Microplastics exposure causes premature ovarian aging via inducing mitochondrial dysfunction

Microplastics exposure causes premature ovarian aging via inducing mitochondrial dysfunction | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Microplastics exposure causes premature ovarian aging via inducing mitochondrial dysfunction Xing Duan, Li-Shu Li, Wen-Jiao Liu, Tong-Tong Duan, Qian-Yin Xu, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6298826/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Ovarian aging represents a significant risk factor for female infertility, primarily due to a decline in ovarian reserve and diminished oocyte quality. However, whether microplastics (MPs) exposure poses a potential factor in inducing ovarian aging and the underlying mechanisms remain poorly understood. In this study, we demonstrated that MPs exposure accelerates ovarian aging, characterized by elevated expression of senescence markers (p16 and p21) and increased fibrosis. Mechanistic analysis indicated that MPs exposure led to ovarian inflammatory damage and mitochondrial dysfunction, which contribute to reduced ovarian reserve. Furthermore, MPs exposure compromised oocyte maturation competence and embryo development by triggering mitochondrial dysfunction and DNA damage. Multi-omics analysis identified L-arginine as a crucial metabolite that protects oocytes from MPs-induced aging. Notably, L-arginine supplementation significantly mitigated the aging phenotypes induced by MPs, as evidenced by reduced ovarian fibrosis, inflammatory damage, and mitochondrial dysfunction, alongside enhanced oocyte quality. These findings provide theoretical support for reproductive protection against MPs and inform environmental risk assessments. Biological sciences/Cell biology/Senescence Biological sciences/Developmental biology/Germline development Health sciences/Diseases/Reproductive disorders Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 1. Introduction The widespread use of plastics has significantly contributed to industrialization and socio-economic development 1 . However, the high physicochemical stability of plastics makes them difficult to degrade, resulting in the accumulation of plastic waste in the environment 2 . Microplastics (MPs), defined as plastic particles smaller than 5 mm in diameter, represent emerging environmental pollutants that are widely distributed in water, air, and soil and the atmosphere 3 . Importantly, MPs have been shown to migrate from the environment into human body through ingestion, inhalation or food chain transfer, raising concerns regarding their potential toxicity due to their extensive exposure pathways 4 . Recent research has shown that MPs are capable of crossing biological barriers and exerting toxic effects on vital organs, such as the brain, liver, kidney, lung and heart 5 . These adverse effects are linked to oxidative stress, chronic inflammation, cytotoxicity and disturbances in metabolic processes and energy homeostasis in biological systems 6 . Consequently, the potential health risks associated with MPs have emerged as a significant public health concern. Microplastics (MPs) have been reported to induce a range of toxic effects, including neurotoxicity, cardiotoxicity, hepatotoxicity, immunotoxicity, metabolic disorders, and even cancer 7 . Exposure to MPs has been shown to trigger autophagy and apoptosis by impairing mitochondrial function and elevating levels of reactive oxygen species (ROS) 8 . Moreover, MPs disrupt mitochondrial activity and cause DNA damage by interfering with glycolysis and tricarboxylic acid (TCA) cycle processes, contributing to renal toxicity 9 . In the liver, MPs exposure has been linked to injury and fibrosis through the promotion of proinflammatory cytokine release, mitochondrial dysfunction, and ferroptosis 10 Recently, increasing attention has been directed toward the reproductive toxicity of microplastics (MPs). Studies have revealed that MPs are capable of penetrating reproductive tissue barriers, including the placental barrier, epithelial barrier, and blood-testis barrier, leading to their accumulation in reproductive organs 11 . MPs have been detected in human follicular fluid, where their presence is associated with a reduction in follicles number, diminished oocyte quality, and decreased pregnancy and fertility 12 . Furthermore, MPs have been shown to cross the placental barrier in pregnant mice, resulting in metabolic disorders and reproductive system impairments in offspring 13 . Recent studies have also indicated that MPs disrupt the gut microbiota homeostasis and metabolic processes, ultimately leading to organismal damage 14 . However, limited research has explored the impact of MPs on ovary metabolism process in mammals, and the specific mechanisms underlying this damage remain unclear. Identifying key metabolites involved in MPs-induced ovarian dysfunction could offer critical insights for the prevention and treatment of reproductive toxicity caused by MPs. Premature ovarian aging (POA), characterized by a progressive decline in ovarian reserve and oocyte quality, ultimately leading to female infertility 15 . This condition has become a pressing public health concern. Environmental factors are increasingly recognized as critical contributors to ovarian reserve depletion and may influence ovarian function during both prenatal development and adulthood. Key environmental toxicants, such as phthalates, bisphenol A, and tobacco, have been implicated in this process 16 . For instance, tobacco smoke has been shown to disrupt follicular development by increasing apoptosis and autophagy, inducing DNA damage, and impairing the connections between oocytes and granulosa cells, all of which contribute to ovarian dysfunction and infertility 17 . Women exposed to cigarette smoke also exhibit a significant reduction in ovarian volume compared to non-exposed women of the same age 18 . Additionally, benzo[a]pyrene preferentially accumulates in the ovary, resulting in a reduced number of primordial follicles and infertility in mice 19 . Similarly, perfluorooctanoic acid has been shown to induce POA by impairing NAD + synthesis and mitochondrial function in adult zebrafish 20 . Although it has been demonstrated that MPs exposure reduced the ovary size and lifespan in Drosophila melanogaster, and MPs exposure induced the inflammation of ovaries and reduced the quality of oocyte in mice 21 , 22 , whether the MPs triggered the occurrence of POA and the potential mechanism remain unclear, especially whether MPs exposure changed the metabolism process of ovary still clarified. This study investigated the detrimental effects of MPs on ovarian function and oocyte quality in mice. Our findings revealed that exposure to MPs induced POA in mice, which characterized by increased senescence markers (p16 and p21) and fibrosis. In addition, we also observed MPs-exposure caused mitochondrial dysfunction, oxidative stress, inflammation, autophagy, and DNA damage in both the ovary and oocytes. Furthermore, we utilized multi-omics analysis to elucidate the signaling pathways and metabolites affected by MPs in the ovary. Notably, MPs exposure significantly disrupted L-arginine synthesis, and in vivo supplementation with L-arginine partially restored ovarian function and oocyte quality by enhancing mitochondrial fitness. These results offer a novel strategy for mitigating the toxic effects of MPs on the female reproductive system. 2. Materials and Methods 2.1 Animal Model ICR mice were purchased from Viton Lever and housed under specific pathogen-free conditions. Four-week-old female mice were used in the experiments and provided with a suitable environment and sufficient water and food. Based on previous studies on microplastic exposure in rodents 23 after one week of acclimatization, each mouse was randomly grouped and orally garaged with 5mg/kg body weight / day of MPs or an equivalent volume of purified water for 4 weeks (n = 50 for each group). Regarding the rescue treatment with L-arginine, we referred to studies on the protective effects of L-arginine 24 . Experimental mice were orally administered 200 mg/kg of L-arginine (dissolved in PBS, Cat#: A8096, Sigma-Aldrich) or an equal volume of PBS every day for four consecutive weeks. All animal experiments were approved by the Laboratory Animal Welfare and Ethics Committee of Southeast University. 2.2 Microplastics FITC-polystyrene (FITC-PS) and polystyrene (PS) particles were purchased from Tianjin BaseLine New Material Technology Company (China). Scanning electron microscopy (SEM, Nova Nano 450, FEI) was used to characterize the primary size and shape of the different MPs. The dried MPs were mixed thoroughly with potassium bromide (KBr) at a mass ratio of 1:100 and finely ground. The surface composition of MPs was then analyzed using an IRPrestige-21 spectrometer (Shimadzu Corporation, Japan). MPs were dispersed in ultrapure water by sonication to determine the hydrodynamic size and zeta potential prior to dynamic light scattering analysis (Zetasizer, Malvern, UK). 2.3 Mouse Oocyte Collection and Culture Germinal vesicles (GV) were collected from experimental mice after intraperitoneal injection of 5 IU pregnant horse serum gonadotropin (PMSG, Sigma) and cultured in Milrinone-free M16 medium for 14 h at 37°C, 5% CO2. To collect ovulated oocytes, female mice were first injected with 5 IU PMSG for 44–48 h, followed by 5 IU hCG. After 15–16 h, the oviductal jugular was collected and treated with 0.3% hyaluronidase (Sigma-Aldrich) to remove the oviductal cells. 2.4 Western Blotting Proteins were extracted from ovaries with RIPA lysis buffer (Beyotime, P0013C), and protein concentration was adjusted by BCA quantification (Beyotime, P0010S) and denatured at 95°C for 10 min. Samples were separated on 10% polyacrylamide gels (20325ES62, YEASEN) and blotted onto PVDF membranes (Immobilon-P, Millipore). Incubation was done in 5% skimmed milk for 1 hr, followed by overnight incubation at 4°C with primary antibodies. The membrane was then washed 3 times with TBST for 5 min each, incubated with the secondary antibody for 2 h at room temperature, and washed again with TBST. Color was developed using the Hyper Signal ECL chemiluminescence kit (4A Biotech Co., Beijing, China), and chemiluminescent signals were captured using a Tanon 4600 chemiluminescence imaging system (Tanon, Beijing, China). The primary and secondary antibodies used are detailed in the Supplementary Table S1 . 2.5 Gene Expression Quantification For RNA expression analysis, total RNA was extracted from ovaries using Trizol (Invitrogen, USA) and resuspended in nuclease-free water. Reverse transcription was performed using the QuantiTect Reverse Transcription Kit (Qiagen NV). Relative quantitative analysis was performed using the 2X Universal SYBR Green Fast qPCR Mix (Abclonal, China, RK21203) on a QuantStudio instrument (Applied Biosystems, Carlsbad, CA, USA). The relative transcript abundance was analyzed via the 2 −ΔΔCT method.Mouse Gapdh was chosen as a standardized control. Gene-specific primer sequences are shown in Supplemental Table S2 . 2.6 Immunofluorescence Oocytes were fixed in 4% paraformaldehyde solution in phosphate-buffered saline (PBS) for 30 min and then permeabilized with 0.5% Triton X-100 (Solarbio, 9002-93-1, China) for 20 min. After being blocked with PBS containing 1% bovine serum albumin for 1 h at room temperature, the samples were incubated with primary antibody at 4°C overnight, followed by incubation with secondary antibody for 1 hour at room temperature. The DNA was then stained with Hoechst 33342 in PBS at a concentration of 10 µg/ml. Samples were then fixed on slides and visualized using a confocal laser scanning microscope (FV 3000, Olympus, Japan). The ovary sections were dewaxed in xylene and rehydrated through graded ethanol concentrations. Antigen retrieval was performed in a microwave oven for 4 minutes in 10 mM sodium citrate buffer (pH 6.0), repeated three times. After antigen retrieval, the sections were blocked for 30 minutes with 10% (v/v) normal donkey serum at room temperature. The subsequent steps were carried out in accordance with the previously established protocol for cell immunofluorescence. The primary and secondary antibodies used are detailed in the Supplementary Table S1 . 2.7 Histological Analysis For histological analysis, ovaries were collected and fixed in 4% paraformaldehyde, embedded in paraffin, sectioned (5 µm thick), and stained with H&E at the Animal Histopathology Core Facility of Southeast University (China). Follicle counting estimated the number of follicles at various stages within each ovary by double-blind assessment of the number of follicles at each level in the H&E-stained images of complete ovarian sections. The criteria used to classify the follicles are as follows: Primordial follicles consist of a singular layer of squamous granulosa cells, pre-antral follicles consist of cuboidal granulosa cells layer and a thecal layer, antral follicles consist of an oocyte surrounded by a fluid-filled antrum and layers of granulosa cells, and atretic follicles exhibit dissolved oocyte cytoplasm with collapsed follicle walls. Tissue sections were dewaxed and stained with Masson's trichrome following the instructions of the commercial Masson staining kit (ServiceBio, Wuhan, China). The histological evaluation showed an enhanced blue coloration, indicating increased levels of collagen and fibrosis. 2.8 Gut Microbiota Analysis Intestinal content samples (approximately 200 mg each) were collected from control and MPs group mice, rapidly frozen in liquid nitrogen, and stored at -80°C. Microbial samples were sent to Shanghai Majorbio Bio-Pharm Technology for high-throughput sequencing of the 16S rRNA gene. Subsequently, genomic DNA was extracted using the OMEGA Soil DNA Kit (Omega Bio-Tek, Norcross, GA, USA) following the manufacturer’s protocol. The V3-V4 region of the bacterial 16S rRNA gene was amplified using the forward primer 338F (5'-ACTCCTACGGGAGGCAGCA-3') and the reverse primer 806R (5'-GGACTACHVGGG TWTCTAAT-3'). The amplified products were purified using Vazyme VAHTSTM DNA Clean Beads (Vazyme, Nanjing, China) and quantified using the Quant-it PicoGreen dsDNA Assay Kit (Invitrogen, Carlsbad, CA, USA). Sequencing was performed on the Illumina NovaSeq platform. Raw sequencing data were demultiplexed to remove primer sequences, followed by quality filtering, denoising, merging, and chimera removal using the DADA2 plugin. The obtained bacterial sequence fragments were classified into ASVs and compared with the Greengenes microbial gene database. Finally, microbiome bioinformatics analysis was conducted using QIIME2 and R (v4.3.1) to calculate and visualize diversity, taxonomic composition, and differential abundance. 2.9 Metabolomics Analyses To perform non-targeted metabolite profiling, mouse ovarian samples were prepared, and the supernatant was extracted from the ovarian homogenate using pre-chilled 70% methanol buffer for LC-MS/MS analysis. All samples were acquired by MetWare Biotechnology Co., Ltd., and analyzed by the LC-MS/MS system following the instrument’s operating protocol. The converted LC-MS data files were processed using XCMS (Scripps, La Jolla, CA) for peak picking, peak alignment, gap filling, and sample normalization. The processed peak information was matched against a self-constructed internal database and public databases to obtain metabolite identification results. Statistical analyses, including OPLS-DA and DESeq2, were performed using R (v4.3.1) to identify metabolite features with significant differences between groups. The differential metabolites were then mapped to the KEGG pathway database. 