Zearalenone Exposure Affects Mouse Oocyte Meiotic Maturation and Granulosa Cell Proliferation Yan-Jun Hou,1* Cheng-Cheng Zhu,1* Yin-Xue Xu,1 Xiang-Shun Cui,2 Nam-Hyung Kim,2 Shao-Chen Sun1 1
College of Animal Science and Technology, Nanjing Agriculture University, Nanjing, 210095, China
2
Department of Animal Sciences, Chungbuk National University, Cheongju, 361–763, Korea
Received 16 December 2013; revised 28 March 2014; accepted 4 April 2014 ABSTRACT: Zearalenone (ZEN) is a metabolite of Fusarium and is a common contaminant of grains and foodstuffs. ZEN acts as a xenoestrogen and is considered to be cytotoxic, tissue toxic, and genotoxic, which causes abortions and stillbirths in humans and animals. Since estrogens affect oocyte maturation during meiosis, in this study we investigated the effects of ZEN on mouse oocyte meiotic maturation and granulosa cell proliferation. Our results showed that ZEN-treated oocyte maturation rates were decreased, which might be due to the disrupted cytoskeletons: (1) ZEN treatment resulted in significantly more oocytes with abnormal spindle morphologies; (2) actin filament expression and distribution were also disrupted after ZEN treatment, which was confirmed by the aberrant distribution of actin regulatory proteins. In addition, cortical granule-free domains (CGFDs) were disrupted after ZEN treatment, which indicated that ZEN may affect mouse oocyte fertilization capability. ZEN reduced mouse granulosa cell proliferation in a dose-dependent manner as determined by MTT assay and TUNEL apoptosis analysis, which may be another cause for the decreased oocyte maturation. Thus, our results demonstrated that exposure to zearalenone affected oocyte meiotic maturation and granulosa cell proliferation in mouse. C 2014 Wiley Periodicals, Inc. Environ Toxicol 00: 000–000, 2014. V
Keywords: Zearalenone; oocyte; cytoskeleton; granulosa cells; apoptosis
Additional Supporting Information may be found in the online version of this article. Correspondence to: S.-C. Sun; e-mail: [email protected] *These authors contributed equally to this work. Contract grant sponsor: National Basic Research Program of China. Contract grant number: 2014CB138503 Contract grant sponsor: Fundamental Research Funds for the Central Universities. Contract grant number: KJQN201402 Contract grant sponsor: the National Natural Science Foundation of China. Contract grant number: 31301860 Contract grant sponsor: BioGreen 21 Program, RDA, Republic of Korea. Contract grant number: PJ009594; PJ009080; PJ00909801 Published online 00 Month 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/tox.21995
INTRODUCTION Mycotoxin contamination is a worldwide food safety problem. Zearalenone (ZEN), a nonsteroidal mycotoxin, is produced by fusarium and is known to have toxic effects and cause reproductive system disorders. ZEN grows on grains, primarily on corn and hay, particularly they are exposed to high moisture levels during storage. ZEN has been associated with hyper-estrogenism and other reproductive disorders in swine and cattle (Balaban et al., 2012). In humans, ZEN exposure has been associated with epidemics of premature thelarche (Fowler et al., 2012). Thus, the toxic effects of ZEN and its metabolites on female reproductive performance are worldwide food safety problem (Pitt et al., 2013).
C 2014 Wiley Periodicals, Inc. V
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In the ovary, follicle-stimulating hormone (FSH) and luteinizing hormone (LH) promoted the estrogen synthesis. Furthermore, granulosa cell gene expression, promoting proliferation, antrumformation, oocyte maturation, and the follicle grows are regulated by FSH and LH (UlloaAguirre et al., 2007). While, the biological activities of estrogens are mediated by nuclear estrogen receptors (ERs), which interact directly with estrogen response elements (ERE) in the promoters of target genes and recruit various coactivators to mediate transcriptional regulation (McDonnell and Norris, 2002). ZEN is structurally similar to 17b-estradiol and has potent estrogenic activity to match the ERs, while for rodents intrauterine exposure to xenoestrogens-affected oocyte integrity, increased the length of the reproductive cycle, and reduced the oocyte ovulation rate and corporalutea formation in animals at sexual maturity, which resulted in smaller litters among offspring (Maranghi et al., 2008). ZEN causes several physiological alterations of the ovaries and reproductive tract (Oliver et al., 2012; Zhu et al., 2012), and it has also been shown that ZEN reduced the quantity of healthy follicles in pigs, which may result in premature oocyte depletion in adulthood (Schoevers et al., 2012). Although it was shown that ZEN was toxic to female reproductive system, till now the mechanisms by which ZEN causes oocyte damage remain poorly understood. In this study we investigated the effects of ZEN on graunlosa cell proliferation and the cytoskeletal integrity in mouse oocytes to determine possible cytotoxic ZEN mechanisms on oocytes. And our results showed that ZEN induced the apoptosis of graunlosa cells, affected spindle morphology, actin filament expression, and cortical granule free domain formation of mouse oocytes.
dissecting microscope to release MGCs. Cell suspensions were placed inT75 flasks that contained DMEM/F12 (GIBCO, UK) (1:1) with 10% (v/v) fetal bovine serum (GIBCO, UK), and ZEN was added to culture medium for a final concentration of 5–160 mM for granulosa cell. All the cell suspensions were incubated at 37 C with 5% CO2 for 7 days.
Oocyte Selection and Culture GV oocytes were harvested from the ovaries of ICR mice, and then cultured in M2 medium (Sigma-Aldrich) under paraffin oil at 37 C and 5% CO2. Samples were then used for immunostaining.
Antibodies and Chemicals Rabbit polyclonal anti-mDia1 and mouse monoclonal profilin-1 antibodies were from Abcam (Cambridge, UK). Rabbit polyclonal anti-g-tubulin and rabbit polyclonal antiROCK antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Hoechst 33342, anti-a-tubulin-FITC, and phalloidin-TRITC (actin) were from Sigma-Aldrich (St. Louis, MO). Alexa Fluor 488 and 568 goat anti-rabbit and anti-mouse secondary antibodies were from Invitrogen (Carlsbad, CA).
Real-Time Quantitative PCR Analysis
MATERIALS AND METHODS
Total RNA was isolated from cells using TRIZOL (Invitrogen), and reactions were run with M-MLV RT according to the manufacturer’s protocol (Promega). RT-PCR was done using SYBR Premix Ex Taq (Takara) in a reaction volume of 20 lL. Primer sequences used are given in Supporting Information (Supporting Information Table S1).
Culture Media
Confocal Microscopy
ZEN, DMSO, M2 medium, and DMEM-F12 medium were from Sigma Chemical Company (St. Louis, MO). ZEN was dissolved in DMSO, with the final concentration of the solvent not more than 0.01% of the culture medium. ZEN was added to culture medium for a final concentration of 10 mM or 50 mM in M2 for oocyte culture. Control medium contained the same final concentration of DMSO. All media were equilibrated in a CO2 incubator for at least 2 h before use.
