Tubacin alleviate deoxynivalenol-exposure caused mouse oocyte maturation arrest

To investigate the effects of deoxynivalenol on mouse oocyte maturation, varying concentrations of DON (1, 2, 3, and 6 μM) were supplemented in the oocyte in vitro maturation medium (Fig. 1A,B). A dose-dependent decrease of polar body extrusion (PBE) rate was observed in DON-exposed oocytes compared with controls, showing a statistically significant reduction in the 2 μM DON group (Fig. 1C). Complete meiotic arrest was induced by 6 μM DON, as evidenced by the absence of first polar body extrusion, whereas co-treatment with Tubacin significantly restored PBE rates and meiotic failure (Fig. 1C). No additional benefit was observed with higher Tubacin concentrations than 4 μM Tubacin in DON-exposed oocytes (Fig. 1C). Treatment of oocytes with 4 µM Tubacin alone exerted a mild inhibitory effect on meiotic progression (Fig. 1C). Importantly, this modest off-target effect was significantly outweighed by Tubacin’s robust rescue of DON-induced defects, confirming its therapeutic benefit. Based on these findings, 2 μM DON and 4 μM Tubacin were selected for subsequent investigations of meiotic arrest mechanisms.

Fig. 1

Tubacin supplementation alleviated meiotic progression defects in deoxynivalenol-exposed oocytes. A Schematic diagram shown the treatment strategy of control, DON, and DON + Tubacin oocytes. B Representative light microscopy images showing oocytes in control, DON, and DON + Tubacin oocytes. Scale bar, 200 μm. C Quantification of the percentage of oocytes that undergo PBE in vitro (One-way ANOVA with Šidák correction for multiple comparisons, *** P < 0.001). D Representative immunofluorescence images of oocytes in varying treatments. Green, (microtubule); blue, DNA (DAPI). Scale bar, 10 μm. E Quantification of the percentage of oocytes that assembled bipolar spindle (One-way ANOVA with Šidák correction for multiple comparisons, ** P < 0.01, *** P < 0.001). F Representative immunofluorescence images demonstrating spindle assembly in mouse metaphase I oocytes from control, DON, and DON + Tubacin. Green, microtubule (β-Tubulin); red, aMTOCs (Pericentrin); blue, DNA (DAPI). Scale bar, 10 μm. G Representative immunofluorescence images demonstrating spindle assembly in mouse metaphase I oocytes from control, DON, and DON + Tubacin. Green, microtubule (β-Tubulin); red, aMTOCs (γ-Tubulin); blue, DNA (DAPI). Scale bar, 10 μm. H Quantitative analysis of mean γ-Tubulin fluorescence intensity for oocytes. Data were presented as the mean ± SD. Numbers indicate the individual oocytes quantified. **** P < 0.0001 by One-way ANOVA with Šidák correction for multiple comparisons. I Representative immunofluorescence images of oocytes after treatment. Green, ROS; DIC, field of differential interference contrast (DIC). Scale bar, 10 μm. J Quantitative analysis of mean ROS fluorescence intensity for oocytes after treatment. Data were presented as the mean ± SEM. Numbers indicate the individual oocytes quantified. **** P < 0.0001 by One-way ANOVA with Šidák correction for multiple comparisons

To elucidate the cellular basis of DON-induced meiotic failure, immunofluorescence analysis was performed to assess spindle architecture and chromosomal organization. Control oocytes displayed characteristic barrel-shaped spindles with chromosomes aligned at the metaphase plate, whereas severe spindle disassembly and chromosomal misalignment were observed in DON-exposed oocytes (Fig. 1D, E). Remarkably, Tubacin co-treatment was found to restore normal spindle morphology and chromosome alignment (Fig. 1D, E).

