Zearalenone Exposure Disrupts Blood–Testis Barrier Integrity through Excessive Ca2+-Mediated Autophagy

Zearalenone (ZEA), a common mycotoxin in grains and animal feeds, has been associated with male reproductive disorders. However, the potential toxicity mechanism of ZEA is not fully understood. In this study, in vivo and in vitro models were used to explore the effects of ZEA on the blood–testis barrier (BTB) and related molecular mechanisms. First, male BALB/C mice were administered ZEA orally (40 mg/kg·bw) for 5–7 d. Sperm motility, testicular morphology, and expressions of BTB junction proteins and autophagy-related proteins were evaluated. In addition, TM4 cells (mouse Sertoli cells line) were used to delineate the molecular mechanisms that mediate the effects of ZEA on BTB. Our results demonstrated that ZEA exposure induced severe testicular damage in histomorphology and an ultrastructural, time-dependent decrease in the expression of blood–testis barrier junction-related proteins, accompanied by an increase in the expression of autophagy-related proteins. Additionally, similar to the in vitro results, the dose-dependent treatment of ZEA increased the level of cytoplasmic Ca2+ and the levels of the autophagy markers LC3-II and p62, in conjunction with a decrease in the BTB junction proteins occludin, claudin-11, and Cx43, with the dislocation of the gap junction protein Cx43. Meanwhile, inhibition of autophagy by CQ and 3-MA or inhibition of cytoplasmic Ca2+ by BAPTA-AM was sufficient to reduce the effects of ZEA on the TM4 cell BTB. To summarize, this study emphasizes the role of Ca2+-mediated autophagy in ZEA-induced BTB destruction, which deepens our understanding of the molecular mechanism of ZEA-induced male reproductive disorders.


Introduction
Zearalenone (ZEA), also known as the F-2 or RAL mycotoxin, is produced by fungi belonging to the Fusarium family, which show strong estrogenic activity [1,2]. ZEA has been proved to induce reproductive toxicity, immunotoxicity, genotoxicity, hepatotoxicity, and so forth [1,3,4]. As the molecular structure of ZEA is similar to that of estrogen [5], it is particularly harmful to the reproductive system. A previous study illustrated that the presence of ZEA in the diet of piglets caused significant increases in vulva size, the genital

Histomorphological and Ultrastructural Changes in Testis after ZEA Exposure
Subsequently, we examined the histological morphology of the testis after ZEA exposure through hematoxylin and eosin (H&E) staining. As shown in Figure 2A, compared with the control group, after 5 d of ZEA treatment, the testes of mice showed obvious thinning of the seminiferous lumen, loose seminiferous epithelium, and vacuolar changes among cells. After 7 d of ZEA treatment, sloughing of spermatogenic cells into the lumen was observed. Then, we conducted transmission electron microscopy on the testes of the mice in the control group and those in the 7 d ZEA treatment group. After ZEA exposure, mitochondrial swelling and degeneration, destroyed BTB, and autophagosome accumulation were observed in the testes ( Figure 2B).

Histomorphological and Ultrastructural Changes in Testis after ZEA Exposure
Subsequently, we examined the histological morphology of the testis after ZEA exposure through hematoxylin and eosin (H&E) staining. As shown in Figure 2A, compared with the control group, after 5 d of ZEA treatment, the testes of mice showed obvious thinning of the seminiferous lumen, loose seminiferous epithelium, and vacuolar changes among cells. After 7 d of ZEA treatment, sloughing of spermatogenic cells into the lumen was observed. Then, we conducted transmission electron microscopy on the testes of the mice in the control group and those in the 7 d ZEA treatment group. After ZEA exposure, mitochondrial swelling and degeneration, destroyed BTB, and autophagosome accumulation were observed in the testes ( Figure 2B). Testicular histomorphological changes in mice after treatment with ZEA. The morphologies of spermatogenic cells and Sertoli cells were normal, and elongated spermatids can be seen in the seminiferous tubule in the corn-oil-treated group (a,b); compared with the corn-oil-treated group, after 5 d of ZEA exposure, the testes of mice showed obvious thinning of the seminiferous lumen, loose seminiferous epithelium, and vacuolar changes between cells (black arrow, (c,d)); after 7 d of ZEA exposure, the testes showed sloughing of spermatogenic cells into the lumen (red arrow, (e,f)).

