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Exposure of oocytes to the Fusarium toxins zearalenone and deoxynivalenol causes 1

aneuploidy and abnormal embryo development# 2

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The mycotoxins zearalenone and deoxynivalenol produced by Fusarium species impair 4

oocyte maturation by altering meiotic spindle formation leading to reduced embryo 5

development and aneuploid embryos after fertilization 6

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Hassan Malekinejad1,4, Eric J. Schoevers2, Ineke J.J.M. Daemen2, Carla Zijlstra3, Ben 8

Colenbrander2, Johanna Fink-Gremmels1 and Bernard A.J. Roelen2* 9

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1Department of Veterinary Pharmacology, Pharmacy and Toxicology, Faculty of 11

Veterinary Medicine, Utrecht University, Yalelaan 104, 3584 CM Utrecht, The 12

Netherlands; 2Department of Farm Animal Health, Faculty of Veterinary Medicine, 13

Utrecht University, Yalelaan 104, 3584 CM Utrecht, The Netherlands; 3Department of 14

Biochemistry and Cell Biology, Faculty of Veterinary Medicine, Utrecht University, 15

Yalelaan 2, 3584 CM Utrecht, The Netherlands 16

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4Present address: Department of Pharmacology and Toxicology, Faculty of Veterinary 18

Medicine, Urmia University, PO Box 1177, Urmia, Iran. 19

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Keywords: Zearalenone/ Deoxynivalenol/ food safety/ mixoploidy/ reproduction 21

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Grant support 24

#This study was financially supported by the Iranian Ministry of Sciences, Research and 25

Technology 26

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* Correspondence to: Bernard A.J. Roelen, Department of Farm Animal Health, Faculty 28

of Veterinary Medicine, Utrecht University, Yalelaan 104, 3584 CM, Utrecht, The 29

Netherlands. Telephone: +31 30 2533352, E-mail: b.a.j.roelen@vet.uu.nl 30

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ABSTRACT 31

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Fungi of the Fusarium species can infect food and feed commodities and produce the 33

mycotoxins zearalenone (ZEA) and deoxynivalenol (DON). Since both toxins have been 34

reported to reduce fertility, the mechanisms of ZEA and DON on inhibition of oocyte 35

maturation was examined. Porcine oocytes were matured in the presence of ZEA (a 36

pseudoestrogen), estradiol and DON. ZEA, estradiol and DON inhibited oocyte 37

maturation with DON being more toxic at equimolar concentrations than ZEA. Different 38

ratios of ZEA/DON did not lead to a more severe inhibition of oocyte maturation. Both 39

mycotoxins caused abnormal formation of the meiotic spindle. 40

The developmental competence of oocytes matured in the presence of mycotoxins was 41

further investigated after in vitro fertilization. Presence of ZEA during maturation 42

reduced the percentages of oocytes that cleaved and formed a blastocyst. Maturation in 43

the presence of equimolar concentrations of DON was not compatible with development. 44

Of the blastocysts that had developed after maturation in the presence of mycotoxins, 45

ploidy of the blastomeres was analysed with fluorescent in situ hybridisation. All 46

blastocysts, even of the control group, contained at least one blastomere with abnormal 47

ploidy, but embryos from oocytes that were exposed to mycotoxins contained more 48

polyploid blastomeres. It is concluded that ZEA and DON can lead to abnormal spindle 49

formation leading to less fertile oocytes and embryos with abnormal ploidy and that the 50

effects of ZEA and DON are not synergistic. 51

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INTRODUCTION 52

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Zearalenone (ZEA) is a mycotoxin, primarily produced by Fusarium fungi, with an 54

unique non-steroidal resorcyclic acid lactone structure. This structure resembles many 55

characteristics of steroid hormones and allows ZEA to bind to both types of estrogen 56

receptors (ERs), ER-α and ER-β, where it acts as an agonist and partial antagonist to 57

estradiol [1-3]. Fungi of Fusarium species can infect maize, wheat, rice and barley crops 58

and their resistant toxins can be transferred into consumer products. ZEA has been found 59

to induce estrogenic effects, often reported as hyperestrogenism, in all laboratory animal 60

species tested, as well as in farm animals, particularly in pigs. In humans, exposure to 61

ZEA has been associated with epidemics of premature thelarche [4]. Species-differences 62

in the susceptibility to ZEA exposure have been associated with differences in the hepatic 63

and extra-hepatic metabolism of ZEA that is catalysed by hydroxysteroid hydrogenases 64

