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Departments of Veterinary Pharmacology, Pharmacy and Toxicology,4 Farm Animal Health,5 and Biochemistry and Cell Biology,6 Faculty of Veterinary Medicine, Utrecht University, 3584 CM Utrecht, The Netherlands
ABSTRACT
Fungi of the Fusarium species can infect food and feed commodities and produce the mycotoxins zearalenone (ZEA) and deoxynivalenol (DON). Since both toxins have been reported to reduce fertility, the mechanisms of ZEA and DON on inhibition of oocyte maturation were examined. Pig oocytes were matured in the presence of ZEA (a mycotoxin with estrogenlike activity), 17beta-estradiol, and DON (all 3.12 µmol/L). Zearalenone, 17beta-estradiol, and DON inhibited oocyte maturation and caused approximately 34% of the oocytes to form an aberrant spindle. Different ratios of ZEA:DON did not lead to a more severe inhibition of oocyte maturation. Both mycotoxins caused abnormal formation of the meiotic spindle. The developmental competence of oocytes matured in the presence of mycotoxins was further investigated after in vitro fertilization. Presence of ZEA (3.12 µmol/L) during maturation reduced the percentages of oocytes that cleaved and formed a blastocyst to about 12%, compared with 25% of control oocytes. Maturation in the presence of equimolar concentrations of DON was not compatible with development. The ploidy of blastomeres from blastocysts derived from mycotoxin-exposed oocytes was analyzed with fluorescent in situ hybridization. All blastocysts, even those from the control group, contained at least one blastomere with abnormal ploidy, but the variation in the percentages of aneuploid blastomeres was significantly larger in embryos from oocytes exposed to mycotoxins. It is concluded that ZEA and DON can lead to abnormal spindle formation, leading to less fertile oocytes and embryos with abnormal ploidy, and that the effects of ZEA and DON are not synergistic.
deoxynivalenol, early development, embryo, food safety, in vitro fertilization, meiosis, mixoploidy, reproduction, toxicology, zearalenone
Zearalenone (ZEA), a mycotoxin primarily produced by Fusarium fungi, has a unique nonsteroidal resorcyclic acid lactone structure. This structure resembles many characteristics of steroid hormones and allows ZEA to bind to both types of estrogen receptors (ERs), ESR1 and ESR2, where it acts as an agonist and partial antagonist to estradiol [1–3]. Fungi of Fusarium species can infect maize, wheat, rice, and barley crops, and their resistant toxins can be transferred into consumer products. Zearalenone has been found to induce estrogenic effects, often reported as hyperestrogenism, in all laboratory animal species tested, as well as in farm animals, particularly in pigs. In humans, exposure to ZEA has been associated with epidemics of premature thelarche [4]. Species differences in the susceptibility to ZEA exposure have been associated with differences in the hepatic and extrahepatic metabolism of ZEA that are catalyzed by hydroxysteroid hydrogenases [5]. Zearalenone is converted primarily into two isomeric metabolites, alpha-zearalenol (
-ZOL) and beta-zearalenol (ß-ZOL). Different lines of evidence, including receptor binding and cell proliferation assays with estrogen-dependent MCF-7 cells, have indicated that -ZOL has a higher estrogenic potency compared with the parent ZEA, whereas ß-ZOL has a lower potency [5]. The species-specific sensitivity observed in clinical trials and in field studies correlates with the rate of bioconversion into -ZOL and identified the pig as the most sensitive farm animal species. Common clinical symptoms in young, premature pigs comprise vulva swelling, enlarged nipples, and an enlarged uterus, whereas in cycling sows, a decreased fertility, increased number of resorptions, and a reduced litter size as well as changes in the levels of circulating estrogen and progesterone levels have been described. In boars, enlarged nipples and reduced testis weights have been observed [6–8].
A common feature of many Fusarium species is that besides their ability to produce ZEA, they can produce nonestrogenic sesquiterpenoid trichothecenes. At present, more than 180 individual trichothecenes are described, the most frequently occurring being deoxynivalenol (DON). Deoxynivalenol exerts proinflammatory effects by inducing cytokine and chemokine expression in mononuclear phagocytes [9–11]. In pigs, which again seem to be the most sensitive species, inflammatory alterations occur particularly in the gastrointestinal tract, hampering nutrient transport and resulting in reduced weight gain. Moreover, at high doses DON has proemetic effects, whereas at lower concentrations feed intake is decreased, contributing to impaired growth and performance. The sensitivity of animals to DON increases when they are exposed at the same time to infectious agents, as co-exposure results in a concomitant inflammatory response [12].
