https://orcid.org/0009-0007-2723-3516
Figures
Abstract
This study investigates the protective effects of nicotinamide mononucleotide (NMN) against aluminum-induced testicular dysfunction in mice. While previous research has demonstrated the general protective role of NMN in testicular function and highlighted the interaction between aluminum and the NLRP3 inflammasome, the precise mechanisms through which NMN mitigates aluminum-induced reproductive toxicity remain unclear. Our findings show that NMN protects Sertoli cells by inhibiting NLRP3-mediated pyroptosis, which subsequently improves spermatogenesis and testosterone synthesis. We also identify WT1 and GATA4 as key regulators involved in maintaining Sertoli cell integrity and function under aluminum-induced stress. This work provides new insights into the molecular mechanisms of aluminum-induced male infertility and underscores NMN’s potential as a therapeutic strategy for preventing reproductive dysfunction caused by environmental toxicants, such as aluminum. Aluminum exposure disrupts spermatogenesis, yet no effective pharmacological interventions exist to treat aluminum-induced male infertility. In this study, we established an aluminum toxicity model in C57BL/6J mice through intragastric administration of AlCl3. We assessed the therapeutic effects of NMN by evaluating testicular histopathology, sperm quality, and serum reproductive hormone levels. Hematoxylin and eosin (H&E) staining revealed significant damage, including reduced seminiferous tubule diameter, disorganized germ cell layers, and a decreased number of germ cells and sperm. Sperm motility was significantly reduced, while the proportion of abnormal sperm increased. However, NMN treatment partially reversed these impairments. Transcriptomic analysis revealed significant upregulation of genes involved in the NLRP3 inflammasome and apoptosis pathways (e.g., NLRP3, GSDMD, caspase-1, and IL-1β) in the AlCl3-exposed group. NMN treatment mitigated testicular damage, suppressed NLRP3-mediated pyroptosis in Sertoli cells, and restored serum testosterone levels. Additionally, NMN treatment preserved the expression of key testicular proteins, including WT-1 GATA4, and vimentin. In summary, our study uncovers a novel mechanism by which aluminum exposure impairs spermatogenesis via NLRP3-mediated pyroptosis in testicular cells. We also demonstrate for the first time that NMN can ameliorate aluminum-induced reproductive dysfunction by inhibiting this pathway, offering a potential therapeutic strategy for aluminum-associated male infertility.
Citation: Nong W, Huang Q, Dou S, Wei Y, Song Y, Wang J, et al. (2026) Nicotinamide mononucleotide improves spermatogenesis in aluminium-exposed mice by inhibiting NLRP3-mediated pyroptosis. PLoS One 21(1): e0339020. https://doi.org/10.1371/journal.pone.0339020
Editor: Suresh Yenugu, University of Hyderabad, INDIA
Received: May 27, 2025; Accepted: December 1, 2025; Published: January 22, 2026
Copyright: © 2026 Nong et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: “Transcriptome data are available in the Sequence Read Archive under accession number PRJNA1379538. Uncropped and unedited blots are provided in S1 Raw Images. All other Supporting information files are accessible as part of the Supporting information.”.
Funding: This work was supported by the National Natural Science Foundation of China( No.82060293),and the 2022 Scientific Research and Technology Development Program of Baise City (No. 20224124). The funders (National Natural Science Foundation of China and Baise City Scientific Research Program) provided the financial support for this work but did not participate in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Aluminum (Al) is an abundant metal that is considered a potentially hazardous environmental contaminant due to its extensive industrial use and anthropogenic emissions. Its increasing application across diverse industries has been associated with various adverse health outcomes related to occupational and environmental exposure [1]. Aluminum salts are also commonly used as adjuvants in pediatric vaccines, raising ongoing concerns regarding their biological safety [2,3]. Aluminum can enter the human body through dermal absorption, inhalation, ingestion, or intramuscular injection, exerting toxic effects on multiple organ systems, particularly the male reproductive system [4]. The testes are among the primary targets of aluminum toxicity, and even low-level exposure can cause significant reproductive dysfunction, including reduced sperm quality, testicular damage, and hormonal disturbances [4,5,6].
Previous studies have demonstrated that aluminum activates glial cells, sensitizing the DDX3X–NLRP3 pyroptosis pathway and promoting the release of proinflammatory cytokines such as interleukin (IL)-1β and IL-18, which exacerbate neuroinflammation [7]. Animal studies have also shown increased serum levels of TNF-α and IL-6 in aluminum-treated rats, along with histopathological alterations in the testes and epididymides [8]. Similarly, Lokman et al. reported elevated testicular expression of IL-1β and NF-κB in aluminum-exposed rats [9]. These findings suggest that inflammation-related mechanisms, particularly NLRP3-mediated pyroptosis, play a critical role in aluminum-induced male reproductive impairment. However, effective pharmacological strategies to prevent or treat aluminum-induced infertility remain lacking.
Exposure to heavy metals such as lead, cadmium, mercury, and aluminum is widely recognized as a major environmental contributor to male infertility. These metals can accumulate in the testes, epididymis, and accessory glands, inducing oxidative stress, mitochondrial dysfunction, and disruption of the hypothalamic–pituitary–gonadal axis, ultimately impairing spermatogenesis and sperm motility [10]. Testicular injury induced by heavy metals has been linked to increased production of reactive oxygen species (ROS) and inflammatory cytokines, resulting in germ cell apoptosis and reduced sperm function [11]. Moreover, aluminum has been shown to activate the NLRP3 inflammasome, thereby promoting testicular inflammation and pyroptotic cell death [12]. Chronic exposure to heavy metals is therefore a major threat to male reproductive health, as it damages Sertoli and Leydig cells and disrupts spermatogenic homeostasis. Elucidating the mechanisms by which aluminum impairs spermatogenesis and exploring effective therapeutic interventions—such as nicotinamide mononucleotide (NMN)—is thus essential for preventing metal-induced male infertility.