Confocal microscopy was done as previously described (Sun et al., 2010). Briefly, oocytes were fixed in 4% paraformaldehyde (w/v) in phosphate-buffered saline (PBS) at room temperature (RT) for 30 min and then placed in a membrane permeabilization solution [0.5% albumin (w/v)-supplemented PBS] at RT for 20 min. After blocking with 1% bovine serum albumin (BSA, w/ v) at RT for 1 h, oocytes were incubated with a primary antibody (mouse profilin-1 at 1:100, rabbit mDia1 at 1:50, rabbit ROCK at 1:50, or rabbit g-tubulin at 1:50) at 4 C overnight or at RT for 4 h. Oocytes were washed three times in wash buffer [0.1% Tween 20 (v/v) and 0.01% Triton X-100 (v/v) in PBS] and then labeled with an Alexa Fluor secondary antibody (1:100) at RT for 1 h. After staining with 1 mg/mL of phalloidin-TRITC at RT for 30 min and washing three times with wash buffer, samples were stained with Hoechst 33342 (10 mg/mL in
Primary Mouse Granulosa Cell (MGC) Culture Mice were intraperitoneally injected with 10U PMSG and sacrificed 48 h later. Ovaries were obtained from superovulated mice, transferred into petri dishes (35 3 15 mm2) filled with PBS, and then pricked with a syringe under a surgical
Environmental Toxicology DOI 10.1002/tox
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Fig. 1. ZEN effects on the maturation competence of mouse oocytes in vitro. A: GVBD (germinal vesicle breakdown) oocyte morphology and the GVBD rate after 3-h culture for ZEN-treated oocytes. B: Morphology and the rate of MII (metaphase II) oocytes after 12-h culture after maturation for ZEN-treated oocytes. Bar 5 50 lm.
PBS) for 10 min. Some oocytes were stained with 5 mg/ mL of lectin-FITC or anti-a-tubulin-FITC (1:200) at RT for 2 h and then stained with Hoechst 33342 (10 mg/mL in PBS) for 10 min. Oocytes were mounted on glass slides and examined with a Zeiss LSM 700 Meta scanning confocal microscope (Carl Zeiss, Jena, Germany).
MTT Assay Test for Granulosa Cell Proliferation Granulosa cells were cultured in the same medium as above in 96-well plates. After culture for 96 h, cells were treated with different concentrations of ZEN ranging from 0 to 160 mM for
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Fig. 2. ZEN effects on spindle morphology and actin expression in mouse oocytes. A: Mycotoxin effects on oocyte spindle morphology. Spindles exhibited no poles, multiple poles, or disrupted poles for ZEN-treated oocytes. B: Rate of abnormal spindle formation after ZEN treatment. C: Localization of g-tubulin after ZEN treatment. In control oocytes, gtubulin was localized at spindle poles, whereas in ZEN-treated oocytes there was disrupted g-tubulin localization. Bar 5 20 lm. D: Actin expression decreased after ZEN treatment. Actin caps were present in control oocytes, but actin formation was disrupted in ZEN-treated oocytes. Actin, red; DNA, blue. Actin fluorescence intensity histograms for both membranes and cytoplasm decreased in ZEN-treated oocytes. E: ZEN effects on profilin-1, ROCK (Rho kinase), and mDia1 protein expression. A loss in profilin-1 and mDia1 localization was observed after ZEN treatment. Profilin-1, green; mDia1, green; ROCK, red; DNA, blue. Histograms for the statistical analysis of profilin-1, ROCK, and mDia1 fluorescence intensities in the membrane and cytoplasm of control and ZENtreated oocytes. Bar 5 20 lm. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
24 h. Then, granulosa cell proliferation was determined as previously described using an MTT assay (Mosmann 1983).
Death Detection Kit (Roche Applied Science, Shanghai, China). Images were obtained using a laser scanning confocal microscopy (Zeiss 710, Germany).
TUNEL Assay The apoptosis rates were determined by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) assay. The detailed procedure was performed according to the protocol of the In Situ Cell
Environmental Toxicology DOI 10.1002/tox
Statistical Analysis At least three replicates were performed for each treatment and results were given as means 6 SEMs. Statistical comparisons were made by analysis of variance (ANOVA),
ZEN EFFECTS ON OOCYTES
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followed by Duncan’s multiple comparisons test. p < 0.05 was considered significant. Fluorescence intensity was quantified by Image J software (NIH, Bethesda, MD); 30 oocytes were analyzed for each experiment. Experiments were repeated in triplicate.
RESULTS ZEN Decreases Mouse Oocyte Developmental Competence We next examined the developmental competence of mouse oocytes after ZEN treatment. Most control oocytes underwent germinal vesicle breakdown (GVBD) after culture for 3 h. However, the GVBD rate was significantly decreased for ZENtreated oocytes: 84.73 6 5.34% (n 5 632) for controls vs. 74.23 6 5.29% (n 5 627) with 10 mM ZEN and 47.93 6 7.05% (n 5 641) with 50 mM ZEN [p < 0.05; Fig. 1(A)]. The polar body extrusion by oocytes was also significantly affected after ZEN treatment: 74.50 6 2.27% (n 5 203) of control oocytes matured to metaphase II (MII) after 12 h culture in vitro as compared with 52.92 6 3.40% (n 5 210) with 10 mM ZEN and 16.06 6 1.11% (n 5 224) with 50 mM ZEN [Fig. 1(B)].
ZEN Disrupts Meiotic Spindle Morphology and Actin Expression in Mouse Oocytes Next, we investigated meiotic spindle morphology after ZEN treatment. The majority of control MI and MII oocytes showed well-aligned chromosomes and normal spindle morphologies (“spindle”-like and two poles). However, a large proportion of oocytes showed misaligned chromosomes and disrupted spindle morphologies after ZEN treatment. Spindles showed no poles, multiple poles, or disrupted poles with ZEN treatment [Fig. 2(A)]. Rates of abnormal; spindle morphology: 1.45 6 1.45% (n 5 227) for controls vs. 20.28 6 0.28% (n 5 208) with 50 mM ZEN [p < 0.05; Fig. 2(B)]. To confirm disrupted spindle assembly, we stained for g-tubulin, a centrosome protein. In control oocytes, gtubulin was localized at spindle poles, whereas in ZENtreated oocytes g-tubulin localization was disrupted. These results showed that ZEN disrupted cytoskeletal elements in mouse oocytes [Fig. 2(C)]. We next examined the distributions of actin filaments. Actin expression in mouse oocytes decreased after ZEN treatment [Fig. 2(D)]. In ZEN-treated MI oocytes, actin fluorescence intensity at the plasma membrane was significantly lower (80.19 6 7.85) than that in control oocytes (128.60 6 12.87; n 5 30, p < 0.05), whereas there was no difference for cytoplasmic actin expression between the controls and ZEN-treated oocytes [23.43 6 4.34 vs. 16.48 6 2.70; p > 0.1; Fig. 2(D)]. To confirm the disruption of actin filaments, we stained for the actin regulatory proteins profilin-1, ROCK, and
Fig. 3. ZEN effects on CGFD (cortical granule free domain) formation in mouse oocytes. A: In oocytes from untreated mice, CGs were absent at the cortex where chromosomes were located during MI (metaphase I). Conversely, in oocytes from ZEN-treated mice, CGs were distributed throughout the entire cortex. Z-stacks correspond to different planes of an oocyte. Arrowhead indicates a CGFD. CG, green; DNA, blue. B: Percentage of oocytes with decreased CGFD after ZEN treatment. Bar 5 20 lm. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
mDia1. There was decreased expression of profilin-1, ROCK, and mDia1 at meiotic spindles in ZEN-treated oocytes as compared with control oocytes. This was supported by quantifying profilin-1, ROCK, and mDia1 fluorescence intensities, which were decreased in ZEN-treated oocytes (28.27 6 2.44 for profilin-1, 40.85 6 3.81 for ROCK, and 29.95 6 1.95 for mDia1) compared to controls [38.52 6 3.51 for profilin-1, 58.06 6 3.86 for ROCK, and 42.03 6 2.87 for mDia1; p < 0.05; Fig. 2(E)].