Effects of ZEA on the Expressions of BTB and Autophagy-Related Regulating Proteins in Mice Testes
Based on the above models for mice, the effects of ZEA on BTB-related protein expressions in testes were evaluated. As shown in Figure 3, similar to its effects on BTB, ZEA increased the autophagy-related proteins p62, Beclin-1, ATG5, and LC3 and decreased the BTB-related junction proteins occludin, claudin-11, and Cx43 in a time-dependent manner. These results suggest that autophagy may be involved in ZEA-induced BTB disruption. Based on the above models for mice, the effects of ZEA on BTB-related protein expressions in testes were evaluated. As shown in Figure 3, similar to its effects on BTB, ZEA increased the autophagy-related proteins p62, Beclin-1, ATG5, and LC3 and decreased the BTB-related junction proteins occludin, claudin-11, and Cx43 in a timedependent manner. These results suggest that autophagy may be involved in ZEAinduced BTB disruption. After exposure, the expressions of p62, Beclin-1, ATG5, LC3, occludin, claudin-11, and Cx43 were evaluated. * p < 0.05 and ** p < 0.01, compared with the control group.

Effects of ZEA on the Expression of BTB-Related Regulating Proteins in TM4 Cells
As shown in Figure 4A, the viability of TM4 cells decreased with an increase in the ZEA concentration, indicating the toxic effect of ZEA on TM4 cells. Sertoli cells are integral for maintaining the integrity of BTB. Therefore, to confirm the in vivo results and test whether these BTB-related regulating proteins are involved in ZEA-induced Sertoli cell BTB disruption, the expressions of p62, LC3, junction protein occludin, claudin-11, and Cx43 were evaluated for TM4 cells. As shown in Figure 4B, similar to the in vivo results, an increase was observed in the expressions of the autophagy-related proteins p62 and LC3, in conjunction with a decrease in the BTB-related junction protein occludin, claudin-11, and Cx43 in TM4 cells in a dose dependent manner with ZEA. In addition, Figure 4C demonstrates that the localization of Cx43 at the interface of TM4 cells significantly decreased. The observed cytoskeleton fracture by ZEA suggests that Cx43 may enter the cell to induce degradation. After exposure, the expressions of p62, Beclin-1, ATG5, LC3, occludin, claudin-11, and Cx43 were evaluated. * p < 0.05 and ** p < 0.01, compared with the control group.

Effects of ZEA on the Expression of BTB-Related Regulating Proteins in TM4 Cells
As shown in Figure 4A, the viability of TM4 cells decreased with an increase in the ZEA concentration, indicating the toxic effect of ZEA on TM4 cells. Sertoli cells are integral for maintaining the integrity of BTB. Therefore, to confirm the in vivo results and test whether these BTB-related regulating proteins are involved in ZEA-induced Sertoli cell BTB disruption, the expressions of p62, LC3, junction protein occludin, claudin-11, and Cx43 were evaluated for TM4 cells. As shown in Figure 4B, similar to the in vivo results, an increase was observed in the expressions of the autophagy-related proteins p62 and LC3, in conjunction with a decrease in the BTB-related junction protein occludin, claudin-11, and Cx43 in TM4 cells in a dose dependent manner with ZEA. In addition, Figure 4C demonstrates that the localization of Cx43 at the interface of TM4 cells significantly decreased. The observed cytoskeleton fracture by ZEA suggests that Cx43 may enter the cell to induce degradation.