NMN, a key precursor in the biosynthesis of nicotinamide adenine dinucleotide (NAD⁺) [13], is widely used to restore NAD⁺ levels and regulate cellular metabolism. NAD⁺ plays a vital role in controlling inflammation and oxidative stress. In models of intervertebral disc degeneration, NMN has been shown to attenuate IL-1β–induced inflammatory responses by inhibiting the NF-κB pathway in a SIRT1-dependent manner, thereby preventing apoptosis in nucleus pulposus cells [14,15]. Moreover, NMN suppresses the expression of IL-6, IL-1β, and TNF-α by inhibiting NF-κB p65 activation, reducing UV-induced skin inflammation [16]. It also inhibits doxorubicin-induced NLRP3 inflammasome activation, restores antioxidant capacity by increasing glutathione (GSH) and superoxide dismutase (SOD) activity, and reduces oxidative markers such as malondialdehyde (MDA) and ROS [17]. Our previous work demonstrated that NMN restored supporting cell function and cytoskeletal integrity in diabetic mice, significantly improving testicular weight and sperm quality [18]. However, whether NMN can alleviate aluminum-induced reproductive dysfunction remains unclear.
In this study, we established an AlCl3-induced mouse model to evaluate the protective effects of NMN against aluminum-related testicular dysfunction. We hypothesize that NMN ameliorates aluminum-induced reproductive impairment by inhibiting NLRP3-mediated pyroptosis, providing a potential therapeutic strategy for aluminum-induced male infertility.
Methods and materials
Construction of an aluminum exposure mouse model
A total of 30 mature male C57BL/6J mice, aged 8 weeks and weighing 21−23 g, were purchased from Silaik Jingda Animal Company (License No.: SCXK (Xiang) 2019−0004). The animal experimental protocol was approved by the SCU Laboratory Animal Welfare and Ethics Committee (NO: 2021USA0628). Prior to the experiment, the mice underwent a 1-week acclimatization period. The housing conditions were maintained at a room temperature of 25 ± 2 °C, with a relative humidity of 50% to 70%, and a 12-hour light/dark cycle. During the experimental period, the mice had ad libitum access to food and water. The mice were randomly divided into three groups: the control group, the aluminum exposure group, and the aluminum exposure plus NMN group, with 10 mice in each group. The aluminum exposure group received an intragastric administration of 20 mg/kg AlCl3 (AlCl3·6H2O, sourced from Shanghai Aladdin Biochemical Technology Company, Lot No. A112509). The aluminum exposure plus NMN group received 20 mg/kg AlCl3 followed by an administration of 500 mg/kg NMN (Batch No. XJY01210615, Shenzhen Xigia Biotechnology Co., Shenzhen, China) 30 minutes later. The control group received an equivalent volume of ultrapure water via intragastric administration once daily for a duration of 6 weeks.
Animal anesthesia, sacrifice, and welfare
All surgical procedures (e.g., for tissue collection) and final sacrifice were performed under anesthesia induced by intraperitoneal injection of urethane (1.0 g/kg). At the end of the experiment, all mice were euthanized by cervical dislocation under deep anesthesia to ensure death without pain. Every effort was made to minimize animal suffering. This included monitoring the mice twice daily for signs of distress (e.g., reduced mobility, abnormal posture, or significant weight loss), providing soft bedding, and ensuring ad libitum access to food and water in a controlled environment throughout the study [18–21].
Sperm analysis
After six weeks of treatment, the mice were anesthetized, and relevant tissues were collected to calculate various parameters. One side of the epididymis was placed in 1.5 mL of 37°C physiological saline and thoroughly cut open. The sample was incubated at 37°C for 15 minutes to allow the sperm to swim out completely. The sperm suspension was then transferred to a counting chamber for analysis. Sperm morphology was examined using a rapid staining kit (Solarbio, China), and sperm counts and the number of abnormal sperm were quantitatively analyzed under a microscope using the counting chamber. A total of 200 sperm were counted, and the percentage of abnormal sperm was calculated. Specific observational methods were conducted according to the previously described procedures [22].
Histological analysis of testicular and epididymal tissues
Testicular and epididymal tissues were fixed in 4% formaldehyde for 24 hours, followed by paraffin embedding and sectioning into 5 µm thick slices for histological analysis. Hematoxylin and eosin (H&E) staining was performed to examine the morphology of the seminiferous tubules and epididymis in the testicular tissue sections. Subsequently, 50 seminiferous tubules were randomly selected from each group under a microscope, and their areas and diameters were measured and statistically analyzed.
Serum detection of reproductive hormones in mice
After anesthetizing the mice, blood was collected by excising the eyeballs. The blood was allowed to stand at room temperature (23 ± 2 °C) for 30 minutes and then centrifuged at 4 °C at 3000 rpm for 15 minutes. The serum supernatant was separated by the Northern Beijing Institute of Biotechnology for radioimmunoassay to measure testosterone levels. An enzyme-linked immunosorbent assay (ELISA) kit was used to determine testosterone levels in the cell culture supernatant according to the manufacturer’s instructions. The protein content was measured at 450 nm.
Transcriptome sequencing and bioinformatic analysis of mouse testes
Testicular RNA sequencing was conducted by Novogene (Beijing, China). In brief, to assess the purity and concentration of RNA, a NanoPhotometer® spectrophotometer (Implen Inc., CA, USA) and a Qubit® RNA Assay Kit in a Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) were utilized. Following the manufacturer’s instructions, an RNA sequencing library was created using the NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (California, USA), with each sample containing 3 µg of RNA. Index codes were incorporated into the attribute sequences of each sample. The indexed samples were clustered using the TruSeq PE Cluster Kit v3 cBot-HS (Illumina, California, USA) on the cBot clustering generation system. The prepared libraries were sequenced on the Illumina HiSeq platform, generating paired-end reads of 150 bp. Differential expression analysis between groups was performed using the DESeq2 R package (1.10.1). DESeq2 adjusted the p-values of the genes to determine which genes exhibited differential expression. Statistical enrichment of differentially expressed genes (DEGs) in KEGG pathways was examined using the Cluster Profiler R package (http://www.genome.jp/kegg/).
