Targeting the CMKLR1‐Mediated Signaling Rebalances Immunometabolism State in Middle‐Age Testicular Macrophages

ABSTRACT

Age‐related male hypogonadism is often associated with obesity‐related metabolic disorders and impaired regulation of the testicular anti‐inflammatory microenvironment. However, how adipose‐mediated signals intersect with local testicular immunity remains unclear. In this study, single‐cell RNA sequencing (scRNA‐seq) of human and mouse testes identified a conserved CD206^LoMHCII^Hi macrophage subset, which undergoes CMKLR1‐mediated metabolic reprogramming toward glycolysis and pro‐inflammatory state during middle age. This immunometabolic shift is further found to impair spermatogenesis. CMKLR1 is identified as a viable target in vivo for restoring immunometabolic balance in aging testes. Systemic administration of a newly developed CMKLR1 antagonist peptide (P12C5) or non‐pharmacological intervention such as high‐intensity interval training (HIIT) rescued spermatogenesis in middle‐aged humans and mice, and reversed the pro‐inflammatory immunometabolic phenotype in testicular macrophages. Together, these findings validate CMKLR1 as a key modulator of testicular immunometabolism and a therapeutic target for mitigating age‐related immunometabolic dysfunction.

In middle age, testicular CMKLR1⁺ macrophages exhibited a pro‐inflammatory immunometabolic profile, mediated by adipose signals associated with high BMI. However, inhibition of CMKLR1 signaling, either through Cmklr1 genetic ablation or treatment with a CMKLR1 antagonist peptide, can reverse this phenotype. Notably, high‐intensity interval training (HIIT), as a non‐pharmacological intervention, also restored immunometabolic homeostasis by targeting CMKLR1⁺ testicular macrophages and mitigating the pro‐inflammatory state.

1. Introduction

Low‐grade chronic inflammation associated with aging, termed inflammaging, is one of the crucial hallmarks of mammalian aging [1, 2]. Tissue‐resident macrophages (TRMs) play essential roles in maintaining innate immune balance and tissue homeostasis [3, 4]. In the testes, an immune‐privileged organ, minimizing inflammatory responses is crucial for normal testicular function [5]. However, with increasing age, this balance is disrupted due to the activation of macrophages and elevated levels of pro‐inflammatory cytokines, accompanied by increased macrophage‐derived oxidative stress [6, 7]. Recent studies categorize testicular macrophages based on their differential CD206 and MHCII expression and their region‐specific distribution [8, 9, 10]. Among these subsets, the CD206^LoMHCII^Hi macrophage subset resides in the peritubular compartment and near Leydig cells within the interstitium [8, 10, 11], an area that constitutes the germ cell niche and modulates spermatogenesis. Although their precise role in the spermatogenic niche remains to be fully elucidated, testicular CD206^LoMHCII^Hi macrophages are likely reshaping the local immune landscape progressively during aging [6], thereby impairing spermatogenesis and steroidogenesis, negatively impacting male fertility and healthspan [6, 7].

Notably, age‐related hypogonadism is positively associated with BMI, suggesting a mechanistic link between systemic metabolic signals and male reproductive aging [12, 13]. Yet crosstalk between systemic metabolic signals and the local testicular immunity remains poorly understood. CMKLR1 is a G‐protein‐coupled receptor [14], also known as an adipose signal receptor for both the adipokine chemerin and lipid‐derived Resolvins. This receptor is tightly linked to increased BMI and obesity‐associated inflammation [15, 16]. This receptor is broadly expressed on macrophages in various tissues [17, 18, 19, 20]. Although the role of CMKLR1 signaling in macrophage‐mediated inflammation has been explored in some organs, its context‐specific function in testicular macrophages remains insufficiently defined [4, 21, 22, 23]. Notably, in murine and human testes, CMKLR1 is expressed in the interstitial compartment [24], a region enriched with testicular macrophages.

Beyond its role in immune regulation, CMKLR1 has been shown to influence cellular metabolism, modulating glycolysis, insulin sensitivity, lipogenesis, fatty acid oxidation (FAO), and oxidative phosphorylation (OXPHOS) in various cell types [25, 26, 27]. Macrophage metabolic reprogramming is defined as the dynamic shift in cellular metabolism in response to microenvironmental cues, which shapes their immune functions [28, 29]. Typically, classically activated macrophages (M1) rely primarily on lipid accumulation and glycolysis to support pro‐inflammatory responses, while alternatively activated macrophages (M2) rely primarily on fatty acid oxidation (FAO) and oxidative phosphorylation (OXPHOS) to promote tissue repair and inflammation resolution. Since macrophage metabolic reprogramming plays critical roles in chronic inflammation, tumor invasion, and tissue homeostasis, TRMs represent a potential target for studying immunometabolism [30, 31, 32, 33].

These considerations have led us to hypothesize that an adipose signal‐driven CMKLR1‐mediated immunometabolic regulatory pathway underlies the metabolic and functional reprogramming of testicular macrophages. Previous studies have shown that age‐related obesity emerges in middle‐aged male C57BL/6 mice, indicating adipose‐associated systemic metabolic signaling [34]. For example, as lipid‐derived mediators, Resolvins are known to modulate the immunometabolic state of brain‐resident microglia (brain TRMs); however, the role of adipose signal receptors in TRMs and particularly in male gonadal TRMs, remains largely unexplored [35].

In this study, we investigated CMKLR1‐driven immunometabolic reprogramming of testicular macrophages in the context of middle age. By integrating single‐cell transcriptomic analyses across male mice and humans, we identified age‐dependent alterations in the immunometabolic landscape of testicular TRMs, with particular emphasis on the role of CMKLR1 driving macrophage metabolic reprogramming. In addition, we employed a novel CMKLR1 antagonist peptide (P12C5), which restored immunometabolic homeostasis in aging testicular TRMs. Furthermore, we evaluated high‐intensity interval training (HIIT) as a non‐pharmacological strategy to rebalance immunometabolic homeostasis during aging. Collectively, our findings highlight CMKLR1 as a feasible therapeutic target for mitigating pro‐inflammatory immunometabolism and age‐related spermatogenic decline in the testis.

2. Results

2.1. Testicular CD206^LoMHCII^Hi Macrophages Exhibit Pro‐Inflammatory State and Glycolysis Metabolism Activation in High BMI Aging Men

To address the immunometabolic traits of testicular macrophages in young and aging populations, we conducted single‐cell transcriptomic analysis by using the publicly available GEO dataset (GSE182786) [36]. We analyzed three cohorts: young men with normal BMI (Young Adult Group, YG), older men with normal BMI (Older Group 1, OG1), and older men with high BMI (Older Group 2, OG2) (Figure S1A). We identified 267 testicular macrophages in YG, 449 in OG1, and 996 in OG2 (Figure S1B).

Testicular macrophages were annotated by using the markers CD206 and MHCII (HLA‐DOA), consistent with previous flow cytometric studies in mice [8, 9, 10]. We found that these markers were conserved in humans, allowing us to categorize testicular macrophages into two main subclusters: CD206 ^Lo MHCII ^Hi and CD206 ^Hi MHCII ^Lo (Figure S1C,D). DEG analysis comparing OG1 vs. YG and OG2 vs. YG revealed that CD206 ^Lo MHCII ^Hi macrophages in OG2 displayed the most pronounced transcriptomic changes (Figure S1E). Specifically, 619 genes were upregulated in CD206 ^Lo MHCII ^Hi macrophages in OG2 compared to YG (Figure S1E). Among these, we noticed that S100A8, which is an inflammation‐associated signal [37], was highly elevated (Figure S1E). Meanwhile, Lactate Dehydrogenase A (LDHA) was significantly upregulated (Figure S1E). KEGG pathway analysis of these genes highlighted enrichment for antigen presentation, phagosome formation, proteasome activity, and oxidative phosphorylation, all of which are associated with age‐related inflammatory and phagocytic processes, as well as immunometabolic status changes (Figure S1F). Downregulated genes were enriched in ribosomal function, indicating potential disruption of protein synthesis and cellular homeostasis (Figure S1F).