2.10 RNA-seq Analysis Total RNA was extracted from mouse ovarian samples using TRIzol reagent (Invitrogen). The concentration and integrity of the RNA were assessed using Qubit 2.0 and Agilent 2100 (Novogene, China), respectively. Subsequently, the RNA-Seq transcriptome library was constructed using the Illumina TruSeq library construction kit (Illumina, California, USA) according to the manufacturer's instructions. The library was then sequenced by Repugene Technology Co., Ltd. (Hangzhou, China) on an Illumina Nova6000 platform. Raw data quality was controlled using Fastp, and the quality of the processed data was verified using FastQC. STAR was employed to align the qualified sequencing data to the reference genome. FeatureCounts was used to quantify gene abundance and transcription levels. Differentially expressed genes (DEGs) were identified through DESeq2 analysis conducted in R (v4.3.1), defined as transcripts with a p-value < 0.05 and |log2 FC| ≥ 1. Functional enrichment and annotation analyses of DEGs were performed using the Kyoto Encyclopedia of Genes and Genomes (KEGG) ( https://www.genome.jp/kegg/ ) and Gene Ontology (GO) ( http://www.geneontology.org/ ). 2.11 Statistical Analysis Statistical analysis was performed with GraphPad Prism software 9 (La Jolla, USA). Comparisons of data were performed by one-way analysis of variance (ANOVA) with Tukey's multiple comparison test or two-tailed unpaired t-test. p values < 0.05 were considered significant. 3. Results 3.1. Characterization of MPs The selected MPs were fully characterized before administration in vitro and in vivo . Scanning electron microscopy (SEM) was used to analyze the morphology and size of the MPs (Fig. 1 A). The results showed that MPs exhibited a nearly spherical morphology with the average diameter of 498.73 ± 39.187 nm (Fig. 1 A). Since surface charge of MPs plays an important role in deciding the biological effects on organism, we performed zeta potential analysis of the MPs in culture medium M16. The results showed that the stability of MPs in culture medium was lower than in water (Fig. 1 B). Additionally, dynamic light scattering (DLS) analysis indicated a slightly larger diameter of MPs in water (Fig. 1 C), likely due to particle aggregation in the diluent. The structural characterization of the MPs was further conducted using Fourier-transform infrared (FTIR) spectroscopy (Fig. 1 D). A peak at 3025.76 cm⁻¹ was associated with the stretching vibrations of unsaturated hydrocarbon groups on the benzene ring (-CH). Absorption peaks near 2921 and 3026 cm⁻¹ confirmed the presence of methylene and unsaturated hydrocarbon groups. Peaks at 1494 and 1693 cm⁻¹ corresponded to skeletal vibrations of the benzene ring, while the peak at 1450 cm⁻¹ was attributed to methylene bending vibrations. Additionally, peaks at 695 and 753 cm⁻¹ were linked to out-of-plane bending vibrations of unsaturated hydrocarbon groups on the benzene ring. These FTIR results confirmed that the MPs were polystyrene, synthesized through the polymerization of styrene. Finally, the double peaks around 2400 cm⁻¹ were identified as the asymmetric stretching vibrations of C = O bonds, likely originating from environmental carbon dioxide. 3.2. MPs exposure induced ovarian aging and reduced oocyte quality To evaluate the effects of MPs exposure on ovarian function, mice were orally exposed to fluorescently labeled MPs through drinking water for 4 weeks at dose of 5mg/kg body weight / two day. As illustrated in Fig. S1 A, the body weight was not changed in MPs-exposed mice, while the ovary organ coefficient was significantly reduced, as evidenced by a decreased in ovarian size. Consist with previous study, we confirmed that fluorescently labeled MPs can penetrated ovary and localized in the granulosa cells (Fig. 1 E). As expected, MPs exposure induced remarkable ovary dysfunction, characterized by a reduction in antral and secondary follicles, an increase in atretic follicles (Fig. 1 F). Additionally, we observed a downregulation in the mRNA expression levels of genes associated with follicle maturation and development, including NOBOX , GDF9 , and BMP15 , as well as genes related to hormone secretion, such as ERα , C/EBPα , and STAR , further confirming the impairment of ovarian function (Fig. S1 B). Western blot analysis showed elevated level of senescence markers P21 and P16 (Fig. 1 G), indicative of ovarian aging. Masson’s staining further demonstrated a marked increase in collagen fiber content (Fig. 1 H), suggesting severe ovarian fibrosis following MPs exposure. To investigate the underlying mechanisms of aberrant ovarian follicle development, we performed immunofluorescent staining and the results indicated a significant increase in γH2AX signals in MPs-treated ovaries (Fig. 1 I), pointing to DNA damage. Since DNA damage is often associated with the onset of apoptosis, our subsequent western blot and RT-qPCR resulting showing elevated Bax expression and decreased Bcl2 levels (Fig. 1 J& S1 C). The release of Cyt-C is a hallmark of the mitochondrial pathway initiating apoptosis, while the upregulation of P-ATM reflects the activation of the DNA damage response. These changes collectively indicate the cellular stress and apoptotic processes induced by MPs exposure (Fig. 1 J). As a consequence, the number of super-ovulated oocyte was significantly reduced, with an increased percentage of fragmentated oocytes in MPs exposed group (Fig. 1 K). Although meiotic resumption, as shown by germinal vesicle breakdown (GVBD), was slightly affected, polar body extrusion (PBE) rates were significantly reduced after MPs exposure (Fig. S1 D). Immunofluorescence (IF) staining showed spindle defects in metaphase II (MII) oocytes, including failure of spindle anchoring and a higher incidence of abnormal spindle morphology (Fig. 1 L). In vivo fertilization experiment demonstrated that MPs-treated oocytes could be fertilized but failed to progress to the 4-cell embryo stage (Fig. 1 M). Given the importance of communication between oocyte and cumulus cells through transzonal projections (TZPs) in maintaining oocyte quality, TZPs was examined by staining phalloidin. The results showed a significantly reduced in the number of TZPs in the cumulus-oocyte complexes (COCs) derived from MPs-exposed mice (Fig. 1 N). Additionally, calcein-acetoxymethyl (C-AM) staining demonstrated impaired gap junction communication between oocytes and cumulus cells (Fig. 1 O), as indicated by a markedly diminished fluorescent signal in oocytes from MPs-treated mice. 3.3. Transcriptomic analysis of the molecular mechanism of MPs-induced ovarian dysfunction To investigate the mechanisms underlying the ovarian toxicity induced by MPs, transcriptomic analysis was performed to examine the differences in mRNA expression (Fig. 2 A). As shown in Fig. 2 B, Principal component analysis (PCA) revealed distinct gene expression patterns between the control and MPs-exposed groups. Heatmap analysis indicated significant differences in the transcriptomic profiles of the ovaries between the control and MPs exposure groups (Fig. 2 C). Volcano plot analysis identified significant transcriptomic changes, with 505 differentially expressed genes (DEGs) being upregulated and 194 DEGs downregulated in the MPs exposure group (Fig. 2 D). To identify the potential pathways affected by MPs, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis was conducted. Microplastic exposure led to alterations in the expression of genes related to mitochondrial energy metabolism pathways, such as ascorbate and aldose metabolism, ABC transporters, and various inflammation-related pathways, including the TNF signaling pathway, IL-17 signaling pathway, chemokine signaling pathway, and cytokine-cytokine receptor interaction. Furthermore, pathways related to Biosynthesis of cofactors and the metabolism of arginine, alanine, glutamate, and aspartate were also disrupted (Fig. 2 E). Additionally, Gene Ontology (GO) analysis of DEGs, encompassing cellular components, biological processes, and molecular functions, revealed that these DEGs were involved in processes such as calcium-mediated signaling, regulation of small molecule metabolic processes, and cell chemotaxis, as well as activities related to ATP hydrolysis activity, monoatomic ion channel activity, and cytokine receptor binding (Fig. 2 F). Collectively, these findings suggest that these pathways and biological processes are closely associated with inflammation, mitochondrial function, and nutrient metabolism. To further explore the trends of these pathway alterations, Gene Set Enrichment Analysis (GSEA) based on KEGG-listed pathways was conducted. The results indicated that the upregulated gene sets in the MPs treatment group were associated with TNF signaling pathway and Cytokine-Cytokine Receptor Interaction in Mus musculus. In contrast, the downregulated gene sets in the MPs treatment group were involved in ATP-dependent chromatin remodeling and arginine and proline metabolism, further indicating that MPs exposure increases cellular inflammation and disrupts energy metabolism (Fig. 2 G). Given that the involvement of “inflammation” in MPs-exposed ovaries and the function of inflammasomes in aging process, we performed IF and western blot to examine the NLRP3 protein expression and the result showed that MPs exposure remarkably increased inflammasome NLRP3 expression compared with that of control group (Fig. 2 H-I). Besides, MPs also significantly increased the inflammatory cytokines IL-10, IL1α and TNFα comparted with the control group, suggesting that MPs exposure cause substantial inflammatory damage to the ovaries (Fig. 2 I). 3.4 MPs exposure induced mitochondrial dysfunction and autophagy in ovaries Given that mitochondria dysfunction-induced elevated reactive oxygen species (ROS) level could acts as activator to NLRP3 activation, we performed western blot and RT-qPCR to examine the effect of MPs on mitochondrial function and fitness. As shown in Fig. 3 A, our results demonstrated that MPs exposure significantly reduced the protein levels of antioxidative markers NRF2 and SOD2. Similarly, the RT-qPCR results confirmed a notable decrease in the mRNA levels of NRF2, SOD2 and HO-1 in MPs-exposed ovaries (Fig. 3 B). Furthermore, western blot analysis revealed that MPs exposure also remarkably reduced the protein levels of SIRT1, SIRT3 and FOXO1 (Fig. 3 C), indicating that MPs exacerbate oxidative stress in ovaries. Subsequently, we also observed an upregulation in the expression of ER stress-related genes such as GRP78 and CHOP(Fig. 3 E), indicating that the oxidative stress induced by MPs in the ovaries subsequently triggered the occurrence of ER stress. To further determine the potential mechanism underlying MPs-induced oxidative stress, we examined the expression of proteins involved in mitochondrial fission and fusion. As shown in Fig. 3 F, mitochondrial fusion-related proteins MFN1 and OPA1 were significantly downregulated in the MPs-exposed group, whereas mitochondrial fission-related proteins FIS1 and DRP1 were markedly upregulated. Consistently, the RT-qPCR results also confirmed that defect of mitochondrial dynamics following MPs exposure (Fig. 3 G). Furthermore, the downregulation of TFAM and PGC1α, key regulators of mitochondrial biogenesis (Fig. 3 H-I), further supports the disruption of mitochondrial function following MPs exposure. Additionally, we accessed mitophagy, a form of autophagy specifically targeting damaged mitochondria. MPs exposure was found to induce mitophagy in the ovaries, as evidenced by increased protein expression of PINK1 and Parkin (Fig. 3 J). Consistently, RT-qPCR results also indicated that MPs exposure activated mitophagy in the ovaries (Fig. 3 K). Given that impaired mitochondrial function often led to defects in autophagosome-lysosome fusion, we further analyzed autophagic marker LC3β, a direct indictor of autophagy. Western blot results revealed MPs exposure significantly increased the protein level of LC3β, suggesting heightened autophagic activity (Fig. 3 L). Interestingly, MPs exposure also significantly reduced the expression of lysosome-associated membrane protein 2 (LAMP2) (Fig. 3 L), indicating impaired lysosomal function and disrupted autophagic flux. Notably, MPs exposure led to elevated protein levels of P62 (Fig. 3 L), suggesting a blockage in autophagosome degradation. This observation was further supported by RT-qPCR results, which revealed significant increases in the mRNA levels of LC3β , ATG7 , Beclin1 , and P62 following MPs exposure (Fig. 3 M). 3.5 MPs exposure in vitro perturb oocyte meiotic progress and mitochondrial function To investigate the effects of MPs exposure on oocyte quality, we performed in vitro experiment by treating the oocyte with different concentration of MPs (30 µg/ml, 50 µg/ml and 100 µg/ml). As shown in Fig. 4 A& S2 A, MPs exposure slightly reduced the oocyte GVBD rate but significantly reduced the rate of PBE in a concentration-dependent manner. Immunofluorescence staining revealed that MPs exposure disrupted the spindle assembly and chromosomes misalignment in metaphase I (MI) oocytes, characterized by reduced spindle size and the formation of multipolar spindles (Fig. 4 B). Quantitative analysis further confirmed abnormalities in spindle morphology and chromosomes alignment. Live cell staining demonstrated that MPs exposure significantly increased cytosolic Ca2 + and ROS levels (Fig. 4 C-D), along with upregulation of ER-tracker and γH2A.X staining, indicating ER stress and DNA damage (Fig. S2 B-C). Consistently, reduced protein levels of SOD2 were observed in MPs-treated oocytes, accompanied by increased acetylation of SOD2 (Fig. 4 E-F), which is associated with impaired antioxidative capacity. Given that excessive ROS can induce mitochondrial dysfunction, we examined the mitochondrial mass using Mito-Tracker staining. The results showed a significant reduction in mitochondrial fluorescent intensity signals following MPs treatment (Fig. 4 G). Additionally, mitochondrial transcription factor A (mtTFA), which is critical for regulating mitochondria biogenesis and mtDNA transcription, was significantly downregulated in MPs-treated oocytes, suggesting compromised mitochondrial function (Fig. 4 H). Interestingly, MPs exposure also disrupted mitochondrial fission-fusion process in oocytes, as evidenced by increased DRP1 expression and reduced MFN1 protein level (Fig. 4 I-J). Since mitophagy, a specialized form of autophagy, mediates the removal of dysfunctional mitochondria to maintain mitochondrial quantity and quality, we further investigated whether MPs exposure activated mitophagy in oocytes. As expected, MPs treatment significantly upregulated PINK1 protein level (Fig. 4 K), a key marker of mitophagy. In line with in vivo experiment, MPs exposure also induced autophagy in oocyte, as evidenced by increased lysosome cluster foci and LC3β expression level (Fig. 4 L-M). Therefore, these findings indicated that MPs exposure impaired oocyte quality by disrupting mitochondrial function. 