Effect of Autophagy on the Damage of BTB-Related Protein in TM4 Cells Induced by ZEA
In order to determine the relationship of autophagy in this pathological process, the regulation of BTB-related proteins in TM4 cells was evaluated using CQ (a classical lysosome inhibitor). The concentration of CQ was selected as 5 µmol/L by CCK8 and Western blotting ( Figure 5A,B). As shown in Figure 5C, the expressions of occludin, claudin-11, and Cx43 in the CQ-and-ZEA-combined treatment group were significantly higher than those in the ZEA-only treatment group. In addition, after TM4 cells were treated with ZEA and 3-MA for 24 h, LC3-II fluorescence was evenly distributed in the cytoplasm of TM4 cells in the control group and the group with 3-MA treatment alone, no obvious fluorescent autophagosome structure was observed, and Cx43 was evenly distributed on the cell membrane. In contrast, ZEA-treated fluorescent LC3-II autophagosomes significantly increased, and Cx43 was no longer evenly present around the cells, indicating that Cx43 may enter the cell and cause degradation through autophagy. After the inhibition of autophagy by 3-MA-combined treatment, it was found that fluorescent autophagosomes significantly decreased, and the localization of Cx43 was normal ( Figure 5D).

Effect of Autophagy on the Damage of BTB-Related Protein in TM4 Cells Induced by ZEA
In order to determine the relationship of autophagy in this pathological process, the regulation of BTB-related proteins in TM4 cells was evaluated using CQ (a classical lysosome inhibitor). The concentration of CQ was selected as 5 μmol/L by CCK8 and Western blotting ( Figure 5A,B). As shown in Figure 5C, the expressions of occludin, claudin-11, and Cx43 in the CQ-and-ZEA-combined treatment group were significantly higher than those in the ZEA-only treatment group. In addition, after TM4 cells were treated with ZEA and 3-MA for 24 h, LC3-II fluorescence was evenly distributed in the cytoplasm of TM4 cells in the control group and the group with 3-MA treatment alone, no obvious fluorescent autophagosome structure was observed, and Cx43 was evenly distributed on the cell membrane. In contrast, ZEA-treated fluorescent LC3-II autophagosomes significantly increased, and Cx43 was no longer evenly present around the cells, indicating that Cx43 may enter the cell and cause degradation through autophagy. After the inhibition of autophagy by 3-MA-combined treatment, it was found that fluorescent autophagosomes significantly decreased, and the localization of Cx43 was normal ( Figure 5D).  Figure 6A. BAPTA-AM can significantly reduce the increase in intracellular calcium induced by ZEA. Apart from this, treatment with BAPTA-AM significantly inhibited the decrease in cell viability induced by ZEA ( Figure 6B). These results strongly suggest that BAPTA-AM can effectively mitigate the cytotoxic damage caused by ZEA. After treatment with ZEA and BAPTA-AM, the expression of autophagy and BTB-related proteins was detected by Western blotting. As shown in Figure 6C, BAPTA-AM can effectively reduce the increase in the autophagic proteins p62 and LC3 caused by ZEA and increase the expressions of the BTB-related proteins occludin, claudin-11, and Cx43. These findings demonstrate that Ca 2+ -mediated autophagy may be involved in ZEA-induced BTB disruption.

Effect of Ca 2+ -Mediated Autophagy on the Damage to BTB-Related Proteins in TM4 Cells Induced by ZEA
BAPTA-AM is a specific chelator of Ca 2+ . After pretreatment with 5 μmol/L BAPTA-AM, cells were treated with 20 μmol/L ZEA for 24 h. Changes in Ca 2+ were detected using flow cytometry, as shown in Figure 6A. BAPTA-AM can significantly reduce the increase in intracellular calcium induced by ZEA. Apart from this, treatment with BAPTA-AM significantly inhibited the decrease in cell viability induced by ZEA ( Figure 6B). These results strongly suggest that BAPTA-AM can effectively mitigate the cytotoxic damage caused by ZEA. After treatment with ZEA and BAPTA-AM, the expression of autophagy and BTB-related proteins was detected by Western blotting. As shown in Figure 6C, BAPTA-AM can effectively reduce the increase in the autophagic proteins p62 and LC3 caused by ZEA and increase the expressions of the BTB-related proteins occludin, claudin-

