Introduction

Aging is a complex natural process characterized by the degradation of cellular structures and disruption of physiological functions1. Cell senescence leads to reduced cell division capacity and impacts tissue repair, immune function, and cognitive function2,3,4,5,6, playing a crucial role in processes like embryonic development, tissue repair, and tumor suppression7,8,9. Cellular senescence is triggered by various stresses, including chromosomal instability, telomere shortening, oxidative stress (OS), and DNA damage10,11. OS refers to the excessive production of reactive oxygen species (ROS), leading to damage of cellular components such as lipids, proteins, and DNA, thereby impairing normal cellular function12,13. Recent studies have identified a novel phenomenon termed Redox Stress Response Resistance (RRR) in aging cells and nematode models, resembling insulin resistance14. Notably, in acute myeloid leukemia (AML), following the loss of PRMT5 functionality, cells characterized by reduced ATF4 levels exhibit both increased OS and cellular aging15. Research indicates that OS accelerates cellular damage and senescence, and significantly impacts fertility16. In approximately 40–50% of cases of male-factor infertility, factors related to OS are considered key contributors to the decline in male fertility17,18,19.

The testis, a key organ in the male reproductive system, is the site of spermatogenesis, an important physiological process involving germ cells and various somatic cells, including Leydig cells (LCs), Sertoli cells, and peritubular myoid cells20. Testicular aging alters spermatogenesis, leading to hormonal imbalances and a range of age-related diseases. With advancing age, testicular fibrosis becomes increasingly prominent, leading to a reduction in the size of the seminiferous tubules. Concurrently, there is a progressive decline in the quantity of seminiferous epithelial cells. Additionally, the basement membrane undergoes thickening, and the architecture of the seminiferous epithelium becomes disrupted. Notably, different cell types within the testis exhibit heterogeneous responses to aging21. Although OS is a major cause of cellular senescence22,23, the specific mechanisms by which OS induces testicular cell senescence remain unclear.

Recent studies across multiple species, including silkworms, nematodes, and fruit flies, have revealed that the FoxO protein can upregulate antioxidant genes and suppress mitochondrial genes. By modulating its target gene OSER1, FoxO reduces OS and extends lifespan24. FoxO signaling pathway is activated in response to various stress signals, including OS, and it modulates the expression of genes involved in cellular physiological events such as apoptosis, cell-cycle control, glucose metabolism, and longevity. polo-like kinase 3 (PLK3), implicated in the FoxO signaling pathway, is suggested to play a role in cell cycle regulation. This protein is a pivotal regulatory factor in the transition between the G1/S and G2/M phases and is also involved in initiating apoptosis under stress conditions25,26. Furthermore, PLK3 modulates the hypoxia signaling pathway by directly phosphorylating HIF-1α, influencing cellular responses to hypoxia27. Collectively, these findings indicate that PLK3 is intricately linked to cellular senescence.

Due to the significant biological similarities between pigs and humans, pigs are increasingly being utilized as important models for studying human diseases28,29,30. Single-cell transcriptomics and cross-species comparative analysis have revealed distinct molecular changes in the testes of pigs during puberty, discovering an early onset of meiotic abnormalities in pigs. They have also identified a unique subtype of porcine spermatogonial stem cells similar to the human transcriptomic state 0, indicating the unique value of the pig model in simulating human testis development31. Breeding boars, after prolonged use, experience a decline in testosterone levels, which leads to reduced libido and reproductive capacity, ultimately leading to their elimination. This process closely mirrors testicular aging, making pigs particularly suitable for modeling male testicular aging, especially given their longer lifespan compared to short-lived small mammals such as rodents. Therefore, it is crucial to study the physiological changes in LCs within the testes of breeding boars during the aging process.

In this study, the relationship between ROS levels and testicular aging in boars was assessed. An in vitro senescence model of porcine LCs was established using hydrogen peroxide (H₂O₂) to mimic the process of OS-induced aging. Potential molecular mechanisms underlying this process were uncovered through transcriptome sequencing, and relevant hub genes were identified. Additionally, the impact of silencing the key gene polo-like kinase 3 (PLK3) on the antioxidant capacity and proliferation of porcine LCs in the senescence model was investigated. These findings provide new insights into the mechanisms of testicular aging and suggest potential therapeutic approaches for related diseases.

Results

Aging testicular somatic cells and ROS accumulation in aged Shaziling boars

Hematoxylin–eosin (HE) staining was performed to analyze the histological structure of testicular tissues from boars at different physiological stages. In D1 boars, the seminiferous tubules in the testes were underdeveloped, primarily filled with Sertoli cells and spermatogonium. By 60 days of age, the tubules had significantly enlarged and started to mature, with the presence of a few spermatogenic cells. At 150 days, the seminiferous tubules exhibited more advanced development, indicating active spermatogenesis. However, some tubules showed sloughed immature spermatogenic cells in their lumens and loose cell connections within the epithelium.