Considering the critical role of microtubule organizing centers (MTOCs) in bipolar spindle formation, the spatial distribution of MTOC-associated proteins was systematically characterized through multi-color immunofluorescence imaging. Pericentrin and γ-Tubulin were detected as tightly focused signals at spindle poles in control oocytes (Fig. 1F, G). In contrast, DON exposure caused the dispersion of these MTOC markers, with both Pericentrin and γ-Tubulin showing abnormal distribution patterns or reduced expression (Fig. 1F-H). This defective localization was effectively rescued by Tubacin co-treatment (Fig. 1F-H).

Given the energy-intensive nature of the oocyte maturation process and the known association between mitochondrial dysfunction and oxidative stress, intracellular reactive oxygen species (ROS) levels were quantitatively assessed using fluorescence-based assays. DON-exposed oocytes exhibited significantly elevated ROS signals compared to controls when measured using DCFH-DA fluorescence (Fig. 1I, J). Furthermore, mitochondrial membrane localization and ATP levels were assessed. Tubacin was found to restore mitochondrial membrane potential and ATP levels impaired by DON treatment, indicating restoration of mitochondrial function (Fig. S1A-C). These collective findings demonstrate that Tubacin mitigates DON-induced spindle defects and microtubule disorganization while reducing oxidative stress in maturing oocytes.

Mechanistic insights into DON-induced microtubule destabilization

To investigate DON-induced cytoskeletal alterations, immunofluorescence analysis was conducted for key microtubule dynamics regulators and spindle assembly factors. The fluorescence intensity of microtubule nucleator TPX2 was significantly reduced in DON-exposed oocytes compared to controls (Fig. 2A, B), and co-treatment with Tubacin restored TPX2 localization patterns to control levels. Notably, aberrant cytoplasmic accumulation of spindle stabilizer KIF11 was observed in DON-treated oocytes, which was rescued by Tubacin supplementation (Fig. 2C, D). These results demonstrate that Tubacin counteracts DON-induced cytoskeletal destabilization through coordinated regulation of microtubule-associated factors. Moreover, molecular docking revealed potential binding of DON to multiple sites on the inside part of α/β-tubulin dimers (Fig. 2E), suggesting structural interference with microtubule polymerization dynamics and microtubule stabilization.

Fig. 2

Mechanism of DON-induced microtubule destabilization. A Representative immunofluorescence images demonstrating spindle assembly in mouse metaphase I oocytes from control, DON, and DON + Tubacin. Green, TPX2; red, microtubule (β-Tubulin); blue, DNA (DAPI). Scale bar, 10 μm. B Quantitative analysis of the mean intensity of TPX2. Data were presented as the mean ± SEM (One-way ANOVA with Šidák correction for multiple comparisons, **** indicates P < 0.0001). C KIF11 spindle pole localization. Green, KIF11; red, microtubule (β-Tubulin); blue, DNA (DAPI). Scale bar, 10 μm. D Quantitative analysis of the mean intensity of KIF11. Data were presented as the mean ± SEM (One-way ANOVA with Šidák correction for multiple comparisons, ** indicates P < 0.01, **** indicates P < 0.0001). E Molecular docking of DON with a/β-Tubulin dimer. F Cold-stable microtubule preservation. Green, microtubule (β-Tubulin); blue, DNA (DAPI). Scale bar, 10 μm. G Spindle length measurements. Data were presented as the mean ± SEM (One-way ANOVA with Šidák correction for multiple comparisons, ** indicates P < 0.01, * ** indicates P < 0.001). H Representative immunofluorescence images demonstrating spindle assembly in mouse metaphase I oocytes from control, DON, and DON + Tubacin. Green, microtubule (β-Tubulin); red, ace-α-Tubulin; blue, DNA (DAPI). Scale bar, 10 μm. I Acetylation intensity quantification. Data were presented as the mean ± SEM (One-way ANOVA with Šidák correction for multiple comparisons, **** indicates P < 0.0001)

Given the central role of KIF11 in microtubule stabilization, cold-induced disassembly assays were performed to evaluate microtubule resilience. Cold-resistant microtubule lattice and spindle length were significantly reduced in DON-exposed oocytes following 10-min cold treatment, while Tubacin co-treatment preserved microtubule stability (Fig. 2F, G). Consistently, acetylated α-tubulin levels showed a significant reduction in DON-exposed oocytes, and Tubacin restored microtubule acetylation to normal levels (Fig. 2H, I). This comprehensive analysis establishes microtubule acetylation as a critical regulatory node in DON-induced meiotic defects, suggesting pharmacological stabilization of microtubule post-translational modifications as a potential therapeutic approach for toxin-associated fertility impairments.

