Mechanisms of Leydig Cell Aging and Obesity-Related Hypogonadism in Men: A Review

Physiological Functions of Testicular Leydig Cells

Testicular Leydig cells exist in the Leydig space between the spermatogenic tubules and are essential for synthesizing and secreting testosterone [1]. Testosterone, the primary male sex hormone, is crucial for male gonadal development, sperm production, and sexual health [2]. It also plays key roles in erectile function, semen quality, muscle mass, bone density, and cardiovascular health [3]. About 95% of testosterone is produced by Leydig cells, with the remaining 5% secreted by the adrenal glands [4].

Testosterone synthesis is regulated by the hypothalamic-pituitary-gonadal (HPG) axis. The hypothalamus secretes gonadotropin-releasing hormone (GnRH), which triggers the pituitary gland to release luteinizing hormone (LH). LH binds to receptors (LH-R) on Leydig cells, activating the cAMP signaling pathway, which in turn activates protein kinase A (PKA) and induces the expression of steroid acute regulatory protein (StAR). Cholesterol, the precursor for testosterone synthesis, is primarily derived from low-density lipoproteins (LDL) and high-density lipoproteins (HDL). LDLs are taken up by cells through LDL receptors and degraded in lysosomes to release cholesterol. HDLs are transported directly to Leydig cells via scavenger receptor class B type I (SR-BI). StAR functions on the outer mitochondrial membrane, binding to the translocator protein (TSPO) and facilitating the transport of cholesterol from the outer to the inner mitochondrial membrane. Cholesterol is converted to pregnenolone by cytochrome P450 side-chain cleavage enzyme (CYP11A1). Pregnenolone is then translocated to the endoplasmic reticulum, where it is converted to progesterone by 3β-hydroxysteroid dehydrogenase (3β-HSD) and finally to testosterone by 17β-hydroxysteroid dehydrogenase (17β-HSD) [5].

As men age, the function of testicular Leydig cells declines, leading to lower testosterone levels and impaired sexual function. Recent studies show that factors like obesity and lipid metabolism disorders can cause age-related changes in Leydig cells, impairing the expression of proteins involved in testosterone synthesis and disrupting their function. Therefore, preserving the normal function of Leydig cells is essential for male health. Further research into the pathogenesis of obesity-related LOH is crucial for improving men’s quality of life and developing new approaches for the early prevention and treatment of related diseases.

Definition and Epidemiology of Late-Onset Hypogonadism (LOH)

LOH, formerly referred to as “male menopause” or “partial androgen deficiency in aging males”, was officially named late-onset hypogonadism by the International Society for the Study of the Aging Male (ISSAM) in 2002 [6]. LOH is clinically characterized by decreased serum testosterone levels and reduced androgen sensitivity, significantly affecting the quality of life and mental health in middle-aged and elderly men. Symptoms include reduced sexual desire, erectile dysfunction, muscle loss, osteoporosis metabolic decline, anemia, and psychological issues such as low mood, anxiety, and depression [6].

LOH is a common clinical syndrome in aging men, although epidemiological data are limited. Prevalence rates vary between 7% and 40% across different countries, ethnic groups, and criteria [7]. A community-based survey reported a 9.1% prevalence of LOH in men aged 40–79 years, based on 3 criteria: reduced sexual function, decreased morning erections, and erectile dysfunction [8]. Among diagnosed LOH patients, the prevalence is 40% in men aged 45 and older and 50% in those aged 80 and older [9].

Over the past 2 decades, the global prevalence of overweight and obesity has risen, reaching 38% in 2023 [10,11]. Obesity-induced lipotoxicity can damage multiple organs, including gonadal function [12]. Epidemiological studies show that circulating testosterone levels decline by 0.5–15% annually in men over 30, while free testosterone levels decrease by 2–3% per year. In obese individuals, testosterone levels are further reduced [13].The European Male Aging Study (EMAS) found that obese men with a BMI over 30 kg/m2 have 30% lower testosterone levels than men with a BMI below 25 kg/m2 [14]. Our epidemiological investigations also reveal that different obesity-related metabolic phenotypes affect testosterone levels.