The seminiferous tubules displayed noticeable differences in aged boars compared to those in 150-day-old boars. Notably, the tubule size has significantly enlarged, and the basement membrane layer has thickened. Concurrently, there is a reduction in the number of spermatogenic cell layers, and the central lumen showed significant empty spaces, indicating reduced sperm production or impaired spermatogenesis, likely caused by the loss of spermatogenic cells. Additionally, compared to 150-day-old boars, LCs in aged boars appeared less plump, with indistinct nuclei and a sparser distribution. These changes may contribute to decreased testosterone levels, reduced libido, and lower reproductive performance in aged boars (Fig. 1A).

Fig. 1
figure 1

Pronounced senescence in aged boar testes. (A) The histological analysis of testicular tissue from pigs of different ages. 1-day-old (D1), 60-days-old (D60), 150-days-old (D150), and aged boars (over 5 years old), scale = 50 μm. (B) SA-β-gal staining indicating cellular senescence in Sertoli cells and most LCs of aged boars. Pink represents the nucleus; blue indicates SA-β-gal staining. Scale bar = 100 μm. (C) Accumulation of reactive oxygen species (ROS) observed in all testicular cells of aged boars. Blue represents the nucleus; red indicates ROS. Scale bar = 100 μm.

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To further investigate cellular aging in testicular tissues, SA-β-gal staining was performed to detect senescent cells. Figure 1B showed pronounced ageing in Sertoli cells and LCs from aged boars, while D150 boars exhibited nearly no senescent cells. Additionally, ROS accumulation was significantly higher in the testes of aged boars than in D150 boars (Fig. 1C).

These results suggest that the increased proportion of senescent LCs and Sertoli cells, accompanied by elevated ROS levels, serves as a marker of aging in boars. As the primary cells responsible for testosterone secretion, the senescence of LCs induced by ROS accumulation may lead to functional disorders, directly contributing to the decline in boar fertility. The mechanisms underlying LC senescence are, therefore, the present study’s focus.

H2O2 exposure inhibits cell proliferation and promotes cellular senescence in LCs

H2O2, a common ROS, is frequently used to induce OS and is a classical agent for creating in vitro cellular aging models32. After a 24 h exposure to 200 µM H2O2, intracellular ROS levels in LCs were significantly elevated, indicating that H2O2 has successfully entered the LCs (Fig. 2A). SA-β-gal was used to analyze the difference in the number of senescent cells between the H-LC and C-LC groups, thereby evaluating the role of ROS accumulation in inducing senescence in LCs. As shown in Fig. 2B, SA-β-gal activity was significantly higher in LCs from the H-LC group compared to the C-LC group, along with a noticeable reduction in cell numbers under identical fields of view. Additionally, the CCK-8 assay revealed that H2O2 exposure significantly reduced cell proliferation ability (P < 0.001) (Fig. 2C). Given that mitochondrial dysfunction is a hallmark of aging, JC-1 staining was used to assess the mitochondrial membrane potential. The H-LC group exhibited a marked decrease in JC-1 aggregates and an increase in JC-1 monomers, indicating significant mitochondrial membrane potential loss (Fig. 2D). It is widely recognized that cellular senescence leads to cell cycle arrest, with p53, p16, and p21 serving as key regulators of the cell cycle and as markers of senescence33. RT-qPCR was performed to detect the expression of p53, p16, and p21, and their expression levels were significantly upregulated in the H-LC group (P < 0.001) (Fig. 2E). The above results indicate that the model was successfully established.

Fig. 2
figure 2

H2O2 exposure induced senescence in LCs. (A) Intracellular ROS levels. Scale bar = 200 μm. (B) SA-β-gal Activity. The treated group (H-LC) exhibited a more pronounced SA-β-gal activity, indicated by a stronger blue color, compared to the control group (C-LC), which hardly showed any blue color. Scale bar = 50 μm (200x); Scale bar = 20 μm (400x). (C) After exposure to 200 µM H2O2 for 24 h, the cell proliferation ability of LCs was significantly reduced. (D) Mitochondrial Membrane Potential: Cells exposed to H2O2 displayed an increase in JC-1 monomers and a decrease in JC-1 aggregates, confirming mitochondrial damage caused by OS. Scale bar = 100 μm. (E) Expression of Senescence Markers. The expression of p53, p16, and p21, which are key markers of cell cycle arrest and senescence, significantly increased in LCs treated with H2O2. *, ** and *** represent P < 0.05, P < 0.01, and P < 0.001 respectively.

