TY - JOUR
AU - Lei,, Minggang
AB - Abstract Zearalenone (ZEA) has been proved to be toxic, particularly to the reproductive system of gilts. The effect of ZEA on gilts during embryo implantation window period is of particular interests. Here, we observed window stage dysontogenesis of gilts treated with ZEA. In endometrial tissues and cells, autophagosomes increased significantly and mitochondria were damaged with increasing ZEA concentration. Addition of autophagy inhibitor confirmed that ZEA blocks the autophagic flow in the fusion of autophagosomes and lysosomes. In conclusion, ZEA exposure during embryo implantation results in endometrium inflammation by activating autophagy while blocking autophagy flow at the same time, leading to the significant accumulation of autophagosomes. The aforementioned effects of ZEA induce the apoptosis of primary endometrial cells through the caspase3 pathway, which would break the uterus environment balance and finally lead to embryo implantation failure and dysontogenesis in gilts. endometrium, autophagy, apoptosis, implantation, reproduction toxicity The mycotoxin of zearalenone (ZEA) in cereals is one of the important hazardous substances (Sohn et al., 1999). Zearalenone has the chemical structure similar to that of estrogen, and can affect the function of reproductive system in mammals, particularly in gilts. For instance, ZEA can competitively bind to animal estrogen receptors (ERs), which in turn affects the growth, estrus cycle, embryo maturation, implantation, and normal pregnancy maintenance of animals (Cozzini and Dellafiora, 2012). The reproductive toxicity of excessive estrogen to gilts mainly causes false and abnormal estrus, dysplasia of vulva and genital, decrease in fertility, increase in embryo absorption, decrease in embryo volume, and changes of progesterone and estrogen levels (Gao et al., 2017; Prouillac et al., 2012). The decline of birth rate caused by moldy feed can lead to great economic loss in production practice (Díaz-Llano and Smith, 2006). For example, the decline of birth rate caused by moldy feed can lead to great economic loss in production practices (Rogowska et al., 2019; Zinedine et al., 2007). The association between moldy feed and issues during the embryo implantation period has been neglected by researchers. Embryonic implantation window is highly regulated by estrogen, which should be maintained at a suitable threshold. If the estrogen level is higher than the threshold value, the embedding window will be closed immediately, resulting in the failure of the uterus to accept embryonic implantation. The estrogen level below the threshold is unfavorable for implantation, but slightly lower estrogen level would be beneficial to the temporal extension of the embryo implantation window (Franczak, 2008; Ma et al., 2003). At the same time, Ványi et al. found that the estrogenic toxicity can lead to hyperplasia of the endometrium and vaginal epithelium (Ványi et al., 1994). The appropriate thickness of endometrial epithelial layer during embryo implantation is beneficial for the embryo to move into the cornua uteri and implant successfully (Zhao et al., 2012). These findings indicate that estrogen is significant for the regulation of embryo implantation. The normal development of embryo is also regulated by estrogen. As an exogenous estrogen, ZEA can affect the secretion of estrogen and the growth of the embryo. There are many reports on the research of toxic effects in rodents but relatively few in embryo implantation of gilts. ZEA can also cause damage to the immune system, digestive system, and reproductive system as a mycotoxin. By affecting the function of uterus, ovary, and embryonic development, ZEA can lead to abnormal development of oocytes and ovaries in gilts, and also can cause apoptosis and injury of pig ovary granulosa cells cultured in vitro (Zhu et al., 2012). Zhang et al. (2017) found that exposure to 10 or 30 μM of ZEA in vitro led to significant reduction of germ cell numbers in newborn mouse ovaries (Zhang et al., 2017). Researchers tested the ovary of gilts exposed to ZEA during pregnancy and lactation and found that the number of follicles decreased with increasing ZEA concentration, which may lead to a quick losses of mature follicles (Schoevers et al., 2012). However, there are few studies concerning the effect of ZEA on embryo implantation, which is key to successful pregnancy. According to related literatures, the number of ovules from each ovulation of gilts is 20–25 and the fertilization rate can reach above 95%. However, 30%–50% of the fertilized oocytes die in the early and middle stages of embryonic development. The embryonic mortality rate is the highest and can reach as high as 20%–25% at the early stage of pregnancy (Spencer and Bazer, 2004a,b), which accounts for over 60% of the total number of dead fertilized oocytes, and is mainly concentrated during 12–18 days of pregnancy. These results indicate that early pregnancy, namely the implantation window, is critical to the reproductive efficiency of gilts. The synchronicity of the development stage of blastocyst and endometrial receptivity and the interaction between blastocyst and uterine epithelium are crucial to embryonic implantation (Carson et al., 2000; Paria, 2002). This process is regulated by genes, homologous transcription factors, cytokines, growth factors, and other factors through autocrine, paracrine, and juxtacrine (Norwitz et al., 2001). Recent studies have also shown that “just-in-time” implantation is crucial for the successful pregnancy of both humans and animals (Song et al., 2002; Wilcox et al., 1999). The subtle damage of ZEA to the reproductive system of gilts may affect the time of embryo implantation and lead to implantation failure. The toxicity of ZEA on the embryo implantation is reflected not only by the status of the embryo, but also by the disorder of the internal environment in endometrium, which affects the transport of nutrients, energy, and immune factors between the mother and embryo, and eventually leads to dysontogenesis. In this study, we exposed gilts to different concentrations of ZEA during embryo implantation window period and studied the effects of ZEA on this process from the perspectives of tissue morphology of endometrium, ultrastructures of tissues, and