV-Hmgcs2-treated mice compared to AAV-mCherry-treated mice (Fig. 6j, k). The increase in Insl3 concentrations and decrease in serum LH concentrations further suggested the improved LCs function in AAV-Hmgcs2-treated mice (Fig. 6l, m). These results indicate that overexpression of Hmgcs2 alleviates senescence and improves the function of LCs in vivo.

We then investigated whether overexpression of Hmgcs2 in LCs could improve spermatogenesis. Four weeks after AAV-Hmgcs2 injection, histological analysis revealed enlarged diameters of seminiferous tubules and increased thickness of seminiferous epithelium (Fig. 6n–p). Sperm analysis demonstrated that Hmgcs2 overexpression significantly increased sperm concentration in aged mice. However, no significant differences were observed in sperm motility and progressive motility between two groups (Fig. 6q–t). Last, we validated the changes in FOXO3a-related inflammation genes through quantitative RT-PCR analysis, revealing that inflammation-related genes were significantly upregulated in aged mice, and Hmgcs2 overexpression reduced the expression of these genes (Supplementary Fig. 9b–e). Overall, these data suggest that enhancing ketogenesis is sufficient to alleviate LCs senescence and testicular aging in aged mice.

Oral BHB treatment improves testicular function in aged mice

We next investigated whether dietary BHB has a beneficial effect on testicular aging in aged mice (18-month-old). A 10 ml BHB salt formulation (KetoForce) was administered in 290 ml of drinking water and mix thoroughly. The KetoForce solution consists of BHB salt with 1.6 g sodium and potassium per 11.7 g BHB. Control cohorts received water supplemented with molar equivalents of the salts presented in the BHB supplement (Fig. 7a). All treated animals consumed comparable amounts of food and water (Fig. 7b). The result showed that oral BHB increased BHB levels in both the serum and testes of aged mice (Fig. 7c). Consistent with in vitro results, Western blot analysis revealed that BHB increased the levels of H3K9ac and FOXO3a in aged testicular tissue, similar to the levels observed in young testes (Fig. 7d, e).

**Fig. 7: Oral administration of BHB partially alleviates testicular aging in aged mice.**

After eight weeks of treatment, oral BHB resulted in a significant decrease in the number of SA-β-gal⁺ LCs in the testes of aged mice (Fig. 7f). Immunofluorescence analysis revealed a decrease in the senescence marker p21 in LCs from the BHB-treated mice (Fig. 7g). Quantitative RT-PCR analysis showed that oral BHB significantly downregulated p21 and Cxcl10 (Fig. 7h, i). Additionally, serum and intratesticular testosterone levels were significantly increased in the BHB-treated group (Fig. 7j, k). Furthermore, the increase in Insl3 concentrations and the decrease in serum LH concentrations confirmed the improvement of endocrine function of testes in BHB-treated mice (Fig. 7l, m). However, sperm analysis showed no significant differences in sperm concentration, sperm motility, or progressive motility between control and experimental cohorts (Fig. 7n–q). Subsequently, we validated the changes in FOXO3a-related inflammation genes through quantitative RT-PCR analysis, revealing that inflammation-related genes were significantly upregulated in aged mice and BHB supplementation reduced the expression of these genes (Supplementary Fig. 9f–i). Collectively, these results suggest that dietary BHB can alleviate the senescence of LCs and mitigate testicular aging.

Discussion

While previous studies have addressed the mechanisms of testicular aging^41,42, a comprehensive understanding of its driving forces remains elusive. Given that senescent cells drive organ dysfunction and aging process, we first aimed to characterize the senescent profile of testicular cells during aging in mice. Utilizing SA-β-gal staining, we observed that senescent cells predominantly localize to the testicular interstitium. Among interstitial cells, LCs produce testosterone and are thus critical for reproductive function and general health⁴³. LCs dysfunction leads to testosterone deficiency, arrested spermatogenesis, and infertility^44,45. By co-staining SA-β-gal with specific cell markers, we identified LCs as the earliest cell type to exhibit signs of cell senescence in the testis, which is evident as early as 8 months of age in mice. Our histological analysis further revealed a notable increase in p21, a well-established marker of senescence, in LCs from aged mice⁴⁶. Accordingly, LCs produce a large amount of ROS when synthesized testosterone, making them vulnerable to ROS-induced damage⁴⁷. Moreover, LCs are long-living cells that exist throughout the male’s life, which exposes LCs to age-accumulated damage⁴³. Based on these findings, we speculated that the intrinsic properties of LCs render them particularly susceptible to aging insults.

To further investigate the characteristics of LCs in aging, we conducted scRNA-seq analysis, revealing notable transcriptomic changes in aged LCs. Intriguingly, Hmgcs2, the gene encoding the rate-limiting enzyme in ketone bodies production²⁷, was significantly downregulated in aged LCs. Consistently, intratesticular ketone bodies (BHB and AcAc) concentrations were markedly decreased in aged testes compared to those in young testes. Ketone bodies are produced in the liver during conditions of reduced carbohydrate availability and serve as an alternative fuel source for peripheral tissues including brain, heart and skeletal muscle^28,48,49. However, their role in the testes has not been systematically studied. Surprisingly, the concentration of ketone bodies in the testes were more than tenfold higher than that in serum, indicating that intratesticular ketogenesis is likely a key event for maintaining testicular homeostasis. Recent studies have reported that local ketogenesis is essential for tissue repair^27,50. For example, proximal tubular epithelial cells in the kidney could produce ketone bodies to attenuate podocyte damage and proteinuria in diabetic mice⁵¹. Similarly, in the mammalian small intestine, the expression of Hmgcs2 distinguishes self-renewing Lgr5⁺ stem cells (ISCs) from differentiated cell types. Loss of Hmgcs2 depletes ketone bodies levels in Lgr5⁺ ISCs and skews their differentiation toward secretory cell fates²⁷. Our present study revealed that silencing Hmgcs2 expression in LCs impaired ketogenesis, consequently inducing premature senescence of LCs and accelerating testicular aging. Interestingly, this knockout specifically affected ketone body levels within the testis without altering serum levels. These results strongly suggest that the early onset of LCs senescence is likely attributed to declined ketogenesis.

