Exposure to Zinc oxide nanoparticles during pregnancy induces oocyte DNA damage and affects ovarian reserve of mouse offspring

Zinc oxide nanoparticles (nZnO) have been shown to have higher toxic effects likely due to their ion-shedding ability and low solubility under neutral conditions. In order to investigate whether exposure to nZnO during embryonic development affects ovary development, 12.5 day post coitum (dpc) fetal mouse ovaries were cultured in the presence of nZnO for 6 days. We found that the nanoparticles (NPs) accumulated within the oocyte cytoplasm in a dose dependent manner, caused DNA damage and apoptosis, and result in a significant decrease in oocyte numbers. No such effects were observed when the ovaries were incubated in the presence of ZnSO4 or bulk ZnO as controls. In addition, we injected intravenously 16 mg/kg body weight nZnO in 12.5 dpc pregnant mice on two consecutive days and analyzed the ovaries of fetuses or offspring at three critical periods of oogenesis: 17.5 dpc, 3 days post-partum (dpp) and 21 dpp. Evidence of increased DNA damage in pachytene oocytes in fetal ovaries and impaired primordial follicle assembly and folliculogenesis dynamics in the ovaries of the offspring were found. Our results indicate that certain types of NPs affect pre- and post-natal oogenesis in vitro and in vivo.


INTRODUCTION
The widespread application of metal nanoparticles has greatly increased the chance of environmental exposure. Furthermore, NPs are found in everyday consumer products including paintings and electronic devises [1][2][3][4]. For this reason, potential toxicological effects of NPs have received increasing attention. It is believed that during embryo/fetal development is particularly vulnerable to the exposure of NPs due to the fact that they can penetrate the placental barrier and cause embryo toxicity [5]. The reproductive and developmental toxicity of carbon and silver NPs have been recently reviewed [6]. Meanwhile, the toxicity of nZnO has been investigated in various organs and tissues, including the testes in animal models [7][8][9]. Some of these studies demonstrated that nZnO may adversely impact the female reproductive system and fertility [10,11]. In this regard, we recently found that exposure of chick oocytes to nZnO inhibits their developmental AGING capabilities following fertilization [12]. Little is known, however, of the consequences of maternal exposure to nZnO during the prenatal period and its effects on oogenesis in offspring.
Primordial germ cell (PGC) proliferation and fetal oocyte degeneration before and early after birth are regarded as important processes responsible for the establishment of a finite pool of primordial follicles. In mice, PGCs mitotically divide until 13.5 dpc at which time they reside in the fetal gonads and after which they immediately enter meiosis. Failure to properly form and repair meiotic DNA double-strand break (DSBs) result in gametes with alterations to chromosome structure or aneuploidy, which can lead to development disorders [13]. From 17.5 dpc to 4 dpp germ cell cysts undergo a process called "breakdown" which is associated with oocytes undergoing waves of programmed cell death (PCD). Therefore, oocytes with errors during chromosomal crossover might be eliminated by oocyte quality control mechanisms. Finally, another mechanism demonstrated to function as a developmental check point for oocytes, involves excessive retrotransposon activation (in particular of LINE-1) occurring as a consequence of the bulk demethylation taking place in PGCs before the beginning of meiosis [14]. As a consequence of PCD, only about one-third of germ cells produced following PGC/oogonia proliferation are present within the ovaries early after birth [15]. Alteration of meiosis and primordial follicle formation may increase the number of poor quality oocytes and lead to increased PCD, decreased ovarian reserve and pathologies such as premature ovarian inefficiency (POI) [16]. Although recent evidence indicates that postnatal neo-oogenesis is possible in some mammalian species [17], it is still largely accepted that the oocyte population forming the primordial follicles, during late pregnancy or early post birth, constitute a finite pool. It is therefore of great importance for mammalian females to efficiently establish the ovarian reserve during the pre-and early postnatal period and to maintain it for the rest of their reproductive lifespan.
In the present work, we studied whether maternal exposure to nZnO during embryo development affects oocyte DNA integrity and the establishment of the ovarian reserve in female offspring and using an in vitro ovary culture system investigated the possible underlying mechanisms.