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H2O2 exposure reduces antioxidant capacity

Notably, we also observed that H2O2 exposure led to a significant decrease in intracellular SOD (P < 0.05), T-AOC and GSH-Px activity (P < 0.01) (Fig. 3A, B and D), accompanied by a marked increase in intracellular MDA levels (P < 0.05) (Fig. 3C). In summary, ROS accumulation inhibited LC proliferation while simultaneously reducing their antioxidant activity.

Fig. 3
figure 3

H2O2 exposure reduces antioxidant capacity. (A) SOD activity. (B) T-AOC content. (C) MDA content. (D) GSH-Px activity. The values are expressed as mean ± SEM (n = 3 biological repetitions). *, ** and *** represent P < 0.05, P < 0.01, and P < 0.001 respectively.

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H2O2 exposure induces differential gene expression in LCs

To explore the molecular mechanisms driving LC senescence, transcriptome sequencing was performed on both H-LC and C-LC group. After statistics and filtering, an average of 28,557,676 clean reads were produced for each sample. The average base quality for Q20 and Q30 were 98.83% and 96.2%, respectively. Clean reads were aligned to the reference genome (Sscrofa11.1) using STAR, with an average mapping rate of 97.66% (Supplementary Table S1). This indicates the reliability of the transcriptome data. Principal Component Analysis (PCA) revealed a clear distinction between H-LC and C-LC group, indicating significant differences in gene expression patterns (Fig. 4A). In total, 601 DEGs were identified between C-LC and H-LC, with 371 upregulated and 230 downregulated (Fig. 4B and C).

Fig. 4
figure 4

Transcriptome analysis of LCs After H2O2 exposure. (A) Principal component analysis (PCA) illustrates a clear separation between H-LC (H2O2-treated LCs) and C-LC (control LCs), n = 3 biological repetitions. (B), (C) Volcano plots depict the differentially expressed genes between C-LC and H-LC. A total of 601 genes were identified, with 371 genes upregulated and 230 genes downregulated.

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Enrichment analysis

H2O2 exposure significantly altered the expression patterns of 601 genes in LCs, followed by GO and KEGG enrichment analyses34. DEGs were mainly enriched in ten GO terms (level 2) related to biological process category, including cell cycle, post-translational protein modification, macromolecule catabolic process, and intracellular transport. In terms of molecular function, DEGs were mainly enriched in protein kinase activity, transferase activity, transferring phosphorus-containing groups, ubiquitin-protein transferase activity, and acyltransferase activity. For cellular component, DEGs were chiefly linked to components like the microtubule cytoskeleton, Golgi apparatus, and various protein complexes (Fig. 5A). Moreover, H2O2 treatment induced notable alterations in a subset of GO terms associated with cellular responses and regulatory mechanisms (Fig. 5B): regulation of the mitotic cell cycle, microtubule motor activity, protein ubiquitination, and response to type II interferon. Notably, the genes involved in these processes were significantly downregulated in aged LCs. Conversely, some genes related to specific cellular components and functions were significantly upregulated in the aged LCs (Fig. 5C). This includes terms such as intracellular transport, microtubule cytoskeleton organization, and Golgi apparatus components.

Fig. 5
figure 5

GO Enrichment analysis of DEGs induced by H2O2 exposure. (A) Overall GO enrichment analysis of DEGs after H2O2 exposure. (B) GO analysis of upregulated DEGs in control LCs (C-LC). (C) GO analysis of upregulated DEGs in aged LCs (H-LC).

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The 601 DEGs were enriched in a multitude of KEGG pathways, which are displayed across 5 classes in Fig. 6A. Interestingly, pathways related to aging in the Cellular Processes class were significantly enriched. These include the p53 signaling pathway, Autophagy-animal, Cell cycle, and Cell senescence. In Environmental Information Processing, many classic pathways related to the regulation of cell proliferation and cycle were enriched, including the MAPK signaling pathway, Wnt signaling pathway, and PI3K-Akt signaling pathway. Furthermore, Fig. 6B shows the pathways with the highest enrichment in each category. Among all KEGG pathways, we selected the top 10 pathways based on significance for display, with MAPK, Endocytosis, and Thermogenesis being the pathways with the most gene enrichment (Fig. 6C). Notably, the network diagram indicates that MAPK, cell cycle, PI3K-Akt, and Regulation of actin cytoskeleton are key pathways altered by H2O2 exposure (Fig. 6D).

Fig. 6
figure 6

KEGG pathway enrichment analysis of DEGs induced by H2O2 exposure (A) Overview of the distribution of 601 DEGs across 5 classes of KEGG pathways. (B) Display of pathways with the highest enrichment scores in each category. (C) Top 10 KEGG pathways. (D) Network diagram illustrating the interconnectivity of key pathways altered by H2O2 exposure.