cells and the activity of endometrial cells. Besides, the mechanism underlying the effects were further investigated. MATERIALS and METHODS Chemicals and reagents ZEA, 3-Methyladenine and rapamycin were purchased from Sigma-Aldrich (St. Louis, Missouri). Bafilomycin A1 was purchased from APExBIO Technology, and fetal bovine serum (FBS) was obtained from Gibco (Invitrogen). Dulbecco’s minimal essential medium Nutrient mixture F-12 (DMEM/F-12) was purchased from Gibco. Animals and treatments All animal experimental procedures were performed in accordance with the National Research Council Guide (Guide for the Care and Use of Laboratory Animals, 2011), and approved by the Scientific Ethic Committee of Huazhong Agricultural University on August 7, 2015. The project identification code is HZAURA-2015-006. Landrace × Large White gilts were chosen from the animal breeding center of the Huazhong Agricultural University Academy of Animal Science (Wuhan, China) and were acclimated to the second estrus prior to experimentation. The gilts weighted approximately 120 ± 5 kg were randomly divided into 4 groups with 5 replicates, and feed twice a day with 1.25 kg of feed. In the third estrus, the gilts were subjected to artificial insemination with the same concentration of semen (100 million/ml) from a common boar. Then, the pregnant gilts at gestation day (GD) 7–14 were individually housed and provided with free access to water and diets containing different concentrations of ZEA (0.03, 1.02, 2.03, 10.08 mg/kg) detected with ELISA. The 4 groups were named as follows: 0, 1, 2, and 10 mg/kg groups. After GD14, the gilts were fed diets without ZEA until GD21 when they were slaughtered. The tissue samples were collected, and the weights of the organs and body were measured. Feeding of 1 and 2 mg/kg ZEA was meant to explore the estrogen toxicity of low-concentration ZEA, whereas high concentration (10 mg/kg) was used to evaluate the mycotoxin toxicity. Cell culture and treatments Primary endometrial (PE) cells were separated using 10% collagen 1 from the endometrium of pigs and cultured in DMEM/F-12 supplemented with 10% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin at 37°C in a humidified 5% CO2 incubator. The cells were dispersed with 0.25% trypsin (w/v) and 0.52 mM EDTA. Zearalenone were diluted in to 0, 5, 10, or 20 μg/ml with serum-free medium. Primary endometrial cells were treated for 24 h with 0, 5, 10, or 20 μg/ml ZEA, respectively. Cell viability assay The RealTime Cellular Analysis (RTCA) system (Roche, Swiss) was used for real-time cell monitoring to detect the migration, cytotoxicity, and proliferation of cells following the methods of a previous study (Roshan Moniri et al., 2015). Primary endometrial cells (1.5 × 104 cells/well) were seeded in E-Plate and treated with ZEA (0, 5, 10, and 20 μg/ml). The impedance signals were recorded every 5 min over a period of 72 h. Reactive oxygen species determination Relative fluorescence units of intracellular dye (ROS, reactive oxygen species was measured using membrane-permeable CM-H2DCFDA 5-(6)-chloromethyl-2′, 7′-dichlorodihydrofluorescein diacetate). Cells were treated for 24 h with ZEA (0, 5, 10, and 20 μg/ml). We strictly followed the instructions of the fluorescent probe assay kit. Finally, 1 × 104 cells were collected for further analyses in the selected gate by flow cytometry (FCM) (Beckman) with excitation =488 nm, emission = 525 ± 20 nm (Kim et al., 2010). Cell apoptosis Apoptosis of cells was detected by Annexin V-FITC (fluorescein isothiocyanate) Apoptosis Detection Kit (Keygen, P R China) strictly following the instruction manual. Cells were treated for 24 h with ZEA (0, 5, 10, and 20 μg/ml) and control (set up with dimethyl sulfoxide [DMSO]), ZEA 10 μg/ml, ZEA (10 μg/ml) + Baf A1 (100 nM), ZEA (10 μg/ml) + 3-Methyladenine (3MA) (5 μM), respectively. All cells were collected and finally 1 × 104 cells were collected for further analyses in the selected gate by FCM with excitation = 488 nm, emission = 585 ± 21 nm. Cell mitochondrial membrane potential detection Cell mitochondrial membrane potential (MMP) of porcine endometrium cells was measured by MMP assay kit with JC-1 (Beyotime, P R China) strictly following the instruction manual. Cells were treated for 24 h with ZEA (0, 5, 10 and 20 μg/ml) and control (set up with DMSO), ZEA 10 μg/ml, ZEA (10 μg/ml) + Baf A1 (100 nM), ZEA (10 μg/ml) + 3MA (5 μM), respectively. All cells were collected and finally 1 × 104 cells were collected for further analyses in the selected gate by FCM (Beckman) with excitation = 488 nm, emission = 525 ± 20 nm. Protein isolation and Western blotting The endometrial tissues and the treated cells were lysed in Radio Immunoprecipitation Assay buffer with 1 mM phenylmethanesulfonyl fluoride (Beyotime). The concentration of total proteins was quantified using a bicinchoninic acid Kit (Roche). For each sample, the same amount of proteins was loaded on 12% SDS-PAGE gels and resolved by electrophoresis, followed by transferring onto nitrocellulose membrane. Then, the membranes were nonspecifically blocked with blocking buffer (5% fat-free dry milk in 0.1% Tween-20 TBST buffer) and incubated with primary antibodies and the blots were incubated with appropriate secondary antibodies, respectively. Finally, specific proteins were conjugated to ECL (BioRad) substrate according the manufacturer’s instructions. Antibodies used included mouse anti-GAPDH (GB13002, Servicebio; 1:25 000), rabbit anti-ACTIN (GB13001, Servicebio; 1:2000), rabbit anti-Bcl2 (WL01556, Wanleibio; 1:2000), rabbit anti-BAX (GB11007, Servicebio; 1:2000), rabbit anti-CASP3 (BS1518, Bioworld; 1:800), rabbit anti-LC3B (12135, PTG; 1:1000), and rabbit anti-P62 (sequestosome 1) (18420, PTG; 1:1000). Virus infection, laser confocal imaging, and live-cell fluorescence imaging To mark autophagy and autophagy lysosome formation process, PE cells were transduced with 100 multiplicities of infection (MOI) of adenovirus mRFP-GFP-LC3 (HanBio, Shanghai, China) for 6–8 h before being treated with ZEA. The nucleus was stained with DAPI. Autophagy was observed under the laser scanning confocal microscope (LSM 800, Zeiss, Germany) or fluorescence microscope (Olympus BX 51, Japan). We evaluated the number of Green Fluorescent Protein (GFP) and Red Fluorescent Protein (mRFP) puncta (puncta/per cell was calculated) to determine the autophagic flux. Fluorescence microscopy of live cells could provide a range of tools to virtually investigate any cellular process under the microscope. We observed the formation of autophagy for 48 h in the cells through live-cell imaging system (LSM 800, Zeiss) which was surrounded by constant temperature (37°C) and atmosphere (5% CO2 and 95% air) (Ettinger and Wittmann, 2014). The filter sets used were as follows: GFP, 488 nm (excitation); and RFP, 561 nm (excitation). The GFP and mRFP puncta were represented as mean and standard error of mean from 2 to 5 independent experiments and from at least 10 to 15 cells in each experiment. Electron microscopy observation The endometrial tissues were immediately removed and cut into 1 mm3 small cubes and endometrial cells were collected. After that, the samples were fixed with 2.5% glutaraldehyde fixing agent mixed with phosphate buffered saline at 4°C (PBS, 0.1 M, pH 7.4) for 24 h, and rinsed in the same PBS and fixed in 1% buffered osmium tetroxide, and washed. The samples were dehydrated in ethanol and permeated with propylene oxide-Araldite compound before they were embedded in Araldite. We sectioned the cubes by ultramicrotome. The sample sections (50 nm) were stained with 1% uranyl acetate and lead citrate, and examined and photographed using a H-7000FA transmission electron microscope (TEM) (H-7000FA, HITACHI, Japan). Histological analysis For histology analysis, tissue samples (uterus, ovary, spleen, and embryo) were fixed in 4% paraformaldehyde and then embedded in paraffin. The sections were stained with H&E according to the standard protocols. Histological analysis was performed by Olympus microscope (BX53, Olympus, Japan) with Pannoramic Viewer system (1.15.3). Statistics The data were expressed as the means ± standard deviation. Each experiment included at least triplicate treatments. Statistical significance was detected using Student’s t test (2-tailed) for absolute values or Fisher’s exact test (2-tailed) for category values in Graphpad Prism or Microsoft Excel. All boxplots showing the error bar indicate that there were significant differences between the compared groups (p < .05). RESULTS As shown in Figures 1A and 1B, on 21st day of pregnancy of gilts, the embryos, with a size of a soybean grain and wrapped in the placenta, had been preliminarily formed and the internal blood vessels were faintly visible. To avoid the degradation of embryo proteins and RNA due to long time exposure in air, 10 embryos were randomly taken from each group and determined on a glass slide by measuring the head-to-tail and back-to-abdomen lengths. The results showed that the embryo size in 10 mg/kg ZEA group was smaller than that of the control group (p<.05), and the embryo size in 1 and 2 mg/kg ZEA groups was slightly larger than that of 0 mg/kg group (p>.05). Interestingly, some poorly developed embryos were observed in the 10 mg/kg group. As shown in Figure 1C, the number of implanted embryos showed no statistical differences among the 4 groups. As shown in Figure 1E, the number of embryos and corpora luteal in the ovaries were statistically analyzed and showed a linear relationship, which means, the number of embryos was positively correlated with that of corpora luteal (p<.01). Figure 1. Open in new tabDownload slide Zearalenone toxicity on embryos. A and B, Fetus size decreased and congenital deficiency increased obviously with increasing ZEA concentration during implantation, n = 5 (0 mg/kg: 95.38 ± 34.30, 1 mg/kg: 76.25 ± 25.78, 2 mg/kg: 88.76 ± 29.28, 10 mg/kg: 67.25 ± 17.63). C, No statistical difference was observed in the number of successfully implanted embryos in gilts, n = 5 (0 mg/kg: 11.4 ± 1.36, 1 mg/kg: 11.4 ± 1.36, 2 mg/kg: 11.00 ± 2.00, 10 mg/kg: 11.20 ± 1.94) (p > .05). D, No significant difference was found in luteal number among different groups, n = 5 (p > .05). E, The number of embryos was positively correlated with that of corpora luteal, n = 5 (p<.01). 0, 1, 2, 10 indicate 0, 1, 2, 10 mg/kg concentration of ZEA. Values are expressed as means ± SD. Figure 1. Open in new tabDownload slide Zearalenone toxicity on embryos. A and B, Fetus size decreased and congenital deficiency increased obviously with increasing ZEA concentration during implantation, n = 5 (0 mg/kg: 95.38 ± 34.30, 1 mg/kg: 76.25 ± 25.78, 2 mg/kg: 88.76 ± 29.28, 10 mg/kg: 67.25 ± 17.63). C, No statistical difference was observed in the number of successfully implanted embryos in gilts, n = 5 (0 mg/kg: 11.4 ± 1.36, 1 mg/kg: 11.4 ± 1.36, 2 mg/kg: 11.00 ± 2.00, 10 mg/kg: 11.20 ± 1.94) (p > .05). D, No significant difference was found in luteal number among different groups, n = 5 (p > .05). E, The number of embryos was positively correlated with that of corpora luteal, n = 5 (p<.01). 0, 1, 2, 10 indicate 0, 1, 2, 10 mg/kg concentration of ZEA. Values are expressed as means ± SD. As shown in Figures 2A and 2B, the effect of ZEA on endometrium and ovary was determined by Hematoxylin and Eosin (H&E) staining. Three uterine sites were randomly chosen to be stained with H&E and observed; the thickness of endometrium was measured equidistantly, and the average thickness was statistically analyzed. The results showed that the thickness of the epithelial cell layer increased with increasing ZEA concentration, and was significantly higher than that of 0 mg/kg group (p<.05). At the same time, the number of erythrocytes in endometrial capillaries showed a significant increase in a dose-dependent manner. As shown in Figure 2C, vessels could be clearly observed in the endometrium with H&E staining. We found that the structure of vascular wall in the 0 mg/kg ZEA groups was dense and uniform. However, in the experimental groups, the vascular epithelial cells showed a decrease in number and alterations in arrangement, and the vascular wall became thinner with increasing ZEA concentration, particularly in the 10 mg/kg group. Figure 2. Open in new tabDownload slide Pathological sections of the uterus and ovarian tissues. A and B, ZEA treatments increased the thickness of the lining of the endometrial epithelium (0 mg/kg: 21.60 ± 2.35 μm, 1 mg/kg: 31.92 ± 2.09 μm, 2 mg/kg: 39.54 ± 5.93 μm, 10 mg/kg: 34.33 ± 4.24 μm) (p < .05) (erythrocyte, black arrow; epithelial layer, black line). C, Variations of vascular epithelial cells after the ZEA treatment. D, Primordial follicles showed slight dysplasia (black arrow). E, The number of granulose-lutein cells was obviously decreased (black arrow). 