Ketone bodies include AcAc and its redox partner BHB²⁸. While AcAc showed protective effects against liver fibrosis and Duchenne muscular dystrophy^52,53, it did not confer benefits to senescent LCs in our study. In contrast, BHB exhibited a geroprotective effect on senescent LCs. BHB serves not only a ketogenic metabolite for energy supply but also acts as an epigenetic modifier, including H3K9 acetylation or Kbhb^35,36. However, BHB treatment did not increase H3K9 Kbhb in MLTC-1 cells, thereby excluding the possibility that BHB functions as an β-hydroxybutyryl donor for lysine β-hydroxybutyrylation of H3K9. Notably, BHB treatment dose-dependently enhanced H3K9ac modifications, suggesting that BHB acts as endogenous histone deacetylase inhibitor to modulate H3K9 acetylation. In addition, BHB inhibited HDAC activity in MLTC-1 cells, comparable to the pharmacological HDAC inhibitor vorinostat. Consistent with this, co-treatment with HDAC inhibitor vorinostat and BHB did not further alleviate senescence or increase progesterone concentrations, indicating redundancy between BHB signaling and HDAC inhibition. Our study further confirmed that HDAC1 plays a significant role in LCs senescence, as inactivation of HDAC1 by shRNA undermined the therapeutic effect of BHB on LCs senescence. Foxo3a is ubiquitously expressed across the body and associated with healthspan and longevity^54,55. It has been reported that downregulation of Foxo3a is associated with physiological vascular aging in aged primates⁵⁶. Similarly, we found that Foxo3a is downregulated in senescent MLTC-1 cells, which can be reversed by BHB treatment. Meanwhile, we found that treatment of MLTC-1 cells with BHB led to increased H3K9ac acetylation at the Foxo3a promoters. However, upon Hdac1 knockdown, BHB failed to enhance Foxo3a levels, indicating that BHB upregulates Foxo3a expression by inhibiting HDAC1. Moreover, loss of Foxo3a could directly trigger MLTC-1 cells senescence, which could not be alleviated by BHB treatment, supporting the notion that BHB alleviates LCs senescence by promoting Foxo3a expression.

Notably, the present study revealed that overexpression of Hmgcs2 in the LCs of aged mice significantly increased BHB levels and mitigated testicular dysfunction, indicating targeted interventions for LCs senescence showed promise in delaying testicular aging. Previous studies have reported different interventions to induce ketogenesis, including fasting, ketogenic diet, and direct oral BHB administration^57,58. Dietary interventions to induce ketosis, such as fasting and ketogenic diet, are not costly and appear relatively safe^59,60. Unfortunately, dietary interventions most frequently fail as therapies due to inability of individuals to adhere to the diets⁶¹. Our findings provide evidence that oral BHB treatment effectively increases testosterone levels and partially mitigates testicular aging, without the need for the significant lifestyle changes required by fasting or ketogenic diets. This approach offers a more feasible therapeutic option for testicular aging. Our results support the notion that either enhancing endogenous ketone body production in LCs or supplementing with exogenous ketone bodies can effectively alleviate aging in mouse testes. Considering the potential health risks associated with exogenous testosterone replacement therapy^7,8,9,10, the use of ketone body supplementation offers a viable and promising alternative for counteracting testicular aging.

In the present study, we did not perform fertility assessments due to limited availability of aged mice. However, our study found that enhancing BHB levels achieved significant improvements in the testosterone levels in aged mice. Notably, the sperm concentrations were significantly restored in AAV-Hmgcs2 treated mice. Due to these remarkable enhancements in testosterone production and sperm parameters, we speculate the treated aged mice may exhibit improved fertility, which needs further evaluation in future studies. Furthermore, the role of Foxo3a in the testis warrants further investigation. Foxo3a is widely recognized as a broad-spectrum transcription factor involved in regulating various physiological and pathological processes, including inhibit inflammation, oxidative stress, and promote DNA damage repair, all of which may contribute to testicular aging^62,63,64. However, Foxo3a regulates distinct downstream target genes in a cell type-dependent manner⁶⁵, its role in testicular aging remains to be clarified in future studies.

In conclusion, our study demonstrated that LCs are the most susceptible cells to aging in the testis and identified Hmgcs2 as a vital downregulated gene in LCs with aging. Moreover, knockout of Hmgcs2 in young LCs impaired ketogenesis in testis, driving senescence of LCs and inducing testicular aging. Conversely, the activation of ketogenesis, as enhanced by genetic manipulation or dietary intervention, alleviates LCs senescence and restores testicular functions, positioning ketogenesis enhancement as a promising therapeutic strategy for delaying testicular aging. Our findings deepen the understanding of testicular aging by highlighting the pivotal role of ketogenesis in maintaining testicular homeostasis, thereby suggesting specific avenues for understanding and potentially mitigating testicular aging.

Methods

Animals

All mice were bred on a C57BL/6 background and raised in a specific pathogen-free (SPF) environment. Wild-type mice were purchased from GemPharmatech (Foshan, Guangdong, China). The Hmgcs2^flox mouse line was generated using CRISPR/Cas9 technology by GemPharmatech (Nanjing, Jiangsu, China). Male mice were identified by PCR performed on DNA isolated from tail samples. All mice were housed in the Sun Yat-sen University Animal Center under specific conditions of constant temperature (24 ± 1 °C), relative humidity (50–60%), and a 12 h light/12 h dark cycle. They were provided ad libitum access to food and water throughout the study. The diet was purchased from Xietong Pharmaceutical Bio-Engineering Co.,Ltd. (Jiangsu, CAT# XT-SL-2KG). The animal use protocol has been reviewed and approved by the Institutional Animal Care and Use Committee (IACUC), Sun Yat-Sen University (Approval No. SYSU-IACUC-2022-001335). Strict adherence to animal welfare and ethical considerations was ensured throughout the study. The primers for genotyping are listed in the Supplementary Table 1.

Sample Preparation for Single-Cell RNA Sequencing

2-month-old and 24-month-old mice were anesthetized with Avertin (250 mg/kg) by i.p. injection. After dissection, the testes were immediately placed in ice-cold saline. After fully washing the testes with phosphate-buffered saline (PBS), the tunica albuginea was carefully removed. The testes were mechanically minced into pieces using microscopic forceps and digested with accutase (Invitrogen, USA) at 37 °C for 5 minutes. The accutase activity was halted by adding DMEM/F12 supplemented with 10% fetal bovine serum (Gibco, USA), and the samples were rinsed twice with PBS. The obtained cell suspension was filtered through a 45 μm strainer and centrifuged. At last, cells were resuspended in PBS containing 0.1% BSA (Thermo Fisher Scientific, USA) at a concentration of ~1000 cells/μL for single-cell sequencing.

Single Cell RNA-seq Performance, Library Preparation and Sequencing

The BD Rhapsody system was used to capture the transcriptomic information. Single-cell capture was achieved by randomly distributing a single-cell suspension across microwells using a limited dilution approach. Beads with oligonucleotide barcodes were added in excess to ensure that a bead paired with a cell in each microwell. Cell-lysis buffer was added to allow poly-adenylated RNA molecules to hybridize to the beads. The beads were collected into a single tube for reverse transcription. Whole transcriptome libraries were prepared using the BD Rhapsody single-cell whole-transcriptome amplification workflow. Sequencing libraries were quantified using a High Sensitivity DNA chip (Agilent, USA) on a Bioanalyzer 2200 and the Qubit High Sensitivity DNA assay (Thermo Fisher Scientific, USA). The library for each sample was sequenced using an Illumina sequencer (Illumina, CA).

