The possible molecular mechanisms of bisphenol A action on porcine early embryonic development

Bisphenol A (BPA) is an environmental contaminant widely used in the plastic industry. BPA has been demonstrated to be an endocrine disruptor and has an adverse effect on the embryonic development of mammals. However, the mechanism of action of BPA is limited. In this study, we investigated the role and mechanism of BPA in porcine embryonic development. First, the parthenotes were treated with different concentrations of BPA. We found that blastocyst formation was impaired and the parthenotes were arrested at the 4-cell stage after treatment with 100 μm BPA. Second, ROS increased following the addition of BPA, which further caused mitochondrial damage, and cytochrome c was released from the mitochondria to induce apoptosis. The adaptive response was demonstrated through LC3 immunofluorescence staining and by assessing autophagy-related gene expression. In addition, BPA caused DNA damage through the p53-p21 signaling pathway. Thus, our results indicate that BPA displays an adverse effect on porcine early embryonic development through mitochondrial and DNA damage.


BPA exposure results in reactive oxygen species (ROS) generation. It has been demonstrated that
ROS can be generated when exposed to environmental contaminants. Oxidative stress can also occur in a biological system when exposed to environmental contaminants and be defined as an imbalance between production and depletion of ROS 18 . Therefore, in this study, ROS levels were examined after BPA exposure. As shown in Fig. 2A, the generation of ROS increased in the treatment group compared with that in the control group. The relative green fluorescence intensity that induced ROS production was significantly increased (Fig. 2B). The mRNA expression level of oxidative stress-related genes (Gpx1, Tfam, and Mnsod) was also examined. The Gpx1 and Tfam mRNA levels significantly decreased compared with that in the control group. The Mnsod expression level showed no significant variation (Fig. 2C).
Effect of BPA exposure on apoptosis. Apoptosis was examined after BPA exposure by using the TUNEL assay. The apoptotic effect was calculated as the ratio between the number of TUNEL-positive nuclei and the total cell number. As shown in Fig. 3A, more TUNEL-positive nuclei were observed in the treatment group. The ratio of the TUNEL-positive nuclei significantly increased after BPA exposure (Fig. 3B). The expression of apoptosis-related genes was also analyzed after treatment with BPA. The expression of both anti-apoptotic genes, Bcl2 and Bcl-xl, significantly reduced (Fig. 3C,D), suggesting that BPA exposure may induce apoptosis.
The release of cytochrome c from the mitochondria into the cytoplasm initiates apoptosis. The co-localization of cytochrome c and mitochondria was examined using immunofluorescence staining. The release of cytochrome c from the mitochondria is shown in Fig. 3D after exposure to BPA. As shown in Fig. 3E, the control group showed obvious co-localization of mitochondria and cytochrome c in the whole blastocyst compared with the treatment group. The spots inside of white box displayed the cytochrome c which was released from the mitochondria. More cytochrome c was released from the mitochondria in the treatment group. Therefore, BPA exposure caused the release of cytochrome c and induced the initiation of apoptosis.
BPA exposure increases P53 and P21 expression in the blastocyst. In order to detect the mechanism of the negative effects of BPA on embryonic development, the activation of the p53 pathway was examined. The activation of p53 can result in poor developmental potential 19 . Therefore, we showed that BPA treatment could cause an increased expression of p53 (Fig. 4A,B). The expression of p21, a known downstream target of p53, was also examined. The fluorescence intensity of p21 increased after embryos were treated with BPA (Fig. 4C,D). These results show that BPA exposure impaired the preimplantation embryo development through the p53-p21 pathway.
BPA exposure results in a decrease of pluripotency markers. Embryonic development is associated with the embryo quality. As the key regulator of pluripotency, OCT4 expression was examined using immunofluorescence staining. The relative fluorescent density of OCT4 decreased in the BPA-exposed group compared with that in the control group (P < 0.05) (Fig. 5A,B). To confirm the effect of BPA on pluripotency, the expression of three pluripotent-related genes was examined. As shown in Fig. 5C, the mRNA expression level of Oct4, Sox2, and Nanog significantly reduced (P < 0.05), which is consistent with the results of immunofluorescence staining. Next, we detected the differentiation where there was no difference between the control and BPA-exposed groups (Fig. 5D,E). These results indicate that BPA exposure can decrease embryo quality.
BPA exposure causes autophagy. Oxidative stress can induce cell autophagy. Therefore, the expression level of LC3 was examined after treatment with BPA. Immunofluorescence staining showed a significant increase in fluorescence intensity (Fig. 6A,B). Consistent with this, the mRNA expression level of autophagy-related genes also significantly increased (Fig. 6C).

BPA exposure causes the alternation of methylation.
To evaluate the effect of embryo exposure to BPA on epigenetic modification, the level of 5-methyl-cytosine (5mC) was examined. As shown in Fig. 7A,B, we found that 5mC was co-localized with DNA and the relative intensity decreased compared with that in the control (P < 0.05). The mRNA expression of DNA methyltransferases (Dnmt3a and Dnmt3b) was analyzed using RT-PCR. mRNA levels were found to be significantly decreased after exposure to BPA (Fig. 7C).

