Authors: Yi Wen, Tingwei Pan, Lu Liu, Jinning Wang, Kexin Yang, Zhushan Fu, Wenhao Li, XinMiao Wang, Zhiru Shen, Yan Li, Yinke Du, Yu Deng
Categories: Research, Polystyrene nanoplastics, Manganese, Male reproductive toxicity, m6A, Mdm2-p53 Pathway, Baicalin
Source: Journal of Nanobiotechnology
Authors: Yi Wen, Tingwei Pan, Lu Liu, Jinning Wang, Kexin Yang, Zhushan Fu, Wenhao Li, XinMiao Wang, Zhiru Shen, Yan Li, Yinke Du, Yu Deng
Nanoplastics (NPs) pollution has become a pressing global environmental issue. NPs possess the ability to adsorb heavy metals, thereby acting as vectors that facilitate the entry of these toxic substances into living organisms. However, the synergistic toxic effects of NPs and heavy metals, particularly with regard to male reproductive health, remain poorly understood. This study establishes in vivo and in vitro models to assess the effects of single and co-exposure to polystyrene nanoplastics (PS-NPs, 0.1 μm) and manganese (Mn) on male reproductive function. Our results reveal that co-exposure leads to a synergistic toxic effect, aggravating testicular damage, sperm abnormalities, and hormone disruption. Mechanistically, PS-NPs and Mn collaboratively suppress the RNA-binding protein YTHDC2, which in turn impairs Mdm2 transcription and translation in an m^6^A-dependent manner. This disruption results in cell cycle arrest via the Mdm2-p53 pathway, ultimately hindering spermatogenesis. Notably, baicalin, a natural compound, effectively targets YTHDC2 and mitigates the reproductive toxicity induced by co-exposure. These findings provide the novel evidence of the synergistic reproductive toxicity of PS-NPs and Mn in male mammals, offering new insights into their combined toxic effects and highlighting the potential of baicalin as a therapeutic intervention.
The online version contains supplementary material available at 10.1186/s12951-025-03535-3.
According to the World Health Organization (WHO), infertility affects approximately 17.5% of the global population [1], with male reproductive factors contributing to nearly half of the associated risks in recent years [2]. The decline in male reproductive capacity is attributed to multiple factors, including genetic deficiencies [3], unhealthy lifestyles [4], and environmental exposures [5]. The global environmental concern surrounding microplastic pollution has grown considerably, and it is now acknowledged worldwide as a new class of pollutant [6]. Various types of microplastics (MPs) are present in soil and freshwater environments [7]. Physical and chemical degradation of plastics in the environment can produce MPs (< 5 mm) or even nanoplastics (NPs) (< 1 μm). Prolonged exposure to environmentally relevant concentrations of NPs can adversely affect testicular volume, structure, and morphology [8], as well as impair sperm quality and male hormone levels [9]. In real-world environments, it is common to encounter simultaneous exposure to NPs and various other pollutants. Consequently, the research has shifted from investigating singular toxic effects to exploring the combined toxicity of NPs, which is of greater practical significance. Due to their high surface area-to-volume ratio, ubiquity, and persistence, NPs can adsorb a wide range of contaminants, including polycyclic aromatic hydrocarbons, pesticides, antibiotics, and heavy metals through both endogenous and exogenous pathways [10]. Studies have shown that NPs, when serving as carriers for heavy metal ions, usually contain metals such as manganese (Mn), cadmium (Cd), and nickel (Ni) present on their surfaces, with interactions occurring among these components [11]. Mn, a widely distributed heavy metal in the environment, has been well-documented for its reproductive toxicity in males [12]. Given its cumulative nature, persistence in the environment, and potential to cause multi-organ damage, further investigation into Mn exposure is of significant importance. In industrial settings, Mn is commonly used as a coloring agent and additive in plastic products, facilitating the likelihood of direct interaction with and NPs. In agriculture and fisheries, Mn-containing fertilizers and feed are adsorbed by NPs in water, significantly enhancing biological exposure to Mn-NPs composites. Beyond occupational and environmental sources, Mn and NPs frequently co-occur in daily life. The United States Geological Survey (USGS) has reported that Mn concentrations in tap water exceed permissible limits in multiple countries [13]. In both terrestrial and aquatic environments, MPs and Mn readily coexist, forming MP-Mn composites that enter biological systems, potentially altering metal bioavailability and toxicity [14–16]. Despite growing concerns, the specific biological effects and mechanisms underlying the combined toxicity of Mn and NPs remain unclear.
Epigenetic mechanisms have been shown to significantly influence male reproductive toxicity induced by environmental pollutants [17]. Recent studies have highlighted the importance of m^6^A methylation modifications in spermatogenesis [18]. YTHDC2, a key protein involved in RNA methylation, is recognized as the most significant member of the YTH domain family. Notably, it is the only member within this family that possesses a domain capable of undergoing degradation [19]. Predominantly expressed in the germ cells of the testis, YTHDC2 interacts with the meiotic-specific protein MEIOC, crucial for the meiotic division of germ cells [20]. Mutations in YTHDC2 result in abnormal meiotic division and subsequent reproductive defects. Similarly, YTHDC2 has been shown to promote the transition from mitosis to meiosis in murine germ cells. In the absence of YTHDC2, male germ cells fail to progress through meiotic prophase and instead undergo aberrant mitotic divisions, ultimately leading to cell death [21]. While considerable research has focused on the role of YTHDC2 during the meiotic phase, its functions during the mitotic phase of spermatogonia remain underexplored [22].
In mammals, spermatogenesis is accompanied by various stages of spermatogenic cell differentiation, including spermatogonia differentiation, initiation of meiosis, and chromosome association, recombination, and segregation in spermatocyte meiosis [23]. This process is tightly coordinated with the cell cycle, in which regulatory proteins A, B, D, and E play critical roles [24, 25]. Murine Double, Minute 2 (Mdm2), encoded by the mouse double minute 2 gene, is crucial for its p53-binding and ubiquitin-protein ligase activities, regulating p53 degradation [26]. Research has shown that apoptosis in spermatogonia is regulated through the Mdm2-p53 signaling pathway [27, 28]. However, studies on the regulatory of the Mdm2-p53 signaling pathway in germ cells concerning the cell cycle are lacking. The exact mechanism by which co-exposure to PS-NPs and Mn affects the Mdm2-p53-Cyclins axis remains unclear and deserves further investigation.
In this study, we established a co-exposure model of PS-NPs and Mn at environmentally relevant concentrations to evaluate their combined impact on male reproductive toxicity. We specifically investigated the role of YTHDC2-mediated m^6^A modification in regulating the cell cycle, elucidating its contribution to spermatogenic disruption induced by PS-NPs and Mn co-exposure. By identifying novel molecular targets, this study provides a theoretical foundation for developing potential interventions to prevent and mitigate NPs and Mn-induced male reproductive toxicity. Additionally, our findings contribute to a deeper understanding of the toxicological mechanisms underlying NPs and Mn co-exposure, offering new insights into the environmental etiology of infertility.
