AMBRA1 activation alleviates zearalenone-induced swine testicular cell ferroptosis by facilitating mitophagy

Abstract

Background

Mycotoxin contamination poses a major challenge to public health and has garnered increasing attention across the world in recent decades. Zearalenone (ZEA), as one of the most prevalent contaminants, induces reproductive toxicity and then poses potential threats to animal health. Autophagy/beclin-1 regulator 1 (AMBRA1) is a protein critical for autophagy induction, and can enhance mitophagy by co-localizing with LC3. However, the potential health risk caused by ZEA in male germ cells of animals is unclear. This study aimed to investigate the underlying mechanisms of ZEA-induced swine testicular (ST) cell injury and to clarify the role of AMBRA1 in this process.

Methods

We established ST cell models to explore the effects of AMBRA1 on ferroptosis induced by ZEA. Multiple experimental approaches were applied to assess cell viability, mitochondrial dysfunction, oxidative stress, iron accumulation, and mitophagy. Mechanistic insights were further validated using AMBRA1 overexpression, RNA-seq, molecular docking, western blotting, immunofluorescence, and qRT-PCR analyses.

Results

In this study, ZEA induced mitochondrial structural damage and impaired mitochondrial function, leading to excessive ROS generation and loss of mitochondrial membrane potential. We also found that ZEA disrupted the iron homeostasis and thus led to the accumulation of ferrous iron, which further induce ferroptosis. In addition, ZEA reduced autophagy activity and autophagic flux, ultimately suppressing mitophagy. Of note, AMBRA1 overexpression effectively relieved ZEA-induced ferroptosis through restoration of mitophagy in ST cells.

Conclusions

In conclusion, our study demonstrated that ZEA targeted the AMBRA1, leading to down-regulation of AMBRA1 expression, which in turn inhibited mitophagy and thus resulted in ferroptosis in ST cells. Given the potential role of AMBRA1 in ST cells, our results uncover a previously unrecognized mechanism in which AMBRA1-mediated mitophagy functions as a crucial defense target against ferroptosis in testicular cells. Importantly, our results propose a unique insight which AMBRA1 as a promising therapeutic target for counteracting mycotoxin-induced testicular injury in animals.

Supplementary Information

The online version contains supplementary material available at 10.1186/s40104-026-01359-1.

Introduction

Mycotoxins are ubiquitous natural contaminants that persist in the environment and pose significant risks to animal health [1, 2]. The Food and Agriculture Organization estimates that approximately a quarter of the global food crops are contaminated by mycotoxin to some extent, resulting in a loss of 1 billion metric tons of agricultural commodities each year [3]. Among these, zearalenone (ZEA) is one of the most frequent mycotoxins generated by Fusarium species, and is commonly detected in cereal crops and feeds around the world [4]. Surveys have indicated that about 10 million tonnes of grain cereals are contaminated with ZEA annually, exceeding upper limit of ZEA contamination in European Union [5]. More critically, worldwide surveillance data have demonstrated that the detection rate of ZEA increased from 47% to 79% within feed raw materials during the period from 2020 to 2021 [6]. The widespread presence of ZEA is connected to its high stability, which hinders its removal from foodstuffs or animal feed. In addition, ZEA has the potential for environmental persistence and bioaccumulation in the food chain [7]. A series of research showed that toxicity caused by ZEA consisted primarily of hepatotoxic, nephrotoxicity, reproductive toxicity, developmental toxicity, carcinogenic, and immunotoxicity [8]. ZEA is structurally similar to endogenous estrogen which exerts estrogenic effects, and leads to hormonal imbalance and pathological changes in the reproductive organ [9]. Some studies have elucidated that ZEA causes morphological and functional changes in the male animal reproductive system such as impaired spermatogenesis and degrade sperm quality [10]. The male animal reproductive toxicity induced by ZEA has been widely studied, but its molecular mechanism remains unclear. Therefore, it is necessary to systematically understand the potential mechanisms of ZEN-induced male animal reproductive toxicity.

Ferroptosis as a non-apoptotic form of cell death is triggered by excessive reactive oxygen species (ROS) accumulation and a disruption in redox reactions [11]. Alterations in iron homeostasis and lipid peroxidation are closely related to ferroptosis, which leads to decreased capacity of intracellular lipid repair systems and abnormal enzyme reactions [12]. A large amount of evidence revealed that ferroptosis is involved in multiple male reproductive diseases, especially in spermatogenic dysfunction, testicular damage and blood-testis barrier disruption [13, 14]. A recent study illustrated that environmental stress resulted in ferroptosis and severe oxidative stress in developing mice, thereby impairing spermatogenesis and testicular dysfunction [15]. Nuclear factor erythroid 2-related factor 2 (NRF2) is a pivotal factor to regulate the responses of cellular anti-oxidant and primarily controlled via the KEAP1-NRF2 stress response pathway [16]. Under physiological conditions, KEAP1 restrains the transcriptional activity of NRF2. Under oxidative stress, NRF2 is released from KEAP1 and translocates into the nucleus to promote its activation [17]. Accumulating evidence indicates that NRF2 as a strong ferroptosis suppressor protein modulates the levels of genes associated with ferroptosis. Recent researches suggested that NRF2 signaling activation could suppress ferroptosis process in renal tubular epithelial cells, implying that NRF2 has protective functions against ferroptosis [18]. However, the specific mechanism by which ferroptosis and NRF2 is involved in ZEA-induced male reproductive toxicity remains unknown.