ZEN Disrupts CGFD Distributions The formation of cortical granule-free domains (CGFDs) is an indicator of oocyte polarity. In control oocytes, cortical granules (CGs) were absent in the area close to chromosomes, whereas CGs were uniformly localized at the plasma membrane in ZEN-treated oocytes [Fig. 3(A)]. Compared with controls (75.75 6 4.35%; n 5 116), the rate of CGFD formation was significantly decreased with 10 mM ZEN (57.56 6 7.11%; (n 5 107) and with 50 mM ZEN [29.50 6 5.15%; n 5 112; p < 0.05; Fig. 3(B)].
ZEN Causes Reduced GC Proliferation and Increased GC Apoptosis Granulosa cells (GCs) were treated with different concentrations of ZEN. After 24 h, the numbers of adherent cells among ZEN-treated GCs were reduced as compared with
Environmental Toxicology DOI 10.1002/tox
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Fig. 4. ZEN effects on the proliferation and apoptosis of mouse granulosa cells. A: Granulosa cell morphologies after ZEN treatment. Bar 5 20 lm. B: MTT assay was used to determine mouse granulosa cell proliferation (changes in absorbance). C: Relative mRNA expression analysis used the 22DD Ct method. BIM and FasL mRNA expression levels were normalized to that of controls. Results are means 6 SDs of three independent experiments. *p < 0.05; **p < 0.01. D: ZEN caused granulosa cells apoptosis increased. Apoptotic images were visualized by TUNEL assay. TUNEL-positive cells display green staining in the nuclei, which merged with the HOCHEST counterstaining (blue). The TUNEL positive rates were determined by averaging numbers of TUNEL-positive staining nuclei from three randomly chosen visual fields in each coverslip. Bar 5 20 lm. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
controls [Fig. 4(A)]. An MTT assay was used to determine the effects of ZEN on GC proliferation. This showed that GC proliferation was reduced by about 50% after exposure to ZEN at doses of 10–20 lM, which indicated a median lethal ZEN concentration of 10–20 lM [Fig. 4(B)]. To explore possible mechanisms by which ZEN reduced GC proliferation, BIM, and FasL mRNA expressions were assessed after treating GCs with ZEN [Fig. 4(C)]. At ZEN doses of 20, 40, 80,120, and 160 lM, the BIM mRNA levels were increased by 3.26 6 0.89, 3.05 6 1.03, 16.27 6 6.34, 20.95 6 6.75, and 55.08 6 8.38 fold, respectively, as compared with that in controls (0 lM ZEN and DMSO). Compared to controls, FasL mRNA levels were significantly enhanced with 80, 120, and 160 lM ZEN by
Environmental Toxicology DOI 10.1002/tox
131.80 6 70.73, 172.90 6 93.59, and 466.20 6 150.70 fold, respectively (p < 0.01). To further support our hypothesis that ZEN was responsible for being the result of DNA degradation rather than bactericidal cell death in dead cells, we used the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) assay. We observed a qualitative increase in TUNEL-positive cells when analyzed by microscopy in the treatment groups while the control group (0.57 6 0.57%) showed no fluorescence single. The TUNEL-positive cells proportions were 3.34 6 0.87%, 7.97 6 1.64%, 31.64 6 10.05%, 71.50 6 9.83%, 82.02 6 4.02%, and 92.67 6 7.33% in 5, 10, 20, 40, 80, 120 lM ZEN groups, respectively [Fig. 4(D)].
ZEN EFFECTS ON OOCYTES
DISCUSSION In this study we investigated the effects of ZEN on granulosa cells and oocytes. We found that ZEN had toxic effects in terms of granulosa cell proliferation and the disruption of meiotic spindles and actin filament distributions in mouse oocytes. We first found that oocyte meiotic maturation was affected by exposure to ZEN. The polar body extrusion rates were decreased; in particular, we found that a large proportion of oocytes were arrested at the GV stage and could not enter germinal vesicle breakdown (GVBD), which indicated the low developmental competence of mouse oocytes after ZEN treatment. The results were similar with previous work which showed that ZEN had a negative effect on the quality of bovine, gilt, and porcine oocytes (Minervini et al., 2001; Alm et al., 2006; Schoevers et al., 2010). Moreover, ZEN and its metabolites also affected blastocyst formation in sows (Long et al., 1992). To investigate the cause of reduced oocyte developmental competence, we first examined the mouse oocyte actin cytoskeleton and microtubules. The cytoskeleton was critical for oocyte cytokinesis and involved extensive rearrangements of actin filaments at the plasma membrane and spindle assembly (Roth and Hansen, 2005). In oocytes, microtubules form the meiotic spindle that drives chromosome congression and segregation, while actin filaments regulate meiotic spindle movements and initiate cytokinesis for small polar body extrusion (Sun and Kim, 2013). Moreover, they also participate in the formation of oocyte polarity (Sun et al., 2011b; Yi and Li, 2012). Thus, cytoskeletal integrity ensures complete oocyte competence, and oocyte cytoskeletal integrity may critically affect the fates of oocyte maturation and embryo development (Eichenlaub-Ritter et al., 2002; Luvoni et al., 2012). We demonstrated that spindle morphology was aberrant and actin filament distributions were disrupted with ZEN treatment. The effect of ZEN on the spindle formation might be direct, and this was confirmed by the disrupted localization of g-tubulin; while the aberrant actin expression was confirmed by the abnormal distributions of the actin related proteins profilin1, ROCK and mDia1, which indicated that the damage to oocyte quality induced by ZEN might be caused by disrupting cytoskeletal integrity. Our results showed that the disruption of cytoskeletal integrity might be one reason for the low oocyte developmental competence. Another phenotype we observed was that the distribution of cortical granules (CGs) was abnormal after ZEN treatment. CGs are produced by Golgi complexes and remodel the embryo surface after fertilization by exocytosis (Kimura and Kimura, 2012). Previous results showed that actin microfilament disruption affected cortical granules migration (Liu et al., 2010), and a disruption of cortical granule-free domains (CGFD) is characteristic of oocyte polarity loss (Sun et al., 2011a). Therefore, our results indicated that
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exposure to ZEN might affect oocyte polarity and subsequent fertilization. We next examined the effects of ZEN on granulosa cells. The granulosa cell apoptosis increased in a dose-dependent manner after ZEN treatment. This result was similar to those in recent studies that showed that ZEN induced porcine granulosa cell apoptosis through a mitochondrial signaling pathway (Zhu et al., 2012), and identical results were also found in somatic cells: ZEN was reported causing DNA strand breaks in HEK293 cells (Gao et al., 2013), and induces sister chromatin exchange and chromosomal aberration in CHO cells (Hassen et al., 2007). ZEN is responsible for DNA degradation rather than bactericidal cell death in dead cells. Granulosa cells are critical for the mammalian oocyte maturation, the apoptosis of granulosa cells might be another reason for the reduced competence of mouse oocyte maturation. In summary, we demonstrated that ZEN affected the proliferation of mouse granulosa cells and oocyte meiotic maturation using an in vitro model. The authors thank Wenxing Sun and Ming Shen for helpful advice.