RESULTS nZnO penetrate into fetal ovaries and increase DNA damage and apoptosis in fetal oocytes
To determine if nZnO could penetrate into the fetal mouse ovary, we cultured 12.5 dpc ovaries in vitro in the presence of ZnSO 4 , bZnO or 10 and 20 µg/ml nZnO, for 6 days (ensure the molar weight of zinc is equal). Observations under an inverted microscope did not reveal any evident morphological alterations in ovaries incubated in the presence of nZnO, ZnSO 4 or bZnO ( Figure S2). Examination of ovary tissue sections immunostained for the oocyte specific protein MVH (a marker of germ cells) under a confocal reflection microscope, indicated that nZnO, but not ZnSO 4 or bZnO, were accumulated in the ovaries in a dose independent manner (Figures 1A and Figure S3). In TEM sections, nZnO were observed in the oocyte cytoplasm ( Figures 1B and Figure S4). The use of a TEM equipped with EDX, confirmed the presence of nZnO but not dye particles within the oocytes ( Figure  1C). Although information about the endocytosis capabilities of fetal oocytes is lacking, this is likely to occur for nZnO since this is the usual route of internalization for similar NPs by cells [18].
The analysis of Bax and Bcl-2 transcripts and their encoded proteins showed that their ratio were increased significantly in the ovaries cultured in the presence of nZnO, thus representing evidence of ongoing apoptosis. However, the ratio was not altered in the presence of ZnSO 4 or bZnO (Figure 2A-2C). Moreover, the number of TUNEL-positive cells was significantly increased in ovaries incubated in the presence of 20 µg/ml nZnO in comparison to the untreated controls ( Figure 2E and 2F). Double immunofluorescence on tissue sections of ovaries cultured for 6 days indicated that the number of MVH positive oocytes stained for γH2AX increased in the nZnO treated ovaries ( Figure 2G and 2H). It is interesting that the amount of MVH protein decreased in the ovaries cultured in the presence of nZnO in a dose dependent manner, but not when ZnSO 4 or bZnO were added to the culture medium ( Figure 2D). Similarly, the number of MVH positive oocyte decreased in the ovaries cultured in presence of nZnO ( Figure 2I).

Exposure of nZnO increase DSBs in fetal oocytes both in vitro and in vivo
We confirmed that nZnO penetrate fetal ovaries and increase DNA damage and apoptosis of fetal mouse oocytes (Figure 2A-2I). We next analyzed cytospreads and confirmed a higher number of strongly positive γH2AX oocytes both at the pachytene and the diplotene stages in the ovaries exposed to nZnO ( Figure 3A and 3B). Moreover, western blot (WB) analysis revealed that the overall γH2AX expression level increased in the whole ovaries ( Figure 3C). Immunofluorescence for RAD51, an enzyme involved in homologous DNA recombination and repair [19], showed a slight but not significant increase in the number of oocytes showing AGING fluorescent foci in the nZnO treated ovaries ( Figure 3D and 3E); RAD51 protein amount in the whole ovaries showed similar results as revealed by WB analysis ( Figure 3C). Despite these adverse effects of nZnO on the fetal ovaries and in particular on the oocytes, immunofluorescence analysis for SCP3 in cytospreads did not reveal alterations in the meiotic entry and progression within the exposed oocytes ( Figure S5A and S5B). Likewise, qRT-PCR did not show significant variation in the mRNA level of genes encoding proteins involved in meiotic processes ( Figure S5C).
In order to investigate the effect of nZnO exposure on fetal oocytes in vivo, we intravenously injected 12.5 dpc pregnant mice with 16 mg/kg body weight nZnO on two consecutive days. At 17.5 dpc, fetuses were collected and ovaries dissected. The number and body weight of fetuses from mothers exposed to nZnO trended lower, but was not statistically significant ( Figure S6A). Double immunofluorescent analysis of oocyte cytospreads with antibodies against the synaptonemal protein SCP3 and γH2AX, demonstrated that nZnO exposure significantly increased DSBs in oocytes at the AGING pachytene, but not at the diplotene stage, in comparison to untreated controls ( Figure 4A and 4B). Enhanced DNA damage in the whole ovaries exposed to nZnO was also suggested by the increased level of γH2AX expression evaluated by WB ( Figure 4C). To sum up, exposure of nZnO increase DSBs in fetal oocytes both in vitro and in vivo (Figures 3, 4 and Figure S5D).

AGING
The adverse effects on primordial follicle assembly and ovarian reserve after pregnant mothers exposed to nZnO The immunofluorescent analysis of the 3 dpp ovaries of pups delivered by mothers exposed to nZnO, showed that the number of MVH positive oocytes was significantly decreased in comparison to untreated controls ( Figure 5A-5C). Furthermore, the oocyte specific proteins MVH, NOBOX, and LHX8 and the transcripts Mvh and Lhx8 in these ovaries also showed decreased expression ( Figure 5D and 5E). NOBOX fluorescence intensity in single oocytes ( Figure 5F), the mRNA expression of Figα and Sohlh2, and the body weight of fetuses from mothers exposed to nZnO trended lower, but were not significant different ( Figures S6B and  S6C). Other parameters such as DNA damage (double positive MVH and γH 2 AX oocytes), and BAX and BCL-2 protein expression in the whole ovaries were not altered by nZnO exposure (Figures S6D and S6E).
We next investigated whether nZnO exposure altered the dynamics of the first folliculogenesis wave in the offspring ovaries. To this aim, we scored the number of oocytes present in primordial, primary, secondary and antral follicles in 21 dpp ovaries of the offspring of the nZnO exposed mothers ( Figure 6A). In Figure 6B, it was shown that whereas no significant difference was detected in the percentage of primordial follicles, primary follicles and antral follicles, the percentage of AGING secondary follicles was significantly higher in nZnO exposed ovaries than in the untreated controls (P < 0.05), suggesting accelerated follicle dynamics. Interestingly, in such ovaries, the total number of oocyte and primordial follicles was decreased although not significantly ( Figure 6C and 6D).
Oocytes obtained from nZnO exposed ovaries of adult offspring (4-6 weeks), showed normal meiotic maturation capabilities evaluated by the rates of germinal vesicle breakdown (GVBD) and PBE (polar body exclusion) after 4 h and 12 h of in vitro culture ( Figure  6E, Figure S6F and S6G). Moreover, both in control and the nZnO exposed group, most oocytes showed normal spindle morphologies at the MI stage ( Figure 6F and Figure S6H). Finally, no obvious differences in the F 2 of control and nZnO exposed groups with regards to the number of fetuses and sex ratios were observed ( Figure 6G and Figure S6I-S6J).