To further assess macrophage function, we compared the expression of specific inflammatory markers among groups. IL1B expression was significantly upregulated in OG2 CD206 ^Lo MHCII ^Hi macrophages, while it remained low in both YG and OG1 (Figure S1G). Similarly, the expression of TGFB1, a cytokine that participates in immune response and tissue homeostasis [38, 39], was significantly higher in OG2 than YG and OG1 (Figure S1G). In contrast, no significant changes in IL1B and TGFB1 were observed in CD206 ^Hi MHCII ^Lo macrophages (Figure S1G). Human testicular CD206 ^Lo MHCII ^Hi macrophages showed increased expression of the macrophage polarization marker STAT3 (Figure S1H). In addition to validating classical M1 and M2 macrophage markers such as MHCII and CD206, we also examined the expression of other commonly reported M1/M2‐associated genes, including CD64 (FCGR1A) and CD163, to comprehensively assess the transcriptional profiles and polarization states of macrophage subsets. The M1 marker CD64 was significantly elevated in OG2 CD206 ^Lo MHCII ^Hi macrophages compared to OG1 and YG (Figure S1H). Conversely, the M2 marker CD163 was significantly upregulated in OG1 but markedly downregulated in OG2 in CD206 ^Lo MHCII ^Hi macrophages (Figure S1H). No significant change of M1 and M2 marker expression was observed in CD206 ^Hi MHCII ^Lo macrophage (Figure S1G). In summary, these results indicate that the pro‐inflammatory phenotype in high BMI aging men is prominent, and this population is particularly vulnerable to metabolic dysregulation.

In OG2, the expression of the hypoxia‐inducible transcription factor HIF1A was significantly elevated in CD206 ^Lo MHCII^Hi macrophage subset rather than CD206 ^Hi MHCII^Lo macrophages (Figure S1I). To further characterize the immunometabolic profile of testicular tissue‐resident macrophages (TRMs), we analyzed the expression of key metabolic enzymes. In CD206 ^Lo MHCII ^Hi macrophages from OG2, genes involved in glucose uptake and anaerobic glycolysis (SLC2A3, HK1, PFKL, PKM, and LDHA) were significantly upregulated compared to OG1 and YG (Figure S2A), indicating enhanced glycolytic activity. Additionally, lipolytic pathway genes such as LIPA and CPT1B were markedly elevated (Figure S2B), suggesting a coordinated upregulation of both glycolysis and lipid catabolism in these cells. In contrast, the CD206 ^Hi MHCII ^Lo macrophages in OG2 showed a significant reduction in PFKL and the lipogenic enzyme FASN, and otherwise displayed minimal changes in immunometabolic gene expression (Figure S2C,D). Together, these findings indicated that testicular CD206 ^Lo MHCII ^Hi macrophages in high‐BMI aging men exhibit more profound alterations in glucometabolic pathways, potentially contributing to their heightened pro‐inflammatory phenotype.

2.2. Testicular CMKLR1⁺ Macrophages Exhibit Pro‐Inflammatory Property in High‐BMI Aging Men

To characterize the transcriptomic features of CMKLR1 positive macrophages in male cohorts, testicular macrophages were stratified into CMKLR1⁺ and CMKLR1 ⁻ populations (Figure 1A–C). Both subsets expressed canonical macrophage markers. Notably, in OG2, CMKLR1⁺ macrophages displayed elevated expression of the M1 marker CD64 and decreased expression of the M2 marker CD163, accompanied by upregulation of the polarization‐associated transcription factor STAT3 (Figure 1D). These pro‐inflammatory features were absent in CMKLR1 ⁻ macrophages (Figure 1D).

FIGURE 1.

Transcriptional and metabolic features of human testicular CMKLR1⁺ macrophages. (A) t‐SNE visualization showing the distribution of CMKLR1⁺ and CMKLR1 ⁻ macrophages in human testicular tissue. (B) Violin plots showing log‐normalized expression of macrophage lineage markers, including macrophage markers CD64 (FCGR1A) and CD14. (C) Feature plots showing expression of macrophage marker CD68, CMKLR1, and CCRL2. (D) Violin plots comparing log‐normalized expression of nuclear transcription marker STAT3, M1 marker CD64, and M2 marker CD163 between CMKLR1⁺ and CMKLR1 ⁻ macrophages across age groups. (E) Violin plot of chemoattractant CCL2 expression in CMKLR1⁺ vs. CMKLR1 ⁻ macrophages across age groups. (F) Violin plot showing expression of anaerobic glycolysis marker LDHA across age groups. (G) Violin plot showing expression of oxidative phosphorylation (OXPHOS) marker NDUFA3 across age groups. (H) t‐SNE plot displaying CMKLR1⁺CD206 ^Lo MHCII ^Hi and CMKLR1⁺CD206 ^Hi MHCII ^Lo macrophage subclusters across age groups. (I) Feature plots showing expressions of CD206 and MHCII (HLA‐DOA). (J) Violin plots of CD206 and MHCII expressing level in CMKLR1⁺CD206 ^Lo MHCII ^Hi macrophages. (K) Volcano plot illustrates differentially expressed genes (DEGs) in CMKLR1⁺CD206 ^Lo MHCII ^Hi macrophages across age groups. (L) Violin plots showing expression of pro‐inflammatory cytokine IL1B and tissue‐repairing cytokine TGFB1 in CMKLR1⁺CD206 ^Lo MHCII ^Hi macrophages across age groups. (M) Violin plots showing expression of other canonical M1 marker (CD64) and canonical M2 marker (CD163) in CMKLR1⁺CD206 ^Lo MHCII ^Hi macrophages across age groups. (N) Violin plots showing expression of anaerobic glucose metabolism enzymes in CMKLR1⁺CD206 ^Lo MHCII ^Hi macrophages across age groups.

In OG2, CMKLR1⁺ macrophages exhibited increased expression of the chemoattractant CCL2, a trend not observed in CMKLR1 ⁻ macrophages (Figure 1E). Moreover, CMKLR1⁺ macrophages in OG2 showed upregulated expression of the anaerobic glycolysis enzyme LDHA and reduced expression of the oxidative phosphorylation marker NDUFA3, in comparison to both OG1 and YG cohorts (Figure 1F,G). CMKLR1 ⁻ macrophages did not display similar metabolic alterations (Figure 1F,G). These results suggest that CMKLR1⁺ macrophages acquire a pro‐inflammatory phenotype and immunometabolic signature in the aging population with elevated BMI.

