Oocyte exposure to ZnO nanoparticles inhibits early embryonic development through the γ-H2AX and NF-κB signaling pathways

The impacts of zinc oxide nanoparticles on embryonic development following oocyte stage exposure are unknown and the underlying mechanisms are sparsely understood. In the current investigation, intact nanoparticles were detected in ovarian tissue in vivo and cultured cells in vitro under zinc oxide nanoparticles treatment. Zinc oxide nanoparticles exposure during the oocyte stage inhibited embryonic development. Notably, in vitro culture data closely matched in vivo embryonic data, in that the impairments caused by Zinc oxide nanoparticles treatment passed through cell generations; and both gamma-H2AX and NF-kappaB pathways were involved in zinc oxide nanoparticles caused embryo-toxicity. Copper oxide and silicon dioxide nanoparticles have been used to confirm that particles are important for the toxicity of zinc oxide nanoparticles. The toxic effects of zinc oxide nanoparticles emanate from both intact nanoparticles and Zn2+. Our investigation along with others suggests that zinc oxide nanoparticles are toxic to the female reproductive system [ovaries (oocytes)] and subsequently embryo-toxic and that precaution should be taken regarding human exposure to their everyday use.

Austin et al. found that intravenous injection of Ag NPs (10 nm) into pregnant mice resulted in notable silver Research Paper www.impactjournals.com/oncotargetaccumulation in the maternal liver, spleen, and visceral yolk sac, and might suppress embryonic growth [1].Parivar et al. observed that TiO 2 NPs caused significant changes in chondrocytes in the following developmental stages: resting, proliferating, hypertrophy, degenerating, perichondrium, and mesenchymal cells [9].Park et al. investigated the effects of chitosan nanoparticles on mouse embryonic development, and indicated that they lowered the expression of both trophectoderm-associated genes and pluripotent marker genes.Yamashita et al. found that SiO 2 and TiO 2 nanoparticles caused pregnancy complications in mice with both types being found in the placenta, fetal liver, and fetal brain [63].Hong et al. found significant reductions in fetal weights along with an increase in abnormalities after administration of ZnO NPs at a rate of 400 mg/kg/day; however, no significant difference was found in the Zn content of fetal tissue between the control and 400 mg/kg/day groups [18,19].Tsyganova et al. reported that Au NPs penetrated the rat placental barrier in vivo; however, no morphological changes took place in the liver, kidneys, spleen, and brain of fetuses [30].Prasek et al. found that Pt NPs did not inhibit the growth and development of chicken embryos; however, it did induce apoptosis and cause a reduction in the number of proliferating cells in brain tissue [44].
In almost all published articles based on studies of nanoparticle embryo-toxicity, the embryos were tested directly (fertilized eggs of aquatic animals, or chickens) or indirectly (pregnant mouse or rat).
Although ZnO NPs are useful and novel material for a broad range of aspects, many reports have indicated that they may have adverse effects on organisms [72][73][74][75][76][77] and specifically on the reproductive systems [78,79].Nanoparticle safety, however, is not fully understood, particularly the effects and underlying mechanisms of ZnO NPs on embryonic development following oocyte stage exposure.Our results demonstrated that ZnO NPs inhibited embryo development during oocyte stage exposure, and that the γ-H2AX and NF-κB pathways might be crucial in this inhibition.Our investigation, along with others, suggests that ZnO NPs are toxic to female reproductive systems [ovaries (oocytes)] and are subsequently embryo-toxic.Precautions should therefore be taken in situations of human exposure.

Characterization of ZnO, CuO, and SiO2 NPs
ZnO, CuO, and SiO 2 NPs were manufactured by Beijing DK Nano Technology Co. LTD (Beijing, China).The ultra-structure of ZnO NPs and their characterization in cells and tissues have been published in our recent articles [Supplementary Figure 1] [80,81].Morphologically, the ZnO NPs were nearly spherical with a milky white color.The size, surface area, and density were approximately 30 nm, 50 m 2 /g, and 5.606 g/cm 3 , respectively (Supplementary Figure 1).The hydrodynamic diameter, polydispersity index and zeta potential for ZnO NPs in phosphate buffered saline (PBS) was 148 ± 26 nm, 0.089 μ2/T 2 , or −27.6 ± 2.3 mV respectively.The morphology and size of CuO and SiO 2 NPs were determined using transmission electron microscopy (TEM) and X-ray Diffractometer (Figure 1).The CuO NPs were nearly spherical and black in color.The size, surface area, and density were approximately 30 nm, 13.1 m 2 /g, and 6.4 g/cm 3 , respectively (Figure 1A and 1B).The SiO 2 NPs were nearly spherical and white in color.Their size, surface area, and density were approximately 30 nm, 600 m 2 /g, and 2.4 g/cm 3 , respectively (Figure 1C and 1D).The hydrodynamic diameter, polydispersity index and zeta potential for CuO NPs in phosphate buffered saline (PBS) was 189 ± 42 nm, 0.072 μ2/T 2 , or −43.2 ± 1.7 mV respectively.The hydrodynamic diameter, polydispersity index and zeta potential for SiO 2 NPs in phosphate buffered saline (PBS) was 137 ± 31 nm, 0.104 μ2/T 2 , or −25.4 ± 2.1 mV respectively.

