Real-life exposure to Fusarium toxins deoxynivalenol and zearalenone triggers apoptosis and activates NLRP3 inflammasome in bovine primary theca cells

Cattle are deemed less susceptible to mycotoxins due to the limited internal exposure resulting from rumen microbiota activity. However, the significant amounts of Fusarium mycotoxins deoxynivalenol (DON) and zearalenone (ZEN) frequently detected in bovine follicular fluid samples suggest that they could affect ovarian function. Both mycotoxins trigger several patterns of cell death and activate the NLRP3 inflammasome in the intestine. In vitro studies have reported a number of adverse effects on bovine oocytes. However, the biological relevance of such findings with regard to realistic concentrations of DON and ZEN in bovine follicular fluid is still not clear. Hence, it is important to better characterize the effects of dietary exposure to DON and ZEN on the bovine ovary. Using bovine primary theca cells, this study investigated the effects of real-life patterns for bovine ovary exposure to DON and ZEN, but also DON metabolite DOM-1, on cell death and NLRP3 inflammasome activation. Exposure to DON starting from 0.1 μM significantly decreased theca cell viability. The kinetics of phosphatidylserine translocation and loss of membrane integrity showed that ZEN and DON, but not DOM-1, induce an apoptotic phenotype. qPCR analysis of the expression of NLRP3, PYCARD, IL-1β, IL-18, and GSDMD in primary theca cells at concentrations of mycotoxin previously reported in cow follicular fluid clearly indicated that DON and DOM-1 individually and in mixture, but not ZEN, activate NLRP3 inflammasome. Altogether, these results suggest that real-life dietary exposure of cattle to DON may induce inflammatory disorders in the ovary.


Introduction
Mycotoxins frequently contaminate food and feed materials, posing a threat to human and animal health (Alassane-Kpembi et al. 2019).Cattle are deemed less susceptible to mycotoxins than monogastrics because the rumen microbiota can degrade or deactivate toxic molecules, limiting the internal exposure to mycotoxins (Gallo et al. 2015).However, the intrinsic sensitivity of ruminant cells may be the same or even higher than monogastrics, as suggested by a comparative study addressing the sensitivity of peripheral blood mononuclear cells isolated from bovine, poultry, and swine to the Fusarium mycotoxin deoxynivalenol (DON) (Novak et al. 2018).Moreover, some mycotoxins possess antimicrobial properties through which they can modify the ruminal microbiota, thereby reducing its detoxification capacity (Debevere et al. 2020;Hartinger et al. 2022).Hence, it is important to better characterize the health risks associated with the dietary exposure of ruminants to mycotoxins.DON and zearalenone (ZEN) are among the most important mycotoxins produced by Fusarium fungi with regard to their occurrence and toxicity (Pierron et al. 2016).Exposure to DON causes feed rejection, vomiting, and immune alterations in animals (EFSA CONTAM Panel 2017a; Yang et al. 2020).At the molecular level, DON induces ribotoxic stress, which disrupts normal cell function by impairing macromolecule synthesis and activating critical cellular kinases involved in the transduction of signals relating to proliferation, differentiation, inflammation, and apoptosis (Payros et al. 2016).DON induces several types of cell death patterns.We reported both the mitochondrial and the death receptor pathways to be involved in the apoptosis induced by DON (Payros et al. 2021).More recently, pyroptosis, another pattern of programmed cell death that relates to DON-induced inflammation, has also been described (Mao et al. 2022).As far as ZEN is concerned, reproductive toxicity in animals due to its estrogenic activity is the most studied aspect (EFSA CONTAM Panel 2017b; Liu and Applegate 2020).ZEN binds to estradiol receptors in the absence or presence of low levels of estrogen, causing morphological and biological changes especially in the reproductive organs (Zinedine et al. 2007).At the cellular level, ZEN also induces oxidative damage resulting from the accumulation of reactive oxygen species that can act as signaling molecules in a number of pathological conditions, including inflammation (Ben Salah-Abbès et al. 2021).
Both ZEN and DON have been shown to induce intestinal inflammation through activation of the NLRP3 inflammasome (Fan et al. 2018;Qu et al. 2022).In the inflammatory response mechanisms, inflammasomes are cytoplasmic multiprotein complexes comprising a sensor protein and inflammatory caspases, the activation of which results in secretion of IL-1β and IL-18, as well as pyroptotic cell death (Zheng et al. 2020).While appropriate inflammasome activation is crucial for the sensing and control of foreign pathogens or tissue damage, aberrant activation may entail uncontrolled tissue responses that can contribute to various pathogenic processes (Li et al. 2021).Especially, activation of the inflammasome in the microenvironment of the ovary has been associated with ovarian aging and alteration of the number and quality of oocytes (Huang et al. 2019;Navarro-Pando et al. 2021).Moreover, the ovulation failure and development of follicular cysts in cows with cystic ovarian disease has shown to involve alteration of the expression of pro-inflammatory cytokines in theca and granulosa cells (Baravalle et al. 2015).
Not only DON and ZEN but also DOM-1, the de-epoxy metabolite of DON, have been detected in ovarian follicular fluid of cows, suggesting that following ingestion, significant amounts of these mycotoxins can be transported to the ovary and may locally interfere with reproductive function in cattle (Takagi et al. 2008;Winkler et al. 2015).Several in vitro studies have reported a number of adverse effects in ZEN-treated bovine oocytes, including alteration of nuclear maturation and developmental ability after fertilization (Minervini et al. 2001;Takagi et al. 2008;Yousef et al. 2023).Likewise, in vitro exposure to DON has been shown to increase apoptosis of bovine theca and granulosa cells and to decrease progesterone and estradiol production (Guerrero-Netro et al. 2015, 2017;Pizzo et al. 2016Pizzo et al. , 2015)).However, the biological relevance of such findings with regard to the real-life concentrations of ZEN and DON in bovine follicular fluid is still not clear.
Using bovine primary theca cells, the purpose of this study was to investigate the effects of real-life exposure to DON, ZEN, and DOM-1 on cell death and NLRP3 inflammasome activation in the bovine ovary.