To further delineate the heterogeneity within CMKLR1⁺ macrophages, we performed subclustering based on the co‐expression of CD206 and MHCII. This analysis revealed two distinct populations: CMKLR1⁺CD206 ^Lo MHCII ^Hi and CMKLR1⁺CD206 ^Hi MHCII ^Lo macrophages (Figure 1H), with annotations and expression levels validated [9] (Figure 1I–J). Differentially expressed gene (DEG) analysis showed substantial transcriptomic divergence between OG1 and OG2 compared to the control group YG (Figure 1K). Particularly, in OG2, CMKLR1⁺CD206 ^Lo MHCII ^Hi macrophages exhibited 786 upregulated and 73 downregulated genes (Figure 1K), including a robust upregulation of CCL2 (Figure 1K). Furthermore, in OG2, CMKLR1⁺CD206 ^Lo MHCII ^Hi macrophages showed a clear pro‐inflammatory signature, including elevated IL1B, increased CD64, and reduced CD163 expression (Figure 1L,M). This inflammatory phenotype was coupled with enhanced glycolytic activity, as evidenced by upregulation of key glycolytic genes including SLC2A3, HK1, PFKL, PKM, and LDHA (Figure 1N). By contrast, CMKLR1⁺CD206 ^Hi MHCII ^Lo macrophages in OG2 exhibited minimal immunometabolic changes compared to YG (Figure 1K). However, the CMKLR1⁺CD206 ^Hi MHCII ^Lo macrophages exhibited a modest upregulation of the M2 marker CD163 in OG2, a pattern not observed in YG or OG1 (Figure S3A–3C).

2.3. Genetic Ablation of Cmklr1 Promotes an Anti‐Inflammatory Immunometabolism Profile in Testicular Macrophages of Adult Male Mice

Our previous studies have unveiled that Cmklr1 regulates lipid accumulation and testicular function in male mouse models [4, 40]. In this study, reduced adipocyte size was observed in Cmklr1 ^−/− male mice in comparison with Cmklr1 ^+/+ male controls (Figure S4A–D), consistent with previous studies [21, 41].

To investigate the immunometabolic traits of Cmklr1 ^⁺/⁺ vs. Cmklr1 ^−/− testicular macrophages and evaluate the role of Cmklr1 in immune‐metabolic regulation, we performed in vivo validation in adult male mice (Figure 2A). The gene‐editing strategy and genotyping results were described in the Methods section and validated (Figure 2B). Sub‐clustering analysis identified the major testicular macrophages (Figure 2C,D). A violin plot confirmed successful knockout of Cmklr1 in testicular macrophages at the transcriptomic level (Figure 2E).

FIGURE 2.

CMKLR1 deficiency confers anti‐inflammatory and immunometabolism transcriptomic profiles in testicular macrophages of young adult mice. (A) Schematic overview of the experimental workflow. (B) Genotyping results of male testicular tissue from 16‐week‐old Cmklr1^⁺/⁺ and Cmklr1^−/− C57BL/6 mice. (C) t‐SNE visualization of testicular macrophages from wild‐type and Cmklr1^−/− mice, combined with feature plot showing log‐normalized expression of Cmklr1 in testicular macrophages of 16‐week‐old Cmklr1^⁺/⁺ and Cmklr1^−/− mice. (D) The log‐normalized expression levels of Cmklr1 in 16‐week‐old Cmklr1^⁺/⁺ and Cmklr1^−/− mice testicular macrophages. (E) Heatmap of log‐normalized expression levels of representative pro‐inflammatory and anti‐inflammatory genes in testicular macrophages. (F) Heatmap showing expression profiles of canonical M1 and M2 macrophage markers. (G) Heatmap of key genes involved in glucose metabolism. (H) Heatmap of genes associated with lipid metabolism.

Cmklr1 ^−/− testicular macrophages exhibited significantly decreased expression of pro‐inflammatory markers such as Il1b and Tnf, and significantly increased expression of anti‐inflammatory cytokine Il4 (Figure 2E). Meanwhile, testicular macrophages showed significantly reduced expression of pro‐inflammatory cytokines, including Il1b and Tnf, along with elevated expression of the anti‐inflammatory cytokine Il4 (Figure 2E). Notably, Il10, another key anti‐inflammatory mediator, was paradoxically downregulated (Figure 2E). Furthermore, M2 markers (Cd206 and Cd163) were upregulated, while the M1 marker MHCII was downregulated (Figure 2F). MHCII and Cd206 showed the most substantial alterations. Glycolysis‐associated rate‐limiting enzymes (Hk1, Pfkp, Pkm, and Ldha) were upregulated in Cmklr1 ^−/− macrophages, while the average expression level of Pfkl was reduced (Figure 2G). Importantly, fatty acid transporter Cd36, lipid droplet lipase Lipa, and fatty acid oxidation (FAO)‐associated genes, including Cpt1a, Cpt1b, Cpt1c, and Cpt2, were uniformly upregulated in Cmklr1 ^−/− macrophages (Figure 2H). Intriguingly, the expression of hormone‐sensitive lipase Lipe was significantly reduced in Cmklr1 ^−/− testicular macrophages (Figure 2H). Together, these findings suggest that CMKLR1 plays an important role in creating an anti‐inflammatory and lipid‐inclined metabolic regulatory environment in testicular macrophages in adult male mice.

2.4. Testicular Cmklr1⁺Cd206^LoMHCII^Hi Macrophages Exhibit Pro‐Inflammatory Immunometabolism State in Middle‐Aged C57BL/6 Mice

Based on the established formula for adult age equivalence between mice and humans [42], 33‐ and 57‐week‐old male mice correspond approximately to 33‐ and 47‐year‐old (46.8 yrs old) humans, respectively (Figure 3A).

FIGURE 3.

Cmklr1 ⁺ Cd206 ^Lo MHCII ^Hi Testicular Macrophages Display Pro‐inflammatory Immunometabolism Transcriptomic Features in Middle‐Aged Male C57BL/6 Mice. (A) Schematic illustration of the experimental workflow. Testes and epididymis were included in the tissue collection. (B) Representative image of age‐related seminiferous tubules atrophy from SDY to SDO groups, with quantification of average seminiferous tubule diameters (SDY: 227.60 ± 2.20 µm; SDO: 197.56 ± 1.02 µm) and surface area (SDY: 35195.11 ± 647.09 µm²; SDO: 24611.08 ± 618.95 µm²). Each plot represents the average value from 20 tubules of a mouse testis cross‐sectional slice; bars represent the mean ± SD of three biological replicates (N = 3) per group. Homogeneity of variance was confirmed before analysis. Statistical significance was determined by an unpaired t‐test; Scale bar: 100 µm. (C) Feature plots showing the expression of macrophage markers in testicular macrophages. (D) Violin plots showing log‐normalized expression of pro‐inflammatory (Il1b) and tissue‐repairing (Tgfb1) cytokines in testicular macrophages. (E) Violin plots showing log‐normalized expression of macrophage polarization markers in testicular macrophages. (F) t‐SNE plot showing the distribution of Cmklr1⁺ and Cmklr1 ⁻ macrophages in SDY and SDO C57BL/6 mouse testis. (G) Split t‐SNE plots of testicular macrophages in SDY and SDO groups. (H) Volcano plot of significantly different expressed genes in SDY and SDO groups. (I) Violin plots showing log‐normalized expression of macrophage polarization markers (MHCII and Cd206) in testicular Cmklr1 ⁺ macrophages. (J) Violin plots showing log‐normalized expression of anaerobic glycolytic enzyme markers in testicular Cmklr1 ⁺ macrophages. (K) t‐SNE plot showing the distribution of Cmklr1⁺ macrophage subpopulations in SDY and SDO C57BL/6 mouse testis. (L) Feature plots showing expression of MHCII (H2‐Aa) and Cd206 in testicular macrophages. (M) Violin plot showing expression of the M2 marker Cd206 in Cmklr1⁺Cd206^LoMHCII^Hi macrophages. (N) Violin plot showing expression of the OXPHOS gene Ndufc2 in Cmklr1⁺Cd206^LoMHCII^Hi macrophages. (O) Violin plot showing expression of the FAO gene Cpt1b in Cmklr1⁺Cd206^LoMHCII^Hi macrophages. Statistical significance was indicated by ^∗ p < 0.05, ^∗∗ p < 0.01, ^∗∗∗ p < 0.001, ^∗∗∗∗ p < 0.0001; ns, not significant.