Inhibition of chicken embryonic development by ZnO NPs
Previously, we found that 10-200 mg/kg (diet) ZnO NPs or 10-200 mg/kg (diet) ZnSO 4 treatments did not decrease hen body weight or egg production [80].Intact NPs were detected in ZnO NP treated hen ovarian tissues (Supplementary Figure 1).ZnO NP or ZnSO 4 treatments had little effect on Zn content in ovarian tissues compared to control treatments (Supplementary Figure 2).The major difference between ZnO NPs and ZnSO 4 treatments was that the ZnO-NP-200 mg/kg treatment significantly decreased egg yolk lipid content when compared with the ZnSO 4 -200 mg/kg treatment [80].Therefore, ZnSO 4 -200 mg/kg and ZnO-NP-200 mg/kg treatments were used in the current investigation.The concentrations of ZnO NPs or ZnSO 4 used in our studies were based on the diet.If the concentration of 200 mg/kg of diet was calculated based on animal body weight (BW), it was calculated to be around 20 mg/kg BW.Therefore, the concentration was lower than that used in other embryo-toxic studies (200-400 mg/kg BW) [18,19].
After a 24-wk trial, hens were artificial inseminated.Before hatching, 200 eggs per treatment were used to determination the fertilization rate; this was approximately 98% and it was the same for control, ZnSO 4 -200 mg/kg and ZnO-NP-200 mg/kg treatments (Figure 2A).However, 7 d after hatching, ZnO-NP-200 mg/kg increased embryonic developmental failure rate (lethality) by 15% when compared with the ZnSO 4 -200 mg/kg treatment or control, which suggested that the ZnO-NP-200 mg/kg treatment inhibited chicken embryonic development (Figure 2B).www.impactjournals.com/oncotarget

Blockage of γ-H2AX and NF-κB pathways by ZnO NPs in chicken embryos
In order to explore the underlying mechanism of ZnO NP inhibition of chicken embryonic development, the protein levels of many pathways were investigated in 3 d and 5 d embryos.It was found that the protein level of γ-H2AX in embryos was significantly decreased by the ZnO-NP-200 mg/kg treatment (Figure 2C).Phosphorylation enzymes were responsible for the de-phosphorylation of γ-H2AX; therefore, these phosphorylation enzymes were subsequently monitored.It was found that the protein levels of phosphorylation enzymes PP4C, PP4R3β, PP4R2, PP2ACα, and PP2A in embryos were increased by ZnO NPs treatment (Figure 2D).Another important factor, NF-κB, was also reduced by ZnO NPs (Figure 2E).ZnO NPs decreased both NF-κB and γ-H2AX protein levels, which suggested that this treatment might decrease cell proliferation or increase apoptosis.Protein levels of PCNA and apoptosis markers were analyzed by WB.ZnO NPs treatment dramatically reduced PCNA protein levels and increased caspase-8 in chicken embryos (Figure 2F).

Disturbance in the γ-H2AX pathway due to ZnO NPs in ovarian cells (CKO-K1 cells)
To verify the phenomenon seen in embryos, hamster ovarian cells (CHO-K1) were cultured and treated with ZnSO 4 -12.5 μg/mL or ZnO-NP-12.5 μg/mL for 24 h.After the 24 h treatment, cells [passage 0 (P 0 )] were collected and half were used for analysis and half were passaged.Following passage and 24 h of growth, the cells [passage 1 (P 1 )] were collected and half were used for analysis and half were passaged again.Subsequently, using the same method, P 2 and P 3 cells were collected.Intact NPs were found in ZnO NPs treated CHO-K1 cells (P 0 ) and confirmed by EDS (Figure 3A and 3B).The cellular concentration of Zn was increased by ZnSO 4 and ZnO NPs treatments (P 0 cells).Further, the concentration of Zn was higher in the ZnO-NP-12.5μg/mL treatment than that in the ZnSO 4 -12.5 μg/mL treatment (Figure 3C).However, these two treatments produced a similar cell growth inhibition of approximately 15% (Figure 3D).ZnSO 4 did not stimulate γ-H2AX protein level; however, ZnO NPs significantly increased the protein level of γ-H2AX after 24 h of treatment (P 0 cells).However, after the passages, γ-H2AX was dramatically reduced (Figure 3E).This indicated that ZnO NPs might cause DNA damage after 24 h treatment.Therefore, the DNA damage related proteins ATM (Ataxia telangiectasia mutated), ATR (ATM-and Rad3-related), and DNA-PK (DNA-dependent protein kinase) were monitored.It was found that ATM and DNA-PK were elevated after a 24-h ZnO NP treatment (P 0 cells; Figure 4A and 4B); however, they were not changed in P 2 or P 3 cells.ATR was not altered by these treatments.Subsequently, TUNEL assay and apoptotic markers were used to monitor apoptosis.More positive cells were detected in ZnO NPs treated cells than that in the control or ZnSO 4 treatment (Figure 5A) by TUNEL assay.Furthermore, the p53 protein level was also increased by ZnO NPs after 24 h treatment (P 0 ; Figure 5B).In P 0 cells, γ-H2AX was increased to repair the DNA damage.However, γ-H2AX was decreased gradually from P 1 to P 3 cells to lower than that in the control treatment, which indicated that γ-H2AX itself might also be suppress by ZnO NPs.Phosphorylation enzymes were also determined and it was found that PP4C and PP6C were gradually elevated from P 1 to P 3 cells and PP2ACα was also increased from P 0 to P 3 cells in ZnO NPs treatment (Figure 6A-6C).The proliferation of the cells was determined by EdU assay and it was found that EdU positive cells were decreased in P 0 and P 1 cells (Figure 6D) due to ZnO NPs treatment.However, ZnSO 4 did not induce DNA damage, apoptosis rate, or γ-H2AX protein level.

