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Comparison of the toxic effects of different mycotoxins on porcine and mouse oocyte meiosis

Background Aflatoxin B1 (AFB1), deoxynivalenol (DON), HT-2, ochratoxin A (OTA), zearalenone (ZEA) are the most common mycotoxins that are found in corn-based animal feed which have multiple toxic effects on animals and humans. Previous studies reported that these mycotoxins impaired mammalian oocyte quality. However, the effective concentrations of mycotoxins to animal oocytes were different. Methods In this study we aimed to compare the sensitivity of mouse and porcine oocytes to AFB1, DON, HT-2, OTA, and ZEA for mycotoxin research. We adopted the polar body extrusion rate of mouse and porcine oocyte as the standard for the effects of mycotoxins on oocyte maturation. Results and Discussion Our results showed that 10 μM AFB1 and 1 μM DON significantly affected porcine oocyte maturation compared with 50 μM AFB1 and 2 μM DON on mouse oocytes. However, 10 nM HT-2 significantly affected mouse oocyte maturation compared with 50 nM HT-2 on porcine oocytes. Moreover, 5 μM OTA and 10 μM ZEA significantly affected porcine oocyte maturation compared with 300 μM OTA and 50 μM ZEA on mouse oocytes. In summary, our results showed that porcine oocytes were more sensitive to AFB1, DON, OTA, and ZEA than mouse oocytes except HT-2 toxin.


INTRODUCTION
Mycotoxins are secondary metabolites produced by fungi, while the most agriculturally common mycotoxins known today include aflatoxins (AF), deoxynivalenol (DON), T-2, ochratoxin A (OTA), zearalenone (ZEA) (Grajewski et al., 2012). These mycotoxins have multiple toxic effects on human and animal health in a very low dose, which draws worldwide attention.
Aflatoxins are the mycotoxins which widely exist in corn-based animal feed. Considering the toxic potency and carcinogenic action, Aflatoxin B1 (AFB1) is the most important AF (Kew, 2013). It causes multiple effects including mitochondrial permeability transition, DNA damage (Shi et al., 2015), oxidative stress (Singh, Maurya & Trigun, 2015), apoptosis (Peng et al., 2016), the defects in skeletal muscle development in different models which contained 75 mg/ml of penicillin, 50 mg/ml of streptomycin, 0.5 mg/ml of FSH, 0.5 mg/ml of LH, 10 ng/ml of the epidermal growth factor, and 0.57 mM cysteine was used for oocyte maturation. M2 and M16 culture medium were from Sigma-Aldrich (Merck; St. Louis, MO, USA).

Oocytes collection and culture
We followed the guidelines of Animal Research Institute Committee of Nanjing Agricultural University to conduct the experiments (SYXK-Su-20170007). Germinal vesicle-intact oocytes of mice that obtained from the ovaries of three-to five-week old ICR mice were collected in M2 medium and cultured with M16 medium (Sigma Chemical Co., St. Louis, MO, USA) under the paraffin oil. These oocytes of mice were placed at 37 C with 5% CO 2 for 12 h to observe the polar body extrusion.
Ovaries of Duroc pigs were purchased at a local slaughterhouse of Feng Yong Food Industry. After slaughter, ovaries were placed in a thermos bottle which contained 0.9% physiological saline and then delivered to our laboratory within 2 h. The temperature of the thermos bottle was close to 38 C. Once the ovaries were delivered, they were washed with sterile saline. We aspirated follicular fluids from 3 to 6 mm antral follicles with a 10 ml disposable syringe and an 18 G needle. Cumulus oocyte complexes (COCs) with intact and compact cumulus were selected for maturation. These oocytes were placed at 38.5 C with 5% CO 2 for 44 h to observe the polar body extrusion.
Toxin treatment AFB1, DON, HT-2, OTA, and ZEA were dissolved and stored at 50 mM in DMSO and then diluted into different concentrations with M199 or M16 maturation medium. The GV oocytes were then cultured with these mycotoxins to analyze the maturation rate by the polar body extrusion index. The same quantity of DMSO was added in the control group.