Toxins
Certified Biopure standard grade de-epoxy deoxynivalenol (DOM-1) in acetonitrile (Romer Labs, Vancouver, BC, Canada) reconstituted at 10 mM in dimethylsulfoxide (DMSO) and purified deoxynivalenol (DON) and zearalenone (ZEN) purchased from Sigma-Aldrich (Oakville, ON, Canada) and dissolved in DMSO at 50 mM and 25 mM, respectively, were stored at -20 ℃ before dilution in complete culture media.

Ovary collection and extraction of bovine theca cells
Unless otherwise stated, culture media and supplements were purchased from Wisent (Wisent Bioproducts, St-Bruno, QC, Canada).Bovine ovaries obtained at a local slaughterhouse from adult cows irrespective of stage of the estrous cycle were transported to the laboratory at 30 ℃ in a phosphatebuffered saline (PBS) containing penicillin (100 IU) and streptomycin (100 μg/mL).Four-to six-millimeter diameter follicles were bisected, and the theca "shells" were peeled from the stroma and incubated for digestion in a solution of 1 mg/mL collagenase type IV (Sigma-Aldrich, Oakville ON, Canada) and 100 μg/mL trypsin inhibitor (Sigma-Aldrich) as previously described (Guerrero-Netro et al. 2017).The resulting cell suspension was filtered through a 150-mesh steel sieve, centrifuged (800 g for 10 min), and the cell pellets were re-suspended in PBS before being subjected to an osmotic shock treatment to remove red blood cells.The theca cells were then washed and re-suspended in McCoy 5A culture medium supplemented with 100 IU/mL penicillin, 1 μg/mL Fungizone, 10 ng/mL bovine insulin, 2 mM L-glutamine, 10 mM HEPES, 5 μg/mL transferrin, 5 ng/mL sodium selenite, and 0.1% BSA.Overall cell viability was assessed using the trypan blue dye exclusion staining, and the cells were seeded on the suitable tissue culture plates for subsequent experiments.