Zearalenone and DON also have been reported to inhibit oocyte nuclear maturation [13], but the mechanisms of toxicity are unknown. Since feeds are generally contaminated with both mycotoxins ZEA and DON, it is important to know how cells behave when exposed to both toxins simultaneously.
All chemicals for the preparation of culture media were purchased from Sigma Chemical Company (St. Louis, MO) unless otherwise indicated. Oocyte maturation medium (OMM) was M199 (Gibco BRL, Paisley, United Kingdom) supplemented with 2.2 mg/ml NaHCO3, 0.1% (w/v) polyvinyl alcohol, and 100 µM cysteamine [14, 15]. Recombinant human FSH (Organon, Oss, The Netherlands) was added to a final concentration of 0.05 IU/ml.
17ß-Estradiol, ZEA, DON, -ZOL, and ß-ZOL were individually added to final concentrations of 0.312, 3.12, or 31.2 µmol/L, and a combination of ZEA and DON was added to give a final concentration of 0.156 or 1.56 µmol/L each. All of the test compounds were dissolved in ethanol, with the final concentration of the solvent not more than 0.01% of the culture medium. Control medium contained the same final concentration of solvent. All media, except media containing HEPES, were equilibrated in a CO2 incubator for at least 2 h before use.
Selection and Culture of Cumulus-Oocyte Complexes
Ovaries were collected from cyclic sows at a slaughterhouse and transported to the laboratory in a thermo flask within 2 h of death. Isolation and selection of cumulus-oocyte complexes (COCs) was as described previously [16]. After selection, 35–50 COCs were transferred to a four-well culture dish (Falcon Becton Dickinson) containing 500 µl OMM, cultured for 20 h in OMM with FSH, washed in OMM, and further cultured for 10, 20, or 24 h in OMM without FSH. As test compounds, estradiol, ZEA, DON, -ZOL, or ß-ZOL was present in the OMM during the complete culture period. COCs cultured for 40 or 44 h in OMM without test compounds, but with FSH during the first 20 h, served as controls. In vitro maturation culture was performed at 38.5°C in a humidified atmosphere of 5% CO2 in air.
In vitro fertilization and in vitro culture were performed as described previously [16]. For in vitro fertilization, fresh extended semen from two randomly selected boars was used, and groups of 50 oocytes were incubated with 5 x 105 sperm cells/ml.
Assessment of Nuclear Maturation
Oocytes were fixed with 4% (w/v) formaldehyde in PBS, washed with PBS, stained with 2.5% (w/v) 4,6-diamino-2-phenyl-indole (DAPI; Molecular Probes, Leiden, The Netherlands), and mounted on slides. The nuclear state of the stained oocytes was assessed under a fluorescence microscope. Oocytes in which diffuse or slightly condensed chromatin could be identified were classified as being at the germinal vesicle (GV) stage. Oocytes possessing clumped or strongly condensed chromatin that formed an irregular network of individual bivalents (prometaphase) or a metaphase plate but no polar body were classified as being at the metaphase I (MI) stage. Oocytes with either a metaphase plate and a polar body or with two bright chromatin spots were classified as being at the MII stage. Oocytes with dispersed or condensed chromatin and no clear spindle formed by microtubuli were categorized as with an aberrant nucleus.