REFERENCES Alm H, Br€ussow K-P, Torner H, Vanselow J, Tomek W, D€anicke S, Tiemann U. 2006. Influence of Fusarium-toxin contaminated feed on initial quality and meiotic competence of gilt oocytes. Reproductive Toxicol 22:44–50. Balaban B, Barut T, Urman B. 2012. Assessment of Oocyte Quality. Practical Manual of In Vitro Fertilization, Springer. Chapter 13, 105–119. Eichenlaub-Ritter U, Shen Y, Tinneberg H-R. 2002. Manipulation of the oocyte: Possible damage to the spindle apparatus. Reproductive Biomed Online 5:117–124. Fowler PA, Bellingham M, Sinclair KD, Evans NP, Pocar P, Fischer B, Schaedlich K, Schmidt J-S, Amezaga MR, Bhattacharya S. 2012. Impact of endocrine-disrupting compounds (EDCs) on female reproductive health. Mol Cell Endocrinol 355:231–239. Gao F, Jiang L-P, Chen M, Geng C-Y, Yang G, Ji F, Zhong L-F, Liu X-F. 2013. Genotoxic effects induced by zearalenone in a human embryonic kidney cell line. Mutat Res/Genetic Toxicol Environm Mutagenesis 755:6–10. Hassen W, Ayed-Boussema I, Oscoz AA, De Cerain Lopez A, Bacha H. 2007. The role of oxidative stress in zearalenonemediated toxicity in Hep G2 cells: Oxidative DNA damage, gluthatione depletion and stress proteins induction. Toxicology 232:294–302. Kimura K, Kimura A. 2012. Rab6 is required for the exocytosis of cortical granules and the recruitment of separase to the granules during the oocyte-to-embryo transition in Caenorhabditis elegans. J Cell Sci 125:5897–5905. Liu S, Li Y, Feng HL, Yan JH, Li M, Ma SY, Chen ZJ. 2010. Dynamic modulation of cytoskeleton during in vitro
Environmental Toxicology DOI 10.1002/tox
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maturation in human oocytes. Am J Obstetrics Gynecol 203:151.e1–151.e7.
exposed to heat shock during maturation. Reproduction 129: 235–244.
Long G, Turek J, Diekman M, Scheidt A. 1992. Effect of zearalenone on days 7 to 10 post-mating on blastocyst development and endometrial morphology in sows. Veterinary Pathol Online 29:60–67.
Schoevers E, Fink-Gremmels J, Colenbrander B, Roelen B. 2010. Porcine oocytes are most vulnerable to the mycotoxin deoxynivalenol during formation of the meiotic spindle. Theriogenology 74:968–978.
Luvoni G, Tessaro I, Apparicio M, Ruggeri E, Luciano A, Modina S. 2012. Effect of vitrification of feline ovarian cortex on follicular and oocyte quality and competence. Reproduction Domestic Animals 47:385–391.
Schoevers EJ, Santos RR, Colenbrander B, Fink-Gremmels J, Roelen BAJ. 2012. Transgenerational toxicity of Zearalenone in pigs. Reproduct Toxicol 34:110–119.
Maranghi F, Tassinari R, Moracci G, Macrı C, Mantovani A. 2008. Effects of a low oral dose of diethylstilbestrol (DES) on reproductive tract development in F1 female CD-1 mice. Reproduct Toxicol 26:146–150.
Sun SC, Xu YN, Li YH, Lee SE, Jin YX, Cui XS, Kim NH. 2011a. WAVE2 regulates meiotic spindle stability, peripheral positioning and polar body emission in mouse oocytes. Cell Cycle 10:1853–1860.
McDonnell DP, Norris JD. 2002. Connections and regulation of the human estrogen receptor. Science 296:1642–1644.
Sun SC, Kim NH. 2013. Molecular mechanisms of asymmetric division in oocytes. Microsc Microanal 19:883–897.
Minervini F, Dell’Aquila M, Maritato F, Minoia P, Visconti A. 2001. Toxic effects of the mycotoxin zearalenone and its derivatives on in vitro maturation of bovine oocytes and 17bestradiol levels in mural granulosa cell cultures. Toxicol In Vitro 15:489–495.
Sun SC, Lee SE, Xu YN, Kim NH. 2010. Perturbation of Spc25 expression affects meiotic spindle organization, chromosome alignment and spindle assembly checkpoint in mouse oocytes. Cell Cycle 9:4552–4559.
Mosmann T. 1983. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J Immunological Methods 65:55–63. Oliver W, Miles J, Diaz D, Dibner J, Rottinghaus G, Harrell R. 2012. Zearalenone enhances reproductive tract development, but does not alter skeletal muscle signaling in prepubertal gilts. Animal Feed Sci Technol 174:79–85. Pitt J, Taniwaki MH, Cole M. 2013. Mycotoxin production in major crops as influenced by growing, harvesting, storage and processing, with emphasis on the achievement of Food Safety Objectives. Food Control, Elsevier 32:205–215. Roth Z, Hansen PJ. 2005. Disruption of nuclear maturation and rearrangement of cytoskeletal elements in bovine oocytes
Environmental Toxicology DOI 10.1002/tox
Sun SC, Wang ZB, Xu YN, Lee SE, Cui XS, Kim NH. 2011b. Arp2/3 complex regulates asymmetric division and cytokinesis in mouse oocytes. PLoS One 6:e18392. Ulloa-Aguirre A, Zarinan T, Pasapera AM, Casas-Gonzalez P, Dias JA. 2007. Multiple facets of follicle-stimulating hormone receptor function. Endocrine 32:251–263. Yi K, Li R. 2012. Actin cytoskeleton in cell polarity and asymmetric division during mouse oocyte maturation. Cytoskeleton 69:727–737. Zhu L, Yuan H, Guo C, Lu Y, Deng S, Yang Y, Wei Q, Wen L, He Z. 2012. Zearalenone induces apoptosis and necrosis in porcine granulosa cells via a caspase-3-and caspase-9dependent mitochondrial signaling pathway. J Cell Physiol 227:1814–1820.
