Deoxynivalenol and Zearalenone: Different Mycotoxins with Different Toxic Effects in the Sertoli Cells of Equus asinus

(1) Background: Deoxynivalenol (DON) and zearalenone (ZEA) are type B trichothecene mycotoxins that exert serious toxic effects on the reproduction of domestic animals. However, there is little information about the toxicity of mycotoxins on testis development in Equus asinus. This study investigated the biological effects of DON and ZEA exposure on Sertoli cells (SCs) of Equus asinus; (2) Methods: We administered 10 μM and 30 μM DON and ZEA to cells cultured in vitro; (3) Results: The results showed that 10 μM DON exposure remarkably changed pyroptosis-associated genes and that 30 μM ZEA exposure changed inflammation-associated genes in SCs. The mRNA expression of cancer-promoting genes was remarkably upregulated in the cells exposed to DON or 30 μM ZEA; in particular, DON and ZEA remarkably disturbed the expression of androgen and oestrogen secretion-related genes. Furthermore, quantitative RT-PCR, Western blot, and immunofluorescence analyses verified the different expression patterns of related genes in DON- and ZEA-exposed SCs; (4) Conclusions: Collectively, these results illustrated the impact of exposure to different toxins and concrete toxicity on the mRNA expression of SCs from Equus asinus in vitro.


Introduction
Deoxynivalenol (DON) and zearalenone (ZEA) are Fusarium mycotoxins produced by Fusarium fungi [1]. DON frequently occurs in the feed of livestock in combination with ZEA [2]. It is inevitable that grains will be contaminated by mycotoxins such as DON or ZEA during growth, storage, and processing [3,4]. Previous in vitro studies demonstrated that DON or ZEA exposure is linked to reproductive disorders in farm animals [5][6][7][8], and the toxins cause major economic loss in domestic animal industries [5,9]. In addition, ZEA causes reproductive disorders and demonstrates species-specific, organ-targeted oestrogenic activity in farm animals [10]. The effects include ovarian atrophy in pigs [11], follicular haematomas in horses [5], and reproductive failure in domestic animals [12][13][14][15]. However, the specific toxicity of DON and ZEA on reproduction in male Equus asinus is still unclear.
Equus asinus is a domestic animal that serves as a pet and draft animal and is important in mule and milk production worldwide [16,17]. According to the statistics of the Food and Agriculture Organization of the United Nations (FAO), in 2019, there were 50,583,572 Equus asinus in the world, of which 2,600,700 were in China (http://www.fao.org/faostat/ zh/#data/QA; accessed on 7 June 2021). China has a long history of raising Equus asinus for agriculture and transportation [8,18,19]. Moreover, Equus asinus is the major source of the Chinese traditional tonic E Jiao, the production of which was worth 17.8 billion dollars in 2020. Previous in vitro investigations indicated that DON or ZEA exposure may influence the genomic stability of Equus asinus and porcine granulosa cells (GCs) [7,20]. In addition, horses fed oats contaminated with ZEA have a high incidence of follicular haematomas [5,21]. The aberrant development of Sertoli cells is also related to some

Western Blot Analysis
Proteins isolated from SCs were used for Western blot analysis in accordance with previous standard methods [36,37]. Proteins from SCs in each treatment group were separated through 10% SDS-PAGE and transferred to PVDF membranes. The membranes were incubated overnight at 4 • C with primary antibodies (Table 1), rinsed three times with TBST, and incubated for 2 h at 37 • C with secondary antibodies (Sangon Biotech, D110058) in TBST. Related proteins were detected using AlphaImager ® (ProteinSimple, 92-13824-00, San Jose, CA, USA) HP. The intensity of all bands was quantified with GAPDH as the internal control using ImageJ software.

TUNEL Staining
The apoptosis rates of SCs were evaluated using a TUNEL BrightRed Apoptosis Detection Kit (Vazyme, A11302, Nanjing, China). Briefly, SCs were fixed for 2 h with 4% paraformaldehyde after 72 h of exposure to 0, 10, or 30 µM DON and ZEA. After the TUNEL reaction, the cells were observed under fluorescence microscopy in accordance with the manufacturer's instructions. TUNEL-positive cells were detected and counted under fluorescence microscopy (Olympus, XB51, Tokyo, Japan). More than 2000 SCs were obtained from each group and counted. Three biological replicates were used for analysis of the TUNEL-positive cell ratio.