[5]. ZEA is converted primarily into two isomeric metabolites, alpha zearalenol (α-ZOL) 65

and beta-zearalenol (β-ZOL). Different lines of evidence, including receptor binding and 66

cell proliferation assays with estrogen-dependent MCF-7 cells, have indicated that α-67

ZOL has a higher estrogenic potency as compared to the parent ZEA, whereas β-ZOL has 68

a lower potency [5]. The species-specific sensitivity observed in clinical trials and in field 69

studies correlates with the rate of bioconversion into α-ZOL, and identified the pig as the 70

most sensitive farm animal species. Common clinical symptoms in young, premature pigs 71

comprise vulva swelling, enlarged nipples and an enlarged uterus, whereas in cycling 72

sows, a decreased fertility, increased number of resorptions and a reduced litter size as 73

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well as changes in the levels of circulating estrogen and progesterone levels have been 74

described. In boars, enlarged nipples and reduced testes weight have been observed [6-8]. 75

A common feature of many Fusarium species is that besides their ability to produce ZEA, 76

non-estrogenic sesquiterpenoid trichothecenes can be produced. At present, more than 77

180 individual trichothecenes are described, the most frequently occurring being 78

deoxynivalenol (DON). DON exerts pro-inflammatory effects by inducing cytokine and 79

chemokine expression in mononuclear phagocytes [9-11]. In pigs, which again seem to 80

be the most sensitive species, inflammatory alterations occur particularly in the gastro-81

intestinal tract, hampering nutrient transport and resulting in reduced weight gain. 82

Moreover, at high doses DON has pro-emetic effects whereas at lower concentrations 83

feed intake is decreased, contributing to impaired growth and performance. The 84

sensitivity of animals to DON increases when these are exposed at the same time to 85

infectious agents, as co-exposure results in a concomitant inflammatory response [12]. 86

ZEA and DON also have been reported to inhibit oocyte nuclear maturation [13] but the 87

mechanisms of toxicity are unknown. Since in practice feeds are generally contaminated 88

with both mycotoxins ZEA and DON, it is important to know how cells behave when 89

exposed to both toxins simultaneously. Here we demonstrate that exposure of porcine 90

oocytes to the mycotoxins ZEA and DON leads to meiotic spindle abnormalities resulting 91

in a high percentage of early embryonic death and embryos with chromosome 92

abnormalities. Synergistic toxicity of ZEA and DON on oocyte maturation was not 93

observed. 94

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MATERIAL AND METHODS 95

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Culture media 97

All chemicals for the preparation of culture media were purchased from Sigma Chemical 98

Company (St Louis, MO, USA) unless otherwise indicated. Oocyte maturation medium 99

(OMM) was M199 (Gibco BRL, Paisley, UK) supplemented with 2.2 mg/ml NaHCO3, 100

0.1% (w/v) polyvinyl alcohol (PVA) and 100 µM cysteamine [14, 15]. Recombinant 101

human FSH (Organon, Oss, The Netherlands) was added to a final concentration of 0.05 102

IU/ml. 103

Estradiol, ZEA, DON, α-ZOL and β-ZOL were individually added to final concentrations 104

of 0.312, 3.12 or 31.2 µM and a combination of ZEA and DON was added to give a final 105

concentration of 0.156 or 1.56 µM each. All the test compounds were dissolved in 106

ethanol with the final concentration of the solvent not more than 0.01% of the culture 107

medium. Control medium contained the same final concentration of solvent. 108

All media, except media containing HEPES, were equilibrated in a CO2 incubator for at 109

least 2 h before use. 110

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Selection and culture of cumulus-oocyte complexes 112

Ovaries were collected from cyclic sows at a slaughterhouse and transported to the 113

laboratory within 2 h in a thermo flask. Isolation and selection of cumulus-oocyte 114

complexes (COCs) was as described [16]. After selection, 35-50 COCs were transferred 115

to a 4-well culture dish (Falcon, Becton Dickinson, UK) containing 500 µl of OMM and 116

cultured for 20 h in OMM with FSH, washed in OMM, and further cultured for 10, 20 or 117

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24 h in OMM without FSH. As test compounds estradiol, ZEA, DON, α-ZOL or β-ZOL 118

were present in the OMM during the complete culture period. COCs cultured for 40 or 44 119

h in OMM without test compounds served as controls. IVM culture was performed at 120

38.5ºC in a humidified atmosphere of 5% CO2 in air. 121

In vitro fertilization (IVF) and in vitro culture (IVC) were as described [16]. 122

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Assessment of nuclear maturation 124