Cytotoxicity test
Theca cells were plated at a density of 30,000 cells per well in 100 μL McCoy 5A modified medium (Wisent Bioproducts, St-Bruno, QC, Canada) in 96-well flat-bottom cell culture plates (Costar, Cambridge, MA, USA).The cells were cultured in an incubator at 37 °C in 5% CO 2 for 48 h, after which 70% of the cell culture medium was replaced by a medium containing graded concentrations of mycotoxins DON or DOM-1 or ZEN, resulting in final mycotoxin concentrations in wells of 10, 1, 0.1, 0.01, 0.001, or 0 μM.After 24 h of mycotoxin exposure, the cell viability was assessed through resazurin reduction quantification using the CellTiter Blue fluorescent cell viability assay (Promega, Madison, WI) according to the manufacturer's instructions.Briefly, 20 µL CellTiter Blue reagent was added to each well, and the cell culture plates were incubated using standard cell culture conditions for 4 h, and the fluorescence (ex/em: 560/590) was recorded in a SpectraMax i3 plate reader (Molecular Devices, San Jose, CA).

Real-time apoptosis and necrosis assay
Apoptosis and necrosis were analyzed simultaneously using the RealTime-Glo™ Annexin V Apoptosis and Necrosis Assay (Promega, Madison, WI, Cat#: JA1011).The assay measures the exposure of phosphatidylserine on the outer leaflet of the cell membrane during the apoptotic process and contains a cell-impermeant pro-fluorescent DNA dye that generates a fluorescent signal upon loss of membrane integrity.
Theca cells were seeded at a density of 30,000 cells per well in 100 μL McCoy 5A modified medium (Wisent Bioproducts, St-Bruno, QC, Canada) in white clear-bottom 96-well plates (Costar, Cambridge, MA, USA) and allowed stabilized in a CO 2 incubator.After 48 h, 50μL of culture medium was removed, and the same volume of a 4X mycotoxin containing fresh medium was added.Meanwhile, a 2X solution of the RealTime-Glo™ Annexin V Apoptosis and Necrosis Assay detection reagents was prepared in culture medium, according to the manufacturer's instructions.A total of 100μL of the 2X reagent solution was added in each well, resulting in a total of 200μL of liquid per well, ensuring a final mycotoxin concentration of 1 μM.The plates were then incubated at 37 °C in 5% CO 2 , and measurements of membrane integrity as fluorescence (ex/em: 485 ± 10/525 ± 10) and phosphatidylserine translocation as a luminescence signal produced by the Annexin V-dependent assembly of two fragments of a luciferase were performed at 2 h, 6 h, 24 h, 48 h, and 72 h.

Total RNA extraction and real-time PCR
The expression of the inflammasome-related genes NLPR3, PYCARD, IL-1β, IL-18, and GSDMD in bovine thecal cells following exposure to the mycotoxins was analyzed using specific primers (Table 1).The theca cells were seeded into 24-well tissue plates (Costar, Cambridge, MA, USA) at a density of 250,000 viable cells in 1 mL and cultured at 37 °C in 5% CO 2 , for 48 h, after which 70% of the cell culture medium was replaced by a medium containing mycotoxins DON or DOM-1 or ZEN.After 72 h of mycotoxin exposure (ZEN 0.01 μM or 10 μM; DON 0.01 μM or 10 μM; DOM-1 1 μM or 10 μM; DON 0.01 μM + DOM-1 1 μM), the medium was removed and total RNA was extracted using PureZOL™ RNA isolation reagent (Bio-Rad) according to the manufacturer's instructions.Following purity and concentration check on a NanoDrop spectrophotometer, total
Real-time PCR was performed on a CFX96 Touch Real-Time PCR Detection System (Bio-Rad) using qPCR Mastermix with Supergreen (Wisent Bioproducts, St-Bruno, QC, Canada) following the manufacturer's protocol.Melting curve was analyzed to verify PCR product specificity, and the amplicons were sequenced to verify authenticity.Samples were run in duplicate, and data were expressed relative to YWHAZ as housekeeping gene and normalized to a calibrator sample using the Pfaffl method with correction for amplification efficiency (Pfaffl 2001).