Compared to individuals with normal weight and metabolism, obese men with normal metabolism, normal-weight men with metabolic disorders, and obese men with metabolic disorders have progressively lower testosterone levels. The lowest levels were observed in obese men with metabolic disorders [15–17]. Impaired gonadal function leads to metabolic disorders, with both conditions often coexisting with diabetes, cardiovascular disease, metabolic syndrome, and osteoporosis [18–20].

Research on Testicular Leydig Cell Senescence and LOH

Aging, a physiological process, leads to a decline in the function of various organs, including the brain, heart, testes, ovaries, lungs, liver, pancreas, bones, muscles, adipose tissue, and skin [21]. It is also a significant risk factor for many diseases [22]. Testicular aging refers to the gradual decline in the structure and function of the testes with age. Key changes include thickening and fibrosis of the basement membrane, as well as a decrease in the number and function of Leydig cells, Sertoli cells, and germ cells, which impair male gonadal function [23].

Cell senescence refers to the irreversible cessation of cell division, leading to a permanent state of stagnation. While it promotes tissue remodeling during development and after injury, it can also reduce tissue regeneration, impair function, and contribute to inflammation and tumorigenesis [24]. Characteristics of cell senescence include cell cycle arrest, resistance to apoptosis, and the senescence-associated secretory phenotype (SASP) [25]. SASP refers to a combination of cytokines, chemokines, extracellular matrix proteases, growth factors, and other signaling molecules secreted by senescent cells. SASP has pro-inflammatory effects and promotes tumor development [26], and it also interacts with the immune response [27]. Markers of cell senescence include SA-β-gal expression, elevated levels of P16, P21, and P53, and increased ROS production [28]. The prevalence of aging-related diseases, including Alzheimer disease, lupus, cancer, and atherosclerosis, is increasing [29–32]. Cell senescence plays a critical role in tissue renewal, normal embryonic development, and cancer prevention [33]. Few studies have focused on testicular Leydig cell senescence, and further research is needed.

Recent advances in single-cell transcriptomics technology have enabled extensive analysis of aging-related changes in different cell types across various tissues and organs. In 2022, research teams led by Jingtao Guo, Guanghui Liu (Institute of Zoology, Chinese Academy of Sciences), and Jigang Wang (Institute of Qinghaosu Research, China) created single-cell transcriptomic landscapes of aged testes in humans, primates, and mice. These studies found that aging leads to a decline in the number of testicular Leydig cells, Sertoli cells, and peritubular myocytes, disrupting testicular tissue structure, thickening the basement membrane, and causing metabolic imbalance and inflammation. These changes can contribute to the decline of male gonadal function [34–36]. In aged mice, reactive oxygen species (ROS) levels increase in testicular Leydig cells, leading to cell apoptosis and shortened telomeres [35]. In aged primates, the number of differentially expressed genes (DEGs) associated with aging is highest in Sertoli cells, with a significant loss of Leydig cells, disrupting the testicular microenvironment and affecting spermatogenesis and testosterone synthesis [34]. In aged men, key components of the hedgehog (HH) signaling pathway, including PTCH1/2 and HHIP, regulate cholesterol transport and testosterone synthesis in Leydig cells, thereby influencing testosterone levels. HIP reduces testosterone levels by inhibiting HGF and FGF family members, which decreases cholesterol transport and testosterone synthesis [36]. Collectively, these studies highlight the critical role of testicular Leydig cell senescence in the decline of gonadal function.

Senescence of testicular Leydig cells can alter the testicular microenvironment, reducing serum testosterone levels and impairing germ cell development and spermatogenesis, ultimately leading to gonadal dysfunction [37,38]. In the steroidogenesis pathway, several changes related to decreased testosterone synthesis have been identified in senescent Leydig cells, including reduced cAMP production in response to LH, lower expression of cholesterol transport proteins (StAR and TSPO), and reduced activity of steroidogenic enzymes in the mitochondria and smooth endoplasmic reticulum [39,40]. Increased ROS and reduced antioxidant capacity disrupt oxidative balance, a hallmark of Leydig cell senescence [41]. Guo’s research group also explored the link between obesity and testicular aging using single-cell genomics. They found that obese elderly men had further declines in testosterone levels, along with exacerbated dysregulation of related gene expression [36]. Therefore, further research on the mechanisms of Leydig cell senescence in LOH and the role of obesity in this process is crucial.

