Spermidine supplementation protects porcine oocytes from triclosan-induced meiotic and fertilization defects by attenuating oxidative stress-mediated apoptosis

Spermidine supplementation protects porcine oocytes from triclosan-induced meiotic and fertilization defects by attenuating oxidative stress-mediated apoptosis | 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 Research Article Spermidine supplementation protects porcine oocytes from triclosan-induced meiotic and fertilization defects by attenuating oxidative stress-mediated apoptosis Yang Gao, Dandan Zhang, Kaixiang Tan, Mengting Wu, Qixiang Tai, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7023955/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Oocytes are highly susceptible to environmental pollutants, with triclosan (TCS)—a pervasive antimicrobial—known to bioaccumulate and impair reproductive function. However, mechanisms driving TCS-induced oocyte degeneration and effective protective approaches remain unclear. This study investigates the protective effects of spermidine against TCS-induced meiotic disruption and fertilization defects in porcine oocytes, exploring its antioxidant and anti-apoptotic mechanisms. Porcine germinal vesicle (GV) oocytes were exposed in vitro to graded TCS concentrations (0.5–5 μM) with or without spermidine supplementation. Meiotic maturation, reactive oxygen species (ROS) production, DNA damage, mitochondrial function, apoptosis, and fertilization competence were assessed using immunofluorescence, fluorescence quantification, and mitochondrial distribution analyses. TCS exposure disrupted meiotic progression, causing spindle defects, chromosome misalignment, mitochondrial dysfunction, elevated ROS, DNA damage, and apoptosis, reducing maturation and fertilization rates. Spermidine significantly reversed these effects by stabilizing cytoskeletal architecture, lowering oxidative stress, and inhibiting apoptosis, thereby improving oocyte quality and developmental competence. Spermidine effectively attenuates TCS-induced meiotic and fertilization impairments by mitigating oxidative stress-mediated apoptosis, offering promising intervention strategies to preserve oocyte quality under environmental toxicant exposure. Spermidine Triclosan Porcine oocytes Oxidative stress Apoptosis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction With the rapid acceleration of industrialization and urbanization, a vast array of exogenous chemical pollutants is continuously released and widely distributed in aquatic systems, soils, and biological organisms, increasingly threatening human health. Oocytes, as critical and non-renewable cells within the female reproductive system, are highly susceptible to environmental toxicants (He et al., 2023 ). Emerging evidence suggests that even low-dose exposure to environmental pollutants can disrupt oocyte meiotic progression, cytoplasmic maturation, and energy metabolism (H. Zhang et al., 2020 ), ultimately compromising developmental competence and fertility (Y. Wang et al., 2023 ). Triclosan (TCS) is a broad-spectrum synthetic phenolic antimicrobial agent widely used since the 1970s due to its potent antibacterial properties. It is commonly incorporated into products such as toothpaste, soaps, cosmetics, plastics, and medical devices (Howse et al., 2019 ; Machado et al., 2019 ; Sinicropi et al., 2022 ). Owing to its lipophilicity and chemical stability, TCS is persistent in the environment and resistant to rapid degradation, resulting in its widespread presence in water, soil, and biota (Jiang et al., 2024 ; Dar et al., 2022 ). Extensive monitoring studies have detected TCS in human blood, urine, breast milk, and cord blood, indicating significant bioaccumulation potential and chronic exposure risk via water sources and food chains (Arnot et al., 2018 ; Luan et al., 2023 ; D. Zhang & Lu, 2023 ). Animal studies confirm that TCS accumulates in adipose tissue and reproductive organs, potentially disrupting hormone levels and metabolic homeostasis (Li et al., 2023 ; Du et al., 2021 ). Both in vivo and in vitro evidence demonstrate that TCS exerts multi-level reproductive toxicity. In male models, TCS exposure causes testicular structural abnormalities, declines in sperm quantity and quality, and androgen disruption (Adegbola et al., 2024 ). Although data on female reproductive toxicity remain limited, emerging studies suggest that TCS interferes with estrogen receptor signaling, impairing ovarian function and oocyte development (Gan et al., 2024 ). Oxidative stress induced by TCS, characterized by elevated reactive oxygen species (ROS), decreased mitochondrial membrane potential, and increased apoptosis, is considered a principal mechanism underlying its reproductive toxicity (Kosińska & Szychowski, 2024 ). Given the widespread use and environmental persistence of TCS, concerns about its ecological and human health risks are growing, especially regarding long-term exposure during pregnancy or reproductive age, which may affect embryonic development and reproductive system function via maternal-fetal transfer (Bai et al., 2020 ). Consequently, multiple countries and regions have started restricting or banning certain TCS-containing products to mitigate potential public health hazards. Despite reports on TCS effects on male reproductive health, its comprehensive toxicological mechanisms impacting oocyte maturation, mitochondrial homeostasis, and apoptosis remain unclear, and effective protective strategies are scarce. Spermidine is a naturally occurring polyamine involved in key physiological processes including cell proliferation, antioxidant stress response (Shi et al., 2022 ), autophagy activation, and regulation of cell death (Yuan et al., 2021 ). Increasing evidence demonstrates that spermidine exerts significant cytoprotective effects by scavenging reactive oxygen species (ROS), stabilizing mitochondrial function, and modulating the autophagy-apoptosis signaling axis (L. Wang et al., 2025 ; Madeo et al., 2018 ). In reproductive biology, spermidine has been shown to maintain ovarian reserve function, enhance oocyte developmental competence, and alleviate ovarian aging (Y. Zhang et al., 2023 ). Mechanistically, these effects are primarily mediated through regulation of intracellular redox balance and promotion of autophagy to clear damaged cellular components, thereby preserving cellular homeostasis (Hofer et al., 2022 ). Furthermore, spermidine protects germ cells from diverse internal and external environmental stresses by inhibiting inflammatory responses and apoptosis (Liu et al., 2020 ; Han et al., 2022 ). However, systematic studies investigating whether spermidine can mitigate oocyte toxicity induced by environmental pollutants, particularly triclosan (TCS), remain lacking. Addressing this knowledge gap is critical for advancing protective strategies to safeguard reproductive cells and reproductive health under environmental pollutant exposure. Programmed cell death (apoptosis) is central to the oocyte’s response to environmental stress (Chesnokov et al., 2024 ). Studies have demonstrated that mitochondrial dysfunction and oxidative stress induced by environmental pollutants can activate the intrinsic apoptotic pathway (Kong et al., 2025 ), involving BAX activation, cytochrome c release, and caspase cascade (Sun et al., 2022 ), leading to oocyte maturation failure and fertilization defects (Stringer et al., 2023 ). During metaphase II (MII) of meiosis, even subtle elevations in apoptotic signaling can cause loss of mitochondrial membrane potential (Bock & Riley, 2023 ), impaired ATP production (Estudillo et al., 2021 ), and dysregulated cortical reaction (Sagvekar et al., 2019 ), thereby compromising subsequent embryonic development (Pei et al., 2023 ). Notably, TCS has been reported to induce apoptosis by disrupting the Bcl-2/BAX ratio and activating caspase-3 (W. Wang et al., 2024 ). However, experimental evidence addressing whether targeted modulation of apoptotic networks can reverse TCS-induced oocyte toxicity is still lacking (Park et al., 2020 ). Therefore, using porcine oocytes as a model to simulate environmental TCS exposure, this study systematically evaluates its effects on meiotic progression, mitochondrial function, oxidative stress, DNA damage, and apoptosis. Furthermore, we investigate whether spermidine supplementation can alleviate these toxic effects through antioxidative and cellular homeostasis mechanisms. Our findings aim to provide novel insights into the mechanistic basis of TCS-induced oocyte toxicity and establish a theoretical foundation for developing small-molecule-based reproductive protection strategies and potential therapeutic targets. Materials and Method Chemicals and animals All chemicals and reagents used in this study were purchased from Sigma-Aldrich (St. Louis, MO, USA), unless otherwise stated. Porcine ovaries were obtained from discarded samples of prepubertal gilts (6 months old, ~100 kg body weight) at licensed slaughterhouses. No live animals were sacrificed specifically for this study. All procedures involving the collection, handling, and use of animal tissues were conducted in strict accordance with institutional guidelines for animal care and use, as well as internationally recognized animal welfare standards. All animal procedures were approved by the Animal Care and Use Committee of Anhui Agricultural University (IACUC approval number: AHAU20208025) and conducted in strict accordance with the Guide for the Care and Use of Laboratory Animals to ensure animal welfare and humane endpoints. Compound Preparation and Experimental Design Triclosan (TCS; Cat# PHR1338, Sigma-Aldrich, USA) and spermidine (SPD; Cat# S2626, Sigma-Aldrich, USA) were each dissolved in anhydrous dimethyl sulfoxide (DMSO) to prepare 100 mM stock solutions, aliquoted and stored at −20°C. Prior to use, TCS stock solutions were diluted in oocyte maturation medium to final working concentrations of 0.5, 1, 2, and 5 μM, while SPD stock was first dissolved in cell culture-grade water and then diluted to 10, 20, and 50 μM. Concentration ranges were selected based on prior literature and preliminary experiments to ensure biological relevance. Porcine oocytes were randomly assigned into three groups during in vitro maturation (IVM): Control (no treatment), TCS exposure (continuous exposure to TCS at specified concentrations), and combined TCS and SPD treatment (same TCS concentrations plus SPD at indicated doses). All groups were cultured for 44 hours at 38.5°C under humidified 5% CO₂ atmosphere. The final DMSO concentration in all culture media was maintained consistently at ≤0.1% (v/v) across all groups to exclude solvent-related effects. Oocyte maturation assessment A total of 1000 cumulus–oocyte complexes (COCs) were collected and subjected to five independent replicates. After 44 hours of in vitro maturation (IVM), cumulus expansion was evaluated based on the morphological characteristics of the surrounding cumulus cells (X. Wang et al., 2023). To assess nuclear maturation, COCs were incubated in Dulbecco’s phosphate-buffered saline (DPBS; Gibco, Grand