Mitochondria are essential organelles within cell, which functional integrity, abundance, and quality are indispensable for sustaining cellular bioenergetics and metabolic homeostasis, thereby supporting cell growth and proliferation [19]. Furthermore, the dysfunction of mitochondria can induce high levels of ROS, mitophagy defects and oxidative injury [20], highlighting the critical importance of timely clearance of aged or damaged mitochondria to maintain cellular homeostasis. Autophagy is a process reliant on lysosomal degradation catabolic process that maintains cellular homeostasis by degrading damaged proteins and organelles [21]. However, both insufficient and excessive autophagy can be detrimental, causing autophagic cell death [22]. Mitophagy is a specific autophagic that selectively removes dysfunctional mitochondria to preserve mitochondrial balance and broadly contains classical and nonclassical pathways [23]. Among them, the PINK1/Parkin signaling is a classic pathway, where mitochondrial depolarization leads to PINK1 accumulation on the outer mitochondrial membrane, the recruitment of the E3 ubiquitin ligase Parkin and the ubiquitination of outer membrane protein, thereby initiating autophagosome formation through the LC3 conjugation [24, 25]. Autophagy/Beclin-1 regulator 1 (AMBRA1) as a critical of autophagy regulator, has unique characteristics in regulating proliferation and autophagy [26]. Some studies showed that AMBRA1 is essential for regulating PINK1-PRAKIN-dependent autophagy as well as independent autophagy [27]. Meanwhile, AMBRA1 interacts with PRAKIN, and the activation of the PINK1-PRAKIN axis can induce autophagy [28]. Importantly, AMBRA1 are implicated in regulation of mitophagy and redox homeostasis, which are essential for maintaining mitochondrial integrity [29]. Efficient mitochondrial is fundamental to spermatogenesis, steroidogenesis, and germ cell survival. Accumulating evidence have indicated that mitochondrial dysfunction and oxidative stress are closely associated with impaired spermatogenesis and testicular injury. Dysregulation of AMBRA1-mediated autophagy impaired mitochondrial clearance, leading to excessive ROS accumulation and disrupting cellular metabolism, thereby contributing to testicular injury, defective spermatogenesis and the development of male reproductive disorders [30, 31]. Nevertheless, it is ambiguous that whether ZEA-induced male reproductive function impairment is related to AMBRA1-dependent mitophagy.

The testes are crucially reproductive organs in male animals that is responsible for the production of sperm and androgen. Moreover, the testes are very sensitive to harmful substances, which can damage the blood-testis barrier, leading to testicular dysfunction. ZEA has adverse effects on the male animal reproductive function, decreasing germ cell numbers and altering the morphology of the testicles, thereby affecting fertility. Several studies have demonstrated that ferroptosis, which is an iron-dependent form of regulated cell death characterized by lipid peroxidation, is closely correlated with testicular function impairment and testicular oxidative stress [32]. Recent studies indicate that efficient mitophagy can alleviate ferroptosis by eliminating damaged mitochondria, preserving mitochondrial function and mitigating oxidative damage in male reproductive tissues [33]. Therefore, the link between mitophagy and ferroptosis is critical for maintaining male reproductive health. Swine are the most susceptible to the toxic impacts of ZEA among species [34] and swine testicular (ST) cells isolate from swine fetal testes are potentially a suitable model to study male animal reproductive toxicity [35]. Therefore, ST cells were used as the study object to pay attention to the impact of ZEA on male reproductive toxicity and its mechanisms in this study. Although AMBRA1 as an essential protein for the induction of mitophagy contributes to mitophagy, but the role of AMBRA1 in male animal reproductive toxicity under ZEA exposure is not well illustrated. The present study was designed to clarify the role of AMBRA1 in ZEA-induced male animal reproductive toxicity, offering a bran-new perspective on the protective strategies of public health.

Materials and methods

Cell culture

ST cells were obtained from ATCC (Manassa, USA) and incubated in DMEM/F12 (MeilunBio, China) containing 10% FBS (Cellmax, China) and 1% penicillin–streptomycin (Biochannel, China) at 37 °C in an atmosphere containing 5% CO₂. ZEA (NCS Testing Technology Co., Ltd., Beijing, China) was dissolved in DMSO and administered to the ST cells for 24 h. The final DMSO concentration in the culture medium does not exceed 0.1% (v/v), a content previously verified to have no detectable impact to assay results compared with the solvent-free control. The dose of ZEA was based on CCK8 assay. Cellular stimulations, such as 100 nmol/L Rapa (MCE, New Jersey, USA), 5 mmol/L 3-MA (MCE, New Jersey, USA), 20 nmol/L BafA1 (MCE, New Jersey, USA), 10 μg/mL PEA (MCE, New Jersey, USA), and 10 μg/mL E64d (MCE, New Jersey, USA) were applied to treat ST cells in accordance with a standard experiment instruction.

Cell viability assay

Cell viability was assessed using the CCK-8 assay kit (APExBIO, Houston, USA). In brief, ST cells were seeded in a 96-well plate (Jet Biofil, Guangzhou, China) and allowed to adhere. After exposure various concentrations of ZEA for 24 h, the absorbance was measured at 450 nm using a microplate spectrophotometer (Multiskan Sky, Thermo Fisher Scientific, Shanghai, China).

RNA-seq

As previously described [36, 37], the standard procedure of RNA sequencing analysis includes RNA extraction, library preparation and data processing. Following the designated treatments, RNA-seq analyses was performed by Shanghai Zhongke New Life Biotechnology Co., Ltd. (Shanghai, China). The detailed procedures are given in Supplementary Methods 1.1.

Transmission electron microscopy (TEM)

TEM analysis was performed as described [38, 39]. In brief, treated and control ST cells were collected and then fixed with 2.5% glutaraldehyde. Then, the cells were post-fixed with 1% osmium tetroxide, followed by dehydration. Samples were infiltrated, embedded, ultrathin sectioned, stained with citric acid, and washed according to established protocols. The ultrastructure was examined under a TEM (HT7650, Hitachi, Japan).

Cell transfection

The AMBRA1 overexpression vector (pcDNA3.1-AMBRA1) was obtained from Sangon Biotech Co., Ltd. (Shanghai, China). pcDNA3.1-AMBRA1 and lipofectamine 3000 (Invitrogen, USA) were separately diluted in Opti-MEM and then combined to form transfection complexes. ST cells were transfected with these complexes to induce AMBRA1 overexpression. The negative control consisted of cells transfected with empty pcDNA3.1.