This review focuses on the pathogenesis of obesity-induced testicular Leydig cell senescence and late-onset hypogonadism (LOH), and current therapeutic strategies to mitigate this process. The aim of this study was to understand the interaction between metabolic dysfunction and gonadal aging by synthesizing recent advances in oxidative stress, inflammation, and cellular aging mechanisms, thereby providing new insights into future treatment of aging-related diseases.

Research Advances in Mechanisms of Testicular Leydig Cell Senescence

The function of testicular Leydig cells is influenced by factors such as age, obesity, metabolic syndrome (MetS), diabetes, and cardiovascular diseases [42]. Reviews suggest that MetS can reduce testosterone synthesis and lead to hypogonadotropic hypogonadism in men through mechanisms like HPG axis disruption, mitochondrial-endoplasmic reticulum stress, aromatase activity, and estrogen overproduction [43]. However, there has been no specific research on the mechanisms of testicular Leydig cell senescence. In 2023, a review article in Cell highlighted the roles of oxidative stress, inflammation, mitochondrial dysfunction, autophagy, and endoplasmic reticulum stress in the aging process [44,45]. Here, we discuss perspectives on the mechanisms underlying testicular Leydig cell senescence (Figure 1).

Oxidative Stress and Inflammation

Oxidative stress and inflammation synergistically drive Leydig cell senescence. Age-related REDOX imbalances (increased pro-oxidants versus decreased antioxidants) and hypoxia-induced ROS overproduction [46] impair mitochondrial function through HIF-1α accumulation, while Nrf2/Keap1 pathway dysfunction exacerbates oxidative damage and decreases testosterone synthesis [47]. Chronic oxidative stress triggers DNA damage and telomere shortening, impeding testicular function and reducing male fertility [48]. In addition, oxidative stress can increase SASP secretion (IL-6, TNF-α) [49]. Obesity/MetS can also further exacerbate inflammation through elevated CRP/CCL2 [50,51] and activation of the p53/p21 pathway to form LOH [52,53]. Obesity exacerbates oxidative stress and inflammation by activating the P38 MAPK pathway, leading to increased expression of aging markers P16 and P21 and decreased expression of testosterone synthesizing proteins, and specific knockout of P38 MAPK can reverse the decline in Leydig cell function caused by obesity and aging, thereby increasing male testosterone levels [54].

Mitochondrial Dysfunction

Mitochondria play a pivotal role in testosterone synthesis [55]. Alterations in mitochondrial membrane potential, structural damage, and oxidative stress directly disrupt testosterone biosynthesis [56]. Obesity induces mitochondrial dysfunction through excessive ROS production and upregulation of CypD, reducing StAR expression and testosterone synthesis. Prolonged hypoxia also triggers Leydig cell apoptosis and mitochondrial dysfunction, lowering testosterone levels [57].

Autophagy

Autophagy is a process by which cells degrade damaged proteins and organelles via lysosomes and is essential for maintaining cellular homeostasis, but excessive autophagy can lead to oxidative damage and cell death [58]. Mitophagy is involved in early embryonic development, stem cell maintenance, differentiation, apoptosis, and inflammation, and is linked to various pathological conditions, including neurodegenerative diseases, cardiovascular disorders, metabolic dysregulation, and cancer [59]. Studies have shown that endocrine disruptors can induce Parkin-mediated mitophagy, disrupting steroid hormone synthesis [60]. Obesity impairs late-stage autophagy through abnormal fatty acid accumulation, damaging Leydig cell steroidogenesis [61]. Khawar et al demonstrated that loss of Sirt1 function hinders autophagy and cholesterol uptake, reducing testosterone synthesis [62]. In a 2024 study, Deng et al reported that long-term high-fat diets decrease lysosomal acidity in Sertoli cells, impairing autophagy and phagocytosis, and leading to symptoms associated with late-onset hypogonadism (LOH) [63].