3.6 Metabolomic profiling identifies decreased arginine levels in ovaries of MPs-exposed mice To examine the effects of MPs exposure on ovarian metabolism, we performed a metabolomics analysis using liquid chromatography-tandem mass spectrometry (LC-MS/MS) (Fig. 5 A). OPLS-DA model presented that the six biological replicates within each group were closely clustered, while a distinct separation was evident between the control and MPs-treated ovaries (Fig. 5 B). The heatmap generated by hierarchical clustering reveals significant differences in the ovarian metabolome between the MPs-treated group and the control group, indicating a distinct metabolite profile (Fig. 5 C). Furthermore, based on the criteria of VIP > 1 and P-value < 0.05, volcano plot analysis showed that 345 annotated metabolites exhibited significant alterations following microplastic treatment, including 185 upregulated and 160 downregulated metabolites (Fig. 5 D). To explore the potential impacts of these differential metabolites, KEGG pathway enrichment analysis was performed. The results indicated that pathways related to mitochondrial energy metabolism (Citrate cycle, Pentose phosphate pathway, Pyruvate metabolism), inflammation-related pathways (Arachidonic acid metabolism, Histidine metabolism), and those involved in Biosynthesis of cofactors, Tryptophan metabolism, Arginine and Proline metabolism may be implicated in the effects of MPs (Fig. 5 E). Additionally, Metabolite Set Enrichment Analysis (MSEA) revealed the presence of relevant pathways such as Histidine metabolism, Tryptophan metabolism, Pentose and glucuronate interconversions, Arginine biosynthesis, and Glutathione metabolism, suggesting that MPs exposure led to significant changes in the levels of numerous metabolites in the ovary (Fig. 5 F). Notably, we observed that arginine, a non-essential amino acid involved in various cellular processes, was downregulated as a result of MPs exposure, and this was present in multiple enriched pathways, including Arginine and Proline metabolism and D-Amino acid metabolism (Fig. 5 G). Similarly, the violin plot obtained from the metabolomics analysis shows a significant reduction in arginine levels in the ovaries of the MPs-treated group (Fig. 5 H). To further investigate the correlation between the transcriptomic and metabolomic profiles, we constructed a co-expression network and generated a nine-quadrant plot to reveal the relationships between genes and metabolites (Fig S3 A). Based on the Pearson correlation coefficient and P-value, a clustering heatmap of differentially expressed genes (DEGs) and differentially altered metabolites was generated. The results showed that arginine was positively correlated with Wnt2 , Mcmdc2 , Khdrbs2 , Gulo , and Ctse , and negatively correlated with A2ml1 , Rnf128 , and Slc6a17 . Additionally, 6-Phosphogluconic acid exhibited positive correlations with Rubcnl , Cyp4f18 , and Gal3st2c , and negative correlations with H2ac10 and Slc39a2 (Fig. 5 I). Furthermore, we mapped the DEGs and differentially altered metabolites to the KEGG database and identified common pathways, including the Pentose phosphate pathway, Glutathione metabolism, Arachidonic acid metabolism, and others. Notably, pathways such as Arginine biosynthesis and Arginine and Proline metabolism were also included (Fig. 5 J). These results collectively indicate that arginine may serve as a potential key metabolite involved in ovarian damage induced by MPs exposure. 3.7 MPs exposure induces intestinal arginine depletion and transport dysfunction in mice The type and composition of gut microbiota play a crucial role in the metabolic activity of the host. Therefore, we assessed the general profiles of the gut microbiota through 16S rDNA sequencing of fecal samples from both groups. In the analysis of microbial diversity, the abundance-based coverage estimator (ACE) index was used to measure the number of microbial species, while the Shannon index reflected the microbial richness; the Simpson index was used to describe the microbial diversity in the samples. Mice fed MPs exhibited a trend toward lower bacterial abundance and diversity at the ASVs level, although no significant difference was observed (Fig. 6 A). In beta diversity, Principal Coordinate Analysis (PCoA) revealed that MPs altered the overall structural composition of the gut microbiota (Fig. 6 B). The taxonomic composition of the gut microbiomes at each level was further analyzed. At the phylum level, MPs increased the relative abundance of Proteobacteria and decreased the relative abundance of Actinobacteria (Fig. 6 C). At the class level, MPs increased the relative percentage of Deltaproteobacteria and decreased the relative percentage of Clostridia and Bacteroidia (Fig. 6 C). Similarly, at the order level, the MPs group exhibited an increased content of Desulfovibrionales and a decrease in Bacteroidales and Coriobacteriales, while at the family level, Desulfovibrionaceae was the most affected bacteria by MPs (Fig. 6 C). Additionally, based on the Lefse cladogram, 35 different bacterial taxa at various taxonomic levels were identified as differential microbiota from phylum to genus (Fig. 6 D). Interestingly, Proteobacteria was recognized as a characteristic differential microbiota, which has been shown to significantly consume arginine, thus affecting the host's gut microbiota 25 . In Fig. 6 E, we provide examples of the differential microbiota between groups, where Desulfovibrio, significantly more abundant in MPs-exposed mice, belongs to the Proteobacteria phylum. Furthermore, the beneficial bacteria Lactobacillus and Adlercreutzia showed a decreasing trend following MPs exposure. Correspondingly, MPs exposure led to abnormal changes in the levels of inflammatory cytokine proteins, including TNFα and IL10, in the gut (Fig. 6 F). RT-qPCR results confirmed the elevation of inflammation levels in the small intestine (Fig. S3 B). Furthermore, the reduction in protein and mRNA levels of ZO-1 and Occudin, along with the abnormal changes in arginine-related transporters, indicated that the intestinal mucosal arrier was disrupted and arginine absorption was affected (Fig. 6 G-H& S3 C). These results collectively suggest that MPs exposure induces dysbiosis in the gut microbiota, which in turn impacts the metabolic state of the host. 3.8 L-arginine alleviated MPs-induced premature ovarian aging in mice To investigate whether MPs-induced premature ovarian aging is associated with L-arginine, dietary supplementation with L-arginine via drinking water was administered. The results showed that L-arginine supplement significantly restored follicles development, as evidenced by the reduced atretic follicle (Fig. 7 A). To further evaluate whether L-arginine could alleviate MPs-induced ovarian aging, western blot analysis showed that L-arginine significantly decreased the protein levels of P21 and P16 (Fig. 7 B), key markers of cellular senescence. Similarly, Masson’s staining demonstrated a marked reduction in collagen fiber contents in MPs-exposed ovaries following L-arginine treatment (Fig. 7 C). Additionally, L-arginine restored the mRNA expression levels of chemokines such as TGFβ , IFNγ , CXCL1 , and CXCL3 compared to the MPs-treated group (Fig. 7 D), suggesting alleviation of MPs-induced ovarian aging. L-arginine also mitigated ovarian inflammation, as evidenced by reduced protein expression of inflammatory markers, such as TNFα, IL-1α and NLRP3 in MPs-exposed ovaries (Fig. 7 E). Given the critical roles of SIRT1/FOXO1 signal pathway in maintaining ovarian function, we accessed the protein expression of SIRT1 and FOXO1. L-arginine supplementation remarkably increased their expression levels in MPs-exposed ovaries (Fig. 7 F). Collectively, the results demonstrated that L-arginine supplementation effectively restored ovarian function in MPs-treated mice. We also examined the effects of L-arginine on oocyte quality. L-arginine supplementation partially rescued meiotic progression, as shown by improvements in GVBD and PBE (Fig. 7 G). Consistently, in vitro supplementation with L-arginine also partially rescued MPs-induced reductions in PBE(Fig. S4 B). Furthermore, L-arginine significantly reduced ROS levels and improved mitochondrial membrane potential in MPs-treated oocytes (Fig. 7 H). Immunofluorescence staining revealed that L-arginine supplementation corrected MPs-induced spindle assembly defects and chromosomes misalignment (Fig. 7 I). These observations indicated that L-arginine alleviates MPs-induced meiotic defects during oocyte maturation by enhancing mitochondrial function. Discussion Ovarian aging is a natural process associated with biological aging. however, its prevalence has increased in recent decades due to genetic and environmental factors, severely impacting female 26 . Studies have shown that exposure to environmental factors, such as bisphenol A and phthalates, is strongly linked to accelerated ovarian aging, leading to diminished ovarian reserve, reduced oocyte quality, and impaired folliculogenesis 27 . MPs have been shown to have toxic effects on multiple organs and systems, including the digestive, nervous, reproductive, and cardiovascular systems 6 , 28 . In the present study, we observed that MPs could penetrate reproductive tissue barriers and accumulated in the ovaries, consistent with previous findings 29 . Notably, our results demonstrated that MPs exposure is a potential inducer of ovarian aging in mice, as evidenced by elevated expression of senescence markers (p16 and p21), and enhanced secretion of senescence-associated secretory phenotype (SASP) factors. Furthermore, MPs exposure reduced the number of antral and secondary follicles while increasing atretic follicles. Consistently with earlier studies, MPs exposure compromised oocyte quality, indicated by impaired oocyte maturation, reduced embryonic developmental competence, and disrupted gap junction communication between oocytes and cumulus cells 12 . These findings highlight the detrimental effects of MPs on ovarian function, suggesting that MPs exposure is a significant risk factor for premature ovarian aging. To uncover the molecular mechanisms underlying MPs-induced ovarian dysfunction, we performed transcriptomic sequencing of MPs-exposed ovaries. The analysis revealed substantial alterations in gene expression, with a significant enrichment of genes related to inflammation and mitochondrial function. Previous studies have shown that MPs exert their harmful effects primarily through mechanisms such as inflammatory damage, oxidative stress, metabolic disruption, and immunotoxicity 30 , 31 . Chronic inflammation resulting from the immune system's inability to eliminate MPs may increase the risk of disease 32 . For example, MPs exposure has been shown to induce inflammatory damage in the lungs, characterized by increased secretion of inflammatory cytokines and neutrophil recruitment 33 . Similarly, in fish, microplastics exposure has been linked to immunotoxicity, oxidative stress, genotoxicity, DNA damage, and reduced growth and reproductive quality 34 . Based on these findings and our sequencing data, we hypothesized that MPs exposure induces inflammatory damage in the ovaries. Indeed, our results demonstrated that MPs exposure significantly activated the NLRP3 inflammasome and altered the expression of inflammation-related proteins, including IL-10, IL-1α and TNFα. Previous studies have shown that NLRP3 activation and IL-1 secretion are closely associated with ovarian aging, and deletion of these genes can delay ovarian aging and preserve fertility in mice by reducing proinflammatory cytokines and apoptotic signaling 35 , 36 . These results suggested that MPs-induced ovarian aging might be related with the constantly activation of NLRP3 and IL1β. Mitochondrial dysfunction is another hallmark of ovarian aging. Targeting mitochondrial dysfunction through therapeutic approaches, such as antioxidants, metabolic improvement, biogenesis promotion, mitophagy enhancement or mitochondrial replacement can efficiently mitigate aged-related ovarian dysfunction and improve oocyte quality 37 Our RNA sequencing data, supported by GO and KEGG analyses, revealed that MPs exposure impaired mitochondrial function. Specifically, MPs exposure induced oxidative stress and disrupted mitochondria dynamics, as evidenced by altered expression of proteins involved in mitochondrial fission/fusion (DRP1, MFN1) and mitophagy (PINK1, Parkin, LC3β) in ovaries and oocytes. There findings suggested that MPs-induced mitochondrial dysfunction contributes to ovarian aging in mice. Metabolic disorder has been reported to play a critical role in ovarian aging and infertility 38 . Numerous metabolisms undergo significant changes during ovarian aging. For examine, NAD+, a coenzyme involved in redox reactions, has been identified as a key metabolite in aging across various tissues 39 . Supplementation with NAD + or its precursor NMN has been shown to alleviate aging-induced ovarian dysfunction and improve oocyte quality, thereby enhancing fertility, suggesting potential of metabolic factors as therapeutic or preventative targets for aging-related disease 40 . We thus performed untargeted metabolomics to examine the metabolic changes associated with MPs exposure. Our analysis revealed a significant reduction in L-arginine levels in ovaries from MPs-treated mice. L-arginine, a versatile amino acid and a central intestinal metabolite in mammals, serves as a precursor of multiple metabolic pathways involved in regulating of cell division and growth 41 . Further investigation revealed that the decreased ovarian L-arginine levels were associated with gut microbiota dysbiosis and impaired intestinal epithelial cell transport capacity. Consistently, RNA-seq analysis confirmed that L-arginine synthesis was disrupted in the ovaries of MPs-treated mice. Previous study showed that L-arginine supplementation can prevent diabetic cardiomyopathy by improving mitochondrial fitness and homeostasis 42 . This suggests that the mitochondrial dysfunction induced by MPs exposure may be associated with the decline in L-arginine levels in ovarian tissue. Additionally, Arginine pathway metabolites are dysregulated in aging 43 , and targeting L-arginine metabolism has been proposed as a promising therapeutic strategy to maintain the intestinal homeostasis and cognitive function during aging 44 . Furthermore, supplementation with L-arginine has been reported to improve oocyte maturation and embryo development rates in cattle 45 . In line with these findings, our study demonstrated that L-arginine supplementation significantly alleviated MPs-induced ovarian aging. This effect was particularly evident in its ability to improve mitochondrial function and reduce inflammatory damage, thereby promoting oocyte maturation both in vitro and in vivo. These results suggest that L-arginine is a critical metabolite for regulating reproductive health, especially in the context of MPs-induced ovarian dysfunction. In conclusion, this study provides a comprehensive analysis of the toxic effects of MPs on reproductive health, demonstrating that MPs exposure induces ovarian toxicity in mice. This is characterized by hallmarks of ovarian aging, including increased atretic follicles, fibrosis, inflammatory damage, mitochondrial dysfunction, and reduced oocyte quality. Through multi-omics analysis, we identified L-arginine as a key metabolite for preserving ovarian function and oocyte quality during MPs-induced ovarian aging. Notably, L-arginine supplementation alleviated the adverse effects of MPs exposure by improving mitochondrial function, reducing inflammation, and promoting oocyte maturation. These findings provide important clinical insights into the potential application of L-arginine as a therapeutic strategy to mitigate female reproductive disorders caused by MPs exposure. Declarations Acknowledgements We thank Dr. Xing Duan’s laboratory member for discussions and constructive suggestions. Author contributions X.D. designed the experiments and supervised the study. LS.L, WJ.L and QY.X performed all experiments and analyzed the data. LS.L, TT.D and XL.L contributed the data collection. LS.L, JY.N, JX.Z and X.Z contributed to images processing and discussion. X.D. wrote the manuscript. Conflict of interest statement The authors declare no conflict of interest. Funding This research is supported by the Fundamental Research Funds for the Central Universities (4025002407). 