Consistent with a recent study [34], middle‐aged male mice exhibited progressive expansion of white adipose tissue, characterized by increased body weight and adipocyte size (Figure S5A–C), which is analogous to high‐BMI aging human cohorts. As male mice entered middle age, they exhibited features of age‐related hypogonadism, including histologically evident seminiferous tubule atrophy (Figure 3B) and a significant decline in epididymal sperm nuclei count (Figure S6A,B).

Single‐cell sequencing identified the testicular macrophage population (Figure 3C). At the transcriptomic level, testicular macrophages from middle‐aged testes displayed upregulation of the pro‐inflammatory cytokine Il1b and downregulation of cytokine Tgfb1 (Figure 3D). For tissue validation, we performed co‐localization of the testicular macrophages with pro‐inflammatory marker IL1β and assessed tissue‐wide β‐galactose staining in both the SDY and SDO groups (Figure S7). Elevated IL1β expression was observed in SDO testicular macrophages, whereas the surrounding Leydig cells increased β‐galactose signal in bright‐field imaging (Figure S7). Quantitative assessment confirmed an increased IL1β‐positive cell signal and ratio in the testicular interstitial part of the SDO, compared with the SDY group (Figure S8A,B).

Consistently, both M1 (MHCII) and M2 (Cd206) markers were significantly reduced in the sedentary older (SDO) group (Figure 3E). Based on Cmklr1 expression level, testicular macrophages were subclustered into Cmklr1⁺ macrophages and Cmklr1⁻ macrophages (Figure 3F). Split t‐SNE plot revealed that the Cmklr1‐ macrophage subpopulation specifically appeared in the SDO group (Figure 3G). In the meantime, cytokines (S100a4 and S100a11) and chemokines (Cxcl2 and Ccl5) were significantly up‐regulated in pro‐inflammatory Cmklr1+ testicular macrophages from SDO group (Figure 3H).

In middle‐aged male mice, Cmklr1⁺ macrophages exhibited a marked reduction in M2 marker Cd206, which was critical for anti‐inflammatory regulation (Figure 3I). Consistent with a shift toward a pro‐inflammatory metabolic state, Cmklr1⁺ macrophages showed elevated expression of key glycolytic enzymes, including Hk1, Pkm, and Ldha, indicative of enhanced anaerobic glycolysis (Figure 3J). Based on studies of testicular macrophages and age‐related changes in testicular macrophages [10, 43], Cmklr1⁺ macrophages were categorized into Cmklr1⁺Cd206 ^Hi MHCII ^Lo, Cmklr1⁺Cd206 ^Lo MHCII ^Hi , Cmklr1⁺Cd206 ^Lo MHCII ^Lo subclusters (Figure 3K,L). Cmklr1⁺Cd206 ^Lo MHCII ^Lo subcluster was specifically detected in SDO. Cmklr1⁺Cd206 ^Hi MHCII ^Lo and Cmklr1⁺Cd206 ^Lo MHCII ^Hi subclusters displayed an age‐related decline in the M2 marker (Figure 3M). The testicular Cmklr1⁺Cd206 ^Lo MHCII ^Hi subcluster displayed reduced expression of OXPHOS Complex I subunit Ndufc2 (Figure 3N). In the meantime, Cmklr1⁺Cd206 ^Lo MHCII ^Hi subcluster displayed decreased expression of FAO β‐oxidation gene Cpt1b (Figure 3O).

In the murine model, macrophages predominantly expressed Cmklr1 in vivo [44]. We assessed the homogeneity of the Cmklr1⁺Cd206 ^Lo MHCII ^Hi subcluster by the method of ROGUE [45]. Cmklr1⁺Cd206 ^Lo MHCII ^Hi subcluster was a highly homogeneous monocyte‐macrophage subcluster (ROGUE value = 0.909) (Figure S12A,B). Peritubular macrophages were associated with the secretion of IL1β (Figure S7) in the aging process, a mediator to promote secondary cellular senescence [46]. Transcriptomic signature indicated a reduced phagocytic capacity (Figure S12C), a feature associated with accelerated tissue deterioration [47].

Recent studies have unveiled that FABP4 is correlated with increased pro‐inflammatory status in aging macrophages, and is negatively associated with CMKLR1 expression [40, 48, 49]. For immunometabolic correlative validation, histological analysis also revealed that peritubular testicular macrophages exhibited increased FABP4⁺ signal intensity (Figure S9A), and the FABP4⁺ macrophage ratio was increased in SDO (Figure S9B).

2.5. CMKLR1 Antagonist P12C5 Rebalances Testicular Inflammaging and Rescues Degenerated Spermatogenesis

To assess the therapeutic potential of CMKLR1 inhibition in testicular aging, 12‐month‐old male C57BL/6 mice were administered either the CMKLR1 antagonist peptide P12C5 (N = 5) or a scrambled control peptide (N = 5) by intraperitoneal injection for four weeks (Figure 4A). P12C5 administration led to a significant reduction in body weight compared with controls, accompanied by improved glucose tolerance (Figure 4B–D). Histological analysis of randomly chosen samples revealed smaller adipocytes in subcutaneous, gonadal, and brown adipose tissues following P12C5 treatment (Figure S10A–D). However, thermogenic UCP1 expression in WAT and BAT remained unchanged (Figure S11A–D).

FIGURE 4.

CMKLR1 antagonist P12C5 restores gonadal and metabolic function in middle‐aged male mice. (A) Schematic of the experimental workflow. (B) Body weight change of 4 weeks in Scramble and P12C5 groups relative to baseline (Day 0) body weight. Each plot represented the mean, and bars represent mean ± SD of five biological replicates (N = 5) per group. Statistical significance was determined by the Mann‐Whitney U‐test. (C) Blood glucose levels of glucose tolerance test (GTT) in the Scramble and P12C5 groups after 4 weeks of administration. Each plot represented the mean, and bars represent mean ± SD of five biological replicates (N = 5) per group. Statistical significance was determined by Mann‐Whitney U‐test. (D) Area under the curve (AUC) of GTT response in both groups. Box plots display the median, interquartile range (IQR), and 1.5× whiskers. Statistical significance was determined by the Mann‐Whitney U‐test. (E) Ratio of testes to body weight in P12C5‐ and scramble‐treated mice. Box plots show the median, IQR, and 1.5× whiskers. P < 0.05 by Mann–Whitney test. (F) Representative images showing rescue of seminiferous tubule atrophy in P12C5‐treated mice compared with Scramble controls. Scale bar, 100 µm. (G) Quantification of average seminiferous tubules diameters in Scramble and P12C5 groups (Scramble: 197.88 ± 0.68 µm; P12C5: 213.21 ± 1.49 µm) and surface area (Scramble: 24916.89 ± 820.61 µm²; P12C5: 29403.41 ± 899.87 µm²). Each plot represents the average value from 20 tubules of an individual mouse testis cross‐sectional slice, bars represent mean ± SD of three biological replicates (N = 3) per group. Homogeneity of variance was confirmed before analysis. Statistical significance was determined by an unpaired t‐test; Scale bar: 100 µm. (H) t‐SNE plot of Cmklr1⁺ testicular macrophages in 12‐week‐old mice treated with P12C5 or scramble control. (I) Violin plots of log‐normalized expression of pro‐inflammatory cytokine Il1b in Cmklr1⁺ macrophages. (J) Heatmap of average log‐normalized expression of immunometabolism genes in Cmklr1 ⁺ Cd206 ^Lo MHCII ^Hi macrophages from P12C5‐ and scramble‐treated mice. (K) Same as (J), highlighting additional representative genes within Cmklr1 ⁺ Cd206 ^Lo MHCII ^Hi macrophages. (L) Heatmap of average log‐normalized immunometabolism gene expression in Cmklr1 ⁺ Cd206 ^Hi MHCII ^Lo macrophages following P12C5 or scramble treatment. (B‐C, D‐E, G, K‐L) Statistical significance was indicated by ^∗ p < 0.05, ^∗∗ p < 0.01, ^∗∗∗ p < 0.001, ^∗∗∗∗ p < 0.0001; ns, not significant.