Oocytes were fixed with 4% (w/v) formaldehyde in phosphate buffered saline (PBS), 125

washed with PBS, stained with 2.5% (w/v) 4,6-diamino-2-phenyl-indole (DAPI) 126

(Molecular Probes, Leiden, Netherlands) and mounted on slides. The nuclear state of the 127

stained oocytes was assessed under a fluorescence microscope. Oocytes in which diffuse 128

or slightly condensed chromatin could be identified were classified as being at the 129

germinal vesicle (GV) stage. Oocytes possessing clumped or strongly condensed 130

chromatin which formed an irregular network of individual bivalents (prometaphase) or a 131

metaphase plate but no polar body, were classified as being at the metaphase (M) I stage. 132

Oocytes with either a metaphase plate and a polar body or with two bright chromatin 133

spots were classified as being at the MII stage. Oocytes with dispersed or condensed 134

chromatin and no clear spindle formed by microtubuli were categorized as with an 135

aberrant nucleus. 136

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Assessment of microtubule organization 138

Denuded oocytes were permeabilized for 1 h at 39ºC in a microtubule stabilising solution 139

as described [16]. The oocytes were then fixed with 4% (w/v) paraformaldehyde in PBS. 140

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Fixed cells were washed with PBS and incubated for 5 min in PBS with 2% (v/v) goat 141

serum. To stain microtubules, cells were incubated for 1 h with monoclonal anti-α-142

tubulin antibody (DAKO, Glostrup, Denmark) diluted 1:100 in PBS with 2% (v/v) goat 143

serum, washed with PBS containing 0.1% (v/v) Tween-20 (PBT), incubated for 1 h with 144

tetramethyl-rhodamine isothiocyanate (TRITC)-labelled goat anti-mouse secondary 145

antibody (DAKO) diluted 1:100 in PBS with 2% (v/v) goat serum and finally washed 146

with PBT. To stain DNA oocytes were incubated with PBS containing 3 µM Sytox-green 147

(Molecular Probes) for 10 min. Stained oocytes were mounted with anti-fade mounting 148

medium (Vectashield, VectorLab, Burlingame, CA, USA) and were examined using 149

confocal laser scanning microscopy (CLSM; Leica TCS MP, Heidelberg, Germany) 150

mounted on an inverted microscope (Leica DM IRBE) equipped with a 100x immersion 151

objective. Argon-Krypton- ion lasers were used for simultaneous excitation of Sytox-152

green and TRITC using 488/568 nm excitation barrier filter combinations. Fluorescence 153

of Sytox-green and TRITC was recorded sequentially. 154

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Fluorescent in situ hybridization 156

Blastocysts were prepared as described for bovine embryos [17]. In short, embryos were 157

washed in lysis buffer (0.01N HCl, 0.1% Tween20) and transferred to a droplet of lysis 158

buffer on a Superforst slide (Menzel Gläzer, Braunschweig, Germany). Blastomere nuclei 159

were dispersed by gently blowing over the surface of the slide. Cells were fixed in 3:1 160

methanol:acetic acid overnight (4°C), baked at 60°C for 3 h and stored at –20°C until use. 161

Three BAC clones from a porcine BAC library [18] were used as probes for fluorescent 162

in situ hybridization (FISH). Clones 192B9 and 375B12 are located near the centromeric 163

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region of chromosome 7 (SSC7p1.1) and clone 498D8 is located near the centromeric 164

region of chromosome 14 (SSC14q1.1). DNA from clones 192B9 and 375B12 was 165

labeled with digoxigenin-11-dUTP, and DNA from clone 498D8 with biotin-16-dUTP, 166

both using a DIG–Nick translation Mix (Roche, Diagnostics GmbH, Germany). 167

In principle, FISH was performed as previously described [19]. Labeled DNA was 168

precipitated simultaneously in the presence of ssDNA and pig DNA, with the latter as 169

competitor, dissolved in hybridization solution (50% deionized formamide, 10% dextran 170

sulphate, 2xSSC, 50 mM sodium phosphate), and denatured by boiling for 7 min. The 171

probes were prehybridized to the competitor for 60 min at 37°C. Final concentrations 172

were 2.5 ng/µl for both SSC7 probes, 5 ng/µl for the SSC14 probe, 1µg/µl ssDNA and 173

500 µg/µl of fragmentated total pig DNA. 174

Slides containing blastocyst nuclei were treated with RNase (100µg/ml, 30 min, 37°C), 175

digested with 0.1 µg/ml proteinase K (4-6 min, 37°C) and dehydrated. The chromosomal 176