Assessment of Microtubule Organization
Denuded oocytes were permeabilized for 1 h at 39°C in a microtubule stabilizing solution as described previously [16]. The oocytes were then fixed with 4% (w/v) paraformaldehyde in PBS. Fixed cells were washed with PBS and incubated for 5 min in PBS with 2% (v/v) goat serum. To stain microtubules, cells were incubated for 1 h with monoclonal anti--tubulin antibody (DAKO, Glostrup, Denmark), diluted 1:100 in PBS with 2% (v/v) goat serum, washed with PBS containing 0.1% (v/v) Tween-20 (PBT), incubated for 1 h with tetramethyl-rhodamine isothiocyanate (TRITC)-labeled goat anti-mouse secondary antibody (DAKO) diluted 1:100 in PBS with 2% (v/v) goat serum, and finally washed with PBT. To stain DNA, oocytes were incubated with PBS containing 3 µM Sytox Green (Molecular Probes) for 10 min. Stained oocytes were mounted with antifade mounting medium (Vectashield; Vector Laboratories, Burlingame, CA) and were examined using confocal laser scanning microscopy (CLSM; Leica TCS MP, Heidelberg, Germany) mounted on an inverted microscope (Leica DM IRBE) equipped with a 100x immersion objective. Argon-krypton ion lasers were used for simultaneous excitation of Sytox Green and TRITC using 488/568-nm excitation barrier filter combinations. Fluorescence of Sytox Green and TRITC was recorded sequentially.
Fluorescent In Situ Hybridization
Blastocysts were prepared as described for bovine embryos [17]. In short, embryos were washed in lysis buffer (0.01 N HCl, 0.1% Tween-20) and transferred to a droplet of lysis buffer on a Superforst slide (Menzel Gläzer, Braunschweig, Germany). Blastomere nuclei were dispersed by gently blowing over the surface of the slide. Cells were fixed in 3:1 methanol:acetic acid overnight (4°C), baked at 60°C for 3 h, and stored at –20°C until use.
Three BAC clones from a porcine BAC library [18] were used as probes for fluorescent in situ hybridization (FISH). Clones 192B9 and 375B12 are located near the centromeric region of chromosome 7 (SSC7p1.1), and clone 498D8 is located near the centromeric region of chromosome 14 (SSC14q1.1). DNA from clones 192B9 and 375B12 was labeled with digoxigenin-11-dUTP, and DNA from clone 498D8 with biotin-16-dUTP, both using a DIG–Nick translation Mix (Roche Diagnostics GmbH).
In principle, FISH was performed as previously described [19]. Labeled DNA was precipitated simultaneously in the presence of ssDNA and pig DNA, with the latter as competitor, dissolved in hybridization solution (50% deionized formamide, 10% dextran sulphate, 2x SSC, 50 mM sodium phosphate), and denatured by boiling for 7 min. The probes were prehybridized to the competitor for 60 min at 37°C. Final concentrations were 2.5 ng/µl for both SSC7 probes, 5 ng/µl for the SSC14 probe, 1 µg/µl of ssDNA, and 500 µg/µl of fragmentated total pig DNA.
Slides containing blastocyst nuclei were treated with RNase (100 µg/ml, 30 min, 37°C), digested with 0.1 µg/ml proteinase K (4–6 min, 37°C), and dehydrated. The chromosomal DNA was denatured by applying 100 µl of 70% formamide/2x SSC on the slide, covered with a cover slip and placed on an 80°C hot plate for 3 min. Next, the slides were dehydrated in an ice-cold series of ethanol and air dried. Hybridization solution (3 µl) containing the preannealed probes was applied to the area of the glass slide where embryonic nuclei were present. This area was cover slipped and sealed with rubber cement. Hybridization was carried out overnight at 37°C in a moist chamber. Following hybridization, slides were washed twice in 2x SSC, three times in 50% formamide/2x SSC, three times in 2x SSC, all at 42°C, and placed in 4x SSC/Tween-20 at room temperature for 5 min. Subsequently, the slides were incubated in 4x SSC containing 5% nonfat dry milk (10 min, 37°C). Specific hybridization sites of the biotinylated probe were visualized using avidin-FITC (Vector Laboratories). Hybridization sites of the digoxigenin-labeled probes were detected using mouse anti-DIG conjugated with Cy3 (Jackson ImmunoResearch Laboratories, Bar Harbor, ME). Nuclear DNA was counterstained with 20 ng DAPI (Serva, Heidelberg, Germany) in 1 ml antifade solution (Vectashield).
DAPI, Cy3, and FITC fluorescence images of individual nuclei were captured using a Leica DMRA fluorescent microscope equipped with the GENUS Image Analysis software of Applied Imaging.