Under these improved systemic metabolic conditions, testicular atrophy was alleviated in P12C5‐treated mice (Figure 4E). Histological examination revealed attenuation of seminiferous tubule degeneration and partial restoration of spermatogenic cell layers (Figure 4F,G). Consistently, epididymal spermatid nuclei counts were partially recovered following P12C5 treatment compared with the scrambled control (Figure S6C,D). These results indicated that CMKLR1 inhibition can partially mitigate spermatogenic decline in aging males, potentially through systemic metabolic improvement and regulation of local immune‐microenvironment homeostasis.

To explore the immunometabolic effects of CMKLR1 blockade, testicular macrophages were classified into two subsets: Cmklr1⁺Cd206 ^Lo MHCII ^Hi and Cmklr1⁺Cd206 ^Hi MHCII ^Lo populations (Figure 4H,I). Intriguingly, Il1b was upregulated in the Cmklr1⁺Cd206 ^Hi MHCII ^Lo population after P12C5 treatment, whereas the Cmklr1⁺Cd206 ^Lo MHCII ^Hi population exhibited reduced expression of Il1b (Figure 4J). Metabolic gene expression profiling revealed that Cmklr1⁺Cd206 ^Lo MHCII ^Hi macrophages exhibited reduced glycolytic activity (Slc2a3, Hk1, Pfkl, Pfkp, Pkm, and Ldha), together with upregulated expression of genes related to lipogenesis (Acly, Acaca, Dgat1), lipid uptake (Cd36), and fatty acid oxidation (Cpt1a) (Figure 4K). Interestingly, the Cmklr1⁺Cd206 ^Hi MHCII ^Lo macrophages exhibited significantly decreased expression of glycolytic enzyme Pfkp, alongside increased expression of lipogenic genes (Acaca, Dgat1) and lipolytic genes (Lipe and Cpt1a) (Figure 4L). This immunometabolic state change was coupled with a significant decline of M1 markers (Cd64, Cd80, and MHCII) in Cmklr1⁺Cd206 ^Hi MHCII ^Lo macrophages (Figure 4L).

For tissue‐level validations, we observed significantly reduced IL1β signal intensity and IL1β⁺ cell ratio in the testicular interstitial compartment after P12C5 i.p. administration, in comparison with the scrambled control (Figure S8C,D). P12C5 treatment also reduced the FABP4⁺ macrophage ratio in the testicular interstitium (Figure S9C,D).

Together, these results demonstrated that pharmacological inhibition of CMKLR1 rebalanced systemic and testicular macrophage immunometabolism, alleviated local inflammaging, and restored spermatogenic capacity by shifting macrophage metabolism from a glycolytic and pro‐inflammatory state toward oxidative and lipid‐utilizing phenotypes.

2.6. Middle Age Testicular Cmklr1+Cd206^LoMHCII^Hi Macrophages Acquire M2‐Like Traits after HIIT

Long‐term exercise has been reported to enhance stem cell proliferation, ameliorate inflammation, and alleviate age‐ and obesity‐ related testicular spermatogenic decline [50, 51, 52]. To test this, two distinct exercise patterns, moderate‐intensity continuous training (MICT) and high‐intensity interval training (HIIT), were applied to middle‐aged male mice (Figure 5A). Compared to age‐matched sedentary controls (SDO group), both MICT and HIIT significantly enlarged the seminiferous tubules’ surface area and tubular diameter (Figure 5B,C).

FIGURE 5.

High‐Intensity Interval Training (HIIT) Reverses Testicular Inflammaging and Restores Spermatogenesis Via the Cmklr1‐Dependent Immunometabolism Pathway. (A) hematic overview of the experimental workflow. (B) Representative H&E‐stained images of seminiferous tubules from middle‐aged sedentary (SDO), moderate‐intensity continuous training (MICTO), and high‐intensity interval training (HIITO) C57BL/6 mice. Scale bar: 100 µm. (C) Representative image of testis seminiferous tubules cross‐sectional slice of SDO, MICTO, and HIITO groups, with quantification of average seminiferous tubules diameters (SDO: 199.18 ± 1.48 µm; MICTO: 223.40 ± 2.24 µm; HIITO: 223.12 ± 1.54 µm) and surface area (SDO: 27149.34 ± 416.07 µm²; MICTO: 34318.07 ± 882.58 µm²; HIITO: 34702.17 ± 473.77 µm²). Each plot represents the average value from 20 tubules of a mouse testis cross‐sectional slice; bars represent mean ± SD of three biological replicates (N = 3) per group. The Brown–Forsythe test was used to confirm homogeneity of variance prior to analysis. Statistical significance among groups was evaluated using one‐way ANOVA followed by Tukey's post hoc multiple comparisons test; Scale bar: 100 µm. (D) t‐SNE plot showing testicular Cmklr1+ and Cmklr1‐ macrophage clusters in SDO, MICTO, and HIITO groups. (E) The dot plot of Cmklr1 in each cell cluster. (F) Feature plots of testicular macrophage markers in testicular macrophage. (G) Violin plots showing log‐normalized expression of pro‐inflammatory cytokine Il1b and anti‐inflammatory Tgfb1 in testicular macrophages across experimental groups. (H) Violin plots of macrophage polarization markers in testicular macrophages. (I) Violin plots of M2 marker Cd206 in testicular Cmklr1+ and Cmklr1‐ macrophage macrophages. (J) Violin plots of glycolytic gene Hk1 in testicular Cmklr1+ and Cmklr1‐ macrophage macrophages. (K) Violin plots of OXPHOS Complex II subunit genes in testicular Cmklr1+ and Cmklr1‐ macrophage macrophages. (L) t‐SNE plot showing testicular Cmklr1+ macrophage subclusters in SDO, MICTO, and HIITO groups. (M) Feature plots of testicular macrophage markers in testicular Cmklr1+ macrophage. (N) Violin plots of Sdhb in testicular Cmklr1+ macrophages subclusters. (O) Violin plots of M2 marker Cd206 in testicular Cmklr1+ macrophages subclusters.

As observed in aging mice, two major testicular macrophage populations were identified: Cmklr1⁺ and Cmklr1⁻ macrophages (Figure 5D,E). Il1b expression was significantly downregulated in testicular macrophages from HIIT‐treated mice compared to SDO (Figure 5G). Meanwhile, the Tgfb1 showed no significant change (Figure 5G). Furthermore, the expression of Cd206 was significantly increased after HIIT compared to SDO (Figure 5H,I). Only HIIT resulted in decreased glycolytic enzyme expression (Hk1) in Cmklr1⁺ macrophages, with no such changes observed in Cmklr1 ⁻ macrophages (Figure 5J). Succinate metabolism is critical for the macrophage pro‐inflammatory and anti‐inflammatory switch [28]. Notably, HIIT resulted in significant upregulation of multiple oxidative phosphorylation (OXPHOS)‐related genes (Sdhb and Sdhc) (Figure 5K).