Inhibition of the NF-κB pathway by ZnO NPs in CKO-K1 cells
Protein levels of NF-κB (p65) were also detected using WB.It was found that NF-κB (p65) was significantly and similarly decreased by the ZnO NPs treatment of P 0 to P 3 cells.However, ZnSO 4 did not alter NF-κB in all these passaged cells (Figure 7A).A20 (TNFAIP3) was found to be an inhibitor of NF-κB, and it was found that ZnO NPs dramatically increased A20 in all passaged cells (P 0 to P 3 ; Figure 7B).However, A20 was not altered in ZnSO 4 treated cells.The data here might suggest that ZnO NPs treatment blocked the NF-κB pathway through A20.

Impairment of γ-H2AX not NF-κB by CuO or SiO2 NPs in CHO-K1 cells
It is still not clear, and it is even controversial whether the toxic effects of ZnO NPs come from Zn 2+ or intact nanoparticles.The properties of CuO NPs and ZnO NPs are similar as they can be dissolved in acidic or neutral solutions to release ionic forms of Zn or Cu.However, SiO 2 NPs differ in that they cannot be dissolved in acidic or neutral solutions.Therefore CuO NPs and SiO 2 NPs were used to verify the impact of ZnO NPs.If CuO and SiO 2 NPs produce similar effects to ZnO NPs, this would suggest that the effects of the latter come from intact nanoparticles; if similar results are not achieved, it would suggest that the effects of ZnO NPs come from Zn 2+ .Intact CuO NPs were detected in treated cells (Figure 8A) and confirmed by EDS (Figure 8B).After a 24-h treatment, the copper content in the CuO-NP-5 μg/mL treatment was much higher than that in the control cells (Figure 8C). Figure 8E showed intact SiO 2 NPs in CHO-K1 cells after a 24-h treatment and this was confirmed by EDS (Figure 8F).The content of Si in the SiO 2 -NP-5 μg/mL treatment was higher than that in the control cells (Figure 8G).Cell growth inhibition was similar (16%) for both CuO-NP-5 μg/mL and SiO 2 -NP-5 μg/mL treatments (Figure 8D and 8H).γ-H2AX was elevated by CuO-NP-5 μg/mL and SiO 2 -NP-5μg/mL after 24 h treatments (P 0 cells); subsequently it was gradually decreased during passages (from P 1 to P 3 cells; Figure 8I).However, NF-κB was not altered by the CuO-NP-5 μg/ mL or SiO 2 -NP-5 μg/mL treatment, and a similar level was maintained during passages (from P 0 to P 3 cells; Figure 8J).
Since γ-H2AX was altered by CuO-NP-5 μg/mL and SiO 2 -NP-5 μg/mL treatments, ATM, ATR and DNA-PK were determined in CHO-K1 cells after treatment.It was found that ATM and DNA-PK were increased in P 0 cells in both CuO-NP-5 μg/mL and SiO 2 -NP-5 μg/mL treatments (Figure 9A and 9B).TUNEL positive cells were found at higher levels in P 0 and P 1 cells, in both the CuO-NP-5 μg/mL and SiO 2 -NP-5 μg/mL treatments, than that in the control treatment (Figure 9C).The protein level of p53 was higher in P 0 and P 1 cells in the CuO-NP-5 μg/mL treatment than that in the control treatment, while it was higher in P 0 cells in the SiO 2 -NP-5μg/mL treatment than that in the control (Figure 9D).
Protein levels of PP4C, PP6C, and PP2ACα were elevated in both CuO-NP-5 μg/mL and SiO 2 -NP-5 μg/ mL treatments (Figure 10A-10C).EdU positive cells were decreased in P 0 cells in both CuO-NP-5 μg/mL and SiO 2 -NP-5 μg/mL treatments (Figure 10D).Altogether,   ZnO-NP-12.5 μg/mL, CuO-NP-5 μg/mL, and SiO 2 -NP-5 μg/mL treatments altered γ-H2AX in a similar pattern in the four passages cells (P 0 to P 3 ); however, only the ZnO-NP-12.5μg/mL treatment decreased NF-κB in the four passages cells (P 0 to P 3 ).These findings suggest that intact NPs from ZnO, CuO, and SiO 2 NP treatments block the γ-H2AX pathway while just Zn 2+ upsets the NF-κB pathway.Clearly, the toxic effects of ZnO NPs emanate from both intact nanoparticles and Zn 2+ .These in vitro culture findings matched well with in vivo embryonic data, suggesting that impairment caused by ZnO NP treatment can pass through cell generations, and that the γ-H2AX and NF-κB pathways were involved in the embryotoxicity of ZnO NPs.