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Analysis of DEGs in OS response

A PPI network analysis of the 601 DEGs was performed using the STRING online database (http://string-db.org), and 15 key genes were identified. (Fig. 7A). To further explore the biological functions and potential mechanisms of action of these genes under specific experimental conditions, we compared their expression patterns between the two different sample groups (Fig. 7B). PLK3 was significantly upregulated in aged LCs, while the other 14 genes were downregulated.

Fig. 7
figure 7

Analysis of core differential genes and their expression patterns. (A) Protein-protein interaction (PPI) analysis presenting the Top 15 core genes among the 601 DEGs. (B) Expression profiles of the Top 15 core genes across six samples.

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PLK3 alleviates OS in LCs induced by H2O2

In other studies, PLK3 has been implicated in cell cycle arrest and responses to various stresses through pathways such as p53, PI3K/AKT, and RAS signaling35,36,37. In this context, as one of the potential candidates identified in our transcriptome analysis, PLK3 may be closely associated with aging induced by cellular stress. To investigate whether PLK3 is a crucial factor in cellular senescence under OS, three siRNAs were designed to knockdown PLK3 expression in LCs, and the most effective siRNA was selected for further experiments (Fig. 8A). Furthermore, Western blot experiments also indicated that the expression of PLK3 protein was downregulated as a result of the interference (Supplementary Fig. S2). Following PLK3 knockdown, the LCs in both the si-PLK3 (named as si-PLK3 group) and control groups (named as si-NC group) were exposed to H2O2. As shown in Fig. 8B, LCs transfected with si-PLK3 exhibited higher cell viability following H2O2 exposure compared to the si-NC group (Fig. 8B). Furthermore, ROS levels in the si-PLK3 group were significantly lower than those in the si-NC group after H2O2 treatment (Fig. 8C). Additionally, T-AOC, GSH-Px and SOD levels in the si-PLK3 group were significantly higher compared to the si-NC group (Fig. 8D). However, no significant changes were observed in the MDA content. These results indicated that PLK3 upregulation during aging may contribute to reduced LC proliferation and decreased intracellular antioxidant activity.

Fig. 8
figure 8

Effects of PLK3 knockdown on LCs under OS. (A) Knockdown efficiency of PLK3 in LCs. (B) Increased cell proliferation ability in si-PLK3 treated LCs under 200 µM H2O2 stress after 24 h. (C) Reduced ROS levels in si-PLK3 LCs compared to si-NC after H2O2 treatment. Scale bar = 200 μm. (D) Enhanced T-AOC, GSH-Px and SOD activity in si-PLK3 LCs with no change in MDA content post-H2O2 treatment. The values are expressed as mean ± SEM (n = 3 biological repetitions). *, ** and *** represent P < 0.05, P < 0.01, and P < 0.001 respectively.

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Discussion

Cellular senescence is a physiological state in which cells permanently cease proliferation after a certain number of divisions. Testicular aging can lead to a decline in male sexual desire and reproductive capacity. Recent studies have revealed molecular mechanisms behind the depletion of spermatogonial stem cell reserves, meiotic disorders, and impaired spermatogenesis in aging testes, with Sertoli cells being particularly susceptible to aging38. Single-cell transcriptome sequencing has shown a disruption in the balance between undifferentiated and differentiated spermatogonial stem cells in the testes of aged mice39, which may be related to the functional decline of Sertoli cells. Similarly, in the testes of men with idiopathic germ cell aplasia, dysregulation of parental imprint genes and DNA damage were found in peritubular myoid cells and LCs40. These findings indicate that Sertoli cells and LCs play crucial roles in testicular aging and male reproductive health, making them key targets for investigating testicular aging mechanisms. Factors associated with OS are recognized as key contributors to the decline in male fertility rates17,18,19. However, while OS is closely linked to aging and may cause testicular aging by damaging the male reproductive system, the underlying molecular mechanisms still need to be investigated.

Given the physiological and anatomical similarities between pigs and humans, using pigs as a model for studying male reproductive aging in humans offers certain advantages28,29,30. In our research group’s preliminary studies, the early appearance of sperm in Shaziling boars suggested an earlier time of sexual maturity. The molecular mechanisms underlying testicular growth and development in these boars were elucidated, revealing several parallels with human testicular biology41,42,43. This discovery provides a time-efficient medical model for research, which helps us to simulate the physiological changes of human reproductive aging more effectively.

In studies on human and animal models, including mice, monkeys, and dogs, it is commonly observed that the number of Sertoli cells and LCs in the testes decreases with age, along with a histological phenotype of interstitial cell atrophy44,45,46,47. These studies have provided us with a preliminary understanding of the impact of aging on the male reproductive system. However, they have limitations in the in-depth exploration of cell type details, particularly in the cellular-level analysis of aging mechanisms. In this study, we expanded our research on testicular aging in Shaziling boars, aiming to provide more information for understanding the potential mechanisms of aging by analyzing specific changes in cell types during the aging process.