Single Cell RNA Sequencing quality control

The Seurat package (version 4.3.1)⁶⁶ was utilized for basic data processing. Briefly, cells that met the following criteria were retained: (1) total number of expressed genes (nFeature RNA) between 300 and 8000; (2) total unique molecular identifier (UMI) count (nCount RNA) < 60,000; and (3) mitochondrial gene percentage (Percent.mt) <50%. Cells not meeting these thresholds were excluded from analysis to reduce potential technical noise and improve the quality of the dataset. Then the data were log-normalized and scaled. Variable genes were identified by the ‘FindVariableGenes’ function. Next, principal component analysis (PCA) was performed, and the top 30 principal components (PCs) were used for t-distributed stochastic neighbor embedding (tSNE) dimension reduction and clustering. The DoubletFinder package (version 2.0.4) was employed to calculate and remove doublets. Then the data were pre-processed again and used for subsequent analyses.

Cell-type identification

After quality control, canonical correlation analysis (CCA) was employed for data integration and to remove batch effects. Cells were visualized using tSNE algorithm. Cell types were identified based on the indicated marker genes (refer to the Results section). Marker genes for each cluster were determined using the ‘FindAllMarkers’ function with the Wilcoxon rank-sum test. Only genes with |avg_logFC | > 0.4, min.pct > 0.5, and an adjusted P value < 0.05 were considered as marker genes. Gene enrichment analysis of cell type-specific markers, based on the Gene Ontology (GO) database, was conducted using clusterProfiler (Version 4.8.3)⁶⁷.

Pseudotime analysis

The Seurat object of LCs was converted to a CellDataSet object to facilitate import into the monocle package (v2.28.0). Pseudotime analysis was performed using the default settings. Aging-associated DEGs of LCs were identified using the ‘FindMarkers’ function with default settings. LCs were arranged according to pseudotime and visualized using the pheatmap package (version 1.0.12).

Gene set enrichment analysis

Gene Set Enrichment Analysis (GSEA) on Gene Ontology (GO) terms was performed using the ‘gseGO’ function in the clusterProfiler R package (v4.8.3). Enrichment was considered significant for terms with adjusted P value < 0.05 and |Normalized Enrichment Score (NES)| > 1. The results were visualized using ggplot2 (version 3.5.0).

Bulk RNA-seq

Raw RNA sequencing data from vehicle- or Hymeglusin-treated MLTC-1 cells were pre-processed using fastp (version 0.23.4) and FastQC (version 0.12.1). Paired-end clean reads were mapped to the mm10 mouse reference genome using HISAT2 (version 2.2.1). Gene expression profiles were quantified using featureCounts (version 2.0.6) to obtain read counts for subsequent analysis. Differential gene expression between groups was analyzed using the DESeq2 package (version 1.40.2). Differentially expressed genes (DEGs) were selected based on a |log2(Fold Change)| > 1 and an adjusted P value < 0.05 for statistical significance.

Ketone bodies measurement

The concentration of BHB in the blood was quantitatively measured using a blood ketone meter (Abbott, USA). Blood samples were collected from the tail vein of mice using standard procedures. A small incision was made using a sterile scalpel and the tip of the strip was then brought into contact with a blood droplet from the tail vein. The strip drew the whole blood into the blood channel via capillary action. The meter began measuring the BHB concentration once the channel was completely filled.

To detect the concentration of AcAc in the blood, samples were collected from the mandibular vein of the mice and left to clot for 1 hour. The samples were then centrifuged at 5000 g for 10 minutes, and the supernatant serum was collected for subsequent analyses. Supernatants were tested according to the protocol provided by the colorimetric kit (Abcam, USA) manufacturer.

To detect the concentrations of ketone bodies in the testes, colorimetric kits were used to analyze AcAc (Abcam, USA) and BHB (Cayman, USA). Testes were collected and placed in 1 ml of saline with two 4 mm beads (Easybio, China). The tissues were homogenized using a high-throughput tissue lyser at a frequency of 50 Hz for 5 minutes. The homogenate was then centrifuged at 10000 g for 3 minutes, and 100 μl of the supernatant was collected and diluted tenfold with saline. The concentrations of ketone bodies were detected according to the manufacturer’s instructions.

To detect intracellular ketone bodies concentrations, colorimetric kits were used to analyze AcAc (Abcam, USA) and BHB (Cayman, USA). The cell culture medium was discarded, the cells were washed three times with saline, and then 1 ml of 80% methanol solution (pre-cooled at −20 °C for 6 hours) was added. After lysis, a cell scraper (NEST, China) was used to transfer the mixture to a 1.5 ml centrifuge tube. The mixture was vortexed for 5 minutes, then centrifuged at 13,000 g for 10 minutes at 4 °C, and the supernatant was collected for subsequent detection. Supernatants were tested according to the protocol provided by the colorimetric kit manufacturer.

Isolation of LCs

Isolation of LCs from wild-type mice was performed as previously described by our group⁶⁸. In brief, the tunica albuginea was carefully removed, and the testes were mechanically minced and enzymatically dissociated using 200 μg/mL DNase I (Gibco, USA) and 1 mg/mL Type IV Collagenase (Gibco, USA) in Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12; 1:1, Gibco, USA) at 37 °C for 15 minutes with gentle agitation (100 cycles/min). The collagenase activity was halted by adding DMEM/F12 supplemented with 10% fetal bovine serum (Gibco, USA), and the samples were rinsed twice with PBS. The resulting cell suspension was filtered through a 45 μm strainer to obtain a single-cell suspension. LCs were subsequently sorted using flow cytometry (Beckman Coulter, USA). For isolation of LCs from AAV-infected testes, the single-cell suspension was prepared according to the above method. GFP⁺ or mCherry⁺ LCs were subsequently sorted using flow cytometry (Beckman Coulter, USA).

Production of lentivirus

HEK293T cells were cultured in DMEM (Corning, USA) supplemented with 10% FBS, 1% Penicillin-Streptomycin (Gibco, USA), and 1% GlutaMAX (Gibco, USA). To package lentivirus for the construction of a HEK-293 reporter cell line, HEK293T cells were seeded in a T75 flask (NEST, China). When cells reached 90% confluency, a mixture of 4.5 µg of lentiviral transfer plasmid, 3 µg of psPAX, and 1.5 µg of pMD2.G was co-transfected using Lipo8000™ Transfection Reagent (Beyotime, China). Twelve hours after transfection, the media was replaced. At 48 and 72 hours post-transfection, the supernatant was collected and then filtered through a 0.45 µm sterile filter (Millipore, USA). Subsequently, the lentivirus was centrifuged at 50,000 g for 90 minutes, resuspended in 0.5 mL PBS, and stored at −80 °C.