DISCUSSION
Researchers have explored the effects of nanoparticles on embryo development; however, these studies have only investigated the effects (phenomenon) on embryos , and not on oocytes.Therefore, the impacts of nanoparticles on embryonic development due to oocyte stage exposure are as yet unknown, and the underlying mechanisms are sparsely understood.ZnO NPs are commonly used in almost every aspect of our lives especially in sunscreen, cosmetics, and biocides [72][73][74][75][76].Even though many reports indicate that ZnO NPs cause adverse effects on reproductive systems and embryonic development [3, 7, 14, 15, 18-20, 26, 40, 47, 62], their safety is not fully understood, particularly the impacts and underlying mechanisms of ZnO NPs on embryonic development due to oocyte stage exposure.
In the current investigation, hens were exposed to ZnO NPs, and after fertilization their impacts on embryonic development and the underlying mechanisms were explored.Results indicated that ZnO NPs inhibited embryonic development by increasing embryo lethality; however, the fertilization rate was not suppressed.Further mechanistic study found that γ-H2AX in embryos was decreased with ZnO NPs treatment.It has been reported that nanoparticles can induce ROS and DNA damage, which consequently activate ATM to induce H2AX phosphorylation (γ-H2AX) [82][83][84][85].ZnO NPs induce DNA damage and increase γ-H2AX to inhibit cell growth [82,85].However, many recent investigations suggest that γ-H2AX is not simply a specific DNA DSB marker and its role is not restricted to the DNA damage response [86].Reports suggest that it involves a multitude of biological processes during cell division [86,87], stem cell biology [88][89][90], angiogenesis [91][92][93], and aging [94][95][96].The de-phosphorylation of γ-H2AX by phosphatases is an important event in complete DNA repair after exogenous DNA damage [97][98][99][100].Two families of phosphatases are involved in the de-phosphorylation of γ-H2AX: firstly, the PP2A family of serine/threonine phosphatases including four distinct catalytic proteins PP2ACα, PP2ACβ, PP4C, and PP6C [97,99], and secondly the PP4 phosphatase complex containing PP4C, PP4R2, and PP4R3β [97,99].In the current study, these phosphatases were determined, and it was found that PP4C, PP4R3β, PP4R2, PP2ACα, and PP2A were elevated by ZnO NPs in embryos (Figure 2D).Transcription factor NF-κB (nuclear factor-kappa B) is a critical regulator of multiple biological functions: cell growth, cell survival, innate and adaptive immunity, and others [101][102][103][104].In the current study it was found that ZnO NPs treatment decreased NF-κB in embryos.Vyas et al. (2015) found that iron oxide nanoparticles could enhance the effects of the anticancer drug, doxorubicin, through elevation in apoptosis and down regulation of both pro-inflammatory mediators IL-6 and NF-κB [105].Sarkar et al. (2015) reported that Ag NPs exposure suppressed mycobacterium tuberculosisinduced expression of a subset of NF-κB mediated genes to suppress mycobacterium tuberculosis-induced NF-κB activation and host immune responses in macrophage cells [106].A20 (TNFAIP3) has been originally described as a zinc finger protein which is a TNF-inducible gene and acts to protect cells from TNF cytotoxicity [107,108].
During later experiments, it has been found that A20 acts as a negative regulator of NF-κB signaling involving IL-1b, TNFα, TRAF1/TRAF2, RIP/TRAF2, and other factors [109][110][111].Kim and Jeong (2015) reported that ZnO NPs induced the expression of A20 and reduce NF-κB activation to increase anti-inflammatory effects [112].Feng et al. (2016) found that A20 suppressed the expression of inflammatory cytokines induced by Ag NPs [113].In the current study, ZnO NP treatment elevated   A20 and decreased NF-κB in embryos.Since the NF-κB pathway is very important for cell survival and growth, the blockage of this pathway might be one of the major reasons for the inhibition of embryonic development caused by ZnO NPs.
ZnO NPs exposure may damage DNA replication and repairmen machinery in hen oocytes, which impairs the embryos to inhibit embryonic development.In order to confirm this phenomenon, normal hamster ovarian cells (CHO-K1) were used to determine the impact of ZnO NPs exposure on γ-H2AX and NF-κB pathways after a 24-h treatment and three cell passages.γ-H2AX was elevated after a 24-h ZnO NP exposure, but it was gradually decreased during progressive cell passages to a level much lower than that in the control treatment; this indicated that ZnO NP treatment caused DNA damage and DNA repairmen/replication machinery failure.Furthermore, the phosphatases PP4C, PP6C, and PP2ACα were induced in P 2 and P 3 cells, which coordinated well with changes of γ-H2AX.NF-κB was diminished after a 24-h ZnO NPs exposure and remained at similar low levels during cell passages while A20 was present at higher levels in all passaged cells than in the control treatment.These in vitro results verified that ZnO NPs treatment blocked both γ-H2AX and NF-κB pathways in vivo (embryos).
ZnO NPs can be endocytosed into cells and dissolved to release Zn 2+ to significantly increase intracellular Zn 2+ levels [114,115].It is known that locally high levels of intracellular Zn 2+ have toxic effects [114,115].Furthermore, ZnO NPs internalized into cells remain as intact NPs for a long time and these induce different toxic effects when compared with Zn 2+ [116][117][118][119].We have already found that ZnO NPs and ZnSO 4 (sole Zn 2+ provider) produced a significantly different impact on gene and protein expression in vitro and in vivo with intact NPs being detected in cells and animal tissues [117][118][119].Intact ZnO NPs can be found in hen ovarian, liver, spleen, and uterine tissues [80,81,[117][118][119].In order to investigate whether the blockage of γ-H2AX and NF-κB pathways by ZnO NP treatment was due to intact NPs or Zn 2+ , CuO NPs and SiO 2 NPs were used in the current investigation.The size of ZnO, CuO, and SiO2 NPs is similar at around 30 nm.The properties of CuO NPs are similar to ZnO NPs in that they can be dissolved in cells, animal tissues, or even solutions [25,64].However, SiO 2 NPs are different from ZnO and CuO NPs in that SiO 2 NPs cannot be dissolved in vivo or in vitro culture [63].If ZnO, CuO, and SiO 2 NPs produce similar effects on γ-H2AX and NF-κB pathways, it would suggest that these effects are from intact NPs; if not, the effects would emanate from Zn 2+ .In the current study it was found that γ-H2AX was increased in CHO-K1 cells after 24 h CuO NP and SiO NP treatments, and it was decreased with progressive cell passages; a similar trend was seen with ZnO NP treatment.This indicated that γ-H2AX blockage was most likely caused by intact NPs (ZnO, CuO, or SiO 2 NPs).However, NF-κB was not altered by CuO NPs or SiO 2 NPs treatments, which suggested that NF-κB blockage was due to Zn 2+ .It seems clear that the toxic effects of ZnO NPs emanate from both intact NPs and Zn 2+ .The blockage of γ-H2AX and NF-κB pathways might increase apoptosis and reduce cell proliferation as indicated by the apoptosis markers and cell proliferating assay in ZnO NP, CuO NP, and SiO NP treatments.