Fluorescent PS with diameters of 0.1 μm, 1 μm, 5 μm were obtained from Tianjin Baseline ChromTech Research Centre. PS-NPs (2.5% w/v, 5 mL) was purchased from macklin which is 0.1 μm.
The morphology of PS particles with different particle sizes was observed using scanning electron microscopy (SEM). PS was incubated with MnCl2 solution for 24 h at 4 °C, followed by centrifugation at 14,000 rpm for 20 min at 4 °C. After removing the supernatant, the pellet was collected and dried in a constant temperature oven. Subsequently, the samples were analyzed using a SEM (HITACHI Regulus 8100, Japan). Mn distribution on the surface of the PS particles was detected using Energy Dispersive X-ray Spectroscopy (AZtecLive Ultim Max 100, UK) in conjunction with the SEM. The hydrodynamic diameter and surface charge of the particles in dd H2O were measured using a ZETA VIEW system (Particle Metrix, Germany).
PS (0.1 μm, 1 μm, and 5 μm) were incubated with 2 mg/mL MnCl2 for 24 h. The mixture was centrifuged at 14,000 rpm for 20 min at 4 °C. The supernatant was collected, diluted, and analyzed for Mn concentration using ICP-MS. The Mn adsorbed on PS was quantified using the D-value method.
C57BL/6 (6–8 weeks, male) were purchased from the Laboratory Animal Centre of China Medical University, Shenyang, China (SPF grade, certificate no. SCXK2022–0001). Animals were housed in cages with a 12-h light/dark cycle at 25 °C ± 1 °C and 55% ± 5% humidity. Food and water were provided ad libitum. All experiments followed the guidelines of the Animal Care and Use Committee of China Medical University, with efforts made to minimize animal use and suffering. Mice were randomly divided to eleven groups based on their weight. Mice were exposed by gavage. The first part included the control group, PS-NPs (5.3 mg/mL) group, MnCl2 (56 mg/kg) group, and PS-NPs + Mn group. The dose of PS-NPs used in this study was determined based on concentrations reported in previous studies. Comparable doses of PS-NPs have been utilized in earlier animal experiments to investigate the reproductive toxicity of microplastics [8, 29, 30]. At these exposure levels, significant toxicological effects have been observed in mice, including reduced testosterone levels and pathological alterations in testicular tissue. Therefore, a concentration of 5.3 mg/mL was selected for the exposure model, as it is considered sufficient to induce measurable toxic effects while minimizing the risk of nonspecific toxicity associated with excessively high concentrations. In this study, the exposure dose of Mn was determined based on previously reported observations of Mn-induced reproductive toxicity in mice, in combination with dose extrapolations derived from literature on environmentally relevant Mn exposure concentrations [31–33]. The second part included the Adenovirus negative control (ADV-NC) group, YTHDC2 over expression (ADV-YTHDC2) group, ADV-NC + PS-NPs + Mn group, ADV-YTHDC2 + PS-NPs + Mn group. The third part included the control group, PS-NPs + Mn group, PS-NPs + Mn + Baicalin (50 mg/kg) group.
Adenovirus contains green fluorescent protein (GFP), ADV-YTHDC2, and ADV-NC, with a titer of 4E + 10TU/mL. It was synthesized by Shanghai Genechem Co., Ltd. After mice were anaesthetised using isoflurane inhalation, the procedure was carried out through an abdominal incision, through which the testes were exposed, and the adenovirus was injected into the testicular spermatogonium tubules using a microinjection feeder needle under small animal Doppler ultrasound guidance (5 µL in each testis, injection rate 0.5 µL/min) [34, 35]. After injection, the tubules were closed with sterile sutures. The whole procedure was performed on a heating pad to ensure that the mice were at normal body temperature.
The GC-1 spg and GC-2 spg cell lines were obtained from Wuhan Pricella Biotechnology Co., Ltd. and cultured in DMEM with 10% fetal bovine serum. The cells were grown at 37 °C in a 5% CO2 incubator.
Cells were incubated for 24 h in media with various Control, PS-NPs (20 µg/mL), MnCl2 (400 µM), and PS-NPs + Mn. For YTHDC2 overexpression or si-Mettl3 knockdown, cells in the Control and PS-NPs + Mn groups were transfected accordingly. For the FTO inhibitor MA2 [36] (100 µM) treatment, cells were divided into four Control, MA2, PS-NPs + Mn, and MA2 (pretreated for 6 h) + PS-NPs + Mn. Similarly, for the methylation inhibitor 3-deazaadenosine [37] (3DAA, 50 µM) group, cells were cultured under Control, 3DAA, PS-NPs + Mn, and 3DAA (pretreated for 12 h) + PS-NPs + Mn conditions. For a specific Mdm2-p53 pathway inhibitor Nutlin-3a [38] (10 µM) intervention, cells were treated with PS-NPs + Mn alone or pretreated with Nutlin-3a for 12 h before PS-NPs + Mn exposure. For the natural compound interventions, cells were exposed to PS-NPs + Mn alone or pretreated for 12 h with Baicalin (5 µM), C3G (50 µM), Rutin (5 µM), or Curcumin (5 µM) before PS-NPs + Mn exposure.
The GC-1 cell density was measured and adjusted using the Countstar IC1000 automated cell counter (5,000 cells per well in a 96-well plate) to ensure consistent seeding density across all groups. A cell-free control group was included to correct for background absorbance, and a negative control group treated with DMSO was used to confirm that the solvent itself had no effect on cell viability. The GC-1 cells were exposed to different times and concentrations of MnCl2, PS-NPs, PS-NPs + Mn, Baicalin, C3G, Rutin, and Curcumin. As described previously [39], absorbance values were recorded for at least 6 wells in each group.
The epididymis was excised to measure sperm motility. The cauda epididymis was minced in normal saline and incubated at 37 °C for 15 min. Sperm count, motility and malformation rate were assessed using the WLJY-9000 Digital Color Sperm Quality Testing System. The sperm suspension was then smeared onto clean slides and stained with a sperm staining solution. Each treatment group was analyzed, and at least 1000 sperm were counted per group. Sperm morphology was evaluated according to the method described by Linder et al. [40].
Testicular tissue and GC-1 cells were lysed, and protein concentrations were quantified. Western blotting was performed as described previously [41]. The dilutions used for the primary antibodies were as anti-YTHDC2 (1:1000), anti-MDM2 (1:1000), anti-p53 (1:500), anti-p21 (1:1000), anti-CCND1 (1:1000), anti-CDK1 (1:1000), and anti-GAPDH (1:10000). HRP-conjugated goat anti-rabbit and anti-mouse secondary antibodies were used at 10000. Immunoreactive bands were visualized using chemiluminescence reagents and analyzed with ImageJ.