Discussion
People increasingly focus on the adverse effect of BPA because of its worldwide use and being detected in food and water consumed by people and animals. Accumulating evidence implicates that environmental toxicity impacts metabolism, the immune system, and reproduction. Several environmental factors that can perturb embryonic development have been reported. These factors have an effect on conditions or techniques used in assisted reproductive technologies (ART). Although the number of babies that are conceived through ART is growing, these individuals represent a relatively small percentage (1-4%) of babies born in industrialized countries 20 . Despite a number of studies have reported the toxic effect of BPA, the mechanism of BPA adverse effect is unknown. In this study, the mechanism of the negative effects of BPA on porcine early embryonic development was determined. Our results demonstrate that BPA treatment leads to the arrest of embryonic development at  the 4-cell stage. In addition, the mechanism of BPA toxicity was explored by assessing DNA damage, apoptosis, autophagy, and epigenetic modification.
Previously, some studies have shown that BPA elicits its function through estrogen-mediated pathways by binding estrogen receptors, therefore, regulating gene expression. Sometimes, BPA shows a dual effect, which can exert pro-and anti-estrogenic effects 21,22 . In the present study, we found that BPA causes the embryonic development arrest. Other previous studies have demonstrated that BPA reduced blastocyst development and metabolism in bovine and mouse embryos 17,23 . The adverse effect of BPA exposure during oocyte maturation leads to alterations in subsequent embryonic development 10 . Most studies have focused on the function of BPA and regarded it as a xenoestrogen; therefore, the mechanism of BPA is via the estrogen-mediated effect.
In this study, we found that BPA modulates oxidative stress in porcine embryonic development. In vitro produced embryo displays low quality and small scale production. The major obstacles in in vitro embryo development are the production of excessive free radicals and exposure to oxidative stress. When ROS production exceeds the antioxidant capacity of embryos, oxidative stress occurs 24,25 . The production of ROS is particular critical in early embryonic development, and excessive ROS will induce apoptosis and metabolic disorders 26,27 .  Several studies have implicated a close relationship between BPA toxicity and the generation of ROS, which results in oxidative stress in tissues 28,29 . We also showed that embryos suffered oxidative stress, which was demonstrated by the increased production of ROS and oxidative stress-related gene mRNA expression of Gpx1 and Tfam. Gpx1 plays a role in the detoxification of hydrogen peroxide, and Tfam can stabilize mitochondria and thus affects oxidative stress 30,31 .
Excessive ROS production leads to different kinds of injuries including mitochondria. In the present study, we observed that BPA causes the generation of ROS. It also resulted in mitochondrial damage, as observed by the release of cytochrome c from mitochondria. Cytochrome c is typically localized between the inner and outer membranes of mitochondria. However, during apoptosis, it is released into the cytoplasm, where it binds to apoptotic protease activating factor 1 (APAF1). The release of cytochrome c from mitochondria is, therefore, an important event in apoptosis initiation 32 . The apoptosis related genes (Bcl-2, Bcl-xl) mRNA expression confirms the effect of BPA on apoptosis The anti-apoptotic Bcl-2 members prevent mitochondrial protein release 33,34 . The result of Bcl2 and Bcl-xl mRNA expression analysis confirmed the effect of BPA on apoptosis.
BPA also can cause DNA damage, which may occur via BPA-induce ROS production in porcine parthenotes 35,36 . However, the effect of BPA exposure on DNA damage has not been extensively investigated. In this study, we found that the possible mechanism of BPA-induce DNA damage may be from the p53 pathway. DNA damage-induced phosphorylation of p53 results in the growth suppression. An increase in p53 and p21 levels were observed in BPA-treated embryos. These results suggest that BPA exposure results in DNA damage through oxidative stress.
As described above, BPA induced an increase in ROS levels, which leads to detrimental effects, such as mitochondrial and DNA damage. Generation of ROS is critical for cell survival and death, as well as autophagy. Among the various molecular components and signaling cascades related to autophagy induction, cellular redox status has been regarded as a critical mediator or regulator of autophagy 37,38 . Our results implicate that BPA-induced autophagy, which was demonstrated by the increased level of LC3 and alteration of the mRNA expression of autophagy-related genes. Autophagy is a cellular mechanism, through which cells digest organelles, part of the cytoplasm, or proteins to circumvent nutrient deprivation or accumulation of damaged proteins/organelles. The importance of autophagy has mostly been attributed to its ability to remove damaged organelles and altered proteins resulting from programmed cell death [39][40][41] . Autophagy regulation and development are closely related to cell death machinery, especially in the regulation of apoptosis. In this study, BPA exposure led to mitochondrial and DNA damage, this causes autophagy as a protective effect.
Accumulating evidence indicates that BPA treatment may alter the epigenome, including DNA methylation and histone modification. Previous studies have suggested that BPA treatment disrupts the methylation status 22,42 . The reduced expression of 5mC and Dnmt genes observed in the present study was caused by BPA exposure. DNA methylation is modulated by DNMTs, which results in the formation of 5mC. DNA methylation can correlate with gene transcription.
The embryonic developmental potential is influenced by pluripotency 43 . In addition, excessive DNA damage can induce apoptosis and decrease pluripotency-related gene expression in pigs 44 . In this study, the reduced expression of pluripotency-related genes might be induced by DNA damage and apoptosis.
In conclusion, the toxic effect of BPA on porcine embryonic development was confirmed in this study. BPA exposure to embryos caused increased levels of ROS resulting in oxidative stress. The oxidative stress caused mitochondrial and DNA damage, which leads to autophagy. In addition, BPA exposure can result in the alteration of DNA methylation, which may affect the health of offspring (Fig. 8).