RNA Extraction, Reverse Transcription, and RNA-Seq
RNAex pro reagent (AG, AG21101, Beijing, China) was used to extract total RNA from cultured DON-and ZEA-exposed SCs in accordance with the manufacturer's instructions. Then, mRNA was reverse transcribed into first-strand cDNA with a cDNA synthesis kit (TransGen, AT311-03, Beijing, China), referring to a previous study [35,38]. The Novogene Company performed RNA sequencing with the 4000 platform (Beijing, China).

Identification of Differentially Expressed Genes
The NovoMagic and R Bioconductor/DESeq2 packages were used to identify differentially expressed genes (DEGs) between different groups of SCs (0, 10, and 30 µM DON and ZEA treatment groups). Raw counts for differential expression analysis were obtained by using NovoMagic online and checked by using our own normalisation approach [35,39]. Data for differential expression analysis were previously normalised through other methods to prevent possible biases [40,41]. The log2|fold change|was disallowed as the filter condition because the sequencing design contained biological replicates for each group. Adjusted p < 0.01 was considered statistically significant.

GO and KEGG Enrichment Analysis
The functional profiles of DEGs were analysed through GO functional enrichment and KEGG pathway analysis by using NovoMagic. NovoMagic is the analysis platform developed by Novogene Company that can visualise GO and KEGG analysis results for DEGs online. In GO analysis, genes can be categorised as molecular function, biological process, and cellular component. NovoMagic was applied to visualise the KEGG analysis results. The log2|fold change|value of DEGs reflects the active status of enriched signalling pathways. Adjusted p < 0.05 was considered statistically significant.

Quantitative Real-Time PCR
Total RNA extraction and cDNA reverse transcription were performed as previously described. A SYBR ® Green Premix Pro Taq HS qPCR Kit was used to perform quantitative real-time PCR (RT-qPCR) on a LightCycler ® 96 RT-PCR instrument (Roche, Germany). RT-qPCR was performed under the following cycling conditions: 30 s at 95 • C; 40 cycles at 95 • C (5 s), 60 • C (30 s), and 72 • C (30 s); melting at 95 • C (1 s), 65 • C (15 s), and 95 • C (1 s); and a final cooling step at 4 • C. The RT-qPCR primers used in this study are listed in Table 2. GAPDH was used as the reference gene for the normalisation of mRNA expression in SCs. Gene expression was quantified through the 2−∆∆CT method. The expression level of each gene is expressed as the mean ± standard deviation (SD), which was calculated from the data of at least three independent biological replicates.

Scanning Electron Microscopy (SEM)
The treated SCs were washed with PBS 3 times, centrifuged for 10 min at 3000 rpm at 4 • C and fixed with 2.5% glutaraldehyde overnight. Next, tert-butanol was used to separate the glutaraldehyde. After being air-dried, the slides were critical-point dried, mounted on stubs, sputter-coated with a thin layer of conductive metal, gold, and palladium, and viewed by SEM (Hitachi HT7700, Hitachi, Ltd., Tokyo, Japan).

Statistical Methods
Data are presented as the mean ± SD. The statistical significance of different effects among the control, DON and ZEA exposure groups of SCs was determined through oneway ANOVA for multiple comparisons. All analyses were conducted using GraphPad Prism analysis software (San Diego, CA, USA). All experiments were repeated at least three times, and the results were considered significant at p < 0.05.