Total RNA was isolated with trizol reagent following the manufacturer’s instructions. The RNA quantity and quality were assessed by reverse transcription followed by quantitative PCR, using a SYBR Green PCR kit. β-actin was used as the internal control to normalize the target gene mRNA expression. Gene expression was determined by calculating the ^ΔΔ^Ct values. The sequences of the primers used in the analysis are provided in Supplementary Table S1.
GC-1 cells were treated with Control-YTHDC2-Vector, Control-YTHDC2-OE, PS-NPs + Mn-YTHDC2-Vector and PS-NPs + Mn-YTHDC2-OE (n = 3). Total RNA was extracted. After quality inspection, mRNA was fragmented and converted into cDNA libraries. RNA sequencing was performed on the Illumina Novaseq™ 6000 and clean reads were saved in FASTQ format. Differential gene expression was analyzed using thresholds of P < 0.05 and |log2FC|>1.
RIP sequencing of YTHDC2 was performed to identify its target transcripts and examine any changes in their expression levels. Total RNA was extracted from GC-1 cells, and the subsequent RIP sequencing was conducted by Shanghai Yunxu Biotechnology Co., Ltd.
For the treatment or exposure conditions, cells were incubated with 2 µM actinomycin D for durations of 0, 3, and 6 h. After incubation, total RNA was extracted from the cells and analyzed by RT-qPCR [33].
To assess protein expression levels, cells subjected to treatment or exposure were incubated with 50 µg/mL Cycloheximide for varying durations of 0, 60, and 120 min. After incubation, cell lysates were collected for subsequent protein quantification assays.
Immunoprecipitation for m^6^A and YTHDC2 was performed using total RNA, with 500 ng of RNA designated as the input sample. The procedure was carried out following the established protocol from previous studies [42].
Immunofluorescence analysis of testicular tissue and GC-1 cells. The primary antibody rabbit anti-YTHDC2 (1:100), mouse anti-Mdm2 (1:100), mouse anti-DDX4 (1:100), and rabbit anti-SYCP3 (1:100) were employed. The secondary antibody FITC labeled goat anti-rabbit IgG (1:200) and Cy3 labeled goat anti-mouse IgG (1:200) were used in the study. Nuclei were stained with DAPI.
Sperm smears were prepared by placing 5–20 µL of semen on a clean slide and spreading with a second slide. After air-drying or fixation in Diff-Quik Fixative for 15–20 s, slides were stained in Diff-Quik I for 10–20 s and Diff-Quik II for 5–10 s. Excess dye was removed by brief rinsing in distilled water (10–15 dips). Slides were air-dried vertically and examined under a light microscope.
Paraffin-embedded testicular tissue slices (5 μm) were deparaffinized in xylene (10 min) and rehydrated through a gradient ethanol series (100%, 90%, 70%, 2 min). Tissue sections were stained using a H&E staining kit. Afterward, the sections were mounted and examined under a microscope for any structural alterations. Spermatogenesis was evaluated based on the Johnson score, which was used to assess the degree of spermatogenic damage [43]. To avoid subjective factors influencing the results, two experienced pathologists evaluated the images.
PAS staining, a method that detects glycogen in the sperm acrosome [44]. Testicular tissue sections were stained using the hematoxylin/periodic acid-Schiff (H/PAS) technique. After dehydration through graded alcohols, sections were rinsed in distilled water for 1 min and oxidized in periodic acid for 10 min. They were then immersed in Schiff reagent for 15 min, washed, and counterstained with hematoxylin for 3 min. Finally, the sections were fixed and sealed.
The level of testosterone in serum was quantified using an QuicKey Pro Mouse T (Testosterone) Elisa Kit. Each sample was analyzed in triplicate within the same assay to ensure consistency and reliability of the results.
The cells were collected and subsequently washed with PBS. After washing, they were treated with trypsin for digestion. The supernatant was discarded to ensure cell collection. Pre-cooled 70% ethanol was then added, and the cells were gently resuspended by pipetting. The cells were fixed by incubation overnight. A propidium iodide solution was added and the mixture was incubated at 37 °C for 30 min. Cell cycle arrest was assessed using flow cytometry. The cell cycle distribution was analyzed, and the proportions of cells in the G0/G1, S, and G2/M phases were calculated.
The molecular structures of the proteins YTHDC2 and MDM2 were retrieved from the Protein Data Bank (PDB) database (https://www.rcsb.org/), while the structural data for the small molecule ligands, including baicalin, C3G, rutin, and curcumin, were sourced from the PubChem database (https://pubchem.ncbi.nlm.nih.gov/). The Chem3D software was employed to preprocess the molecular structures of YTHDC2 and MDM2. This process involved several steps, including dehydrogenation, amino acid modifications, energy optimization, and fine-tuning of force field parameters, all aimed at achieving the lowest energy conformation for the ligand structure. The molecular docking procedure was carried out through the Vina plug-in integrated within AutoDock Tools, where the target proteins were subjected to docking with the corresponding active ligand structures. The binding affinity (kcal/mol) was used as an indicator of the ligand-receptor interaction strength, with lower affinity values indicating more stable binding.
SRAMP is a database for predicting mammalian m^6^A modification sites [45]. The SRAMP database was used to predict the m^6^A modification site of mdm2.
The data regarding the expression location of YTHDC2 in the mouse testis are sourced from the Male Health Atlas and the Human Testis Non-Obstructive Azoospermia Atlas, accessible at http://www.malehealthatlas.cn/.
Data were expressed as mean ± standard deviation (SD), with each experiment repeated at least three times. Statistical analyses were conducted using SPSS version 22 and GraphPad Prism. For comparisons involving a single factor, one-way ANOVA was first performed to assess overall group differences. When appropriate, two-way ANOVA was applied, followed by Tukey’s multiple comparison test to identify specific group differences. For data that did not meet the assumptions of normality and homogeneity of variance, the Kruskal–Wallis H test was employed. Statistical significance was set at P < 0.05.
SEM was used to examine the morphology of PS of different sizes, revealing that these particles were spherical, uniformly sized, and well dispersed (Fig. 1a). However, significant inter-particle adhesion was observed following Mn adsorption (Fig. 1b). Dynamic light scattering (DLS) was used to measure the particle size of PS-NPs/PS-MPs after Mn adsorption. The average diameters were found to be 97.3 nm, 1260 nm, and 5344 nm, respectively, which were comparable to those observed prior to Mn adsorption (Fig. 1c). And the surface charge of PS NPs + Mn (0.1 μm) is -32.81 ± 0.59 mV (Fig. 1d). To assess cellular uptake, we exposed two types of spermatogenic cells (spermatogonia and spermatocytes) to fluorescently labeled PS (0.1 μm, 1 μm, and 5 μm) for 24 h. Our results demonstrated that 0.1 μm PS-NPs penetrated spermatogenic cells the most (Fig. 1e-f and Figure S1). To investigate the interaction between PS and Mn, we first validated the pretreatment, adsorption characteristics, and cellular uptake of these particles in germ cells. Mn was incubated with 0.1 μm, 1 μm and 5 μm PS for 24 h, and the highest Mn adsorption observed on 0.1 μm PS-NPs (Fig. 1g). EDX analysis of PS-NPs + Mn confirmed the even distribution of Mn within the particles, indicating that Mn was enriched on the surface of the PS-NPs (Fig. 1h). Based on these findings, 0.1 μm PS-NPs were selected for subsequent cellular and animal studies.