Tubacin protects against defects induced by DON in TUBB8 oocyte-specific knockin mouse model

Meiotic spindle assembly and maintenance of the architecture stability in human oocytes are mainly mediated by the microtubule encoding the primate-specific gene TUBB8, whereas murine spindle assembly predominantly depends on microtubule-organizing centers (MTOCs). This interspecies divergence highlights the critical need for experimental models that more faithfully replicate human reproductive physiology; thus, we generated a TUBB8 oocyte-specific knockin (TUBB8-KI) mouse model (Fig. 3A). DON exposure recapitulated meiotic defects in knockin oocytes, with Tubacin demonstrating comparable rescue efficacy to wild-type controls (Fig. 3B, C). No significant differences in spindle morphology or maturation rates were observed between control oocytes and TUBB8-KI oocytes (Fig. 3D), establishing its reliability for human-relevant reproductive toxicity evaluations. To investigate TUBB8 expression patterns in knock-in mice and its influence on endogenous tubulin, metaphase-I oocytes from wild-type and TUBB8-KI mice were collected for Smart-seq2 RNA-seq analysis. TUBB8 expression was moderate relative to other tubulin isotypes following knock-in (Fig. S2A, 2 C). Critically, the overall transcriptomic profile remained largely unaffected by TUBB8 knock-in. Only 11 genes were differentially expressed in knock-in oocytes versus controls, including one down-regulated and ten up-regulated genes (Fig. S2B). Among these, mRNA levels of endogenous tubulins Tubb4b and Tuba3a were elevated (Fig. S2B), suggesting compensation for increased total β-Tubulin levels due to exogenous TUBB8 insertion. This may reflect upregulation of α-Tubulin and β-Tubulin transcripts to maintain the balance of the abundance of α/β-tubulin heterodimers, ensuring cellular functional capacity.

Fig. 3

Pharmacological rescue of DON-induced defects in humanized TUBB8-knockin oocytes. A TUBB8- knockin strategy. B Representative light microscopy images showing oocytes in control, TUBB8-KI, TUBB8-KI + DON, and TUBB8-KI + DON + Tubacin oocytes. C Quantification of the percentage of oocytes that undergo PBE in vitro (One-way ANOVA with Šidák correction for multiple comparisons, ns = no significance, *** indicates P < 0.001). D Representative immunofluorescence images in mouse metaphase II oocytes from control and TUBB8-KI oocytes. Green, microtubule (β-Tubulin); red, TUBB8-HA; magenta, F-actin; blue, DNA (DAPI). Scale bar, 10 μm. E Representative immunofluorescence images of oocytes in varying treatments. Green, (microtubule); blue, DNA (DAPI). Scale bar, 10 μm. F Quantification of the percentage of oocytes that assembled bipolar spindle (One-way ANOVA with Šidák correction for multiple comparisons, *** indicates P < 0.001, **** indicates P < 0.0001). G Representative immunofluorescence images in mouse metaphase I oocytes from TUBB8-KI, TUBB8-KI + DON (DON), and DON + Tubacin. Green, DCTN1; red, microtubule (TUBB8-HA); blue, DNA (DAPI). Scale bar, 10 μm. H Mean intensity of DCTN1. Data were presented as the mean ± SEM (One-way ANOVA with Šidák correction for multiple comparisons, ** indicates P < 0.01, *** indicates P < 0.001). I Representative immunofluorescence images in mouse metaphase I oocytes from TUBB8-KI, TUBB8-KI + DON (DON), and DON + Tubacin. Green, KIF11; red, microtubule (TUBB8-HA) KIF11, blue, DNA (DAPI). Scale bar, 10 μm. J Quantitative analysis of the mean intensity of KIF11. Data were presented as the mean ± SEM (One-way ANOVA with Šidák correction for multiple comparisons, ** indicates P < 0.01, **** indicates P < 0.0001)