We adhered to the scoring criteria previously proposed [17], with minor modifications. In a given nucleus, specific hybridization signals were considered to reflect the true chromosome constitution if the signals were of similar size, shape, and intensity and were more than a diameter of a single signal apart. For each nucleus, red and green fluorescent signals were recorded separately. For further analysis, signals were grouped, and a nucleus was considered diploid if two green or two red signals (2 + 2, 2 + 1, or 2 + 0) were detected; haploid if one green and one red spot (1+1) were detected; and triploid if 3 + 3, 3 + 2, 3 + 1, or 3 + 0 signals were observed. Nuclei with higher ploidy were classified accordingly. Nuclei lacking signals, such as 1 + 0 or 0 + 0, were recorded as false negative. Damaged nuclei, in which fluorescent signals could not be scored, were recorded as such. The percentage of false-aneuploid interphase nuclei was determined in normal (2n) lymphocyte nuclei and was used as normal cutoff. Thus, an embryo was only considered mixoploid if the percentage of haploid, triploid, or tetraploid nuclei exceeded the normal cutoff.
Statistical analysis was conducted with SPSS software (SPSS Inc., Chicago, IL) using an analysis of logistic regression following a binomial distribution. The data concerning nuclear development were analyzed by the model: Ln (
/1 – ) = + treatment, where = frequency of positive outcome, and = the intercept. Treatment was an independent categorical variable in this model. P values <0.05 were considered significant.
Estradiol and Mycotoxins Disturb Nuclear Maturation
In fluid of antral follicles, the concentration of estradiol reaches approximately 89 ng/ml [20, 21], and it is assumed that in the ovary maturing oocytes are exposed to this concentration of estradiol. Previous results from our group have, however, established that exposure of oocytes to estradiol results in aberrant meiotic spindle formation, at least in the cattle [22]. Since ZEA has an estrogenic action in several cell types, the effect of oocyte exposure to ZEA was investigated. Therefore, pig COCs were cultured in normal maturation medium for 44 h in the presence of 17ß-estradiol, ZEA, or DON (all 0.312 µmol/L), after which the nuclear morphology of the oocytes was examined. At the start of the culture, most (>98%) of the oocytes were at the germinal vesicle (GV) stage of meiosis (data not shown). The majority of oocytes cultured in control maturation medium reached the MII stage after 44 h of culture. Exposure to estradiol, however, significantly reduced the percentage of oocytes that reached the MII stage (Fig. 1A). Simultaneously, the percentage of oocytes with nuclear aberrations was strongly increased in the presence of estradiol (Fig. 1B). Culture of COCs in the presence of either ZEA or DON also caused a significant decrease in the percentages of oocytes that reached the MII stage (Fig. 1A) and an increase in the percentages of oocytes with an aberrant nucleus (Fig. 1B). When oocytes were exposed to ZEA and DON simultaneously (0.156 µmol/L each), an even greater increase in the percentage of oocytes with an aberrant nuclear morphology was observed (Fig. 1B). The percentage of oocytes with nuclear aberrations after exposure to ZEA plus DON was similar to that of oocytes exposed to estradiol (Fig. 1B). No significant differences were observed in the percentages of oocytes that were at the GV or MI stages after 44 h of culture in the presence of mycotoxins or estradiol (data not shown).
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To further examine the toxicity of ZEA on oocyte maturation, COCs were exposed to different concentrations of ZEA and its direct metabolites -ZOL and ß-ZOL. All three compounds decreased the percentages of oocytes that reached the MII stage but induced nuclear malformations in a concentration-dependent manner, with ZEA and -ZOL being the most effective at lower concentrations (Fig. 2).
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Different Ratios of ZEA:DON Exposure Do Not Lead to Differences in Oocyte Maturation
Since it has been described that in different products, such as maize and wheat, mycotoxins can occur in different ratios, the effect of exposure to two different ratios of ZEA:DON (i.e., high ZEA:low DON and low ZEA:high DON) on oocyte nuclear maturation was examined. Similar to the exposure of oocytes to ZEA and DON, exposure to two different ratios of ZEA:DON resulted in a decrease of the percentage of oocytes that reached the MII stage and an increase in the percentage of oocytes exhibiting aberrant nuclei (Fig. 3). No significant differences were observed in the percentages of oocytes at GV and MI stages after 44 h of culture (not shown). Importantly, no differences in oocyte nuclear maturation were observed when oocytes were exposed to the two ratios of ZEA and DON, indicating that different ratios of the mycotoxins do not lead to enhanced or reduced toxicity.