Quantitative real-time PCR (qRT-PCR)
Real-time quantitative reverse transcription PCR (qRT-PCR) was performed to detect mRNA expression levels. Total RNA was extracted from mouse testicular tissue using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Subsequently, RNA was reverse transcribed into complementary DNA (cDNA) using the TransGen One-Step gDNA Removal and cDNA Synthesis Super Mix (TransGen, Beijing, China). GAPDH was used as a reference for mRNA expression. Real-time quantitative PCR was conducted on an ABI7900 PCR system (Applied Biosystems, Foster City, CA, USA) using SYBR Green qPCR Master Mix (Thermo Fisher Scientific, Cat. No. 4309155). The primer sequences are listed in Table 1.
Western blot analysis
The testicular lysate was prepared by combining 300 μL of RIPA lysis buffer (CWBIO, Beijing, China) with 6 μL of PMSF (Solarbio) and three steel balls with a diameter of 3.2 mm in a 1.5 mL EP tube. The tissue was homogenized using a tissue lyser (Tissuelyser-24). The protein concentration was determined using a BCA protein assay kit (CWBIO), following denaturation of the proteins by heating at 100°C for 10 minutes. Denatured proteins were separated by 10% SDS-PAGE and subsequently transferred to a polyvinylidene fluoride membrane. The membrane was blocked for 2 hours with PBST (phosphate-buffered saline containing 0.1% Tween-20) supplemented with 5% non-fat dry milk. After washing with PBST, the membrane was incubated overnight at 4°C with primary antibodies against Tubulin, Vimentin, WT-1 GATA4, NLRP3, GSDMD, caspase-1, and IL-1β. Subsequently, the membrane was incubated for 2 hours at room temperature with HRP-conjugated affinity-purified goat anti-mouse IgG (H + L) or goat anti-rabbit IgG (H + L). Finally, ECL (CW0049M, CWBIO) was added, and the protein bands were detected using the T-5500 chemiluminescence imaging system. The antibodies used in the experiment are listed in Table 2.
Immunohistochemical analysis
Paraffin sections were hydrated using an ethanol gradient and subjected to high-pressure incubation for 3 minutes in a pH 6.0 citrate buffer to retrieve antigens. Endogenous peroxidase blocking agent was applied, followed by washing with PBS. A nonspecific staining blocking agent was then added, along with the corresponding primary antibody solution. The samples were covered with a wet box lid and incubated overnight at 4°C. The primary antibody solution was washed out with PBS, and a secondary antibody was introduced and incubated for 20 minutes. Following another PBS wash to remove excess secondary antibody, DAB chromogen was applied for staining. After thorough washing with water to remove residual DAB, the samples were stained with hematoxylin, washed, blued, dehydrated, and mounted. Each group was analyzed and counted for the number of positive cells in 50 seminiferous tubules under a microscope. Antibodies used are listed in Table 2.
Statistical analysis
Data were analyzed using GraphPad Prism 8.0 (GraphPad Software, La Jolla, USA). Continuous variables are expressed as mean ± standard deviation (SD), and one-way analysis of variance (ANOVA) was employed. For pairwise comparisons between groups, the Student-Newman-Keuls (SNK) test was used when variances were homogeneous, while the Games-Howell test was applied when variances were heterogeneous. A p-value of ≤ 0.05 was considered statistically significant.
Results
Effects of NMN treatment on testicular and epididymal morphology and sperm quality in aluminum-exposed mice
An aluminum exposure model was established via oral AlCl3 administration to investigate NMN’s effects on body weight, testicular weight, and semen quality in aluminum-exposed mice. After one week of acclimation feeding, male mice were randomly assigned to groups. The AlCl3 intervention group exhibited reduced body weight, which gradually increased after NMN supplementation, aligning with the control group. Testicular weighing revealed reduced weights in the AlCl3-treated group (0.16 ± 0.01 g, p < 0.01) compared to the control group. NMN supplementation increased testicular weight (0.19 ± 0.01 g, p < 0.01). Sperm quality assessment in the AlCl3-treated group revealed decreased motility, reduced sperm count, and increased abnormal sperm proportion, with more pronounced abnormalities than the control group. All parameters improved after NMN supplementation. NMN treatment significantly increased sperm count in aluminum-exposed mice, improved body weight, testicular weight, and sperm motility, while reducing both the proportion and severity of abnormal sperm (Table 3).
Effects of NMN on reproductive hormones in aluminum-exposed mice
To evaluate the impact of aluminum exposure on reproductive health in mice, we measured reproductive hormones. Results showed that serum testosterone levels decreased in the AlCl3 intervention group compared to controls, but increased after NMN supplementation (Fig 1A). Similar to testosterone, serum FSH and LH concentrations were significantly reduced in the AlCl3-treated group. Following NMN treatment, female hormones FSH and LH concentrations remained significantly decreased (Fig 1B, C), potentially related to feedback regulation of the HPG axis.
A) Serum testosterone; B) Serum testosterone FSH; C) Serum LH. Testosterone (T), Follicle-stimulating hormone(FSH), luteinizing hormone (LH); **p < 0.01.
Effects of NMN treatment on the morphology of testes and epididymis in mice exposed to aluminum
The morphology of the testes and epididymis was assessed across different groups of mice. In the control group, the testes exhibited normal structural integrity, with intact seminiferous tubules and orderly arrangement of germ cells at various stages of division. A significant number of differentiated and mature sperm were visible within the lumen (Fig 2A, B). In contrast, aluminum-exposed mice showed atrophy of the seminiferous tubules, disorganized structures, and a reduced number of germ cells with sparse arrangement (Fig 2A). Notably, very few sperm were observed in the epididymis of aluminum-exposed mice (Fig 2B, E and F). However, following NMN treatment, these pathological changes showed varying degrees of improvement (Fig 2A–F).