Further sub‐clustering of Cmklr1⁺ macrophages revealed three subclusters: Cmklr1⁺Cd206 ^Lo MHCII ^Hi, Cmklr1⁺Cd206 ^Hi MHCII ^Lo and Cmklr1⁺Cd206 ^Lo MHCII ^Lo macrophages (Figure 5L,M). Importantly, HIIT selectively enhanced expression of OXPHOS gene Sdhb in Cmklr1⁺Cd206 ^Lo MHCII ^Hi macrophages (Figure 5N) and the M2 marker Cd206 (Figure 5N). Moreover, HIIT significantly increased Cd206 expression in both Cmklr1⁺Cd206 ^Lo MHCII ^Hi and Cmklr1⁺Cd206 ^Hi MHCII ^Lo subtypes (Figure 5O). Collectively, these results demonstrated that long‐term HIIT reversed the pro‐inflammatory transcriptional signature of Cmklr1⁺Cd206 ^Lo MHCII ^Hi macrophages, further enhanced OXPHOS activity, and supported the role for exercise in mitigating age‐related testicular dysfunction.

3. Discussion

In middle‐aged men (around their 50s), testicular macrophages exhibit altered immune profiles [53], which positively correlate with body mass index (BMI) [36]. Among the multiple mechanisms that maintain testicular immune privilege, tissue‐resident macrophages are essential regulators that help sustain the anti‐inflammatory milieu [10]. However, during aging, testicular macrophages undergo a shift toward a pro‐inflammatory state, leading to impaired spermatogenesis and disrupted testicular homeostasis [6]. Despite this, mechanisms linking systemic metabolism and testicular macrophage inflammation have remained largely unexplored.

Previous studies have revealed an intricate interplay between immune and metabolic regulation in adipocytes, which is mediated by CMKLR1 [21]. Extending this finding, our work identified a parallel mechanism of immunometabolic crosstalk in testicular macrophages. Here, we demonstrated that CMKLR1‐mediated signaling regulated the immunometabolic state of testicular macrophages in mice. Cmklr1 expression in the murine testis is enriched in macrophages in vivo [44]. Single‐cell transcriptomic analysis revealed that genetic ablation of Cmklr1 promoted an anti‐inflammatory phenotype, characterized by increased expression of Cd206 and Cd163 and decreased production of IL‐1β and TNF. In addition, CMKLR1 has been implicated in the regulation of both lipid and glucose metabolism [27, 40]. In macrophages, enhanced lipogenesis and glycolysis (Warburg effect) drove a pro‐inflammatory state, whereas lipolysis and oxidative phosphorylation (OXPHOS) were associated with anti‐inflammatory polarization [29]. Consistent with this, Cmklr1 ^−/− testicular macrophages exhibited enhanced lipid utilization, establishing CMKLR1 as an indispensable regulator of macrophage immunometabolism.

In human single‐cell datasets, CMKLR1 signaling was conserved, highlighting its translational relevance. CMKLR1⁺ testicular macrophages displayed an age‐ and BMI‐ associated pro‐inflammatory immunometabolic phenotype, whereas CMKLR1 ⁻ macrophages did not show obvious immunometabolic changes. These findings correspond to the age‐related adipose expansion observed in both middle‐aged male mice (7–12 months) and aging men with elevated BMI, linking systemic adiposity with local inflammaging. Cmklr1⁺ macrophages upregulated inflammation‐related chemokines and cytokines (e.g., CCL2 in humans, and S100a4, S100a11, Cxcl2, Ccl5 in mice) [54, 55, 56], and these chemokines and cytokines are identified as key mediators of inflammaging.

Testicular macrophages consist of two conserved major subtypes (CD206 ^Hi MHCII ^Lo and CD206 ^Lo MHCII ^Hi) in both humans and mice [9, 43]. In our study, CMKLR1 was expressed in both subsets. Aging, particularly in the context of high BMI, induced pronounced metabolic reprogramming in CMKLR1⁺CD206 ^Lo MHCII ^Hi macrophages (Cmklr1⁺Cd206 ^Lo MHCII ^Hi in mice), driving them toward a pro‐inflammatory, glycolytic phenotype characterized by increased IL1B, glycolytic enzymes (SLC2A3, HK1, PKM, LDHA), and M1 marker CD64, alongside decreased M2 marker CD163. Correspondingly, in mice, Cmklr1⁺Cd206 ^Lo MHCII ^Hi macrophages upregulated glycolysis‐related genes while downregulating transcripts involved in lipolysis and OXPHOS. These results indicated that age‐related adiposity synergistically reprograms Cmklr1⁺Cd206 ^Lo MHCII ^Hi testicular macrophages toward a Warburg‐like and pro‐inflammatory state, contributing to impaired spermatogenesis in both species. Flow cytometric analyses have identified Cd206 ^Lo MHCII ^Hi macrophages as a unique cell cluster [9, 57] in testicular macrophage subtypes. In line with this, our single cell transcriptomic results indicated that the Cmklr1⁺Cd206 ^Lo MHCII ^Hi subset was also a highly homogeneous macrophage subcluster. Through in vivo validation, we found that this immunometabolic shift appeared to be orchestrated through CMKLR1 signaling, thereby linking systemic metabolic cues to local immunometabolic regulation in the testis.

The CMKLR1‐blocking peptide P12C5 improved systemic metabolism (glucose tolerance and body weight) and partially restored spermatogenesis in middle‐aged mice. The pro‐inflammatory role of CMKLR1‐mediated signaling has also been reported in other tissues, including the brain, artery, and muscle [58, 59]. Consistently, inhibition of CMKLR1 in testicular macrophages reduced inflammatory activation and restored lipid‐metabolic signatures (increased Cpt1a, Acaca, Dgat1, and Cd206), supporting CMKLR1 inhibition as a feasible anti‐inflammatory intervention in aging.

On the other hand, we also explored exercise as a non‐pharmacological therapeutic strategy [61, 62, 63]. In our study, high‐intensity interval training (HIIT), a regimen combining both aerobic and anaerobic components, exhibited superior anti‐inflammatory effects. HIIT specifically targeted the Cmklr1⁺ macrophage subset, inducing pronounced metabolic reprogramming: it significantly reduced the glycolytic rate‐limiting enzyme Hk1, while enhancing anti‐inflammatory immunometabolism features, such as the M2 Marker Cd206 and Complex II genes. Specifically, Cmklr1⁺Cd206 ^Lo MHCII ^Hi macrophages displayed significant up‐regulation of these anti‐inflammatory immunometabolic traits. Consistently, a recent study shows that high‐intensity exercise enhances Resolvin E1 production and targets CMKLR1 to reduce inflammation‐associated pain in mice [64]. Our findings identified testicular CMKLR1⁺ macrophages as key responders to HIIT‐induced systemic signals, mediating the beneficial effects of exercise on the testicular immune environment.

Nevertheless, the use of whole‐body Cmklr1 knockout mice has inherent limitations. Although global Cmklr1 deletion may also affect other metabolic tissues (e.g. adipocytes and pancreatic β‐cells) [21, 65]. Our in vivo validation and the observed CMKLR1‐dependent immunometabolic and inflammatory changes in testicular peritubular macrophages support a macrophage‐intrinsic role for CMKLR1 signaling. Future studies employing myeloid‐specific Cmklr1 conditional knockout models will help further delineate its cell‐type‐specific functions.

Collectively, these findings indicated that CMKLR1 acted as an adipose‐signal‐responsive modulator of testicular inflammaging by shaping macrophage immunometabolic programming. Targeting CMKLR1, either through specific receptor blockade or via lifestyle interventions like HIIT, could be a feasible strategy to restore immunometabolic balance in the aging testis.