CONCLUSIONS
In summary, intact NPs were detected in ovarian tissue in vivo and cultured cells in vitro under ZnO NPs treatments.ZnO NPs treatment inhibited embryo development following oocyte stage exposure, and the γ-H2AX and NF-κB pathways involve in the inhibition of embryo development.Notably, the in vitro cultural data closely matched in vivo embryonic data indicating that impairments caused by ZnO NPs treatment can pass through cell generations, and that γ-H2AX and NF-κB pathways were involved in the embryotoxicity of ZnO NPs.The current study clearly shows that the toxic effects of ZnO NPs emanate from both intact NPs and Zn 2+ .This study, along with others, suggests that ZnO NPs are toxic to the female reproductive system [ovaries (oocytes)] and subsequently embryo-toxic.Therefore, precautions should be taken against human exposure during daily life.

Characterization of ZnO, CuO and SiO 2 NPs
ZnO NPs were synthesized by Beijing DK Nano Technology Co. LTD (Beijing, China) as reported in our recent publications [80,81].The characteristics of ZnO NPs (morphology, size, agglomeration, etc.) were determined by transmission electron microscopy (TEM; JEM-2100F, JEOL Inc., Japan) and dynamic light scattering (DLS) particle size analyzer (Nano-Zetasizer-HT, Malvern Instruments, Malvern, UK).CuO and SiO2 NPs were manufactured by Beijing DK Nano Technology Co. LTD (Beijing, China) too.The morphology and size of CuO and SiO 2 NPs were characterized using transmission electron microscopy (TEM; JEM-2100F, JEOL Inc., Japan) and a D8 Advance Powder X-ray Diffractometer (Bruker AXS, Karlsruhe, Germany).The hydrodynamic diameter, polydispersity index and zeta potential were determined in phosphate buffered saline (PBS) after 30 min sonication.