Apoptosis Rates and DEGs of Equus asinus SCs Exposed to DON and ZEA
First, we examined the purity of isolated SCs. The isolated cells were identified as SOX9 (a specific Sertoli cell marker)-positive [42] using immunohistochemistry methods (purity of isolated SCs > 97%) ( Figure 1A,B). Then, we exposed the SCs to 10 or 30 µM DON and ZEA for 72 h of in vitro culture ( Figure 1C). As shown in Figure 2, flow cytometry analysis was used to investigate the effects of DON and ZEA on cell apoptosis (Figure 2A). The results showed that the apoptosis rate was significantly increased under DON and ZEA treatment (10 µM DON: 16.60% ± 1.39%; 30 µM DON: 22.05% ± 1.11%; 10 µM ZEA: 8.06% ± 0.49%; 30 µM ZEA: 14.58% ± 1.42%) relative to that under the control treatment (0 µM DON: 4.47% ± 0.24%; 0 µM ZEA: 4.72% ± 0.31%; p < 0.05 or p < 0.01; Figure 2B,C). Interestingly, 10 µM DON exposure remarkably increased the apoptosis rate of SCs compared with 30 µM ZEA treatment. From Figure S1, the percentages of TUNEL-positive SCs also remarkably increased under DON and ZEA treatment (10 µM DON: 22.72% ± 1.79%; 30 µM DON: 64.15% ± 3.15%; 10 µM ZEA: 15.36% ± 2.79%; 30 µM ZEA: 24.17% ± 2.22%) relative to those under the control treatment (0 µM DON: 3.07% ± 0.14%; 0 µM ZEA: 3.32% ± 0.61%; p < 0.01; Figure S1).    Nine libraries from the three groups were sequenced, and 715,715,682 raw reads (GEO accession number: GSE172037), with 703,892,136 clean reads, were obtained. Then, we performed RNA-seq analysis to confirm the effects of DON and ZEA exposure on SCs ( Figure 3). We screened a total of 9393 and 6065 DEGs in the DON and ZEA treatment groups, respectively, based on the research criterion FDR < 0.05 ( Figure 3F). We found that 3300 and 3251 genes were up-and downregulated under 10 µM DON treatment, while 4841 and 3764 genes were up-and downregulated under 30 µM DON treatment, respectively ( Figure 3A,B). Furthermore, we identified 2816 and 3131 DEGs that were upand downregulated under 30 µM ZEA treatment, while only 391 and 412 DEGs were upand downregulated under 10 µM ZEA treatment, respectively ( Figure 3C,D). Meanwhile, we selected some DEGs between the mycotoxin and control groups with degrees greater than 20 to form a heat map ( Figure 3E).   We annotated the functional interactions of genes that were differentially expressed between the control and DON or ZEA treatment groups by using the STRING database to investigate the potential effects of mycotoxin exposure on SCs. Search Tool for the Retrieval of Interacting Genes/Proteins (STRING, https://string-db.org/; accessed on 7 June 2021) is a database of protein-protein interaction. This database contains the direct and physically related interactions between known and predicted protein and genes. The R Bioconductor/STRINGdb was applied for PPI of interested DEGs [43].