Endoplasmic Reticulum (ER) Stress

The endoplasmic reticulum (ER) is responsible for protein synthesis and folding, and its homeostasis disruption induces ER stress [64]. Hypoxia and high-cholesterol diets can trigger ER stress, reducing the expression of StAR and 3β-HSD and inhibiting testosterone synthesis [65]. ER stress activates the CHOP and Caspase-12 pathways, leading to Leydig cell apoptosis [66]. Studies have found that a high-cholesterol diet causes cholesterol accumulation in Leydig cells and increases the expression of ER stress-related proteins BiP and ATF6. This is accompanied by reduced expression of steroidogenic enzymes, such as StAR, 3β-HSD, Cyp11a1, and Cyp17a1, resulting in decreased testosterone production [67]. Gao et al discovered that elevated ER stress, disrupted biological rhythms, reduced Leydig cell testosterone synthesis, and lower steroid gene expression and serum testosterone levels in aged male mice can contribute to LOH [68]. Other studies have shown that inhibiting ER stress can restore testosterone levels, while high-fat diets and iron overload syndrome impair testosterone biosynthesis through ER stress [69].

Treatment of LOH

NO FERTILITY REQUIREMENT:

Exogenous testosterone supplementation is a common treatment to maintain normal testosterone levels. Testosterone replacement therapy (TRT) is the standard treatment for LOH, restoring normal testosterone levels and improving hypogonadism symptoms [2]. Commonly used forms of testosterone replacement include oral testosterone undecanoate, intramuscular testosterone undecanoate injection, and transdermal preparations such as testosterone gel and patches [70]. However, long-term use can lead to adverse effects, including lipid metabolism disorders and cardiovascular diseases [66].

FERTILITY REQUIREMENT:

For LOH patients seeking fertility, common treatments include gonadotropins, aromatase inhibitors, and selective estrogen receptor modulators (SERMs), all of which stimulate testosterone secretion from Leydig cells and improve spermatogenesis [67]. Exogenous gonadotropins, such as human chorionic gonadotropin (hCG), human menopausal gonadotropin (hMG), highly purified FSH (hpFSH), and recombinant human FSH (rhFSH), are administered as intramuscular or subcutaneous injections to stimulate spermatogenesis and endogenous testosterone production in Leydig cells [68]. Aromatase inhibitors, such as anastrozole and letrozole, block the conversion of androgens to estrogens, commonly used to treat male infertility and hypogonadism. They stimulate spermatogenesis and increase testosterone levels [69,71]. Selective estrogen receptor modulators (SERMs), such as clomiphene citrate (CC), increase hormone levels by stimulating the pituitary gland and GnRH release. This enhances GnRH pulse frequency and amplitude, promoting the release of FSH and LH, which boosts sperm production and testosterone synthesis in Leydig cells [72].

Senolytic Therapy

Senolytic therapy is an emerging anti-aging treatment that uses small-molecule drugs to eliminate senescent cells, alleviating symptoms of age-related diseases and delaying aging [73]. Oral dasatinib and quercetin (D+Q) are classic senolytic therapies that selectively induce apoptosis in senescent cells [74].

Studies show that senolytic therapy can enhance the treatment of aging-related diseases and reduce the recurrence and mortality of conditions like atherosclerosis, diabetes, cancer, bone loss, and metabolic disorders [75,76]. Palmer et al used a transgenic mouse model treated with senolytics (D+Q) to examine how eliminating senescent cells affects obesity-induced adipose tissue dysfunction and glucose homeostasis [77].