Animal study design (diets and treatments) and sample collection
All animal experimental procedures followed the regulations of the animal ethics committee of the www.impactjournals.com/oncotargetQingdao Agricultural University and were reported in our recent publication [80].All hens (Jinghong-1 strain) were housed in a ventilated and conventional caged commercial poultry house with a lighting program of 16:8 light/dark and ad-lib food and water.The formulation of the basal diet (corn-soybean base) has been previously reported [Supplementary Table 1] [80,118].The experimental feeding time was from 6 wks to 32 wks of age (Supplementary Figure 3A).ZnO NPs or ZnSO 4 was added into the diet and the concentration of Zn (mg/ kg) addition was based on diet.The two treatments were ZnSO 4 -200 mg/kg and ZnO-NP-200 mg/kg of diet.A total of 400 pullets were randomly assigned to the two treatments, with five replicates per treatment and forty animals per replicate.After 24 wks treatments, the hens were artificially inseminated with fresh, diluted semen 0.03 mL/hen providing about 210 million sperm (Supplementary Figure 3A).Eggs were collected and stored at 13°C and 75% relative humidity for 5 days until placed in incubators.The fertilization rate was detected by propidium iodide staining of the germinal disc.Egg yolks were separated from the albumen and placed into 0.9% NaCl solution.After visual fertility examination germinal discs seeming to be infertile were removed from the vitelline membrane, put into 0.9% NaCl solution and stained on a slide with propidium iodide (PI; P4170, Sigma-Aldrich Ltd).In case of fertile egg, propidium iodide stains the nucleus of the dividing embryonic cells which appear in lighting points [120,121].The embryonic development failure rate was determined by candling eggs after 7 d of incubation and breaking out eggs to differentiate early dead from infertile [120][121][122].At 3 d and 5 d incubation, 50 embryos/treatment were collected and stored at −80°C.At the age of 30, 31 and 32 wks, the experiments were repeated three times.
In vitro mechanism study CHO-K1 were cultured and treated with ZnSO 4 , ZnO NPs, CuO NPs or SiO 2 NPs for 24 h (Supplementary Figure 3B).After 24h treatment, the cells (passage 0 (P 0 )) were collected and half were used for analysis and half were passaged.Following passage and 24 h growth the cells (passage 1 (P 1 )) were collected and half were used for analysis and half were passaged.Similarly, P 2 and P 3 cells were collected (Supplementary Figure 3B).The cells collected were for detection of NPs presentation in cells, cellular element concentrations, protein levels by western blotting or IHF analysis.

Detection of ZnO NPs in cultural cells using transmission electron microscopy (TEM) and energy disperse spectroscopy (EDS)
Sample preparation procedures for detecting nanoparticles are reported in our recent publication [80,81].Briefly, cell samples were collected and fixed for 2 h in 2% glutaraldehyde made in sodium phosphate buffer (pH 7.2).Specimens were then washed extensively to remove the excess fixative and subsequently post-fixed in 1% OsO 4 for 1 h in the dark.Specimens were then dehydrated in an increasingly graded series of ethanol and infiltrated with increased concentrations of Spur's embedding medium in propylene epoxide.Subsequently the specimens were polymerized in embedding medium for 12 h at 37°C, 12 h at 45°C, and 48 h at 60°C.Fifty nanometer sections were cut on a Leica Ultracut E equipped with a diamond knife (Diatome, Hatfield, PA), and collected on form var-coated, carbon-stabilized Mo grids.The section-containing grids were stained with uranyl acetate, air dried overnight, and imaged on a JEM-2010F TEM (JEOL Ltd., Japan).The presence of ZnO NPs in the tissues was confirmed by using X-Max N 80 TLE EDS (Oxford Instruments, U.K.).

Measurement of Zn concentration in cultured cells
After 24 h treatment with ZnO NPs, ZnSO 4 , CuO NPs or SiO 2 NPs, cells (2 × 10 6 cells/treatment) were lysed in 500 µL of 0.4% Triton X-100 in PBS, then the lysate was diluted to 2mL with 0.1% Triton X-100.Samples were determined by inductively coupled plasma optical emission spectroscopy (ICP-OES, Optima 2100, Perkin-Elmer, Shelton, CT, USA) [81].The voltage for the ion www.impactjournals.com/oncotargetlens was set at 6 V; the gas flow rate in the spray chamber was 0.88 L/min; the power output for the RF generator was 1100 W; the auxiliary gas flow rate was 1.2 L/min; and the nebulizer gas flow rate of the plasma was 16 L/min.All the certified reference materials (in solution) were purchased from the National Institute of Metrology (Beijing, China).Blank controls underwent the same procedures.All procedures were performed in triplicate [81].