ROS measurement

The levels of ROS were assessed using a ROS detection kit (Beijing Boxbio Science & Technology Co., Ltd., Beijing, China). In brief, ST cells were exposed to DCFH-DA for the indicated time at 37 °C, followed by washing with serum-free medium to remove excess probe and resuspending in PBS. A flow cytometry (Solaibao, China) was applied to detect the fluorescence intensity and data analysis was performed by using FlowJo software.

Mitochondrial membrane potential (MMP) analysis

As previously described [25, 40], The JC-1 fluorescent probe (Beyotime, Shanghai, China) was carried out to assesse MMP. The fluorescence signals were examined under a fluorescence microscope (Leica, Germany).

MitoSOX assay

According to a previous method [41, 42], the production of superoxide in mitochondria was determined using the MitoSOX™ Red mitochondrial superoxide indicator (Invitrogen, USA). Specifically, ST cells were cultured with MitoSOX™ Red working solution at 37 °C for 30 min. After washing with PBS to remove excess dye, the images were visualized with a fluorescence microscope (Leica, Germany).

Lipid peroxidation detection

BODIPY fluorescent probe (Invitrogen, USA) was utilized for cell staining to evaluate lipid peroxidation within lipid droplets. Fluorescence signals were visualized with a fluorescence microscope (Leica, Germany), and quantitative analysis was subsequently performed.

Ferric ion concentration measurement

Cytoplasmic and mitochondrial ferrous ion levels were determined using the FerroOrange and Mito-FerroGreen fluorescent probes, respectively (Dojindo Molecular Technologies, Japan). Briefly, ST cells were incubated with the corresponding probe solutions at 37 °C for 30 min under light-protected conditions. A fluorescence microscope (Leica, Germany) was employed to obtain images.

Transfection with Mito-Keima adenovirus

Mitophagy was assessed using the fluorescent protein Mito-Keima via adenoviral transfection (Hanbio Technology Co., Ltd., Shanghai, China) in accordance with the instructions. Concisely, ST cells were cultured under standard conditions and transfected with Mito-Keima adenovirus. Fluorescence images were captured by using a confocal microscope (Leica, Germany) at excitation wavelengths of 440 nm and 550 nm.

Transfection with mRFP-GFP-LC3 adenovirus

To monitor autophagy flux, ST cells were infected with mRFP-GFP-LC3 adenovirus (Hanbio Technology Co., Ltd., Shanghai, China). The images were acquired using a confocal microscope (Leica, Germany) and the numbers of yellow and red LC3 puncta were quantified to assess autophagic flux.

qRT-PCR

The extraction of total RNA from ST cells was carried out using TRIzol reagent (Invitrogen, USA), followed by reverse transcription into cDNA by using a RT-PCR Kit (TransGen Biotech, China) as previously described [43, 44]. The QuantStudio 5 system (ABI) was used to conduct real-time PCR. Relevant qRT-PCR primers sequences are shown in Table S1. β-Actin served as a reference gene to standardize the levels of the target genes. The data were computed with 2^−ΔΔCt method.

Western blot

ST cells were lysed in RIPA solution (APExBIO, Houston, USA) supplemented with PMSF (Seven, Beijing, China) and protease inhibitor cocktail (MedChemExpress, USA) for the purpose of extracting total proteins as reported previously [45, 46]. The same amount of protein was electrophoresed on SDS-PAGE gels (Yeason, China), followed by transferring to nitrocellulose membranes. After blocking with 5% skim milk, the primary antibodies [β-actin rabbit monoclonal antibody (GeneTex, USA); TFRC, TF, PCBP1, ACSL4, LPCAT3, SLC3A2, NRF2, GPX4, HO-1, CAT, P62, Beclin1, LC3, AMBRA1, TOMM20, PINK1, and Parkin (ABclonal, China); P53, SLC40A1, and KEAP1 (Bioss, China); SLC7A11 and NQO1 (Proteintech, USA); COX2 (Affinity, USA)] were incubated overnight at 4 °C before incubation with the secondary antibodies (Zhongshan Jinqiao Biotechnology Co., Ltd., Beijing, China) for visualization. Image acquisitions were captured with an Amersham Imager (GE, Switzerland) and Image J was used to quantify protein bands.

Immunofluorescence (IF) analysis

The procedure for IF staining was performed as previously documented [47, 48]. The ST cells were fixed with 4% formaldehyde for 15 min and were then permeabilized with 0.5% Triton X-100 for 10 min. The ST cells were blocked with 1% BSA for 30 min at 37 °C and incubated with corresponding primary antibodies overnight at 4 °C. Then, the cells were exposed to respective secondary antibodies for 1 h. The nucleus was counterstained with DAPI (Beyotime, China) and images were visualized using a laser scanning fluorescence microscopy (Leica, Germany).

Cellular thermal shift assay (CETSA)

CETSA experiments were carried out according to the protocol described [49]. Briefly, ST cells exposed to ZEA were harvested, and resuspended in PBS that contained complete protease inhibitor mix, followed by ultrasonic lysis. The cell suspensions were equally distributed into PCR microtubes and incubated for 30 min. Afterward, each cell suspension sample was heated separately at the specified temperatures for 3 min and centrifuged at 20,000 × g for 30 min at 4 °C. The supernatants were collected and analyzed by western blotting to assess protein stability.

Molecular docking and molecular dynamics simulations

Molecular docking was executed using AutoDock software, and simulation of molecular dynamics were performed using GROMACS 2020 according to the method established previously [50, 51]. In addition, the result was visually analyzed by use of the PyMol. The detailed procedures are shown in Supplementary Methods 1.2.

Statistical analysis

The analysis of experiment data was generated using GraphPad Prism version 9.5. The measurement data are presented as the mean ± SD and all tests contained repetition a minimum of three times. One-way ANOVA was used for multiple group comparison, and Student’s t-tests was used for two-group comparisons. Pairwise comparisons were carried out using a Tukey post-hoc test. The value of P < 0.05 was indicated statistically significant.