Construction of gene knockdown cell line

MLTC-1 cells were cultured in RPMI 1640 (Corning, USA) supplemented with 10% FBS (Gibco, USA), 1% Penicillin-Streptomycin (Gibco, USA), and 1% GlutaMAX (Gibco, USA). Sh-RNA sequences (Supplementary Table 2) were cloned into pLKO.1-copGFP-PURO plasmids by Singke Biotech. The plasmids were used for the production of lentiviral vectors. At 70% confluence, the cells were transfected with lentivirus containing shRNA sequences in the presence of 8 µg/mL polybrene. Four days post-transduction, the cells were digested with 0.25% trypsin at 37 °C for 3 minutes and then filtered through a 45 μm strainer to obtain a single-cell suspension. GFP⁺ cells were subsequently sorted using flow cytometry (Beckman Coulter, USA) for further experiments.

RNA extraction, cDNA synthesis, and quantitative RT-PCR

Total RNA was extracted from testes or cells using the RNeasy Mini Kit (Yibin, China) according to the manufacturer’s protocol. RNA purity was assessed by measuring the 260/280 ratio (1.87–1.93) and the 260/230 ratio (2.18–2.34) using a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific, USA). Reverse transcription was performed using the NovoScript® 1st Strand cDNA Synthesis Kit (Novoprotein, China). Quantitative RT-PCR was carried out using SYBR PCR Master Mix (Roche, CH) and a LightCycler 480 Detection System (Roche, CH). Primer validation was performed by generating a melting curve to confirm a single peak, thereby ruling out the possibility of non-specific product or primer dimer formation. β-actin served as an internal control, and target gene expression levels were calculated using the ΔCt method and expressed relative to β-actin. The primers are listed in the Supplementary Table 3.

Induce cell premature senescence and treatment with AcAc or BHB

To induce premature senescence, H₂O₂ (Hengjian, China) was added to the medium at a concentration of 100 μM. After 24 hours of exposure to H₂O₂, BHB or AcAc was added to the culture medium. All groups were analyzed on the fourth day. Doxorubicin (MCE, USA) was dissolved in DMSO, stored at −20 °C, and then added to the culture medium at a concentration of 150 nM with a 24-hour interval between treatments. 24 hours after adding doxorubicin, BHB or AcAc was added to the culture medium. All groups were analyzed on the fifth day. For inducing premature senescence by radiation, cells were exposed to 8 Gy of ionizing radiation using an RS 2000 X-ray Biological Irradiator (Rad Source, USA) at a dose rate of 1.185 Gy/min. 3 days post-irradiation, BHB or AcAc was added to the culture medium. Subsequently, the cells were cultured under standard conditions. 3-Hydroxybutyric acid sodium (MCE, USA) and Lithium acetoacetate (Sigma, USA) were both dissolved in saline, stored at −80 °C, and added to the culture medium at a concentration of 5 mM.

ChIP-qPCR

A ChIP assay kit (CST, USA) was employed to assess the binding of H3K9ac to the promoters of Foxo3a. MLTC-1 cells were seeded in a T75 flask (NEST, China). Following treatment, cells at 70% confluence were fixed with 1% formaldehyde on ice to cross-link proteins to chromatin DNA. After washing, the chromatin DNA was enzymatically sheared to produce DNA fragments ranging from 200 to 1,000 bp. Subsequently, DNA recovery was performed according to the manufacturer’s protocol. The recovered DNA from the reverse cross-linking was utilized for qPCR analysis. An equal amount of sheared DNA without antibody precipitation was processed for reverse cross-linking and utilized as an input control. The primers are listed in the Supplementary Table 4.

Western blot

For protein extraction from cultured cells, cells were washed three times with ice-cold PBS. Subsequently, the cells were lysed directly in RIPA buffer (Beyotime, China) for 30 min, followed by centrifugation at 13000 g for 10 min at 4 °C. Each supernatant was recovered as a total cell lysate. For protein extraction from tissue, testes were grinded in liquid nitrogen and subsequently lysed with RIPA Buffer (Beyotime, China). The lysates were placed on ice for 30 minutes and then centrifuged at 12,000 g for 15 minutes at 4 °C. Supernatants containing proteins were collected, and protein concentrations were determined using Pierce™ BCA Protein Assay Kits (Thermo Fisher, USA). The lysates were resolved by SDS polyacrylamide gel electrophoresis and then transferred onto 0.45 µm pore-sized polyvinylidene fluoride membranes (Millipore, USA). Subsequently, the membranes were blocked with 5% milk (Phygene, China), incubated with primary antibodies overnight at 4 °C, washed with tris-buffered saline Tween-20 (TBST), and incubated with HRP-conjugated secondary antibodies for 1 hour at room temperature. Finally, the signals were visualized using an ECL substrate (NCM, China). Immunoreactivity was semi-quantitatively detected by ChemiDoc Imaging Systems (Bio-Rad, USA). The primary and secondary antibodies are listed in the Supplementary Table 5.

Immunofluorescence staining

For immunofluorescence staining of sections, the tissues were fixed in 4% paraformaldehyde (PFA; Phygene, China) at 4 °C for 4 hours, dehydrated with 30% sucrose (Sangon Biotech, China), and sectioned at a thickness of 10 μm. The sections underwent antigen retrieval using Citrate Antigen Retrieval Solution (Beyotime, China), followed by permeabilization with 0.2% Triton X-100 (Sigma, USA) for 15 minutes. After blocking with 2% BSA (Sigma, USA) and 10% goat serum (Boster, China) in PBS for 45 minutes at room temperature, sections were incubated overnight with primary antibodies. Subsequently, sections were washed with PBS five times, incubated with appropriate secondary antibodies for 60 minutes at room temperature, and counterstained with DAPI (Gibco) for 5 minutes. Images were captured using an LSM800 confocal microscope (Zeiss, Germany) or a Nikon C2 confocal microscope (Nikon, Japan). For immunofluorescence staining of cells, the cells were seeded in a 24-well plate and fixed in 4% PFA (Phygene, China) for 15 minutes. Fixed cells were permeabilized with PBS containing 0.2% Triton X-100 for 15 minutes at room temperature. Non-specific antibody binding was blocked with 2% BSA and 10% goat serum in PBS for 45 minutes at room temperature, followed by overnight incubation with relevant primary antibodies at 4 °C. Next, cells were washed three times with PBS and incubated with appropriate secondary antibodies for 45 minutes at room temperature. Finally, the nuclei were counterstained with DAPI for 5 minutes, and images were captured using an LSM800 confocal microscope (Zeiss, Germany) or a Nikon C2 confocal microscope (Nikon, Japan). The primary and secondary antibodies are listed in the Supplementary Table 6.