TUNEL assay
The apoptotic cells were determined by the Dead End Colorimetric TUNEL assay kit (Promega, Madison, WI, USA) with CHO-K1 cells after ZnSO 4 , ZnO NPs, CuO NPs and SiO 2 NPs treatments according to the manufacturer's protocols.This assay is detecting the DNA fragmentation by labeling the terminal end of nucleic acids.The stained sections were visualized with a Nikon Eclipse TE2000-U fluorescence microscope (Nikon, Inc., Melville, NY), and the captured fluorescent images were analyzed using MetaMorph software [118].

Western blotting.
Embryo and CHO-K1 cell samples were lysed in RIPA buffer containing a protease inhibitor cocktail from Sangong Biotech, Ltd. (Shanghai, China).Protein concentration was determined using a BCA kit (Beyotime Institute of Biotechnology, Shanghai, PR China) [81].The information for the primary antibodies (Abs) is present in Supplementary Table 2. GAPDH and Actin were used as loading controls.Secondary donkey anti-goat Ab (Cat no.A0181) was purchased from Beyotime Institute of Biotechnology, and goat anti-rabbit (Cat no.: A24531) Abs were bought from Novex ® by Life Technologies (USA).
Fifty micrograms of total protein per sample were loaded onto 10% SDS polyacrylamide electrophoresis gels.The gels were transferred to a polyvinylidene fluoride (PVDF) membrane at 300 mA for 2.5 h at 4 o C. Subsequently, the membranes were blocked with 5% bovine serum albumin (BSA) for 1 h at room temperature (RT), followed by three washes with 0.1% Tween-20 in TBS (TBST).The membranes were incubated with primary Abs diluted at 1:500 in TBST with 1% BSA overnight at 4 o C.After three washes with TBST, the blots were incubated with the HRP-labeled secondary goat anti-rabbit or donkey antigoat Ab respectively for 1 h at RT.After three washes, the blots were imaged [81].

Immunofluorescent staining
The collected cells were fixed in 4% paraformaldehyde for 1 h, then the cells were spread onto poly-L-lysine coated microscope slides and airdry.After three washings with PBS (5 min each) cells were incubated with 2% (v/v) Triton X-100 in PBS for 1 h at RT.Then, after three washes with PBS, the cells were blocked with 1% (wt/v) BSA and 1% goat serum in PBS for 30 min at RT, then incubation with primary antibodies diluted in blocking solution overnight at 4°C.The information for the primary antibodies (Abs) is present in Supplementary Table 2.The following morning, after three washes with PBS Tween 20 (0.5%) the slides were incubated with Alexa Fluor 546 goat anti-rabbit IgG (1:200) for 30 min in darkness at RT.The negative controls samples were incubated with secondary antibody and without primary antibody.Slides were washed with PBS Tween-20 three times and then incubated with DAPI (4.6-diamidino-2-phenylindole hydrochloride, 100 ng/ml) as nuclear stain for 5 min.After brief wash with ddH 2 O, the slides were covered with an anti-fading mounting medium (Vector, Burlingame, USA).Fluorescent images were obtained with Leica Laser Scanning Confocal Microscope (LEICA TCS SP5 II, Germany) [118].

Figure 2 :
Figure 2: Inhibition of chicken embryonic development by ZnO NPs.(A) Fertilization rate; (B) Embryo development failure rate; (C) Decrease in γ-H2AX by ZnO NPs using WB analysis; (D) Elevation of the protein levels of de-phosphorylation enzymes PP4C, PP4R3β, PP4R2, PP2ACα, and PP2A by ZnO NPs using WB analysis; (E) Reduction in NF-κB major component p65 and increase in A20 by ZnO NPs treatment using WB analysis; (F) Decrease in PCNA while elevation in caspase 8 by ZnO NPs treatment using WB analysis; N³5.

Figure 3 :
Figure 3: ZnO NPs in CHO-K1 cells and the impacts on cell growth and γ-H2AX.(A) TEM photo of ZnO NPs in CHO-K1 cells indicated by the red arrow; (B) EDS image of ZnO NPs in CHO-K1 cells, three Zn peaks shown; (C) Concentration of Zn in ZnO NPs or ZnSO 4 treated CHO-K1, a,b,c, same letter means no difference, different letters mean the groups have difference, p < 0.05; (D) Effects of ZnO NP and ZnSO 4 on CHO-K1 cell growth; (E) Alteration of γ-H2AX by ZnO NPs using WB analysis; N ≥ 3.

Figure 4 :
Figure 4: IHF images for ATM and DNA-PK in ZnO NPs treated CHO-K1 cells.(A) Increase in the protein level of ATM in P 0 and P 1 cells by ZnO NPs treatment.Red: ATM staining; Blue: DAPI staining for nucleus; (B) Elevation of the protein levels of DNA-PK in P 0 and P 1 cells by ZnO NPs treatment.Red: DNA-PK staining; Blue: DAPI staining for nucleus.Scale bar: 25 mm; N ≥ 3. a-c Means not sharing a common superscript are different.