Island, NY, USA) supplemented with 100 μg/mL penicillin G, 75 μg/mL streptomycin sulfate, and 0.1% hyaluronidase. Following gentle pipetting to remove cumulus cells, denuded oocytes were observed under an inverted microscope (Zeiss, Germany). Oocytes were classified into two categories based on the presence of the first polar body: immature (no polar body) and mature (with first polar body visible). Immunofluorescence Staining To assess the developmental quality of porcine oocytes, samples were fixed and permeabilized at room temperature for 30 minutes in PHEM buffer containing 2% paraformaldehyde and 0.5% Triton X-100 (composition: 60 mM PIPES, 25 mM HEPES, 10 mM EGTA, 4 mM MgSO₄, pH 6.9). After fixation, oocytes were washed three times in PBS containing 0.1% Tween-20, 5 minutes per wash. Subsequently, samples were blocked overnight at 4°C with 2% bovine serum albumin (BSA). Oocytes were then incubated with anti–α-tubulin primary antibody (1:200, diluted in PBS containing 2% BSA and 0.1% Tween-20) overnight at 4°C. On the following day, Alexa Fluor 488–conjugated secondary antibody (1:1000) was applied at room temperature for 1 hour in the dark. Nuclei were counterstained with Hoechst 33342 for 10 minutes, followed by washing and mounting. Fluorescence images were captured using a laser scanning confocal microscope. Measurement of Reactive Oxygen Species (ROS) Levels To quantify intracellular ROS levels in porcine oocytes, oocytes were incubated in Dulbecco’s PBS containing 10 μM DCFH-DA probe (Beyotime, China) at 37 °C in 5% CO₂ for 30 minutes in the dark. After incubation, the cells were gently washed three times (5 minutes each) with PBS containing 0.1% BSA to remove unbound probes. The oocytes were then transferred onto glass slides and imaged using a Zeiss LSM 700 META confocal microscope. Imaging parameters, including excitation wavelength, laser power, and gain, were kept constant to ensure comparability. The mean fluorescence intensity within defined regions of interest (ROIs) was measured using ImageJ software to reflect relative ROS levels. Annexin-V Staining Apoptosis in porcine oocytes was detected using an Annexin-V-FITC staining kit (Beyotime, China). Oocytes were washed twice with PBS at room temperature (5 minutes each), then incubated in the dark for 30 minutes in a staining solution containing 10 μL Annexin-V-FITC and 90 μL binding buffer. After staining, the oocytes were gently washed with PBS and transferred to glass-bottom dishes for imaging using a Zeiss LSM 700 META confocal microscope. Imaging parameters were standardized across all samples. Fluorescence signals from Annexin-V-FITC were used to evaluate early-stage apoptosis in the oocytes. Sperm-binding assay Mature porcine oocytes were obtained through in vitro maturation (IVM), while sperm were purified from thawed semen using the swim-up method. Oocytes and sperm were co-incubated at a 1:1 ratio for 1 hour at 38.5°C in an atmosphere containing 5% CO₂ to facilitate sperm binding. Following incubation, oocytes were gently washed three times (5 minutes each) with PBS containing 0.1% BSA to remove unbound sperm. The oocytes were then stained with Hoechst 33342 for 10 minutes, washed, and transferred to glass-bottom culture dishes for imaging. Fluorescence images were captured using a Zeiss LSM 700 META confocal microscope under identical imaging settings for all samples. The number of sperm bound to each oocyte was counted to evaluate sperm-binding capacity. Image Acquisition and Analysis Tissue sections and oocyte samples were stained under the same immunofluorescence conditions and imaged on the same day using a Zeiss laser scanning confocal microscope (LSM 700, Zeiss, Germany). To reduce batch effects, imaging parameters including laser power, scan speed, pinhole size, gain, and offset were kept constant. Images were exported in TIFF format and analyzed with ImageJ software (v1.53o, NIH, USA). Background fluorescence was removed by applying a consistent threshold. Regions of interest (ROIs), such as the cytoplasm or nucleus of each oocyte or selected tissue area, were defined using the “ROI Manager” tool. Total fluorescence intensity within each ROI was measured and normalized to the area to calculate the average fluorescence intensity (mean gray value/μm²). At least 20 oocytes or equivalent tissue ROIs per group were analyzed, and the average value was used for statistical comparison. Statistical Analysis All experiments were independently repeated at least three times, with the number of porcine oocytes indicated in the figure legends. Due to limited sample size, oocytes from multiple experiments were combined for proportion analysis. Differences between groups were compared using Fisher’s exact test, and proportions are presented as standard error of the mean (SEM). Statistical analyses were performed using GraphPad Prism version 9.4.1. Details of tests and significance are provided in the figure legends. Results Spermidine supplementation mitigates triclosan-induced meiotic failure and cumulus expansion impairment in porcine oocytes Given that the environmental pollutant triclosan (TCS) may impair oocyte maturation competence, we first added various concentrations of TCS (0.5, 1, 2, and 5 µM) to the in vitro maturation (IVM) medium to evaluate its effects on porcine oocyte meiotic progression. As shown in Fig. 1 A, the control cumulus–oocyte complexes (COCs) exhibited robust cumulus cell expansion, indicative of normal oocyte development. In contrast, COCs in the TCS-treated groups showed varying degrees of impaired cumulus expansion, with more pronounced inhibition at higher concentrations. To further confirm whether TCS disrupts oocyte meiotic maturation, we quantified the polar body extrusion (PBE) rate. TCS exposure significantly decreased PBE in a dose-dependent manner (Fig. 1 B). Specifically, 2 µM and 5 µM TCS treatments markedly reduced the PBE rate (Control: 82.0 ± 1.2%, n = 144; 0.5 µM: 80.1 ± 1.9%, n = 141, P > 0.05; 1 µM: 77.1 ± 1.1%, n = 140, P < 0.1; 2 µM: 57.7 ± 3.4%, n = 142, P < 0.001; 5 µM: 57.1 ± 3.4%, n = 142, P < 0.001). Based on these results, 2 µM was selected for subsequent experiments. We next examined whether spermidine (SPD), an autophagy-regulating factor, could alleviate TCS-induced oocyte maturation defects. Different concentrations of SPD (10, 20, or 50 µM) were supplemented into the IVM system containing 2 µM TCS. As shown in Fig. 1 C, SPD supplementation significantly improved PBE rates, with the most pronounced rescue observed at 50 µM (Control: 80.8 ± 2.8%, n = 118, P 0.05; TCS + 20 µM SPD: 67.2 ± 1.5%, n = 119, P < 0.1; TCS + 50 µM SPD: 76.0 ± 1.1%, n = 117, P < 0.001). Furthermore, quantitative analysis of the cumulus expansion area revealed that SPD supplementation markedly alleviated TCS-induced defects in cumulus cell expansion (Control: 7643.0 ± 581.4 µm², n = 65; TCS: 2480.0 ± 133.5 µm², n = 88, P < 0.001; TCS + SPD: 4237.0 ± 192.3 µm², n = 67, P < 0.01; Fig. 1 D). Taken together, these findings suggest that SPD exerts a protective effect against TCS-induced meiotic arrest and cumulus expansion defects in porcine oocytes. Spermidine ameliorates triclosan-induced defects in spindle assembly and chromosome organization during porcine oocyte meiosis To investigate the effects of triclosan (TCS) exposure on porcine oocyte meiotic maturation and the potential protective mechanisms of spermidine (SPD), we first examined spindle morphology and chromosome alignment in MII-stage oocytes. As shown in Fig. 2 A, oocytes from the control group exhibited typical barrel-shaped spindles with well-aligned chromosomes at the metaphase plate. In contrast, oocytes exposed to TCS showed pronounced spindle disorganization, disrupted microtubule aggregation, and increased frequencies of chromosome misalignment and abnormal clustering. Quantitative analysis revealed that the incidence of spindle defects was significantly elevated in the TCS group compared to controls (Control: 19.8 ± 1.0%, n = 106; TCS: 62.3 ± 1.7%, n = 103; P < 0.001) (Fig. 2 B), accompanied by a significant increase in chromosome misalignment (Control: 20.8 ± 0.7%; TCS: 63.2 ± 1.7%; P < 0.001) (Fig. 2 C). Notably, supplementation with SPD markedly attenuated these abnormalities, reducing the rates of spindle and chromosome defects to 29.4 ± 0.6% and 28.4 ± 0.7%, respectively (n = 102; P < 0.001), suggesting that SPD can effectively mitigate TCS-induced meiotic impairments. Given that microtubule stability is critical for proper spindle assembly, we further assessed the levels of acetylated α-tubulin as a marker of microtubule integrity. TCS exposure significantly decreased the fluorescence intensity of acetylated α-tubulin (Control: 44.8 ± 0.6, n = 35; TCS: 20.1 ± 0.5, n = 35; P < 0.001), whereas SPD treatment substantially restored the acetylation levels (33.9 ± 0.5, n = 35; P < 0.001) (Fig. 3 A, B). These findings suggest that SPD alleviates TCS-induced meiotic defects by preserving microtubule stability, thereby promoting proper spindle organization and chromosome alignment during oocyte maturation. Spermidine ameliorates triclosan-induced disruption of actin cytoskeleton during porcine oocyte maturation To examine the impact of triclosan (TCS) exposure on the actin cytoskeleton during porcine oocyte meiosis, we performed immunofluorescence staining using phalloidin-TRITC. In control oocytes, actin filaments were strongly and continuously distributed along the plasma membrane and within the cytoplasm. In contrast, oocytes exposed to TCS exhibited markedly reduced and discontinuous actin signals, indicating cytoskeletal disruption (Control: 26.7 ± 0.4, n = 39; TCS: 9.7 ± 0.4, n = 39; P < 0.001). Notably, supplementation with spermidine (SPD) significantly restored actin filament intensity to levels comparable to controls (TCS + SPD: 25.3 ± 0.5, n = 39; P < 0.001) (Fig. 4 A-B). These findings suggest that TCS disrupts actin cytoskeletal dynamics during oocyte maturation, potentially impairing meiotic progression, while SPD supplementation effectively rescues actin integrity and supports proper meiotic development. Spermidine restores mitochondrial function and cortical granule protein ovastacin distribution in triclosan-exposed porcine oocytes Mitochondria are the primary energy source of oocytes, generating substantial ATP to support meiotic maturation. To investigate whether triclosan (TCS) exposure impairs oocyte meiosis via mitochondrial dysfunction, we performed MitoTracker Red staining to assess mitochondrial localization and distribution. Immunofluorescence analysis revealed that TCS exposure markedly altered mitochondrial distribution in porcine oocytes: mitochondria in control oocytes were continuously and evenly distributed along the cortex with strong fluorescence intensity, whereas TCS-treated oocytes exhibited abnormal clustered mitochondrial aggregation with uneven distribution and significantly reduced fluorescence intensity (Fig. 5 A). Quantitative analysis confirmed a significant decrease in mitochondrial fluorescence intensity in the TCS group compared to controls (Control: 40.1 ± 0.6, n = 33; TCS: 5.0 ± 0.3, n = 34; P < 0.001) (note: these values represent fluorescence intensity, not percentages). Spermidine (SPD) supplementation substantially alleviated this defect, partially restoring mitochondrial fluorescence intensity (TCS + SPD: 19.8 ± 0.7, n = 33; P < 0.001) (Fig. 5 B). These findings indicate that TCS exposure induces mitochondrial dysfunction and mislocalization in oocytes, which can be partially rescued by SPD treatment. Furthermore, ovastacin (ASTL), a key enzyme localized in cortical granules, plays a crucial role in preventing polyspermy and ensuring monospermic fertilization. To evaluate ovastacin distribution dynamics, immunofluorescence staining using anti-ASTL antibody was performed. As shown in Fig. 5 C, ovastacin was evenly and continuously distributed in the cortex of control oocytes, whereas TCS exposure caused aberrant localization and significantly reduced fluorescence intensity of ovastacin. SPD supplementation markedly mitigated these defects (Control: 24.5 ± 0.2, n = 34; TCS: 5.2 ± 0.2, n = 34; TCS + SPD: 16.0 ± 0.2, n = 34; P < 0.001) (Fig. 5 D). These results suggest that SPD partially restores TCS-induced ovastacin mislocalization, thereby reducing polyspermy risk and preserving normal fertilization capacity of oocytes. Spermidine supplementation alleviates triclosan-induced impairment of oocyte fertilization and early embryonic development in porcine models To further evaluate the protective effects of spermidine (SPD) against triclosan (TCS)-induced reproductive toxicity, sperm-oocyte binding assays were performed. Sperm heads were labeled with Hoechst 33342, and two-cell stage embryos were used as a negative control to confirm binding specificity. Quantitative analysis revealed a significant reduction in the number of sperm bound to the zona pellucida of oocytes exposed to TCS compared to controls (Control: 126.3 ± 1.6, n = 34; TCS: 48.1 ± 1.5, n = 34; P < 0.001). Notably, SPD co-treatment significantly restored sperm binding capacity (TCS + SPD: 99.1 ± 1.5, n = 34; P < 0.001 vs. TCS alone) (Figs. 6 A, B). Consistently, in vitro fertilization (IVF) results showed that TCS exposure significantly decreased the two-cell embryo formation rate (Control: 54.9 ± 1.4%, n = 104; TCS: 23.6 ± 1.2%, n = 106; P < 0.001), whereas SPD supplementation markedly improved embryonic developmental competence (TCS + SPD: 47.7 ± 1.2%, n = 109; P < 0.001 vs. TCS alone) (Figs. 6 C, D). Collectively, these data demonstrate that SPD effectively mitigates TCS-induced impairment of oocyte fertilization capacity and early embryonic development. Spermidine Supplementation Mitigates Triclosan-Induced Mitochondrial Dysfunction, Oxidative Stress, and Meiotic Failure in Porcine Oocytes Mitochondrial dysfunction often leads to excessive accumulation of reactive oxygen species (ROS), triggering oxidative stress. To clarify the impact of triclosan (TCS) exposure on oxidative stress in oocytes, we performed fluorescence staining with the DCFH-DA probe to quantitatively assess ROS levels. Results showed weak green fluorescence in control oocytes, indicating low ROS levels, whereas TCS-exposed oocytes exhibited significantly enhanced ROS signals (Control: 5.4 ± 0.3, n = 36; TCS: 21.5 ± 0.4, n = 38; P < 0.001). Notably, supplementation with spermidine (SPD) markedly reduced ROS levels, restoring them close to control levels (TCS + SPD: 11.3 ± 0.3, n = 34; P < 0.001) (Figs. 7 A, B). Excessive ROS accumulation can induce DNA double-strand breaks, resulting in DNA damage. To further assess DNA damage, γH2A.X immunofluorescence staining was performed to monitor DNA damage accumulation in oocytes. The TCS-exposed group showed significantly elevated γH2A.X-positive signals compared to controls (Control: 8.9 ± 0.3, n = 35; TCS: 26.6 ± 0.4, n = 33; P < 0.001), while SPD supplementation substantially attenuated this damage signal (TCS + SPD: 12.7 ± 0.4, n = 34; P < 0.001) (Figs. 7 C, D). Persistent DNA damage often triggers cell cycle arrest and apoptosis. Early apoptosis was assessed by Annexin-V staining. Control oocytes exhibited minimal Annexin-V signal on the cell membrane (3.6 ± 0.2, n = 34), whereas TCS exposure significantly increased Annexin-V positivity (18.0 ± 0.3, n = 34; P < 0.001). SPD treatment significantly decreased Annexin-V positive rates (9.0 ± 0.3, n = 34; P < 0.001) (Figs. 7 E, F). Collectively, these results demonstrate that SPD effectively alleviates TCS-induced ROS overaccumulation, DNA damage, and early apoptosis, suggesting its potential protective role against environmental toxicant-induced oocyte injury. Discussion Triclosan (TCS), widely used and persistent in the environment, has raised considerable concern; however, its toxic effects on reproductive cells and underlying mechanisms remain incompletely understood. Accumulation and chronic exposure to environmental TCS may impair reproductive function and threaten offspring health (C. Wang et al., 2018 ). Therefore, elucidating the molecular mechanisms by which TCS disrupts oocyte maturation and identifying effective protective strategies are of critical importance. This study utilized porcine oocytes, which closely resemble human oocytes physiologically, to systematically delineate the mechanisms underlying TCS-induced meiotic disruption and cumulus cell abnormalities. Notably, we provide the first evidence that the natural polyamine spermidine (SPD) markedly alleviates TCS-induced oocyte damage, highlighting its potential application in protecting against environmental reproductive toxicity. We established a porcine oocyte TCS exposure model to systematically assess its toxic effects on meiotic progression and cumulus cell development. To better mimic environmental exposure, a short-term multi-dose regimen (0.5, 1, 2, and 5 µM) was applied to comprehensively evaluate TCS effects on in vitro oocyte maturation. Environmental and biological monitoring studies report TCS concentrations within this range (Park et al., 2020 ), indicating the environmental relevance of our experimental design. Cumulus cell expansion and first polar body extrusion are key indicators of oocyte maturation (Richani et al., 2021 ). Results demonstrated that TCS significantly inhibited cumulus expansion in a dose-dependent manner and markedly reduced polar body extrusion rates at concentrations ≥ 2 µM, suggesting meiotic maturation impairment consistent with previous reports (Zhao et al., 2025 ). Introducing SPD as a potential intervention partially rescued TCS-induced suppression of cumulus expansion and polar body extrusion, implying a protective role during oocyte maturation. At the subcellular level, we systematically investigated the molecular mechanisms of TCS toxicity and the mitigating effects of SPD. SPD significantly ameliorated TCS-induced spindle assembly defects and chromosome misalignment, promoting normal meiotic progression. Additionally, SPD restored TCS-disrupted actin cytoskeleton organization, recovering cortical integrity and continuity. Considering the crucial role of actin dynamics in polar body extrusion, cytoplasmic maturation, and subsequent embryonic development (Pelzer et al., 2023 ), SPD-mediated actin repair further supports its protective potential on oocyte structure and function. Moreover, proper localization of cortical granules and the key enzyme ovastacin is essential for preventing polyspermy and ensuring monospermic fertilization (Zafar et al., 2021 ). We found that TCS exposure markedly disrupted ovastacin distribution in oocytes, likely impairing zona pellucida modification via cortical reaction interference and reducing fertilization competence. SPD supplementation significantly restored ovastacin localization and expression, suggesting protection via cytoskeletal stabilization or modulation of exocytosis. This restoration correlated with improved sperm binding and enhanced early embryonic development post in vitro fertilization, underscoring SPD’s potential to alleviate environmental toxicant-induced oocyte injury. Mechanistically, TCS exposure significantly induced intracellular reactive oxygen species (ROS) accumulation and activated apoptosis-related pathways, disrupting oocyte developmental homeostasis. Immunofluorescence analysis confirmed that TCS markedly increased the DNA damage marker γH2AX, indicating induction of DNA double-strand breaks, a critical factor underlying meiotic failure and oocyte developmental impairment. Conclusions In summary, triclosan (TCS) markedly impairs both nuclear and cytoplasmic maturation of oocytes by inducing oxidative stress and DNA damage. Supplementation with spermidine (SPD) effectively alleviates these toxic effects, promoting spindle assembly, accurate chromosome alignment, and restoration of actin cytoskeleton integrity, thereby significantly enhancing oocyte maturation quality. These findings suggest that SPD holds potential as a protective agent against oocyte developmental toxicity induced by environmental pollutants. This study not only advances our understanding of the toxicological mechanisms underlying environmental pollutant impacts on oocyte quality but also provides a theoretical foundation and practical guidance for developing targeted interventions to safeguard oocyte quality in human assisted reproductive technologies and embryonic engineering. Declarations CRediT authorship contribution statement M.Q.Z., Y.H.Z., and Y.Z.: Conceptualization, Methodology, Supervision. Y.G., D.Z., and K.T.: Investigation, Data curation, Formal analysis. C.Z., M.W., Q.T., G.Z., and J.C.: Software, Validation, Visualization. M.Q.Z., D.Z., and Y.G.: Writing – original draft, Writing – review & editing. We sincerely thank Z. Cui , C. Zhou , and Y. Miao for their valuable support and insightful suggestions, and for kindly providing the antibodies used in this study. Funding This work was supported by the National Key Research and Development Program of China (2023YFD13000502), the National Natural Science Foundation of China (32272881, 32402767), and the Natural Science Foundation of Anhui Province (2308085QC82). Data availability All data supporting the findings of this study are available within the main text or the supplementary materials. Additional data will be made available upon reasonable request. Conflict of interest The authors declare no competing financial interests or personal relationships that could influence the work reported in this paper. Author declaration The authors confirm that all necessary institutional approvals related to ethical use of experimental animals were obtained and are clearly stated in the manuscript. We also confirm compliance with institutional and national regulations regarding intellectual property and publication policies. Consent to publish All authors have reviewed and approved the final version of the manuscript and consent to its submission for publication. References Adegbola, C. A., Akhigbe, T. M., Adeogun, A. E., Tvrdá, E., Pizent, A., & Akhigbe, R. E. (2024). A systematic review and meta-analysis of the impact of triclosan exposure on human semen quality. Frontiers in Toxicology , 6 . https://doi.org/10.3389/ftox.2024.1469340 Arnot, J. A., Pawlowski, S., & Champ, S. (2018). A weight-of-evidence approach for the bioaccumulation assessment of triclosan in aquatic species. 