H&E staining

The testes were collected, fixed overnight in Bouin’s solution (Sigma, USA), dehydrated in 75% ethanol, embedded in paraffin, and then sectioned into 4 μm-thick slices. Subsequently, the paraffin-embedded sections were deparaffinized using xylene and then gradually rehydrated with a series of ethanol concentrations. For histological analysis, the prepared sections were stained with haematoxylin and eosin (H&E). The stained sections were examined using KF-PRO-120-H1 slide scanner (KFBIO, China).

SA-β-gal staining

According to the manufacturer’s protocol for the senescence-associated β-galactosidase kit (Beyotime, China), cells seeded in 12-well plates were washed with PBS and subsequently fixed in β-galactosidase staining fixative (Beyotime, China) for 15 minutes. After washing, the cells were incubated overnight with the working solution at 37 °C. Senescent cells were observed and counted under an DMi8 microscope (Leica, Germany) from three random fields of view. For SA-β-gal staining of tissue sections, the O.C.T (optimal cutting temperature compound)-embedded testicular tissues were cryosectioned at a thickness of 10 μm, and stored at −80 °C. Before SA-β-Gal staining, sections were thawed at RT and rinsed in PBS for 1 min, fixed in in β-galactosidase staining fixative (Beyotime, China) at RT for 15 min. The sections were then stained according to the manufacturer’s protocol for 6 hours. After staining, the sections were retrieved using Citrate Antigen Retrieval Solution (Beyotime, China), permeabilized with 0.2% Triton X-100 (Sigma, USA) for 15 minutes, blocked with 2% BSA (Sigma, USA) and 10% goat serum (Boster, China) in PBS for 45 minutes at room temperature, and then subsequently incubated overnight with primary antibodies. Following incubation, the sections were washed five times with PBS, incubated with appropriate secondary antibodies for 60 minutes at room temperature, and images were captured using DMi8 microscope (Leica, Germany).

Computer-aided semen analysis

Semen samples were analysed as previously reported³⁰. In brief, two cauda epididymides were harvested from each mouse, incised with micro scissors, and then incubated in 1 mL DMEM/F12 containing 0.5% BSA for 15 min at 37 °C to allow for sperm release. The tissue was removed, and sperm samples were analysed using a Hamilton Thorne’s Ceros II system. At least six fields were assessed for each sample, and the sperm concentration and percentages of motile and progressively motile spermatozoa were determined.

Sex hormone assays

Sex hormone concentrations were assayed as previously reported by our group⁶⁹. In brief, blood was collected from the mandibular vein of the mice and left to clot for 1 hour. The samples were then centrifuged at 5000 g for 10 minutes, and the supernatant serum was collected for subsequent analyses. For intratesticular testosterone concentration detection, testes were extracted from the mice and placed in 1 ml of saline with two 4 mm beads (Easybio, China). A high-throughput tissue crusher was used to crush the testicular tissue at a frequency of 50 Hz for 5 minutes. Subsequently, the sample was centrifuged at 10,000 g for 3 minutes, and 100 μl of the supernatant was collected and diluted tenfold with physiological saline. Serum and testes samples were collected at the indicated time points and stored at −80 °C until analysis. Testosterone levels were measured using a chemiluminescent immunoassay (CLIA) (KingMed Diagnostics Group Co, Ltd, China). The minimum detectable dose of testosterone is 0.01 ng/mL. The concentration of serum LH and FSH was calculated based on the instructions provided by the manufacturer using an Elisa kit (Cloud-clone, China). The concentration of serum Insl3 was calculated based on the instructions provided by the manufacturer using an Elisa kit (Phoenix, USA). To detect the ability of cells to secrete progesterone, MLTC-1 cells were washed three times with PBS, and then fresh RPMI 1640 medium containing 10% FBS and 1% GlutaMAX was added. After 4 hours, the culture supernatant was collected for subsequent detection, and the cells were digested with 0.25% trypsin for cell counting using a computer-assisted system (Nexcelom, USA).

Gene delivery in animal models

For Hmgcs2 overexpression, the full-length mouse Hmgcs2 cDNA was cloned into the AAV-CAG vector. Wild-type aged mice (15-month-old) were intratesticularly injected with AAVDJ-Hmgcs2 for Hmgcs2 overexpression. For conditional knockout (CKO), the full-length Cre cDNA was inserted into the AAV-CAG vector. Eight-week-old male Hmgcs2^fl/fl mice were intratesticularly injected with AAVDJ-Cre for conditional knockout. Following sequencing-based verification, the constructed vector was custom-packaged, purified, and titrated by Packgene Bioscience (Guangzhou, China). The viruses were stored at −80 °C until use. For gene delivery, mice were anesthetized with Avertin (250 mg/kg) via intraperitoneal injection. The injection site was sterilized with ethanol and a topical application of povidone-iodine. The mice testes were stabilized through the scrotum with fingers, and AAV particles were injected into the testes using an insulin syringe (20 μL per testis; 2 × 10⁹ genome copies per testis). After injection, the needle was retained inside the tissue for 30 seconds to allow the virus to disperse within the testis.

BHB supplementation in aged mice

Wild-type aged mice (18-months-old) received sodium/potassium salt supplementation in their drinking water. The KetoForce solution is a BHB salt with 1.6 g of sodium and potassium per 11.7 g of BHB. 10 ml BHB salt formulation (KetoForce) was administered in 290 ml of drinking water for BHB supplementation. For the control group, 3.2 g of sodium chloride and 2.4 g of potassium chloride were added to 250 ml of water. Both the experimental group and the control group were supplemented with equivalent sodium and potassium.

Statistical analysis

All data were subjected to statistical analysis using IBM SPSS Statistics version 25.0 software (IBM SPSS Statistics, Armonk, NY, USA) and the results were visualized using GraphPad Prism 9 software (GraphPad Software, La Jolla, CA, USA). The bioinformatics data were analyzed using R (version 4.0.3). Gene Ontology (GO) enrichment analysis was assessed using a one-sided hypergeometric test with Benjamini-Hochberg (BH) correction. Gene Set Enrichment Analysis (GSEA) was performed using a two-sided permutation test with BH correction. Differentially expressed genes (DEGs) between Leydig cells (LCs) from young and old groups were identified using a two-sided Wilcoxon rank-sum test with Bonferroni correction. For experiments excluding RNA-seq data, statistical differences between samples were assessed with t-tests, one-way analysis of variance (ANOVA), Mann-Whitney U test, or Kruskal-Wallis test. Differences were considered significant when p < 0.05, and p values were indicated in figures. The detailed statistical analyses have been provided in the Source Data file.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

The scRNA-seq data generated in this study have been deposited in the GEO database under accession code GSE270931. The bulk RNA-seq data generated in this study have been deposited in the GEO database under accession code GSE287203. The rest of the data generated in this study are included in the published article and supplementary files. Source data are provided with this paper.