Fig. 1Characterization of PS-NPs/PS-MPs and PS-NPs + Mn/PS-MPs+ Mn. (a and b) SEM image of PS-NPs/PS-MPs and PS-NPs + Mn/PS-MPs + Mn. (c) The particles size of PS-NPs + Mn/PS-MPs + Mn (DLS). (d) Zeta Potential of PS-NPs + Mn (0.1 μm). (e and f). The entry of fluorescent microplastics with particle sizes of 0.1 μm, 1 μm, and 5 μm into spermatogenic cells. e: GC-1 spg. f: GC-2 spg. The magnification was set to ×200. (g) Adsorption of heavy metal Mn by microplastics with particle sizes of 0.1 μm, 1 μm, and 5 μm. (h) Elemental distribution mapping of C, Mn, and O in PS-NPs and PS-NPs + Mn (scale bar, 100 nm)
To assess the potential impact of co-exposure on the reproductive toxicity of PS-NPs and Mn in vivo, mice were subjected to 8 weeks of gavage with Mn, PS-NPs, or a combination of PS-NPs + Mn. During this period, testicular volume was monitored every three weeks (Fig. 2a). After exposure, the testes exhibited varying degrees of volume reduction, with the most pronounced decrease observed in the PS-NPs + Mn co-exposure group (Fig. 2b and c). Additionally, the testis coefficient in the PS-NPs + Mn group was significantly decreased compared with the single-exposure groups (Figure S2a). Concurrently, we measured Mn accumulation in the testis and observed notably elevated levels in the co-exposure group (Fig. 2d). However, a decreasing trend in Mn levels was observed compared to the group exposed to Mn alone, although the difference was not statistically significant. This suggests that the adsorption of Mn by PS-NPs may influence the absorption and distribution of Mn within the testes. Subsequently, we evaluated sperm viability and testicular morphology. Exposure to either PS-NPs or Mn alone significantly reduced sperm count and motility (Fig. 2e and Figure S2b), whereas co-exposure to PS-NPs + Mn exacerbated these declines. Sperm smear staining further revealed morphological abnormalities including loss of the head and body, small heads, and headless sperm (Fig. 2h). The proportion of abnormal spermatozoa was significantly elevated in the co-exposure group compared to the single-exposure groups (Fig. 2g and Figure S2d). Furthermore, testosterone - a critical androgen for spermatogenesis - were more substantially reduced in the co-exposure group compared to the PS-NPs or Mn single-exposure groups (Fig. 2f). The H&E staining analysis revealed varying degrees of testicular tissue damage across exposure groups. Treatment with PS-NPs alone resulted in disordered arrangement of spermatogenic cells, while Mn exposure led to significant cellular disarray, deep nuclear staining, and spermatogonia loss. Co-exposure to PS-NPs + Mn resulted in more severe cellular loss in the seminiferous tubules and fewer spermatozoa in the luminal space, indicating a synergistic toxic effect on testicular structure (Figure S2c and S2e). PAS staining revealed a progressive decline in germ cell numbers within the seminiferous tubules, accompanied by a significant reduction in sperm count, with the most pronounced effects observed in the PS-NPs + Mn co-exposure group (Fig. 2i). Additionally, DDX4, a key marker of spermatogenic cells, exhibited noticeably reduced expression, as determined by laser scanning confocal microscopy (Fig. 2j and l). Furthermore, SYCP3, a protein critical for meiotic chromosome recombination and segregation, showed reduced expression and fluorescence intensity under PS-NPs + Mn exposure (Fig. 2k and l), potentially leading to meiotic failure. These results highlight the sensitivity of spermatogenic cells in mouse testes to PS-NPs + Mn exposure.
Fig. 2Co-exposure to PS-NPs + Mn causes spermatogenesis blockage and spermatogenic cell abnormalities in mice. (a) Schematic of PS-NPs and PS-NPs + Mn exposure. (b-c) Typical images of testicular ultrasound (b). Testicular volume statistics (c). (d) The extent of Mn accumulation in the testis. (e) Sperm counts. (f) Serum T levels. (g) The malformation rate of sperm. (h) Representative images of sperm. (i) Testicular images with PAS staining. The magnification was set to ×200, scale bar = 50 μm. (j) Number of DDX4-positive cells in testicular tissue. (k) Number of SYCP3-positive cells in testicular tissue. (l) The levels of spermatogenic cell marker (DDX4), spermatocyte marker (SYCP3) in testicular tissues were measured by immunofluorescence. The magnification was set to ×400, scale bar = 25 μm
To identify potential target genes following PS-NPs + Mn exposure, we performed RNA-seq analysis on GC-1 cells from the treated group (Fig. 3a). Based on cell viability results, we selected 400 µM MnCl2 and 20 ug/mL PS-NPs for both separate and combined toxicity treatments on GC-1 cells (Figure S3a-d). Differentially expressed genes (DEGs) analysis post-exposure revealed 1470 up-regulated and 555 down-regulated genes, based on |log2FC|>1, p < 0.05. Gene ontology (GO) functional enrichment analysis of the DEGs indicated significant alterations in N6-methyladenosine-containing RNA binding, with YTHDC2 showing the most pronounced differential expression (Fig. 3b and c). The mRNA and protein levels of IGF2BP1-3 ware inconsistent with sequencing results (Figure S3h-i). Given that YTHDC2 is highly expressed in testicular tissue and plays a crucial role in spermatogenesis, we further explored its function using data from the Male Health Atlas Database. Among various testicular cell types, YTHDC2 was preferentially expressed in spermatogenic cells (Figure S3e-g), and its expression was consistently detected across all spermatogonium stages (Fig. 3d-f). To further investigate the relevance of YTHDC2 in human reproductive health, we analyzed testicular samples from patients with non-obstructive azoospermia (NOA) and found that YTHDC2 expression was significantly reduced compared to that in healthy individuals (Fig. 3g). This finding suggests that YTHDC2 plays a critical role in the early stages of spermatogenesis. We next examined whether YTHDC2 expression was altered following PS-NPs + Mn exposure. qPCR and Western blotting confirmed YTHDC2 downregulation in PS-NPs + Mn-exposure GC-1 cells (Fig. 3h and j) and testicular tissue (Fig. 3i and k). Immunofluorescence staining further corroborated these findings, LCSM images further demonstrated a marked decrease in YTHDC2 expression (Fig. 3l-o). Taken together, these results suggest YTHDC2 is sensitively downregulated in response to PS-NPs + Mn exposure in both in vitro and in vivo models, suggesting a potential role in mediating PS-NPs + Mn-induced reproductive toxicity.