Mechanistic analyses revealed that DON exposure abolished the polarized distribution of MTOC-associated components, as evidenced by the diffuse cytoplasmic localization or diminished expression of Pericentrin and DCTN1, respectively (Fig. 3E-H). Tubacin treatment effectively rescued their characteristic bipolar enrichment (Fig. 3E-H), suggesting the restoration of MTOC-mediated spindle organization. Furthermore, DON-induced suppression of KIF11 was observed to correlate with impaired bipolar spindle formation (Fig. 3I-J). Tubacin intervention not only normalized KIF11 expression levels but also re-established its canonical bipolar localization pattern (Fig. 3I-J), providing direct evidence of spindle stability rescue through pharmacological modulation.

DON-exposure inhibits mRNA translation in oocytes

Low-input Smart-seq2 profiling was performed to characterize DON-induced transcriptional alterations in maturing oocytes. Spearman correlation analysis revealed clear segregation of experimental groups with high inter-replicate consistency (Fig. 4A). The mRNA profiling in control, DON exposure, and DON + Tubacin oocytes was shown in Fig. 4B. DON exposure induced 432 differentially expressed genes (DEGs, 416 upregulated and 16 downregulated), while Tubacin co-treatment reduced DEGs to 112 (49 upregulated, 63 downregulated) (Fig. 4C and Fig. S3A), demonstrating partial restoration of transcriptional homeostasis through microtubule stabilization. It was well documented that DON inhibits protein synthesis primarily by interfering with ribosome function [25]. Indeed, the structural analysis demonstrated high-affinity binding of DON to the peptidyl transferase center (PTC) within the 28S rRNA of ribosomal 60S subunits (Fig. 4D), providing evidence for impaired peptide bond formation and translation elongation. This ribosomal targeting correlated with abnormal maternal mRNA accumulation (Table S2), suggesting DON-mediated inhibition of maternal mRNA decay during meiotic progression.

Fig. 4

Deoxynivalenol-exposure inhibit mRNA translation in oocytes. A Heatmap of the Spearman correlation coefficients of total transcripts among control, DON, and DON + Tubacin oocytes. B The number of differentially expressed genes (DEGs). C The heatmap illustration transcriptome landscape for displaying gene expression in control, DON, and DON + Tubacin oocytes. D Structural data of DON inhibits peptide bond formation by binding to the peptidyl transferase center of the ribosomal large subunit (60S), suggesting its potential interference with the translation elongation process. E Volcano plot analysis showing DEGs (downregulated, blue; upregulated, red) in DON-exposed oocytes compared with controls. F gene ontology (GO) pathway enrichment analysis with decreased expression genes in Cell Component, Molecular Function, and Biological Process. G Selected biological processes and gene with differentially expressed. Shown are selected differentially expressed (Fold change and P-adj values). H Representative immunofluorescence images of oocytes in varying treatments. Green, (microtubule); red, F-actin; blue, DNA (DAPI). Scale bar, 10 μm. I F-actin intensity quantification. Data were presented as the mean ± SEM (One-way ANOVA with Šidák correction for multiple comparisons, *** indicates P < 0.001, **** indicates P < 0.0001)