0, 1, 2, 10 indicate 0, 1, 2, and 10 mg/kg concentrations of ZEA. Scale bar = 50 μm. Values are expressed as means ± SD for n = 5. Figure 2. Open in new tabDownload slide Pathological sections of the uterus and ovarian tissues. A and B, ZEA treatments increased the thickness of the lining of the endometrial epithelium (0 mg/kg: 21.60 ± 2.35 μm, 1 mg/kg: 31.92 ± 2.09 μm, 2 mg/kg: 39.54 ± 5.93 μm, 10 mg/kg: 34.33 ± 4.24 μm) (p < .05) (erythrocyte, black arrow; epithelial layer, black line). C, Variations of vascular epithelial cells after the ZEA treatment. D, Primordial follicles showed slight dysplasia (black arrow). E, The number of granulose-lutein cells was obviously decreased (black arrow). 0, 1, 2, 10 indicate 0, 1, 2, and 10 mg/kg concentrations of ZEA. Scale bar = 50 μm. Values are expressed as means ± SD for n = 5. The ovary is also one of the target organs of ZEA toxicity. As shown in Figure 2D, observation of the ovarian cortex and medulla exposed to ZEA showed that the ovary granulosa cells had no obvious change, but the ovarian primordial follicles exhibited slight dysplasia. As shown in Figure 2E, the number of granulose-lutein cells in 10 mg/kg group was obviously smaller than that in the 0 mg/kg group. A precisely timed synchronization between blastocyst implantation and endometrial development is essential for the initiation of pregnancy. Thus, a successful implantation requires a healthy endometrial lining. We isolated the PE cells and treated them with ZEA at 0, 5, 10, and 20 μg/ml concentrations. RealTime Cellular Analysis system was used to detect the effects of ZEA on cell viability. As shown in Figure 3A, 5 μg/ml ZEA did not obviously affect the proliferation rate and growth of PE cells. Although in 10 or 20 μg/ml ZEA group, the growth rate was nearly zero in the subsequent growth process. Besides, the cell index decreased sharply in 10 and 20 μg/ml ZEA groups after 3 h of ZEA treatment. As shown in Figures 3B and 3C, electron microscopy observation was performed for the PE cells after the addition of 0, 10, and 20 μg/ml ZEA for 24 h. The results were consistent with those of electron microscopy observation of endometrium: the amount of autophagosomes increased along with the increasing ZEA concentration. We speculated that ZEA causes apoptosis, so we detected apoptosis by FCM. As shown in Figures 3D and 3E, the results showed that cell apoptosis was increased significantly in a dose-dependent manner. Because it was observed that mitochondria were swollen in both endometrial tissues and PE cells under electron microscopy, we examined the MMP using the JC-1 method by FCM. As shown in Figures 3F and 3G, the MMP loss was obvious in 10 and 20 μg/ml ZEA groups (*p < .05, **p < .01). Figure 3. Open in new tabDownload slide Zearalenone showed significant toxicity on PE cells. A, D, and F, PE cells were treated for 24 h with 0, 5, 10, 20 μg/ml ZEA, respectively. A, RTCA system was used to detect the effects of ZEA on cell viability, n = 4–6. B and C, PE cells were treated for 24 h with ZEA at 0, 10, 20 μg/ml concentrations and observed with electron microscopy (autophagy, tail arrows; mitochondria, triangle arrow); scale bar = 1 μm, n = 4–6 (0 mg/kg: 10.50 ± 3.50, 10 mg/kg: 15.33 ± 4.60, 20 mg/kg: 22.50 ± 5.68). D and E, FCM showed that cell apoptosis was increased significantly in a dose-dependent manner, n = 3. F and G, MMP was detected by FCM, n = 3 (*p < .05, **p < .01, ***p < .001). Values are expressed as means ± SD. Figure 3. Open in new tabDownload slide Zearalenone showed significant toxicity on PE cells. A, D, and F, PE cells were treated for 24 h with 0, 5, 10, 20 μg/ml ZEA, respectively. A, RTCA system was used to detect the effects of ZEA on cell viability, n = 4–6. B and C, PE cells were treated for 24 h with ZEA at 0, 10, 20 μg/ml concentrations and observed with electron microscopy (autophagy, tail arrows; mitochondria, triangle arrow); scale bar = 1 μm, n = 4–6 (0 mg/kg: 10.50 ± 3.50, 10 mg/kg: 15.33 ± 4.60, 20 mg/kg: 22.50 ± 5.68). D and E, FCM showed that cell apoptosis was increased significantly in a dose-dependent manner, n = 3. F and G, MMP was detected by FCM, n = 3 (*p < .05, **p < .01, ***p < .001). Values are expressed as means ± SD. To examine the state of endometrium cells after ZEA treatment, we collected fresh endometrial tissues of the gilts respectively from 0, 1, 10 mg/kg groups. The tissues were washed with 4°C PBS and fixed with 2.5% glutaraldehyde, which were then observed for cell ultrastructure using TEM ultramicrotomy. As shown in Figure 4A, the internal environment of endometrial cells exposed to ZEA was changed. The number of intracellular vacuoles in endometrial cells of the 1, 10 mg/kg ZEA groups increased in a dose-dependent manner compared that of 0 mg/kg ZEA group. Besides the changes in cell structure, the number of autophagosomes in endometrial cells also increased with increasing ZEA concentration (p<.05) as shown in Figures 4B and 4D. Figure 4. Open in new tabDownload slide Zearalenone destroyed endometrial tissue cells under the TEM. A, The number of intracellular vacuoles increased in the treatment groups in a dose-dependent manner; scale bar =2 μm. B and D, Electron micrographs of autophagosome fractions at ×5000 magnification; scale bar = 1 μm. Zearalenone caused significant increases in autophagy in endometrial tissue cells (0 mg/kg: 6.75 ± 1.48, 1 mg/kg: 13.63 ± 2.78, 10 mg/kg: 25.00 ± 5.25) (p < .01). C and E, Electron micrographs of chondriosome at ×5000 magnification; scale bar = 1 μm. High concentration ZEA caused significant increases in mitochondrial swelling (0 mg/kg: 0.09 ± 0.02, 1 mg/kg: 0.29 ± 0.07, 10 mg/kg: 0.71 ± 0.0.9) (p < .01). 0, 1, and 10 indicate 0, 1, and 10 mg/kg concentrations of ZEA. Values are expressed as means ± SD, n = 5. Figure 4. Open in new tabDownload slide Zearalenone destroyed endometrial tissue cells under the TEM. A, The number of intracellular vacuoles increased in the treatment groups in a dose-dependent manner; scale bar =2 μm. B and D, Electron micrographs of autophagosome fractions at ×5000 magnification; scale bar = 1 μm. Zearalenone caused significant increases in autophagy in endometrial tissue cells (0 mg/kg: 6.75 ± 1.48, 1 mg/kg: 13.63 ± 2.78, 10 mg/kg: 25.00 ± 5.25) (p < .01). C and E, Electron micrographs of chondriosome at ×5000 magnification; scale bar = 1 μm. High concentration ZEA caused significant increases in mitochondrial swelling (0 mg/kg: 0.09 ± 0.02, 1 mg/kg: 0.29 ± 0.07, 10 mg/kg: 0.71 ± 0.0.9) (p < .01). 