DEGs Involved in GO Classification and KEGG Pathways
We applied the NovoMagic and R packages to annotate the GO enrichment functions of DEGs in each group ( Figure 4A-C). We found that DEGs in DNA metabolic process, immune system process, DNA repair, and apoptotic process ( Figure 4A) were remarkably enriched in SCs exposed to 10 µM and 30 µM DON. Meanwhile, DEGs involved in the small molecular metabolic process, immune system process, DNA metabolic process, and cell cycle were remarkably enriched in SCs exposed to 10 µM and 30 µM ZEA ( Figure 4B). Moreover, DEGs were significantly enriched in the regulation of apoptotic processes in SCs exposed to 30 µM ZEA ( Figure 4C). In addition, upregulated DEGs in SCs under 10 µM DON treatment were significantly enriched in the immune system and apoptotic processes (p < 0.001). Downregulated DEGs in SCs exposed to 10 µM ZEA were significantly enriched in the steroid biosynthesis process [8]. We applied the NovoMagic and R packages to annotate the GO enrichment functions of DEGs in each group ( Figure 4A-C). We found that DEGs in DNA metabolic process, immune system process, DNA repair, and apoptotic process ( Figure 4A) were remarkably enriched in SCs exposed to 10 μM and 30 μM DON. Meanwhile, DEGs involved in the small molecular metabolic process, immune system process, DNA metabolic process, and cell cycle were remarkably enriched in SCs exposed to 10 μM and 30 μM ZEA ( Figure 4B). Moreover, DEGs were significantly enriched in the regulation of apoptotic processes in SCs exposed to 30 μM ZEA ( Figure 4C). In addition, upregulated DEGs in SCs under 10 μM DON treatment were significantly enriched in the immune system and apoptotic processes (p < 0.001). Downregulated DEGs in SCs exposed to 10 μM ZEA were significantly enriched in the steroid biosynthesis process [8]. We used NovoMagic and the clusterProfiler R package to identify extremely affected KEGG pathways to obtain insight into the function of DEGs ( Figure 4D-F). DEGs in SCs exposed to 10 μM and 30 μM DON were significantly enriched in the PI3K/AKT [35], MAPK, and TNF signalling pathways ( Figure 4D). Meanwhile, DEGs in SCs treated with 10 μM and 30 μM ZEA were significantly enriched in the regulation of the cell cycle and P53 signalling pathway ( Figure 4E). In addition, we identified DEGs that were significantly enriched in the PI3K/AKT [35], MAPK, and Hippo signalling pathways af- We used NovoMagic and the clusterProfiler R package to identify extremely affected KEGG pathways to obtain insight into the function of DEGs ( Figure 4D-F). DEGs in SCs exposed to 10 µM and 30 µM DON were significantly enriched in the PI3K/AKT [35], MAPK, and TNF signalling pathways ( Figure 4D). Meanwhile, DEGs in SCs treated with 10 µM and 30 µM ZEA were significantly enriched in the regulation of the cell cycle and P53 signalling pathway ( Figure 4E). In addition, we identified DEGs that were significantly enriched in the PI3K/AKT [35], MAPK, and Hippo signalling pathways after 30 µM ZEA treatment ( Figure 4F).
The results of GO and KEGG pathway analyses revealed that POLD1, Caspase1, GS-DMD, CCL17, and PRDX4, which are involved in DNA metabolism, pyroptosis, and inflammation processes, were differentially expressed in SCs exposed to 10 µM and 30 µM DON. The CDK1, CCNB2, ESR1, and NOX1 genes involved in the cell cycle and steroidrelated signalling pathways were changed in SCs exposed to 30 µM ZEA. WNT2, MSH6, RAF, and Cyclin D1 (CCND1), genes involved in cancer processes, were also differentially expressed after exposure to 10 µM and 30 µM DON.