Fig. 3Co-exposure to PS-NPs + Mn leads to down-regulation of the binding protein YTHDC2. (a) Schematic of PS-NPs + Mn treated GC-1 spg cells. (b) GO enrichment of differential genes after Co-exposure to PS-NPs and Mn (Molecular Function). (c) Scatterplot of differential genes (Ythdc2, Igf2bp1, Igf2bp2, and Igf2bp3) in the “N6-methyladenosine-containing RNA binding” pathway. (d) Mouse germ cell lineage atlas. (e) YTHDC2 expression in germ cell lineage. (f) YTHDC2 expression levels in germ cell lines at different stages. (g) YTHDC2 expression in normal and NOA. (h) Relative mRNA level of YTHDC2 in GC-1 spg. (i) Relative mRNA level of YTHDC2 in testis. (j) Relative protein level of YTHDC2 in GC-1 spg. (k) Relative protein level of YTHDC2 in testis. (l and n) The levels of YTHDC2 in GC-1 was measured by immunofluorescence. The magnification was set to ×200, scale bar = 50 μm. (m and o) The levels of YTHDC2 in testicular tissues was measured by immunofluorescence. The magnification was set to ×200, scale bar = 50 μm
To verify the functional role of YTHDC2 as a key mediator in the pathway of PS-NPs + Mn-induced reproductive toxicity, we constructed a testicular YTHDC2 overexpression model using ultrasound-guided testicular injection (Fig. 4a). Adenovirus fluorescence is expressed in both the ADV-YTHDC2 and ADV-NC groups, confirming successful viral transduction (Figure S4a). Quantitative analysis demonstrated a significant increase in both YTHDC2 mRNA and protein levels in the ADV-YTHDC2 group when compared to the ADV-NC group, confirming the successful establishment of the overexpression model (Figure S4b and S4c). As shown in Fig. 4b-c, Ythdc2 overexpression did not significantly affect testicular volume. However, in the PS-NPs + Mn-ADV-NC group, sperm count and viability decreased, and the rate of sperm malformation increased compared to the ADV-NC group. Conversely, in the PS-NPs + Mn-ADV-YTHDC2 group, sperm count and viability increased, and the rate of sperm malformation decreased compared to the PS-NPs + Mn-ADV-NC group (Fig. 4d-f and i). Additionally, testosterone levels were higher in the PS-NPs + Mn-ADV-YTHDC2 group than in the PS-NPs + Mn-ADV-NC group (Fig. 4g). Histological analysis further supported these findings. H&E staining indicated that YTHDC2 overexpression significantly alleviated spermatogenic cell damage (Fig. 4h and j). PAS staining results also showed a significant rebound in sperm count within the seminiferous tubules (Fig. 4k). Collectively, these results demonstrate that upregulation of YTHDC2 in testis effectively mitigates PS-NPs + Mn-induced male reproductive toxicity and spermatogenic dysfunction.
Fig. 4YTHDC2 in the testis resists spermatogenesis disorders caused by PS-NPs + Mn exposure. (a) Schematic of ADV-YTHDC2 over-expression in testis. (b) Typical images of testicular ultrasound. (c) Testicular volume statistics. (d) Sperm counts. (e) Sperm motility. (f) The malformation rate of sperm. (g) Serum T levels. (h) Histological score. (i) Representative images of sperm. (j) Testicular images with HE staining. The magnification was set to ×200, scale bar = 50 μm. (k) Testicular images with PAS staining. The magnification was set to ×200, scale bar = 50 μm
To explore the potential molecular mechanisms underlying YTHDC2-mediated spermatogenesis dysfunction following co-exposure to PS-NPs and Mn, we established a YTHDC2-overexpressing GC-1 cell line, treated it with PS-NPs + Mn, and subsequently performed transcriptome sequencing analysis (Figure S5a). Gene ontology (GO) functional enrichment analysis (Fig. 5a) and KEGG pathway analysis (Fig. 5b) were conducted on differentially expressed genes (DEGs) with |log2FC|>1 and p < 0.05, revealing significant enrichment in the p53 signaling and cell cycle pathway. To further examine the impact on cell cycle progression, flow cytometry was performed to assess cell cycle distribution. The results demonstrated an increased proportion of G1-phase cells in PS-NPs + Mn-exposed groups, indicating a G1-phase arrest. Notably, this arrest was alleviated in PS-NPs + Mn-YTHDC2-treated cells, where the G1-phase distribution returned to near-normal levels (Fig. 5c and d). To identify downstream target molecules regulated by YTHDC2, RNA immunoprecipitation sequencing (RIP-seq) was performed (Figure S5b and S5c). The top 10 pathways associated with YTHDC2 protein binding included the cell cycle and p53 signaling (Fig. 5e). Furthermore, seven genes (Mdm2, Cdk1, Ccne1, Ccnd1, Ccne2, Ccnb1, and Cdkn1a) were identified from RNA-seq data and enriched in both the cell cycle and p53 signaling pathway, modulated by PS-NPs + Mn exposure and directly regulated by YTHDC2 (Figure S5d). The Venn diagram illustrates the intersection of DEGs from RNA-seq, RIP-seq and MeRIP-seq analyses, revealing four YTHDC2 target Mdm2, Cdk1, Ccne1, and Ccnd1 (Fig. 5f and Figure S5e). To further explore the functional relationships among these target molecules, we performed protein-protein interaction (PPI) prediction, which indicated significant interactions among them (Figure S5f). To experimentally validate the potential binding of these target mRNAs to the YTHDC2 protein, we conducted YTHDC2-RIP qPCR. The results confirmed that Mdm2, Cdk1, and Ccnd1 directly interact with YTHDC2 (Fig. 5g). Further functional analysis in YTHDC2-overexpressing GC-1 cells exposed to PS-NPs + Mn, using qPCR and Western blot assays, demonstrated that YTHDC2 regulates the expression of Mdm2 and Ccnd1, while Cdk1 expression remained unchanged (Fig. 5h-i and Figure S5g-h). Notably, in GC-1 cells overexpressing YTHDC2, a significant reduction in Mdm2 mRNA levels was observed (Fig. 5h), accompanied by a marked increase in its protein levels (Fig. 5i), suggesting a potential post-transcriptional regulatory mechanism. To gain a deeper understanding of the structural basis underlying YTHDC2 regulation of MDM2, we conducted structure-based modeling to predict the interaction dynamics between YTHDC2 and MDM2. The results of the docking analysis demonstrated that YTHDC2 interacts with the intracellular domain of MDM2, binding at several distinct sites (Fig. 5j). To