Statistical analysis
Data are presented as means (n = 3). The concentration-response curves were made by GraphPad Prism 5. At least three biological replicates were used for each analysis. Each replicate was done by an independent experiment at the different time. Results are given as means ± SEM, and two groups were compared by student t-test. A p-value of <0.05 was considered significant.

Effects of DON on mouse and porcine oocyte maturation
We next examined the effects of DON on mouse and porcine oocytes. Mouse oocytes were cultured for 12 h with 1, 2, 3, and 4 mM DON. Our results showed that DON affected mouse oocyte maturation. The average polar body extrusion rate of the control group was 76.73 ± 3.24% (n = 162) ( Fig. 2A), when mouse oocytes cultured with 1 mM AFB1, there was no significantly difference between the control group and 1 mM group (76.01 ± 2.35% n = 139, p > 0.05). While the rate of polar body extrusion in mouse oocytes was significantly decreased with 2 mM DON treatment (44.38 ± 4.87%, n = 173, p < 0.05), 3 mM DON treatment (16.30 ± 4.00%, n = 199, p < 0.001), 4 mM DON treatment (2.28 ± 0.69%, n = 189, p < 0.001), compared with the control group. We cultured the porcine oocytes for 44 h with 0.5, 1, 2, 3 mM DON. The polar body extrusion rate was 72.05 ± 2.6% (n = 195) in the control group of porcine oocytes, which was close to mouse oocytes. At the concentration of 0.5 mM, the control group and 0.5 mM treatment group (68.42 ± 4.55% n = 159, p > 0.05) showed no significant difference. However, the rate of polar body extrusion in porcine oocytes was significantly decreased with 1 mM DON treatment (46.29 ± 3.89%, n = 176, p < 0.05), 2 mM DON treatment (17.02 ± 4.87%, n = 145, p < 0.01), 3 mM DON treatment (3.29 ± 1.81%, n = 132, p < 0.001) (Fig. 2B). To compare the sensitivity of mouse and porcine, we also analyzed the rate of same concentrations. The rates of control groups were close to each other, while at the concentrations of 1 mM (p < 0.001), 2 mM (p < 0.01), 3 mM (p < 0.01), polar body extrusion rates of mouse oocytes and porcine oocytes showed significant difference (Fig. 2C), indicating that compared with mouse oocytes, porcine oocytes were more sensitive to DON.

Effects of OTA on mouse and porcine oocyte maturation
We next examined the effects of OTA on mouse and porcine oocytes. Mouse oocytes were cultured for 12 h with 200, 300, 400, and 600 mM OTA. The average polar body extrusion rate of the control group was 80.23 ± 3.87% (n = 169) (Fig. 4A) (Fig. 4B).
With the same concentration of OTA treatment, the rate of porcine oocyte polar body extrusion was lower than mouse oocytes (Fig. 4C), indicating that compared with mouse oocytes, porcine oocytes were more sensitive to OTA. Effects of ZEA on mouse and porcine oocyte maturation The last we examined was the effects of ZEA on mouse and porcine oocytes. Mouse oocytes were cultured for 12 h with 10, 50, 100, and 200 mM ZEA. The average polar body extrusion rate of the control group was 81.29 ± 6.06% (n = 155) (Fig. 5A), when the concentration was 10 mM, the average rate of MII oocytes was 74.52 ± 4.92% (n = 154) which showed no significant difference with the control groups (p > 0.05). While the rate of polar body extrusion in mouse oocytes was significantly decreased with 50 mM ZEA treatment (54.35 ± 3.9%, n = 128, p < 0.05), 100 mM ZEA treatment (26.23 ± 8.00%, n = 150, p < 0.05), 200 mM ZEA treatment (0.00 ± 0.00%, n = 132, p < 0.01). For porcine oocytes, the average polar body extrusion rate of the control groups was 77.85 ± 9.51% (n = 175), while the 5 mM ZEA groups (60.45 ± 1.65%, n = 199, p > 0.05) showed no significant differences with the control groups. However, the rate of polar body extrusion in porcine oocytes was significantly decreased with the 10 mM groups (51.42 ± 2.73%, n = 190, p < 0.05), 30 mM ZEA treatment (19.10 ± 4.49%, n = 147, p < 0.05) and 50 mM ZEA treatment (4.08 ± 4.08%, n = 148, p < 0.01) (Fig. 5B). Our results showed that with the same concentration of ZEA treatment, the rate of porcine oocyte polar body extrusion was lower than mouse oocytes. Control groups of mouse and porcine oocytes showed no significant difference (p > 0.05). However, there were significant difference between mouse oocytes and porcine oocytes at 10 mM (p < 0.01) and 50 mM (p < 0.001) (Fig. 5C), indicating that compared with mouse oocytes, porcine oocytes were more sensitive to ZEA.