DNA was denatured by applying 100 µl of 70% formamide/2xSSC on the slide, covered 177

with a cover slip and placed on a 80°C hot plate for 3 min. Next, the slides were 178

dehydrated in an ice-cold series of ethanol and air dried. Three µl of hybridization 179

solution, containing the preannealed probes, were applied to the area of the glass slide 180

where embryonic nuclei were present. This area was cover slipped and sealed with rubber 181

cement. Hybridization was carried out overnight at 37°C in a moist chamber. Following 182

hybridization, slides were washed twice in 2xSSC, three times in 50% formamide/2xSSC, 183

three times in 2xSSC, all at 42°C, and placed in 4xSSC/Tween20 at room temperature for 184

5 min. Subsequently, the slides were incubated in 4xSSC containing 5% non fat dry milk 185

(10 min, 37°C). Specific hybridization sites of the biotinylated probe were visualised 186

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using avidin-FITC (Vector). Hybridization sites of the digoxigenin labeled probes were 187

detected using mouse-anti-DIG conjugated with Cy3 (Jackson ImmunoResearch Lab, Bar 188

Harbor, ME, USA). Nuclear DNA was counterstained with 20 ng DAPI (Serva, 189

Heidelberg, Germany) in 1 ml antifade solution (Vectashield, Vector). 190

DAPI, Cy3 and FITC fluorescence images of individual nuclei were captured using a 191

Leica DMRA fluorescent microscope equipped with the GENUS Image Analysis 192

software of Applied Imaging. 193

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Scoring criteria 195

We adhered to the scoring criteria previously proposed [17], with minor modifications. 196

In a given nucleus, specific hybridization signals were considered to reflect the true 197

chromosome constitution if the signals were of similar size, shape and intensity and were 198

more than a diameter of a single signal apart. For each nucleus red and green fluorescent 199

signals were recorded separately. For further analysis, signals were grouped and a 200

nucleus was considered diploid if two green or two red signals (2+2, 2+1 or 2+0) were 201

detected, haploid if one green and one red spot (1+1) were detected, and triploid if 3+3, 202

3+2, 3+1 or 3+0 signals were observed. Nuclei with higher ploidy were classified 203

accordingly. Nuclei lacking signals, such as 1+0 or 0+0, were recorded as false negative. 204

Damaged nuclei, in which fluorescent signals could not be scored, were recorded as such. 205

The percentage of false-aneuploid interphase nuclei was determined in normal (2n) 206

lymphocyte nuclei, and used as normal cut-off. Thus, an embryo was only considered 207

mixoploid if the percentage of haploid, triploid or tetraploid nuclei exceeded the normal 208

cut-off. 209

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Statistical analysis 211

Statistical analysis was conducted with SPSS software (SPSS Inc, Chicago, IL, USA) by 212

using an analysis of logistic regression following a binomial distribution. The data 213

concerning nuclear development were analysed by the model: Ln (π/1-π) = α + treatment, 214

where π = frequency of positive outcome, and α = the intercept. Treatment was an 215

independent categorical variable in this model. P values < 0.05 were considered as 216

significant. 217

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RESULTS 218

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Estradiol and mycotoxins disturb nuclear maturation 220

In fluid of antral follicles, the concentration of estradiol reaches approximately 89 ng per 221

ml [20, 21] and it is assumed that in the ovary maturing oocytes are exposed to this 222

concentration of estradiol. Previous results from our group have however established that 223

exposure of oocytes to estradiol results in aberrant meiotic spindle formation, at least in 224

the bovine [22]. Since ZEA has an estrogenic action in several cell types, the effect of 225

oocyte exposure to ZEA was investigated. Therefore, porcine COCs were cultured in 226

normal maturation medium for 44 h in the presence of estradiol, ZEA, or DON (all 0.312 227

µM), after which the nuclear morphology of the oocytes was examined. At the start of the 228

culture, most (>98%) of the oocytes were at the germinal vesicle (GV) stage of meiosis 229

(data not shown). The majority of oocytes cultured in control maturation medium reached 230

the MII stage after 44 h of culture. Exposure to estradiol however significantly reduced 231

the percentage of oocytes that reached the MII stage (Fig. 1A). Simultaneously, the 232

percentage of oocytes with nuclear aberrations was strongly increased in the presence of 233

estradiol (Fig. 1B). Culture of COCs in the presence of either ZEA or DON also caused a 234

significant decrease in the percentages of oocytes that reached the MII stage (Fig. 1A) 235

and an increase in the percentages of oocytes with an aberrant nucleus (Fig. 1B). When 236

oocytes were exposed to ZEA and DON simultaneously (0.156 µM each), an even bigger 237

increase in the percentage of oocytes with an aberrant nuclear morphology was observed 238