Discussion
Zearalenone is present in a wide range of human foods, and studies have shown that zearalenone can cause male sterility and neurological dysfunction [30]. The purpose of this study was to investigate the effects of ZEA on BTB and the related in vivo and in vitro molecular mechanisms. In order to establish an in vivo model of ZEA-induced male reproductive injury, mice were administered ZEA orally at a dosage of 40 mg/kg·bw by gavage for 5 and 7 d according to previous reports to simulate a high-exposure scenario [12,31]. Reportedly, the contamination of ZEA in grains and animal feeds can range from 4 to 11,192 μg/kg [32]. Furthermore, the dose for the lowest observed effect level for developmental and reproductive toxicity has been reported to be approximately 20 mg/kg·bw in rodents [33]. Considering the sensitivity of pigs to ZEA [34] and the differences between rodents, pigs, and ruminants, we chose a ZEA dosage of 40 mg/kg·bw and implemented it in the present study. Our data show that ZEA exposure decreased sperm motility. Hyperchromatic nuclei were found in the lumen of the ZEA treatment group, indicating that spermatogenic cells fell into the lumen. Meanwhile, we observed mitochondrial swelling and degeneration, destroyed BTB, and autophagosome accumulation in the testis after ZEA exposure, which is supported by previous studies [31,35], indicating that the mouse-based model of ZEA-induced male reproductive injury is valid. The reason for the spermatogenesis impairment in immature testis after ZEA exposure remains unclear. BTB is a well-known prerequisite for spermatogenesis. When junctional proteins are abnormally expressed in Sertoli cells, spermatogenic cells do not show differentiation or meiosis, resulting in infertility [36][37][38]. We also found that autophagic proteins increased, and BTB-related proteins decreased with an increase in and Cx43 using Western blot analysis. TM4 cells were exposed to 20 µmol/L ZEA for 24 h with or without BAPTA-AM pretreatment (* p < 0.05 and ** p < 0.01, compared with the control group; # p < 0.05 and ## p < 0.01, compared with the ZEA-treated groups).