Results

ZEA induced structural and functional damage to mitochondria of ST cells

To evaluate the potential effects of ZEA on male reproductive function, we constructed ZEA exposure models in ST cells (Fig. 1A). The results demonstrated that the cells viability showed a progressive downward trend with the increasing concentration of ZEA, with 10 μmol/L ZEA selected for subsequent experiments (P < 0.05; Fig. 1B). Transcriptomic analysis revealed marked alterations in gene expression profiles following ZEA treatment, as evidenced by PCA and volcano plots (Fig. 1C and D). Subsequently, all DEGs were conducted for GO, KEGG pathway analyses and GSEA (Fig. 1E–G). Our present study proved that ZEA exposure caused pronounced mitochondrial injury, including cristae disruption, severe vacuolization, decreased mitochondria volume density and increased Flameng score (Fig. 1H). Excessive ROS production is a major driver of lipid peroxidation and change the integrity of mitochondrial membranes. Our results also demonstrated that ZEA exposure markedly increased ROS accumulation (Fig. 1J). Furthermore, the result showed the decreased MMP levels after ZEA treatment (Fig. 1I). Collectively, these findings indicated that ZEA exposure induced both structural and functional mitochondrial impairment, ultimately driving oxidative injury and mitochondrial dysfunction in ST cells.

Fig. 1.

ZEA induced structural and functional damage to mitochondria of ST cells. A The ST cells were treated with ZEA. B CCK-8 assay. C PCA analysis. D Volcano plots analysis. E KEGG analysis. F GO analysis. G GSEA analysis. H TEM analysis; volume density of mitochondria; Flameng score; yellow: mitochondria. I MMP level. J ROS production. Data are presented as the mean ± SD. Symbol for the significance of differences between the CON group and ZEA group: **P < 0.01, ***P < 0.001

ZEA induced ferroptosis in ST cells

Our research results from KEGG and GSEA found alterations in glutathione enrichment (Fig. 1E and G). To further verify our hypothesis, we subsequently examined classical ferroptosis-related parameters. Western blot analysis showed an upward trend in the protein levels of TF, TFRC, ACSL4, LPCAT3, COX2, and P53, accompanied by a downward trend in PCBP1 and SLC7A11, SLC3A2, and SLC40A1 in ZEA exposure group (P < 0.01; Fig. 2A and B). Consistently, IF analysis revealed elevated fluorescence intensity of TFRC and P53, while SLC7A11 fluorescence intensity was reduced (Fig. 2G–I). In addition, ZEA treatment enhanced Mito-FerroGreen and FerroOrange signal intensity, suggesting a strongly enhancement in the levels of mitochondrial and intracellular ferrous iron (Fig. 2C and D). The data revealed that the levels of mitochondrial ROS and lipid peroxidation were significantly up-regulated after ZEA exposure (Fig. 2E and F). These findings indicated that ZEA exposure could lead to ST cell injury by inducing ferroptosis.

Fig. 2.

ZEA induced ferroptosis in ST cells. A Expression of ferroptosis-related proteins. B Quantitation of the relative expression of ferroptosis-related proteins. C Intracellular iron level. D Mitochondrial iron level. E Mitochondrial ROS level. F Lipid peroxidation level. G Representative IF images of SLC7A11. H Representative IF images of TFRC. I Representative IF images of p53. Data are presented as the mean ± SD. Symbol for the significance of differences between the CON group and ZEA group: **P < 0.01, ***P < 0.001

ZEA restrained NRF2/KEAP1/SLC7A11 signaling pathway in ST cells

We investigated whether ZEA-induced ferroptosis plays a role in modulating antioxidant responses through the Nrf2 signaling pathways. IF analysis revealed a marked reduction in nuclear NRF2 accumulation in ZEA-treated ST cells (Fig. 3A). In addition, the result showed that the protein expression of NRF2, GPX4, HO-1, NQO1 and CAT were obviously reduced, but the protein expression of KEAP1 was obviously enhanced (P < 0.01; Fig. 3B and C). At the transcript level, qPCR analysis revealed that ZEA exposure reduced the mRNA expression of NRF2 and its downstream antioxidant genes, such as TXNRD1, CAT, GCLC, GCLM, SOD1, SOD2, SOD3, NQO1, HO-1, and GPX4, whereas KEAP1 expression was upregulated (P < 0.01; Fig. 3D and Fig. S1). This was consistent with the decreases in the fluorescence intensities of its downstream targets, NQO1 and SOD2 (Fig. 3F and G). The result of correlation analysis revealed that ZEA-induced ferroptosis was tightly associated with the decrease of NRF2 protein level (Fig. 3E). Additionally, the parameters of ferroptosis indicators were assessed by using PCA, which further certified that ZEA exposure influenced the expression of ferroptosis indicators (Fig. 3H). Collectively, these results suggested that ZEA exposure suppressed NRF2 signaling pathway, resulting in a decline in antioxidant capacity and thus contributing to ferroptosis in ST cells.

Fig. 3.