Cellular and Molecular Effects of DON and ZEA Exposure on SCs
Exposure to 10 and 30 µM DON may lead to the pyroptosis of SCs (Figures 5-7), and DON and ZEA might induce inflammation and endocrine effects in SCs through different molecular mechanisms (Figures 8-11).
As shown in Figure 5, the number of immunofluorescence-positive genes, such as Caspase1 ( Figure 5A-C) and GSDMD ( Figure 5D-F), remarkably increased in the 10 µM and 30 µM DON groups, whereas the genes showed no significant differences in SCs treated with 10 µM and 30 µM ZEA relative to those in the control group. Moreover, exposure to 10 µM and 30 µM DON significantly upregulated the mRNA abundance and protein levels of Caspase1 ( Figure 6A-C) in SCs. In addition, the mRNA abundance and protein levels of GSDMD ( Figure 6B) and GSDMD-N ( Figure 6D) were increased after the treatment of 10 µM and 30 µM DON. Representative SEM images indicated that SCs treated with 10 µM and 30 µM DON undergo membrane perforation and produce apoptotic body-like cell protrusions prior to plasma membrane rupture [44] (Figure 7). However, there were no significant differences in SCs treated with 10 µM ZEA compared with the control group ( Figure 7).
Immunohistochemical results of SCs exposed to the mycotoxin indicated that the number of CCL17-positive cells remarkably increased in the 10 µM and 30 µM DON exposure groups ( Figure 8A-C), while there was a significantly increased number of IL10RA-positive cells in the 10 µM and 30 µM ZEA treatment groups ( Figure 8D-F). The mRNA abundance and protein levels of CCL17 and IL10RA were significantly upregulated in SCs exposed to 10 µM and 30 µM DON (p < 0.05 or p < 0.01; Figure 9A,C) and ZEA (p < 0.05 or p < 0.01; Figure 9B,D) relative to those in SCs under the control treatment.
As shown in Figure 10, the number of immunofluorescence-positive genes, such as AR, remarkably decreased in the 10 µM and 30 µM DON exposure groups ( Figure 10A-C), whereas the expression of ESR1 genes was significantly increased in SCs treated with 10 µM and 30 µM ZEA ( Figure 10D-F) relative to those in the control group. Moreover, exposure to 10 µM and 30 µM DON significantly downregulated the mRNA abundance and protein levels of the AR gene ( Figure 11A,C), while ZEA exposure significantly upregulated the expression of the ESR1 gene ( Figure 11B-D) in SCs.   treated with 10 μM and 30 μM DON undergo membrane perforation and produce apoptotic body-like cell protrusions prior to plasma membrane rupture [44] (Figure 7). However, there were no significant differences in SCs treated with 10 μM ZEA compared with the control group (Figure 7).  els were normalised to GAPDH. The exposure time was 50 s. The results are presented as the means ± SD. All experiments were repeated at least three times. ** p < 0.01.  Our RT-qPCR and Western blot results indicated that SCs under 10 µM and 30 µM DON treatment exhibited significantly lower MSH6 mRNA and protein levels than those under the control treatment (p < 0.05 or p < 0.01) ( Figure 14A,D), whereas the CDK1 and CCNB2 genes were significantly upregulated in the 10 µM and 30 µM ZEA exposure groups ( Figure 14B,C,E,F). We conducted more RT-qPCR to evaluate the expression of different transcripts in the pathways of SCs among the control, DON and ZEA treatments ( Figure S3). Cells 2021, 10, x FOR PEER REVIEW 15 of 27 Immunohistochemical results of SCs exposed to the mycotoxin indicated that the number of CCL17-positive cells remarkably increased in the 10 μM and 30 μM DON exposure groups ( Figure 8A-C), while there was a significantly increased number of IL10RA-positive cells in the 10 μM and 30 μM ZEA treatment groups ( Figure 8D-F). The mRNA abundance and protein levels of CCL17 and IL10RA were significantly upregulated in SCs exposed to 10 μM and 30 μM DON (p < 0.05 or p < 0.01; Figure 9A,C) and ZEA (p < 0.05 or p < 0.01; Figure 9B,D) relative to those in SCs under the control treatment.   As shown in Figure 10, the number of immunofluorescence-positive genes, such as AR, remarkably decreased in the 10 μM and 30 μM DON exposure groups ( Figure 10A-C), whereas the expression of ESR1 genes was significantly increased in SCs treated with

Discussion
Fusarium mycotoxins have been implicated in poor reproductive performance in domestic animals, including male Equus asinus [5,7,8,32,39,[45][46][47]. In vitro reports with DON or ZEA demonstrated that mycotoxins are able to directly affect the reproductive [47][48][49][50], endocrine [51,52], and immune systems [53][54][55], as well as inheritance [32,45]. Previous research demonstrated that the function of SCs is essential in the processes of normal spermatogenesis and testis development [30][31][32]. Moreover, present findings suggested that individual or mixtures of Fusarium toxins had cytotoxic effects on porcine Sertoli and Leydig cells [56,57]. However, additive effects were not always observed for the mixtures of Fusarium toxins [56,[58][59][60]. The present study was designed to investigate the effects of mycotoxin DON and ZEA treatment on pyroptosis, viability, the cell cycle, cell secretion, and cell inflammation in cultured SCs. We used immature SCs cultured in vitro and RNA-seq methods to compare the toxic effects of DON and ZEA to Equus asinus SCs. This is the first study to describe the differences in the transcriptomes of SCs between DON and ZEA exposure. Our results provide a basic database for mycotoxins in SC studies of Equus asinus.

