Abstract
T-2 toxin is a mycotoxin produced by phytopathogenic fungi of the Fusarium genus and has many well-studied deleterious effects on mammalian cells and reproductive tract. Despite the wide scale studies, the effects on preimplantation stage embryos are lacking. The aim of our study was to investigate the impact of T-2 on the cleavage stage of mouse embryos with regard to development to blastocysts and nuclear chromatin status.
Six-weeks-old BDF1 female mice were superovulated and placed together overnight with mature males. Zygotes were flushed 20 h after human chorionic gonadotropin injection and divided randomly into treated (supplemented with 0.5, 0.75, and 1 ng/ml T-2) and nontreated (control) groups. Embryos were cultured in vitro for 96 h. Developmental stage was evaluated in the 72nd- and 96th-h for assessment of development dynamics. At the end of culture period, blastocysts from treated and control groups with normal morphology were selected for nuclear chromatin analysis. Blastocysts were categorized (grade A, B, and C) depending on the proportion of blasomeres with micronuclei and/or lobulated nuclei.
Our data show significant decrease in the proportions of blastocysts in the 0.75 and 1 ng/ml toxin-supplemented groups compared with the control group. Blastocyst rate did not differ in embryos treated with 0.5 ng/ml T-2 but 24 h delay was found in blastocoel formation in all the treated groups. Only grade A (21.1%) and B (78.9%) blastocysts were found in low-toxin-contaminated group similar to the control ones (50–50%). Grade C embryos appeared in the 0.75 ng/ml (10%) treated group and the rate increased significantly (33.3%) in the highest contaminated group.
T-2 mycotoxin has a harmful effect on early embryo development which results in decreased blastocyst proportion, delayed blastulation, and increased rate of chromatin damage.
Introduction
T-2 toxin (T-2) is a type A trichothecene mycotoxin produced by phytopathogenic fungi of the Fusarium genus (mainly by Fusarium sporotrichoides and Fusarium poae), which can infect crop plants such as wheat, barley, and rice in the field and/or during storage (Glenn, 2007). T-2 has been implicated in serious poisoning episodes affecting humans and farm animals and is considered a significant toxicological hazard (Sudakin, 2003; Wannemacher and Wiener, 1997).
Many studies have shown the adverse effect of T-2 mycotoxin on physiological processes of different types of cells. Trichothecenes are lipophilic compounds that can pass into the cell and after binding to the peptydil transferase, the protein synthesis and indirectly the DNA and RNA syntheses are inhibited (Thomson and Wannemacher, 1990). Oxidative stress is also involved in the toxicities of trichothecene mycotoxins including T-2 toxin. The T-2 toxin-induced oxidative stress may cause DNA damage and apoptosis in the cell (Chaudhari et al., 2009; Wu et al., 2011). Immunosuppressive effect of the toxin was also found in both in vivo and in vitro studies (Gutleb et al., 2002). It is well documented that T-2 can cause reproductive disorders both in male and female individuals. These abnormalities are examples for retarded ovulation and decreased progesterone production (Huszenicza et al., 2000), disrupted corpus luteum maturation (Ványi et al., 1995), inhibited granulosa cell proliferation (Caloni et al., 2009), and reduced sperm motility (Kovács et al., 2011).
Numerous evidences were found about the fetotoxic effect of T-2 mycotoxin. The toxin passes through the placenta and may adversely affect the development of fetus (Lafarge-Frayssinet et al., 1990). Thymus atrophy was observed after T-2 toxin exposure in pregnant mice (Holladay et al., 1993). The apoptosis-inducing effect of the toxin was shown in developing fetal central nervous system (Ishigami et al., 1999). Rousseaux and Schiefer (1987) reported bone malformation, absence of bones, and wavy and fused bones in mouse offspring following T-2 mycotoxin administration. Sehata et al. (2004) found abnormalities in the blood–brain barrier and fetal brain lesions in rats.
Despite the wide scale studies about the toxin, the effect of T-2 and the mode of action how it influences the early embryo development still remains unclear. The aim of our study was to investigate embryo toxicity of T-2 via the in vitro development of preimplantation mouse embryos and its effect on the nuclear chromatin status.