4. Conclusion

We identified a conserved CMKLR1⁺CD206^LoMHCII^Hi testicular macrophage subset across humans and mice that was responsive to adiposity‐associated signals in middle age. This subset acquired a pro‐inflammatory immunometabolic profile with testicular aging. The testicular macrophage pro‐inflammatory immunometabolic status can be reversed by treatment with CMKLR1 antagonist peptide or HIIT, thereby restoring an anti‐inflammatory phenotype in the middle‐aged murine model.

5. Experimental Sections

5.1. Animal Ethics Statement

All animal procedures were approved by the Institutional Animal Care and Use Committees (IACUC) of the Shenzhen Institute of Advanced Technology (SIAT) and Peking University Shenzhen Graduate School (PKUSZ), under the protocols SIAT‐IACUC‐20250414‐YYS‐NLDXZX‐ZJ‐A2822‐01 and AP20230915‐01, respectively. To generate the exercise‐intervened aging male rodent model, 30‐week‐old SPF C57BL/6 wild‐type (WT) male mice (N = 32) were purchased from BesTest Biotechnology Co., Ltd. (Zhuhai, Guangdong, China). For CMKLR1 functional validation, 16‐week‐old SPF C57BL/6 Cmklr1 ^+/+ (N = 3) and Cmklr1 ^−/− (N = 3) male mice were housed and obtained from the Laboratory Animal Research Center of PKUSZ. To evaluate the effect of CMKLR1 antagonist treatment, 12‐month‐old SPF C57BL/6 WT male mice (N = 10) were purchased from Charles River (China). All mice were housed at the SIAT Laboratory Animal Research Center under controlled conditions (12 h:12 h light/dark cycle, 30–70% humidity, and temperature 22–24 °C). Mice had ad libitum access to a standard maintenance diet (crude fat content 4%–6%, total calory 3.0–3.4 kcal/g) and water. After one week of acclimatization, mice were randomly grouped and ear‐tagged. All procedures were performed to minimize animal suffering. Animals were euthanized under isoflurane anesthesia prior to tissue collection.

5.2. Animal Models

5.2.1. Long‐Term Exercise Model

A total of 24 SPF 31‐week‐old male C57BL/6 mice were randomly assigned to:

1. Sedentary Older group (SDO, N = 8)

2. Moderate‐Intensity Continuous Training Older group (MICTO, N = 8)

3. High‐Intensity Interval Training Older group (HIITO, N = 8)

From 32 weeks of age, the MICTO and HIITO mice underwent treadmill training (ZHPT/5S, Zhenghua, Anhui, China), while SDO mice were placed in empty cages as controls. At 33 weeks of age, training is initiated. All training groups underwent one week of treadmill adaptation (5 m/min, 10 min/day, 0°). During the subsequent 24‐week training period:

1. Sedentary: Set in vacant chambers with 0 velocity.

2. MICT: Warm‐up (10 m/min, 1 min), followed by 45 min of continuous running at 17 m/min.

3. HIIT: Warm‐up (10 m/min, 1 min), followed by 9 cycles of 4 min at 15 m/min (moderate) and 4 min at 25 m/min (high).

After the main session, all mice underwent 1 min of recovery at 10 m/min.

Training volumes (distance) were matched between groups. Body weight was measured twice weekly throughout. At the end of 24 weeks, mice were sacrificed for serum and tissue collection.

5.2.2. Aging Model

To evaluate aging‐related changes, 30‐week‐old male C57BL/6 mice (N = 8) were housed under standard conditions for acclimatization (designated as Sedentary Younger group, SDY). At 32 weeks, SDY mice remained sedentary for one week and were sacrificed at 33 weeks for sample collection. Body weight was monitored three times per week. The SDO group served as aged controls for cross‐sectional comparison.

5.2.3. Genotyping

The Cmklr1 knockout allele (4117 bp deletion) was validated in previous studies [40]. Genomic DNA was extracted from tail tips and analyzed by PCR and gel electrophoresis. PCR product sizes: 495 bp for wild‐type, 293 bp for knockout alleles.

5.2.4. CMKLR1 Antagonist Treatment Model

To assess the in vivo effects of the CMKLR1 antagonist peptide P12C5 (FYSHSMPRLPPA) and Scramble control (SPYFRMHPLPSA), ten 12‐month‐old C57BL/6 male mice were randomly divided into:

1. 12C5‐treated group (N = 5)

2. Scramble peptide control group (N = 5)

Both peptides were synthesized by GL Biotech (Shanghai, China), stored at −20 °C in 50:50 DMSO/ddH₂O (2 mg/mL), and diluted 100× in saline before injection (final concentration: 20 µg/mL). The working solution was sterilized using a 0.22‐µm syringe‐driven filter (Millex, Merck). Mice were intraperitoneally injected with 0.2 mg/kg, three times per week. Body weight was monitored regularly prior to dosing (11:00 am–12:00 pm).

5.2.5. Intraperitoneal Glucose Tolerance Test (IPGTT)

Mice were fasted overnight (14–16 h), weighed, and blood was collected from the tail tip for baseline glucose (t = 0) using Accu‐Chek Performa (Roche). Then, 2 g/kg of 20% glucose was intraperitoneally injected. Glucose levels were measured at 15, 30, 60, and 120 min post‐injection. The working solution was sterilized using a 0.22‐µm syringe‐driven filter (Millex, Merck). Mice were refed post‐experiment. AUC (area under the curve) analysis was performed for glucose tolerance.

5.2.6. Body Weight Monitoring

Body weight was recorded twice weekly (randomly selected weekdays between 11:00 am–12:00 pm). After IPGTT, weights were recorded on the following Thursday and Friday.

5.3. Histological Analyses

5.3.1. Hematoxylin–Eosin (HE) Staining

Testes, epididymis, white adipose tissue (sWAT and gWAT), and brown adipose tissue (BAT) were collected from at least three mice per group. Tissues were fixed in 4% paraformaldehyde (PFA) at 4 °C for one week, dehydrated, cleared with xylene, embedded in paraffin, sectioned (5 µm), and stained with HE. Seminiferous tubule diameter and epithelial thickness were measured by light microscopy. Selection criteria for tubules followed established standards [66].

5.3.2. Immunofluorescence (IF) Staining

Testes were fixed in 4% paraformaldehyde (PFA) at 4°C for 12–16 h, followed by sequential dehydration in 10%, 20%, and 30% sucrose solutions until the tissues sank. Samples were then embedded in OCT compound (Sakura Tissue‐Tek, Cat.4583) and frozen at −80°C. Frozen sections (5 µm thick) were prepared using a cryostat (Leica CM1950) and mounted on glass slides (CITOTEST, 188105 W).

For immunofluorescence staining, sections were air‐dried for 10 min and permeabilized with 0.5% Triton X‐100 in 5% BSA 1xPBS buffer for 2 h at room temperature. Sections were incubated with primary antibodies (see Table S1) at 4°C overnight, washed three times with PBS, and subsequently incubated with appropriate fluorophore‐conjugated secondary antibodies (see Table S1) for 2 h at room temperature in the dark. Nuclei were counterstained with DAPI (1:1000) for 5 min, and slides were mounted using glycerol. Images were captured using a fluorescence microscope (OLYMPUS, BX53).

5.3.3. Senescence‐Associated β‐Galactosidase (SA‐βGal) Staining

Frozen testicular sections (5 µm thick) were equilibrated to room temperature and washed three times with 1× PBS (5 min each) to remove residual OCT compound. Senescence‐associated β‐galactosidase (SA‐βGal) activity was detected using the commercial detection kit (Biosharp, BL133A) according to the manufacturer's instructions. Sections were incubated at 37°C for 16 h, followed by three washes with 1× PBS (5 min each) to remove residual reagents. Subsequent co‐localization IF staining with other immunometabolic markers was performed as described in Section 4.3.2.