validate this interaction at the cellular level, we examined the spatial co-localization of YTHDC2 and MDM2 using immunofluorescence staining. Our results demonstrated significant co-localization of YTHDC2 and MDM2 in GC-1 cell line. Notably, co-exposure to PS-NPs + Mn led to a marked reduction in co-localization, providing strong anatomical evidence for the functional coupling of YTHDC2 and MDM2 in germ cells (Fig. 5k). This finding is consistent with the role of YTHDC2 as an m^6^A reader protein, which has been shown to facilitate mRNA decay and enhance translation. Given this function, we investigated the impact of YTHDC2 on both the degradation and translation of Mdm2. As shown in Fig. 5l, YTHDC2 overexpression in GC-1 cells under PS-NPs + Mn exposure significantly increased Mdm2 mRNA degradation. Moreover, YTHDC2 overexpression also promoted Mdm2 protein translation (Fig. 5m). Since the interaction between Mdm2 and p53 plays a key role in cell cycle regulation, we further examined the expression levels of p53 and its downstream effector p21, both of which are involved in the p53 signaling pathway. Exposure to PS-NPs + Mn resulted in elevated expression levels of p53 and p21, whereas YTHDC2 overexpression markedly attenuated this effect (Figure S5i-j and Fig. 5n). We also examined the expression of target molecules for both individual and co-exposure, and the changes were most pronounced in the co-exposure group (Figure S5k). In conclusion, co-exposure to PS-NPs + Mn downregulated YTHDC2 expression, thereby influencing both the translation and degradation of Mdm2, ultimately modulating the p53 signaling pathway and cell cycle progression. Fig. 5YTHDC2 inhibiton mediates PS-NPs+Mn-induced down-regulation of the Mdm2-p53 pathway. (a) RNA-seq: GO analysis of DEGs with |log2FC|>1 and p < 0.05, comparing the YTHDC2-vector+PS-NPs+Mn group to the YTHDC2-OE+PS-NPs+Mn group, as determined by RNA-seq with a |log2FC|>1 and p < 0.05, (b) KEGG pathway enrichment analysis of DEGs with |log2FC|>1 and p < 0.05, comparing the YTHDC2-vector+PS-NPs+Mn group to the YTHDC2-OE+PS-NPs+Mn group, represented as a bubble chart. (c and d) cell-cycle phase. (e) RIP-seq: KEGG enrichment bubble chart of YTHDC2 binding gene. (f) Overlap of RNA-seq genes, Ythdc2 RIP-seq gene, and genes containing m6Am6A. (g) RIP assays were performed with the YTHDC2 antibody, followed by qPCR analysis. (h) Relative mRNA level of Mdm2 in GC-1 spg. (i) Relative protein level of Mdm2 in GC-1 spg. (j) Docking model of YTHDC2 (white) with MDM2 (blue). (k) Co-localization of YTHDC2 and MDM2 in GC-1 Spg. The magnification was set to ×200, scale bar = 50 μm. (l) Act-D analysis of stability of Mdm2 mRNA. (m) CHX analysis of the stability of Mdm2 protein. (n) Relative protein levels of p53, Ccnd1 and p21 in GC-1 spg
To further elucidate the molecular mechanism by which YTHDC2 regulates Mdm2, we predicted three m^6^A methylation high confidence sites within the Mdm2 mRNA sequence using the SRAMP online tool (Fig. 6a and Figure S6a). To validate these m^6^A sites, we performed methylated RNA immunoprecipitation (meRIP) followed by qPCR. The meRIP-qPCR results revealed significant enrichment at sites P1 and P2, indicating the presence of m^6^A modifications within the sequence (Fig. 6b and c). To assess whether Mdm2 mRNA expression is influenced by m^6^A modification, we modulated total m^6^A methylation by silencing Mettl3 (Figure S6b and S6c) and by treating the cells with the methylation inhibitor 3-deazaadenosine (3DAA) and the FTO inhibitor MA2. The results demonstrated a significant decrease in Mdm2 expression when m^6^A levels were reduced by si-Mettl3 (Fig. 6d) and 3DAA treatment (Fig. 6e). Conversely, Mdm2 expression was upregulated upon treatment with MA2, which enhances m^6^A methylation (Fig. 6e). Importantly, the relative upregulation of Mdm2 induced by YTHDC2 overexpression was significantly attenuated by 3DAA treatment (Fig. 6f), suggesting that YTHDC2 regulates Mdm2 stability through m^6^A-dependent mechanisms. In conclusion, co-exposure to PS-NPs + Mn downregulated YTHDC2 expression, thereby disrupting m^6^A-mediated regulation of Mdm2 translation and degradation, subsequently altering the translation and degradation of Mdm2 in depend m^6^A manner. This dysregulation ultimately led to cell cycle arrest via the Mdm2-p53 signaling pathway (Fig. 6g).
Fig. 6YTHDC2 regulates Mdm2 in an m^6^A-dependent manner. Methylation modification sites of Mdm2 were predicted using the m^6^A site prediction tool. (b) The MeRIP quantitative PCR verified the presence of m^6^A modification at the P1-3 sites. (c) The m^6^A site of Mdm2 mRNA. (d) Western blot results showing the relative protein levels of Mdm2 in Mettl3 knocked down GC-1 Spg. (e) The relative protein level of Mdm2 in GC-1 Spg treated with 3DAA and MA2. (f) The relative protein level of Mdm2 in YTHDC2-overexpressing GC-1 Spg treated with or without 3-DAA. (g) The molecular mechanism of YTHDC2 regulating cell cycle
To better elucidate the regulatory role of YTHDC2 in the Mdm2-p53 pathway and its impact on cell cycle progression, we conducted experiments using GC-1 cells. Initially, we treated the cells with a YTHDC2 overexpression plasmid and subsequently exposed them to PS-NPs + Mn. To counteract YTHDC2-induced modulation of the Mdm2-p53 pathway during PS-NPs + Mn exposure, we employed Nutlin-3a, a specific Mdm2-p53 pathway inhibitor. Our results demonstrated that Nutlin-3a treatment led to significantly increased p53 and p21 protein expression levels compared to the group subjected to PS-NPs + Mn and YTHDC2 overexpression (Fig. 7a-d). Additionally, Ccnd1 protein expression was further downregulated in the Nutlin-3a-treated group (Fig. 7e and f). Furthermore, when we co-transfected the cells with YTHDC2 overexpression plasmid and treated them with Nutlin-3a during PS-NPs + Mn exposure, we observed a notable exacerbation of PS-NPs + Mn-induced G1 phase cell cycle arrest compared to the control group (Fig. 7g and h). Our findings suggest that YTHDC2 regulates the Mdm2-p53 pathway and play a critical role in PS-NPs + Mn-induced spermatogonium cell cycle arrest.