A) HE staining of mouse testis; B) HE staining of mouse epididymis; C) Diameter of mouse spermatogenic tubule; D)Mouse spermatogenic tubule area; E-F) Spermatogonium and spermatocyte count. **p < 0.01.
Impact of NMN intervention on the function of testicular sertoli cells in aluminum-exposed mice
Sertoli Cells play a crucial role in spermatogenesis. To investigate whether the function and structure of testicular Sertoli Cells in aluminum-exposed mice are compromised, we employed qRT-PCR, Western blotting, and immunohistochemistry to assess the changes in mRNA and protein expression of WT-1 GATA4, and Vimentin. Compared to the control group, the mRNA expression levels of SCs marker genes (GATA4, WT-1 Vimentin) were significantly reduced in the testes of aluminum-exposed mice. However, NMN treatment significantly increased the expression of GATA4, WT-1 and Vimentin mRNA in the testes compared to aluminum-exposed mice (Fig 3A–C). The results of Western blot analysis of SCs marker gene expression corroborated our qRT-PCR findings (Fig 3D–G). Immunohistochemical analysis of testes from different treatment groups revealed a decrease in the number of SCs in the testes of aluminum-exposed mice compared to the control group (Fig 3H). Red arrows indicate cells with positive signals. The number of vimentin-positive cells was significantly higher in the AlCl3 + NMN group compared to the AlCl3 group (Fig 3H).
A–C) qPCR validation of NMN intervention effects on GATA4, WT-1 and Vimentin mRNA expression in aluminum-exposed mouse testes; D–G) Western Blotting assessment of NMN intervention effects on testicular supporting cell function in aluminum-exposed mice; H) Immunohistochemical detection of Vimentin, ZO-1, and Occludin expression in mouse testicular tissue; I–L) qPCR validation of NMN intervention effects on ZO-1, Occludin, TNF-α, and IL-6 in aluminum-exposed mouse testes. *p < 0.05, **p < 0.01.
To assess the effects of aluminum exposure and NMN treatment on blood-testis barrier (BTB) integrity, immunohistochemical staining was used to detect the expression of tight junction proteins occludin and ZO-1 in mouse testes. As shown in Fig 3H, both occludin and ZO-1 expression were significantly reduced in the AlCl3 group, indicating impaired BTB structure. qPCR results were consistent with this finding (Fig 3I, J). Conversely, NMN administration restored their expression levels to near-control group levels. These findings indicate that aluminum exposure compromises blood-testis barrier integrity, while NMN supplementation protects and stabilizes tight junction dynamics within Sertoli cells.
Supplementation with NMN can alter the transcriptional profile in mice exposed to aluminum
To further investigate the molecular mechanisms by which NMN improves spermatogenesis in aluminum-exposed mice, we conducted RNA sequencing (RNA-seq) analysis on testicular tissue. Principal component analysis (PCA) of all expressed genes revealed significant changes in the transcriptome of the testicular tissue from mice treated with AlCl3. Notably, NMN treatment partially ameliorated these changes (Fig 4A). Visualization through a volcano plot (Fig 4B) showed that compared to the Ctrl, the AlCl3 treatment group had 809 upregulated genes and 733 downregulated genes. Following NMN supplementation, the AlCl3 treatment group exhibited 1,083 upregulated genes and 712 downregulated genes (Fig 4B). Venn diagram analysis and clustered heatmap analysis (Fig 3C) were employed to integrate data across groups. Notably, the AlCl3 treatment group displayed 233 differentially expressed genes (DEGs) that were upregulated compared to the control group, while these genes were downregulated in the AlCl3 + NMN group. Conversely, 350 DEGs that were downregulated in the AlCl3 treatment group were upregulated in the AlCl3 + NMN group. Our findings suggest that these genes may play a crucial regulatory role in NMN’s ability to alleviate spermatogenic impairment caused by aluminum exposure. Further clustering analysis through heatmap visualization revealed that genes such as NLRP3, GSDMD, caspase-1, and IL-1β were upregulated in the AlCl3 treatment group compared to the control group but were improved following NMN treatment (Fig 4D). To deepen our understanding of the biological logic behind these changes in gene expression, we conducted KEGG enrichment analysis, which indicated that these DEGs were primarily enriched in signaling pathways related to NOD-like receptors, apoptosis, and oxidative stress (Fig 4E). This molecular mechanism elucidates how NMN improves reproductive dysfunction in aluminum-exposed mice by inhibiting NLRP3-mediated pyroptosis in germ cells.
Testicular tissues from the control, high aluminum dose, and NMN intervention groups were analyzed through RNA sequencing. A) PCA analysis of testis gene expression is shown, with each point representing a sample. B) Volcano map comparison of gene expression between pairs (n = 3). C) Venn diagrams illustrate the differentially expressed genes between groups, and heat maps of the differentially expressed genes in clusters. D) Cluster analysis of genes related to the NOD-like receptor signaling pathway. E) KEGG pathway enrichment analysis of differentially expressed genes across the three groups.
NMN intervention alleviates inflammatory responses and pyroptosis levels in testicular tissues of aluminum-exposed mice
To further evaluate inflammatory responses, we detected TNF-α and IL-6 expression levels in testicular tissues via qRT-PCR. As shown in Fig 3K, L, compared with the control group, mRNA expression levels of both TNF-α and IL-6 were significantly elevated in the AlCl3 group. Notably, NMN treatment significantly reduced the expression levels of these cytokines, restoring them to levels comparable to the control group. Transcriptomic analysis of testicular tissue revealed that AlCl3 treatment significantly upregulated the expression of inflammation-related and pyroptosis-associated factors. Key regulatory molecules and effectors of testicular pyroptosis (including NLRP3, GSDMD, caspase-1, and IL-1β) showed an upward trend in the AlCl3-treated group, but their expression levels were significantly reduced after NMN intervention. Expression levels of NLRP3, caspase-1, GSDMD, and IL-1β in testicular tissues across groups were detected via qRT-PCR, Western blotting, and IHC techniques, revealing expression trends consistent with transcriptomic analysis. As shown in Fig 5A, qRT-PCR results indicated significantly elevated mRNA expression levels of key regulatory molecules and effector factors (NLRP3, caspase-1, GSDMD, and IL-1β) in the testes of AlCl3-treated mice, which exhibited a downregulated trend after NMN intervention. Western blot results corroborated the qRT-PCR analysis trend (Fig 4B, C). Furthermore, immunohistochemical results confirmed the presence of pyroptosis-related factor expression in testicular tissue, which was significantly elevated in the AlCl3-treated group compared to the control group and decreased following NMN intervention (Fig 5D).