Discussion
Fusarium mycotoxins have been implicated in poor reproductive performance in domestic animals, including male Equus asinus [5,7,8,32,39,[45][46][47]. In vitro reports with DON or ZEA demonstrated that mycotoxins are able to directly affect the reproductive [47][48][49][50], endocrine [51,52], and immune systems [53][54][55], as well as inheritance [32,45]. Previous research demonstrated that the function of SCs is essential in the processes of normal spermatogenesis and testis development [30][31][32]. Moreover, present findings suggested that individual or mixtures of Fusarium toxins had cytotoxic effects on porcine Sertoli and Leydig cells [56,57]. However, additive effects were not always observed for the mixtures of Fusarium toxins [56,[58][59][60]. The present study was designed to investigate the effects of mycotoxin DON and ZEA treatment on pyroptosis, viability, the cell cycle, cell secretion, and cell inflammation in cultured SCs. We used immature SCs cultured in vitro and RNA-seq methods to compare the toxic effects of DON and ZEA to Equus asinus SCs. This is the first study to describe the differences in the transcriptomes of SCs between DON and ZEA exposure. Our results provide a basic database for mycotoxins in SC studies of Equus asinus.
Pyroptosis is a newly discovered type of regulated necrotic cell death induced by inflammasomes, such as Caspase1 and Caspase11 (in mouse cells) [61,62]. Both Caspase1 and Caspase11 can induce pyroptosis by processing Gasdermin D (GSDMD), yielding an Nterminal fragment that forms pores on the plasma membrane, leading to cell death [63,64]. Unlike apoptosis, pyroptosis is a highly specific type of inflammation that has been proven to be strongly associated with cancer [65]. Our results showed that the mRNA and protein expression levels of Caspase1 and GSDMD-N in Equus asinus SCs remarkably increased upon exposure to 10 µM DON. Moreover, representative scanning electron microscopy images indicated that SCs treated with DON undergo membrane perforation and produce apoptotic body-like cell protrusions prior to plasma membrane rupture [44]. Some SCs also showed initial changes in the substructure of the plasma membrane characterised by focal disappearance of membrane structure and partial loss of continuity, as described for necrotic cells [66]. These results indicated that the canonical pyroptosis pathways in SCs had been activated and that SCs possessed the typical pyroptosis appearance [67] and severe cellular inflammation.
Furthermore, the mRNA abundance and protein levels of CCL17 in the SCs remarkably increased after exposure to DON. High levels of CCL17 have been found in SCs in seminoma tumours [68]. Therefore, DON treatment may promote the expression of the hallmarks of tumour formation given its effect on pyroptosis-related genes and CCL17 expression. ZEA is a non-steroidal oestrogenic mycotoxin. In contrast, depending on the molecular structure, ZEA may have binding affinities to oestrogen receptors and, therefore, mimic oestrogenic effects in SCs. We found that exposure to 30 µM ZEA significantly increased the mRNA abundance and protein levels of ESR1, ESR2, and IL10RA in SCs. This phenomenon indicates that exposure to ZEA may lead to inflammation and affect the endocrine function of SCs. A total of 6551 and 803 genes were differentially expressed in SCs under the control of 10 µM DON and ZEA treatment. Furthermore, we identified 8605 and 5947 DEGs in the SCs under 30 µM DON and ZEA treatment, respectively. In summary, the above results suggest that DON treatment may lead to pyroptosis and promote the expression of oncogenes, while exposure to ZEA results in inflammation and affects endocrine disruption in cells. Furthermore, Equus asinus SCs are more sensitive to DON exposure than SCs treated with the same dose of ZEA.