Figure 5 :
Figure 5: IHF images for TUNEL assay and p53 levels in ZnO NPs treated CHO-K1 cells.(A) Increase in the TUNEL positive cells in P 0 and P 1 cells by ZnO NPs treatment.Red: TUNEL staining; Blue: DAPI staining for nucleus; (B) Elevation of the protein levels of p53 in P 0 and P 1 cells by ZnO NPs treatment.Red: p53 staining; Blue: DAPI staining for nucleus.Scale bar: 25 μm; N ≥ 3. a-c Means not sharing a common superscript are different.

Figure 6 :
Figure 6: IHF images for de-phosphorylation enzymes PP4C, PP6C, PP2ACα and EdU analysis after ZnO NPs treatment.(A) IHF staining for PP4C, increase in the PP4C protein levels in P 1 , P 2 and P 3 cells.Red: PP4C staining; Blue: DAPI staining for nucleus; (B) IHF staining for PP6C, elevation of the PP6C protein levels in P 1 , P 2 and P 3 cells.Red: PP6C staining; Blue: DAPI staining for nucleus; (C) IHF staining for PP2ACα, increase in the PP2ACα protein levels in P 0 , P 1 , P 2 and P 3 cells.Red: PP2ACα staining; Blue: DAPI staining for nucleus; (D) EdU analysis, decrease in the EdU positive cells in P 0 cells.Green: P EdU staining; Blue: DAPI staining for nucleus; Scale bar: 25 mm; N ≥ 3. a-c Means not sharing a common superscript are different.

Figure 7 :
Figure 7: NF-κB major component p65 and A20 in ZnO NPs treated CHO-K1 cells.(A) reduction in NF-κB major component p65 by ZnO NPs treatment; (B) Increase in A20 by ZnO NPs in CHO-K1 cells using IHF staining; Scale bar: 25 mm; N ≥ 3. a-b Means not sharing a common superscript are different.

Figure 8 :
Figure 8: Impairment of γ-H2AX not NF-κB by CuO or SiO2 NPs in CHO-K1 cells.(A) TEM photo of CuO NPs in CHO-K1 cells indicated by the red arrow; (B) EDS image of CuO NPs in CHO-K1 cells, two Cu peaks have shown; (C) Concentration of Cu in CuO NPs treated CHO-K1, *p < 0.05; (D) Effects of CuO NPs on CHO-K1 cell growth; (E) TEM photo of SiO 2 NPs in CHO-K1 cells indicated by the red arrow; (F) EDS image of SiO 2 NPs in CHO-K1 cells; (G) Concentration of Si in SiO 2 NPs treated CHO-K1, *p < 0.05; (H) Effects of SiO 2 NPs on CHO-K1 cell growth; (I) Decrease in γ-H2AX by CuO and SiO 2 NPs using WB analysis; (J) No alteration in NF-κB major component p65 by CuO NPs or SiO 2 NPs treatment using WB analysis; N ≥ 3.

Figure 9 :
Figure 9: IHF images for ATM, DNA-PK, TUNEL assay and p53 levels in CuO and SiO2 NPs treated CHO-K1 cells.(A) Increase in the protein levels of ATM in P 0 and P 1 cells.Red: ATM staining; Blue: DAPI staining for nucleus; (B) Elevation of the protein levels of DNA-PK in P 0 and P 1 cells.Red: DNA-PK staining; Blue: DAPI staining for nucleus.Scale bar: 25 μm.(C) Increase in TUNEL positive cells in P 0 and P 1 cells.Red: TUNEL staining; Blue: DAPI staining for nucleus; (D) Elevation of the protein levels of p53 in P 0 and P 1 cells.Red: p53 staining; Blue: DAPI staining for nucleus.Scale bar: 25 μm; N ≥ 3. a-b Means not sharing a common superscript are different.

Figure 10 :
Figure 10: IHF images for de-phosphorylation enzymes PP4C, PP6C, PP2ACα and EdU analysis in CuO and SiO2 NPs treated cells.(A) IHF staining for PP4C, increase in the PP4C protein levels in P 1 , P 2 and P 3 cells.Red: PP4C staining; Blue: DAPI staining for nucleus; (B) IHF staining for PP6C, elevation of the PP6C protein levels in P 1 , P 2 and P 3 cells.Red: PP6C staining; Blue: DAPI staining for nucleus; (C) IHF staining for PP2ACα, increase in the PP2ACα protein level in P 0 , P 1 , P 2 and P 3 cells.Red: PP2ACα staining; Blue: DAPI staining for nucleus; (D) EdU analysis, decrease in the EdU positive cells in P 0 cells.Scale bar: 25 μm; N ≥ 3. a-d Means not sharing a common superscript are different.