Statistical analysis
All statistical analyses were performed with Prism software 8.0.2 for Windows (GraphPad).The reported values are the means ± standard error of the mean (SEM) of at least three independent experiments, each with triplicate wells per experimental condition.Data that did not follow a normal distribution (Shapiro-Wilk test) were log-transformed prior to statistical analysis.Student's t-tests or one-way ANOVAs followed by Tukey's multiple comparison tests were run for comparisons between the experimental conditions.Differences with a p value < 0.05 were considered statistically significant.

Low doses of DON alter the viability of bovine theca cells
Dietary exposure of dairy cows to the mycotoxins ZEN and DON in the range of the current guidance values resulted in the presence not only of both mycotoxins but also DOM-1, the de-epoxy metabolite of DON in the follicular fluid (Winkler et al. 2015).Using the CellTiter Blue test, the effects of graded concentrations of these mycotoxins on the viability of bovine theca cells were evaluated.After a 24-h exposure, the highest toxicity on the theca cells was observed with DON which caused a statistically significant decrease in cell viability starting from 0.1 μM (p < 0.01), with IC 50 = 0.63 ± 0.07 μM (Fig. 1).A significant reduction of cell viability was also observed in ZEN (p < 0.01) and DOM-1 (p < 0.05) treatments, but only at the highest tested dose (10 µM).Compared with DOM-1, 10 µM ZEN had a stronger effect on the inhibition of theca cell viability.

ZEN and DON, but not DOM-1, induce an apoptotic phenotype in bovine theca cells
To explore the mechanisms behind the observed cell death, we performed in parallel the kinetics of phosphatidylserine translocation and loss of membrane integrity in the theca cells exposed to ZEN or DON or DOM-1.Using the RealTime-Glo™ Annexin V Apoptosis and Necrosis Assay, luminescence, meaning the translocation of phosphatidylserine, and fluorescence, meaning the loss of membrane integrity, were sequentially recorded throughout a 72-h exposure of the theca cells to 1 µM of each mycotoxin (Fig. 2).Apoptotic cell death is characterized by a delay between phosphatidylserine translocation and loss of membrane integrity, whereas necrotic cell death is defined by concomitant phosphatidylserine translocation and loss of membrane integrity (Kupcho et al. 2019).The results indicate that within 72 h of cell culture, both the fluorescence and luminescence signal in even the control cells increased slowly because of the mechanism of normal primary cell apoptosis.However, in the process of toxin exposure, all ZEN, DON, and DOM-1 induced higher luminescence signals within 2 h (p < 0.01 or p < 0.001).Compared with the DOM-1 condition, there was a clear temporal lag between phosphatidylserine translocation and loss of membrane integrity in the ZEN and DON conditions, which indicates that necrosis observed is secondary to a primary apoptotic process.Indeed, ZEN and DON induced a significant necrosis signal starting only from 24 h (p < 0.05), while the necrotic signal in DOM-1 condition was already higher at 2 h (p < 0.05).This kinetic difference in the onset of the apoptosis and necrosis signals induced by ZEN and DON, but not by DOM-1, is the hallmark of the apoptotic phenotype.