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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-7023955","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":483973255,"identity":"ccb15cba-085e-4386-a698-9b18063b7780","order_by":0,"name":"Yang Gao","email":"","orcid":"","institution":"Hefei Normal University","correspondingAuthor":false,"prefix":"","firstName":"Yang","middleName":"","lastName":"Gao","suffix":""},{"id":483973258,"identity":"6afb9448-ed7f-4ba8-98c3-075eca2cde4f","order_by":1,"name":"Dandan Zhang","email":"","orcid":"","institution":"General Hospital of WanBei Coal Group","correspondingAuthor":false,"prefix":"","firstName":"Dandan","middleName":"","lastName":"Zhang","suffix":""},{"id":483973260,"identity":"3e61e002-1502-478b-92d1-97603ea81d45","order_by":2,"name":"Kaixiang Tan","email":"","orcid":"","institution":"Anhui Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Kaixiang","middleName":"","lastName":"Tan","suffix":""},{"id":483973263,"identity":"e309d3a1-4867-4d4c-a946-59cfbcb17f4a","order_by":3,"name":"Mengting Wu","email":"","orcid":"","institution":"Hefei Normal University","correspondingAuthor":false,"prefix":"","firstName":"Mengting","middleName":"","lastName":"Wu","suffix":""},{"id":483973264,"identity":"afd1a7fc-2962-4b2b-b0e9-92676c15f4f3","order_by":4,"name":"Qixiang Tai","email":"","orcid":"","institution":"Hefei Normal University","correspondingAuthor":false,"prefix":"","firstName":"Qixiang","middleName":"","lastName":"Tai","suffix":""},{"id":483973265,"identity":"c0aeb469-694a-4d8a-b39c-717367b43e8d","order_by":5,"name":"Guilan Zhu","email":"","orcid":"","institution":"Hefei Normal University","correspondingAuthor":false,"prefix":"","firstName":"Guilan","middleName":"","lastName":"Zhu","suffix":""},{"id":483973266,"identity":"cdd2e4b0-a134-49b7-b1cf-1d581e9e13ea","order_by":6,"name":"Jinwu Chen","email":"","orcid":"","institution":"Hefei Normal University","correspondingAuthor":false,"prefix":"","firstName":"Jinwu","middleName":"","lastName":"Chen","suffix":""},{"id":483973267,"identity":"5108a342-753e-4c2e-a022-54a1e5c87f52","order_by":7,"name":"Changyin Zhou","email":"","orcid":"","institution":"Guangdong Second Provincial General Hospital","correspondingAuthor":false,"prefix":"","firstName":"Changyin","middleName":"","lastName":"Zhou","suffix":""},{"id":483973268,"identity":"4d365972-32f0-43cd-ae8e-278fd878e698","order_by":8,"name":"Yong Zhu","email":"","orcid":"","institution":"Hefei Normal University","correspondingAuthor":false,"prefix":"","firstName":"Yong","middleName":"","lastName":"Zhu","suffix":""},{"id":483973269,"identity":"c88c5a2c-2941-43d9-bbb2-75ea209cb54f","order_by":9,"name":"Yunhai Zhang","email":"","orcid":"","institution":"Anhui Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Yunhai","middleName":"","lastName":"Zhang","suffix":""},{"id":483973270,"identity":"ea85b92c-067f-4ddb-8196-572fc406d1e4","order_by":10,"name":"Mianqun Zhang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABAElEQVRIiWNgGAWjYDACCSSS4QPDAWQuEVoYZ5CgBQKYeYjRIj+7+dnDrzkW8ubsOWaPbWruyBscYD54m4fBLg+XFsY5x8yNZbdJGO7seWNunHPsmeGGA2zJ1jwMycW4tDBLJJhJS26TYNxwI8dMOoftMOOGAzxm0kAXJjbg0MImkf4NpMUerMXi32H7DQf4v+HVwiORYyb5cZtEIlgLY9vhRKAtbHi1SEjklEkzbpNI3nDmWZlkb9/h5JmH2Ywt5xgk49QiPyN9m+TPbXW2G44nb5P48e2wbd/x5oc33lTY4dQCDgIeMJUA44IIAzzqgYDxB4qWUTAKRsEoGAVoAABPTlaYAPwYJgAAAABJRU5ErkJggg==","orcid":"","institution":"Anhui Agricultural University","correspondingAuthor":true,"prefix":"","firstName":"Mianqun","middleName":"","lastName":"Zhang","suffix":""}],"badges":[],"createdAt":"2025-07-02 00:53:11","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7023955/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7023955/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":86703158,"identity":"149c4ce1-f12e-4699-a216-815b057d85b9","added_by":"auto","created_at":"2025-07-14 16:40:55","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":19055236,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of SPD supplementation on the in vitro maturation of TCS-exposed porcine oocytes. (A) Representative images of in vitro matured oocytes in control, TCS-exposed and SPD-supplemented oocytes. Cumulus cell expansion of COCs and polar body extrusion of DOs were imaged by the confocal microscope. Scale bars, 350 μm (a-c); 150 μm (d-f); 40 μm (g-i). (B) Polar body extrusion rates of control (n = 144) and different concentrations of TCS-exposed oocytes (0.5 μM: n = 141; 1 μM: n = 140; 2 μM: n = 142; 5 μM: n = 142) after culture for 44 h in vitro. (C) Polar body extrusion rates of control (n = 118), TCS-exposed (n = 114) and different concentrations of SPD-supplemented oocytes (10 μM: n = 114; 20 μM: n = 119; 50 μM: n = 117) after culture with 2 μM TCS for 44 h in vitro. (D) The cumulus expansion area for COCs was recorded in control, TCS-exposed and SPD-supplemented oocytes. Data were presented as mean percentage (mean ± SEM) of at least three independent experiments. *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001, ns, no significance.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-7023955/v1/a532bb4b2dacb8e094abe650.png"},{"id":86703153,"identity":"c0c9fac5-1464-4fd9-b396-fd1ee44be724","added_by":"auto","created_at":"2025-07-14 16:40:55","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":312109,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of SPD supplementation on the spindle/chromosome structure in TCS-exposed porcine oocytes. (A) Representative images of spindle morphologies and chromosome alignment in control, TCS-exposed and SPD-supplemented oocytes. Scale bar, 5 μm. (B) The rate of abnormal spindle morphology in control (n = 106), TCS-exposed (n = 103) and SPD-supplemented (n = 102) oocytes was counted. (C) Statistics of the rate of chromosome misalignment in control (n = 106), TCS-exposed (n = 103) and SPD-supplemented (n = 102) oocytes. Data were presented as mean ± SEM of at least three independent experiments. **P \u0026lt; 0.01, ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-7023955/v1/703b52831ba1cf6646b6b079.png"},{"id":86703156,"identity":"249f1d6a-1e72-432b-b94b-57ae001e0be2","added_by":"auto","created_at":"2025-07-14 16:40:55","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":3958121,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of SPD supplementation on the acetylation level of α-tubulin in TCS-exposed porcine oocytes. (A) Representative images of acetylated α-tubulin in control, TCS-exposed and SPD-supplemented oocytes. Scale bar, 5 μm. (B) Quantitative analysis of the fluorescence intensity of acetylated α-tubulin in control (n = 35), TCS-exposed (n = 35) and SPD-supplemented (n = 35) oocytes. Data were presented as mean ± SEM of at least three independent experiments. ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-7023955/v1/91fa74b0c0d29e924e2116a5.png"},{"id":86703431,"identity":"48a8c4cf-b63a-4ff2-9efe-181081da587a","added_by":"auto","created_at":"2025-07-14 16:48:55","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1316593,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of SPD supplementation on the actin cytoskeleton in TCS-exposed porcine oocytes. (A) Representative images of actin filaments in control, TCS-exposed and SPD-supplemented oocytes. Scale bar, 35 μm. (B) Quantitative analysis of the fluorescence intensity of actin signals on the membrane in control (n = 39), TCS-exposed (n = 39) and SPD-supplemented (n = 39) oocytes. (C) The graphs showed the fluorescence intensity profiling of actin filaments in control, TCS-exposed and SPD-supplemented oocytes. Pixel intensities were measured along the lines which were drawn across the oocytes. Data were presented as mean ± SEM of at least three independent experiments. ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-7023955/v1/55637f7c342179ee5586cb69.png"},{"id":86703432,"identity":"ea01e2dc-dbd4-4b7f-af71-ba9fd770b348","added_by":"auto","created_at":"2025-07-14 16:48:55","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":7954105,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of SPD supplementation on the mitochondrial distribution and dynamics of ovastacin in TCS-exposed porcine oocytes. (A) Representative images of mitochondria in control, TCS-exposed and SPD-supplemented oocytes. Scale bar, 35 μm. (B) Quantitative analysis of the fluorescence intensity of mitochondriain in control (n = 33), TCS-exposed (n = 34) and SPD-supplemented (n = 33) oocytes. (C) Representative images of ovastacin distribution in control, TCS-exposed and SPD-supplemented oocytes. Scale bar, 35 μm. (D) Quantitative analysis of the fluorescence intensity of ovastacin in control (n = 34), TCS-exposed (n = 34) and SPD-supplemented (n = 34) oocytes. Data were presented as mean ± SEM of at least three independent experiments. ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-7023955/v1/701156fd09d6c2abfac9f3ba.png"},{"id":86703157,"identity":"a20fd4b6-649f-4948-b4e7-ef220497c461","added_by":"auto","created_at":"2025-07-14 16:40:55","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1606674,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of SPD supplementation on the sperm binding and fertilization ability in TCS-exposed porcine oocytes. (A) Representative images of sperm binding to the matured oocytes or 2-cell embryos in control, TCS-exposed and SPD-supplemented oocytes. Scale bar, 35 μm. (B) The number of sperm binding to the surface of zona pellucida surrounding control (n = 34), TCS-exposed (n = 34) and SPD-supplemented (n = 34) oocytes was counted. (C) Representative images of 2-cell embryos developed from in vitro fertilized control, TCS-exposed and SPD-supplemented oocytes. Scale bars, 150 μm (d-f). (D) The fertilization rate was recorded in control (n = 104), TCS-exposed (n = 106) and SPD-supplemented (n = 109) oocytes. Data were presented as mean ± SEM of at least three independent experiments. **P \u0026lt; 0.01, ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-7023955/v1/dcd55878b7063824d548bb3a.png"},{"id":86703433,"identity":"2c707016-4ba0-43fb-8f67-f8e88d062901","added_by":"auto","created_at":"2025-07-14 16:48:55","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":466055,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of SPD supplementation on the ROS levels, DNA damage accumulation and occurrence of\u0026nbsp;apoptosis in TCS-exposed porcine oocytes. (A) Representative images of ROS levels in control, TCS-exposed and SPD-supplemented oocytes. Scale bar, 35 μm. (B) Quantitative analysis of the fluorescence intensity of ROS in control (n = 36), TCS-exposed (n = 38) and SPD-supplemented (n = 34) oocytes. (C) Representative images of DNA damage in control, TCS-exposed and SPD-supplemented oocytes. Scale bar, 5 μm. (D) Quantitative analysis of the fluorescence intensity of γH\u003csub\u003e2\u003c/sub\u003eA.X signals in control (n = 35), TCS-exposed (n = 33) and SPD-supplemented (n = 34) oocytes. (E) Representative images of apoptotic oocytes in control, TCS-exposed and SPD-supplemented oocytes. Scale bar, 35 μm. (F) Quantitative analysis of the fluorescence intensity of Annexin-V signals in control (n = 34), TCS-exposed (n = 34) and SPD-supplemented (n = 34) oocytes. Data were presented as mean ± SEM of at least three independent experiments. ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-7023955/v1/085db88593d465d80a3bb479.png"},{"id":87117709,"identity":"f9ca02d0-ddb7-4e4f-96c3-f963b3173a03","added_by":"auto","created_at":"2025-07-20 02:31:43","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":33059167,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7023955/v1/6cced41d-3ab0-4c8e-9110-1772fe7b534c.pdf"},{"id":86703180,"identity":"7e1f5800-33d6-44f1-910e-715ae91a46ad","added_by":"auto","created_at":"2025-07-14 16:40:56","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":66696284,"visible":true,"origin":"","legend":"","description":"","filename":"GraphicalAbstract.tif","url":"https://assets-eu.researchsquare.com/files/rs-7023955/v1/6345b4e325074b494f69e5d2.tif"}],"financialInterests":"No competing interests reported.","formattedTitle":"Spermidine supplementation protects porcine oocytes from triclosan-induced meiotic and fertilization defects by attenuating oxidative stress-mediated apoptosis","fulltext":[{"header":"Introduction","content":"\u003cp\u003eWith the rapid acceleration of industrialization and urbanization, a vast array of exogenous chemical pollutants is continuously released and widely distributed in aquatic systems, soils, and biological organisms, increasingly threatening human health. Oocytes, as critical and non-renewable cells within the female reproductive system, are highly susceptible to environmental toxicants (He et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Emerging evidence suggests that even low-dose exposure to environmental pollutants can disrupt oocyte meiotic progression, cytoplasmic maturation, and energy metabolism (H. Zhang et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), ultimately compromising developmental competence and fertility (Y. Wang et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eTriclosan (TCS) is a broad-spectrum synthetic phenolic antimicrobial agent widely used since the 1970s due to its potent antibacterial properties. It is commonly incorporated into products such as toothpaste, soaps, cosmetics, plastics, and medical devices (Howse et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Machado et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Sinicropi et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Owing to its lipophilicity and chemical stability, TCS is persistent in the environment and resistant to rapid degradation, resulting in its widespread presence in water, soil, and biota (Jiang et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Dar et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Extensive monitoring studies have detected TCS in human blood, urine, breast milk, and cord blood, indicating significant bioaccumulation potential and chronic exposure risk via water sources and food chains (Arnot et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Luan et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; D. Zhang \u0026amp; Lu, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Animal studies confirm that TCS accumulates in adipose tissue and reproductive organs, potentially disrupting hormone levels and metabolic homeostasis (Li et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Du et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Both in vivo and in vitro evidence demonstrate that TCS exerts multi-level reproductive toxicity. In male models, TCS exposure causes testicular structural abnormalities, declines in sperm quantity and quality, and androgen disruption (Adegbola et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Although data on female reproductive toxicity remain limited, emerging studies suggest that TCS interferes with estrogen receptor signaling, impairing ovarian function and oocyte development (Gan et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Oxidative stress induced by TCS, characterized by elevated reactive oxygen species (ROS), decreased mitochondrial membrane potential, and increased apoptosis, is considered a principal mechanism underlying its reproductive toxicity (Kosińska \u0026amp; Szychowski, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Given the widespread use and environmental persistence of TCS, concerns about its ecological and human health risks are growing, especially regarding long-term exposure during pregnancy or reproductive age, which may affect embryonic development and reproductive system function via maternal-fetal transfer (Bai et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Consequently, multiple countries and regions have started restricting or banning certain TCS-containing products to mitigate potential public health hazards. Despite reports on TCS effects on male reproductive health, its comprehensive toxicological mechanisms impacting oocyte maturation, mitochondrial homeostasis, and apoptosis remain unclear, and effective protective strategies are scarce.\u003c/p\u003e\u003cp\u003eSpermidine is a naturally occurring polyamine involved in key physiological processes including cell proliferation, antioxidant stress response (Shi et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), autophagy activation, and regulation of cell death (Yuan et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Increasing evidence demonstrates that spermidine exerts significant cytoprotective effects by scavenging reactive oxygen species (ROS), stabilizing mitochondrial function, and modulating the autophagy-apoptosis signaling axis (L. Wang et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Madeo et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). In reproductive biology, spermidine has been shown to maintain ovarian reserve function, enhance oocyte developmental competence, and alleviate ovarian aging (Y. Zhang et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Mechanistically, these effects are primarily mediated through regulation of intracellular redox balance and promotion of autophagy to clear damaged cellular components, thereby preserving cellular homeostasis (Hofer et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Furthermore, spermidine protects germ cells from diverse internal and external environmental stresses by inhibiting inflammatory responses and apoptosis (Liu et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Han et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). However, systematic studies investigating whether spermidine can mitigate oocyte toxicity induced by environmental pollutants, particularly triclosan (TCS), remain lacking. Addressing this knowledge gap is critical for advancing protective strategies to safeguard reproductive cells and reproductive health under environmental pollutant exposure.\u003c/p\u003e\u003cp\u003eProgrammed cell death (apoptosis) is central to the oocyte\u0026rsquo;s response to environmental stress (Chesnokov et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Studies have demonstrated that mitochondrial dysfunction and oxidative stress induced by environmental pollutants can activate the intrinsic apoptotic pathway (Kong et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), involving BAX activation, cytochrome c release, and caspase cascade (Sun et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), leading to oocyte maturation failure and fertilization defects (Stringer et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). During metaphase II (MII) of meiosis, even subtle elevations in apoptotic signaling can cause loss of mitochondrial membrane potential (Bock \u0026amp; Riley, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), impaired ATP production (Estudillo et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), and dysregulated cortical reaction (Sagvekar et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), thereby compromising subsequent embryonic development (Pei et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Notably, TCS has been reported to induce apoptosis by disrupting the Bcl-2/BAX ratio and activating caspase-3 (W. Wang et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). However, experimental evidence addressing whether targeted modulation of apoptotic networks can reverse TCS-induced oocyte toxicity is still lacking (Park et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eTherefore, using porcine oocytes as a model to simulate environmental TCS exposure, this study systematically evaluates its effects on meiotic progression, mitochondrial function, oxidative stress, DNA damage, and apoptosis. Furthermore, we investigate whether spermidine supplementation can alleviate these toxic effects through antioxidative and cellular homeostasis mechanisms. Our findings aim to provide novel insights into the mechanistic basis of TCS-induced oocyte toxicity and establish a theoretical foundation for developing small-molecule-based reproductive protection strategies and potential therapeutic targets.\u003c/p\u003e"},{"header":"Materials and Method","content":"\u003cp\u003e\u003cstrong\u003eChemicals and animals\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll chemicals and reagents used in this study were purchased from Sigma-Aldrich (St. Louis, MO, USA), unless otherwise stated. Porcine ovaries were obtained from discarded samples of prepubertal gilts (6 months old, ~100 kg body weight) at licensed slaughterhouses. No live animals were sacrificed specifically for this study. All procedures involving the collection, handling, and use of animal tissues were conducted in strict accordance with institutional guidelines for animal care and use, as well as internationally recognized animal welfare standards. All animal procedures were approved by the Animal Care and Use Committee of Anhui Agricultural University (IACUC approval number: AHAU20208025) and conducted in strict accordance with the Guide for the Care and Use of Laboratory Animals to ensure animal welfare and humane endpoints.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompound Preparation and Experimental Design\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTriclosan (TCS; Cat# PHR1338, Sigma-Aldrich, USA) and spermidine (SPD; Cat# S2626, Sigma-Aldrich, USA) were each dissolved in anhydrous dimethyl sulfoxide (DMSO) to prepare 100 mM stock solutions, aliquoted and stored at −20°C. Prior to use, TCS stock solutions were diluted in oocyte maturation medium to final working concentrations of 0.5, 1, 2, and 5 μM, while SPD stock was first dissolved in cell culture-grade water and then diluted to 10, 20, and 50 μM. Concentration ranges were selected based on prior literature and preliminary experiments to ensure biological relevance. Porcine oocytes were randomly assigned into three groups during in vitro maturation (IVM): Control (no treatment), TCS exposure (continuous exposure to TCS at specified concentrations), and combined TCS and SPD treatment (same TCS concentrations plus SPD at indicated doses). All groups were cultured for 44 hours at\u0026nbsp;38.5°C\u0026nbsp;under humidified 5% CO₂ atmosphere. The final DMSO concentration in all culture media was maintained consistently at ≤0.1% (v/v) across all groups to exclude solvent-related effects.