College of Animal Science and Technology, Nanjing Agriculture University, Nanjing, 210095, China
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Department of Animal Sciences, Chungbuk National University, Cheongju, 361–763, Korea
Received 16 December 2013; revised 28 March 2014; accepted 4 April 2014 ABSTRACT: Zearalenone (ZEN) is a metabolite of Fusarium and is a common contaminant of grains and foodstuffs. ZEN acts as a xenoestrogen and is considered to be cytotoxic, tissue toxic, and genotoxic, which causes abortions and stillbirths in humans and animals. Since estrogens affect oocyte maturation during meiosis, in this study we investigated the effects of ZEN on mouse oocyte meiotic maturation and granulosa cell proliferation. Our results showed that ZEN-treated oocyte maturation rates were decreased, which might be due to the disrupted cytoskeletons: (1) ZEN treatment resulted in significantly more oocytes with abnormal spindle morphologies; (2) actin filament expression and distribution were also disrupted after ZEN treatment, which was confirmed by the aberrant distribution of actin regulatory proteins. In addition, cortical granule-free domains (CGFDs) were disrupted after ZEN treatment, which indicated that ZEN may affect mouse oocyte fertilization capability. ZEN reduced mouse granulosa cell proliferation in a dose-dependent manner as determined by MTT assay and TUNEL apoptosis analysis, which may be another cause for the decreased oocyte maturation. Thus, our results demonstrated that exposure to zearalenone affected oocyte meiotic maturation and granulosa cell proliferation in mouse. C 2014 Wiley Periodicals, Inc. Environ Toxicol 00: 000–000, 2014. V
Keywords: Zearalenone; oocyte; cytoskeleton; granulosa cells; apoptosis
Additional Supporting Information may be found in the online version of this article. Correspondence to: S.-C. Sun; e-mail: [email protected] *These authors contributed equally to this work. Contract grant sponsor: National Basic Research Program of China. Contract grant number: 2014CB138503 Contract grant sponsor: Fundamental Research Funds for the Central Universities. Contract grant number: KJQN201402 Contract grant sponsor: the National Natural Science Foundation of China. Contract grant number: 31301860 Contract grant sponsor: BioGreen 21 Program, RDA, Republic of Korea. Contract grant number: PJ009594; PJ009080; PJ00909801 Published online 00 Month 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/tox.21995
INTRODUCTION Mycotoxin contamination is a worldwide food safety problem. Zearalenone (ZEN), a nonsteroidal mycotoxin, is produced by fusarium and is known to have toxic effects and cause reproductive system disorders. ZEN grows on grains, primarily on corn and hay, particularly they are exposed to high moisture levels during storage. ZEN has been associated with hyper-estrogenism and other reproductive disorders in swine and cattle (Balaban et al., 2012). In humans, ZEN exposure has been associated with epidemics of premature thelarche (Fowler et al., 2012). Thus, the toxic effects of ZEN and its metabolites on female reproductive performance are worldwide food safety problem (Pitt et al., 2013).
C 2014 Wiley Periodicals, Inc. V
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In the ovary, follicle-stimulating hormone (FSH) and luteinizing hormone (LH) promoted the estrogen synthesis. Furthermore, granulosa cell gene expression, promoting proliferation, antrumformation, oocyte maturation, and the follicle grows are regulated by FSH and LH (UlloaAguirre et al., 2007). While, the biological activities of estrogens are mediated by nuclear estrogen receptors (ERs), which interact directly with estrogen response elements (ERE) in the promoters of target genes and recruit various coactivators to mediate transcriptional regulation (McDonnell and Norris, 2002). ZEN is structurally similar to 17b-estradiol and has potent estrogenic activity to match the ERs, while for rodents intrauterine exposure to xenoestrogens-affected oocyte integrity, increased the length of the reproductive cycle, and reduced the oocyte ovulation rate and corporalutea formation in animals at sexual maturity, which resulted in smaller litters among offspring (Maranghi et al., 2008). ZEN causes several physiological alterations of the ovaries and reproductive tract (Oliver et al., 2012; Zhu et al., 2012), and it has also been shown that ZEN reduced the quantity of healthy follicles in pigs, which may result in premature oocyte depletion in adulthood (Schoevers et al., 2012). Although it was shown that ZEN was toxic to female reproductive system, till now the mechanisms by which ZEN causes oocyte damage remain poorly understood. In this study we investigated the effects of ZEN on graunlosa cell proliferation and the cytoskeletal integrity in mouse oocytes to determine possible cytotoxic ZEN mechanisms on oocytes. And our results showed that ZEN induced the apoptosis of graunlosa cells, affected spindle morphology, actin filament expression, and cortical granule free domain formation of mouse oocytes.
dissecting microscope to release MGCs. Cell suspensions were placed inT75 flasks that contained DMEM/F12 (GIBCO, UK) (1:1) with 10% (v/v) fetal bovine serum (GIBCO, UK), and ZEN was added to culture medium for a final concentration of 5–160 mM for granulosa cell. All the cell suspensions were incubated at 37 C with 5% CO2 for 7 days.
Oocyte Selection and Culture GV oocytes were harvested from the ovaries of ICR mice, and then cultured in M2 medium (Sigma-Aldrich) under paraffin oil at 37 C and 5% CO2. Samples were then used for immunostaining.
Antibodies and Chemicals Rabbit polyclonal anti-mDia1 and mouse monoclonal profilin-1 antibodies were from Abcam (Cambridge, UK). Rabbit polyclonal anti-g-tubulin and rabbit polyclonal antiROCK antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Hoechst 33342, anti-a-tubulin-FITC, and phalloidin-TRITC (actin) were from Sigma-Aldrich (St. Louis, MO). Alexa Fluor 488 and 568 goat anti-rabbit and anti-mouse secondary antibodies were from Invitrogen (Carlsbad, CA).
Real-Time Quantitative PCR Analysis
MATERIALS AND METHODS
Total RNA was isolated from cells using TRIZOL (Invitrogen), and reactions were run with M-MLV RT according to the manufacturer’s protocol (Promega). RT-PCR was done using SYBR Premix Ex Taq (Takara) in a reaction volume of 20 lL. Primer sequences used are given in Supporting Information (Supporting Information Table S1).
Culture Media
Confocal Microscopy
ZEN, DMSO, M2 medium, and DMEM-F12 medium were from Sigma Chemical Company (St. Louis, MO). ZEN was dissolved in DMSO, with the final concentration of the solvent not more than 0.01% of the culture medium. ZEN was added to culture medium for a final concentration of 10 mM or 50 mM in M2 for oocyte culture. Control medium contained the same final concentration of DMSO. All media were equilibrated in a CO2 incubator for at least 2 h before use.