Discussion
Zearalenone is present in a wide range of human foods, and studies have shown that zearalenone can cause male sterility and neurological dysfunction [30]. The purpose of this study was to investigate the effects of ZEA on BTB and the related in vivo and in vitro molecular mechanisms. In order to establish an in vivo model of ZEA-induced male reproductive injury, mice were administered ZEA orally at a dosage of 40 mg/kg·bw by gavage for 5 and 7 d according to previous reports to simulate a high-exposure scenario [12,31]. Reportedly, the contamination of ZEA in grains and animal feeds can range from 4 to 11,192 µg/kg [32]. Furthermore, the dose for the lowest observed effect level for developmental and reproductive toxicity has been reported to be approximately 20 mg/kg·bw in rodents [33]. Considering the sensitivity of pigs to ZEA [34] and the differences between rodents, pigs, and ruminants, we chose a ZEA dosage of 40 mg/kg·bw and implemented it in the present study. Our data show that ZEA exposure decreased sperm motility. Hyperchromatic nuclei were found in the lumen of the ZEA treatment group, indicating that spermatogenic cells fell into the lumen. Meanwhile, we observed mitochondrial swelling and degeneration, destroyed BTB, and autophagosome accumulation in the testis after ZEA exposure, which is supported by previous studies [31,35], indicating that the mouse-based model of ZEA-induced male reproductive injury is valid. The reason for the spermatogenesis impairment in immature testis after ZEA exposure remains unclear. BTB is a well-known prerequisite for spermatogenesis. When junctional proteins are abnormally expressed in Sertoli cells, spermatogenic cells do not show differentiation or meiosis, resulting in infertility [36][37][38]. We also found that autophagic proteins increased, and BTB-related proteins decreased with an increase in ZEA exposure time, indicating that BTB damage caused by ZEA is an important factor for the decrease in sperm motility, and autophagy may be involved in the regulation of BTB by ZEA.
CQ is a classical lysosome inhibitor, which inhibits the binding of lysosomes and autophagosomes by inducing the disintegration of the Golgi matrix and nucleosome/lysosome system and blocking the autophagy pathway downstream [39]. Our study found that CQ combined with ZEA can reverse the downregulation of occludin, claudin-11, and Cx43 proteins caused by ZEA. Autophagy is one of the most important degradation pathways in intracellular protein metabolism. Studies have found that external stimuli can promote the endocytosis and degradation of membrane surface proteins by activating the autophagy pathways of epithelial cells: nano-alumina can activate autophagy and mitochondrial damage in brain endothelial cells, thereby downregulating the expression of the tight junction proteins occludin and claudin-5. Pretreatment with Warman penicillin, an inhibitor of phagocytic pathways, can significantly alleviate the downregulation of connexin caused by alumina [40]. In addition, after glucose and oxygen deprivation, mouse brain microvascular endothelial cells (bEND3) can enter the cytoplasm through caveolin-1 targeting the membrane surface connexin claudin-5 and enter LC3-II autophagosomes for degradation. The inhibition of autophagy or blocking of the binding of lysosomes to autophagosomes could prevent the degradation of claudin-5 [41]. In addition, we treated TM4 cells with 3-MA, an inhibitor of the upstream PI3K/Beclin-1/Atg13 complex of autophagy [42], which inhibited autophagy. Moreover, we detected the colocalization of LC3-II and Cx43 by immunofluorescence and found that ZEA can transfer the membrane protein Cx43 to the cytoplasm and cause degradation by activating autophagy pathways, whereas the inhibition of autophagy by 3-MA could significantly inhibit the degradation of Cx43. Interestingly, the results were consistent with those of previous studies that reported that 3-MA could alleviate the increase in intestinal permeability and the decrease and recombination of TJ proteins induced by burns [43]. Overall, these results suggest that autophagy may be involved in ZEA-induced BTB disruption.
Intracellular free Ca 2+ ions are important signaling molecules in cells, participating in various physiological and pathological processes [44]. Free Ca 2+ ions in the cell are input from the outside of the cell mainly through the Na + /Ca 2+ exchanger, the plasma membrane Ca 2+ -ATPase (PMCA). An increase in the intracellular free Ca 2+ concentration is mainly caused by extracellular Ca 2+ ions through the voltage-gated ion channel (VGCC). Ca stores regulate the Ca 2+ receptor-operated channels (ROCs) into cells, and intracellular Ca stores release Ca 2+ ions into cells through IP3 and ryanodine receptor Ca channels [45][46][47]. Many studies have shown that an increase in intracellular Ca 2+ concentrations is the main cause of apoptosis and even death [48,49]. Our results showed that ZEA could increase the concentration of intracellular Ca 2+ in a dose-dependent manner, and the decrease in cell viability induced by ZEA can be effectively alleviated by adding the intracellular Ca integrator BAPTA-AM. These results show that a Ca 2+ overload in TM4 cells may be caused by the release of intracellular Ca stores by ZEA, which is consistent with the results of our previous study [27]. Our study found that BAPTA-AM combined with ZEA can reverse the upregulation of LC3-II and p62 proteins induced by ZEA, indicating that the increase in intracellular Ca may be involved in autophagy induced by ZEA. Interestingly, when we combined BAPTA-AM with ZEA, we found that BAPTA-AM could alleviate the downregulation of tight and gap junction-related proteins induced by ZEA. These results strongly suggest that Ca 2+ can mediate the downregulation of these proteins in this pathological process. Indeed, Sertoli cell BTB-related junction proteins are reportedly the substrates that are indirectly regulated by Ca 2+ [50,51]. Overall, our results strongly suggest that Ca 2+ -mediated autophagy may be involved in ZEA-induced BTB disruption, which could contribute to a better understanding of potential molecular mechanisms of ZEA-induced reproductive toxicity, and possibly a novel potential target for preventive and/or therapeutic treatments.

Conclusions
In summary, our study confirmed that ZEA disrupted BTB by increasing the concentration of Ca 2+ in the cytoplasm and inducing the accumulation of autophagosomes (Figure 7). Thus, the substantial activation of Ca 2+ , elevation in the expression of autophagy-related proteins, and decrease in dislocation of tight and gap junction-related proteins provide a novel molecular mechanism for ZEA-induced BTB disruption and related male reproductive disorders.