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOocyte maturation assessment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA total of 1000 cumulus–oocyte complexes (COCs) were collected and subjected to five independent replicates. After 44 hours of in vitro maturation (IVM), cumulus expansion was evaluated based on the morphological characteristics of the surrounding cumulus cells (X. Wang et al., 2023). To assess nuclear maturation, COCs were incubated in Dulbecco’s phosphate-buffered saline (DPBS; Gibco, Grand Island, NY, USA) supplemented with 100 μg/mL penicillin G, 75 μg/mL streptomycin sulfate, and 0.1% hyaluronidase. Following gentle pipetting to remove cumulus cells, denuded oocytes were observed under an inverted microscope (Zeiss, Germany). Oocytes were classified into two categories based on the presence of the first polar body: immature (no polar body) and mature (with first polar body visible).\u003c/p\u003e\n\u003ch3\u003eImmunofluorescence Staining\u003c/h3\u003e\n\u003cp\u003eTo assess the developmental quality of porcine oocytes, samples were fixed and permeabilized at room temperature for 30 minutes in PHEM buffer containing 2% paraformaldehyde and 0.5% Triton X-100 (composition: 60 mM PIPES, 25 mM HEPES, 10 mM EGTA, 4 mM MgSO₄, pH 6.9). After fixation, oocytes were washed three times in PBS containing 0.1% Tween-20, 5 minutes per wash. Subsequently, samples were blocked overnight at 4°C with 2% bovine serum albumin (BSA). Oocytes were then incubated with anti–α-tubulin primary antibody (1:200, diluted in PBS containing 2% BSA and 0.1% Tween-20) overnight at 4°C. On the following day, Alexa Fluor 488–conjugated secondary antibody (1:1000) was applied at room temperature for 1 hour in the dark. Nuclei were counterstained with Hoechst 33342 for 10 minutes, followed by washing and mounting. Fluorescence images were captured using a laser scanning confocal microscope.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMeasurement of Reactive Oxygen Species (ROS) Levels\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo quantify intracellular ROS levels in porcine oocytes, oocytes were incubated in Dulbecco’s PBS containing 10 μM DCFH-DA probe (Beyotime, China) at 37 °C in 5% CO₂ for 30 minutes in the dark. After incubation, the cells were gently washed three times (5 minutes each) with PBS containing 0.1% BSA to remove unbound probes. The oocytes were then transferred onto glass slides and imaged using a Zeiss LSM 700 META confocal microscope. Imaging parameters, including excitation wavelength, laser power, and gain, were kept constant to ensure comparability. The mean fluorescence intensity within defined regions of interest (ROIs) was measured using ImageJ software to reflect relative ROS levels.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnnexin-V Staining\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eApoptosis in porcine oocytes was detected using an Annexin-V-FITC staining kit (Beyotime, China). Oocytes were washed twice with PBS at room temperature (5 minutes each), then incubated in the dark for 30 minutes in a staining solution containing 10 μL Annexin-V-FITC and 90 μL binding buffer. After staining, the oocytes were gently washed with PBS and transferred to glass-bottom dishes for imaging using a Zeiss LSM 700 META confocal microscope. Imaging parameters were standardized across all samples. Fluorescence signals from Annexin-V-FITC were used to evaluate early-stage apoptosis in the oocytes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSperm-binding assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMature porcine oocytes were obtained through in vitro maturation (IVM), while sperm were purified from thawed semen using the swim-up method. Oocytes and sperm were co-incubated at a 1:1 ratio for 1 hour at 38.5°C in an atmosphere containing 5% CO₂\u0026nbsp;to facilitate sperm binding. Following incubation, oocytes were gently washed three times (5 minutes each) with PBS containing 0.1% BSA to remove unbound sperm. The oocytes were then stained with Hoechst 33342 for 10 minutes, washed, and transferred to glass-bottom culture dishes for imaging. Fluorescence images were captured using a Zeiss LSM 700 META confocal microscope under identical imaging settings for all samples. The number of sperm bound to each oocyte was counted to evaluate sperm-binding capacity.\u003c/p\u003e\n\u003ch3\u003eImage Acquisition and Analysis\u003c/h3\u003e\n\u003cp\u003eTissue sections and oocyte samples were stained under the same immunofluorescence conditions and imaged on the same day using a Zeiss laser scanning confocal microscope (LSM 700, Zeiss, Germany). To reduce batch effects, imaging parameters including laser power, scan speed, pinhole size, gain, and offset were kept constant. Images were exported in TIFF format and analyzed with ImageJ software (v1.53o, NIH, USA). Background fluorescence was removed by applying a consistent threshold. Regions of interest (ROIs), such as the cytoplasm or nucleus of each oocyte or selected tissue area, were defined using the “ROI Manager” tool. Total fluorescence intensity within each ROI was measured and normalized to the area to calculate the average fluorescence intensity (mean gray value/μm²). At least 20 oocytes or equivalent tissue ROIs per group were analyzed, and the average value was used for statistical comparison.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll experiments were independently repeated at least three times, with the number of porcine oocytes indicated in the figure legends. Due to limited sample size, oocytes from multiple experiments were combined for proportion analysis. Differences between groups were compared using Fisher’s exact test, and proportions are presented as standard error of the mean (SEM). Statistical analyses were performed using GraphPad Prism version 9.4.1. Details of tests and significance are provided in the figure legends.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003eSpermidine supplementation mitigates triclosan-induced meiotic failure and cumulus expansion impairment in porcine oocytes\u003c/b\u003e\u003c/p\u003e\u003cp\u003eGiven that the environmental pollutant triclosan (TCS) may impair oocyte maturation competence, we first added various concentrations of TCS (0.5, 1, 2, and 5 \u0026micro;M) to the in vitro maturation (IVM) medium to evaluate its effects on porcine oocyte meiotic progression. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, the control cumulus\u0026ndash;oocyte complexes (COCs) exhibited robust cumulus cell expansion, indicative of normal oocyte development. In contrast, COCs in the TCS-treated groups showed varying degrees of impaired cumulus expansion, with more pronounced inhibition at higher concentrations. To further confirm whether TCS disrupts oocyte meiotic maturation, we quantified the polar body extrusion (PBE) rate. TCS exposure significantly decreased PBE in a dose-dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Specifically, 2 \u0026micro;M and 5 \u0026micro;M TCS treatments markedly reduced the PBE rate (Control: 82.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2%, n\u0026thinsp;=\u0026thinsp;144; 0.5 \u0026micro;M: 80.1\u0026thinsp;\u0026plusmn;\u0026thinsp;1.9%, n\u0026thinsp;=\u0026thinsp;141, P\u0026thinsp;\u0026gt;\u0026thinsp;0.05; 1 \u0026micro;M: 77.1\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1%, n\u0026thinsp;=\u0026thinsp;140, P\u0026thinsp;\u0026lt;\u0026thinsp;0.1; 2 \u0026micro;M: 57.7\u0026thinsp;\u0026plusmn;\u0026thinsp;3.4%, n\u0026thinsp;=\u0026thinsp;142, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001; 5 \u0026micro;M: 57.1\u0026thinsp;\u0026plusmn;\u0026thinsp;3.4%, n\u0026thinsp;=\u0026thinsp;142, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Based on these results, 2 \u0026micro;M was selected for subsequent experiments.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eWe next examined whether spermidine (SPD), an autophagy-regulating factor, could alleviate TCS-induced oocyte maturation defects. Different concentrations of SPD (10, 20, or 50 \u0026micro;M) were supplemented into the IVM system containing 2 \u0026micro;M TCS. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, SPD supplementation significantly improved PBE rates, with the most pronounced rescue observed at 50 \u0026micro;M (Control: 80.8\u0026thinsp;\u0026plusmn;\u0026thinsp;2.8%, n\u0026thinsp;=\u0026thinsp;118, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001; TCS: 55.4\u0026thinsp;\u0026plusmn;\u0026thinsp;3.1%, n\u0026thinsp;=\u0026thinsp;114; TCS\u0026thinsp;+\u0026thinsp;10 \u0026micro;M SPD: 62.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5%, n\u0026thinsp;=\u0026thinsp;114, P\u0026thinsp;\u0026gt;\u0026thinsp;0.05; TCS\u0026thinsp;+\u0026thinsp;20 \u0026micro;M SPD: 67.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5%, n\u0026thinsp;=\u0026thinsp;119, P\u0026thinsp;\u0026lt;\u0026thinsp;0.1; TCS\u0026thinsp;+\u0026thinsp;50 \u0026micro;M SPD: 76.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1%, n\u0026thinsp;=\u0026thinsp;117, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Furthermore, quantitative analysis of the cumulus expansion area revealed that SPD supplementation markedly alleviated TCS-induced defects in cumulus cell expansion (Control: 7643.0\u0026thinsp;\u0026plusmn;\u0026thinsp;581.4 \u0026micro;m\u0026sup2;, n\u0026thinsp;=\u0026thinsp;65; TCS: 2480.0\u0026thinsp;\u0026plusmn;\u0026thinsp;133.5 \u0026micro;m\u0026sup2;, n\u0026thinsp;=\u0026thinsp;88, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001; TCS\u0026thinsp;+\u0026thinsp;SPD: 4237.0\u0026thinsp;\u0026plusmn;\u0026thinsp;192.3 \u0026micro;m\u0026sup2;, n\u0026thinsp;=\u0026thinsp;67, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Taken together, these findings suggest that SPD exerts a protective effect against TCS-induced meiotic arrest and cumulus expansion defects in porcine oocytes.\u003c/p\u003e\u003cp\u003e\u003cb\u003eSpermidine ameliorates triclosan-induced defects in spindle assembly and chromosome organization during porcine oocyte meiosis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo investigate the effects of triclosan (TCS) exposure on porcine oocyte meiotic maturation and the potential protective mechanisms of spermidine (SPD), we first examined spindle morphology and chromosome alignment in MII-stage oocytes. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, oocytes from the control group exhibited typical barrel-shaped spindles with well-aligned chromosomes at the metaphase plate. In contrast, oocytes exposed to TCS showed pronounced spindle disorganization, disrupted microtubule aggregation, and increased frequencies of chromosome misalignment and abnormal clustering. Quantitative analysis revealed that the incidence of spindle defects was significantly elevated in the TCS group compared to controls (Control: 19.