Fig. 7YTHDC2 regulation of Mdm2-p53 pathway alterations involved in PS-NPs + Mn co-exposure-induced cell cycle arrest in spermatogenic cells. (a) Relative mRNA level of p53 in GC-1 spg. (b) Relative protein level of p53 in GC-1 spg. (c) Relative mRNA level of p21 in GC-1 spg. (d) Relative protein level of p21 in GC-1 spg. (e) Relative mRNA level of Ccnd1 in GC-1 spg. (f) Relative protein level of Ccnd1 in GC-1 spg. (g and h) cell-cycle phase
Building on the molecular mechanism outlined previously, we subsequently investigated natural compounds that might mitigate reproductive impairment induced by PS-NPs + Mn through the modulation of YTHDC2. To achieve this, we utilized molecular docking, a computational approach based on structural analysis, to identify natural compounds that may regulate the expression of YTHDC2. As shown in Fig. 8a, four compounds - baicalin, C3G, rutin, and curcumin - exhibited strong binding affinities to YTHDC2, with binding energies of -8.2, -8.0, -7.8, and − 7.0 kcal/mol, respectively. In order to confirm these results, we first pre-treated the GC-1 spg with the natural compounds prior to exposing the cells to PS-NPs + Mn (Figure S7a-d). Western blot analysis revealed that baicalin pre-treatment significantly restored YTHDC2 expression, which was otherwise downregulated by PS-NPs + Mn exposure (Fig. 8b and c). Additionally, the protein levels of key downstream regulators, including MDM2, P53, P21, and CCND1, were markedly altered (Fig. 8b and d-g). To evaluate the in vivo effects, mice were pre-treated with baicalin before receiving PS-NPs + Mn gavage. Baicalin administration improved multiple reproductive parameters, including testicular volume (Fig. 8h and i), sperm count (Fig. 8j), sperm viability (Fig. 8k), sperm malformation rate (Fig. 8l and m), testosterone content (Fig. 8n) and testicular pathological tissue damage (Fig. 8o-q). Collectively, these findings suggest that YTHDC2 is a promising therapeutic target for mitigating PS-NPs + Mn-induced reproductive impairment in vivo.
Fig. 8Baicalin alleviates PS-NPs + Mn-induced male reproductive impairment by targeting YTHDC2. (a) Molecular Docking diagram of Baicalin, C3G, Rutin, Curcumin, and YTHDC2. (b-g) Representative western blot (b) and quantification of YTHDC2 (c), p53 (d), MDM2 (e), CCND1 (f), and p21 (g) in control and PS-NPs + Mn-exposed cells treated with natural compounds (Baicalin, C3G, Rutin, Curcumin). (h) Typical images of testicular ultrasound (i) Testicular volume statistics. (j) Sperm counts. (k) Sperm motility. (l) Representative images of sperm. (m) The malformation rate of sperm. (n) Serum T levels. (o) Histological score. (p) Testicular images with H&E staining. The magnification was set to ×400, scale bar = 25 μm. (q) Testicular images with PAS staining. The magnification was set to ×400, scale bar = 25 μm
Due to human activities and industrial progress, pollution from NPs and heavy metals has become increasingly severe, posing a significant threat to humanity. The adsorptive properties of NPs exacerbate the risks associated with co-exposure to heavy metals by facilitating their transport and bioavailability [46]. Previous studies reported that PS in the marine environment carry heavy metal, including cd, Mn, zinc, and lead, thereby increasing their persistence and potential toxicity [47]. Although research has examined the toxic effects of microplastics on organisms, there is limited understanding of the mechanisms driving the joint toxic effects of simultaneous exposure to NPs and heavy metals. Our study demonstrates that 56 days of exposure to PS-NPs and Mn induces male reproductive toxicity. evidenced by sperm abnormalities and changes in testosterone levels. These findings establish an in vivo mouse model for investigating the reproductive toxicity induced by co-exposure to PS-NPs and Mn, thereby providing a valuable framework for future mechanistic studies.
This study investigates the characteristics, adsorption behavior, and accumulation of PS of different sizes. Consistent with the findings of Xu et al. [29], our research indicates that 0.1 μm particles accumulate more significantly in the testes of mice and can penetrate germ cells. Furthermore, it was found that 0.1 μm NPs possess the highest adsorption capacity for Mn. PS are defined as plastic fragments and particles with a diameter smaller than 5 mm further classified into NPs (< 1 μm) and MPs (1 μm–5 mm) [48]. Wang et al. suggest that the distribution coefficient of pollutant concentration in MPs increases with decreasing particle size for macroaggregates and submicron polystyrene. Conversely, the distribution coefficient for NPs decreases with increasing particle size [49]. According to a report by Shen et al., 0.1 μm NPs particles can accumulate in testicular tissue and in spermatogonium cells, whereas 1 μm PS particles have difficulty entering and accumulating in these tissues and cells [50]. This may be due to the size and charge of microplastic particles, which are critical factors influencing their absorption and toxicity. Smaller and more charged particles tend to exhibit higher toxicity within cells and tissues [51]. Based on these observations, 0.1 μm PS-NPs were selected for subsequent cellular and animal experiments.
Our research examined the harmful effects of exposing individuals to both PS-NPs and Mn. The reproductive toxicity of co-exposure is more severe than that of individual exposure to either Mn or PS-NPs. When NPs and Mn coexist in the environment, their environmental behavior, bioavailability, and toxicity may be influenced through a series of complex interactions. Under conditions of co-exposure, these pollutants have the potential to exert synergistic toxic effects on biological systems. This outcome can be attributed to several key factors. Firstly, Mn exhibits a higher adsorption affinity for MPs/NPs compared to other metal ions [14]. Once adsorbed, heavy metals accumulate on the surface of MPs/NPs either in the form of oxides or through cation exchange, facilitating their migration into soil and aquatic environments. These contaminated MPs/NPs are subsequently ingested by organisms across various trophic levels, ultimately leading to the bioaccumulation of Mn in biological systems [52]. Although MPs/NPs themselves exhibit relatively low inherent toxicity to organisms, their role as carriers for environmental pollutants can significantly enhance toxicity through the release of adsorbed contaminants within biological systems [53]. Furthermore, the strong hydrophobic nature and large specific surface area of MPs/NPs enable them to persistently retain Mn contamination in the environment, thereby prolonging its presence. This extended co-contamination may increase the bioavailability of Mn in soil, expanding its migration and contamination range and further amplifying its toxic effects.
The molecular mechanisms underlying spermatogenic disorders induced by PS-NPs + Mn co-exposure remain elusive. In the present study, using co-staining of testicular tissues with the germ cell maker gene DDX4 [54] and the maker gene SYCP3 of spermatogonia [55], we found that spermatogonia and spermatocytes are affected by exposure to PS-NPs + Mn. As an early developmental stage of the germ cell lineage, spermatogonia possess a high proliferative capacity [56]. Serving as the starting point of spermatogenesis, spermatogonia are particularly susceptible to exogenous chemical insults. Their quantity and condition directly influence the progression of both mitosis and meiosis, ultimately affecting sperm quality [57]. Moreover, as critical precursor cells in the spermatogenic process, their responses to toxic exposures provide valuable insights into the potential impact of early reproductive toxicity on subsequent developmental stages. For these reasons, spermatogonia were selected as the primary focus of the present study.