DON and DOM-1, but not ZEN, activate NLRP3 inflammasome in theca cells
Building from the toxicity scale indicated by the viability assay, we investigated the effects of graded doses of ZEN, DON, and DOM-1 on NLRP3 inflammasome activation in theca cells.The expressions of the pattern recognition receptor encoding gene NLRP3, adaptor protein encoding gene PYCARD, the cytokine encoding genes IL-1β and IL-18, and the pore-forming protein encoding gene GSDMD were analyzed after 72-h exposure of theca cells to the mycotoxins (Fig. 3).The expression of all the NLRP3 inflammasomerelated genes was dramatically increased upon exposure of the theca cells to 1 µM DON (p < 0.001).Even the 0.01 µM dose of DON significantly upregulated the expression of NLRP3, IL-1β, and GSDMD (p < 0.05 or p < 0.01).Albeit to a lesser extent, DOM-1 at 1 µM and 10 µM significantly induced the activation of all the genes except IL-18 (p < 0.05 or p < 0.01 or p < 0.001).Conversely, only the expression of NLRP3 and IL-1β in theca cells was slightly modified following exposure to 10 µM ZEN (p < 0.05).

NLRP3 inflammasome activation induced by low-dose DON in theca cells is potentiated by DOM-1
To better capture the effects of dietary DON on NLRP3 inflammasome activation in the ovary, we analyzed the gene expression of NLRP3, PYCARD, and IL-1β following exposure of bovine theca cells to a mixture of 0.01 μM DON and 1 μM DOM-1, which is within their reported concentration range in bovine follicular fluid (Winkler et al. 2015) (Fig. 4).All the tested genes were upregulated by exposure of theca cells to the mixture for 72 h (p < 0.01 or p < 0.001).Moreover, combining 1 µM DOM-1 to 0.01 µM DON resulted consistently in a significantly higher activation of the tested genes compared with 0.01 µM DON alone, or 1 µM DOM-1 alone (p < 0.05 or p < 0.01).Of note, although the activation of IL-1β by 1 µM DOM-1 alone was not significant, the combined effect with 0.01 µM DON resulted in a significantly higher gene activation compared with DON alone (p < 0.05).