DISCUSSION
In the present study we used the polar body extrusion as the index for oocyte maturation to compare the sensitivity of mouse and porcine oocytes to AFB1, DON, HT-2, OTA, and ZEA. Our results provided the basic database for the mycotoxin on oocyte studies. Mycotoxins are shown to affect human and animal health from multiple aspects, such as immune system, micro-organisms. And recent years, the toxicity of mycotoxins on reproductive system especially on oocytes and sperms were reported. Our previous work found that when 50 mM AFB1 affected COCs growth, especially the polar body extrusion was significantly reduced in porcine oocytes (Liu et al., 2015). However, 10 mM AFB1 significantly increased the proportion of sperm with fragmented DNA in mice (Komsky-Elbaz, Saktsier & Roth, 2018). This indicated that even in the reproductive system, the sensitivity of different cell types or animal models to the mycotoxins is different. Our results showed that 10 mM AFB1 affected porcine oocyte maturation instead of 50 mM AFB1 in mouse oocytes.
A total of 2 mM DON was shown to affect the formation of the meiotic spindle in mouse oocytes (Lan et al., 2018). While a recent study showed that 10 mM DON affected the morphology of pig ovaries with an ex vivo approach (Gerez, Desto & Bracarense, 2017). Our recent study also showed that 3 mM DON exposure altered autophagy/apoptosis and epigenetic modifications in porcine oocytes . In the present study our results showed that 1 mM DON already affected porcine oocyte maturation instead of 2 mM DON in mouse oocytes, which showed similar sensitivity pattern to AFB1. However, HT-2 had different sensitivity pattern compared with AFB1 and DON. HT-2 toxin was shown to affect cytoskeletal dynamics, apoptosis/autophagy, oxidative stress, and epigenetic modifications in mouse oocytes . Our results showed that 10 nM HT-2 affected mouse oocyte maturation while the similar results only occurred at 50 nM HT-2 exposure for porcine oocytes. Further study is still needed to explore the toxic effects of HT-2 toxin in different reproductive cell types like cumulus cells and sperm.
A recent study indicated that OTA significantly impaired oocyte maturation, in vitro fertilization (IVF) rates and inhibited embryonic development in vitro, because OTA could induce caspase-dependent apoptosis with in vivo model (Huang & Chan, 2016). 1-10 mM OTA in the drinking water was adopted in this study. Our results showed that 5 mM OTA affected porcine oocyte maturation instead of 300 mM OTA in mouse oocytes. The big difference for the OTA in mouse oocyte between in vivo and in vitro model needs more study to explain. For ZEA, at the concentration of 30 mM, ZEA was shown to affect porcine oocyte maturation and embryonic development through oxidative stress, autophagy and early apoptosis (Komsky-Elbaz, Saktsier & Roth, 2018); and for mouse oocytes, it affected oocyte quality by altering the epigenetic modification levels (Zhu et al., 2014). Our results showed that 10 mM ZEA affected porcine oocyte maturation instead of 50 mM ZEA in mouse oocytes, which showed similar concentration pattern with AFB1 and DON.

CONCLUSION
In all, our results showed that these five mycotoxins all affected mouse and porcine oocyte quality, however, different sensitivity patterns between mouse and porcine oocytes were found. Generally porcine oocytes were more sensitive to AFB1, DON, OTA, ZEA compared with mouse oocytes except HT-2. Our results provided a basic database for the further studies on mammalian oocytes.