YTHDC2, a protein that binds to m^6^A, plays a crucial role in the process of spermatogenesis. YTHDC2 can affect spermatogenesis both in an m^6^A-dependent and non-m^6^A-dependent manner. Male Health Atlas Database statistically found that YTHDC2 expression is significantly decreased in patients with reproductive disorders, suggesting an important role for YTHDC2 in spermatogenesis [58]. In our study NPs with Mn exposure caused significant downregulation of YTHDC2. Through the creation of an adenovirus YTHDC2 overexpression model, we found that YTHDC2 overexpression mitigated the male reproductive toxicity induced by co-exposure, indicating that YTHDC2 levels may be a potential regulatory target for managing PS-NPs + Mn induced reproductive toxicity. Previous studies have primarily focused on elucidating the role of YTHDC2 during meiosis. However, our preliminary research and single-cell transcriptomic analyses revealed that YTHDC2 remains highly expressed in a subset of spermatogonia that have not yet entered meiosis, suggesting a potential meiosis-independent function of YTHDC2 at the spermatogonium stage [39, 58]. It has been reported that male germ cells lacking YTHDC2 are unable to proceed through meiotic prophase and instead undergo aberrant mitosis [21]. Nevertheless, the functional role and underlying mechanisms of YTHDC2 during the mitotic phase—particularly in spermatogonium proliferation and fate determination—remain poorly understood. Therefore, this study aims to address this knowledge gap and expand our understanding of YTHDC2’s role throughout the entire process of spermatogenesis.
Although YTHDC2 is implicated in spermatogenesis regulation, its downstream effectors are critical in the impairment of spermatogenesis following PS-NPs + Mn exposure. Our RNA-seq experiments revealed that exposure to PS-NPs + Mn caused significant alterations in the p53 pathway and cell cycle in response to YTHDC2 modulation. As reported in previous literature, a reduction in Ythdc2 expression leads to cell cycle arrest in spermatogonium cells, which ultimately results in impaired spermatogenesis [39]. These findings provide evidence for the hypothesis that YTHDC2 plays a direct influence on the spermatogonium cell cycle process. However, further in-depth mechanistic investigations are required to clarify the precise molecular mechanism pathways involved in this regulatory process.
YTHDC2 downregulation prevented the degradation of Mdm2 transcripts, leading to elevated Mdm2 mRNA levels while simultaneously suppressing Mdm2 protein translation, ultimately resulting in decreased Mdm2 protein levels. Mdm2 is a gene crucial for regulating cell growth [39]. The YTHDC2 protein contains a highly conserved YTH domain [59], which is pivotal for m^6^A binding specificity. Unique among its family, YTHDC2 also possesses a helicase domain that enhances RNA binding [60]. Previous studies suggest that YTHDC2 regulates both translation efficiency and mRNA stability through interactions with m^6^A modification. Within mammalian cells, YTHDC2 has been implicated in facilitating mRNA decay either during or post-translation [19]. The YTHDC2-mediated modulation of Mdm2 observed in this study aligns with these concepts. However, the precise molecular mechanisms require further elucidation in future studies.
Mdm2 plays a vital role in the regulation of p53, functioning as a key protein that negatively impacts p53’s stability and transcriptional activity [61]. Additionally, Mdm2 suppresses p53-induced apoptosis, while MDMX may also inhibit p53-induced cell cycle arrest [28]. The p21 protein, a cyclin-dependent kinase (CDK) inhibitor, is transcriptionally regulated by p53, leading to the suppression of CDK activity and preventing cyclin-CDK complex formation, thereby arresting damaged cells in the G1/S phase [62]. Earlier research has indicated that daily exposure to MPs and NPs disrupt testicular MDM2 and p53 protein expression [63]. Consequently, we hypothesize that the reduction of Mdm2 protein due to PS-NPs + Mn exposure weakens its interaction with p53, leading to spermatogonium cell cycle arrest. Furthermore, treatment with Nutlin-3a, a classical inhibitor of the Mdm2-p53 interaction, resulted in increased p53 and p21 mRNA and protein levels, while concurrently reducing CCND1 mRNA and protein expression. This supports the notion that Mdm2 downregulation diminishes its interaction with p53, thereby upregulating p21 and diminishing CCND1 expression, ultimately inducing G1 phase arrest in spermatogonium cell cycle.
Natural compounds have become a major focus in current research. An increasing number of studies have explored their potential roles in mitigating reproductive damage. Baicalin, a flavonoid and the primary active component of scutellaria baicalensis, has been widely recognized for its pharmacological activities, including antipyretic [64], sedative, antibacterial, anti-inflammatory [65], and antioxidant properties, with no significant toxic side effects [66]. Several studies have shown that baicalein can play a protective role against heat stress injury in mice testis. Guo et al. found that baicalein could protect bovine testicular supporting cells and alleviate the reproductive injury caused by heat stress [67]. Consistent with these findings, our study demonstrated that baicalin protects against PS-NPs + Mn-induced spermatogenesis disorders in mice. Specifically, we identified the YTHDC2/MDM2/P53 axis as a critical regulatory pathway through which baicalin exerts its protective effects on reproductive function. Although our study suggests that baicalin may alleviate reproductive toxicity by restoring the expression of YTHDC2, the long-term biological consequences of sustained interaction between baicalin and YTHDC2 remain unclear. Specifically, it has yet to be determined whether this binding interferes with the endogenous RNA recognition or helicase activity of YTHDC2 under physiological conditions. Further investigation is warranted to elucidate these potential effects.
In summary, our result is the first to elucidate the synergistic effects and epigenetic mechanisms underlying PS-NPs and Mn-induced male reproductive toxicity. We found that YTHDC2 was significantly downregulated in spermatogonia, contributing to spermatogenic disruption under co-exposure conditions. Moreover, YTHDC2 promoted Mdm2 mRNA degradation and facilitated protein translation via the m^6^A modification pathway, leading to spermatogonium cell cycle arrest via the YTHDC2-Mdm2-p53 signaling axis. Dysregulation of this axis may be a key driver of PS-NPs + Mn-induced male reproductive toxicity. Meanwhile, baicalin was identified as a potential therapeutic agent capable of targeting YTHDC2 and alleviating reproductive toxicity induced by PS-NPs and manganese. Taken together, these findings deepen our understanding of the combined reproductive toxicity of nanoplastics and heavy metals, and highlight the potential of natural compounds in mitigating their adverse health effects.
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Supplementary Material 1