0, 1, and 10 indicate 0, 1, and 10 mg/kg concentrations of ZEA. Values are expressed as means ± SD, n = 5. As shown in Figures 4C and 4E, when comparing the cell organelles of different groups, we found that the mitochondria of endometrial cells were swollen and ruptured in 1, 10 mg/kg ZEA groups. In 1 mg/kg group, we observed a mild swelling of the mitochondria and damage of the crest. Although the mitochondrial crest was destructed, extremely swollen, and significantly damaged in 10 mg/kg group (p<.05). As the marker protein of autophagy, microtubule-associated protein 1 light chain 3 (LC3) was our primary research object. As shown in Figures 5E and 5F, the expression of LC3 II was increased, suggesting the increase in autophagy in endometrial tissues. However, as a reaction substrate of LC3 II for autophagy, P62 did not decrease along with the increase in LC3 II, indicating that the autophagy flux might not be complete. Figure 5. Open in new tabDownload slide Zearalenone affected the expression of proteins related to autophagy and apoptosis in endometrium during implantation window. A and B, ZEA treatment had no significant effects on Bcl2/Bax (p > .05). C and D, ZEA led to significant increase in cleaved caspase3 in endometrial tissues. E and F, LC3 II expression increased significantly but P62 expression did not decrease after ZEA treatment (*p < .05, **p < .01). Values are expressed as means ± SD for n = 5. Figure 5. Open in new tabDownload slide Zearalenone affected the expression of proteins related to autophagy and apoptosis in endometrium during implantation window. A and B, ZEA treatment had no significant effects on Bcl2/Bax (p > .05). C and D, ZEA led to significant increase in cleaved caspase3 in endometrial tissues. E and F, LC3 II expression increased significantly but P62 expression did not decrease after ZEA treatment (*p < .05, **p < .01). Values are expressed as means ± SD for n = 5. Bcl2/Bax are important regulatory proteins of apoptosis, which were not affected by ZEA at various concentrations. Caspase3 is a caspase protein that plays a central role in the execution-phase of cell apoptosis. As shown in Figures 5A–D, we found that the expression of activated (cleaved) caspase3 increased with increasing ZEA concentration (*p < .05, **p < .01). To further confirm the effect of ZEA on autophagy in PE cells, cells were infected by adenovirus with mRFP-GFP-LC3-AAV to trace the autophagy flux (the MOI results are shown in Supplementary Figure 2). The GFP signal is sensitive to the acid in autolysosomes and lysosomes, whereas the mRFP signal is more stable. The yellow fluorescence dots (merging of green and red fluorescence) and red fluorescence dots in ZEA, ZEA+Rapa and rapamycin treatment groups were significantly increased, but yellow fluorescence dots increased significantly faster than red fluorescence dots, and the red fluorescence dots decreased in ZEA+Rapa treatment groups compared with in Rapa treatment groups. Therefore, the merging of GFP and mRFP fluorescence signals would generate yellow fluorescence, which indicates a compartment has not been fused with the lysosome, such as a phagophore or an autophagosome. In contrast, an mRFP signal without GFP corresponds to an autolysosome. Primary endometrial cells were treated with control (set up with DMSO), 10 μg/ml ZEA, ZEA (10 μg/ml) + Rap (5 μM), and Rap (5 μM) for 12 h (ZEA), Rap concentration gradient is shown in Supplementary Figure 3; ZEA concentration gradient is shown in Supplementary Figure 4. As predicted, the yellow fluorescence dots increased significantly with increasing ZEA concentration. As shown in Figure 6, these results indicated that autophagy was activated. Rap-activated autophagy; however, the ZEA+Rap treatment resulted in significant reduction of mRFP fluorescence, indicating the blocking of autophagy flow (*p < .05, **p < .01). Figure 6. Open in new tabDownload slide Zearalenone-activated autophagy but interfered with autophagy flow. A, PE cells infected with mRFP-GFP-LC3-AAV (MOI 100, 12 h). Cells were fixed and stained with DAPI (blue, cytoblast; yellow, autophagosome; red, autolysosome). The merging of the 3 signals is shown in the right panels. Scale bar = 10 µm. B, Number of mRFP or GFP puncta per cell. The yellow fluorescence dots represent autophagosomes, and RFP fluorescence dots represent autolysosomes (***p < .001).Values are expressed as means ± SD for n = 3. Figure 6. Open in new tabDownload slide Zearalenone-activated autophagy but interfered with autophagy flow. A, PE cells infected with mRFP-GFP-LC3-AAV (MOI 100, 12 h). Cells were fixed and stained with DAPI (blue, cytoblast; yellow, autophagosome; red, autolysosome). The merging of the 3 signals is shown in the right panels. Scale bar = 10 µm. B, Number of mRFP or GFP puncta per cell. The yellow fluorescence dots represent autophagosomes, and RFP fluorescence dots represent autolysosomes (***p < .001).Values are expressed as means ± SD for n = 3. PE cells were also treated with control (set up with DMSO), 10 μg/ml ZEA, ZEA (10 μg/ml) + BafA1 (100 nM), ZEA (10 μg/ml) + 3MA (5 μM), Baf (100 nM), and 3MA (5 μM). Results showed that ZEA and Baf both blocked autophagy flow, indicating that they have synergistic effects on autophagy flow, but 3MA showed no such effects (Supplementary Figure 3). To further explore the effects of simultaneous increasing in autophagy and blocking of autophagy flow, we evaluated the apoptosis and MMP in PE cells. Primary endometrial cells were treated with control (set up with DMSO), ZEA 10 μg/ml, ZEA (10 μg/ml) + BafA1 (100 nM), ZEA (10 μg/ml) + 3MA (5 μM), Baf (100 nM), and 3MA (5 μM). The results showed that the apoptosis and MMP loss of PE cells increased significantly with increasing ZEA concentration (Figs. 7A–G) (*p < .05, **p < .01). It was shown that ZEA and ZEA+BafA1 significantly increased cell apoptosis and MMP loss, and obviously elevated the expression of cleaved caspase3 and LC3 