By studying the Shaziling pigs, we can offer a different perspective on experimental design compared to existing studies, particularly in the analysis of specific cellular changes and aging mechanisms. Currently, cellular senescence is characterized by a set of markers including growth arrest, SA-β-gal activity, and telomere damage, among others, rather than relying on a single biomarker48. A significant accumulation of senescent cells was observed in the testes of aged boars, particularly surrounding the basement membrane of the seminiferous tubules, as evidenced by SA-β-gal staining. Additionally, the aging of Sertoli cells and LCs was found to be accompanied by ROS accumulation. Consistent with other studies38, the senescence of Sertoli cells is a commonly observed phenomenon in aged testes. The aging process in the testes is closely associated with the functional decline of Sertoli cells and LCs. As a core component of the hypothalamic-pituitary-gonadal axis, LCs secrete testosterone, a key hormone influencing male sexual maturation, libido, and fertility. Therefore, this study focuses on the mechanisms of LCs senescence.

Increasing evidence shows that H2O2 can induce OS and cellular senescence32. We have observed that different cells exhibit varying tolerances to H2O2 in other studies. For instance, 400 nM H2O2 did not harm the survival of human dental pulp cells (hDPCs) but induced premature aging in immortalized hDPCs49. A treatment of 600 µmol H2O2 was used on HepG2 cells to establish an in vitro oxidative damage model50. Porcine granulosa cells (GC) were exposed to 200 µM H2O2 for 12 h to simulate the occurrence of OS51. SH-SY5Y cells were treated with 200 µM H2O2 for 2 h to induce oxidative damage52. In our preliminary studies, 200 µM H2O2 effectively induced OS in LCs, leading to ROS accumulation, cellular senescence, and suppressed cell proliferation. The mitochondria of senescent cells exhibit functional impairments, typically characterized by diminished respiratory capability and a decrease in mitochondrial membrane potential (MMP) under homeostatic conditions. A lowered MMP is often associated with an increase in ROS production53. Following the establishment of H2O2-induced senescence in LCs, the transition from JC-1 aggregates to JC-1 monomers confirmed the mitochondrial damage induced by OS. Furthermore, H2O2 markedly induced OS in LCs, resulting in reduced antioxidant capacity and lipid damage. These findings are consistent with prior studies54, confirming OS as a key driver of cellular senescence and validating our in vitro H2O2-induced senescence model in porcine LCs.

Although H2O2 has been extensively studied for its effects on various cell types, its specific impact on testicular LCs warrants further investigation. Therefore, we performed transcriptome analysis on LCs exposed to H2O2 and found that there were 601 DEGs after H2O2 treatment. The enrichment analysis of these differentially expressed genes within the framework of Gene Ontology (GO) terminology revealed that, revealed that partially downregulated DEGs were significantly associated with GO terms related to cellular response and regulatory mechanisms55. This suggests that OS might compromise these fundamental functions by disrupting cellular stress responses and repair mechanisms. Conversely, some upregulated DEGs are linked to specific cellular components and functions, indicating that OS could adversely affect cellular structure and functionality, such as causing damage to cell membranes and mitochondria56,57. These changes resonate with the effects of OS on the proliferative capacity and mitochondrial function of testicular LCs. The integrated analysis of GO terms for biological process, molecular function, and cellular component components suggests that these specific changes may be the cell’s response mechanisms to oxidative damage, as well as regulation of cell signaling and cell cycle58,59. The enrichment analysis provides a comprehensive overview of the molecular shifts within LCs in response to H2O2, highlighting the intricate interplay between upregulated and down-regulated genes and their roles in maintaining cellular homeostasis under OS conditions. A thorough investigation into these regulatory mechanisms may contribute to addressing the damage caused by OS to LCs and provide novel approaches for the prevention and treatment of related diseases.