Methods
Embryo recovery and in vitro culture
Procedures with animals were performed following good veterinary practice for animal welfare according to Hungarian national laws in force. The protocol of the animal experiment was approved by the ethical committee of the Faculty of Veterinary Science, Szent Istvan University of Budapest (40/2013.II.14.). Six-weeks-old BDF1 mice were kept under 12-h light/12-h dark schedule at a temperature of 21°C. Superovulated (Day 1: 7.5 IU equine chorionic gonadotropin intraperitoneally (i.p.); Day 3: 7.5 IU human chorionic gonadotropin (hCG) i.p. (Alvetra und Werfft, Austria)) female mice were placed together overnight with mature males after hCG treatment. One-cell zygotes were obtained 20 h after hCG treatment and transferred to culture medium (Cleavage Medium, Cook Medical, Roskilde, Denmark) supplemented with T-2 mycotoxin (Sigma Aldrich, St Louis, Missouri, USA) in different concentrations (0.5, 0.75, and 1 ng/ml). The embryos obtained were pooled and randomly divided into treated (medium with different concentrations of toxin) and control (medium without toxin) groups. Toxin concentrations were based on our previous studies (Somoskői et al., 2012). Embryos were cultured for 96 h at 37.5°C with 6.5% carbon dioxide and maximal humidity in air. Average embryo number was 19.6/group/repeat.
Embryo development was examined on the 72nd- and 96th-h of culture to assess developmental dynamics. Embryos were classified as uncompacted and/or damaged, morula, and blastocyst (early, mid, and late/expanded stage).
Detection of the nuclear chromatin status
After 96 h, the nuclear chromatin status of blastocysts was examined. To evaluate nuclear chromatin status, embryos were stained with 2.5 μg/ml 4′,6-diamidino-2-phenylindole (DAPI) in Dulbecco’s phosphate-buffered saline (Sigma Aldrich), kept in 4% paraformaldehyde at 4°C in the dark until observation and mounted on microscope slides for microscopy. Nuclear chromatin was observed under a Nikon A1 (Japan) confocal microscope equipped with DAPI filter set on 400× magnification. Image analysis was performed with Image J software (NIH, Bethesda, Maryland, USA). Embryos were classified as normal (grade A) when the presence of a regular-shaped nucleus inside each blastomere was observed. The formation of micronuclei and lobulated nuclei was considered as signs of chromatin damage. Embryos showing affected blastomeres less than 20% were classified as grade B, and embryos with more than 20% affected blastomeres were classified as grade C (Martino et al., 2013).
Statistical analysis
Data were analyzed with R v3.0.0 software. Rates of blastocysts on the 72nd and 96th h and chromatin status were compared between control and treated groups by χ2 test. Analysis of variance with Tukey’s test was used to compare blastocyst proportion after all repeats. Differences at p < 0.05 were considered significant.
Results
After 72 h, 61.5% of control embryos reached the blastocyst stage, whereas no blastocyst was found in any of the treated groups. Then, 24 h later blastocysts appeared in all groups however with significantly lower proportion in the high-toxin-contaminated groups (p < 0.001; Figure 1).
Development dynamics in control and treated groups. χ 2 test: a,b p < 0.001.
All the treated blastocysts showed stage-specific morphology (Fujimori, 2010) and considered normal as the control ones.
After 96 h of culture, the developmental stage of embryos was assessed. Table 1 shows the number and proportion of different embryo stages in the toxin-treated and control groups.
Numbers and ratios of embryos in different developmental stages after 96 h of culture in medium with and without T-2 toxin.
Data show that in the group cultured in medium supplemented with 0.5 ng/ml T-2 toxin, the blastocyst rate was 10% lower than the control group. In the groups in which the embryos were cultured in mediums enriched with 0.75 or 1 ng/ml T-2 toxin decreased blastocyst formation rates were found (by >38% and >57% compared with the control).
In each group, the proportions of blastocysts were assessed after each repeat. Data show that the blastocyst rate in the low-toxin group is similar to the control group. However, in the high-toxin-treated groups significant decrease was detected (Figure 2).
Proportion of blastocysts following 96 h in vitro culture after all replicates (six or seven replicates, 19.6 average embryo/group/repeat). Tukey’s test: a,b p < 0.05.