A) Effect of NMN intervention on the expression of NLRP3,GSDMD, caspase-1 and IL-1β mRAN in the testis of aluminium-exposed mice; B) Western Blotting detected the effects of NMN intervention on the expression of NLRP3 and inflammation-related gene proteins in the testis of mice exposed to aluminum; C)Image J Analysis of the effects of NMN intervention on the expression of NLRP3,GSDMD, caspase-1 and IL-1β proteins in the testis of aluminium-exposed mice; D) Immunohistochemical detection of NMN intervention on the expression of NLRP3 and inflammation-related gene proteins in testis of aluminum-exposed mice.
NMN intervention significantly reverses AlCl3-induced damage to mouse spermatogenic cells
To precisely identify the developmental stage at which germ cell loss occurs, this study employed immunofluorescence staining to systematically detect germ cell-specific markers at different developmental stages. Results showed that the pan-germ cell marker MVH exhibited significantly reduced expression in the AlCl3-exposed group, while recovery to near-normal levels was observed in the AlCl3 + NMN group (Fig 6A). Notably, the pro-meiotic marker STRA8 showed no significant differences among the three groups (control, AlCl3, and AlCl3 + NMN), indicating that spermatocyte differentiation remained relatively intact under these conditions (Fig 6B). Conversely, the meiosis-specific marker SCP3 in spermatocytes was significantly weakened in the AlCl3 group, while NMN intervention effectively restored its expression levels (Fig 6C). These findings suggest that aluminum exposure primarily impacts the meiotic stage of germ cells, while NMN exerts significant cytoprotective effects by maintaining meiotic integrity in spermatocytes.
Immunofluorescence staining was used to detect the expression of germ cell markers at different developmental stages. A) Shows that the pan-germ cell marker MVH was significantly downregulated in the AlCl3-exposed group, with expression restored in the AlCl3 + NMN group; B) The pro-meiotic marker STRA8 showed no significant differences among the control, AlCl3, and AlCl3 + NMN groups; C) Spermatocyte meiosis marker SCP3 showed significantly reduced expression in the AlCl3 group, with expression restored in the AlCl3 + NMN group following NMN intervention. Results indicate aluminum exposure primarily impacts the meiosis stage of germ cells, while NMN maintains spermatocyte meiosis integrity to exert protective effects.
Discussion
Aluminum is a prevalent environmental toxicant that can enter the body via inhalation, ingestion, dermal contact, and intramuscular injection. Accumulation of aluminum can exert toxic effects on various organ systems, with the male reproductive system being one of its primary targets. Exposure to aluminum can lead to impaired fertility and significantly reduced sperm quality [4]. In this study, mice exposed to AlCl3 exhibited severe histological damage, including reduced seminiferous tubule area, morphological disruption, and a decrease in spermatogonia, primary spermatocytes, Sertoli cells, and sperm. Additionally, there was a significant reduction in testicular and epididymal weights, as well as decreased sperm motility and an increase in abnormal sperm. These findings align with previous reports indicating that aluminum exposure impairs spermatogenesis and epididymal sperm maturation. Notably, NMN treatment effectively ameliorated these reproductive damages, suggesting that its protective effects extend beyond cellular and molecular levels to functional sperm outcomes. The improvement in sperm quality likely results from the restoration of Sertoli cell integrity, reduced oxidative stress, and normalization of testosterone levels, all contributing to enhanced spermatogenic efficiency.
Our study investigates the protective role of nicotinamide mononucleotide (NMN) against aluminum-induced testicular dysfunction, focusing on its interaction with the NLRP3 inflammasome. While previous studies have indicated NMN’s protective effects on testicular function [23,24] and the role of NLRP3 in aluminum toxicity [25,26], our research uncovers a novel mechanism by which NMN alleviates aluminum-induced reproductive dysfunction. Specifically, we show that NMN mitigates testicular damage by inhibiting NLRP3-mediated pyroptosis in Sertoli cells, a pathway not fully explored in the context of aluminum exposure.
This study identifies NMN as an effective inhibitor of NLRP3, GSDMD, caspase-1, and IL-1β expression, key components of the inflammasome pathway. By reducing the activation of these inflammatory mediators, NMN restores spermatogenesis, improves testosterone synthesis, and enhances overall testicular function. Furthermore, we highlight the roles of WT1 and GATA4 in maintaining Sertoli cell integrity and their restoration following NMN treatment, contributing to an improved testicular microenvironment and fertility outcomes.
Thus, our findings represent a significant advancement in understanding the molecular mechanisms underlying aluminum-induced reproductive toxicity and provide evidence supporting NMN as a potential therapeutic agent for male infertility caused by environmental toxicants like aluminum.
Sertoli cells (SCs), located within the seminiferous tubules, are crucial for the development and maturation of germ cells [27]. The integrity of the blood-testis barrier (BTB) is essential for spermatogenesis, as it provides a specialized microenvironment for germ cell development and protects against toxic insults. Occludin and ZO-1 are critical components of the Sertoli cell tight junction complex that regulate BTB permeability. Our results show a marked reduction in these junctional proteins following aluminum exposure, suggesting BTB disruption and impaired Sertoli cell junctional communication. NMN treatment effectively restored Occludin and ZO-1 expression, indicating its role in maintaining BTB stability. These findings align with previous evidence that NAD ⁺ -dependent signaling enhances tight junction assembly and antioxidative capacity in epithelial barriers. Therefore, NMN’s protective effects on BTB integrity may represent a key mechanism by which it preserves spermatogenesis under aluminum toxicity.