Furthermore, DEGs were also found to be enriched in numerous KEGG pathways, with particular significance in the MAPK signaling pathway. The MAPK pathway is central to cellular processes such as proliferation, differentiation, migration, and stress response, rapidly enhancing stress resistance by modulating the expression of antioxidant enzymes. Under conditions of OS, the MAPK pathway is commonly activated; excessive activation of ERK can lead to cellular dysfunction and apoptosis60. The activation of JNK may induce the expression of cellular senescence phenotypes61. JNK, once activated, translocates to the nucleus, phosphorylates the transcription factor c-Jun, regulates gene expression, and is involved in cellular stress response, apoptosis, and differentiation62. Prolonged activation of p38 MAPK can accelerate cellular senescence and impair the cell’s stress response and repair capabilities63. The interplay between MAPK pathway activation and signaling pathways associated with aging may further intensify cellular dysfunction. Under OS, the regulation of the actin cytoskeleton significantly impacts cell morphology, migration, and proliferation64. OS influences the polymerization and depolymerization of actin through the production of ROS. Structural changes in actin induced by OS can destabilize the cytoskeleton, affecting cell morphology and migration. This instability may also impact cell division and proliferation, thus promoting the aging process. The network diagram illustrates the intricate nature of intracellular signaling, where distinct pathways intersect through the MAPK signaling pathway, allowing cells to precisely regulate various signals to adapt to fluctuating internal and external conditions. For instance, autophagy and ubiquitin-mediated protein degradation enable cells to adapt to and preserve their physiological functions by clearing damaged organelles, maintaining the balance of intracellular components, and responding to environmental shifts. Under starvation, cells engage in autophagy to degrade non-essential components, generating energy and essential metabolic precursors, which ensures their survival and function65. Cells selectively eliminate damaged mitochondria through a process known as mitophagy, which safeguards against the accumulation of nuclear DNA damage induced by mitochondrial ROS, thus preventing DNA damage-related aging66. The mTOR signaling pathway is a pivotal regulator of cell cycle progression, and its inhibition can initiate autophagy67. Ubiquitin-mediated protein degradation is a prevalent mechanism for cellular regulation of protein levels and functions68. The Ubiquitin-Proteasome System (UPS) is involved in DNA repair and transcription, and its dysfunction is linked to various neurodegenerative diseases69. UPS regulates the misfolding and aggregation of alpha-synuclein, influencing the pathogenesis of Parkinson’s disease (PD)70. Aging, a primary risk factor for most neurodegenerative diseases, may be associated with a decline in UPS function. As age advances, UPS efficiency may diminish, leading to delayed protein degradation and the accumulation of toxic protein aggregates. The interplay between these signaling pathways and the MAPK signaling pathway modulates the cell cycle, protecting cells from stress-induced damage and aging.

In addition, a sophisticated regulatory mechanism exists between the levels of ROS inside cells and antioxidant signaling pathways. This regulation is essential for maintaining cellular redox balance and protecting against damage caused by OS. When confronted with OS-induced damage, cells activate their antioxidant signaling pathways, including Nrf2-ARE, heat shock proteins, and FoxO proteins. These pathways can enhance the transcription and expression of antioxidant genes, such as glutathione reductase and superoxide dismutase. These antioxidant enzymes assist in the elimination of reactive oxygen species within cells, preserve redox balance, and thus mitigate the damage caused by OS to cells. The FoxO signaling pathway is a complex and crucial pathway that plays a significant role in regulating the cell cycle, resistance to OS, and lifespan. It modulates gene expression via post-translational modifications in response to insulin or growth factors71,72. Under OS conditions, the transcriptional activity of the FoxO pathway is often compromised, leading to reduced expression of antioxidant enzymes and DNA repair-related genes73. This decline in transcriptional activity can diminish the cell’s resistance to oxidative damage and accelerate the aging process. The significant enrichment of pathways like MAPK and FoxO suggests that LCs were actively responding to external stress, corroborating our study’s results. A protein-protein inter-action (PPI) network analysis of the DEGs identified the top 15 genes with the most interactions, which are closely related to processes like cell cycle regulation and DNA replication, indicating their potential key role in LCs’ response to OS.

PLK3 is involved in cell cycle arrest and stress responses through various signaling pathways, including p53, PI3K/AKT, and RAS35,36,37. During the OS response, p53 may be a direct target of PLK3. Activation of p53 subsequently promotes the activation of p2174. We hypothesize that PLK3 may be intimately linked to aging induced by cellular stress. Our research findings indicate significant expression levels of PLK3 and p53 in aged LC. To ascertain PLK3’s role in cellular stress response, we performed in vitro experiments. These experiments demonstrated that si-PLK3 treatment reduced ROS levels, enhanced antioxidant capacity, and increased cell viability. This suggests that cells exposed to 200 µM H2O2 for 24 h undergo significant OS, and PLK3 plays a crucial role in the cell’s response to this stress. PLK3 likely regulates the cell cycle by activating the p53 and FoxO pathways, but further research is needed to elucidate the underlying mechanisms.

These findings offer significant insights into the regulatory mechanisms by which LCs respond to OS, identifying potential therapeutic targets to enhance cellular resistance to oxidative damage and promote tissue health and repair.