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0%, n\u0026thinsp;=\u0026thinsp;106; TCS: 62.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.7%, n\u0026thinsp;=\u0026thinsp;103; P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB), accompanied by a significant increase in chromosome misalignment (Control: 20.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7%; TCS: 63.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.7%; P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Notably, supplementation with SPD markedly attenuated these abnormalities, reducing the rates of spindle and chromosome defects to 29.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6% and 28.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7%, respectively (n\u0026thinsp;=\u0026thinsp;102; P\u0026thinsp;\u0026lt;\u0026thinsp;0.001), suggesting that SPD can effectively mitigate TCS-induced meiotic impairments.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eGiven that microtubule stability is critical for proper spindle assembly, we further assessed the levels of acetylated α-tubulin as a marker of microtubule integrity. TCS exposure significantly decreased the fluorescence intensity of acetylated α-tubulin (Control: 44.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6, n\u0026thinsp;=\u0026thinsp;35; TCS: 20.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5, n\u0026thinsp;=\u0026thinsp;35; P\u0026thinsp;\u0026lt;\u0026thinsp;0.001), whereas SPD treatment substantially restored the acetylation levels (33.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5, n\u0026thinsp;=\u0026thinsp;35; P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, B). These findings suggest that SPD alleviates TCS-induced meiotic defects by preserving microtubule stability, thereby promoting proper spindle organization and chromosome alignment during oocyte maturation.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eSpermidine ameliorates triclosan-induced disruption of actin cytoskeleton during porcine oocyte maturation\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo examine the impact of triclosan (TCS) exposure on the actin cytoskeleton during porcine oocyte meiosis, we performed immunofluorescence staining using phalloidin-TRITC. In control oocytes, actin filaments were strongly and continuously distributed along the plasma membrane and within the cytoplasm. In contrast, oocytes exposed to TCS exhibited markedly reduced and discontinuous actin signals, indicating cytoskeletal disruption (Control: 26.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4, n\u0026thinsp;=\u0026thinsp;39; TCS: 9.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4, n\u0026thinsp;=\u0026thinsp;39; P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Notably, supplementation with spermidine (SPD) significantly restored actin filament intensity to levels comparable to controls (TCS\u0026thinsp;+\u0026thinsp;SPD: 25.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5, n\u0026thinsp;=\u0026thinsp;39; P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-B). These findings suggest that TCS disrupts actin cytoskeletal dynamics during oocyte maturation, potentially impairing meiotic progression, while SPD supplementation effectively rescues actin integrity and supports proper meiotic development.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eSpermidine restores mitochondrial function and cortical granule protein ovastacin distribution in triclosan-exposed porcine oocytes\u003c/b\u003e\u003c/p\u003e\u003cp\u003eMitochondria are the primary energy source of oocytes, generating substantial ATP to support meiotic maturation. To investigate whether triclosan (TCS) exposure impairs oocyte meiosis via mitochondrial dysfunction, we performed MitoTracker Red staining to assess mitochondrial localization and distribution. Immunofluorescence analysis revealed that TCS exposure markedly altered mitochondrial distribution in porcine oocytes: mitochondria in control oocytes were continuously and evenly distributed along the cortex with strong fluorescence intensity, whereas TCS-treated oocytes exhibited abnormal clustered mitochondrial aggregation with uneven distribution and significantly reduced fluorescence intensity (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Quantitative analysis confirmed a significant decrease in mitochondrial fluorescence intensity in the TCS group compared to controls (Control: 40.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6, n\u0026thinsp;=\u0026thinsp;33; TCS: 5.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3, n\u0026thinsp;=\u0026thinsp;34; P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (note: these values represent fluorescence intensity, not percentages). Spermidine (SPD) supplementation substantially alleviated this defect, partially restoring mitochondrial fluorescence intensity (TCS\u0026thinsp;+\u0026thinsp;SPD: 19.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7, n\u0026thinsp;=\u0026thinsp;33; P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). These findings indicate that TCS exposure induces mitochondrial dysfunction and mislocalization in oocytes, which can be partially rescued by SPD treatment.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFurthermore, ovastacin (ASTL), a key enzyme localized in cortical granules, plays a crucial role in preventing polyspermy and ensuring monospermic fertilization. To evaluate ovastacin distribution dynamics, immunofluorescence staining using anti-ASTL antibody was performed. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC, ovastacin was evenly and continuously distributed in the cortex of control oocytes, whereas TCS exposure caused aberrant localization and significantly reduced fluorescence intensity of ovastacin. SPD supplementation markedly mitigated these defects (Control: 24.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2, n\u0026thinsp;=\u0026thinsp;34; TCS: 5.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2, n\u0026thinsp;=\u0026thinsp;34; TCS\u0026thinsp;+\u0026thinsp;SPD: 16.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2, n\u0026thinsp;=\u0026thinsp;34; P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). These results suggest that SPD partially restores TCS-induced ovastacin mislocalization, thereby reducing polyspermy risk and preserving normal fertilization capacity of oocytes.\u003c/p\u003e\u003cp\u003e\u003cb\u003eSpermidine supplementation alleviates triclosan-induced impairment of oocyte fertilization and early embryonic development in porcine models\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo further evaluate the protective effects of spermidine (SPD) against triclosan (TCS)-induced reproductive toxicity, sperm-oocyte binding assays were performed. Sperm heads were labeled with Hoechst 33342, and two-cell stage embryos were used as a negative control to confirm binding specificity. Quantitative analysis revealed a significant reduction in the number of sperm bound to the zona pellucida of oocytes exposed to TCS compared to controls (Control: 126.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.6, n\u0026thinsp;=\u0026thinsp;34; TCS: 48.1\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5, n\u0026thinsp;=\u0026thinsp;34; P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Notably, SPD co-treatment significantly restored sperm binding capacity (TCS\u0026thinsp;+\u0026thinsp;SPD: 99.1\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5, n\u0026thinsp;=\u0026thinsp;34; P\u0026thinsp;\u0026lt;\u0026thinsp;0.001 vs. TCS alone) (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, B). Consistently, in vitro fertilization (IVF) results showed that TCS exposure significantly decreased the two-cell embryo formation rate (Control: 54.9\u0026thinsp;\u0026plusmn;\u0026thinsp;1.4%, n\u0026thinsp;=\u0026thinsp;104; TCS: 23.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2%, n\u0026thinsp;=\u0026thinsp;106; P\u0026thinsp;\u0026lt;\u0026thinsp;0.001), whereas SPD supplementation markedly improved embryonic developmental competence (TCS\u0026thinsp;+\u0026thinsp;SPD: 47.7\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2%, n\u0026thinsp;=\u0026thinsp;109; P\u0026thinsp;\u0026lt;\u0026thinsp;0.001 vs. TCS alone) (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC, D). Collectively, these data demonstrate that SPD effectively mitigates TCS-induced impairment of oocyte fertilization capacity and early embryonic development.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eSpermidine Supplementation Mitigates Triclosan-Induced Mitochondrial Dysfunction, Oxidative Stress, and Meiotic Failure in Porcine Oocytes\u003c/b\u003e\u003c/p\u003e\u003cp\u003eMitochondrial dysfunction often leads to excessive accumulation of reactive oxygen species (ROS), triggering oxidative stress. To clarify the impact of triclosan (TCS) exposure on oxidative stress in oocytes, we performed fluorescence staining with the DCFH-DA probe to quantitatively assess ROS levels. Results showed weak green fluorescence in control oocytes, indicating low ROS levels, whereas TCS-exposed oocytes exhibited significantly enhanced ROS signals (Control: 5.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3, n\u0026thinsp;=\u0026thinsp;36; TCS: 21.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4, n\u0026thinsp;=\u0026thinsp;38; P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Notably, supplementation with spermidine (SPD) markedly reduced ROS levels, restoring them close to control levels (TCS\u0026thinsp;+\u0026thinsp;SPD: 11.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3, n\u0026thinsp;=\u0026thinsp;34; P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA, B).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eExcessive ROS accumulation can induce DNA double-strand breaks, resulting in DNA damage. To further assess DNA damage, γH2A.X immunofluorescence staining was performed to monitor DNA damage accumulation in oocytes. The TCS-exposed group showed significantly elevated γH2A.X-positive signals compared to controls (Control: 8.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3, n\u0026thinsp;=\u0026thinsp;35; TCS: 26.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4, n\u0026thinsp;=\u0026thinsp;33; P\u0026thinsp;\u0026lt;\u0026thinsp;0.001), while SPD supplementation substantially attenuated this damage signal (TCS\u0026thinsp;+\u0026thinsp;SPD: 12.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4, n\u0026thinsp;=\u0026thinsp;34; P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC, D).\u003c/p\u003e\u003cp\u003ePersistent DNA damage often triggers cell cycle arrest and apoptosis. Early apoptosis was assessed by Annexin-V staining. Control oocytes exhibited minimal Annexin-V signal on the cell membrane (3.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2, n\u0026thinsp;=\u0026thinsp;34), whereas TCS exposure significantly increased Annexin-V positivity (18.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3, n\u0026thinsp;=\u0026thinsp;34; P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). SPD treatment significantly decreased Annexin-V positive rates (9.