In addition, bioinformatics analyses found that PRDX4 and cell oxidation-reduction related genes were influenced in SCs exposed to 10 µM DON and 30 µM ZEA. Our results indicated that DON might have stronger oxidative toxicity than ZEA in Equus asinus SCs. Moreover, several studies have demonstrated that MSH6 (an important component of the mismatch repair system) is a tumour-related factor that can affect tumorigenesis, proliferation, migration, and invasion effects [69]. It is well known that suppression of MSH6 is associated with a variety of tumours [70,71]. Interestingly, it was found in this study that 10 µM DON exposure significantly decreased the mRNA and protein levels of MSH6 in SCs. Therefore, exposure to 10 µM DON may be potentially mutagenic and carcinogenic. Several studies have indicated that CCNB2 is a regulatory protein involved in mitosis, and its product can combine with CDK1 to form a maturation-promoting factor [72][73][74][75]. CCNB2 overexpression can result in uncontrolled cell growth [76,77].
In this study, we found that exposure to 30 µM ZEA remarkably upregulated the mRNA and protein abundance of CCNB2 in SCs. In contrast, exposure to 10 µM ZEA resulted in CDK1 overexpression in SCs. These results suggest that 30 µM ZEA treatment may promote the expression of oncogenes in SCs. Furthermore, exposure to 10 µM DON resulted in IL32 and NOX1 gene overexpression, while the AR, AIG1, MCM6, and POLD1 genes were suppressed in SCs. In addition, 30 µM ZEA treatment led to PFKM, NOX1, and ESR2 gene overexpression in the cells. In our experiments, we observed a suppressive effect on the mRNA abundance of the AR gene of SCs at concentrations of 10 µM and 30 µM DON treatment, while there was a stimulatory effect on the ESR1 and ESR2 genes of SCs under 30 µM ZEA exposure. The above results validated that DON and ZEA had differential toxic patterns in Equus asinus SCs. Additionally, the exact mechanisms by which DON or ZEA changes cell secretion in Equus asinus SCs remain the subject of further investigation.
We also found proapoptotic effects of DON and ZEA using flow cytometry and TUNEL-positive analysis. A significant reduction in the SCs was observed at toxic concentrations of both mycotoxins. The results demonstrate that the mycotoxins DON and ZEA can stimulate cell apoptosis in Equus asinus SCs. Both mycotoxins seem to transmit their molecular effects by influencing the MAPK signalling cascades and the protein kinase Akt, which could result in translation anomalies. However, it can be assumed that DON and ZEA modulate the process of translation at different molecular levels. Whereas DON mainly had an impact on pyroptosis and androgen disruption and therefore on the biological activity of the Caspase1, GSDMD, CCL17, AR, and AIG1 genes, ZEA increased the abundance of IL 10 RA, CDK1, PFKM, NOX1, and specific bands of the ESR1 and ESR2 genes. Thus, DON and ZEA exerted toxic effects on SCs in a different manner. Further investigations are required to obtain more information about specific signalling cascades that transmit the toxicity of DON and ZEA in Equus asinus SCs.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/cells10081898/s1, Figure S1: DON and ZEA exposure increasing apoptosis rate in cultured SCs. SCs were stained blue with Hoechst33342 solution. TUNEL assay was performed using immunostaining. (A) Immunofluorescent staining of TUNEL and Hoechst33342 of DON treated SCs. Bar indicates 50 µm. (B) The percentages of TUNEL positive SCs exposed to DON. (C) Immunofluorescent staining of TUNEL and Hoechst33342 of ZEA treated SCs. Bar indicates 50 µm. (D) The percentages of TUNEL positive SCs exposed to ZEA. The results are presented as mean ± SD. All experiments were repeated at least three times. p < 0.05; * p < 0.01. Figure S2: Immunofluorescence assay probing the expression of SCs phosphor-NOX1 proteins in DON (A) and ZEA (D) treatment groups. The percentages of positive cells (B/E) and fluorescence intensity (C/F) were analyzed respectively. Bar indicates 50 µm. Data are presented as means ± SD. p < 0.05; * p < 0.01. Figure

Institutional Review Board Statement:
The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Ethics Committee of QINGDAO AGRICULTURAL UNIVERSITY (protocol code DEC 2020-019 and 5 November 2020).

Informed Consent Statement: Not applicable.
Data Availability Statement: Nine libraries from the three groups were sequenced, and 715,715,682 raw reads (GEO accession number: GSE172037) were uploaded to Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/geo/, accessed on 25 July 2021).