Previous research has identified aluminum accumulation in Sertoli cells of exposed mice, with transmission electron microscopy revealing disrupted tight junctions between Sertoli cells [28,29]. Vimentin, a type III intermediate filament, is a major structural protein in the Sertoli cell cytoskeleton. During normal spermatogenesis, Vimentin is the predominant protein in the SC cytoskeleton, and its distribution pattern is directly related to the morphological integrity of the germinal epithelium [30]. The downregulation of Vimentin is associated with decreased spermatogenesis [31]. GATA4 and WT1 are key transcription factors for Sertoli cell differentiation and function. Their downregulation in aluminum-exposed mice indicates impaired Sertoli cell support, which NMN treatment alleviates by restoring their expression [32–37].
Our findings indicate that mice exposed to AlCl3 exhibited a significant downregulation of Vimentin expression, which may disrupt the connections between Sertoli cells and germ cells, leading to significant sloughing of the germinal epithelium [34] and a reduction in spermatogonia, primary spermatocytes, and supporting cells. GATA4 is considered a specific marker for supporting cells during development and adulthood [32,33]. Numerous studies have shown that GATA4 works synergistically with the WT1 gene to promote Sertoli cell function, male sexual differentiation, and testosterone production [36–39]. Our results demonstrate that mRNA and protein expressions of WT1 and GATA4 were significantly reduced in the testicular tissue of mice exposed to aluminum. NMN upregulated the expression levels of WT1 and GATA4 in AlCl3-treated mice, mitigating the destruction of tubule integrity and Sertoli cell connections, ultimately improving the testicular dysfunction induced by AlCl3.
Further immunofluorescence analysis revealed that germ cell loss was most pronounced during the meiotic stage, rather than the pre-meiotic phase. MVH expression was markedly downregulated following aluminum exposure, indicating overall germ cell depletion, while STRA8 expression remained relatively stable across groups, suggesting preserved spermatogonial differentiation. However, the marked reduction in SCP3, a synaptonemal complex protein essential for meiosis, confirmed that aluminum primarily disrupts spermatocyte meiosis. NMN supplementation restored SCP3 expression, implying that NMN preserves meiotic progression by protecting Sertoli cell function and reducing inflammatory and oxidative stress in the testicular microenvironment.
To further investigate the mechanism by which NMN ameliorates reproductive damage due to aluminum exposure, we performed transcriptome sequencing (RNA-seq) analysis on testicular tissue. While NMN’s protective effects on testes in diabetic/aged models and aluminum-NLRP3 links in neural systems have been reported, our work uncovers a previously unrecognized role of NLRP3-GSDMD pyroptosis in aluminum-induced spermatogenesis impairment. Crucially, we identify NMN as an effective inhibitor of this pathway specifically in testicular tissue and establish WT1/GATA4 as key regulators in maintaining the spermatogenic microenvironment upon aluminum exposure (Fig 3). These findings expand the mechanistic understanding of reproductive toxicity and highlight NMN’s translational potential.
The results revealed significant upregulation of NLRP3, GSDMD, caspase-1, and IL-1β in the AlCl3 treatment group compared to the control group, which improved after NMN supplementation. Studies indicate that non-infectious testicular inflammation can also lead to decreased Sertoli cell function [40]. Additionally, high expression of NLRP3 in rat Sertoli cells has been shown to activate caspase-1, promoting the secretion of inflammatory cytokines IL-1β and IL-18 [41]. There is substantial evidence that pyroptosis plays a critical role in male reproductive disorders, with extensive research confirming that the NLRP3 inflammasome is a key regulatory factor in pyroptosis [42]. The NLRP3 inflammasome is a multiprotein complex composed of the intracellular innate immune receptor NLRP3, apoptosis-associated speck-like protein (ASC), and the protease caspase-1. Activation of the NLRP3 inflammasome induces the maturation and secretion of IL-1β and IL-18, subsequently triggering pyroptosis [43,44]. Consistently, our findings indicate that AlCl3-induced inflammation activates NLRP3-mediated pyroptosis in Sertoli cells.
Although caspase-3 and caspase-9 are pivotal in apoptotic cell death, our study focused on caspase-1 due to its specific role in NLRP3 inflammasome-mediated pyroptosis, a process characterized by inflammatory cytokine release and membrane rupture, which aligns with the histopathological observations in aluminum-exposed testes (Fig 2). Notably, caspase-1 activation directly cleaves GSDMD to execute pyroptosis, whereas caspase-3/-9 operate in distinct apoptotic pathways. This mechanistic specificity justifies our emphasis on caspase-1 as the key effector in aluminum-induced testicular injury.
Transcriptome sequencing results showed upregulation of NLRP3, GSDMD, caspase-1, and IL-1β in the AlCl3 treatment group, which were significantly downregulated following NMN supplementation. These results suggest that AlCl3-induced impairment of spermatogenic function in male mice is directly related to testicular inflammatory responses and activation of pyroptosis pathways, with NLRP3, GSDMD, caspase-1, and IL-1β potentially serving as key targets for treating AlCl3-induced testicular dysfunction. Molecular validation through Western blot and quantitative PCR confirmed these findings, showing that NMN treatment significantly reduced the expression levels of these inflammatory mediators. Immunohistochemical results were consistent with the transcriptome sequencing, Western blot, and PCR findings, further demonstrating that NMN improves male reproductive dysfunction caused by aluminum exposure by suppressing NLRP3, GSDMD, caspase-1, and IL-1β expression.
In conclusion, this study established a mouse model.
Conclusion
NMN mitigates NLRP3 inflammasome activation-induced pyroptosis, reduces inflammatory factor expression, promotes testosterone synthesis and secretion, and restores germ cell function, ultimately improving spermatogenesis in aluminum-exposed mice.