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Cell Rep 32 , 107987 (2020). Nüse, B., Holland, T., Rauh, M., Gerlach, R.G. & Mattner, J. L-arginine metabolism as pivotal interface of mutual host–microbe interactions in the gut. Gut Microbes 15 (2023). Fiordelisi, A. , et al. L-Arginine supplementation as mitochondrial therapy in diabetic cardiomyopathy. Cardiovasc Diabetol 23 , 450 (2024). Reisz, J.A. , et al. Arginine metabolism is a biomarker of red blood cell and human aging. Aging Cell 24 , e14388 (2025). He, J. , et al. L-arginine metabolism ameliorates age-related cognitive impairment by Amuc_1100-mediated gut homeostasis maintaining. Aging Cell 23 , e14081 (2024). Dubeibe, D.F. , et al. L-arginine affects the IVM of cattle cumulus-oocyte complexes. Theriogenology 88 , 134-144 (2017). Additional Declarations There is NO Competing Interest. Supplementary Files SupplementaryTableS1AntibodyInformation.pdf Supplementary Table S1 Antibody Information SupplementaryTableS2Listofprimerssequences.pdf Supplementary Table S2 List of primers sequences S1N.jpg Fig. S1. Supplementary Data Related to Fig. 1 S2N.jpg Fig. S2. Supplementary Data Related to Fig. 4 S3N.jpg Fig. S3. Supplementary Data Related to Fig. 5-6 S4N.jpg Fig. S4. Supplementary Data Related to Fig. 7 Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6298826","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":446431844,"identity":"19b07e0d-b543-4af4-b9d5-fa04fe2fea96","order_by":0,"name":"Xing Duan","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA10lEQVRIiWNgGAWjYDCCAyDCgIGBH8JlJkGLZANpWkC6DhCrhe9G8rPHPAV37DafP3xMgqHCOrGB/ewBvFokb6SZG84weJa87UZamgTDmfTEBp68BLxaDG4kmEl8MDicbHaDx0yCse1wYoMEjwEBLenfJBKAWoz7z3+TYPxHlJYcsC12Bgw5bBKMDURokTzzpkxyhsHhBIkbacYWCcfSjdt4cvBr4Tuevk2a589he/7+ww9vfKixlu1nP4NfCwwkNoDIBCBmI0o9ENgTq3AUjIJRMApGIAAAtAJGLgLrKHgAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0002-0538-9102","institution":"Southeast University","correspondingAuthor":true,"prefix":"","firstName":"Xing","middleName":"","lastName":"Duan","suffix":""},{"id":446431845,"identity":"e1e551a1-4df9-4686-9e66-68d7f790b5ed","order_by":1,"name":"Li-Shu Li","email":"","orcid":"","institution":"Southeast University","correspondingAuthor":false,"prefix":"","firstName":"Li-Shu","middleName":"","lastName":"Li","suffix":""},{"id":446431846,"identity":"39d96ac7-a0b3-434e-9fd2-ae50a55a5b0a","order_by":2,"name":"Wen-Jiao Liu","email":"","orcid":"","institution":"Zhejiang A\u0026F University,","correspondingAuthor":false,"prefix":"","firstName":"Wen-Jiao","middleName":"","lastName":"Liu","suffix":""},{"id":446431847,"identity":"df1fbc2c-337b-4938-8a4d-8c4fc13d2c9c","order_by":3,"name":"Tong-Tong Duan","email":"","orcid":"","institution":"Zhejiang A\u0026F University,","correspondingAuthor":false,"prefix":"","firstName":"Tong-Tong","middleName":"","lastName":"Duan","suffix":""},{"id":446431848,"identity":"b6440f25-7519-4152-be63-23c7f65b3c56","order_by":4,"name":"Qian-Yin Xu","email":"","orcid":"","institution":"Southeast University","correspondingAuthor":false,"prefix":"","firstName":"Qian-Yin","middleName":"","lastName":"Xu","suffix":""},{"id":446431849,"identity":"db6b9903-01e2-4a1f-b56b-8d580c06ab22","order_by":5,"name":"Jin-Xing Zhang","email":"","orcid":"","institution":"Zhejiang A\u0026F University","correspondingAuthor":false,"prefix":"","firstName":"Jin-Xing","middleName":"","lastName":"Zhang","suffix":""},{"id":446431850,"identity":"b513c77b-be29-4309-a401-2164c3c803f9","order_by":6,"name":"Jia-Yi Ni","email":"","orcid":"","institution":"Zhejiang A\u0026F University","correspondingAuthor":false,"prefix":"","firstName":"Jia-Yi","middleName":"","lastName":"Ni","suffix":""},{"id":446431851,"identity":"73584691-0162-4dfd-9688-0671bdb390ce","order_by":7,"name":"Xing Zhang","email":"","orcid":"","institution":"Zhejiang A\u0026F University","correspondingAuthor":false,"prefix":"","firstName":"Xing","middleName":"","lastName":"Zhang","suffix":""},{"id":446431852,"identity":"96d17dad-0e8b-4350-b983-f64b942f363f","order_by":8,"name":"Xin-Le Lai","email":"","orcid":"","institution":"Zhejiang A\u0026F University","correspondingAuthor":false,"prefix":"","firstName":"Xin-Le","middleName":"","lastName":"Lai","suffix":""}],"badges":[],"createdAt":"2025-03-25 00:50:39","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6298826/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6298826/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":81175710,"identity":"f3a2e4f1-7ef3-4dfc-bdcb-e8d002e2e50b","added_by":"auto","created_at":"2025-04-23 06:15:10","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":682267,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCharacterization of MPs and its effect on ovarian premature Aging\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Representative SEM images of MPs and quantitative results of their size distribution. Scale bar, 1 μm. (B) Zeta potential of MPs in culture medium and distilled water. (C) Hydrodynamic size distribution of MPs in distilled water. (D) FTIR spectra of MPs. (E) Representative fluorescence images of ovarian sections from control and MPs-treated groups. Scale bar, 20 μm. (F) Representative images of ovarian sections and quantitative analysis of follicle proportions at different developmental stages in control and MPs-treated mice. Scale bar, 0.5 mm. (G) Representative western blot images and quantitative results of P16 and P21 protein levels in ovaries from control and MPs-treated mice. (H) Representative Masson-stained images of ovarian sections and quantitative analysis of positive area ratios in control and MPs-treated mice. Scale bar, 100 μm. (I) Representative images and normalized fluorescence intensity of γH2A.X in ovarian sections from control and MPs-treated mice. Scale bar, 50 μm. (J) Representative Western blot images and quantitative results of Bax, Bcl2, Cyt-C, and p-ATM protein levels in ovaries from control and MPs-treated mice. (K) Representative DIC images of mature oocytes and quantitative analysis of fragmentation rate and cell count after superovulation in control and MPs-treated mice. Scale bar, 80 μm. (L) Representative images of spindle morphology and chromosome alignment in mature oocytes and the rate of abnormal spindle morphology in control vs MPs-treated mice. Scale bar, 10 μm. (M) Representative DIC images of early embryos at the zygote, 2-cell, and 4-cell stages, and quantitative analysis of developmental rates in control and MPs-treated mice. Scale bar, 80 μm. (N) Representative images and quantitative analysis of TZPs in oocytes from control and MPs-treated mice. Scale bar, 10 μm. (O) Representative images of calcein staining and quantitative analysis of the normalized fluorescence intensity ratio of cumulus/oocyte complexes in control and MPs-treated mice. Scale bar, 20 μm. *P \u0026lt; 0.05; **P\u0026lt; 0.01; ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6298826/v1/3ecedafa13bc1c4ca1859958.png"},{"id":81174785,"identity":"5b53ecad-1109-455c-a057-5b46e6d71fcd","added_by":"auto","created_at":"2025-04-23 06:07:11","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":526456,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTranscriptomic Analysis of the Molecular Mechanism of MPs-Induced Ovarian Dysfunction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Schematic diagram of transcriptomic sequencing of ovaries from control and MPs-treated mice. (B) Heatmap of differentially expressed genes (DEGs) between control and MPs-treated groups. (C) Volcano plot of DEGs between control and MPs-treated groups. Blue dots represent downregulated genes, and orange dots represent upregulated genes. (D) KEGG enrichment analysis of DEGs between control and MPs-treated groups. The ordinate denotes the enriched pathways, and the abscissa denotes the Rich factor. The size of the circles denotes the number of genes, and the color denotes the P-value of the indicated pathway. (E) GO annotation analysis of DEGs between control and MPs-treated groups based on molecular function (blue), cellular component (red), and biological process (green). The ordinate indicates the secondary classification of GO terms, and the abscissa indicates the number of DEGs. (F) Gene Set Enrichment Analysis (GSEA) of DEGs between control and MPs-treated groups using a KEGG-based list. (G) Heatmap of DEGs related to arginine and proline metabolism and D-amino acid metabolism between control and MPs-treated groups. (H) Representative images and normalized fluorescence intensity of NLRP3 in ovarian sections from control and MPs-treated mice. Scale bar, 80 μm. (I) Representative western blot images and quantitative results of NLRP3, IL1α, TNFα, and IL10 protein levels in ovaries from control and MPs-treated mice. *P \u0026lt; 0.05; **P \u0026lt; 0.01; ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6298826/v1/fae744b1c7c8043d8fc53d4b.png"},{"id":81174762,"identity":"8623742b-a3e5-4c08-9769-6dc5ac67866b","added_by":"auto","created_at":"2025-04-23 06:07:07","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":284946,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMPs Exposure Induced Mitochondrial Dysfunction and Autophagy in Ovaries\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Representative western blot images and quantitative results of SOD2 and NRF2 protein levels in ovaries from control and MPs-treated mice. (B) Quantitative results of relative gene expression levels of \u003cem\u003eSOD2\u003c/em\u003e, \u003cem\u003eNRF2,\u003c/em\u003e and \u003cem\u003eHO-1 \u003c/em\u003ein ovaries from control and MPs-treated mice. (C) Representative Western blot images and quantitative results of SIRT3, SIRT1, and FOXO1 protein levels in ovaries from control and MPs-treated mice. (D) Quantitative results of relative gene expression levels of \u003cem\u003eSIRT3\u003c/em\u003e in ovaries from control and MPs-treated mice. (E) Quantitative results of relative gene expression levels of \u003cem\u003eGRP78,\u003c/em\u003e \u003cem\u003eCHOP\u003c/em\u003e, \u003cem\u003eFOXO3,\u003c/em\u003e and \u003cem\u003eNOXO1 \u003c/em\u003ein ovaries from control and MPs-treated mice. (F) Representative western blot images and quantitative results of DRP1, MFN1, FIS1, and OPA1 protein levels in ovaries from control and MPs-treated mice. (G) Quantitative results of relative gene expression levels of \u003cem\u003eDRP1\u003c/em\u003e, \u003cem\u003eMFN1\u003c/em\u003e, \u003cem\u003eFIS1\u003c/em\u003e, and \u003cem\u003eOPA1 \u003c/em\u003ein ovaries from control and MPs-treated mice. (H) Representative western blot images and quantitative results of PGC1α protein levels in ovaries from control and MPs-treated mice. (I) Quantitative results of relative gene expression levels of \u003cem\u003eTFAM\u003c/em\u003e and \u003cem\u003ePGC1α \u003c/em\u003ein ovaries from control and MPs-treated mice. (J) Representative western blot images and quantitative results of PINK1 and Parkin protein levels in ovaries from control and MPs-treated mice. (K) Quantitative results of relative gene expression levels of \u003cem\u003ePINK1\u003c/em\u003e, \u003cem\u003ePARKIN\u003c/em\u003e, \u003cem\u003eFUNDC1\u003c/em\u003e, and \u003cem\u003ePTEN \u003c/em\u003ein ovaries from control and MPs-treated mice. (L) Representative western blot images and quantitative results of LAMP2, LC3β, and P62 protein levels in ovaries from control and MPs-treated mice. (M) Quantitative results of relative gene expression levels of \u003cem\u003eLC3α\u003c/em\u003e, \u003cem\u003eLC3β,\u003c/em\u003e \u003cem\u003eATG7\u003c/em\u003e, \u003cem\u003eBECLIN1\u003c/em\u003e, and \u003cem\u003eP62\u003c/em\u003e in ovaries from control and MPs-treated mice. *P \u0026lt; 0.05; **P \u0026lt; 0.01; ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6298826/v1/45cca80bb1d1f41ae1e9db0d.png"},{"id":81174809,"identity":"4104e560-038f-4682-bade-0db4f60c972f","added_by":"auto","created_at":"2025-04-23 06:07:13","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":813268,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMPs Exposure In Vitro Perturbs Oocyte Meiotic Progression and Mitochondrial Function\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Representative DIC images and quantitative analysis of the rate of polar body extrusion (PBE) in oocytes from control and MPs-treated groups. Scale bar, 60 μm. (B) Representative images of spindle morphology and chromosome alignment in oocytes, with quantitative analysis of relative spindle area and chromosome dispersion distance in control and MPs-treated groups. Scale bar, 15 μm. (C) Representative images and normalized fluorescence intensity of ROS staining in oocytes from control and MPs-treated groups. Scale bar, 60 μm.(D) Representative images and normalized fluorescence intensity of Ca²⁺staining in oocytes from control and MPs-treated groups. Scale bar, 15 μm. (E) Representative images and normalized fluorescence intensity of SOD2 in oocytes from control and MPs-treated groups. Scale bar, 15 μm. (F) Representative images and normalized fluorescence intensity of Ace-SOD2 in oocytes from control and MPs-treated groups. Scale bar, 15 μm. (G) Representative images and normalized fluorescence intensity of Mito-Tracker-stained mitochondria in oocytes from control and MPs-treated groups. Scale bar, 15 μm. (H) Representative images and normalized fluorescence intensity of mtTFA in oocytes from control and MPs-treated groups. Scale bar, 15 μm. (I) Representative images and normalized fluorescence intensity of DRP1 in oocytes from control and MPs-treated groups. Scale bar, 15 μm. (J) Representative images and normalized fluorescence intensity of MFN1 in oocytes from control and MPs-treated groups. Scale bar, 15 μm. (K) Representative images and normalized fluorescence intensity of PINK1 in oocytes from control and MPs-treated groups. Scale bar, 15 μm. (L) Representative images and quantitative analysis of lysosome area and normalized fluorescence intensity of Lyso-Tracker Red-stained lysosomes in oocytes from control and MPs-treated groups. Scale bar, 15 μm. (M) Representative images and normalized fluorescence intensity of LC3β in oocytes from control and MPs-treated groups. Scale bar, 15 μm. *P \u0026lt; 0.05; **P \u0026lt; 0.01; ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6298826/v1/49efbc53cf908b7346002834.png"},{"id":81174774,"identity":"7e9480b3-4ab0-4109-b73b-0eb4ceb05ed0","added_by":"auto","created_at":"2025-04-23 06:07:10","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":959512,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMetabolomic profiling identifies decreased arginine levels in ovaries of MPs-exposed mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A-B) Schematic diagram of metabolomic sequencing of ovaries from control and MPs-treated mice. (C) Heatmap of differentially expressed metabolites (DEMs) between control and MPs-treated groups. (D) Volcano plot of DEMs between control and MPs-treated groups. Blue dots represent downregulated metabolites, and orange dots represent upregulated metabolites. (E) KEGG enrichment analysis of DEMs between control and MPs-treated groups. The ordinate denotes the enriched pathways, and the abscissa denotes the Rich factor. The size of the circles denotes the number of metabolites, and the color denotes the P-value of the indicated pathway. (F) Metabolite Set Enrichment Analysis (MSEA) of DEMs between control and MPs-treated groups using a KEGG-based list. (G) Heatmap of DEMs related to arginine and proline metabolism and D-amino acid metabolism between control and MPs-treated groups. (H) Quantitative analysis of arginine levels in ovaries from control and MPs-treated mice based on metabolomic data.(I) Correlation heatmap between DEGs and DEMs in control and MPs-treated groups.