References

Finkelstein, J. S. et al. Gonadal steroids and body composition, strength, and sexual function in men. N. Engl. J. Med 369, 1011–1022 (2013).
Article CAS PubMed PubMed Central Google Scholar
Oatley, J. M. & Brinster, R. L. The germline stem cell niche unit in mammalian testes. Physiol. Rev. 92, 577–595 (2012).
Article CAS PubMed Google Scholar
Basaria, S. Reproductive aging in men. Endocrinol. Metab. Clin. North Am. 42, 255–270 (2013).
Article PubMed Google Scholar
Paul, C. & Robaire, B. Ageing of the male germ line. Nat. Rev. Urol. 10, 227–234 (2013).
Article CAS PubMed Google Scholar
Juul, A. & Skakkebaek, N. E. Androgens and the ageing male. Hum. Reprod. Update 8, 423–433 (2002).
Article CAS PubMed Google Scholar
Ramasamy, R., Wilken, N., Scovell, J. M. & Lipshultz, L. I. Effect of testosterone supplementation on symptoms in men with hypogonadism. Eur. Urol. 67, 176–177 (2015).
Article CAS PubMed Google Scholar
Bhasin, S. et al. Testosterone Therapy in Men With Hypogonadism: An Endocrine Society Clinical Practice Guideline. J. Clin. Endocrinol. Metab. 103, 1715–1744 (2018).
Article PubMed Google Scholar
Cappola, A. R. Testosterone therapy and risk of cardiovascular disease in men. JAMA 310, 1805–1806 (2013).
Article CAS PubMed Google Scholar
Halpern, J. A. & Brannigan, R. E. Testosterone Deficiency. JAMA 322, 1116 (2019).
Article PubMed Google Scholar
Bremner, W. J. Testosterone deficiency and replacement in older men. N. Engl. J. Med 363, 189–191 (2010).
Article CAS PubMed Google Scholar
Suryadevara, V. et al. SenNet recommendations for detecting senescent cells in different tissues. Nat. Rev. Mol. Cell Biol. 25, 1001–1023 (2024).
Article CAS PubMed Google Scholar
Lopez-Otin, C., Blasco, M. A., Partridge, L., Serrano, M. & Kroemer, G. Hallmarks of aging: An expanding universe. Cell 186, 243–278 (2023).
Article CAS PubMed Google Scholar
van Deursen, J. M. The role of senescent cells in ageing. Nature 509, 439–446 (2014).
Article ADS PubMed PubMed Central Google Scholar
Xu, M. et al. Transplanted Senescent Cells Induce an Osteoarthritis-Like Condition in Mice. J. Gerontol. A Biol. Sci. Med Sci. 72, 780–785 (2017).
CAS PubMed Google Scholar
Xu, M. et al. Senolytics improve physical function and increase lifespan in old age. Nat. Med 24, 1246–1256 (2018).
Article CAS PubMed PubMed Central Google Scholar
Palla, A. R. et al. Inhibition of prostaglandin-degrading enzyme 15-PGDH rejuvenates aged muscle mass and strength. Science. https://doi.org/10.1126/science.abc8059 (2021).
Niu, F. et al. Aberrant hyper-expression of the RNA binding protein GIGYF2 in endothelial cells modulates vascular aging and function. Redox Biol. 65, 102824 (2023).
Article CAS PubMed PubMed Central Google Scholar
Perheentupa, A. & Huhtaniemi, I. Aging of the human ovary and testis. Mol. Cell Endocrinol. 299, 2–13 (2009).
Article CAS PubMed Google Scholar
Kaufman, J. M., Lapauw, B., Mahmoud, A., T’Sjoen, G. & Huhtaniemi, I. T. Aging and the Male Reproductive System. Endocr. Rev. 40, 906–972 (2019).
Article PubMed Google Scholar
Johnson, L., Nguyen, H. B., Petty, C. S. & Neaves, W. B. Quantification of human spermatogenesis: germ cell degeneration during spermatocytogenesis and meiosis in testes from younger and older adult men. Biol. Reprod. 37, 739–747 (1987).
Article CAS PubMed Google Scholar
Lipsett, M. B. Physiology and pathology of the Leydig cell. N. Engl. J. Med 303, 682–688 (1980).
Article CAS PubMed Google Scholar
Lee, S. Y. et al. ROS inhibit the expression of testicular steroidogenic enzyme genes via the suppression of Nur77 transactivation. Free Radic. Biol. Med 47, 1591–1600 (2009).
Article CAS PubMed Google Scholar
Alfano, M. et al. Aging, inflammation and DNA damage in the somatic testicular niche with idiopathic germ cell aplasia. Nat. Commun. 12, 5205 (2021).
Article ADS CAS PubMed PubMed Central Google Scholar
Huang, D. et al. A single-nucleus transcriptomic atlas of primate testicular aging reveals exhaustion of the spermatogonial stem cell reservoir and loss of Sertoli cell homeostasis. Protein Cell 14, 888–907 (2023).
PubMed Google Scholar
Deng, Z. et al. Targeting dysregulated phago-/auto-lysosomes in Sertoli cells to ameliorate late-onset hypogonadism. Nat. Aging 4, 647–663 (2024).
Article CAS PubMed Google Scholar
He, S. & Sharpless, N. E. Senescence in Health and Disease. Cell 169, 1000–1011 (2017).
Article CAS PubMed PubMed Central Google Scholar
Cheng, C. W. et al. Ketone Body Signaling Mediates Intestinal Stem Cell Homeostasis and Adaptation to Diet. Cell 178, 1115–1131 e1115 (2019).
Article CAS PubMed PubMed Central Google Scholar
Puchalska, P. & Crawford, P. A. Multi-dimensional Roles of Ketone Bodies in Fuel Metabolism, Signaling, and Therapeutics. Cell Metab. 25, 262–284 (2017).
Article CAS PubMed PubMed Central Google Scholar
Darbey, A. et al. A comparison of in vivo viral targeting systems identifies adeno-associated virus serotype 9 (AAV9) as an effective vector for genetic manipulation of Leydig cells in adult mice. Andrology 9, 460–473 (2021).
Article CAS PubMed Google Scholar
Zhang, S. et al. AAV-mediated gene therapy restores natural fertility and improves physical function in the Lhcgr-deficient mouse model of Leydig cell failure. Cell Prolif. 57, e13680 (2024).
Article CAS PubMed PubMed Central Google Scholar
Gorgoulis, V. et al. Cellular Senescence: Defining a Path Forward. Cell 179, 813–827 (2019).
Article CAS PubMed Google Scholar
Zhang, C. et al. FOXO4-DRI alleviates age-related testosterone secretion insufficiency by targeting senescent Leydig cells in aged mice. Aging (Albany NY) 12, 1272–1284 (2020).
Article CAS PubMed Google Scholar
Sun, T. et al. Characterization of cellular senescence in doxorubicin-induced aging mice. Exp. Gerontol. 163, 111800 (2022).
Article CAS PubMed Google Scholar
Yang, D. et al. NKG2D-CAR T cells eliminate senescent cells in aged mice and nonhuman primates. Sci. Transl. Med 15, eadd1951 (2023).
Article CAS PubMed Google Scholar
Xie, Z. et al. Metabolic Regulation of Gene Expression by Histone Lysine beta-Hydroxybutyrylation. Mol. Cell 62, 194–206 (2016).
Article CAS PubMed PubMed Central Google Scholar
Shimazu, T. et al. Suppression of oxidative stress by beta-hydroxybutyrate, an endogenous histone deacetylase inhibitor. Science 339, 211–214 (2013).
Article ADS CAS PubMed Google Scholar