ZEA restrained NRF2/KEAP1/SLC7A11 signaling pathway in ST cells. A Representative IF images of NRF2. B Expression of NRF2 and its downstream antioxidant proteins. C Quantitation of the relative expression of NRF2 and its downstream antioxidant proteins. D Heatmap of relative mRNA levels of NRF2 and its downstream antioxidant indicators. E Correlation analysis. F Representative IF images of NQO1. G Representative IF images of SOD2. H PCA analysis. Data are presented as the mean ± SD. Symbol for the significance of differences between the CON group and ZEA group: **P < 0.01, ***P < 0.001

ZEA caused AMBRA1-mediated mitophagy inhibition in ST cells

Considering the close association between mitophagy and ferroptosis, we then identified whether ZEA exposure could affect mitophagy in ST cells. Western blot analysis showed that ZEA exposure induced a down-regulation of Beclin1, LC3II/I, AMBRA1, PINK1, and Parkin, and up-regulation of p62 and TOMM20 (Figs. 4A and 5A; P < 0.01). The qPCR analysis was consistent with the protein expression results (P < 0.01; Fig. 4B and Fig. S2). Consistently, the cellular IF further confirmed these findings, showing alterations in the distribution of P62, LC3, AMBRA1, and TOMM20 (Figs. 4C–E and 5B–C). Analysis of autophagic flux have demonstrated that ZEA caused an obvious decrease of yellow and red puncta, indicating an inhibition of autophagic flux (Fig. 4G and H). Notably, the result suggested that ZEA-induced decrease in the LC3II/I ratio was restored in the presence of Rapa (P < 0.001; Fig. 4F). Consistently, we observed that ZEA exposure diminished red puncta after Baf-A1 treatment, and increased both yellow and red puncta in Rapa-treated cells (P < 0.01; Fig. 4G and H). These results collectively suggested that ZEA mainly restrains autophagy initiation rather than enhances autophagosomes clearance in the late phase. Mito-Keima is a reliable indicator for evaluating mitophagy, which was applied to detect the ability of mitophagy. The data indicated that ZEA resulted in a decrease of the red fluorescence and an increase of green fluorescence, indicating that ZEA suppress the activity of mitophagy in ST cells. Notably, Rapa treatment partially restored red fluorescence, indicating recovery of mitophagic flux (Fig. 4I). In addition, we confirmed that ZEA-induced mitophagy inhibition was accompanied by accumulation of acidic puncta (Fig. 4I). Moreover, we constructed a PPI network diagram of the related genes, illustrating the interconnections among ferroptosis, autophagy and mitophagy (Fig. 5I). Given that AMBRA1 is a crucial positive regulator of autophagy initiation and mitochondrial clearance, we further investigated the possibility of direct interaction between ZEA and AMBRA1. Molecular docking result demonstrated a stable binding between ZEA and AMBRA1 (Fig. 5D), binding energy values summarized in Table S2. Furthermore, molecular dynamics simulation clarified that the stability of ZEA and AMBRA1 complex was firmly supported (Fig. 5E–H). Furthermore, CETSA results demonstrated that ZEA exposure markedly enhanced the thermal stability of AMBRA1, further providing evidence that ZEA directly interacts with AMBRA1 (Fig. 5J). Taken together, these findings revealed that ZEA exposure could inhibit autophagy and mitophagy by downregulating AMBRA1 in ST cells.

Fig. 4.

ZEA caused autophagy inhibition in ST cells. A Expression of autophagy-related proteins. B Relative mRNA levels of autophagy genes. C Representative IF images of P62. D Representative IF images of LC3. E Quantitation of p62 and LC3. F The protein levels of LC3II/I. G Quantification of autophagosomes and autolysosomes. H mRFP-GFP-LC3 adenovirus transfection. I Mito-Keima fluorescence. Data are presented as the mean ± SD. Symbol for the significance of differences between the CON group and ZEA group: **P < 0.01 ***P < 0.001. Symbol for the significance of differences between the ZEA group and Rapa+ZEA group: ^###P < 0.001 

Fig. 5.

ZEA caused AMBRA1-mediated mitophagy inhibition in ST cells. A Expression of mitophagy-related proteins. B Representative IF images of AMBRA1 protein. C Representative IF images of TOMM20 protein. D Molecular docking simulation for the ligand–protein binding of ZEA with AMBRA1. E RMSD. F Rg. G Buried SASA. H Hbond Number. I PPI network. J CETSA. Data are presented as the mean ± SD. Symbol for the significance of differences between the CON group and DON group: ***P < 0.001

AMBRA1 overexpression mitigates ZEA-induced suppression of mitophagy in ST cells

To elucidate the functional involvement of AMBRA1 in ZEA-mediated inhibition of mitophagy, ST cells were subjected to transfect with AMBRA1 overexpression constructs (Fig. 6A). As expected, western blot analysis demonstrated that AMBRA1 overexpression markedly reversed the reduction of AMBRA1, PINK1 and Parkin protein levels, and the increase of TOMM20 upon ZEA exposure (P < 0.05; Fig. 6B). Consistent with the findings in western blot, IF analysis revealed comparable changes in AMBRA1 and TOMM20 (Fig. 6C and D). Mito-Keima analysis further confirmed that overexpression of AMBRA1 reversed accumulation of ZEA-induced green fluorescence and markedly increased the red signal, indicating restoration of mitophagic flux (Fig. 6E). Furthermore, we also examined autophagy-related indicators. The data showed that AMBRA1 overexpression significantly relieved ZEA-induced decrease in LC3II/I ratios and Beclin1 protein levels, while preventing the augment in P62 protein levels (P < 0.001; Fig. 6G). Correspondingly, the IF results were consistent with the results of western blot after AMBRA1 overexpression treatment (Fig. 6H and I). The results also revealed that AMBRA1 overexpression effectively mitigated the ZEA-induced reduction in RFP-GFP-LC3 puncta, suggesting that the impaired autophagosome formation was effectively rescued (Fig. 6F and Fig. S5). Given the above, the results provided compelling evidence that AMBRA1 overexpression effectively counteracted ZEA-induced suppression of mitophagy, thereby restoring both mitophagy and autophagy activity in ST cells.

Fig. 6.