(J) Joint KEGG enrichment analysis of DEGs and DEMs between control and MPs-treated groups. *P \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6298826/v1/251b37ed5e9f9b4dcd64d68d.png"},{"id":81174784,"identity":"75e753e9-1a23-45de-8982-51990b82f18a","added_by":"auto","created_at":"2025-04-23 06:07:11","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":534733,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGut Microbiota Analysis in MPs-Treated Mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Alpha diversity analysis of gut microbiota between control and MPs-treated groups. (B) Principal Coordinate Analysis (PCoA) of gut microbiota between control and MPs-treated groups. (C) Composition of gut microbiota at the phylum, class, order, and family levels. (D) Linear Discriminant Analysis Effect Size (LEfSe) analysis to identify key phylotypes in control and MPs-treated groups. (E) Differential gut microbiota between control and MPs-treated groups. (F) Representative Western blot images and quantitative results of IL10 and TNFα protein levels in the intestines of control and MPs-treated mice. (G) Representative Western blot images and quantitative results of ZO-1 and Occludin protein levels in the intestines of control and MPs-treated mice. (H) Quantitative results of relative gene expression levels of CAT1, CAT2, CAT3, and SLC6A20 in the intestines of control and MPs-treated mice. *P \u0026lt; 0.05; **P \u0026lt; 0.01; ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6298826/v1/527b2d536098c7011ac263d9.png"},{"id":81174764,"identity":"21636486-ecfd-4dd9-affc-a934e45f9635","added_by":"auto","created_at":"2025-04-23 06:07:08","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":576668,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eL-Arginine Alleviates MPs-Induced Premature Ovarian Aging in Mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Representative images of ovarian sections and quantitative analysis of follicle proportions at different developmental stages in control, MPs-treated, and L-arginine (Arg)-rescued groups. Scale bar, 150 μm. (B) Representative western blot images and quantitative results of P16 and P21 protein levels in ovaries from control, MPs-treated, and Arg-rescued groups. (C) Representative Masson-stained images of ovarian sections and quantitative analysis of positive area ratios in control, MPs-treated, and Arg-rescued groups. Scale bar, 150 μm. (D) Quantitative results of relative gene expression levels of \u003cem\u003eTNFα\u003c/em\u003e, \u003cem\u003eIFNγ,\u003c/em\u003e \u003cem\u003eCXCL1,\u003c/em\u003e and \u003cem\u003eCXCL3\u003c/em\u003e in ovaries from control, MPs-treated, and Arg-rescued groups. (E) Representative western blot images and quantitative results of IL1α, TNFα, and NLRP3 protein levels in ovaries from control, MPs-treated, and Arg-rescued groups.(F) Representative Western blot images and quantitative results of SIRT1 and FOXO1 protein levels in ovaries from control, MPs-treated, and Arg-rescued groups. (G) Representative DIC images and quantitative analysis of the rate of GVBD and PBE in \u003cem\u003ein vitro\u003c/em\u003e matured oocytes from control, MPs-treated, and Arg-rescued groups. Scale bar, 80 μm. (H) Representative images and normalized fluorescence intensity of ROS and TMRE staining in oocytes from control, MPs-treated, and Arg-rescued groups. Scale bar, 80 μm. (I) Representative images of spindle morphology and chromosome alignment, along with quantitative analysis of spindle abnormality rate and chromosome dispersion distance in oocytes from control, MPs-treated, and Arg-rescued groups. Scale bar, 15 μm. *P \u0026lt; 0.05; **P \u0026lt; 0.01; ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-6298826/v1/bb859c93fb6599db1ae558e4.png"},{"id":81174798,"identity":"7a03079d-8de6-4f35-b782-31204a95524d","added_by":"auto","created_at":"2025-04-23 06:07:12","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":501789,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic diagram of\u003c/strong\u003e \u003cstrong\u003emicroplastics exposure causes premature ovarian aging via inducing mitochondrial dysfunction\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-6298826/v1/b1efa30930df2897037b1222.png"},{"id":83569026,"identity":"dd65664c-ff0c-431f-b6bb-30d0c5d934e4","added_by":"auto","created_at":"2025-05-28 15:55:42","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6009025,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6298826/v1/a0c96764-76ff-4ef0-ae3b-0994b57a8b7b.pdf"},{"id":81174787,"identity":"a90aebc5-4141-4555-97ef-54d56d8c275b","added_by":"auto","created_at":"2025-04-23 06:07:11","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":89433,"visible":true,"origin":"","legend":"Supplementary Table S1 Antibody Information","description":"","filename":"SupplementaryTableS1AntibodyInformation.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6298826/v1/a94d1d589f1cfc565ce99eae.pdf"},{"id":81174767,"identity":"7ac10ea7-1bab-4a81-9a0b-46b76b97b9b4","added_by":"auto","created_at":"2025-04-23 06:07:09","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":81477,"visible":true,"origin":"","legend":"Supplementary Table S2 List of primers sequences","description":"","filename":"SupplementaryTableS2Listofprimerssequences.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6298826/v1/bc8cff92abf0ed2156251edc.pdf"},{"id":81174778,"identity":"0ba7cf7d-ac96-4a30-adff-e13aae489fad","added_by":"auto","created_at":"2025-04-23 06:07:10","extension":"jpg","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":599405,"visible":true,"origin":"","legend":"Fig. S1. Supplementary Data Related to Fig. 1","description":"","filename":"S1N.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6298826/v1/49d35bc1042da89287bf3177.jpg"},{"id":81174765,"identity":"9ca1e0b2-9a95-4f68-a8da-0e549e75a0ff","added_by":"auto","created_at":"2025-04-23 06:07:08","extension":"jpg","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":880232,"visible":true,"origin":"","legend":"Fig. S2. Supplementary Data Related to Fig. 4","description":"","filename":"S2N.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6298826/v1/b4b993771a95e4a428f87503.jpg"},{"id":81174812,"identity":"678d6393-2249-49ba-9835-dd518a13f5cb","added_by":"auto","created_at":"2025-04-23 06:07:13","extension":"jpg","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":476983,"visible":true,"origin":"","legend":"\u003cp\u003eFig. S3. Supplementary Data Related to Fig. 5-6\u003c/p\u003e","description":"","filename":"S3N.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6298826/v1/4fdf7de0682ffaa0b97770a0.jpg"},{"id":81174766,"identity":"8a9af1b9-575a-4462-8a3c-e8ee775c5bba","added_by":"auto","created_at":"2025-04-23 06:07:09","extension":"jpg","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":647004,"visible":true,"origin":"","legend":"\u003cp\u003eFig. S4. Supplementary Data Related to Fig. 7\u003c/p\u003e","description":"","filename":"S4N.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6298826/v1/16d53e73db2e5c189b76440a.jpg"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Microplastics exposure causes premature ovarian aging via inducing mitochondrial dysfunction","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eThe widespread use of plastics has significantly contributed to industrialization and socio-economic development\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. However, the high physicochemical stability of plastics makes them difficult to degrade, resulting in the accumulation of plastic waste in the environment\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Microplastics (MPs), defined as plastic particles smaller than 5 mm in diameter, represent emerging environmental pollutants that are widely distributed in water, air, and soil and the atmosphere\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Importantly, MPs have been shown to migrate from the environment into human body through ingestion, inhalation or food chain transfer, raising concerns regarding their potential toxicity due to their extensive exposure pathways\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Recent research has shown that MPs are capable of crossing biological barriers and exerting toxic effects on vital organs, such as the brain, liver, kidney, lung and heart\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. These adverse effects are linked to oxidative stress, chronic inflammation, cytotoxicity and disturbances in metabolic processes and energy homeostasis in biological systems\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Consequently, the potential health risks associated with MPs have emerged as a significant public health concern.\u003c/p\u003e \u003cp\u003eMicroplastics (MPs) have been reported to induce a range of toxic effects, including neurotoxicity, cardiotoxicity, hepatotoxicity, immunotoxicity, metabolic disorders, and even cancer\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Exposure to MPs has been shown to trigger autophagy and apoptosis by impairing mitochondrial function and elevating levels of reactive oxygen species (ROS)\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Moreover, MPs disrupt mitochondrial activity and cause DNA damage by interfering with glycolysis and tricarboxylic acid (TCA) cycle processes, contributing to renal toxicity\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. In the liver, MPs exposure has been linked to injury and fibrosis through the promotion of proinflammatory cytokine release, mitochondrial dysfunction, and ferroptosis\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e Recently, increasing attention has been directed toward the reproductive toxicity of microplastics (MPs). Studies have revealed that MPs are capable of penetrating reproductive tissue barriers, including the placental barrier, epithelial barrier, and blood-testis barrier, leading to their accumulation in reproductive organs\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. MPs have been detected in human follicular fluid, where their presence is associated with a reduction in follicles number, diminished oocyte quality, and decreased pregnancy and fertility\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Furthermore, MPs have been shown to cross the placental barrier in pregnant mice, resulting in metabolic disorders and reproductive system impairments in offspring\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Recent studies have also indicated that MPs disrupt the gut microbiota homeostasis and metabolic processes, ultimately leading to organismal damage\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. However, limited research has explored the impact of MPs on ovary metabolism process in mammals, and the specific mechanisms underlying this damage remain unclear. Identifying key metabolites involved in MPs-induced ovarian dysfunction could offer critical insights for the prevention and treatment of reproductive toxicity caused by MPs.\u003c/p\u003e \u003cp\u003ePremature ovarian aging (POA), characterized by a progressive decline in ovarian reserve and oocyte quality, ultimately leading to female infertility\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. This condition has become a pressing public health concern. Environmental factors are increasingly recognized as critical contributors to ovarian reserve depletion and may influence ovarian function during both prenatal development and adulthood. Key environmental toxicants, such as phthalates, bisphenol A, and tobacco, have been implicated in this process\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. For instance, tobacco smoke has been shown to disrupt follicular development by increasing apoptosis and autophagy, inducing DNA damage, and impairing the connections between oocytes and granulosa cells, all of which contribute to ovarian dysfunction and infertility\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Women exposed to cigarette smoke also exhibit a significant reduction in ovarian volume compared to non-exposed women of the same age\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Additionally, benzo[a]pyrene preferentially accumulates in the ovary, resulting in a reduced number of primordial follicles and infertility in mice\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Similarly, perfluorooctanoic acid has been shown to induce POA by impairing NAD\u0026thinsp;+\u0026thinsp;synthesis and mitochondrial function in adult zebrafish\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Although it has been demonstrated that MPs exposure reduced the ovary size and lifespan in Drosophila melanogaster, and MPs exposure induced the inflammation of ovaries and reduced the quality of oocyte in mice\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e, whether the MPs triggered the occurrence of POA and the potential mechanism remain unclear, especially whether MPs exposure changed the metabolism process of ovary still clarified.\u003c/p\u003e \u003cp\u003eThis study investigated the detrimental effects of MPs on ovarian function and oocyte quality in mice. Our findings revealed that exposure to MPs induced POA in mice, which characterized by increased senescence markers (p16 and p21) and fibrosis. In addition, we also observed MPs-exposure caused mitochondrial dysfunction, oxidative stress, inflammation, autophagy, and DNA damage in both the ovary and oocytes. Furthermore, we utilized multi-omics analysis to elucidate the signaling pathways and metabolites affected by MPs in the ovary. Notably, MPs exposure significantly disrupted L-arginine synthesis, and in vivo supplementation with L-arginine partially restored ovarian function and oocyte quality by enhancing mitochondrial fitness. These results offer a novel strategy for mitigating the toxic effects of MPs on the female reproductive system.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Animal Model\u003c/h2\u003e \u003cp\u003eICR mice were purchased from Viton Lever and housed under specific pathogen-free conditions. Four-week-old female mice were used in the experiments and provided with a suitable environment and sufficient water and food. Based on previous studies on microplastic exposure in rodents\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e after one week of acclimatization, each mouse was randomly grouped and orally garaged with 5mg/kg body weight / day of MPs or an equivalent volume of purified water for 4 weeks (n\u0026thinsp;=\u0026thinsp;50 for each group). Regarding the rescue treatment with L-arginine, we referred to studies on the protective effects of L-arginine \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Experimental mice were orally administered 200 mg/kg of L-arginine (dissolved in PBS, Cat#: A8096, Sigma-Aldrich) or an equal volume of PBS every day for four consecutive weeks. All animal experiments were approved by the Laboratory Animal Welfare and Ethics Committee of Southeast University.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Microplastics\u003c/h2\u003e \u003cp\u003eFITC-polystyrene (FITC-PS) and polystyrene (PS) particles were purchased from Tianjin BaseLine New Material Technology Company (China). Scanning electron microscopy (SEM, Nova Nano 450, FEI) was used to characterize the primary size and shape of the different MPs. The dried MPs were mixed thoroughly with potassium bromide (KBr) at a mass ratio of 1:100 and finely ground. The surface composition of MPs was then analyzed using an IRPrestige-21 spectrometer (Shimadzu Corporation, Japan). MPs were dispersed in ultrapure water by sonication to determine the hydrodynamic size and zeta potential prior to dynamic light scattering analysis (Zetasizer, Malvern, UK).