Materials and Methods
All chemicals used in this study were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise indicated.
Oocyte collection, in vitro maturation, and embryo culture. Ovaries from prepubertal gilts were obtained from a local slaughterhouse, maintained in saline at 37 °C, and transported to the laboratory. Follicles that were 3-6 mm in diameter were aspirated. Cumulus-oocyte complexes (COCs) that were surrounded by more than three layers of cumulus cells were selected for culture. COCs were isolated from follicles and washed three times with TL-HEPES. COCs were cultured in tissue culture medium 199 (TCM 199) with 10% porcine follicular fluid, 0.1 g/L sodium pyruvate, 0.6 mM L-cysteine, 10 ng/mL epidermal growth factor, 10 IU/mL luteinizing hormone, and 10 IU/mL follicle stimulating hormone at 38.5 °C for 44 h in a humidified atmosphere of 5% CO 2 .
After maturation, cumulus cells were removed with 0.1% hyaluronidase and repeated pipetting. For activation of parthenogenesis, oocytes with polar bodies were selected. They were activated by two direct current pulses of 1.1 kV/cm for 60 µs and then incubated in porcine zygote medium (PZM-5) containing 7.5 μg/mL of cytochalasin B for 3 h. Finally, approximately 90 embryos were cultured in PZM-5 for 8 days at 38.5 °C in a humidified atmosphere of 5% CO 2 . On the fifth day, 10% fetal bovine serum was added to the medium. To determine the effect of BPA on early porcine embryonic development, BPA was added to the medium after activation at final concentrations of 100 or 200 μM. The 100 μM concentration was used in the following experiments as it represents the minimum concentration that induces an effect on blastocyst formation.
Reactive oxygen species (ROS) staining. Blastocysts (n = 39, 3 replicates) were incubated for 15 min in IVC medium containing 10 µM 2′,7′-dichlorodihydrofluorescein diacetate (H 2 DCF-DA) at 37 °C. After incubation, blastocysts were washed three times with IVC medium and transferred to PBS drops covered with paraffin oil in a polystyrene culture dish. The fluorescent signal was captured using an epifluorescence microscope (Nikon Corp., Tokyo, Japan). The fluorescence intensity in the control group was arbitrarily set at 1, and the fluorescence intensities in the treatment groups were then measured and expressed as relative values for the control group.
Terminal deoxynucleotidyl transferase-mediated 2′-deoxyuridine 5′-triphosphate (dUTP) nick-end labeling (TUNEL) assay. After the embryos had been treated with BPA, the blastocysts were collected. The blastocysts (n = 36, 3 replicates) were then fixed in 3.7% paraformaldehyde for 15 min at room temperature and subsequently permeabilized by incubation in 0.5% Triton X-100 for 30 min at 37 °C. The embryos were incubated with fluorescein-conjugated dUTP and the terminal deoxynucleotidyl transferase enzyme (In Situ Cell Death Detection Kit, Roche; Mannheim, Germany) for 1 h at 37 °C, and then washed three times with PBS/ PVA. Embryos were treated with Hoechst 33342 for 5 min, washed three times with PBS/PVA, and mounted onto glass slides. Images were captured using a confocal microscope (Zeiss LSM 710 META, Jena, Germany).
Immunofluorescence and confocal microscopy. Embryos (n = 38, 3 replicates) were fixed in 3.7% paraformaldehyde for 20 min at room temperature, permeabilized with PBS/PVA containing 0.5% Triton X-100 at 37 °C for 1 h, and then incubated in PBS/PVA containing 1.0% bovine serum albumin at 37 °C for 1 h. Subsequently, the embryos were incubated overnight at 4 °C with anti-LC3 (ab58610, 1:100; Abcam, Cambridge, UK), anti-cytochrome C (ab110325, 1:100; Abcam), anti-p53 (sc6243, 1:100; Santa Cruz Biotech, CA, USA) and BPA exposure causes the generation of oxidative stress, which results in DNA and mitochondrial damage. Apoptosis was induced by the release of cytochrome c from the mitochondria because of mitochondrial damage. The DNA damage led to the activation of the p53-p21 pathway. In addition, the oxidative stress induced the adaptive response, autophagy.