AMBRA1 overexpression mitigates ZEA-induced suppression of mitophagy in ST cells A ST cells were treated with AMBRA1 and/or ZEA. B Expression of mitophagy-related proteins. C Representative IF images of AMBRA1. D Representative IF images of TOMM20. E Mito-Keima fluorescence. F mRFP-GFP-LC3 adenovirus transfection. G Expression of autophagy-related proteins. H Representative IF images of p62 protein. I Representative IF images of LC3 protein. Data are presented as the mean ± SD. Symbol for the significance of differences between the NC + CON group and NC + ZEA group: ***P < 0.001. Symbol for the significance of differences between the NC + ZEA group and AMB + ZEA group: ^#P < 0.05, ^##P < 0.01, ^###P < 0.001

AMBRA1 overexpression alleviated ZEA-induced inhibition of NRF2 signal pathway in ST cells

To demonstrate the role of AMBRA1 in ZEA-induced inhibition of NRF2 signaling pathway, we detected the expression of NRF2 and its downstream signaling components. IF analysis confirmed that AMBRA1 overexpression promoted NRF2 nuclear translocation (Fig. 7A). The data also revealed that overexpression of AMBRA1 could attenuate ZEA-induced decrease in NRF2, GPX4, HO-1, NQO1 and CAT protein expression, while simultaneously reversing the abnormal accumulation of KEAP1(P < 0.01; Fig. 7B and D). Consistently, IF assay demonstrated that the fluorescence intensity of SOD2 and NQO1 were markedly increased following AMBRA1 overexpression (Fig. 7E). After that, the NRF2 and its downstream factors were detected using PCA analysis, which further demonstrated that AMBRA1 overexpression effectively restored the ZEA-induced dysregulation of NRF2 signaling pathway (Fig. 7F). The results also revealed that there was a close association between NRF2 and autophagy-related genes (Fig. 7C). We then carried out RDA analyses to certified that ZEA-induced inhibition of NRF2 signaling pathway and mitophagy were positively related to the decrease of AMBRA1 protein level (Fig. 7G). Collectively, these findings demonstrated that AMBRA1 overexpression could restore ZEA-induced inhibition of NRF2 signal pathway in ST cells.

Fig. 7.

AMBRA1 overexpression alleviated ZEA-induced inhibition of NRF2 signal pathway in ST cells. A Representative IF images of NRF2. B Expression of NRF2 and its downstream antioxidant proteins. C Chords diagrams. D Quantitation of the relative protein expression of NRF2 and its downstream antioxidant proteins. E Representative IF images of SOD2 and NQO1. F PCA analysis. G RDA analysis. Data are presented as the mean ± SD. Symbol for the significance of differences between the NC + CON group and NC + ZEA group: ***P < 0.001. Symbol for the significance of differences between the NC + ZEA group and AMB + ZEA group: ^#P < 0.05, ^##P < 0.01, ^###P < 0.001

AMBRA1 overexpression mitigated ZEA-induced ferroptosis by facilitating mitophagy in ST cells

To identify the role of AMBRA1 in ZEA-induced ferroptosis, we detected ferroptosis-related markers in ST cells with or without AMBRA1 overexpression under ZEA exposure. We found that AMBRA1 overexpression reversed ZEA-induced downregulation in the protein expression of PCBP1, SLC3A2, SLC7A11 and SLC40A1, as well as counteracting the enhancement in the protein expression of TF, TFRC, ACSL4, LPCAT3, COX2 and p53 (P < 0.01; Fig. 8A and B). Consistently, we found that AMBRA1 overexpression restored the fluorescence intensities of SLC7A11, p53 and TFRC (Fig. S3). The result indicated that AMBRA1 overexpression ameliorated the ZEA-induced decrease of MMP, suggesting the recovery of mitochondrial function (Fig. S4). In addition, AMBRA1 overexpression markedly attenuated ZEA-induced accumulation of mitochondrial ROS and lipid peroxidation (Fig. 8C and D). Consequently, we further detected the levels of ferrous iron in ST cells and found that both intracellular and mitochondrial ferrous iron levels were significantly decreased following AMBRA1 overexpression, reversing the ZEA-induced iron overload (Fig. 8E and F). As a whole, these findings indicated that AMBRA1 overexpression not only restored ferroptosis-related protein expression but also preserved mitochondrial function and redox homeostasis under ZEA exposure. Importantly, AMBRA1 mitigated ZEA-induced ferroptosis by regulating AMBRA1-induced mitophagy in ST cells (Fig. 9).

Fig. 8.

AMBRA1 overexpression mitigated ZEA-induced ferroptosis by facilitating mitophagy in ST cells. A Expression of ferroptosis-related proteins. B Quantitation of the protein expression of ferroptosis-related proteins. C Mitochondrial ROS level. D lipid peroxidation level. E Intracellular iron level. F Mitochondrial iron level. Data are presented as the mean ± SD. Symbol for the significance of differences between the NC + CON group and NC + ZEA group: **P < 0.01, ***P < 0.001. Symbol for the significance of differences between the NC + ZEA group and AMB + ZEA group: ^#P < 0.05, ^##P < 0.01, ^###P < 0.001

Fig. 9.

The mechanistic of AMBRA1 activation alleviates zearalenone-induced swine testicular cell ferroptosis by facilitating mitophagy

Discussion

ZEA is a mycotoxin with estrogen-like properties that frequently contaminates cereal crops, which has raised concerns about its potential effects on both the ecological environment and animal health [52]. Accumulating evidence has shown that ZEA exerts toxic impacts on multiple organs, especially the kidney, liver, gastrointestinal tract and reproductive system. Among these, the detrimental effects of ZEA on the male reproductive system are severe and should not be overlooked, including testicular germ cell deficiency, impaired sperm function, reduced testosterone levels and reproductive organ damage [53]. Whereas, the potential mechanism of ZEA-induced male animal reproductive dysfunction remains largely unknown. Therefore, we performed in vitro experiments using ST cells to further clarify the mechanisms of ZEA-induced male reproductive function impairment. In this study, we demonstrated that ZEA exposure impaired mitochondrial structure and function, reduced MMP, and promoted excessive ROS generation, thereby causing oxidative stress in the ST cells. Subsequently, our results showed that ZEA exposure induced the abnormal metabolism of glutathione and the excessive accumulation of lipid peroxide, which collectively contributed to the induction of ferroptosis. Moreover, we proved that ZEA-induced ferroptosis was primarily driven by excessive ferrous iron overload and the suppression of mitophagy. Therefore, targeting mitophagy-related regulators may provide new insights into the treatment of ZEA-induced reproductive toxicity. AMBRA1 is a critical autophagy regulator and is recognized as a key factor in maintaining mitochondrial quality control [54]. We found that AMBRA1 overexpression activated mitophagy and thus alleviated ferroptosis, eventually mitigating ZEA-induced ST cell injury. Collectively, targeting AMBRA1-mediated mitophagy may provide a promising intervention strategy for the treatment of male animal reproductive disorders associated with ZEA exposure.