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Mycotoxins Already Cause Nuclear Aberrations Before 30 Hours
To gain insight into the dynamics of the nuclear maturation of oocytes when exposed to mycotoxins, oocytes were incubated for 30 h and 40 h in the presence or absence of ZEA, DON, and estradiol (all 3.12 µM concentrations) and examined for nuclear morphology. Less than 10% of control oocytes had developed to the MII stage after 30 h of culture, and the majority of oocytes were still at the MI stage (Table 1). A significant increase in the percentage of oocytes exhibiting nuclear aberrations was already evident after 30 h in culture with ZEA, DON, and estradiol, with DON causing significantly more aberrations than ZEA. Exposure to ZEA and DON simultaneously, however, resulted in the highest percentage of oocytes with aberrant nuclear morphology after 30 h (Table 1).
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After 40 h of culture, the majority of oocytes cultured in control medium had developed to the MII stage, but exposure to estradiol, ZEA, and DON decreased the percentages of oocytes at the MII stage and increased the percentages of oocytes with nuclear malformations (Table 1). Similar to what had been observed at 30 h, combined exposure to ZEA and DON led to the highest percentage of oocytes exhibiting nuclear aberrations (Table 1).
Different Mycotoxins Lead to Abnormal Spindle Morphology
To further investigate the effects of estradiol, ZEA, and DON (all 3.12 µM concentration) on oocyte nuclear maturation, the morphology of the meiotic spindles was examined in more detail by staining of DNA and microtubules, followed by CLSM. The majority of porcine oocytes matured in control medium for 30 h had reached the MI stage. At this stage, GV breakdown had occurred, and the homologous pairs of chromosomes were aligned in the spindle consisting of microtubules (Fig. 4A). After 40 h of culture, most oocytes had reached the MII stage, where one polar body had already segregated and the remaining chromosomes were aligned at the metaphase plate closely associated with microtubules (Fig. 4B). When pig oocytes were cultured in the presence of estradiol, most nuclei exhibited a normal MI and MII spindle, but in a significant number of oocytes (Table 1), the chromosomes failed to align after 30 h and instead appeared to cluster together after GV breakdown. No or few signs of microtubule formation were observed (Fig. 4C). After 40 h of exposure to estradiol, chromosomes appeared to be more clustered but did not align properly, and microtubules were absent or did not form a normal spindle (Fig. 4D). A similar pattern was observed when oocytes were cultured in the presence of ZEA. In approximately 30% of the oocytes, exposure to ZEA caused clustering of chromosomes, without normal pairwise alignment, closely associated with microtubules that did not form a spindle (Fig. 4, E and F). On the contrary, exposure to DON resulted in tightly associated DNA that formed a clear spindlelike structure. However, the microtubules exhibited a fuzzy appearance instead of forming a normal spindle (Fig. 4G). The pattern was similar after 40 h of culture, although at this stage most of the microtubules had disappeared (Fig. 4H). Exposure of oocytes to both ZEA and DON (1.56 µM each) resulted in nuclear aberrations, of which the morphology appeared to be a combination of the abnormalities observed after exposure to ZEA and DON individually (Fig. 4, I and J).
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Mycotoxins Reduce the Developmental Capacity of Oocytes
To investigate the consequences of mycotoxin exposure (3.12 µM concentration) during maturation of oocytes on developmental capacity, oocytes were fertilized and the percentages of cleaved oocytes and blastocysts determined. Presence of ZEA during maturation significantly reduced the percentage of oocytes that cleaved and formed blastocysts after fertilization compared with oocytes matured in control medium (Fig. 5). A similar reduction in developmental capacity was observed after exposure to estradiol. When oocytes were matured in the presence of DON, the apparent spindle abnormality was not compatible with development, as only a few oocytes cleaved and no blastocysts were formed. Zearalenone and DON presented in equimolar (1.56 µM) concentrations reduced development similarly to ZEA alone (Fig. 5).