Confocal microscopy was done as previously described (Sun et al., 2010). Briefly, oocytes were fixed in 4% paraformaldehyde (w/v) in phosphate-buffered saline (PBS) at room temperature (RT) for 30 min and then placed in a membrane permeabilization solution [0.5% albumin (w/v)-supplemented PBS] at RT for 20 min. After blocking with 1% bovine serum albumin (BSA, w/ v) at RT for 1 h, oocytes were incubated with a primary antibody (mouse profilin-1 at 1:100, rabbit mDia1 at 1:50, rabbit ROCK at 1:50, or rabbit g-tubulin at 1:50) at 4 C overnight or at RT for 4 h. Oocytes were washed three times in wash buffer [0.1% Tween 20 (v/v) and 0.01% Triton X-100 (v/v) in PBS] and then labeled with an Alexa Fluor secondary antibody (1:100) at RT for 1 h. After staining with 1 mg/mL of phalloidin-TRITC at RT for 30 min and washing three times with wash buffer, samples were stained with Hoechst 33342 (10 mg/mL in
Primary Mouse Granulosa Cell (MGC) Culture Mice were intraperitoneally injected with 10U PMSG and sacrificed 48 h later. Ovaries were obtained from superovulated mice, transferred into petri dishes (35 3 15 mm2) filled with PBS, and then pricked with a syringe under a surgical
Environmental Toxicology DOI 10.1002/tox
ZEN EFFECTS ON OOCYTES
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Fig. 1. ZEN effects on the maturation competence of mouse oocytes in vitro. A: GVBD (germinal vesicle breakdown) oocyte morphology and the GVBD rate after 3-h culture for ZEN-treated oocytes. B: Morphology and the rate of MII (metaphase II) oocytes after 12-h culture after maturation for ZEN-treated oocytes. Bar 5 50 lm.
PBS) for 10 min. Some oocytes were stained with 5 mg/ mL of lectin-FITC or anti-a-tubulin-FITC (1:200) at RT for 2 h and then stained with Hoechst 33342 (10 mg/mL in PBS) for 10 min. Oocytes were mounted on glass slides and examined with a Zeiss LSM 700 Meta scanning confocal microscope (Carl Zeiss, Jena, Germany).
MTT Assay Test for Granulosa Cell Proliferation Granulosa cells were cultured in the same medium as above in 96-well plates. After culture for 96 h, cells were treated with different concentrations of ZEN ranging from 0 to 160 mM for
Environmental Toxicology DOI 10.1002/tox
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Fig. 2. ZEN effects on spindle morphology and actin expression in mouse oocytes. A: Mycotoxin effects on oocyte spindle morphology. Spindles exhibited no poles, multiple poles, or disrupted poles for ZEN-treated oocytes. B: Rate of abnormal spindle formation after ZEN treatment. C: Localization of g-tubulin after ZEN treatment. In control oocytes, gtubulin was localized at spindle poles, whereas in ZEN-treated oocytes there was disrupted g-tubulin localization. Bar 5 20 lm. D: Actin expression decreased after ZEN treatment. Actin caps were present in control oocytes, but actin formation was disrupted in ZEN-treated oocytes. Actin, red; DNA, blue. Actin fluorescence intensity histograms for both membranes and cytoplasm decreased in ZEN-treated oocytes. E: ZEN effects on profilin-1, ROCK (Rho kinase), and mDia1 protein expression. A loss in profilin-1 and mDia1 localization was observed after ZEN treatment. Profilin-1, green; mDia1, green; ROCK, red; DNA, blue. Histograms for the statistical analysis of profilin-1, ROCK, and mDia1 fluorescence intensities in the membrane and cytoplasm of control and ZENtreated oocytes. Bar 5 20 lm. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
24 h. Then, granulosa cell proliferation was determined as previously described using an MTT assay (Mosmann 1983).
Death Detection Kit (Roche Applied Science, Shanghai, China). Images were obtained using a laser scanning confocal microscopy (Zeiss 710, Germany).
TUNEL Assay The apoptosis rates were determined by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) assay. The detailed procedure was performed according to the protocol of the In Situ Cell
Environmental Toxicology DOI 10.1002/tox
Statistical Analysis At least three replicates were performed for each treatment and results were given as means 6 SEMs. Statistical comparisons were made by analysis of variance (ANOVA),
ZEN EFFECTS ON OOCYTES
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followed by Duncan’s multiple comparisons test. p < 0.05 was considered significant. Fluorescence intensity was quantified by Image J software (NIH, Bethesda, MD); 30 oocytes were analyzed for each experiment. Experiments were repeated in triplicate.
RESULTS ZEN Decreases Mouse Oocyte Developmental Competence We next examined the developmental competence of mouse oocytes after ZEN treatment. Most control oocytes underwent germinal vesicle breakdown (GVBD) after culture for 3 h. However, the GVBD rate was significantly decreased for ZENtreated oocytes: 84.73 6 5.34% (n 5 632) for controls vs. 74.23 6 5.29% (n 5 627) with 10 mM ZEN and 47.93 6 7.05% (n 5 641) with 50 mM ZEN [p < 0.05; Fig. 1(A)]. The polar body extrusion by oocytes was also significantly affected after ZEN treatment: 74.50 6 2.27% (n 5 203) of control oocytes matured to metaphase II (MII) after 12 h culture in vitro as compared with 52.92 6 3.40% (n 5 210) with 10 mM ZEN and 16.06 6 1.11% (n 5 224) with 50 mM ZEN [Fig. 1(B)].
ZEN Disrupts Meiotic Spindle Morphology and Actin Expression in Mouse Oocytes Next, we investigated meiotic spindle morphology after ZEN treatment. The majority of control MI and MII oocytes showed well-aligned chromosomes and normal spindle morphologies (“spindle”-like and two poles). However, a large proportion of oocytes showed misaligned chromosomes and disrupted spindle morphologies after ZEN treatment. Spindles showed no poles, multiple poles, or disrupted poles with ZEN treatment [Fig. 2(A)]. Rates of abnormal; spindle morphology: 1.45 6 1.45% (n 5 227) for controls vs. 20.28 6 0.28% (n 5 208) with 50 mM ZEN [p < 0.05; Fig. 2(B)]. To confirm disrupted spindle assembly, we stained for g-tubulin, a centrosome protein. In control oocytes, gtubulin was localized at spindle poles, whereas in ZENtreated oocytes g-tubulin localization was disrupted. These results showed that ZEN disrupted cytoskeletal elements in mouse oocytes [Fig. 2(C)]. We next examined the distributions of actin filaments. Actin expression in mouse oocytes decreased after ZEN treatment [Fig. 2(D)]. In ZEN-treated MI oocytes, actin fluorescence intensity at the plasma membrane was significantly lower (80.19 6 7.85) than that in control oocytes (128.60 6 12.87; n 5 30, p < 0.05), whereas there was no difference for cytoplasmic actin expression between the controls and ZEN-treated oocytes [23.43 6 4.34 vs. 16.48 6 2.70; p > 0.1; Fig. 2(D)]. To confirm the disruption of actin filaments, we stained for the actin regulatory proteins profilin-1, ROCK, and
Fig. 3. ZEN effects on CGFD (cortical granule free domain) formation in mouse oocytes. A: In oocytes from untreated mice, CGs were absent at the cortex where chromosomes were located during MI (metaphase I). Conversely, in oocytes from ZEN-treated mice, CGs were distributed throughout the entire cortex. Z-stacks correspond to different planes of an oocyte. Arrowhead indicates a CGFD. CG, green; DNA, blue. B: Percentage of oocytes with decreased CGFD after ZEN treatment. Bar 5 20 lm. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
mDia1. There was decreased expression of profilin-1, ROCK, and mDia1 at meiotic spindles in ZEN-treated oocytes as compared with control oocytes. This was supported by quantifying profilin-1, ROCK, and mDia1 fluorescence intensities, which were decreased in ZEN-treated oocytes (28.27 6 2.44 for profilin-1, 40.85 6 3.81 for ROCK, and 29.95 6 1.95 for mDia1) compared to controls [38.52 6 3.51 for profilin-1, 58.06 6 3.86 for ROCK, and 42.03 6 2.87 for mDia1; p < 0.05; Fig. 2(E)].