Mitochondria are a crucial organelle serving as a central hub for cellular metabolism and signaling within the cell, and its dysfunction is associated with a range of pathological diseases [55]. The mitochondrial quality control mechanism is crucial for sustaining mitochondrial function. Under various stress conditions, mitochondria frequently exhibit apparent alterations, such as fragmentation, swelling and loss of cristae integrity, and accompany by outer membrane rupture and inner membrane damage, ultimately compromising the damage of mitochondrial integrity [56]. The shape of mitochondria and their cristae structure can affect mitochondrial function. Accumulating evidence has demonstrated that AFB1 could induce mitochondrial morphology impairment and enhanced mitochondria-lipid droplet interactions [57]. FB1 exposure induced mitochondrial disorganization, with swelling, vacuolation and disrupted cristae [58]. Consistent with these findings, our study revealed that ZEA exposure induced significant mitochondrial morphological abnormalities. Moreover, mitochondrial dysfunction inhibits oxidative phosphorylation and reduces ATP generation, while excessive accumulation of ROS aggravates oxidative stress and cellular injury [59]. Previous studies have shown that DON is reported to induce mitochondrial injury and trigger oxidative stress in IPEC-J2 cells [60]. It is shown that OTA disrupts the mitochondrial structure and dynamics in cells, thereby resulting in the loss of mitochondrial homeostasis and promoting oxidative stress [61]. In agreement with above findings, our results demonstrated that ZEA exposure reduced MMP and pronouncedly increased ROS, thereby aggravating mitochondrial dysfunction. Overall, these findings further confirms that ZEA exposure can impair mitochondrial integrity and function, thereby contributing to oxidative stress in ST cells.

Ferroptosis involves a unique mechanism of cell death that is driven by iron-dependent lipid peroxidation, which plays a pivotal role in multiple diseases [62]. Growing evidence suggests that alterations in mitochondrial morphology and function are characteristic of ferroptosis, resulting in ROS overproduction and subsequent cell death [63]. Some reports showed distinct features of mitochondrial morphology in ferroptosis cells [64], which are consistent with our results. Interestingly, we also found that ZEA altered the expression of ferroptosis-related indicators and thus induced ferroptosis in ST cells. Emerging evidence have indicated that ferroptosis as a key pathological mechanism in male reproductive disorders disrupted testicular homeostasis and impaired sperm quality [65]. Moreover, environmental pollutants result in ferroptosis through enhancing oxidative stress and weakening antioxidant defense pathways, thereby leading to lipid peroxidation and male reproductive impairments such as sperm reduction, impaired motility, and morphological abnormalities [66]. Glutathione is essential for preserving the redox balance of the cell, and its depletion serves as a critical initiating signal of ferroptosis, thereby impairing antioxidant defense [67]. Ferroptosis is identified by the depletion of glutathione and the GPX4 inactivation, preventing the metabolism of lipid oxides by glutathione reductase catalyzed by GPX4. Consistently, our results suggested that ZEA exposure caused dysregulation of glutathione metabolism and inhibited the expression of GPX4 protein. Ferroptosis is dependent upon intracellular iron, accumulation of lipid ROS, and lipid peroxidation. We also found that ZEA obviously increased cytoplasmic and mitochondrial ROS and ferrous ion levels, and induced lipid peroxidation. NRF2, as a regulator of the cellular antioxidant defense, maintains redox homeostasis by inducing target genes that suppress lipid peroxidation, limit free iron accumulation, and correct oxidative imbalances. NRF2 activation inhibited ferroptosis by enhancing cellular antioxidant defenses through upregulation of glutathione synthesis and GPX4 expression [68]. We discovered that ZEA exposure inhibited NRF2 signaling pathway, thereby weakening the antioxidant capacity of ST cells. Collectively, these observations support the conclusion that ZEA can trigger ferroptosis by inhibiting the NRF2 signaling in ST cells.

Ferroptosis is an autophagic cell death process, and autophagy is a fundamental cellular process that maintains homeostasis through the clearance of misfolded proteins and damaged organelles [69]. Autophagy is indispensable for male reproductive function, particularly in regulating spermatogenesis, sperm maturation and testicular homeostasis [70]. Recent studies have suggested that DON exposure disrupts oocyte maturation and reproductive function by delaying meiotic progression, inducing excessive autophagy and apoptosis, ultimately compromising oocyte quality and fetal development [71]. In our study, ZEA exposure resulted in abnormal levels of autophagy-related proteins and thus inhibited autophagy in ST cells. Mitochondria are crucial organelles for maintaining testicular homeostasis and sperm quality by providing ATP required for spermatogenesis and sperm maturation [72]. Mitophagy as a selective form of autophagy plays a pivotal role in the male reproductive system. Emerging evidence have indicated that a strong association between mitochondrial function and ferroptosis as both processes play critical roles in the regulation of cellular oxidative metabolism [73]. Dysfunctional mitophagy results in the buildup of damaged mitochondria, generating ROS and subsequently causing oxidative stress and damage to intracellular iron metabolism [74]. Accumulating evidence have suggested that dysregulated mitophagy is closely associated with male reproductive impairment [31]. In addition, emerging data have demonstrated that toxicants and environmental stressors can disrupt autophagic flux, thereby contributing to reproductive disorders [75]. Consistently, our results demonstrated that ZEA exposure could suppress mitophagy activation and impair autophagic flux, which may underlie mitochondrial dysfunction. Based on above results, we propose that ZEA inhibits mitophagy and thus induces ferroptosis, ultimately resulting in male reproductive dysfunction.