(Fig. 1B). The percentage of oocytes with nuclear aberrations after exposure to ZEA or 239

DON was similar to that of oocytes exposed to estradiol (Fig. 1B). No significant 240

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differences were observed in the percentages of oocytes that were at the GV or MI stages 241

after 44 h culture in the presence of mycotoxins or estradiol (data not shown). 242

To further examine the toxicity of ZEA on oocyte maturation, COCs were exposed to 243

different concentrations of ZEA and its direct metabolites α-ZOL and β-ZOL. All three 244

compounds decreased the percentages of oocytes that reached the MII stage but induced 245

nuclear malformations in a concentration dependent manner, with ZEA and α-ZOL being 246

the most effective at lower concentrations (Fig. 2). 247

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Different ratios of ZEA/DON exposure do not lead to differences in oocyte maturation 249

Since it has been described that in different products, such as maize and wheat, 250

mycotoxins can occur in different ratios, the effect of exposure to 2 different ratios of 251

ZEA/DON (i.e. high ZEA/ low DON and low ZEA/ high DON) on oocyte nuclear 252

maturation was examined. Similar to the exposure of oocytes to ZEA and DON, exposure 253

to two different ratios of ZEA/DON resulted in a decrease of the percentage of oocytes 254

that reached the MII stage and an increase in the percentage of oocytes exhibiting 255

aberrant nuclei (Fig. 3). No significant differences were observed in the percentages of 256

oocytes at GV and MI stages after 44 h of cultures (not shown). Importantly, no 257

differences in oocyte nuclear maturation were observed when oocytes were exposed to 258

the two ratios of ZEA and DON, indicating that different ratios of the mycotoxins do not 259

lead to enhanced or reduced toxicity. 260

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Mycotoxins cause nuclear aberrations already before 30 h. 262

To gain insight in the dynamics of the nuclear maturation of oocytes when exposed to 263

mycotoxins, oocytes were incubated for 30 h and 40 h in the presence or absence of ZEA, 264

DON and estradiol and examined for nuclear morphology. Less than 10% of control 265

oocytes had developed to the MII stage after 30 h of culture, and the majority of oocytes 266

were still at the MI stage (Table 1). A significant increase in the percentage of oocytes 267

exhibiting nuclear aberrations was already evident after 30 h in culture with ZEA, DON 268

and estradiol with DON causing significantly more aberrations than ZEA. Exposure to 269

ZEA and DON simultaneously however resulted in the highest percentage of oocytes 270

with aberrant nuclear morphology after 30 h (Table 1). 271

After 40 h of culture the majority of oocytes cultured in control medium had developed to 272

the MII stage, but exposure to estradiol, ZEA and DON decreased the percentages of 273

oocytes at the MII stage and increased the percentages of oocytes with nuclear 274

malformations (Table 1). Similar to what had been observed at 30 h, combined exposure 275

to ZEA and DON led to the highest percentage of oocytes exhibiting nuclear aberrations 276

(Table 1). 277

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Different mycotoxins lead to abnormal spindle morphology 279

To further investigate the effects of estradiol, ZEA and DON on oocyte nuclear 280

maturation, the morphology of the meiotic spindles was examined in more detail by 281

staining of DNA and microtubules followed by CLSM. The majority of porcine oocytes 282

matured in control medium for 30 h had reached the MI stage. At this stage, GV 283

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breakdown had occurred, and the homologous pairs of chromosomes were aligned in the 284

spindle consisting of microtubules (Fig. 4A). After 40 h of culture, most oocytes had 285

reached the MII stage, where one polar body had already segregated and the remaining 286

chromosomes were aligned at the metaphase plate closely associated with microtubules 287

(Fig. 4B). When porcine oocytes were cultured in the presence of estradiol, most nuclei 288

exhibited a normal MI and MII spindle, but in a significant number of oocytes (Table 1) 289

the chromosomes failed to align after 30 h and instead appeared to cluster together after 290

GV breakdown. No or little signs of microtubule formation were observed (Fig. 4C). 291

After 40 h exposure to estradiol, chromosomes appeared to be more clustered but did not 292

align properly and microtubules were absent or did not form a normal spindle (Fig. 4D). 293