ZEN Disrupts CGFD Distributions The formation of cortical granule-free domains (CGFDs) is an indicator of oocyte polarity. In control oocytes, cortical granules (CGs) were absent in the area close to chromosomes, whereas CGs were uniformly localized at the plasma membrane in ZEN-treated oocytes [Fig. 3(A)]. Compared with controls (75.75 6 4.35%; n 5 116), the rate of CGFD formation was significantly decreased with 10 mM ZEN (57.56 6 7.11%; (n 5 107) and with 50 mM ZEN [29.50 6 5.15%; n 5 112; p < 0.05; Fig. 3(B)].
ZEN Causes Reduced GC Proliferation and Increased GC Apoptosis Granulosa cells (GCs) were treated with different concentrations of ZEN. After 24 h, the numbers of adherent cells among ZEN-treated GCs were reduced as compared with
Environmental Toxicology DOI 10.1002/tox
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Fig. 4. ZEN effects on the proliferation and apoptosis of mouse granulosa cells. A: Granulosa cell morphologies after ZEN treatment. Bar 5 20 lm. B: MTT assay was used to determine mouse granulosa cell proliferation (changes in absorbance). C: Relative mRNA expression analysis used the 22DD Ct method. BIM and FasL mRNA expression levels were normalized to that of controls. Results are means 6 SDs of three independent experiments. *p < 0.05; **p < 0.01. D: ZEN caused granulosa cells apoptosis increased. Apoptotic images were visualized by TUNEL assay. TUNEL-positive cells display green staining in the nuclei, which merged with the HOCHEST counterstaining (blue). The TUNEL positive rates were determined by averaging numbers of TUNEL-positive staining nuclei from three randomly chosen visual fields in each coverslip. Bar 5 20 lm. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
controls [Fig. 4(A)]. An MTT assay was used to determine the effects of ZEN on GC proliferation. This showed that GC proliferation was reduced by about 50% after exposure to ZEN at doses of 10–20 lM, which indicated a median lethal ZEN concentration of 10–20 lM [Fig. 4(B)]. To explore possible mechanisms by which ZEN reduced GC proliferation, BIM, and FasL mRNA expressions were assessed after treating GCs with ZEN [Fig. 4(C)]. At ZEN doses of 20, 40, 80,120, and 160 lM, the BIM mRNA levels were increased by 3.26 6 0.89, 3.05 6 1.03, 16.27 6 6.34, 20.95 6 6.75, and 55.08 6 8.38 fold, respectively, as compared with that in controls (0 lM ZEN and DMSO). Compared to controls, FasL mRNA levels were significantly enhanced with 80, 120, and 160 lM ZEN by
Environmental Toxicology DOI 10.1002/tox
131.80 6 70.73, 172.90 6 93.59, and 466.20 6 150.70 fold, respectively (p < 0.01). To further support our hypothesis that ZEN was responsible for being the result of DNA degradation rather than bactericidal cell death in dead cells, we used the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) assay. We observed a qualitative increase in TUNEL-positive cells when analyzed by microscopy in the treatment groups while the control group (0.57 6 0.57%) showed no fluorescence single. The TUNEL-positive cells proportions were 3.34 6 0.87%, 7.97 6 1.64%, 31.64 6 10.05%, 71.50 6 9.83%, 82.02 6 4.02%, and 92.67 6 7.33% in 5, 10, 20, 40, 80, 120 lM ZEN groups, respectively [Fig. 4(D)].
ZEN EFFECTS ON OOCYTES
DISCUSSION In this study we investigated the effects of ZEN on granulosa cells and oocytes. We found that ZEN had toxic effects in terms of granulosa cell proliferation and the disruption of meiotic spindles and actin filament distributions in mouse oocytes. We first found that oocyte meiotic maturation was affected by exposure to ZEN. The polar body extrusion rates were decreased; in particular, we found that a large proportion of oocytes were arrested at the GV stage and could not enter germinal vesicle breakdown (GVBD), which indicated the low developmental competence of mouse oocytes after ZEN treatment. The results were similar with previous work which showed that ZEN had a negative effect on the quality of bovine, gilt, and porcine oocytes (Minervini et al., 2001; Alm et al., 2006; Schoevers et al., 2010). Moreover, ZEN and its metabolites also affected blastocyst formation in sows (Long et al., 1992). To investigate the cause of reduced oocyte developmental competence, we first examined the mouse oocyte actin cytoskeleton and microtubules. The cytoskeleton was critical for oocyte cytokinesis and involved extensive rearrangements of actin filaments at the plasma membrane and spindle assembly (Roth and Hansen, 2005). In oocytes, microtubules form the meiotic spindle that drives chromosome congression and segregation, while actin filaments regulate meiotic spindle movements and initiate cytokinesis for small polar body extrusion (Sun and Kim, 2013). Moreover, they also participate in the formation of oocyte polarity (Sun et al., 2011b; Yi and Li, 2012). Thus, cytoskeletal integrity ensures complete oocyte competence, and oocyte cytoskeletal integrity may critically affect the fates of oocyte maturation and embryo development (Eichenlaub-Ritter et al., 2002; Luvoni et al., 2012). We demonstrated that spindle morphology was aberrant and actin filament distributions were disrupted with ZEN treatment. The effect of ZEN on the spindle formation might be direct, and this was confirmed by the disrupted localization of g-tubulin; while the aberrant actin expression was confirmed by the abnormal distributions of the actin related proteins profilin1, ROCK and mDia1, which indicated that the damage to oocyte quality induced by ZEN might be caused by disrupting cytoskeletal integrity. Our results showed that the disruption of cytoskeletal integrity might be one reason for the low oocyte developmental competence. Another phenotype we observed was that the distribution of cortical granules (CGs) was abnormal after ZEN treatment. CGs are produced by Golgi complexes and remodel the embryo surface after fertilization by exocytosis (Kimura and Kimura, 2012). Previous results showed that actin microfilament disruption affected cortical granules migration (Liu et al., 2010), and a disruption of cortical granule-free domains (CGFD) is characteristic of oocyte polarity loss (Sun et al., 2011a). Therefore, our results indicated that
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exposure to ZEN might affect oocyte polarity and subsequent fertilization. We next examined the effects of ZEN on granulosa cells. The granulosa cell apoptosis increased in a dose-dependent manner after ZEN treatment. This result was similar to those in recent studies that showed that ZEN induced porcine granulosa cell apoptosis through a mitochondrial signaling pathway (Zhu et al., 2012), and identical results were also found in somatic cells: ZEN was reported causing DNA strand breaks in HEK293 cells (Gao et al., 2013), and induces sister chromatin exchange and chromosomal aberration in CHO cells (Hassen et al., 2007). ZEN is responsible for DNA degradation rather than bactericidal cell death in dead cells. Granulosa cells are critical for the mammalian oocyte maturation, the apoptosis of granulosa cells might be another reason for the reduced competence of mouse oocyte maturation. In summary, we demonstrated that ZEN affected the proliferation of mouse granulosa cells and oocyte meiotic maturation using an in vitro model. The authors thank Wenxing Sun and Ming Shen for helpful advice.