5.3.4. Immunohistochemistry (IHC) Staining

Testes were fixed in 4% paraformaldehyde overnight at 4°C, dehydrated through graded ethanol, and embedded in paraffin. Sections (5 µm thick) were deparaffinized in xylene, rehydrated through graded ethanol, and rinsed in distilled water. Antigen retrieval was performed by heating sections in citrate buffer (Beyotime, P0081) for 20 min using a microwave oven (medium–high power for 5 min; followed by medium power for 5 min, repeated three times), then allowing the slides to cool in buffer to room temperature. Endogenous peroxidase activity was blocked with 30% hydrogen peroxide in methanol for 30 min and washed via PBS buffer. Nonspecific binding was blocked with 5% BSA PBS buffer for 2 h at room temperature. The sections were then incubated with primary antibodies (See in Table S1) at 4°C overnight, followed by HRP‐conjugated secondary antibodies (See in Table S1) for 2 h at room temperature. Immunoreactivity was visualized using DAB substrate, and nuclei were counterstained with hematoxylin. Slides were dehydrated, cleared, and mounted with neutral resin.

5.4. Single‐Cell RNA Sequencing (scRNA‐seq)

5.4.1. Public Dataset Acquisition

Human testicular scRNA‐seq data were retrieved from GEO database (GSE182786).

5.4.2. Nuclei Isolation from Mouse Testes

Testicular parenchyma (capsule removed) was minced in NST buffer and lysed on ice for 7 min. After staining with Trypan Blue, nuclei were filtered through a 40 µm strainer and washed with ST Wash buffer. Nuclei were resuspended in PBS + 1% BSA for downstream use.

5.4.3. Library Preparation and Sequencing

Nuclear suspensions (700–1200 nuclei/µL) were loaded using Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1 (10× Genomics). Libraries were sequenced on the Illumina NovaSeq 6000 PE150 platform.

5.4.4. Alignment and Quantification

Raw FASTQ files were processed using Cell Ranger v9.0.0, aligned to the mouse genome (GRCm39), and converted to gene‐barcode matrices by Shanghai OEBiotech.

5.4.5. Data Processing and Clustering

Seurat (v5.0.0) was used for quality control (criteria: >200 genes, >1000 UMI, log10GenesPerUMI > 0.7, <20% mitochondrial UMI, <5% hemoglobin UMI), normalization, HVG selection (top 2000), PCA, and t‐SNE clustering. Batch effects were corrected with Harmony (v1.0). Doublets were removed using Doublet Finder (v2.0.3). The subcluster analysis was customized for the study of interest (Source code was publicized on GitHub).

5.4.6. Cell Type Annotation

Nuclei were annotated based on known marker genes, and expression differences were analyzed using Wilcoxon rank‐sum tests. The Loupe Browser (10x Genomics) assisted in visualization and annotation.

5.4.7. Differential Gene Expression Analysis

DEGs were identified using Seurat's Find Markers (|log₂FC| > 1.5, adjusted P < 0.05). Functional enrichment (KEGG pathway analysis) was performed using OE Cloud (https://cloud.oebiotech.com/).

5.4.8. Cell Purity Analysis

To determine whether macrophages were under clustered, we assessed the transcriptional homogeneity of macrophage clusters using ROGUE [45]. ROGUE is an entropy‐based metric used to evaluate the purity of single‐cell populations. A cluster with a ROGUE value greater than 0.9 is considered highly homogeneous.

5.5. Statistical Analysis

For human single‐cell RNA sequencing (SC‐seq) data, box plots are presented displaying the median, interquartile range (IQR), and whiskers extending to 1.5× the IQR. Statistical significance between each pair of groups was assessed using the Wilcoxon rank‐sum test. The following notations indicate levels of significance:

1. Asterisks (*) indicate significance between OG1 and YG.

2. Ampersands (&) indicate significance between OG2 and YG.

3. Hashes (#) indicate significance between OG2 and OG1.

The significance thresholds are defined as follows:

1. ns: not significant (p > 0.05)

2. */ & / #: P < 0.05 and ≥ 0.01

3. ** / && / ##: P < 0.01 and ≥ 0.001

4. *** / &&& / ###: P < 0.001 and ≥ 0.0001

5. **** / &&&& / ####: P < 0.0001

Wilcoxon rank‐sum tests were used to compare log‐normalized gene expression levels between groups. Differentially expressed gene (DEG) analysis was performed using the FindMarkers function in the Seurat package.

For mouse single‐cell sequencing (SN‐seq) data and experimental results, statistical significance relative to the control group was consistently indicated by an asterisk (*), regardless of the specific comparison group. All animal experiments were independently repeated at least three times.

The Mann–Whitney U test was applied to compare metabolic‐related parameters between two independent groups with small sample sizes (N = 5 per group). For tissue validation experiments with smaller sample sizes (N = 3 per group), two‐tailed and unpaired t‐tests were used to compare mean values between experimental and control groups, and homogeneity of variances (F‐Test) was performed. For comparisons among more than two groups, one‐way ANOVA was performed after confirming homogeneity of variances (Brown–Forsythe test was used). When one‐way ANOVA was used, Tukey's post hoc multiple comparisons test was applied. All statistical analyses were performed using Microsoft Excel and GraphPad Prism version 8.0.

Author Contributions

Z.D.Z. conceived the metabolism‐immunometabolism‐testicular axis hypothesis, designed and performed the animal experiments, conducted histological and morphological analyses, analyzed all single‐cell sequencing data, interpreted the results, drafted and edited the manuscript. Z.D.Z. contributed to this work solely. F.F.D. assisted in animal experiments, including blood sample collection. J.C. was responsible for the management of genetically modified mice. T.X.X. managed the project budget and data organization. M.X.L. contributed to the maintenance of experimental equipment. L.G., J.Y. and Y.L. provided guidance on the experimental design of the study. Y.L.Y. assisted with animal experiments, specifically the collection of non‐testicular tissues and organs. Y.L.Y. also supervised the overall progress, interpreted the results, and participated in manuscript editing. J.T. secured funding, supervised the project, and revised the manuscript. J.V.Z., Y.L.Y., and F.F.D. assisted with the funding application, secured funding, and revised the manuscript. J.V.Z. conceived and designed the exercise‐HPG axis modulation plan and provided the experimental platform and antagonist polypeptides, secured funding, supervised the project, interpreted the results, and revised the manuscript.

Funding

National Key R&D Program of China (2024YFA1803001), Shenzhen Medical Research Fund (B2404004), National Natural Science Foundation of China (32571489, 82301907, 82270600), Guangdong Basic and Applied Basic Research Foundation (2024A1515010059, 2024A1515030279 and 2022A1515220204), Shenzhen Science and Technology Program (JCYJ20220818101218040, JCYJ20220818103607015, JCYJ20220818103608017, JCYJ20230807140805011, JCYJ20220818102811025), Research Foundation of Guangdong Provincial Reproductive Science Institute (QD202201), Shenzhen Key Laboratory of Metabolic Health (ZDSYS20210427152400001), Sino‐European Center of Biomedicine and Health.

Conflicts of Interest

The authors declare no competing interests. The P12C5 antagonist peptide used in this study has been patented (ZL 201711387983.9), which is owned by Shenzhen Institute of Advanced Technology (SIAT), with Jian V. Zhang and J. Chen listed as inventors. This study was limited to animal experiments and did not involve any clinical application or claims of therapeutic efficacy.

Supporting information

Supporting File: advs73529‐sup‐0001‐SuppMat.docx.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.