Animal and Treatment
Eighteen adult male BALB/C mice (6 weeks old, from the Comparative Medicine Center of Yangzhou University, China) were housed in a room with a controlled temperature (25 ± 1 • C) and 12 /12 h light/dark cycle. The mice were given free to access to drinking water and mouse chow. The care and use of the animals during this experiment followed the guidelines of the Animal Care and Welfare Committee of Yangzhou University. The license number of the experimental animals was SYXK (Su) 2021-0026, and the time was 26 March 2021 to 25 March 2026. In this study, mice were randomly divided into three groups, and ZEA at a dose of 40 mg/kg·/bw was administered orally by gavage for 5 and 7 d according to previous reports, which was conducted to simulate the high-exposure scenario [12,30]. For the control group, mice were given the same volume of corn oil. After final exposure, the mice were humanely euthanized, and the testes were isolated and weighted.

Sperm Motility
Freshly isolated epididymis was briefly placed in 2 mL normal saline. Six deep cuts were made in each cauda using microscissors to release sperm into the media. The sperm suspension was incubated in a water bath for 20 min; after 10 min of incubation (at 37 • C), a drop of sperm suspension was placed in a preheated blood cell count board. The Integrated Semen Analysis System (ISAS, Paterna, Spain) was used to analyze sperm motility.

Histopathological Observation of the Testes
Simply, testes were fixed in 4% paraformaldehyde for 24 h and then washed in running water for 6 h, dehydrated with ethanol gradient embedded in paraffin, and sectioned. Hematoxylin and eosin staining was used for routine staining. Images were observed under a Leica microscope.

Ultrastructure Observation of the Testes
The experiment was conducted under the guidance of standard technology in the pathology electron microscope equipment of Yangzhou University.

Cell Culture
TM4 cells (mouse Sertoli cell line) were purchased from the American Type Culture Collection (ATCC, Rockefeller, MD, USA). Cells were cultivated in DMEM/F12 and added to a 10% FBS 1% of penicillin and 1 mM glutamine medium and cultured at 37 • C with 5% CO 2 .

Cell Proliferation Assay
After the cells were plated at a density of 1 × 104 per well in a 96-well plate. Control wells, blank wells, and experimental wells were set separately, with 6 replicates in each group. CCK-8 solution (10 µM) was added to each well, the culture plate was placed in an incubator for 2-4 h, and the absorbance was determined at 450 nm.

Measurement of Intracellular Ca 2+
Immediately after the cells were treated according to the processes mentioned above, they were collected and induced with Fluo-3AM at 37 • C in the dark for 30 min. The cells were monitored using a flow cytometer at an excitation wavelength of 488 nm.

Western Blotting Analysis
Freshly isolated testis and TM4 cells were lysed, and the concentration of protein was measured using a BCA protein assay kit, followed by Western blot at a concentration of 10-30 µg. After electrophoresis, proteins were transferred to a polyvinylidene fluoride membrane and incubated in 5% skim milk for 2 h. The membranes were incubated with primary antibodies at 4 • C for 12 h, washed 5 times with TBST, and then incubated with HRP-linked secondary antibodies for 2 h. After washing 5 times with TBST, the protein signals were detected using an ECL detection system and analyzed with the ImageJ software.

Immunofluorescence Analysis
TM4 cells were seeded in a 24-well plate. After treatment, the cells were washed and fixed in 4% paraformaldehyde at 4 • C for 30 min. After washing, they were treated with 0.5% Triton X-100 at room temperature for 15 min and then incubated with 5% BSA sealing solution for 1 h. The cells were then incubated overnight with the corresponding primary antibody. After being washed, the cells were then incubated with fluorescent-labelled secondary antibodies for 1 h in the dark. The localization of Cx43 and LC3 in TM4 cells was detected via confocal fluorescence microscopy.  Institutional Review Board Statement: The authors confirm that the ethical policies of the journal, as noted on the journal's author guidelines page, have been adhered to. The care and use of the animals during this experiment followed the guidelines of the Animal Care and Welfare Committee of Yangzhou University. Approved ID: SYXK (Su) 2021-0026.