A similar pattern was observed when oocytes were cultured in the presence of ZEA. In 294

approximately 30% of the oocytes exposure to ZEA caused clustering of chromosomes, 295

without normal pair wise alignment, closely associated with microtubules that did not 296

form a spindle (Fig. 4E,F). On the contrary, exposure to DON resulted in tightly 297

associated DNA that formed a clear spindle-like structure. However, the microtubules 298

exhibited a fuzzy appearance instead of forming a normal spindle (Fig. 4G). The pattern 299

was similar after 40 h of culture, although at this stage most of the microtubules had 300

disappeared (Fig. 4H). Exposure of oocytes to both ZEA and DON resulted in nuclear 301

aberrations of which its morphology appeared to be a combination of the abnormalities 302

observed after exposure to ZEA and DON individually (Fig. 4I,J). 303

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Mycotoxins reduce the developmental capacity of oocytes 305

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To investigate the consequences of mycotoxin exposure during maturation of oocytes on 306

developmental capacity, oocytes were fertilized and the percentages of cleaved oocytes 307

and blastocysts determined. Presence of ZEA during maturation significantly reduced the 308

percentage of oocytes that cleaved and formed blastocysts after fertilization, compared 309

with oocytes matured in control medium (Fig. 5). A similar reduction in developmental 310

capacity was observed after exposure to estradiol. When oocytes were matured in the 311

presence of DON the apparent spindle abnormality was not compatible with development 312

as only few oocytes cleaved and no blastocysts were formed. ZEA and DON presented in 313

equimolar concentrations reduced development similar to ZEA alone (Fig. 5). 314

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Oocytes exposed to mycotoxins give rise to aneuploid embryos 316

Oocytes that were exposed during maturation to estradiol, ZEA, and a combination of 317

ZEA and DON were fertilized and the developing blastocysts were analysed for ploidy of 318

the blastomeres using FISH for chromosomes 7 and 14. 319

To evaluate the efficiency of simultaneous hybridization and detection of the probes 320

described above on interphase nuclei, probes were also hybridized to chromosome slides 321

prepared from cultured blood lymphocytes of karyotypically normal pigs. Since these 322

slides contained both interphase and metaphase nuclei, these experiments were also used 323

to validate the efficacy of the FISH procedure on metaphase nuclei. 324

The number of blastomeres that composed the blastocyst were similar between the groups 325

of embryos, although the variability in the number of blastomeres was considerable and 326

ranged between 11 and 77 (Fig. 6A). All blastocysts, including that from control oocytes 327

had at least one blastomere with abnormal ploidy. Strikingly, when oocytes were matured 328

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in the presence of ZEA or a combination of ZEA and DON, blastocysts that developed 329

exhibited less blastomeres with normal ploidy (Fig. 6B). Instead, a significant proportion 330

of the blastomeres contained more than two copies of the chromosomes 7 or 14, with 331

some blastomeres even containing 9 to 11 copies of the examined chromosomes (Fig. 332

6C). 333

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DISCUSSION 334

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Both the human and animal diets contain several plant-derived, non-steroidal estrogenic 336

compounds [23] that are either produced by plants (phytestrogens) or by fungi that infect 337

plants (mycestrogens). The most prominent mycestrogen is ZEA and the occurrence of 338

ZEA or its derivatives has been reported worldwide [24]. ZEA has been associated with 339

hyperestrogenism and other reproductive disorders such as impaired fertility in farm 340

animals [8]. ZEA and its derivatives can cross the placenta and are excreted with milk 341

causing exposure of the embryo and neonate [25, 26]. 342

Under physiological conditions, follicular cells produce relatively large amounts of 343

estradiol during the follicular phase of the estrous cycle, and a decline of the estradiol 344

levels concurs with the breakdown of the germinal vesicle in oocytes [20]. Although 345

there are no indications that estradiol is involved in the resumption of meiosis, in vitro 346

maturation experiments demonstrated that estradiol can inhibit nuclear maturation and 347

cause spindle malformations during meiosis [22]. Estrogens can diffuse in and out of 348

cells but in target cells they are bound by the nuclear receptors ER-α and ER-β. 349

Hormone-binding leads to conformational changes of these receptors, allowing binding 350

to specific elements on DNA and in combination with components of the cellular 351

transcription machinery can activate or repress transcription [27]. In the ovaries of 352

various mammalian species, including humans, expression of ER-α has been detected in 353

the germinal epithelium, interstitial cells and theca cells, whereas ER-β expression has 354

been detected in granulosa and cumulus cells [28-31]. In bovine follicles, expression of 355

ER-α mRNA was detected in cumulus cells, and ER-β mRNA expression was detected in 356