Materials and methods

Ethics statement

We confirmed that all experiments in this study were performed in accordance with the relevant guidelines and regulations. Also, all the procedure of the study is followed by the ARRIVE guidelines. All animal procedures were approved by the Ethics Committee of University of South China (Approval No. 2024–059). Animal welfare was ensured at all stages of the study, preventing unnecessary suffering of the boars.

Sample collection

Testes from healthy Shaziling pigs at different stages (including 1-day-old (D1), 60-day-old (D60), 150-day-old (D150), and aged boars (over 5 years old) were provided by the Livestock Breeding Station of Xiangtan (Hunan Province, China). Among these, the D1, D60, and D150 pigs were half-siblings, with three individuals per group (n = 3). For the older boars, four individuals with similar body weights were selected (n = 4). The aged boars, over 5 years old and exhibiting decreased libido and reproductive capacity, were slaughtered using high-voltage electric stunning. Testicular samples were then collected, with the scrotum first disinfected using medical-grade alcohol and subsequently dried with sterile gauze. For the D1, D60, and D150 boars, pretreatment for testicular sample collection included an intramuscular injection of atropine sulfate (0.05 mg/kg) and subsequent anesthesia induction with Zoletil 50 (5 mg/kg) and Xylazine (2 mg/kg). The surgical area was disinfected with medical-grade alcohol, followed by drying with sterile gauze. A 5% iodine tincture (Stary Co., China) was then applied for further antiseptic cleansing. An incision was made in the scrotal skin to allow the testes to be extruded, and the testicles were removed using a ligature method. The entire procedure was performed under sterile conditions. After surgery, the pigs were kept in a clean environment until full recovery. All samples were cut into 3 × 3 cm tissue sections and fixed with 4% paraformaldehyde.

HE staining and ROS detection

Testicular tissues fixed in paraformaldehyde were embedded in paraffin and sectioned into 5 μm-thick slices. The sections were then stained with HE staining according to a previously described protocol75 to observe the histological morphology of testes. The ROS staining procedure involved outlining the tissue sections with a pen, followed by a 5 min treatment with a fluorescence quencher and a 10 min rinse with water. ROS dye was applied to the marked area, and the sections were incubated in a 37 °C dark chamber for 30 min. After incubation, the sections underwent PBS washing and DAPI staining for 10 min to visualize the nuclei. Finally, the sections were mounted and photographed. Cellular ROS production was measured using a Reactive Oxygen Species Assay Kit (S0033S, Beyotime, Shanghai, China). Collect LC cells into centrifuge tubes. Load the cells with the DCFH-DA probe and incubate at 37 °C for 20 min. During incubation, gently invert the tubes 4–5 times to ensure adequate contact between the probe and the cells. After incubation, wash the cells three times with a serum-free cell culture medium. Detect fluorescence using a microplate reader (ThermoFisher Scientific Inc., Denver, CO, USA) with an excitation wavelength of 488 nm and an emission wavelength of 525 nm.

Cell culture and H2O2 exposure

The porcine primary LCs were isolated from from Shaziling piglets using the collagenase digestion method76. The cells were verified using the specific marker genes of LCs, including 3β-hydroxysteroid dehydrogenase (3β-HSD) and cytochrome P450 family 17 subfamily A member 1 (CYP17A1). The LCs were cultured in Dulbecco’s modified Eagle medium/Nutrient Mixture F-12 (DMEM/F-12, Gibco, Grand Island, NY, USA), supplemented with 10% Fetal Bovine Serum (FBS) (Gibco, Detroit, MI, USA) and maintained in a humidified incubator at 37 °C with 5% CO2. To induce OS, the LCs were exposed to 200 µM H2O2 (Sinopharm, Shanghai, China). After a 24 h treatment, the cells were prepared for subsequent experimental procedures.

SA-β-gal staining

Senescence-associated β-galactosidase (SA-β-gal), a well-established marker of cellular aging, was detected using a staining kit (S191073, Wuhan Pinuofei Biological Technology Co., Ltd, Wuhan, China) and provided technical support for this procedure. Cells were first rinsed with PBS, then fixed for 15 min. After three additional PBS rinses, the cells were stained and incubated overnight at 37 °C in a CO2 free environment. Using a photographic microscope (Nikon, Tokyo, Japan), images of stained cells were captured.

Determination of mitochondrial function

The Mitochondrial membrane potential (ΔΨm) was measured using the JC-1 fluorescent probe (C2006, Beyotime, Shanghai, China). The shift from JC-1 aggregates (red fluorescence at 525 nm) to monomers (green fluorescence at 490 nm) was observed under a fluorescence microscope (Leica, Wetzlar, Germany). This transformation directly reflects the dynamic changes in the mitochondrial membrane potential.