Discussion
This study demonstrates that realistic concentrations of the Fusarium mycotoxin DON trigger apoptosis in bovine theca cells and activate the NLRP3 inflammasome.Winkler et al. (2015) and Takagi et al. (2008) estimated real-life exposure of the cow ovary to Fusarium mycotoxins by feeding dairy cows a diet contaminated by DON and ZEN at levels close to the current European Union (EU) guidance values, or by collecting ovaries at a local abattoir in Japan.The concentrations detected following LC/MS/MS analysis of the follicular fluids ranged from 0.1 to 0.4 nM for ZEN, from 0 to 10 nM for DON, and from 0 to 320 nM for DOM-1.In this study, we investigated for concentrations of ZEN, DON, and DOM-1 covering this realistic range, the cytotoxicity and inflammasome activation in bovine ovary using primary theca cells.
All three compounds showed to decrease the viability of theca cells, albeit with different efficacy, ZEN and DOM-1 appearing much less effective than DON.The observed reduction of viability was significant starting from 0.1 µM DON, versus 10 µM for DOM-1 and ZEN.Compared with other farm animal species, few studies investigated the toxicity of mycotoxins for bovine primary cells or cell lines.DON inhibited the proliferation of bovine peripheral blood mononuclear cells at concentrations starting from 0.27 µM and triggered apoptosis in primary granulosa cells starting from 0.33 µM (Daenicke et al. 2011;Guerrero-Netro et al. 2015).The reported cytotoxic concentrations for DON in this study indicate that theca cells may be among the most sensitive bovine cells to DON toxicity.With respect to cell lines that seem globally less sensitive than primary cells, DON significantly altered the viability of bovine mammary and kidney epithelial cells from 0.83 and 33 µM, respectively (Bailey et al. 2019;Wang et al. 2019a).On the other hand, only concentrations of ZEN higher or equal to 5 µM and 200 µM induced significant apoptosis in bovine mammary epithelial and kidney epithelial cells, which is in line with the reported lower cytotoxicity of ZEN compared with DON (Bailey et al. 2019;Fu et al. 2019).
In this study, no IC 50 could be derived for the alteration of the viability of theca cell induced by the de-epoxy metabolite DOM-1 although the highest concentration tested (10 µM) reduced the viability by 12%.This corroborates the residual toxicity of DOM-1 relative to its parent compound DON that was shown in bovine peripheral blood mononuclear and theca cells (Daenicke et al. 2011;Guerrero-Netro et al. 2017;Novak et al. 2018).
Fig. 3 Effects of ZEN, DON, and DOM-1 on the NLRP3 inflammasome activation in theca cells.Expression of NLRP3 inflammasomeassociated gene IL-1β, IL-18, GSDMD, PYCARD, and NLRP3 in theca cells treated with high and low doses of DON (0.01 μM, 1 μM) (A), DOM-1 (1 μM, 10 μM) (B), and ZEN (0.01 μM, 10 μM) (C) for 72 h (n = 3).Compared with the control, *p < 0.05, **p < 0.01, and ***p < 0.001 ◂ To clarify the mechanisms involved in the cytotoxicity observed with DON, ZEN, and DOM-1 in theca cells, we monitored in parallel the kinetics of the phosphatidylserine translocation and the loss of membrane integrity in the exposed cells.The kinetics of membrane phosphatidylserine translocation and loss of membrane integrity are valuable indicators for the mode of cell death, since apoptosis inducers are expected to produce substantial phosphatidylserine translocation prior to the loss of membrane integrity (Molagoda et al. 2019).In contrast with DON and ZEN that are known apoptosis inducers (Cai et al. 2019;Payros et al. 2021), there was no kinetic difference in the phosphatidylserine release and the loss of membrane integrity in the theca cells exposed to DOM-1.A previous study demonstrated the lack of caspase-3 activation by DOM-1 up to 100 µM in the non-transformed porcine intestinal epithelial IPEC-J2 cell line (Springler et al. 2017).Caspase-3 activation is a tipping point for the mammalian cell's entry into the apoptotic pathway, as a convergent point for the extrinsic and intrinsic apoptotic pathways (Nicholson et al. 1995).The results presented here strengthen the hypothesis that the residual cytotoxicity of DOM-1 involves alternative forms of cell death.
Next, we investigated the effects of ZEN, DON, and DOM-1 on inflammasome activation in the bovine ovary.Several publications have reported that the Fusarium mycotoxins ZEN and DON activate the NLRP3 inflammasome.Exposure to 200 µM ZEN was shown to activate NLRP3 inflammasome and pyroptotic cell death in rat INS-1 insulinoma cells (Wang et al. 2019b).NLRP3 inflammasome activation was also shown in pig IPEC-J2 cells and mouse peritoneal macrophages exposed to 8 µg/ mL ZEN and in the colon of mice fed a diet contaminated by 4.5 mg/kg ZEN (Fan et al. 2018).Likewise, the treatment of mice BV2 microglial cells with DON significantly induced the expression of ASC and NLRP3 and stimulated the secretion of IL-1β, highlighting the potential of this mycotoxin to trigger neuroinflammation (Molagoda et al. 2019).Recent experimental results in mice have stressed the NLRP3 inflammasome as a potential therapeutic target for the