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both cumulus cells and oocytes [22]. Binding of estradiol to ER-α occurs via the aromatic 357

ring of estradiol, but the volume of the binding pocket of the receptor is almost twice as 358

big as estradiol, presumably allowing the acceptance of a number of different non-359

steroidal compounds such as ZEA [2]. 360

ZEA can bind to ER-α and ER-β with approximately 7 and 15%, respectively, of the 361

affinity of estradiol [3]. However, transactivation studies demonstrated that ZEA is able 362

to generate a response via the ERs in the same order of magnitude as that of estradiol, the 363

transactivation via ER-α being more efficient than that via ER-β [3]. Since it has also 364

been reported that cells can respond to estradiol independent of ER activation, a so-called 365

non-genomic effect [32], it cannot be excluded that the abnormalities observed after 366

exposure to estradiol or ZEA are not only caused via binding to and activation of ER but 367

also involve non-genomic mechanisms yet to be determined. Indeed it has been reported 368

that estrogen can bind tubulin and inhibit tubulin polymerization [33]. 369

After exposure of oocytes to estradiol, the most commonly observed nuclear aberration 370

was the almost complete absence of a microtubule spindle and the appearance of 371

disorganized chromosomes that were not aligned properly. The morphology of the 372

meiotic spindle after exposure of oocytes to ZEA was reminiscent of that after exposure 373

to estradiol, but not identical. The chromosomes were not properly aligned and although 374

microtubules were clearly present, they did not form an organized spindle. These results 375

suggest that the effects of ZEA include mechanisms other than activation of estrogen 376

receptors. 377

In contrast to ZEA, DON does not bind estrogen receptors. Its toxicity has been 378

associated with inhibition of protein synthesis at the level of the ribosomes and a 379

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ribotoxic stress response involving activation of JNK/p38 kinases and an increase in 380

cytokine and chemokine transcription [12]. In turn, DON induces apoptosis of lymphoid 381

cells [11, 34, 35]. In the oocytes exposed to DON only, no signs of apoptosis were 382

observed. The fuzzy appearance of the microtubules in the meiotic spindle, however, was 383

incompatible with further development. In addition, the great majority of the oocytes that 384

did not form a morphologically normal meiotic spindle was either not fertilized, or did 385

not cleave after fertilization, indicating that DON induced more cellular damage than 386

spindle malformation. 387

Alignment of the chromosomes is a microtubule dependent process and when 388

microtubules were not visible or exhibited a fuzzy appearance, as observed after estradiol 389

and DON exposure, the chromosomes failed to align. Spindle malformations in maturing 390

oocytes can lead to aneuploidy and have serious consequences for fertilisation and 391

embryonic development [36, 37]. Indeed the percentages of blastocysts that were formed 392

after exposure of oocytes to the selected mycotoxins were severely reduced and a 393

significant percentage of the embryos that were formed contained polyploid cells, some 394

cells even containing 11 of chromosomes 7 or 14. The mechanism behind the duplication 395

of chromosomes remains unclear, but the embryos that contained most abnormal cells 396

were those embryos that contained fewest cells, suggesting that in these embryos cells 397

either duplicated DNA without normal cell division, or that the abnormal chromosome 398

content inhibited embryonic development. 399

In conclusion, the mycotoxins ZEA and DON reduce fertility by altering spindle 400

formation during meiosis of the oocyt. Importantly, mycotoxin-induced spindle 401

malformations in the oocyte can result in aneuploid embryos. ZEA and DON are 402

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produced by the same Fusarium species, risk assessment relating to exposure of 403

contaminated food or feeds needs to consider possible additive or synergistic effect of 404

these mycotoxins [38, 39]. In this study, clear differences were seen in the effect of either 405

mycotoxin, and the potency appeared to be an additive rather than reflecting a synergistic 406

interaction. 407

408

Acknowledgement 409

Present and past colleagues of the Department of Farm Animal Health are thanked for 410

helping in collecting oocytes. 411

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REFERENCES 413

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534 535

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FIGURE LEGENDS 535 Figure 1 536

Exposure to mycotoxins inhibits the maturation process of oocytes. Oocytes were 537

incubated in maturation medium (C) with 3.12 µM estradiol (E2), zearalenone (ZEA), 538

deoxynivalenol (DON), or 1.56 µM ZEA + 1.56 µM DON (Z+D), cultured for 44 h and 539

the percentages of oocytes exhibiting a normal metaphase II spindle (A) or an abnormal 540

nucleus (B) were determined. Bars are averages ± s.e.m.; a,b,c indicate significant 541

(p<0.05) differences between groups. 542

543

Figure 2 544

ZEA and its metabolite α-ZOL are more potent inhibitors of maturation than the 545

metabolite β-ZOL. Oocytes were matured for 44 h in the presence of ZEA, α-ZOL and β-546

ZOL at the indicated concentrations and analysed for the percentages of oocytes with an 547