II at the same time, which was not observed in 3MA+ZEA group (*p < .05, **p < .01, ***p < .001). Figure 7. Open in new tabDownload slide Zearalenone inhibited autophagy flow to damage MMP and cause apoptosis. A–G, PE cells were treated with control (set up with DMSO), 10 μg/ml ZEA, ZEA (10 μg/ml) + BafA1 (100 nM), ZEA (10 μg/ml) + 3MA (5 μM), Baf (100 nM), and 3MA (5 μM), and were detected by FCM, respectively. A and B, ZEA has synergistic effects with Baf A1 on apoptosis but has no such effect with 3MA. C and D, MMP was changed by ZEA and Baf A1 synergistically. E and F, The changes in apoptotic proteins were consistent with those in apoptosis. E and G, ZEA and ZEA+BafA1 led to significant increase in LC3 II expression but ZEA + 3MA did not (*p < .05, **p < .01, ***p < .001). Values are expressed as means ± SD for n = 3. Figure 7. Open in new tabDownload slide Zearalenone inhibited autophagy flow to damage MMP and cause apoptosis. A–G, PE cells were treated with control (set up with DMSO), 10 μg/ml ZEA, ZEA (10 μg/ml) + BafA1 (100 nM), ZEA (10 μg/ml) + 3MA (5 μM), Baf (100 nM), and 3MA (5 μM), and were detected by FCM, respectively. A and B, ZEA has synergistic effects with Baf A1 on apoptosis but has no such effect with 3MA. C and D, MMP was changed by ZEA and Baf A1 synergistically. E and F, The changes in apoptotic proteins were consistent with those in apoptosis. E and G, ZEA and ZEA+BafA1 led to significant increase in LC3 II expression but ZEA + 3MA did not (*p < .05, **p < .01, ***p < .001). Values are expressed as means ± SD for n = 3. On one hand, ZEA binds to the ER on the cell membrane as a kind of estrogen and influences cell physiological functions by regulating the expression of the genes and proteins related to estrogen. On the other hand, we hypothesize that ZEA enters the cells and damages the mitochondria, which lead to the loss of MMP and release of ROS. Reactive oxygen species can cause oxidative stress damage to cells and cause apoptosis. Our results showed that ZEA activates autophagy but blocks the autophagy flow in the process of autophagosome fusion with lysosomes. Hence, the cells cannot degrade the damaged organelles and proteins, resulting in the accumulation of a large amount of autophagosomes and lysosomes which finally causing apoptosis. The damage of mitochondrial and the blocking of autophagy flow can cause apoptosis of PE cells through the caspase3 apoptosis pathway. The schematic diagram is shown in Figure 8. Figure 8. Open in new tabDownload slide Schematic diagram of ZEA toxicity mechanism in PE cells. On one hand, ZEA binds to ER to affect cell physiological activity; on the other hand, ZEA damages MMP and mitochondrial function, leading to the block of autophagy flow, which causes apoptosis through caspase3 pathway. Figure 8. Open in new tabDownload slide Schematic diagram of ZEA toxicity mechanism in PE cells. On one hand, ZEA binds to ER to affect cell physiological activity; on the other hand, ZEA damages MMP and mitochondrial function, leading to the block of autophagy flow, which causes apoptosis through caspase3 pathway. DISCUSSION Appropriate embryo implantation directly influences the gestational and postnatal development of the embryos. Endometrial receptivity has a significant effect on the success rate of embryo implantation. It is unclear what levels of ZEA and period tested in this study represent a condition found in a swine operation animals exposed to moldy feed with ZEA. Still, it allowed exploring the toxicity of ZEA during the preimplantation period. We treated gilts with feed containing ZEA during implantation and found that ZEA supplementation causes damage to the endometrium to influence the implantation. Based on the obtained results, we further demonstrated the mechanism underlying the toxicity of ZEA on the endometrium. Our study showed that exposure to ZEA obviously affected the embryo implantation. There was no significant difference in the number of embryos, but the fetus size decreased and congenital deficiency increased obviously with increasing ZEA concentration. High concentrations of ZEA led to more frequent occurrence of weaker embryos, particularly during early embryonic development stage. This suggests that ZEA can affect embryo implantation and embryo development during embryo implantation. Our results were consistent with previous hypothesis that exposure to ZEA in the early pregnancy will affect embryo implantation (Gao et al., 2017; Kunishige et al., 2017). Previous studies have reported that ZEA has little toxicity on embryos because the placental barrier can block the invasion of most ZEA into the embryo. Therefore, we speculate that ZEA is more likely to cause embryo implantation disorder by damaging the endometrium. The molecular mechanisms of this effect would be further discussed in the following sections. ZEA Has Significant Effect on the Ovary During Embryo Implantation During the embryo implantation, the simultaneous development of embryo and endometrium as well as the pregnancy maintenance are precisely controlled by a series of hormones and genes. The combination of estrogen and ER in the maternal endometrium can alter the morphology of the endometrium. It is reported that embryo implantation is regulated by the inflammatory process in response to sequential exposure to estrogen and progesterone, followed by resolution and repair (Lessey and Young, 2014). ZEA can also change the tissue morphology of endometrium by reducing the synthesis of progesterone and luteinizing hormone of the ovary (Etienne and Jemmali, 1982). To maintain the estrogen balance required by the embryo implantation, the body will decrease the expression of ER to balance the effect of excess estrogen caused by ZEA. This mechanism explains why the excess estrogen caused by low concentrations of ZEA would not directly cause embryo implantation failure. However, high concentrations of ZEA will cause severe damage to hormone levels and cell functions, which cannot be regulated by the body. Estrogen can also maintain the corpus luteum during embryo implantation. A recent study has suggested that steroid hormones, especially estrogen and progesterone, play an important roles in supporting endometrial preparation to establish endometrial receptivity (Ozturk and Demir, 2010). The tissue sectioning