Supporting information
S1 Raw Images. Uncropped Western blot images.
This file contains all original, uncropped Western blot scans related to the figures in the main text.
https://doi.org/10.1371/journal.pone.0339020.s001
(PPTX)
S1 File. Abstract Figure.
NMN improves spermatogenesis in aluminum-exposed mice by suppressing inflammation and pyroptosis-related factors.
https://doi.org/10.1371/journal.pone.0339020.s002
(TIF)
References
- 1. Vlasak T, Dujlovic T, Barth A. Aluminum exposure and cognitive performance: A meta-analysis. Science of The Total Environment. 2024;906:167453.
- 2. Shardlow E, Mold M, Exley C. Unraveling the enigma: elucidating the relationship between the physicochemical properties of aluminium-based adjuvants and their immunological mechanisms of action. Allergy Asthma Clin Immunol. 2018;14:80. pmid:30455719
- 3. Janeway CA. Approaching the Asymptote? Evolution and Revolution in Immunology. Cold Spring Harb Symp Quant Biol. 1989;54:1–13.
- 4. Yokel RA. Aluminum reproductive toxicity: a summary and interpretation of scientific reports. Crit Rev Toxicol. 2020;50(7):551–93. pmid:32869713
- 5. Gupta A, Kumar A, Naqvi S, Flora SJS. Chronic exposure to multi-metals on testicular toxicity in rats. Toxicol Mech Methods. 2021;31(1):53–66. pmid:32972309
- 6. Cao C, Zhang H, Wang K, Li X. Selenium-Rich Yeast Mitigates Aluminum-Mediated Testicular Toxicity by Blocking Oxidative Stress, Inhibiting NO Production, and Disturbing Ionic Homeostasis. Biol Trace Elem Res. 2020;195(1):170–7. pmid:31327124
- 7. Hao W, Zhu X, Liu Z, Song Y, Wu S, Lu X, et al. Aluminum exposure induces central nervous system impairment via activating NLRP3-medicated pyroptosis pathway. Ecotoxicol Environ Saf. 2023;264:115401. pmid:37634479
- 8. Khafaga AF. Exogenous phosphatidylcholine supplementation retrieve aluminum-induced toxicity in male albino rats. Environ Sci Pollut Res Int. 2017;24(18):15589–98. pmid:28523611
- 9. Lokman M, Ashraf E, Kassab RB, Abdel Moneim AE, El-Yamany NA. Aluminum Chloride-Induced Reproductive Toxicity in Rats: the Protective Role of Zinc Oxide Nanoparticles. Biol Trace Elem Res. 2022;200(9):4035–44. pmid:34741695
- 10. López-Botella A, Velasco I, Acién M, Sáez-Espinosa P, Todolí-Torró J-L, Sánchez-Romero R, et al. Impact of Heavy Metals on Human Male Fertility-An Overview. Antioxidants (Basel). 2021;10(9):1473. pmid:34573104
- 11. Agarwal A, Baskaran S, Parekh N, Cho C-L, Henkel R, Vij S, et al. Male infertility. Lancet. 2021;397(10271):319–33. pmid:33308486
- 12. Bhattacharya I, Sharma SS, Majumdar SS. Etiology of Male Infertility: an Update. Reprod Sci. 2024;31(4):942–65. pmid:38036863
- 13. Wei C-C, Kong Y-Y, Li G-Q, Guan Y-F, Wang P, Miao C-Y. Nicotinamide mononucleotide attenuates brain injury after intracerebral hemorrhage by activating Nrf2/HO-1 signaling pathway. Sci Rep. 2017;7(1):717. pmid:28386082
- 14. Ji ML, Jiang H, Zhang XJ, Shi PL, Li C, Wu H, et al. Preclinical development of a microRNA-based therapy for intervertebral disc degeneration. Nat Commun. 2018.
- 15.
Shen J, Fang J, Hao J, Zhong X, Wang D, Ren H, et al. SIRT1 inhibits the catabolic effect of IL-1β through TLR2/SIRT1/NF-κB pathway in human degenerative nucleus pulposus cells. 2016;19(E215).
- 16. Zhou X, Du H-H, Long X, Pan Y, Hu J, Yu J, et al. β-Nicotinamide Mononucleotide (NMN) Administrated by Intraperitoneal Injection Mediates Protection Against UVB-Induced Skin Damage in Mice. J Inflamm Res. 2021;14:5165–82. pmid:34675595
- 17. Wan Y, He B, Zhu D, Wang L, Huang R, Zhu J, et al. Nicotinamide mononucleotide attenuates doxorubicin-induced cardiotoxicity by reducing oxidative stress, inflammation and apoptosis in rats. Arch Biochem Biophys. 2021;712:109050. pmid:34610336
- 18. Ma D, Hu L, Wang J, Luo M, Liang A, Lei X, et al. Nicotinamide mononucleotide improves spermatogenic function in streptozotocin-induced diabetic mice via modulating the glycolysis pathway. Acta Biochim Biophys Sin (Shanghai). 2022;54(9):1314–24. pmid:35929593
- 19. Peng H, Huang Y, Wei G, Pang Y, Yuan H, Zou X, et al. Testicular Toxicity in Rats Exposed to AlCl3: a Proteomics Study. Biol Trace Elem Res. 2024;202(3):1084–102. pmid:37382810
- 20. Güvenç M, Cellat M, Gökçek İ, Arkalı G, Uyar A, Tekeli İO, et al. Tyrosol prevents AlCl3 induced male reproductive damage by suppressing apoptosis and activating the Nrf-2/HO-1 pathway. Andrologia. 2020;52(2):e13499. pmid:31828839
- 21. Mills KF, Yoshida S, Stein LR, Grozio A, Kubota S, Sasaki Y, et al. Long-Term Administration of Nicotinamide Mononucleotide Mitigates Age-Associated Physiological Decline in Mice. Cell Metab. 2016;24(6):795–806. pmid:28068222