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Mouse Oocyte Collection and Culture\u003c/h2\u003e \u003cp\u003eGerminal vesicles (GV) were collected from experimental mice after intraperitoneal injection of 5 IU pregnant horse serum gonadotropin (PMSG, Sigma) and cultured in Milrinone-free M16 medium for 14 h at 37\u0026deg;C, 5% CO2. To collect ovulated oocytes, female mice were first injected with 5 IU PMSG for 44\u0026ndash;48 h, followed by 5 IU hCG. After 15\u0026ndash;16 h, the oviductal jugular was collected and treated with 0.3% hyaluronidase (Sigma-Aldrich) to remove the oviductal cells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Western Blotting\u003c/h2\u003e \u003cp\u003eProteins were extracted from ovaries with RIPA lysis buffer (Beyotime, P0013C), and protein concentration was adjusted by BCA quantification (Beyotime, P0010S) and denatured at 95\u0026deg;C for 10 min. Samples were separated on 10% polyacrylamide gels (20325ES62, YEASEN) and blotted onto PVDF membranes (Immobilon-P, Millipore). Incubation was done in 5% skimmed milk for 1 hr, followed by overnight incubation at 4\u0026deg;C with primary antibodies. The membrane was then washed 3 times with TBST for 5 min each, incubated with the secondary antibody for 2 h at room temperature, and washed again with TBST. Color was developed using the Hyper Signal ECL chemiluminescence kit (4A Biotech Co., Beijing, China), and chemiluminescent signals were captured using a Tanon 4600 chemiluminescence imaging system (Tanon, Beijing, China). The primary and secondary antibodies used are detailed in the Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Gene Expression Quantification\u003c/h2\u003e \u003cp\u003eFor RNA expression analysis, total RNA was extracted from ovaries using Trizol (Invitrogen, USA) and resuspended in nuclease-free water. Reverse transcription was performed using the QuantiTect Reverse Transcription Kit (Qiagen NV). Relative quantitative analysis was performed using the 2X Universal SYBR Green Fast qPCR Mix (Abclonal, China, RK21203) on a QuantStudio instrument (Applied Biosystems, Carlsbad, CA, USA). The relative transcript abundance was analyzed via the 2\u003csup\u003e\u0026minus;ΔΔCT\u003c/sup\u003e method.Mouse \u003cem\u003eGapdh\u003c/em\u003e was chosen as a standardized control. Gene-specific primer sequences are shown in Supplemental Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Immunofluorescence\u003c/h2\u003e \u003cp\u003eOocytes were fixed in 4% paraformaldehyde solution in phosphate-buffered saline (PBS) for 30 min and then permeabilized with 0.5% Triton X-100 (Solarbio, 9002-93-1, China) for 20 min. After being blocked with PBS containing 1% bovine serum albumin for 1 h at room temperature, the samples were incubated with primary antibody at 4\u0026deg;C overnight, followed by incubation with secondary antibody for 1 hour at room temperature. The DNA was then stained with Hoechst 33342 in PBS at a concentration of 10 \u0026micro;g/ml. Samples were then fixed on slides and visualized using a confocal laser scanning microscope (FV 3000, Olympus, Japan).\u003c/p\u003e \u003cp\u003eThe ovary sections were dewaxed in xylene and rehydrated through graded ethanol concentrations. Antigen retrieval was performed in a microwave oven for 4 minutes in 10 mM sodium citrate buffer (pH 6.0), repeated three times. After antigen retrieval, the sections were blocked for 30 minutes with 10% (v/v) normal donkey serum at room temperature. The subsequent steps were carried out in accordance with the previously established protocol for cell immunofluorescence. The primary and secondary antibodies used are detailed in the Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Histological Analysis\u003c/h2\u003e \u003cp\u003eFor histological analysis, ovaries were collected and fixed in 4% paraformaldehyde, embedded in paraffin, sectioned (5 \u0026micro;m thick), and stained with H\u0026amp;E at the Animal Histopathology Core Facility of Southeast University (China). Follicle counting estimated the number of follicles at various stages within each ovary by double-blind assessment of the number of follicles at each level in the H\u0026amp;E-stained images of complete ovarian sections. The criteria used to classify the follicles are as follows: Primordial follicles consist of a singular layer of squamous granulosa cells, pre-antral follicles consist of cuboidal granulosa cells layer and a thecal layer, antral follicles consist of an oocyte surrounded by a fluid-filled antrum and layers of granulosa cells, and atretic follicles exhibit dissolved oocyte cytoplasm with collapsed follicle walls.\u003c/p\u003e \u003cp\u003eTissue sections were dewaxed and stained with Masson's trichrome following the instructions of the commercial Masson staining kit (ServiceBio, Wuhan, China). The histological evaluation showed an enhanced blue coloration, indicating increased levels of collagen and fibrosis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Gut Microbiota Analysis\u003c/h2\u003e \u003cp\u003eIntestinal content samples (approximately 200 mg each) were collected from control and MPs group mice, rapidly frozen in liquid nitrogen, and stored at -80\u0026deg;C. Microbial samples were sent to Shanghai Majorbio Bio-Pharm Technology for high-throughput sequencing of the 16S rRNA gene. Subsequently, genomic DNA was extracted using the OMEGA Soil DNA Kit (Omega Bio-Tek, Norcross, GA, USA) following the manufacturer\u0026rsquo;s protocol. The V3-V4 region of the bacterial 16S rRNA gene was amplified using the forward primer 338F (5'-ACTCCTACGGGAGGCAGCA-3') and the reverse primer 806R (5'-GGACTACHVGGG TWTCTAAT-3'). The amplified products were purified using Vazyme VAHTSTM DNA Clean Beads (Vazyme, Nanjing, China) and quantified using the Quant-it PicoGreen dsDNA Assay Kit (Invitrogen, Carlsbad, CA, USA). Sequencing was performed on the Illumina NovaSeq platform. Raw sequencing data were demultiplexed to remove primer sequences, followed by quality filtering, denoising, merging, and chimera removal using the DADA2 plugin. The obtained bacterial sequence fragments were classified into ASVs and compared with the Greengenes microbial gene database. Finally, microbiome bioinformatics analysis was conducted using QIIME2 and R (v4.3.1) to calculate and visualize diversity, taxonomic composition, and differential abundance.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Metabolomics Analyses\u003c/h2\u003e \u003cp\u003eTo perform non-targeted metabolite profiling, mouse ovarian samples were prepared, and the supernatant was extracted from the ovarian homogenate using pre-chilled 70% methanol buffer for LC-MS/MS analysis. All samples were acquired by MetWare Biotechnology Co., Ltd., and analyzed by the LC-MS/MS system following the instrument\u0026rsquo;s operating protocol. The converted LC-MS data files were processed using XCMS (Scripps, La Jolla, CA) for peak picking, peak alignment, gap filling, and sample normalization. The processed peak information was matched against a self-constructed internal database and public databases to obtain metabolite identification results. Statistical analyses, including OPLS-DA and DESeq2, were performed using R (v4.3.1) to identify metabolite features with significant differences between groups. The differential metabolites were then mapped to the KEGG pathway database.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10 RNA-seq Analysis\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted from mouse ovarian samples using TRIzol reagent (Invitrogen). The concentration and integrity of the RNA were assessed using Qubit 2.0 and Agilent 2100 (Novogene, China), respectively. Subsequently, the RNA-Seq transcriptome library was constructed using the Illumina TruSeq library construction kit (Illumina, California, USA) according to the manufacturer's instructions. The library was then sequenced by Repugene Technology Co., Ltd. (Hangzhou, China) on an Illumina Nova6000 platform. Raw data quality was controlled using Fastp, and the quality of the processed data was verified using FastQC. STAR was employed to align the qualified sequencing data to the reference genome. FeatureCounts was used to quantify gene abundance and transcription levels. Differentially expressed genes (DEGs) were identified through DESeq2 analysis conducted in R (v4.3.1), defined as transcripts with a p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and |log2 FC| \u0026ge; 1. Functional enrichment and annotation analyses of DEGs were performed using the Kyoto Encyclopedia of Genes and Genomes (KEGG) (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.genome.jp/kegg/\u003c/span\u003e\u003cspan address=\"https://www.genome.jp/kegg/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and Gene Ontology (GO) (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.geneontology.org/\u003c/span\u003e\u003cspan address=\"http://www.geneontology.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.11 Statistical Analysis\u003c/h2\u003e \u003cp\u003eStatistical analysis was performed with GraphPad Prism software 9 (La Jolla, USA). Comparisons of data were performed by one-way analysis of variance (ANOVA) with Tukey's multiple comparison test or two-tailed unpaired t-test. p values\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Characterization of MPs\u003c/h2\u003e \u003cp\u003eThe selected MPs were fully characterized before administration \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e. Scanning electron microscopy (SEM) was used to analyze the morphology and size of the MPs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). The results showed that MPs exhibited a nearly spherical morphology with the average diameter of 498.73 ± 39.187 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Since surface charge of MPs plays an important role in deciding the biological effects on organism, we performed zeta potential analysis of the MPs in culture medium M16. The results showed that the stability of MPs in culture medium was lower than in water (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Additionally, dynamic light scattering (DLS) analysis indicated a slightly larger diameter of MPs in water (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC), likely due to particle aggregation in the diluent.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe structural characterization of the MPs was further conducted using Fourier-transform infrared (FTIR) spectroscopy (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). A peak at 3025.76 cm⁻¹ was associated with the stretching vibrations of unsaturated hydrocarbon groups on the benzene ring (-CH). Absorption peaks near 2921 and 3026 cm⁻¹ confirmed the presence of methylene and unsaturated hydrocarbon groups. Peaks at 1494 and 1693 cm⁻¹ corresponded to skeletal vibrations of the benzene ring, while the peak at 1450 cm⁻¹ was attributed to methylene bending vibrations. Additionally, peaks at 695 and 753 cm⁻¹ were linked to out-of-plane bending vibrations of unsaturated hydrocarbon groups on the benzene ring. These FTIR results confirmed that the MPs were polystyrene, synthesized through the polymerization of styrene. Finally, the double peaks around 2400 cm⁻¹ were identified as the asymmetric stretching vibrations of C = O bonds, likely originating from environmental carbon dioxide.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.2. MPs exposure induced ovarian aging and reduced oocyte quality\u003c/h2\u003e \u003cp\u003eTo evaluate the effects of MPs exposure on ovarian function, mice were orally exposed to fluorescently labeled MPs through drinking water for 4 weeks at dose of 5mg/kg body weight / two day. As illustrated in Fig.\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA, the body weight was not changed in MPs-exposed mice, while the ovary organ coefficient was significantly reduced, as evidenced by a decreased in ovarian size. Consist with previous study, we confirmed that fluorescently labeled MPs can penetrated ovary and localized in the granulosa cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). As expected, MPs exposure induced remarkable ovary dysfunction, characterized by a reduction in antral and secondary follicles, an increase in atretic follicles (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). Additionally, we observed a downregulation in the mRNA expression levels of genes associated with follicle maturation and development, including \u003cem\u003eNOBOX\u003c/em\u003e, \u003cem\u003eGDF9\u003c/em\u003e, and \u003cem\u003eBMP15\u003c/em\u003e, as well as genes related to hormone secretion, such as \u003cem\u003eERα\u003c/em\u003e, \u003cem\u003eC/EBPα\u003c/em\u003e, and \u003cem\u003eSTAR\u003c/em\u003e, further confirming the impairment of ovarian function (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB). Western blot analysis showed elevated level of senescence markers P21 and P16 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG), indicative of ovarian aging. Masson’s staining further demonstrated a marked increase in collagen fiber content (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH), suggesting severe ovarian fibrosis following MPs exposure.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo investigate the underlying mechanisms of aberrant ovarian follicle development, we performed immunofluorescent staining and the results indicated a significant increase in γH2AX signals in MPs-treated ovaries (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI), pointing to DNA damage. Since DNA damage is often associated with the onset of apoptosis, our subsequent western blot and RT-qPCR resulting showing elevated Bax expression and decreased Bcl2 levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eJ\u0026amp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003eS1\u003c/span\u003eC). The release of Cyt-C is a hallmark of the mitochondrial pathway initiating apoptosis, while the upregulation of P-ATM reflects the activation of the DNA damage response. These changes collectively indicate the cellular stress and apoptotic processes induced by MPs exposure (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eJ). As a consequence, the number of super-ovulated oocyte was significantly reduced, with an increased percentage of fragmentated oocytes in MPs exposed group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eK). Although meiotic resumption, as shown by germinal vesicle breakdown (GVBD), was slightly affected, polar body extrusion (PBE) rates were significantly reduced after MPs exposure (Fig.\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003eS1\u003c/span\u003eD). Immunofluorescence (IF) staining showed spindle defects in metaphase II (MII) oocytes, including failure of spindle anchoring and a higher incidence of abnormal spindle morphology (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eL).\u003c/p\u003e \u003cp\u003eIn vivo fertilization experiment demonstrated that MPs-treated oocytes could be fertilized but failed to progress to the 4-cell embryo stage (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eM). Given the importance of communication between oocyte and cumulus cells through transzonal projections (TZPs) in maintaining oocyte quality, TZPs was examined by staining phalloidin. The results showed a significantly reduced in the number of TZPs in the cumulus-oocyte complexes (COCs) derived from MPs-exposed mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eN). Additionally, calcein-acetoxymethyl (C-AM) staining demonstrated impaired gap junction communication between oocytes and cumulus cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eO), as indicated by a markedly diminished fluorescent signal in oocytes from MPs-treated mice.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Transcriptomic analysis of the molecular mechanism of MPs-induced ovarian dysfunction\u003c/h2\u003e \u003cp\u003eTo investigate the mechanisms underlying the ovarian toxicity induced by MPs, transcriptomic analysis was performed to examine the differences in mRNA expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, Principal component analysis (PCA) revealed distinct gene expression patterns between the control and MPs-exposed groups. Heatmap analysis indicated significant differences in the transcriptomic profiles of the ovaries between the control and MPs exposure groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Volcano plot analysis identified significant transcriptomic changes, with 505 differentially expressed genes (DEGs) being upregulated and 194 DEGs downregulated in the MPs exposure group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). To identify the potential pathways affected by MPs, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis was conducted. Microplastic exposure led to alterations in the expression of genes related to mitochondrial energy metabolism pathways, such as ascorbate and aldose metabolism, ABC transporters, and various inflammation-related pathways, including the TNF signaling pathway, IL-17 signaling pathway, chemokine signaling pathway, and cytokine-cytokine receptor interaction. Furthermore, pathways related to Biosynthesis of cofactors and the metabolism of arginine, alanine, glutamate, and aspartate were also disrupted (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). Additionally, Gene Ontology (GO) analysis of DEGs, encompassing cellular components, biological processes, and molecular functions, revealed that these DEGs were involved in processes such as calcium-mediated signaling, regulation of small molecule metabolic processes, and cell chemotaxis, as well as activities related to ATP hydrolysis activity, monoatomic ion channel activity, and cytokine receptor binding (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). Collectively, these findings suggest that these pathways and biological processes are closely associated with inflammation, mitochondrial function, and nutrient metabolism. To further explore the trends of these pathway alterations, Gene Set Enrichment Analysis (GSEA) based on KEGG-listed pathways was conducted. The results indicated that the upregulated gene sets in the MPs treatment group were associated with TNF signaling pathway and Cytokine-Cytokine Receptor Interaction in Mus musculus. In contrast, the downregulated gene sets in the MPs treatment group were involved in ATP-dependent chromatin remodeling and arginine and proline metabolism, further indicating that MPs exposure increases cellular inflammation and disrupts energy metabolism (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eG).