MII stage meiotic spindle (A) or an abnormal nucleus (B). Bars are averages ± s.e.m.; 548

a,b,c indicate significant (p<0.05) differences between bars within concentration groups. 549

550

Figure 3 551

Exposure to ZEA and DON simultaneously at different ratios do not lead to enhanced or 552

reduced toxicity. Oocytes were matured for 44 h in the presence of high ZEA/ low DON 553

(3.12/0.312 µM) or low ZEA/ high DON (0.312/3.12 µM) and analysed for the 554

percentages of oocytes with an MII stage meiotic spindle (A) or an abnormal nucleus (B). 555

Bars are averages ± s.e.m.; a,b,c indicate significant (p<0.05) differences between 556

groups. 557

558

26

26

Figure 4 559

Meiotic spindle abnormalities after exposure to mycotoxins. Oocytes were matured for 30 560

(A, C, E, G, I) or 40 (B, D, F, H, J) hours with control medium (A, B) or with estradiol 561

(C, D), ZEA (E, F) DON (G, H), all at 0.312 µM, or with a combination of ZEA and 562

DON (0.156 µM each). DNA (green) and microtubules (red) were visualized and 563

analysed with a CLSM. Scale bar represents 5 µM. 564

565

Figure 5 566

Reduced developmental competence after exposure of oocytes to mycotoxins. Oocytes 567

were matured for 40 h in the presence of the indicated components, fertilized and cultured 568

during which the percentages of cleaved oocytes was determined after 2 days (A) and the 569

percentage of blastocysts after 6 days (B). Bars are averages ± s.e.m.; a,b,c indicate 570

significant (p<0.05) differences between groups. 571

572

Figure 6 573

Reduced ploidy after exposure to mycotoxins. After maturation in the absence (control) 574

or presence of estradiol (3.12 µM), ZEA (3.12 µM) or ZEA + DON (1.56 µM each), 575

oocytes were fertilized and the developing blastocysts were analysed for cell number (A), 576

percentage of ploidy (B), and chromosomal abnormalities (C). (A) Numbers of nuclei 577

representing cell numbers in embryos from oocytes matured under various conditions. 578

Each dot represents an individual blastocyst. (B) Box plots of the percentages of diploid 579

cells in blastocysts. The bottom and top of the boxes represent the 25th and 75th 580

percentiles, respectively. Whiskers on the top and bottom of the boxes represent the 581

27

27

largest and smallest observations within 1.5 interquartile ranges. Samples outside these 582

areas are plotted individually. Horizontal lines within boxes represent the median of the 583

observations. (C) Number of nuclei with polyploid numbers of chromosomes 7 or 14 in 584

blastocysts from control oocytes (black bars), oocytes matured in the presence of 585

estradiol (hatched bars), oocytes matured in the presence of zearalenone (densely dotted 586

bars), and oocytes matured in the presence of zearalenone and deoxynivalenol (dotted 587

bars). 588

589

590

591

592

593

28

28

Table 1. Exposure to estradiol, ZEA, DON (3.12 µM), and the combination of ZEA and 593

DON (1.56 µM each) inhibits nuclear maturation of porcine COCs. 594

Exposure n Aberrations (%) GV (%) MI(%) MII(%)

Control

30 h 103 16 (15.5)a 12 (11.7)a 66 (64.1)a 9 (8.7)a

40 h 137 16 (11.7)a’ 13 (9.5)a’ 18 (13.1)a’ 90 (65.7)a’

Estradiol

30 h 85 23 (27.1)b 14 (16.5)a 43 (50.6)b 5 (5.9)a

40 h 71 24 (33.8)b’ 6 (8.5)a’ 11 (15.5)a’ 30 (42.3)b’

ZEA

30 h 108 30 (27.8)b 18 (16.7)a 55 (50.9)b 5 (4.7)a

40 h 92 33 (35.9)b’ 8 (8.7)a’ 11 (12.0)a’ 40 (43.5)b’

DON

30 h 80 27 (33.8)c 18 (22.5)b 33 (41.3)b 2 (2.5)b

40 h 101 33 (32.7)b’ 12 (11.9)a’ 16 (15.9)a’ 40 (39.6)b’

ZEA + DON

30 h 79 35 (44.3)d 13 (16.5)a 27 (34.2)c 4 (5.1)b

40 h 76 33 (43.4)c’ 7 (9.2)a’ 9 (11.8)a’ 27 (35.5)c’

abcd values for 30 h and a’b’c’ values for 40 h exposure in same column with different 595

superscripts differ significantly ( P < 0.05). 596