of ovary showed that ZEA caused decrease in ovary granulosa cells and poor cell status, which in turn lead to a decrease in the production of progesterone by the corpus luteum. Based on our conclusion that there is a linear relationship between the amounts of corpus luteums and embryos, it is clear that successful embryo implantation requires the sufficient secretion of progesterone by the corpus luteum. To sum up, ZEA decreases the secretion efficiency of hormones by reducing the density and quality of granulosa luteal cells and disturbing the balance of embryogenic hormones. Our results are consistent with the resultsthat the feeding of ZEA on seventh day of pregnancy could result in slow follicle growth of gilts (Diekman and Long, 1989). We can infer that ZEA can result in fewer and malformed oocytes. This inference is consistent with some studies of ovary granulosa cells. It has been confirmed that ZEA can cause growth retardation of oocytes in mice by increasing the methylation of DNA and changing the epigenetic modification to alter cellular activity (Zhu et al., 2014). But this inference may not be conclusive because of the small sample size and the angle of ovary H&E section for observation. ZEA Has a Significant Effect on Endometrium During Implantation In the embryo implantation process, the endometrium is the basis of embryo attachment and growth, and is related to the formation of placenta, the transport of nutrients, and the regulation of immunity. Undoubtedly, the health of endometrium is critical to successful implantation (Horcajadas et al., 2007). Therefore, we detected the effects of ZEA on the endometrium from the perspectives of tissue structure, PE cells and cell microstructure. The effects turned out to be very significant. The thickness of endometrial epithelial layer changes periodically with estrogen levels. Most researchers agree that low endometrial thickness is not conducive to embryo implantation, whereas thickness above the normal levels has also certain negative effects (Fang et al., 2016; Gingold et al., 2015). As indicated by the H&E staining, the endometrial epithelial cell layer was significantly thickened, indicating the adverse effects of ZEA on embryo implantation. The hyperemia of endometrial and the decrease in vascular wall thickness can clearly indicate the increase in vascular wall permeability in endometrium. An increase in vascular permeability during embryo implantation would result in an immune inflammatory response, which would lead to the concentration of local immune molecules and cause local immune imbalance in the uterus. The embryos are seen as foreign by the mother’s immune system. Thusly, reduction of local immune response would be more conducive to implantation, but the immune balance should be maintained at a proper level to prevent infection (Robertson and Moldenhauer, 2014). Severe hemorrhagic inflammation was observed in the endometrium of high concentration ZEA group, which would have a serious impact on embryo implantation. Many researchers have confirmed that ZEA could inhibit cell proliferation or cause death of multiple cells such as lymphocyte, peripheral blood mononuclear cell, oocyte, and hemameba (Lioi et al., 2004; Murata et al., 2003). To confirm that ZEA has effect on PE cell viability, we examined the effect of ZEA on cells by determining the cell proliferation and apoptosis. RealTime Cellular Analysis (Dreesen et al., 2013) system was used to detect the real-time status of cells. The results demonstrated that the addition of different concentrations of ZEA had different effects on the cells. No significant differences were found in 5 μg/ml ZEA group compared with the control group, whereas significant differences were found in 10 and 20 μg/ml ZEA groups compared with the control group that the proliferation of cells was significantly inhibited or even stopped. High concentration of ZEA caused cell death in a short period of time, which undoubtedly would have harmful effects on embryo implantation. There were also studies confirming the endometrial cytotoxicity of ZEA. It is found that alpha-zearalenol (alpha-ZEL) at the concentrations of 7.5, 15, and 30 μM could significantly inhibit the proliferation of endometrial cells in pigs, causing the proliferation disorder of the cells (Tiemann, 2003). Consistently, different concentrations of ZEA also caused inhibitory effects on PE cells in this study, and our results are actually more accurate and real-time, and intuitively prove the toxicity of ZEA on PE cells. Many studies of ZEA cytotoxicity have shown that ZEA can cause apoptosis. It is also found that ZEA resulted in apoptosis in sheep’s testicles (Zhu et al., 2012). To determine whether the cell death caused by ZEA is apoptosis, we measured the apoptosis of cells exposed to ZEA by FCM. The results were positive. The number of apoptotic cells increased with increasing in ZEA concentration. Electron microscopy observation also showed that ZEA resulted in the apoptosis of endometrial cells. Our results demonstrated that ZEA can cause endometrial cell apoptosis, which has not been reported before. Bcl2 family proteins participate in cell homeostasis and regulate cell death (Gómez-Fernández, 2014). In this study, the expression of Bcl2/Bax in endometrial tissues was measured, and we found that the increase in ZEA concentration had no effect on its expression, suggesting that Bcl2/Bax is not the main pathway by which apoptosis is increased. Caspase3 is the ultimate executive protein of cell mitochondrial apoptosis pathway and its activation means the occurrence of apoptosis (Han et al., 1997). The expression of cleaved caspase3 was significantly higher in high ZEA concentration groups than in the control group, confirming that cell apoptosis occurs through the caspase3 pathway. Previous studies have also shown that ZEA can cause apoptosis through the caspase3 pathway such as in mice testis, mouse sperm, and A549 (human lung cancer cells) (Li et al., 2016; Long et al., 2016). These findings together demonstrated a common phenomenon that ZEA causes cell apoptosis through the caspase3 pathway. To explore which cellular components are damaged by ZEA, we hypothesized that ZEA destroys the mitochondria based on the result that the mitochondria were badly swollen and broken after ZEA treatment. To