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3, n\u0026thinsp;=\u0026thinsp;34; P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE, F). Collectively, these results demonstrate that SPD effectively alleviates TCS-induced ROS overaccumulation, DNA damage, and early apoptosis, suggesting its potential protective role against environmental toxicant-induced oocyte injury.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eTriclosan (TCS), widely used and persistent in the environment, has raised considerable concern; however, its toxic effects on reproductive cells and underlying mechanisms remain incompletely understood. Accumulation and chronic exposure to environmental TCS may impair reproductive function and threaten offspring health (C. Wang et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Therefore, elucidating the molecular mechanisms by which TCS disrupts oocyte maturation and identifying effective protective strategies are of critical importance. This study utilized porcine oocytes, which closely resemble human oocytes physiologically, to systematically delineate the mechanisms underlying TCS-induced meiotic disruption and cumulus cell abnormalities. Notably, we provide the first evidence that the natural polyamine spermidine (SPD) markedly alleviates TCS-induced oocyte damage, highlighting its potential application in protecting against environmental reproductive toxicity.\u003c/p\u003e\u003cp\u003eWe established a porcine oocyte TCS exposure model to systematically assess its toxic effects on meiotic progression and cumulus cell development. To better mimic environmental exposure, a short-term multi-dose regimen (0.5, 1, 2, and 5 \u0026micro;M) was applied to comprehensively evaluate TCS effects on in vitro oocyte maturation. Environmental and biological monitoring studies report TCS concentrations within this range (Park et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), indicating the environmental relevance of our experimental design. Cumulus cell expansion and first polar body extrusion are key indicators of oocyte maturation (Richani et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Results demonstrated that TCS significantly inhibited cumulus expansion in a dose-dependent manner and markedly reduced polar body extrusion rates at concentrations\u0026thinsp;\u0026ge;\u0026thinsp;2 \u0026micro;M, suggesting meiotic maturation impairment consistent with previous reports (Zhao et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Introducing SPD as a potential intervention partially rescued TCS-induced suppression of cumulus expansion and polar body extrusion, implying a protective role during oocyte maturation.\u003c/p\u003e\u003cp\u003eAt the subcellular level, we systematically investigated the molecular mechanisms of TCS toxicity and the mitigating effects of SPD. SPD significantly ameliorated TCS-induced spindle assembly defects and chromosome misalignment, promoting normal meiotic progression. Additionally, SPD restored TCS-disrupted actin cytoskeleton organization, recovering cortical integrity and continuity. Considering the crucial role of actin dynamics in polar body extrusion, cytoplasmic maturation, and subsequent embryonic development (Pelzer et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), SPD-mediated actin repair further supports its protective potential on oocyte structure and function.\u003c/p\u003e\u003cp\u003eMoreover, proper localization of cortical granules and the key enzyme ovastacin is essential for preventing polyspermy and ensuring monospermic fertilization (Zafar et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). We found that TCS exposure markedly disrupted ovastacin distribution in oocytes, likely impairing zona pellucida modification via cortical reaction interference and reducing fertilization competence. SPD supplementation significantly restored ovastacin localization and expression, suggesting protection via cytoskeletal stabilization or modulation of exocytosis. This restoration correlated with improved sperm binding and enhanced early embryonic development post in vitro fertilization, underscoring SPD\u0026rsquo;s potential to alleviate environmental toxicant-induced oocyte injury.\u003c/p\u003e\u003cp\u003eMechanistically, TCS exposure significantly induced intracellular reactive oxygen species (ROS) accumulation and activated apoptosis-related pathways, disrupting oocyte developmental homeostasis. Immunofluorescence analysis confirmed that TCS markedly increased the DNA damage marker γH2AX, indicating induction of DNA double-strand breaks, a critical factor underlying meiotic failure and oocyte developmental impairment.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn summary, triclosan (TCS) markedly impairs both nuclear and cytoplasmic maturation of oocytes by inducing oxidative stress and DNA damage. Supplementation with spermidine (SPD) effectively alleviates these toxic effects, promoting spindle assembly, accurate chromosome alignment, and restoration of actin cytoskeleton integrity, thereby significantly enhancing oocyte maturation quality. These findings suggest that SPD holds potential as a protective agent against oocyte developmental toxicity induced by environmental pollutants. This study not only advances our understanding of the toxicological mechanisms underlying environmental pollutant impacts on oocyte quality but also provides a theoretical foundation and practical guidance for developing targeted interventions to safeguard oocyte quality in human assisted reproductive technologies and embryonic engineering.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCRediT authorship contribution statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eM.Q.Z., Y.H.Z., and Y.Z.: Conceptualization, Methodology, Supervision.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eY.G., D.Z., and K.T.: Investigation, Data curation, Formal analysis.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eC.Z., M.W., Q.T., G.Z., and J.C.: Software, Validation, Visualization.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eM.Q.Z., D.Z., and Y.G.: Writing – original draft, Writing – review \u0026amp; editing.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe sincerely thank \u003cstrong\u003eZ. Cui\u003c/strong\u003e, \u003cstrong\u003eC. Zhou\u003c/strong\u003e, and \u003cstrong\u003eY. Miao\u003c/strong\u003e for their valuable support and insightful suggestions, and for kindly providing the antibodies used in this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Key Research and Development Program of China (2023YFD13000502), the National Natural Science Foundation of China (32272881, 32402767), and the Natural Science Foundation of Anhui Province (2308085QC82).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data supporting the findings of this study are available within the main text or the supplementary materials. Additional data will be made available upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing financial interests or personal relationships that could influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor declaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors confirm that all necessary institutional approvals related to ethical use of experimental animals were obtained and are clearly stated in the manuscript. We also confirm compliance with institutional and national regulations regarding intellectual property and publication policies.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to publish\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors have reviewed and approved the final version of the manuscript and consent to its submission for publication.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAdegbola, C. A., Akhigbe, T. M., Adeogun, A. E., Tvrd\u0026aacute;, E., Pizent, A., \u0026amp; Akhigbe, R. E. (2024). A systematic review and meta-analysis of the impact of triclosan exposure on human semen quality. \u003cem\u003eFrontiers in Toxicology\u003c/em\u003e, \u003cem\u003e6\u003c/em\u003e. https://doi.org/10.3389/ftox.2024.1469340\u003c/li\u003e\n \u003cli\u003eArnot, J. A., Pawlowski, S., \u0026amp; Champ, S. (2018). 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Triclosan Caused Oocyte Meiotic Arrest by Modulating Oxidative Stress, Organelle Dysfunctions, Autophagy, and Apoptosis in Pigs. \u003cem\u003eAnimals\u003c/em\u003e, \u003cem\u003e15\u003c/em\u003e(6), Article 6. https://doi.org/10.3390/ani15060802\u003cstrong\u003e\u003c/strong\u003e\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Spermidine, Triclosan, Porcine oocytes, Oxidative stress, Apoptosis","lastPublishedDoi":"10.21203/rs.3.rs-7023955/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7023955/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eOocytes are highly susceptible to environmental pollutants, with triclosan (TCS)—a pervasive antimicrobial—known to bioaccumulate and impair reproductive function. However, mechanisms driving TCS-induced oocyte degeneration and effective protective approaches remain unclear. This study investigates the protective effects of spermidine against TCS-induced meiotic disruption and fertilization defects in porcine oocytes, exploring its antioxidant and anti-apoptotic mechanisms. Porcine germinal vesicle (GV) oocytes were exposed in vitro to graded TCS concentrations (0.5–5 μM) with or without spermidine supplementation. Meiotic maturation, reactive oxygen species (ROS) production, DNA damage, mitochondrial function, apoptosis, and fertilization competence were assessed using immunofluorescence, fluorescence quantification, and mitochondrial distribution analyses. TCS exposure disrupted meiotic progression, causing spindle defects, chromosome misalignment, mitochondrial dysfunction, elevated ROS, DNA damage, and apoptosis, reducing maturation and fertilization rates. Spermidine significantly reversed these effects by stabilizing cytoskeletal architecture, lowering oxidative stress, and inhibiting apoptosis, thereby improving oocyte quality and developmental competence. Spermidine effectively attenuates TCS-induced meiotic and fertilization impairments by mitigating oxidative stress-mediated apoptosis, offering promising intervention strategies to preserve oocyte quality under environmental toxicant exposure.\u003c/p\u003e","manuscriptTitle":"Spermidine supplementation protects porcine oocytes from triclosan-induced meiotic and fertilization defects by attenuating oxidative stress-mediated apoptosis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-14 16:40:50","doi":"10.21203/rs.3.rs-7023955/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"33325c21-6dc3-4ba6-9d56-9b47f8e18bf1","owner":[],"postedDate":"July 14th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-07-20T02:23:17+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-14 16:40:50","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7023955","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7023955","identity":"rs-7023955","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}