Statistical analysis of the obtained data shows significant negative effect of the T-2 toxin concentration on blastocyst formation (p < 0.001). In the pairwise comparison, we found significant differences in the blastocyst rates between the control and treated groups with high (0.75 and 1 ng/ml) toxin concentration. There was also significant difference in the blastocyst proportions between the high and low (0.5 ng/ml) toxin-supplemented groups. It was found that 1 ng/ml T-2 toxin contamination did not cause further significant decrease compared with 0.75 ng/ml T-2 toxin.
Nuclear chromatin status of treated and untreated control blastocysts embryos was also evaluated (Figure 3). All the examined blastocysts either in control or treated groups were morphologically normal. Only grade A and grade B embryos were found in the control and low–toxin-contaminated groups. Ten percent of the blastocysts in the 0.75 ng/ml T-2 toxin-treated group was categorized as grade C. Significant increase (33.3%) of grade C embryos was found in the group of embryos treated with the highest (1 ng/ml) toxin concentration compared with the control group. Figure 4 shows representative pictures of blastocysts with different grades.
Proportion of grades A, B, and C blastocysts in the control and treated groups. Asterisk (*) means p < 0.05 (χ 2 test) compared with the control.
Representative pictures of grades A, B, and C blastocysts. Arrowheads show micronuclei.
Discussion
Although fetotoxic effect of T-2 toxin is well documented in the advanced stage of gestation, to the best of our knowledge, no data are available on how T-2 mycotoxin affects early preimplantation stage of embryo development. Fang et al. (2012) found that T-2 induces significant increase of DNA fragmentation in 0.5 ng/ml concentration and results in significantly higher reactive oxygen species production above 1 ng/ml exposure in differentiated murine embryonic stem cells after 24 h incubation. Our findings show that the lowest inhibitory dose of T-2 toxin on mouse preimplantation embryos is 0.75 ng/ml. Embryo model is considered to be a more relevant system for assessing toxicological effects on reproduction compared to cell culture. Embryos are simultaneously cytological, and embryological test models being both few cells structures or self-determined systems and whole organisms. Based on the model, it is possible to estimate both cytotoxic and embryotoxic effects after pathogenic influence has occurred (Popov and Protasova, 2011).
The toxin concentration that we used in this study for in vitro culture of embryos can be found in human peripheral blood (0.2–1800 ng/ml) (Berek et al., 2001) and can occur if tolerable daily intake (TDI) dose (100 ng/bw/day) is ingested by a human (70 kg body weight (b.w.)) calculated with 60% absorption rate (EFSA, 2011). Our data show that in a concentration-dependent way the toxin has harmful effect on early embryo development as well as the blastulation dynamics. Whereas the normal cleavage rate is equal to control in the low-toxin-treated group, 1.5-fold concentration caused reduction to less than 40%. However, we found delayed cleavage already on the lowest (0.5 ng/ml) toxin concentration since embryos in this group reached blastocyst stage 24 h later than in the control group.
Micronuclei are small fragments of chromatin separated from the main cell nucleus which are evidence of chromosome breaking or mitotic spindle dysfunction and are frequently produced by genotoxic agents (Heddle et al., 1991). High proportion of micronuclei present poor implantation potential (Jackson et al., 1998) and has been associated with developmental arrest (Moriwaki et al., 2004) and apoptosis (Hnida et al., 2004). Our results show that blastocysts denoted as morphologically normal can contain damaged chromatin in trichothecene-contaminated environment. It may cause disturbed implantation following high toxin impact.
Our data contribute to reveal the mode of action of T-2 mycotoxin, which is the most toxic trichothecene, has an established TDI of 100 ng/kg b.w. for the sum of T-2 and HT-2 toxins (EFSA, 2011) and still has only recommendation values on the presence in cereals and cereal products within the European Union (EC, 2013).
Footnotes
Acknowledgments
We are grateful to Viktor Plattner for performing the confocal microscopy analysis, Zsuzsa Keresztes for animal housing and hormonal treatment, and László Kametler for preparation of toxin solutions.
Conflict of interest
The authors declared no conflicts of interest.
Funding
This work was supported by the Hungarian Scientific Research Fund (OTKA-100810); TÁMOP 4.2.2.A-11/1/KONV-2012-0053; 17586-3/2013/TUDPOL; and NKB-4533/53/2013.