Traditional Chinese Medicine (TCM) Treatment

In traditional Chinese medicine (TCM), LOH is often classified as “impotence” or “deficiency syndrome.” TCM treatments are tailored to different typologies based on individual diagnoses. Formulas such as Kidney-tonifying and Liver-dredging Decoction, Six-Ingredient Rehmannia Decoction, Shiziyuchun Pill, Yijing Formula, and Erxian Decoction are used to treat LOH patients with liver and kidney deficiencies, while Qi Zhen Zi Yin Combination addresses kidney and yin deficiencies [78]. Studies show that Strong Essence Tablet can enhance testosterone synthesis in Leydig cells by regulating the ROS/FOXO4/p53 pathway, alleviate Leydig cell senescence, and improve LOH [79]. Acupuncture and moxibustion therapy are thought to regulate hormone levels, including LH and testosterone. Clinical studies indicate that acupuncture at points such as Guanyuan, Taixi, Xuehai, Shenshu, and Zusanli can improve male fertility in patients with kidney yin deficiency [80]. Research on Chinese herbal components shows that dendrobium leaf extract reduces SHBG levels and increases serum testosterone in elderly LOH rats, alleviating LOH symptoms. Lycium barbarum increases serum testosterone by reducing cell apoptosis and oxidative damage [81]. Lycium barbarum polysaccharides (LBP) reduce testicular oxidative and ER stress in obese mice, improving testosterone levels [82]. Ginsenosides reduce testicular oxidative stress and inflammation, attenuating Leydig cell senescence, promoting testosterone synthesis, and delaying LOH onset and progression [53]. Angelica polysaccharides alleviate oxidative stress and improve serum testosterone in aged mice [83]. Additionally, studies show that acupuncture enhances both male and female reproductive function, improving sperm quality and follicle health [84].

Stem Cell Therapy

With the advancement of biotechnology, stem cell transplantation has become a promising approach for treating LOH. Recent research on stem cell therapies has primarily focused on testicular Leydig stem cells (SLCs), induced pluripotent stem cells (iPSCs), and mesenchymal stem cells (MSCs) [85].

SLCs can self-renew and differentiate into Leydig cells, and are located in the Leydig compartments of the testis. Studies show that SLC transplantation can partially restore testosterone production in aging Leydig cells [86]. Isolated SLCs can generate new Leydig cells to replace senescent ones after transplantation into the testis, thereby boosting testosterone production, which is crucial for treating LOH [87].

iPSCs are artificially induced pluripotent stem cells with unlimited self-renewal and differentiation potential. Research has shown that human iPSCs can be induced into mature Leydig-like cells, maintaining normal testosterone synthesis and secretion [88]. iPSC-derived Leydig-like cells can be used to study Leydig cell differentiation, providing strong evidence for LOH treatment [85].

MSCs are mesenchymal cells that can be isolated from bone marrow, placenta, adipose tissue, and other sources, with self-renewal and differentiation potential [89]. Houdling et al successfully induced human bone marrow-derived mesenchymal stem cells (BMSCs) to differentiate into Leydig cells, opening new possibilities for LOH therapy [90].

Treatment of Obesity-Related Hypogonadism

Obesity-related hypogonadism can be managed through a balanced diet, moderate exercise, and medication, improving gonadal function and overall health. Studies have shown that weight loss can restore testosterone levels [91]. Ding et al found that moderate exercise increases FSH, LH, and testosterone levels by improving the testicular microenvironment and demethylating sperm DNA genes [92]. Caloric restriction therapy promotes weight loss and delays aging by limiting intake while ensuring balanced nutrition [93]. Georgia et al found that dietary restriction reduced adiposity, improved metabolism, increased muscle and bone mass, and alleviated obesity-related hypogonadism [94]. Therefore, obese men with LOH can benefit from weight loss interventions [13]. Metformin improves testicular oxidative stress, boosts testosterone levels, and enhances fertility in obese mice [95]. Resveratrol enhances autophagy and reduces mitochondrial dysfunction in Leydig cells of aging mice, improving testosterone synthesis [96]. GLP-1 analogs, such as liraglutide, improve sex hormone levels and semen quality in obese male mice by activating the testis AC3/cAMP/PKA pathway, enhancing reproductive function [97]. Qu Shin et al found that metabolic surgery is an effective treatment for obesity-induced hypogonadism, increasing testosterone levels and improving male fertility postoperatively [98] (Figure 2).

Outlook and Discussion

PATHOPHYSIOLOGICAL MECHANISM OF OBESITY-RELATED LOH:

Obesity-related LOH typically manifests as hypogonadotropic hypogonadism, characterized by low testosterone levels, reduced sexual desire, erectile dysfunction, muscle atrophy, fatigue, and cognitive decline [1]. Gonadotropins LH and FSH may be normal or low in such cases. Normally, LH and FSH stimulate testosterone production in the testes. However, with age, their secretion declines, leading to reduced testicular stimulation and decreased testosterone synthesis [99]. Obesity-induced Leydig cell senescence further reduces testosterone synthesis, creating a vicious cycle that negatively impacts patients’ quality of life and contributes to LOH development [100]. Recent studies have increasingly focused on the role of Leydig cell senescence in obesity-related LOH. Obesity is not only an independent risk factor for low testosterone but also accelerates Leydig cell aging via various mechanisms, further exacerbating LOH [101].