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Oocytes Exposed to Mycotoxins Give Rise to Aneuploid Embryos
Oocytes that were exposed during maturation to estradiol, ZEA (both 3.12 µmol/L), and a combination of ZEA and DON (1.56 µmol/L each) were fertilized, and the developing blastocysts were analyzed for ploidy of the blastomeres using FISH for chromosomes 7 and 14.
To evaluate the efficiency of simultaneous hybridization and detection of the probes described above on interphase nuclei, probes were also hybridized to chromosome slides prepared from cultured blood lymphocytes of karyotypically normal pigs. Since these slides contained both interphase and metaphase nuclei, these experiments were also used to validate the efficacy of the FISH procedure on metaphase nuclei.
The number of blastomeres that composed the blastocyst were similar between the groups of embryos, although the variability in the number of blastomeres was considerable and ranged between 11 and 77 (Fig. 6A). All blastocysts, including those from control oocytes, had at least one blastomere with abnormal ploidy. Strikingly, when oocytes were matured in the presence of ZEA or a combination of ZEA and DON, blastocysts that developed exhibited fewer blastomeres with normal ploidy (Fig. 6B). Instead, a significant proportion of the blastomeres contained more than two copies of the chromosomes 7 or 14, with some blastomeres even containing 9 to 11 copies of the examined chromosomes (Fig. 6C).
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Both human and nonhuman animal diets contain several plant-derived, nonsteroidal estrogenic compounds [23] that are either produced by plants (phytestrogens) or by fungi that infect plants (mycestrogens). The most prominent mycestrogen is ZEA, and the occurrence of ZEA or its derivatives has been reported worldwide [24]. Zearalenone has been associated with hyperestrogenism and other reproductive disorders, such as impaired fertility in farm animals [8]. Zearalenone and its derivatives can cross the placenta and are excreted with milk, causing exposure of the embryo and neonate [25, 26].
Under physiological conditions, follicular cells produce relatively large amounts of estradiol during the follicular phase of the estrous cycle, and a decline in estradiol levels concurs with the breakdown of the germinal vesicle in oocytes [20]. There are no indications that estradiol is involved in the resumption of meiosis but, in contrast, in vitro maturation experiments have demonstrated that estradiol can disturb nuclear maturation and cause spindle malformations during meiosis [22]. Estrogens can diffuse in and out of cells, but in target cells they are bound by the nuclear receptors ESR1 and ESR2. Hormone binding leads to conformational changes of these receptors, allowing binding to specific elements on DNA and, in combination with components of the cellular transcription machinery, activation or repression of transcription [27]. In the ovaries of various mammalian species, including humans, expression of ESR1 has been detected in the germinal epithelium, interstitial cells, and theca cells, whereas ESR2 expression has been detected in granulosa and cumulus cells [28–31]. In bovine follicles, expression of ESR1 mRNA has been detected in cumulus cells, and ESR2 mRNA expression in both cumulus cells and oocytes, although it is unclear whether this also results in protein expression [22]. Binding of estradiol to ESR1 occurs via the aromatic ring of estradiol, but the volume of the binding pocket of the receptor is almost twice as large as estradiol, presumably allowing the acceptance of a number of different nonsteroidal compounds, such as ZEA [2].
Zearalenone can bind to ESR1 and ESR2 with approximately 7% and 15%, respectively, of the affinity of estradiol [3]. However, transactivation studies have demonstrated that ZEA is able to generate a response via the ERs in the same order of magnitude as that of estradiol, the transactivation via ESR1 being more efficient than that via ESR2 [3]. Since it has also been reported that cells can respond to estradiol independently of ER activation, a so-called nongenomic effect [32], it cannot be excluded that the abnormalities observed after exposure to estradiol or ZEA are not only caused via binding to and activation of ER but also involve nongenomic mechanisms yet to be determined. Indeed, it has been reported that estrogen can bind tubulin and inhibit tubulin polymerization [33].