OS indicators

The total antioxidant capacity (T-AOC) (S0119), superoxide dismutase (SOD) activity (S0101S), and malondialdehyde (MDA) (S0131S) levels in the collected cells was determined using the respective assay kits (Beyotime, Shanghai, China) according to the manufacturer’s protocol. The activity of glutathione peroxidase (GSH-Px) was assessed using the corresponding commercial kits (A005-1-2, Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Protein content was quantified using the BCA protein assay kit (P0010, Beyotime, Shanghai, China).

Real-time quantitative PCR (RT-qPCR)

RNA was isolated from the samples using TRIzol reagent (15596026, Invitrogen, Carlsbad, CA, USA), followed by cDNA synthesis via reverse transcription with the PrimeScript RT Reagent Kit with gDNA Eraser (RR047A, TAKARA, Beijing, China). Primer sequences were designed with the aid of Primer 5.0 software and synthesized by Sangon Biotech Co., Ltd. (Shanghai, China). RT-qPCR was performed on a CFX96 Real-Time PCR Detection System (Bio-Rad, Hercules, Los Angeles, CA, USA). GAPDH was used as an internal control gene to normalize gene expression levels, and the data analysis was performed using the 2−∆∆CT method. The primers used for RT-qPCR are listed in Supplementary Table S2.

RNA sequencing and bioinformatic analysis

LCs treated with H2O2 were named as H-LC, and the control group was named as C-LC. Three biological replicates were included for each group. RNA from both H-LC and C-LC groups was isolated using TRIzol reagent (15596026, Invitrogen, Carlsbad, CA, USA). cDNA was synthesized and sequenced on the Illumina Nova 6000 platform (Illumina, Inc., San Diego, CA, USA) using 150-base pair paired-end sequencing. This article’s RNA-seq data has been deposited at the National Genomics Data Center under accession no. CRA029156 (National Genomics Data Center). Raw reads were processed using Trimmomatic (version 0.39.2) to remove adapter sequences and filter out low-quality reads. The cleaned reads were then aligned to the pig reference genome (Sscrofa11.1) using STAR (version 2.5.2) Genes exhibiting |log2(fold change)| > 1 and an adjusted p-value < 0.05 were considered as DEGs. In order to verify the accuracy of the sequencing results, we selected several genes for RT-qPCR (Supplementary Fig. S1).

Enrichment analysis

Following the identification of DEGs, we employed the same functional enrichment analysis methods as in previous studies41 to conduct GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) analyses of these genes. This was done to explore the biological functions and pathways associated with these genes.

Construction and module analysis of the protein-protein interaction (PPI) network

PPI were used to reveal the patterns and functions of protein interactions within organisms. Specific sets of genes were used to construct and visualize networks through tools like the STRING online database (http://string-db.org) and Cytoscape (version 3.9.1), and further applied modular analysis algorithms to conduct an in-depth investigation of the PPI network.

Cell transfection

Transfect the cells when they reach 50% confluence. After mixing the siRNA and transfection reagent (Lipo2000) (Invitrogen, Carlsbad, CA, USA) in a specified ratio, add the mixture to the cell culture medium and continue incubation for 24 h (siRNA sequence are listed in Supplementary Table S3). Next, assess the interference efficiency of each si-PLK3 in LCs using RT-qPCR and western blotting (The western blotting image is provided in the Supplementary Information.). Subsequently, the transfected cells were induced to experience OS by treatment with H2O2. Inoculate the cells that have been transfected for 24 h onto a cell culture plate and treat them with 200 µM H2O2 for an additional 24 h for various experimental measurements.

Cell viability assay

After cell transfecting, LCs were plated into a 96-well plate at a concentration of 3000 cells per well and exposured to 200 µM H2O2 for 24 h. Subsequently, 10 µL Cell-Counting-Kit-8 (CCK-8, UEL-C6005; UElandy, Suzhou, China) was added to each well, followed by a 2 h incubation at 37 °C. The absorbance was then measured at 450 nm (Molecular Devices, San Jose, CA, USA).

Statistical analysis

GraphPad Prism 8 (GraphPad Software Company, California, USA) was utilized for the creation of statistical illustrations. A t-test was performed to compare the means between the experimental and control groups at a specific time point. Statistically significant differences were defined as p < 0.05. The mean and SEM were determined from data obtained from a minimum of three separate experiments.

Conclusions

This study demonstrated that increased ROS was a key factor contributing to testicular aging in boars, particularly negatively affecting LC function. Sertoli cells and LCs are the most susceptible to aging in testicular cells of aged shaziling boars. By developing an LC senescence model, we identified 601 DEGs associated with OS response, cell cycle regulation, and intracellular processes. PPI network analysis identified PLK3 and determined it to be a key factor in the LCs senescence under OS. These findings provide a vital molecular basis for the development of anti-aging strategies and the extension of health span.