GO enrichment analysis identified significant transcriptional suppression of cytoskeletal regulators and mitochondrial/ribosomal components in DON-exposed oocytes (Fig. 4E,F). Notably, cytoskeleton-related effectors included microtubule dynamics controllers (Tuba4a, Cdk5rap2, Stau2) and actin filament organizers (Unc45a, Ccdc60, Reps2) were significantly up-regulated upon DON exposure, while organelle-associated DEGs encompassed mitochondrial transporters (Prkag2, Slc25a54, Timm50, Tor1b, Mpv17l) and ribosomal biogenesis factors (Rbm41, Gpn1, Rpl36al, Eif3l, Nalf2) were remarkably elevated in DON-induced oocytes. (Fig. 4G). Gene Set Enrichment Analysis (GSEA) revealed up-regulated genes enriched in ribosomal functions and down-regulated genes associated with ubiquitin-mediated proteolysis, suggesting DON potentially impacts ribosomal mRNA translation and ubiquitin-dependent degradation pathways (Fig. S3B). These findings suggest that DON impairs oocyte maturation possibly through disruption of microtubule-mediated transport and organelle-specific functions. DON destabilizes cytoskeletal architecture, and simultaneously impairs energy metabolism and protein synthesis via mitochondrial/ribosomal dysfunction, collectively compromising oocyte developmental competence.

Following Tubacin supplementation, we observed that differentially expressed genes were enriched in the biological process of chromosome separation (Fig. S3C). This is associated with that most DEGs in DON-induced oocytes with maturation arrest were enriched in asymmetric division-related cell cycle progression (Fig. S3D). In addition, the DEGs were involved in GTPse binding and active transmembrane transporter activity, which is consistent with the above observations that Tubacin supplementation in DON-induced oocytes leads to the meiotic progression restored and chromosomal aligned. The protein–protein interaction network (PPI) further demonstrated that Tubacin may enhance the activity of ATPase and electron transfer within the mitochondrial inner membrane (Fig. S3E). Thus, we speculate that Tubacin coordinates mitochondrial bioenergetics, and cytoskeletal remodeling to rescue meiotic competence.

DON exposure resulted in abnormal zygote migration and cleavage

The developmental competence of fertilized oocytes following DON exposure was investigated through pronuclei migration and cleavage analysis (Fig. 5A, B). A dose-dependent impairment of 2-cell embryo formation was observed, with fertilized oocytes exhibiting greater sensitivity to DON toxicity than GV-stage oocytes (Fig. 5B). Complete cleavage arrest occurred at 3 μM DON, and 2 μM Tubacin restored 2-cell rates to 62.5% (Fig. 5B). The activation of zygotic genome activation (ZGA) was further assessed by quantifying the expression for key markers, including Muervl, Hsp70.1, eIF1A and Zscan4d. Tubacin effectively reversed DON-induced dysregulation of Muervl, eIF1A and Zscan4d (Fig. S4A), confirming its role in safeguarding epigenetic reprogramming during early embryogenesis. Furthermore, our data demonstrate that DON exposure severely compromises blastocyst development, while Tubacin co-treatment significantly restores developmental rates (Fig. S4B,C).

Fig. 5

DON exposure resulted in abnormal zygote migration and cleavage. A Representative light microscopy images showing zygotes in control, DON, and DON + Tubacin. Scale bar, 200 μm. B Quantification of the percentage of zygotes that undergo cleavage (One-way ANOVA with Šidák correction for multiple comparisons, ns = no significance, ** indicates P < 0.01, *** indicates P < 0.001). C Representative immunofluorescence images of zygotes in varying treatments. Green, (microtubule); red, F-actin; magenta, ace-a-Tubulin; blue, DNA (DAPI). Scale bar, 10 μm. D F-actin intensity quantification. Data were presented as the mean ± SEM (One-way ANOVA with Šidák correction for multiple comparisons, ** indicates P < 0.01, **** indicates P < 0.0001). E Acetylated α-tubulin levels. Data were presented as the mean ± SEM (One-way ANOVA with Šidák correction for multiple comparisons, *** indicates P < 0.001)

Further investigation revealed both cytoskeletal disruption and microtubule acetylation defects. Tubacin intervention specifically restored acetylated microtubule density and improved cleavage rates as well as F-actin amount and pattern, establishing microtubule stability as the primary determinant of developmental competence (Fig. 5C-E). The partial rescue achieved through pharmacological acetylation enhancement identifies microtubule post-translational modification as a critical therapeutic target for eliminating mycotoxin-induced developmental arrest.