Benjamin, D. I. et al. Fasting induces a highly resilient deep quiescent state in muscle stem cells via ketone body signaling. Cell Metab. 34, 902–918 e906 (2022).
Article CAS PubMed PubMed Central Google Scholar
Yan, P. et al. FOXO3-Engineered Human ESC-Derived Vascular Cells Promote Vascular Protection and Regeneration. Cell Stem Cell 24, 447–461 e448 (2019).
Article CAS PubMed Google Scholar
Tsai, W. B., Chung, Y. M., Takahashi, Y., Xu, Z. & Hu, M. C. Functional interaction between FOXO3a and ATM regulates DNA damage response. Nat. Cell Biol. 10, 460–467 (2008).
Article CAS PubMed PubMed Central Google Scholar
Ohzono, H. et al. Targeting FoxO transcription factors with HDAC inhibitors for the treatment of osteoarthritis. Ann. Rheum. Dis. 82, 262–271 (2023).
Article CAS PubMed Google Scholar
Xia, K. et al. Single-cell RNA sequencing reveals transcriptomic landscape and potential targets for human testicular ageing. Hum. Reprod. 39, 2189–2209 (2024).
Article CAS PubMed PubMed Central Google Scholar
Santiago, J., Silva, J. V., Alves, M. G., Oliveira, P. F. & Fardilha, M. Testicular Aging: An Overview of Ultrastructural, Cellular, and Molecular Alterations. J. Gerontol. A Biol. Sci. Med Sci. 74, 860–871 (2019).
Article CAS PubMed Google Scholar
Zirkin, B. R. & Papadopoulos, V. Leydig cells: formation, function, and regulation. Biol. Reprod. 99, 101–111 (2018).
Article PubMed PubMed Central Google Scholar
Mendonca, B. B., Costa, E. M., Belgorosky, A., Rivarola, M. A. & Domenice, S. 46,XY DSD due to impaired androgen production. Best. Pr. Res Clin. Endocrinol. Metab. 24, 243–262 (2010).
Article CAS Google Scholar
Teerds, K. J. & Huhtaniemi, I. T. Morphological and functional maturation of Leydig cells: from rodent models to primates. Hum. Reprod. Update 21, 310–328 (2015).
Article CAS PubMed Google Scholar
Di Micco, R., Krizhanovsky, V., Baker, D. & d’Adda di Fagagna, F. Cellular senescence in ageing: from mechanisms to therapeutic opportunities. Nat. Rev. Mol. Cell Biol. 22, 75–95 (2021).
Article PubMed Google Scholar
Hanukoglu, I. Antioxidant protective mechanisms against reactive oxygen species (ROS) generated by mitochondrial P450 systems in steroidogenic cells. Drug Metab. Rev. 38, 171–196 (2006).
Article CAS PubMed Google Scholar
Robinson, A. M. & Williamson, D. H. Physiological roles of ketone bodies as substrates and signals in mammalian tissues. Physiol. Rev. 60, 143–187 (1980).
Article CAS PubMed Google Scholar
Matsuura, T. R., Puchalska, P., Crawford, P. A. & Kelly, D. P. Ketones and the Heart: Metabolic Principles and Therapeutic Implications. Circ. Res 132, 882–898 (2023).
Article CAS PubMed PubMed Central Google Scholar
Lin, Y. H. et al. Ketone bodies promote stroke recovery via GAT-1-dependent cortical network remodeling. Cell Rep. 42, 112294 (2023).
Article CAS PubMed Google Scholar
Tomita, I. et al. SGLT2 Inhibition Mediates Protection from Diabetic Kidney Disease by Promoting Ketone Body-Induced mTORC1 Inhibition. Cell Metab. 32, 404–419 e406 (2020).
Article CAS PubMed Google Scholar
Puchalska, P. et al. Hepatocyte-Macrophage Acetoacetate Shuttle Protects against Tissue Fibrosis. Cell Metab. 29, 383–398 e387 (2019).
Article CAS PubMed Google Scholar
Zou, X. et al. Acetoacetate Accelerates Muscle Regeneration and Ameliorates Muscular Dystrophy in Mice. J. Biol. Chem. 291, 2181–2195 (2016).
Article CAS PubMed Google Scholar
Kops, G. J. et al. Forkhead transcription factor FOXO3a protects quiescent cells from oxidative stress. Nature 419, 316–321 (2002).
Article ADS CAS PubMed Google Scholar
Chung, W. H., Dao, R. L., Chen, L. K. & Hung, S. I. The role of genetic variants in human longevity. Ageing Res Rev. 9, S67–S78 (2010).
Article CAS PubMed PubMed Central Google Scholar
Zhang, W. et al. A single-cell transcriptomic landscape of primate arterial aging. Nat. Commun. 11, 2202 (2020).
Article ADS CAS PubMed PubMed Central Google Scholar
Huffman, J. & Kossoff, E. H. State of the ketogenic diet(s) in epilepsy. Curr. Neurol. Neurosci. Rep. 6, 332–340 (2006).
Article CAS PubMed Google Scholar
Veech, R. L., Chance, B., Kashiwaya, Y., Lardy, H. A. & Cahill, G. F. Jr. Ketone bodies, potential therapeutic uses. IUBMB Life 51, 241–247 (2001).
Article CAS PubMed Google Scholar
Xie, S. et al. Dietary ketone body-escalated histone acetylation in megakaryocytes alleviates chemotherapy-induced thrombocytopenia. Sci. Transl. Med 14, eabn9061 (2022).
Article CAS PubMed Google Scholar
Monda, A. et al. Exploring the ketogenic diet’s potential in reducing neuroinflammation and modulating immune responses. Front Immunol. 15, 1425816 (2024).
Article CAS PubMed PubMed Central Google Scholar
Menyhart, O. & Gyorffy, B. Dietary approaches for exploiting metabolic vulnerabilities in cancer. Biochim Biophys. Acta Rev. Cancer 1879, 189062 (2024).
Article CAS PubMed Google Scholar
Zhu, D. et al. Intrapericardial Exosome Therapy Dampens Cardiac Injury via Activating Foxo3. Circ. Res 131, e135–e150 (2022).
Article CAS PubMed PubMed Central Google Scholar
Xiao, Y. et al. Comparative single-cell transcriptomic analysis across tissues of aging primates reveals specific autologous activation of ZNF281 to mitigate oxidative stress in cornea. Aging Cell 23, e14319 (2024).
Article CAS PubMed PubMed Central Google Scholar
White, R. R. et al. FOXO3a acts to suppress DNA double-strand break-induced mutations. Aging Cell 19, e13184 (2020).
Article CAS PubMed PubMed Central Google Scholar
Webb, A. E., Kundaje, A. & Brunet, A. Characterization of the direct targets of FOXO transcription factors throughout evolution. Aging Cell 15, 673–685 (2016).
Article CAS PubMed PubMed Central Google Scholar
Hao, Y. et al. Integrated analysis of multimodal single-cell data. Cell 184, 3573–3587 e3529 (2021).
Article CAS PubMed PubMed Central Google Scholar
Yu, G., Wang, L. G., Han, Y. & He, Q. Y. clusterProfiler: an R package for comparing biological themes among gene clusters. OMICS 16, 284–287 (2012).
Article CAS PubMed PubMed Central Google Scholar
Luo, P. et al. An autofluorescence-based isolation of Leydig cells for testosterone deficiency treatment. Mol. Cell Endocrinol. 535, 111389 (2021).
Article CAS PubMed Google Scholar
Xia, K. et al. Precise Correction of Lhcgr Mutation in Stem Leydig Cells by Prime Editing Rescues Hereditary Primary Hypogonadism in Mice. Adv. Sci. (Weinh.) 10, e2300993 (2023).
PubMed Google Scholar