- 22. Ma M, Zhang N, Hao D-M, Bo L, Yu Y-X. Internal quality control products for computer-assisted sperm analysis: Research and application. Zhonghua Nan Ke Xue. 2020;26(10):895–9. pmid:33382220
- 23. Mou H, Zhang X, Ren F, Deng Y, Chi A, Zhan G, et al. Nicotinamide mononucleotide supplementation ameliorates testicular damage induced by ischemia-reperfusion through reshaping macrophage and neutrophil inflammatory properties. Int Immunopharmacol. 2025;152:114407. pmid:40073809
- 24. Wu H, Huang B, Zhou Z, Lei X, Zhang Y, She W, et al. Nicotinamide mononucleotide ameliorates impaired testicular spermatogenesis in uranium-exposed mice by modulating glycolytic pathways. Toxicol Sci. 2025;206(2):285–98. pmid:40372707
- 25. Yang W, Nong W, Liu K, Lei X, Chen X, Jiang P, et al. Nicotinamide mononucleotide ameliorates ionizing radiation-induced spermatogenic dysfunction in mice by modulating the glycolytic pathway. Acta Biochim Biophys Sin (Shanghai). 2024;57(2):274–85. pmid:39420833
- 26. Li B, Zhang X, Huo S, Zhang J, Du J, Xiao B, et al. Aluminum activates NLRP3 inflammasome-mediated pyroptosis via reactive oxygen species to induce liver injury in mice. Chem Biol Interact. 2022;368:110229. pmid:36354081
- 27. Huo S, Zhang X, Xu J, Zhang J, Du J, Li B, et al. Parkin-mediated mitophagy protects against aluminum trichloride-induced hippocampal apoptosis in mice via the mtROS-NLRP3 pathway. Ecotoxicol Environ Saf. 2023;264:115459. pmid:37703808
- 28. Mok K-W, Mruk DD, Lee WM, Cheng CY. Rictor/mTORC2 regulates blood-testis barrier dynamics via its effects on gap junction communications and actin filament network. FASEB J. 2013;27(3):1137–52. pmid:23288930
- 29.
Maghraoui S, Florea A, Ayadi A, Horea M, Tekaya LJSEJ. Histological and ultrastructural changes observed in testicles, epididymides, seminal vesicles and liver of rat after intraperitoneal exposition to aluminium and indium. 2022.
- 30. Abdel-Moneim AM. Effects of taurine against histomorphological and ultrastructural changes in the testes of mice exposed to aluminium chloride. Arh Hig Rada Toksikol. 2013;64(3):405–14. pmid:24084349
- 31. Griswold MD. Protein secretions of Sertoli cells. Int Rev Cytol. 1988;110:133–56.
- 32. Amlani S, Vogl AW. Changes in the distribution of microtubules and intermediate filaments in mammalian Sertoli cells during spermatogenesis. Anat Rec. 1988;220:143.
- 33. Cameron DF, Snydle FE, Ross MH, Drylie DM. Ultrastructural alterations in the adluminal testicular compartment in men with varicocele. Fertil Steril. 1980;33(5):526–33. pmid:7371882
- 34. Wang CY, Tang MC, Chang WC, Furushima K, Jang CW, Behringer RR, et al. PiggyBac Transposon-Mediated Mutagenesis in Rats Reveals a Crucial Role of Bbx in Growth and Male Fertility. Biol Reprod. 2016.
- 35. McCoard SA, Lunstra DD, Wise TH, Ford JJ. Specific staining of Sertoli cell nuclei and evaluation of Sertoli cell number and proliferative activity in Meishan and White Composite boars during the neonatal period. Biol Reprod. 2001;64(2):689–95. pmid:11159374
- 36. Imai T, Kawai Y, Tadokoro Y, Yamamoto M, Nishimune Y, Yomogida K. In vivo and in vitro constant expression of GATA-4 in mouse postnatal Sertoli cells. Mol Cell Endocrinol. 2004;214(1–2):107–15. pmid:15062549
- 37. Viger RS, Mertineit C, Trasler JM, Nemer M. Transcription factor GATA-4 is expressed in a sexually dimorphic pattern during mouse gonadal development and is a potent activator of the Müllerian inhibiting substance promoter. Development. 1998.
- 38. Lavoie HA. The Role of GATA in Mammalian Reproduction. Exp Biol Med (Maywood). 2003;228:1282–90.
- 39. Tremblay JJ, Viger RS. Transcription factor GATA-4 enhances Müllerian inhibiting substance gene transcription through a direct interaction with the nuclear receptor SF-1. Mol Endocrinol. 1999;13(8):1388–401. pmid:10446911
- 40. Aldahhan RA, Stanton PG, Ludlow H, De Kretser DM, Hedger MP. Experimental cryptorchidism causes chronic inflammation and a progressive decline in Sertoli cell and Leydig cell function in the adult rat testis. Reprod Sci. 2021:1–13.
- 41. Hayrabedyan S, Todorova K, Jabeen A, Metodieva G, Toshkov S, Metodiev MV, et al. Sertoli cells have a functional NALP3 inflammasome that can modulate autophagy and cytokine production. Reports. 2016;6:1–17.
- 42. Zhong O, Ji L, Wang J, Lei X, Huang H. Association of diabetes and obesity with sperm parameters and testosterone levels: a meta-analysis. Diabetol Metab Syndr. 2021.
- 43. Mu Y, Yin T-L, Zhang Y, Yang J, Wu Y-T. Diet-induced obesity impairs spermatogenesis: the critical role of NLRP3 in Sertoli cells. Inflamm Regen. 2022;42(1):24. pmid:35915511
- 44. Hao W, Hao C, Wu C, Xu Y, Wu S, Lu X, et al. Aluminum impairs cognitive function by activating DDX3X-NLRP3-mediated pyroptosis signaling pathway. Food Chem Toxicol. 2021;157:112591. pmid:34614429