this end, we detected the integrity of membrane potential after ZEA treatment (Wang et al., 2013). Mitochondrial membrane potential loss was detected for the PE cells exposed to ZEA. This phenomenon is consistent with the results that ZEA results in MMP loss in RAW 264.7 cells and leads to apoptosis of cells (Yu et al., 2011). Mitochondrial cation equilibrium is critical for regulating numerous cellular processes, including energy metabolism and MMP. Mitochondria undergo rapidly changes in matrix Ca2+ concentration to affect cell survival and aerobic metabolism (Hajnóczky et al., 1995; Jouaville et al., 1999). So, we suspect that ZEA disrupts Ca2+ transport and causes the swelling of mitochondria, which will finally lead to apoptosis. Combined with the result that mitochondria were significantly swollen and damaged, we inferred that the invasion of ZEA into endometrium cells causes mitochondrial damage and metabolic disorder, leading to a lack of energy supply, or stress response to the invasion of exogenous substances of the cells, thusly activating cellular autophagy and causing damage to endometrial cells. We studied the cellular autophagy by electron microscopy, the results showed that ZEA led to a significant increase in the number of autophagosomes and lysosomes in the tissue cells and PE cells, suggesting the occurrence of cell autophagy. We further explored the relative expression of the autophagy marker proteins LC3 and P62to prove that autophagy occurred in the endometrial cells (Klionsky et al., 2016). The expression of protein LC3 increases along with increasing ZEA concentration, particularly in the 10 mg/kg ZEA group, indicating that ZEA causes autophagic injury to endometrial tissues during embryo implantation. But as a degradation substrate of LC3, P62 showed no corresponding decrease. Because P62 protein generally decreases with the increase in LC3 in the complete autophagy flow, we assumed that the autophagy flow of cells was interrupted or blocked. To further confirm this assumption, we infected the PE cells with mRFP-GFP-LC3-AAV and observed the changes in the autophagy flow of cells exposed to ZEA, with the rapamycin being used as the positive control for autophagy. Our results showed that the formation of autolysosomes in cells was decreased, but that of autophagosomes was increased, indicating that ZEA leads to autophagy but inhibits the autophagy flow at the same time, which will inhibit the degradation of autophagosomes and the renewal of the cells. The continuous malignant accumulation of autophagosomes in cells will eventually lead to the death of cells. It is found that ZEA can activate the apoptosis pathway of H9C2 myocardial cells and cause the increase in LC3 expression (Ben Salem et al., 2017). This finding strongly supports our conclusion, which was for the first time proposed in a study about the mechanism of ZEA toxicity on the reproductive system of gilts. To further explore in which stage of the autophagy flow is blocked by ZEA, we used 3MA and BafA1 to block the formation of autophagosomes and autolysosomes in autophagy flow. The results showed that ZEA has a synergistic blocking effect with BafA1but not 3MA. Taken together, we believe that ZEA blocks the autophagy flow after the formation of autophagosomes (blocked by 3MA) and before or at the formation of autolysosomes (blocked by BafA1). In-depth experimental results show that ZEA inhibited autophagy flow and damage MMP and cause apoptosis, these are exactly in line with our conjectures. Ultimately, we speculate that high concentration of ZEA activates autophagy, but it can also block the combination of autophagosomes and lysosomes, which will lead to the disorder of autophagy flow and accumulation of autophagosomes. This is the new finding that has never been reported before. CONCLUSION ZEA has significant influence on the reproductive system of gilts in embryo implantation window period. We here focused on the mechanism of ZEA toxicity on PE cells and embryo implantation process. Zearalenone can inhibit the cell proliferation and cause endometrial hemorrhagic inflammation. In endometrial cells, ZEA activates autophagy and blocks the autophagy flow at the same time, leading to the accumulation of a large amount of autophagosomes. The aforementioned effects of ZEA will finally induce the apoptosis of PE cells through the caspase3 pathway. The mitochondrial damage, autophagy flow blocking and apoptosis of cells are all not conducive to the establishment and maintenance of the embryo implantation in uterus, and may lead to dysontogenesis. In actual production, the breeding management of gilts should be more precisely and strictly controlled. In particular, the contamination of various mycotoxins should be strictly controlled in the feeding of gilts during early pregnancy so as to improve the breeding efficiency and promote the economy efficiency. DEFICIENCY We did not perform a power analysis to determine the biological repeat of our experiment. The number of experimental animals referred to previous studies of ZEA (Dänicke et al., 2005; Goyarts et al., 2007), as well as other studies that used 5 pigs as biological repeats (Kiezun et al., 2017; Zhang et al., 2014). But still, our results are true and reliable. SUPPLEMENTARY DATA Supplementary data are available at Toxicological Sciences online. AUTHORS’ CONTRIBUTIONS M.L. conceived and designed the experiments; L.W. and Q.D. analyzed the data, prepared figure and contributed writing the manuscript; L.W., Q.D., D.G., Y.W., and S.X. collected the samples; L.W., Q.D., and H.R. performed the experiments; M.L. and Q.D. reviewing the manuscript. All authors contributed to the manuscript at various stages. FUNDING This work was supported by the National Basic Research Program of China (2014CB138504) and the National Porcine Industry Technology System (CAR-36). 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Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)
TI - Zearalenone Blocks Autophagy Flow and Induces Cell Apoptosis During Embryo Implantation in Gilts
JO - Toxicological Sciences
DO - 10.1093/toxsci/kfaa018
DA - 2020-05-01
UR - https://www.deepdyve.com/lp/oxford-university-press/zearalenone-blocks-autophagy-flow-and-induces-cell-apoptosis-during-QL4JZtOYFx
SP - 126
VL - 175
IS - 1
DP - DeepDyve
ER -