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eGiven that the involvement of “inflammation” in MPs-exposed ovaries and the function of inflammasomes in aging process, we performed IF and western blot to examine the NLRP3 protein expression and the result showed that MPs exposure remarkably increased inflammasome NLRP3 expression compared with that of control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eH-I). Besides, MPs also significantly increased the inflammatory cytokines IL-10, IL1α and TNFα comparted with the control group, suggesting that MPs exposure cause substantial inflammatory damage to the ovaries (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eI).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.4 MPs exposure induced mitochondrial dysfunction and autophagy in ovaries\u003c/h2\u003e \u003cp\u003eGiven that mitochondria dysfunction-induced elevated reactive oxygen species (ROS) level could acts as activator to NLRP3 activation, we performed western blot and RT-qPCR to examine the effect of MPs on mitochondrial function and fitness. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, our results demonstrated that MPs exposure significantly reduced the protein levels of antioxidative markers NRF2 and SOD2. Similarly, the RT-qPCR results confirmed a notable decrease in the mRNA levels of \u003cem\u003eNRF2, SOD2\u003c/em\u003e and \u003cem\u003eHO-1\u003c/em\u003e in MPs-exposed ovaries (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Furthermore, western blot analysis revealed that MPs exposure also remarkably reduced the protein levels of SIRT1, SIRT3 and FOXO1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eC), indicating that MPs exacerbate oxidative stress in ovaries. Subsequently, we also observed an upregulation in the expression of ER stress-related genes such as GRP78 and CHOP(Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eE), indicating that the oxidative stress induced by MPs in the ovaries subsequently triggered the occurrence of ER stress.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further determine the potential mechanism underlying MPs-induced oxidative stress, we examined the expression of proteins involved in mitochondrial fission and fusion. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eF, mitochondrial fusion-related proteins MFN1 and OPA1 were significantly downregulated in the MPs-exposed group, whereas mitochondrial fission-related proteins FIS1 and DRP1 were markedly upregulated. Consistently, the RT-qPCR results also confirmed that defect of mitochondrial dynamics following MPs exposure (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eG). Furthermore, the downregulation of TFAM and PGC1α, key regulators of mitochondrial biogenesis (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eH-I), further supports the disruption of mitochondrial function following MPs exposure. Additionally, we accessed mitophagy, a form of autophagy specifically targeting damaged mitochondria. MPs exposure was found to induce mitophagy in the ovaries, as evidenced by increased protein expression of PINK1 and Parkin (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eJ). Consistently, RT-qPCR results also indicated that MPs exposure activated mitophagy in the ovaries (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eK). Given that impaired mitochondrial function often led to defects in autophagosome-lysosome fusion, we further analyzed autophagic marker LC3β, a direct indictor of autophagy. Western blot results revealed MPs exposure significantly increased the protein level of LC3β, suggesting heightened autophagic activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eL). Interestingly, MPs exposure also significantly reduced the expression of lysosome-associated membrane protein 2 (LAMP2) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eL), indicating impaired lysosomal function and disrupted autophagic flux. Notably, MPs exposure led to elevated protein levels of P62 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eL), suggesting a blockage in autophagosome degradation. This observation was further supported by RT-qPCR results, which revealed significant increases in the mRNA levels of \u003cem\u003eLC3β\u003c/em\u003e, \u003cem\u003eATG7\u003c/em\u003e, \u003cem\u003eBeclin1\u003c/em\u003e, and \u003cem\u003eP62\u003c/em\u003e following MPs exposure (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eM).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.5 MPs exposure \u003cem\u003ein vitro\u003c/em\u003e perturb oocyte meiotic progress and mitochondrial function\u003c/h2\u003e \u003cp\u003eTo investigate the effects of MPs exposure on oocyte quality, we performed \u003cem\u003ein vitro\u003c/em\u003e experiment by treating the oocyte with different concentration of MPs (30 µg/ml, 50 µg/ml and 100 µg/ml). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eA\u0026amp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003eS2\u003c/span\u003eA, MPs exposure slightly reduced the oocyte GVBD rate but significantly reduced the rate of PBE in a concentration-dependent manner. Immunofluorescence staining revealed that MPs exposure disrupted the spindle assembly and chromosomes misalignment in metaphase I (MI) oocytes, characterized by reduced spindle size and the formation of multipolar spindles (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Quantitative analysis further confirmed abnormalities in spindle morphology and chromosomes alignment. Live cell staining demonstrated that MPs exposure significantly increased cytosolic Ca2 + and ROS levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eC-D), along with upregulation of ER-tracker and γH2A.X staining, indicating ER stress and DNA damage (Fig.\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003eS2\u003c/span\u003eB-C). Consistently, reduced protein levels of SOD2 were observed in MPs-treated oocytes, accompanied by increased acetylation of SOD2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eE-F), which is associated with impaired antioxidative capacity. Given that excessive ROS can induce mitochondrial dysfunction, we examined the mitochondrial mass using Mito-Tracker staining. The results showed a significant reduction in mitochondrial fluorescent intensity signals following MPs treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eG). Additionally, mitochondrial transcription factor A (mtTFA), which is critical for regulating mitochondria biogenesis and mtDNA transcription, was significantly downregulated in MPs-treated oocytes, suggesting compromised mitochondrial function (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eH).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eInterestingly, MPs exposure also disrupted mitochondrial fission-fusion process in oocytes, as evidenced by increased DRP1 expression and reduced MFN1 protein level (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eI-J). Since mitophagy, a specialized form of autophagy, mediates the removal of dysfunctional mitochondria to maintain mitochondrial quantity and quality, we further investigated whether MPs exposure activated mitophagy in oocytes. As expected, MPs treatment significantly upregulated PINK1 protein level (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eK), a key marker of mitophagy. In line with \u003cem\u003ein vivo\u003c/em\u003e experiment, MPs exposure also induced autophagy in oocyte, as evidenced by increased lysosome cluster foci and LC3β expression level (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eL-M). Therefore, these findings indicated that MPs exposure impaired oocyte quality by disrupting mitochondrial function.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.6 Metabolomic profiling identifies decreased arginine levels in ovaries of MPs-exposed mice\u003c/h2\u003e \u003cp\u003eTo examine the effects of MPs exposure on ovarian metabolism, we performed a metabolomics analysis using liquid chromatography-tandem mass spectrometry (LC-MS/MS) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). OPLS-DA model presented that the six biological replicates within each group were closely clustered, while a distinct separation was evident between the control and MPs-treated ovaries (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). The heatmap generated by hierarchical clustering reveals significant differences in the ovarian metabolome between the MPs-treated group and the control group, indicating a distinct metabolite profile (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). Furthermore, based on the criteria of VIP \u0026gt; 1 and P-value \u0026lt; 0.05, volcano plot analysis showed that 345 annotated metabolites exhibited significant alterations following microplastic treatment, including 185 upregulated and 160 downregulated metabolites (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). To explore the potential impacts of these differential metabolites, KEGG pathway enrichment analysis was performed. The results indicated that pathways related to mitochondrial energy metabolism (Citrate cycle, Pentose phosphate pathway, Pyruvate metabolism), inflammation-related pathways (Arachidonic acid metabolism, Histidine metabolism), and those involved in Biosynthesis of cofactors, Tryptophan metabolism, Arginine and Proline metabolism may be implicated in the effects of MPs (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). Additionally, Metabolite Set Enrichment Analysis (MSEA) revealed the presence of relevant pathways such as Histidine metabolism, Tryptophan metabolism, Pentose and glucuronate interconversions, Arginine biosynthesis, and Glutathione metabolism, suggesting that MPs exposure led to significant changes in the levels of numerous metabolites in the ovary (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). Notably, we observed that arginine, a non-essential amino acid involved in various cellular processes, was downregulated as a result of MPs exposure, and this was present in multiple enriched pathways, including Arginine and Proline metabolism and D-Amino acid metabolism (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e5\u003c/span\u003eG). Similarly, the violin plot obtained from the metabolomics analysis shows a significant reduction in arginine levels in the ovaries of the MPs-treated group (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e5\u003c/span\u003eH).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further investigate the correlation between the transcriptomic and metabolomic profiles, we constructed a co-expression network and generated a nine-quadrant plot to reveal the relationships between genes and metabolites (Fig \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eA). Based on the Pearson correlation coefficient and P-value, a clustering heatmap of differentially expressed genes (DEGs) and differentially altered metabolites was generated. The results showed that arginine was positively correlated with \u003cem\u003eWnt2\u003c/em\u003e, \u003cem\u003eMcmdc2\u003c/em\u003e, \u003cem\u003eKhdrbs2\u003c/em\u003e, \u003cem\u003eGulo\u003c/em\u003e, and \u003cem\u003eCtse\u003c/em\u003e, and negatively correlated with \u003cem\u003eA2ml1\u003c/em\u003e, \u003cem\u003eRnf128\u003c/em\u003e, and \u003cem\u003eSlc6a17\u003c/em\u003e. Additionally, 6-Phosphogluconic acid exhibited positive correlations with \u003cem\u003eRubcnl\u003c/em\u003e, \u003cem\u003eCyp4f18\u003c/em\u003e, and \u003cem\u003eGal3st2c\u003c/em\u003e, and negative correlations with \u003cem\u003eH2ac10\u003c/em\u003e and \u003cem\u003eSlc39a2\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e5\u003c/span\u003eI). Furthermore, we mapped the DEGs and differentially altered metabolites to the KEGG database and identified common pathways, including the Pentose phosphate pathway, Glutathione metabolism, Arachidonic acid metabolism, and others. Notably, pathways such as Arginine biosynthesis and Arginine and Proline metabolism were also included (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e5\u003c/span\u003eJ). These results collectively indicate that arginine may serve as a potential key metabolite involved in ovarian damage induced by MPs exposure.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.7 MPs exposure induces intestinal arginine depletion and transport dysfunction in mice\u003c/h2\u003e \u003cp\u003eThe type and composition of gut microbiota play a crucial role in the metabolic activity of the host. Therefore, we assessed the general profiles of the gut microbiota through 16S rDNA sequencing of fecal samples from both groups. In the analysis of microbial diversity, the abundance-based coverage estimator (ACE) index was used to measure the number of microbial species, while the Shannon index reflected the microbial richness; the Simpson index was used to describe the microbial diversity in the samples. Mice fed MPs exhibited a trend toward lower bacterial abundance and diversity at the ASVs level, although no significant difference was observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). In beta diversity, Principal Coordinate Analysis (PCoA) revealed that MPs altered the overall structural composition of the gut microbiota (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). The taxonomic composition of the gut microbiomes at each level was further analyzed. At the phylum level, MPs increased the relative abundance of Proteobacteria and decreased the relative abundance of Actinobacteria (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). At the class level, MPs increased the relative percentage of Deltaproteobacteria and decreased the relative percentage of Clostridia and Bacteroidia (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). Similarly, at the order level, the MPs group exhibited an increased content of Desulfovibrionales and a decrease in Bacteroidales and Coriobacteriales, while at the family level, Desulfovibrionaceae was the most affected bacteria by MPs (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). Additionally, based on the Lefse cladogram, 35 different bacterial taxa at various taxonomic levels were identified as differential microbiota from phylum to genus (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). Interestingly, Proteobacteria was recognized as a characteristic differential microbiota, which has been shown to significantly consume arginine, thus affecting the host's gut microbiota\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. In Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e6\u003c/span\u003eE, we provide examples of the differential microbiota between groups, where Desulfovibrio, significantly more abundant in MPs-exposed mice, belongs to the Proteobacteria phylum. Furthermore, the beneficial bacteria Lactobacillus and Adlercreutzia showed a decreasing trend following MPs exposure. Correspondingly, MPs exposure led to abnormal changes in the levels of inflammatory cytokine proteins, including TNFα and IL10, in the gut (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e6\u003c/span\u003eF). RT-qPCR results confirmed the elevation of inflammation levels in the small intestine (Fig.