A higher estrogenic potency of the ZEA metabolite -ZOL has been demonstrated [5]. Further, in the present study, we found that in maturing oocytes ZEA and -ZOL were more potent in inhibiting oocyte maturation at the lowest concentrations compared with ß-ZOL, although at higher concentrations the inhibition of maturation and occurrence of spindle abnormalities were similar with all three toxins. The activity of hydroxysteroid dehydrogenases in granulosa cells will be important for the occurrence of the hydroxylated products of ZEA in follicular fluid.
After exposure of oocytes to estradiol, the most commonly observed nuclear aberration was the almost complete absence of a microtubule spindle and the appearance of disorganized chromosomes that were not aligned properly. The morphology of the meiotic spindle after exposure of oocytes to ZEA was reminiscent of that after exposure to estradiol, but was not identical. The chromosomes were not properly aligned, and although microtubules were clearly present, they did not form an organized spindle. These results suggest that the effects of ZEA include mechanisms other than activation of estrogen receptors.
In contrast to ZEA, DON does not bind estrogen receptors. Its toxicity has been associated with inhibition of protein synthesis at the level of the ribosomes and a ribotoxic stress response involving activation of MAPK8/MAPK14 kinases and an increase in cytokine and chemokine transcription [12]. In turn, DON induces apoptosis of lymphoid cells [11, 34, 35]. In the present study in the oocytes exposed to DON only, no signs of apoptosis were observed. The fuzzy appearance of the microtubules in the meiotic spindle, however, was incompatible with further development. In addition, the great majority of the oocytes that did not form a morphologically normal meiotic spindle were either not fertilized or did not cleave after fertilization, indicating that DON induced more cellular damage than spindle malformation.
Alignment of the chromosomes is a microtubule-dependent process, and when microtubules were not visible or exhibited a fuzzy appearance, as observed after estradiol and DON exposure, the chromosomes failed to align. Spindle malformations in maturing oocytes can lead to aneuploidy and have serious consequences for fertilization and embryonic development [36, 37]. Indeed, in the present study, the percentages of blastocysts that were formed after exposure of oocytes to the selected mycotoxins were severely reduced, and a significant percentage of the embryos that were formed contained polyploid cells, some cells even containing 11 of chromosomes 7 or 14. The mechanism behind the duplication of chromosomes remains unclear, but the embryos that contained most abnormal cells were those embryos that contained fewest cells, suggesting that in these embryos cells either duplicated DNA without normal cell division or that the abnormal chromosome content inhibited embryonic development.
This study demonstrates that the mycotoxins ZEA and DON reduce fertility by altering spindle formation during meiosis of the oocyte. Importantly, mycotoxin-induced spindle malformations in the oocyte can result in aneuploid embryos. Purified mycotoxins can be less toxic than when occurring in feed, suggesting that mycotoxins act synergistically. Since ZEA and DON are produced by the same Fusarium species, risk assessment relating to exposure of contaminated food or feeds must consider possible additive or synergistic effect of these mycotoxins [38, 39]. In this study, clear differences were seen in the effect of either mycotoxin, and the potency appeared to be an additive rather than a reflection of a synergistic interaction, suggesting that monitoring ZEA and DON contamination in feed can provide a fair prediction of toxicity.
In conclusion, levels of mycotoxin contamination should be carefully monitored in both feed and food, as mycotoxins not only can cause reduced fertility by inhibiting oocyte maturation and embryo development, but may also lead to mixoploid embryos. In this respect it is important to determine the concentrations of mycotoxins that can occur in follicular fluid.
ACKNOWLEDGMENTS
Present and past colleagues of the Department of Farm Animal Health are thanked for helping to collect oocytes.
FOOTNOTES
3Current address: Department of Pharmacology and Toxicology, Faculty of Veterinary Medicine, Urmia University, PO Box 1177, Urmia, Iran. 
1Supported by the Iranian Ministry of Sciences, Research, and Technology.
Correspondence: 2Bernard A.J. Roelen, Department of Farm Animal Health, Faculty of Veterinary Medicine, Utrecht University, Yalelaan 104, 3584 CM, Utrecht, The Netherlands. FAX: 31 30 25348811; e-mail: b.a.j.roelen{at}vet.uu.nl
Received: 9 May 2007.
First decision: 5 June 2007.
Accepted: 23 July 2007.
REFERENCES
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