REFERENCES Alm H, Br€ussow K-P, Torner H, Vanselow J, Tomek W, D€anicke S, Tiemann U. 2006. Influence of Fusarium-toxin contaminated feed on initial quality and meiotic competence of gilt oocytes. Reproductive Toxicol 22:44–50. Balaban B, Barut T, Urman B. 2012. Assessment of Oocyte Quality. Practical Manual of In Vitro Fertilization, Springer. Chapter 13, 105–119. Eichenlaub-Ritter U, Shen Y, Tinneberg H-R. 2002. Manipulation of the oocyte: Possible damage to the spindle apparatus. Reproductive Biomed Online 5:117–124. Fowler PA, Bellingham M, Sinclair KD, Evans NP, Pocar P, Fischer B, Schaedlich K, Schmidt J-S, Amezaga MR, Bhattacharya S. 2012. Impact of endocrine-disrupting compounds (EDCs) on female reproductive health. Mol Cell Endocrinol 355:231–239. Gao F, Jiang L-P, Chen M, Geng C-Y, Yang G, Ji F, Zhong L-F, Liu X-F. 2013. Genotoxic effects induced by zearalenone in a human embryonic kidney cell line. Mutat Res/Genetic Toxicol Environm Mutagenesis 755:6–10. Hassen W, Ayed-Boussema I, Oscoz AA, De Cerain Lopez A, Bacha H. 2007. The role of oxidative stress in zearalenonemediated toxicity in Hep G2 cells: Oxidative DNA damage, gluthatione depletion and stress proteins induction. Toxicology 232:294–302. Kimura K, Kimura A. 2012. Rab6 is required for the exocytosis of cortical granules and the recruitment of separase to the granules during the oocyte-to-embryo transition in Caenorhabditis elegans. J Cell Sci 125:5897–5905. Liu S, Li Y, Feng HL, Yan JH, Li M, Ma SY, Chen ZJ. 2010. Dynamic modulation of cytoskeleton during in vitro
Environmental Toxicology DOI 10.1002/tox
8
HOU ET AL.
maturation in human oocytes. Am J Obstetrics Gynecol 203:151.e1–151.e7.
exposed to heat shock during maturation. Reproduction 129: 235–244.
Long G, Turek J, Diekman M, Scheidt A. 1992. Effect of zearalenone on days 7 to 10 post-mating on blastocyst development and endometrial morphology in sows. Veterinary Pathol Online 29:60–67.
Schoevers E, Fink-Gremmels J, Colenbrander B, Roelen B. 2010. Porcine oocytes are most vulnerable to the mycotoxin deoxynivalenol during formation of the meiotic spindle. Theriogenology 74:968–978.
Luvoni G, Tessaro I, Apparicio M, Ruggeri E, Luciano A, Modina S. 2012. Effect of vitrification of feline ovarian cortex on follicular and oocyte quality and competence. Reproduction Domestic Animals 47:385–391.
Schoevers EJ, Santos RR, Colenbrander B, Fink-Gremmels J, Roelen BAJ. 2012. Transgenerational toxicity of Zearalenone in pigs. Reproduct Toxicol 34:110–119.
Maranghi F, Tassinari R, Moracci G, Macrı C, Mantovani A. 2008. Effects of a low oral dose of diethylstilbestrol (DES) on reproductive tract development in F1 female CD-1 mice. Reproduct Toxicol 26:146–150.
Sun SC, Xu YN, Li YH, Lee SE, Jin YX, Cui XS, Kim NH. 2011a. WAVE2 regulates meiotic spindle stability, peripheral positioning and polar body emission in mouse oocytes. Cell Cycle 10:1853–1860.
McDonnell DP, Norris JD. 2002. Connections and regulation of the human estrogen receptor. Science 296:1642–1644.
Sun SC, Kim NH. 2013. Molecular mechanisms of asymmetric division in oocytes. Microsc Microanal 19:883–897.
Minervini F, Dell’Aquila M, Maritato F, Minoia P, Visconti A. 2001. Toxic effects of the mycotoxin zearalenone and its derivatives on in vitro maturation of bovine oocytes and 17bestradiol levels in mural granulosa cell cultures. Toxicol In Vitro 15:489–495.
Sun SC, Lee SE, Xu YN, Kim NH. 2010. Perturbation of Spc25 expression affects meiotic spindle organization, chromosome alignment and spindle assembly checkpoint in mouse oocytes. Cell Cycle 9:4552–4559.
Mosmann T. 1983. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J Immunological Methods 65:55–63. Oliver W, Miles J, Diaz D, Dibner J, Rottinghaus G, Harrell R. 2012. Zearalenone enhances reproductive tract development, but does not alter skeletal muscle signaling in prepubertal gilts. Animal Feed Sci Technol 174:79–85. Pitt J, Taniwaki MH, Cole M. 2013. Mycotoxin production in major crops as influenced by growing, harvesting, storage and processing, with emphasis on the achievement of Food Safety Objectives. Food Control, Elsevier 32:205–215. Roth Z, Hansen PJ. 2005. Disruption of nuclear maturation and rearrangement of cytoskeletal elements in bovine oocytes
Environmental Toxicology DOI 10.1002/tox
Sun SC, Wang ZB, Xu YN, Lee SE, Cui XS, Kim NH. 2011b. Arp2/3 complex regulates asymmetric division and cytokinesis in mouse oocytes. PLoS One 6:e18392. Ulloa-Aguirre A, Zarinan T, Pasapera AM, Casas-Gonzalez P, Dias JA. 2007. Multiple facets of follicle-stimulating hormone receptor function. Endocrine 32:251–263. Yi K, Li R. 2012. Actin cytoskeleton in cell polarity and asymmetric division during mouse oocyte maturation. Cytoskeleton 69:727–737. Zhu L, Yuan H, Guo C, Lu Y, Deng S, Yang Y, Wei Q, Wen L, He Z. 2012. Zearalenone induces apoptosis and necrosis in porcine granulosa cells via a caspase-3-and caspase-9dependent mitochondrial signaling pathway. J Cell Physiol 227:1814–1820.