\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003eS3\u003c/span\u003eB). Furthermore, the reduction in protein and mRNA levels of ZO-1 and Occudin, along with the abnormal changes in arginine-related transporters, indicated that the intestinal mucosal arrier was disrupted and arginine absorption was affected (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e6\u003c/span\u003eG-H\u0026amp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003eS3\u003c/span\u003eC). These results collectively suggest that MPs exposure induces dysbiosis in the gut microbiota, which in turn impacts the metabolic state of the host.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e3.8 L-arginine alleviated MPs-induced premature ovarian aging in mice\u003c/h2\u003e \u003cp\u003eTo investigate whether MPs-induced premature ovarian aging is associated with L-arginine, dietary supplementation with L-arginine via drinking water was administered. The results showed that L-arginine supplement significantly restored follicles development, as evidenced by the reduced atretic follicle (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e7\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further evaluate whether L-arginine could alleviate MPs-induced ovarian aging, western blot analysis showed that L-arginine significantly decreased the protein levels of P21 and P16 (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e7\u003c/span\u003eB), key markers of cellular senescence. Similarly, Masson’s staining demonstrated a marked reduction in collagen fiber contents in MPs-exposed ovaries following L-arginine treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e7\u003c/span\u003eC). Additionally, L-arginine restored the mRNA expression levels of chemokines such as \u003cem\u003eTGFβ\u003c/em\u003e, \u003cem\u003eIFNγ\u003c/em\u003e, \u003cem\u003eCXCL1\u003c/em\u003e, and \u003cem\u003eCXCL3\u003c/em\u003e compared to the MPs-treated group (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e7\u003c/span\u003eD), suggesting alleviation of MPs-induced ovarian aging. L-arginine also mitigated ovarian inflammation, as evidenced by reduced protein expression of inflammatory markers, such as TNFα, IL-1α and NLRP3 in MPs-exposed ovaries (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e7\u003c/span\u003eE). Given the critical roles of SIRT1/FOXO1 signal pathway in maintaining ovarian function, we accessed the protein expression of SIRT1 and FOXO1. L-arginine supplementation remarkably increased their expression levels in MPs-exposed ovaries (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e7\u003c/span\u003eF). Collectively, the results demonstrated that L-arginine supplementation effectively restored ovarian function in MPs-treated mice.\u003c/p\u003e \u003cp\u003eWe also examined the effects of L-arginine on oocyte quality. L-arginine supplementation partially rescued meiotic progression, as shown by improvements in GVBD and PBE (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e7\u003c/span\u003eG). Consistently, \u003cem\u003ein vitro\u003c/em\u003e supplementation with L-arginine also partially rescued MPs-induced reductions in PBE(Fig.\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003eS4\u003c/span\u003eB). Furthermore, L-arginine significantly reduced ROS levels and improved mitochondrial membrane potential in MPs-treated oocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e7\u003c/span\u003eH). Immunofluorescence staining revealed that L-arginine supplementation corrected MPs-induced spindle assembly defects and chromosomes misalignment (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e7\u003c/span\u003eI). These observations indicated that L-arginine alleviates MPs-induced meiotic defects during oocyte maturation by enhancing mitochondrial function.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eOvarian aging is a natural process associated with biological aging. however, its prevalence has increased in recent decades due to genetic and environmental factors, severely impacting female\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Studies have shown that exposure to environmental factors, such as bisphenol A and phthalates, is strongly linked to accelerated ovarian aging, leading to diminished ovarian reserve, reduced oocyte quality, and impaired folliculogenesis\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. MPs have been shown to have toxic effects on multiple organs and systems, including the digestive, nervous, reproductive, and cardiovascular systems\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. In the present study, we observed that MPs could penetrate reproductive tissue barriers and accumulated in the ovaries, consistent with previous findings\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Notably, our results demonstrated that MPs exposure is a potential inducer of ovarian aging in mice, as evidenced by elevated expression of senescence markers (p16 and p21), and enhanced secretion of senescence-associated secretory phenotype (SASP) factors. Furthermore, MPs exposure reduced the number of antral and secondary follicles while increasing atretic follicles. Consistently with earlier studies, MPs exposure compromised oocyte quality, indicated by impaired oocyte maturation, reduced embryonic developmental competence, and disrupted gap junction communication between oocytes and cumulus cells\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. These findings highlight the detrimental effects of MPs on ovarian function, suggesting that MPs exposure is a significant risk factor for premature ovarian aging.\u003c/p\u003e\u003cp\u003eTo uncover the molecular mechanisms underlying MPs-induced ovarian dysfunction, we performed transcriptomic sequencing of MPs-exposed ovaries. The analysis revealed substantial alterations in gene expression, with a significant enrichment of genes related to inflammation and mitochondrial function. Previous studies have shown that MPs exert their harmful effects primarily through mechanisms such as inflammatory damage, oxidative stress, metabolic disruption, and immunotoxicity\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. Chronic inflammation resulting from the immune system's inability to eliminate MPs may increase the risk of disease\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. For example, MPs exposure has been shown to induce inflammatory damage in the lungs, characterized by increased secretion of inflammatory cytokines and neutrophil recruitment\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Similarly, in fish, microplastics exposure has been linked to immunotoxicity, oxidative stress, genotoxicity, DNA damage, and reduced growth and reproductive quality\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. Based on these findings and our sequencing data, we hypothesized that MPs exposure induces inflammatory damage in the ovaries. Indeed, our results demonstrated that MPs exposure significantly activated the NLRP3 inflammasome and altered the expression of inflammation-related proteins, including IL-10, IL-1α and TNFα. Previous studies have shown that NLRP3 activation and IL-1 secretion are closely associated with ovarian aging, and deletion of these genes can delay ovarian aging and preserve fertility in mice by reducing proinflammatory cytokines and apoptotic signaling \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e,\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. These results suggested that MPs-induced ovarian aging might be related with the constantly activation of NLRP3 and IL1β. Mitochondrial dysfunction is another hallmark of ovarian aging. Targeting mitochondrial dysfunction through therapeutic approaches, such as antioxidants, metabolic improvement, biogenesis promotion, mitophagy enhancement or mitochondrial replacement can efficiently mitigate aged-related ovarian dysfunction and improve oocyte quality\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e Our RNA sequencing data, supported by GO and KEGG analyses, revealed that MPs exposure impaired mitochondrial function. Specifically, MPs exposure induced oxidative stress and disrupted mitochondria dynamics, as evidenced by altered expression of proteins involved in mitochondrial fission/fusion (DRP1, MFN1) and mitophagy (PINK1, Parkin, LC3β) in ovaries and oocytes. There findings suggested that MPs-induced mitochondrial dysfunction contributes to ovarian aging in mice.\u003c/p\u003e\u003cp\u003eMetabolic disorder has been reported to play a critical role in ovarian aging and infertility\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. Numerous metabolisms undergo significant changes during ovarian aging. For examine, NAD+, a coenzyme involved in redox reactions, has been identified as a key metabolite in aging across various tissues\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. Supplementation with NAD + or its precursor NMN has been shown to alleviate aging-induced ovarian dysfunction and improve oocyte quality, thereby enhancing fertility, suggesting potential of metabolic factors as therapeutic or preventative targets for aging-related disease\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. We thus performed untargeted metabolomics to examine the metabolic changes associated with MPs exposure. Our analysis revealed a significant reduction in L-arginine levels in ovaries from MPs-treated mice. L-arginine, a versatile amino acid and a central intestinal metabolite in mammals, serves as a precursor of multiple metabolic pathways involved in regulating of cell division and growth\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. Further investigation revealed that the decreased ovarian L-arginine levels were associated with gut microbiota dysbiosis and impaired intestinal epithelial cell transport capacity. Consistently, RNA-seq analysis confirmed that L-arginine synthesis was disrupted in the ovaries of MPs-treated mice.\u003c/p\u003e\u003cp\u003ePrevious study showed that L-arginine supplementation can prevent diabetic cardiomyopathy by improving mitochondrial fitness and homeostasis\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. This suggests that the mitochondrial dysfunction induced by MPs exposure may be associated with the decline in L-arginine levels in ovarian tissue. Additionally, Arginine pathway metabolites are dysregulated in aging\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e, and targeting L-arginine metabolism has been proposed as a promising therapeutic strategy to maintain the intestinal homeostasis and cognitive function during aging\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. Furthermore, supplementation with L-arginine has been reported to improve oocyte maturation and embryo development rates in cattle\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. In line with these findings, our study demonstrated that L-arginine supplementation significantly alleviated MPs-induced ovarian aging. This effect was particularly evident in its ability to improve mitochondrial function and reduce inflammatory damage, thereby promoting oocyte maturation both in vitro and in vivo. These results suggest that L-arginine is a critical metabolite for regulating reproductive health, especially in the context of MPs-induced ovarian dysfunction.\u003c/p\u003e\u003cp\u003eIn conclusion, this study provides a comprehensive analysis of the toxic effects of MPs on reproductive health, demonstrating that MPs exposure induces ovarian toxicity in mice. This is characterized by hallmarks of ovarian aging, including increased atretic follicles, fibrosis, inflammatory damage, mitochondrial dysfunction, and reduced oocyte quality. Through multi-omics analysis, we identified L-arginine as a key metabolite for preserving ovarian function and oocyte quality during MPs-induced ovarian aging. Notably, L-arginine supplementation alleviated the adverse effects of MPs exposure by improving mitochondrial function, reducing inflammation, and promoting oocyte maturation. These findings provide important clinical insights into the potential application of L-arginine as a therapeutic strategy to mitigate female reproductive disorders caused by MPs exposure.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Dr. Xing Duan\u0026rsquo;s laboratory member for discussions and constructive suggestions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eX.D. designed the experiments and supervised the study. LS.L, WJ.L and QY.X performed all experiments and analyzed the data. LS.L, TT.D and XL.L contributed the data collection. LS.L, JY.N, JX.Z and X.Z contributed to images processing and discussion. X.D. wrote the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest statement\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research is supported by the Fundamental Research Funds for the Central Universities (4025002407).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that all the original data related to the figures and supplementary materials published in this article are available upon rationale request to the corresponding author.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSen, G. 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However, whether microplastics (MPs) exposure poses a potential factor in inducing ovarian aging and the underlying mechanisms remain poorly understood. In this study, we demonstrated that MPs exposure accelerates ovarian aging, characterized by elevated expression of senescence markers (p16 and p21) and increased fibrosis. Mechanistic analysis indicated that MPs exposure led to ovarian inflammatory damage and mitochondrial dysfunction, which contribute to reduced ovarian reserve. Furthermore, MPs exposure compromised oocyte maturation competence and embryo development by triggering mitochondrial dysfunction and DNA damage. Multi-omics analysis identified L-arginine as a crucial metabolite that protects oocytes from MPs-induced aging. Notably, L-arginine supplementation significantly mitigated the aging phenotypes induced by MPs, as evidenced by reduced ovarian fibrosis, inflammatory damage, and mitochondrial dysfunction, alongside enhanced oocyte quality. These findings provide theoretical support for reproductive protection against MPs and inform environmental risk assessments.\u003c/p\u003e","manuscriptTitle":"Microplastics exposure causes premature ovarian aging via inducing mitochondrial dysfunction","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-23 06:07:01","doi":"10.21203/rs.3.rs-6298826/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-aging","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"nataging","sideBox":"Learn more about [Nature Aging](https://www.nature.com/nataging/)","snPcode":"","submissionUrl":"","title":"Nature Aging","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Research","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"89b4d066-6ab2-4e13-887f-a0f1f6452088","owner":[],"postedDate":"April 23rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":47519429,"name":"Biological sciences/Cell biology/Senescence"},{"id":47519430,"name":"Biological sciences/Developmental biology/Germline development"},{"id":47519431,"name":"Health sciences/Diseases/Reproductive disorders"}],"tags":[],"updatedAt":"2025-07-16T08:52:20+00:00","versionOfRecord":[],"versionCreatedAt":"2025-04-23 06:07:01","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6298826","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6298826","identity":"rs-6298826","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}