AMBRA1 is increasingly recognized as a pivotal modulator of autophagy and mitophagy, exerting essential functions in maintaining mitochondrial quality control. Mechanistically, AMBRA1 modulated autophagy function primarily through the regulation of BECN1, thereby contributing to the initiation of autophagy [76]. In addition, AMBRA1 contributes to the clearance of damaged mitochondria through the PINK1/Parkin pathway, and directly interacts with LC3 to induce Parkin-independent mitophagy, thereby maintaining mitochondrial homeostasis [77]. Conversely, AMBRA1 deficiency disrupted mitophagic and autophagy flux, thereby promoting the progressive accumulation of damaged mitochondria and disrupting cellular homeostasis [78]. It has been proven that the silencing of the AMBRA1 gene determines a decrease of primordial germ cells and thus results in the development of all-male progeny, showing a close association between AMBRA1 and male reproductive health [79]. In line with above findings, our study suggested that ZEA exposure significantly reduced the expression of AMBRA1, which was accompanied by a decline in mitophagic flux and thus inhibited mitophagy in ST cells. Inhibition of AMBRA1 expression impairs mitophagy and leads to the accumulation of dysfunctional mitochondria and excessive production of ROS, thereby disrupting iron homeostasis and promoting ferroptosis [80]. NRF2 has a function in managing the redox balance within cells and is crucial in managing numerous important parts of the ferroptosis pathway [81]. Mechanistically, the interaction between AMBRA1 and the NRF2 signaling pathway in the regulation of ferroptosis is likely mediated through mitochondrial quality control and redox homeostasis. Overexpression of AMBRA1 can trigger mitophagy in SH-SY5Y cells to inhibit oxidative stress [82]. Impairment of AMBRA1-dependent mitophagy enhances mitochondrial ROS accumulation, which in turn suppresses NRF2-driven antioxidant responses and promotes ferroptosis. Consistently, we also found that ZEA-induced decrease of AMBRA1 level caused ROS overproduction and increased ferrous iron, eventually inducing ferroptosis in ST cells. Furthermore, overexpression of AMBRA1 activated autophagy and mitophagy, which inhibited the occurrence of ferroptosis. Our findings highlight the central role of AMBRA1 in linking mitophagy to ferroptosis and suggest its potential as a therapeutic target for preventing ZEA-induced male reproductive toxicity in animals.

Although our findings demonstrate that activation of AMBRA1 alleviates ZEA-induced ferroptosis by promoting mitophagy, several limitations should be acknowledged. First, the present study was primarily based on in vitro experiments, therefore, in vivo experiments is required in future studies to confirm these results. Second, while the pig model provides important value, additional experimental models may better capture more extensive reproductive pathological phenotypes. Moreover, future investigations will focus on exploring additional molecular mechanisms through which AMBRA1 confers protection against ZEA-induced reproductive toxicity.

Conclusion

In conclusion, our study demonstrated that ZEA targeted the AMBRA1, leading to down-regulation of AMBRA1 expression, which in turn inhibition mitophagy and thus resulted in ferroptosis in ST cells. Given the potential role of AMBRA1 in ST cells, our results uncover a previously unrecognized mechanism in which AMBRA1-mediated mitophagy functions as a crucial defense target against ferroptosis in testicular cells. Importantly, our results propose a unique insight which AMBRA1 as a promising therapeutic target for counteracting mycotoxin-induced testicular injury. Collectively, such paradigm not only provides a dependable insight in the regulation of testicular function by AMBRA1, but also provides a conceptual framework for developing novel intervention strategies to safeguard male reproductive health in animals.

Abbreviations

AMBRA1

Autophagy/Beclin-1 regulator 1

BafA1

Bafilomycin A1

CCK-8

Cell Counting Kit-8

CETSA

Cellular thermal shift assay

FBS

Fetal bovine serum

IF

Immunofluorescence

LDA

Linear Discriminant Analysis

MCE

MedChemExpress

MMP

Mitochondrial membrane potential

NRF2

Nuclear factor erythroid 2-related factor 2

NMDS

Nonmetric multidimensional scaling

PEA

Pepstatin A

qRT-PCR

Quantitative real-time PCR

Rapa

Rapamycin

ROS

Reactive oxygen species

RNA-seq

RNA-sequencing

SD

Standard deviation

ST

Swine testicular

TEM

Transmission electron microscopy

WB

Western Blot

ZEA

Zearalenone

3-MA

3-Methyladenine

Authors’ contributions

HZY, YSJ, ZXY: conceptualization, methodology, investigation, writing – original draft. LM, YQ and CY: conceptualization, methodology, investigation, writing – original draft. CYH, JFW and CMS: formal analysis, methodology. WJX, SYJ, ZJ, MXY and WYB: validation, formal analysis. ZY and LJL: project administration, conceptualization, writing-review & editing.

Funding

This study received assistance from National Natural Science Foundation of China (Grant No. 32402968), Key Program of Natural Science Foundation of Heilongjiang Province of China (Grant No. ZD2021C003), Outstanding Youth of Natural Science Foundation of Heilongjiang Province of China (Grant No. YQ2023C017), Academic Backbone Project of Northeast Agricultural University (Grant No. 54960212), Heilongjiang Postdoctoral Fund (Grant No. LBH-Z23010), Heilongjiang Postdoctoral Special Fund (Grant No. LBH-TZ2404), China Postdoctoral Science Foundation (Grant No. 2023MD744173) and Postdoctoral Special Funding of China (Grant No. 2024T170118).

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Declarations

Ethics approval and consent to participate

All procedures for experiments were conducted in accordance with the Guidelines for Care and Use of Laboratory Animals of Northeast Agricultural University.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.