Obesity is a key risk factor for LOH, as excess fat tissue aromatizes testosterone into estrogen, further lowering testosterone levels. Chronic low-grade inflammation associated with obesity can accelerate Leydig cell aging, worsening LOH symptoms [102]. Thus, obesity not only predisposes individuals to LOH but also promotes its progression by accelerating Leydig cell aging and enhancing inflammation.

INTERACTION BETWEEN LEYDIG CELL SENESCENCE AND TESTICULAR MICROENVIRONMENT:

As individuals age, Leydig cell senescence not only reduces testosterone synthesis but also increases local inflammation and tissue stiffness by secreting inflammatory and pro-fibrotic factors, further impairing testicular function. Oxidative stress, inflammation, extracellular matrix changes, and fibrosis are typical features of Leydig cell senescence, leading to reduced testosterone synthesis and lower testosterone levels [103]. Pro-inflammatory factors and extracellular matrix components released by senescent cells worsen the local microenvironment, creating a vicious cycle of obesity and LOH. Furthermore, senescent cells affect fat metabolism, exacerbating obesity and worsening LOH symptoms [104].

CURRENT TREATMENT STRATEGIES:

Current standard treatments for LOH include exogenous testosterone, HCG, and aromatase inhibitors [105]. For obesity-related LOH, treatments like diet control, metformin, resveratrol, and anti-aging therapies (eg, dasatinib and quercetin) improve interactions between obesity and aging Leydig cells, reduce inflammation, slow aging, and promote testosterone synthesis, alleviating LOH symptoms [106].

POTENTIAL OF TRADITIONAL CHINESE MEDICINE IN THE TREATMENT OF LOH:

Due to the limited efficacy and potential adverse effects of Western medicine treatments, traditional Chinese medicine has emerged as a complementary option for LOH. TCM improves the endocrine environment, slows aging, and enhances reproductive function by invigorating the kidneys, regulating qi and blood, and activating microcirculation. For example, Yishenshugan Decoction, a classic formula, has gained clinical recognition for improving male sexual function and raising testosterone levels. Research shows that Yishenshugan Decoction not only promotes sex hormone synthesis but also reduces chronic inflammation caused by obesity, improving LOH symptoms [79]. Chinese herbs like wolfberry, yellow essence, and Horny Goat Weed have been shown to enhance testosterone synthesis and sexual function, with mechanisms involving antioxidant, anti-inflammatory, and anti-aging effects [107].

While TCM shows promise in improving LOH symptoms, there is still a lack of high-quality clinical studies to confirm its long-term efficacy and safety. More clinical trials and mechanistic studies are needed to explore the potential of TCM in treating LOH and integrate modern medical theories to optimize treatment strategies.

FUTURE OUTLOOK:

While existing treatments have improved the management of obesity-associated LOH, several challenges remain, such as assessing the ability of stem cell therapy to improve the testicular microenvironment, prioritizing clinical trials of traditional Chinese medicines (such as Yishenshugan Decoction), and optimizing anti-aging targeted therapies (such as nanoparticle delivery) and safety are critical. Regulatory barriers, cost-effectiveness, and long-term safety issues must be addressed to move these therapies from the laboratory to the clinic. Future studies can focus on optimizing the targeting of anti-aging therapy, enhancing the microenvironment adaptability of stem cell therapy, and promoting mechanism research and clinical validation through interdisciplinary cooperation and technological innovation through the combination of traditional Chinese and Western medicine, so as to finally realize the transformation of diagnosis and treatment mode from symptom relief to etiological reversal to provide more accurate and safer treatment options for patients with obesity-related LOH.

Conclusions

In summary, obesity can promote obesity-related LOH by promoting Leydig cell aging, altering the endocrine environment, and increasing chronic inflammation. Understanding the role of testicular Leydig cell aging in obesity-related LOH and exploring early intervention measures for obesity and cell aging are key to improving the quality of life of men.