Download references

Acknowledgements

This work was supported by National Key Research and Development Program of China(2022YFA1104100 to A.P.X.), National Natural Science Foundation of China (82430050 to A.P.X., 32130046 to A.P.X., 82371611 to K.X., 82371609 to C.D., 82171564 to P.L., 82101669 to K.X., 82301847 to H.C., 82171617 to Q.K., 82301796 to P.L.), Key Research and Development Program of Guangdong Province (2019B020235002 to Q.K.), Natural Science Foundation of Guangdong Province (2022A1515010371 to K.X.), Key Scientific and Technological Program of Guangzhou City (2023B01J1002 to A.P.X.), Pioneering talents project of Guangzhou Development Zone (2021-L029 to A.P.X.), Shenzhen Nanshan District Health System Science and Technology Major Project (NSZD2023049 to H.C.), Sanming Project of Medicine in Shenzhen Nanshan (SZSM202103012 to A.P.X.). Figures 1a, 2c, 3a, 4a, 5a, 6a, 7a and Supplementary Fig. 6d, h were created using BioRender (https://app.biorender.com/).

Author information

Author notes

These authors contributed equally: Congyuan Liu, Hao Peng, Jiajie Yu, Peng Luo.

Authors and Affiliations

Center for Stem Cell Biology and Tissue Engineering, Key Laboratory for Stem Cells and Tissue Engineering, Ministry of Education, Sun Yat-sen University, Guangzhou, Guangdong, China
Congyuan Liu, Hao Peng, Chuanfeng Xiong, Hang Fan, Yuanchen Ma, Suyuan Zhang, Lerong Zhao, Yuchen Zhang, Qiong Ke, Tao Wang, Weiqiang Li, Andy Peng Xiang & Kai Xia
National-Local Joint Engineering Research Center for Stem Cells and Regenerative Medicine, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong, China
Congyuan Liu, Hao Peng, Chuanfeng Xiong, Hang Fan, Yuanchen Ma, Suyuan Zhang, Lerong Zhao, Yuchen Zhang, Qiong Ke, Tao Wang, Weiqiang Li, Andy Peng Xiang & Kai Xia
Department of Urology and Andrology, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, Guangdong, China
Jiajie Yu, Cuifeng Yang & Chunhua Deng
Reproductive Medicine Center, The First Affiliated Hospital, Sun Yat-sen University, The Key Laboratory for Reproductive Medicine of Guangdong Province, Guangzhou, Guangdong, China
Peng Luo
Center for Stem Cells Translational Medicine, Shenzhen Qianhai Shekou Free Trade Zone Hospital, Shenzhen, Guangdong, China
Hong Chen & Xiaolu Guo
Brain Cognition and Brain Disease Institute, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, Guangdong, China
Hong Chen & Xiaolu Guo
State Key Laboratory of Ophthalmology, Zhong Shan Ophthalmic Center, Sun Yat-sen University, Guangzhou, Guangdong, China
Wangsheng Ou

Contributions

C.L., H.P., J.Y. and P.L. carried out the experiments and data analysis, design the experimental, and wrote the manuscript. J.Y. and Y.M. analyzed scRNA-seq data. P.L. and H.C. assisted with the animal experiments. S.Z. and H.P. helped with immunofluorescence staining. W.O., C.X., C.Y., L.Z., H.F., X.G. and Y.Z. assisted with animal experiments and genotyping. T.W., Q.K., W.L. and C.D. assisted with the experimental design and revised the manuscript. A.P.X. and K.X. conceived the project, supervised all experiments, and revised the manuscript. All authors fulfill the criteria for authorship.

Corresponding authors

Correspondence to Andy Peng Xiang or Kai Xia.

Ethics declarations

Competing interests

The authors declare no competing interests.

Peer review

Peer review information

Nature Communications thanks Ana Gomes and the other anonymous reviewer(s) for their contribution to the peer review of this work. A peer review file is available.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Source data

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.

Reprints and permissions

About this article

Cite this article

Liu, C., Peng, H., Yu, J. et al. Impaired ketogenesis in Leydig Cells drives testicular aging. Nat Commun 16, 4224 (2025). https://doi.org/10.1038/s41467-025-59591-8

Download citation

Received: 23 July 2024
Accepted: 17 April 2025
Published: 07 May 2025
DOI: https://doi.org/10.1038/s41467-025-59591-8