treatment of ovarian aging and subsequent infertility, firstly as the ablation of NLRP3 improved the pregnancy rate of Nlrp3 −/− mice, and secondly as serum anti-Müllerian hormone (AMH) levels of the mice were inversely correlated with NLRP3 inflammasome activation in the ovaries (Navarro-Pando et al. 2021;Lliberos et al. 2020;Lliberos et al. 2021;D. Wang et al. 2020).The serum AMH level reflects the functional ovarian reserve, which means the pool of growing follicles that potentially can ovulate (Moolhuijsen and Visser 2020).A diminished ovarian reserve is characterized by poor fertility outcomes even when assisted reproductive techniques are used (Cohen et al. 2015) and is associated with a poorer response to superovulation in cattle (Mossa and Ireland 2019).Of note, dietary intake of ZEN below the threshold contamination level permitted in Japan, i.e. < 1 mg/kg, was shown to significantly alter AMH concentrations in female cattle herds (Fushimi et al. 2015).Hence, it appears important to clarify the effects of the concentrations of ZEN and DON, but also DOM-1, that reach the ovaries on in situ inflammasome activation.In our study, the qPCR analysis of bovine primary theca cells exposed to 1 µM DON showed a transcriptional upregulation of the canonical NLRP3 inflammasome components NLRP3, PYCARD (also referred to as ASC), IL-1β, and IL-18, which is a clear indication of the priming of NLRP3 inflammasome (Swanson et al. 2019).Interestingly, we also observed an upregulation of GSDMD which encodes a pore-forming protein known as the executioner of pyroptosis, a proinflammatory type of cell death induced by the activation of the NLRP3 inflammasome (Yu et al. 2021).This was still obvious when the theca cells were exposed to 10 nM, indicating a dose-dependent effect, and suggests that reallife exposure to DON in the bovine ovary can trigger local inflammasome activation.
Exposure of the theca cells to up to 10 µM ZEN did not induce significant upregulation of PYCARD, IL-1β, or IL-18 mRNA abundance, indicating that activation of NLRP3 inflammasome is unlikely at the concentrations ZEN found within the bovine ovary.These contrasting effects of ZEN and DON on bovine theca cells are in line with the previous observations that ZEN, unlike trichothecene mycotoxins, does not activate the NLRP3 inflammasome in LPS-activated human macrophages (Kankkunen et al. 2014;Lee et al. 2021).
Although much less effective than DON, DOM-1, especially at 10 µM, clearly triggered the NLRP3 inflammasome in theca cells.In this study, the lowest concentration of DOM-1 tested that induced a transcriptional upregulation, albeit partially, of the canonical NLRP3 inflammasome components was 1 µM.This concentration is still in the range of the anticipated exposure level of the ovaries of cows fed in compliance with current mycotoxin regulations.It also suggests that the fraction of DON that is converted into DOM-1 may contribute significantly to the overall activation of the inflammasome in the ovary.Hence, to refine the characterization of the actual risk associated with dietary DON, we investigated the effects of DON and DOM-1 in combination on the activation of NLRP3 inflammasome in bovine theca cells.A concentration of DOM-1 likely to reach the bovine ovary clearly potentiated the activation of NLRP3 inflammasome induced by DON.Altogether, the results indicate that even assuming a partial conversion into DOM-1, the DON concentrations expectable in the ovary under practical feeding conditions may trigger inflammatory disorders in the bovine ovary.However, the primary cultures of theca cells used in this study represent an oversimplified model with several limitations including the lack of interplay of theca cells with other ovary structures and possible hyper-responsive state of the theca cells, due to in vitro manipulations.Nonetheless, these findings warrant in vivo investigations of the effects of DON in feed at current EU guidance values on the cattle ovarian health.
In conclusion, this study investigated the effects of reallife exposure to Fusarium mycotoxins DON and ZEN on cell death and NLRP3 inflammasome activation in bovine ovarian theca cells.Low doses of both mycotoxins induced an apoptotic phenotype in bovine theca cells.Moreover, at concentration ranges close to what is anticipated in follicular fluid of dairy cows based on the current EU guidance values, DON but also its metabolite DOM-1 activated the NLRP3 inflammasome in theca cells.Given the connection between ovarian inflammation, aging, and infertility, the impact of DON exposure on the dairy cow fertility deserves more attention.

Fig. 2
Fig. 2 ZEN, DON, and DOM-1 induce apoptosis and necrosis in theca cells.RealTime-Glo™ Annexin V Apoptosis and Necrosis Assay detection of apoptosis and secondary necrosis in 1 μM of ZEN

Fig. 4
Fig. 4 Combined effects of low doses of DON and DOM-1 on the NLRP3 inflammasome activation in bovine theca cells.Expression of the inflammasome-associated genes NLRP3, PYCARD, and IL-1β in theca cells treated with 0.01 μM DON or 1 μM DOM-1, or 0.01 µM

Table 1
Primer sequences for the Bos taurus genes used in this study