DISCUSSION
Nanoparticles are widely used as antibiotic agents in textile, wound dressings, medical devices, and in appliances like refrigerators and washing machines [20]. They are heavily used due to their unique characteristics, such as high hydrophilicity, and ease of synthesis [21,22]. The increasing use of nanomaterials has raised concerns about the potential risks to human health, especially on the reproduction system [5,23,24]. Several studies have addressed the concerns of NPs exposure on reproductive health, however these studies focused on the impact of NPs on germ cell and pregnancy complications [5,20,25] (Figure 7). It is worth AGING noting that fetuses are known to be more sensitive to environmental toxins than adults [26][27][28]. Furthermore, it has been suggested that chemicals can affect the maturation of germ cells, impair fertility, cause cancer, and disrupt the development of offspring [29,30]. The current study proved that nZnO exposure to pregnant mice perturbed fetal oogenesis and follicle development. We investigated several critical periods of oogenesis both in vivo and in vitro and it was found that exposure to nZnO could induce the accumulation of DSBs in fetal oocytes and decrease the ovarian follicle reservoir in fetal ovaries.
In vitro experiment results primarily showed that nZnO had toxicity on fetal oocytes. 12.5 dpc fetal ovaries were isolated and cultured with nZnO in vitro, and nZnO penetrated into the fetal ovaries and caused cytotoxicity to the germ cells. Consistent with previous studies [7,[31][32][33], Exposure to nZnO mainly induced cellular DNA damage and as a consequence resulted in increased expression of apoptosis related proteins in the fetal ovaries. Since several types of cells exist within the fetal ovaries, we next investigated whether the nZnO penetrated and affected particular cell types. Noteworthy, the co-immunostaining analysis revealed that DNA damage signals mainly occurred in the germ cells, and TEM analysis also demonstrated that nZnO exposure caused obvious lesions in fetal oocytes. However, of great concern is that the orchestrated germ cell development is indispensable for successful establishment of the ovarian reservoir. The primordial follicle pool represents the lifelong reproductive reserve in the female ovary and in vitro exposure to nZnO affected fetal oocyte development. In particular, fetal ovary exposure to nZnO increased the accumulation of DSBs as shown by γH2AX expression during the first meiotic prophase (MPI). As a consequence of the cytotoxicity to germ cells it is reasonable to propose that germ cells with a high level of DNA damage underwent apoptosis leading to decreased germ cells in the fetal ovary.

AGING
To future confirm nZnO toxicity on pregnant mice, the germ cell number at 17.5 dpc (germ cells arrived in diplotene of MPI), 3 dpp (ovarian reservior establishment), 21 dpp (puberty), and 4 -6 weeks (adult) [34,35] was compared. It was found that nZnO exposure increased the accumulation of DSBs in 17.5 dpc fetal oocytes. However, when the fetus developed to 3 dpp, the accumulation of DSBs in the oocytes was no longer detectable, and the percentage of primordial follicle (PF) was significantly decreased. The progression of follicular development was significantly altered in 21 dpp mice ovary in the nZnO exposured group with a lower percentage of PFs. In addition, the rate of GVBD, PBE and the percentage of spindle defects were examined. The developmental competence of offspring oocytes showed no difference between the nZnO exposure group and the untreated control group. Considering the fetal oocytes attrition (FOA), a conversed and physiologically normal event of fetal oogenesis [14], it may be proposed that nZnO induced accumulation of DSBs in fetal germ cells led to higher apoptosis. Therefore, the difference in apoptosis by 3 dpp was no longer detectable, while the nZnO exposed group showed a decreased ovarian reservoir. Notably, because of the FOA eliminate the "low-quality" oocytes (defective of DSBs caused by nZnO), thus, the number of oocytes surviving to birth are likely to normal oocytes and determine the fertility [36,37]. In a word, the continual deletion of inferior oocytes might guarantee the quality of oocytes and maximize the chances of reproductive success. Differing from males, females are born with a finite pool of ovarian follicles. Currently, more and more women are suffering from POI which is characterized by estrogen deficiency, menstrual irregularity, elevated serum gonadotropins, low serum estradiol and impaired fertility prior to 40 years of age [38,39]. Thus, the orchestrated establishment of the ovarian follicle reserve is of great importance for female reproductive lifespan and is known to be vulnerable to environmental toxins [40,41]. The accelerated elimination of the primordial follicle pool is of great concern, as exhaustion is irreversible and may cause POI [38,42]. Although the mechanisms underlying this phenomenon were not investigated in this study, we may propose that nZnO exposure during primordial follicle assembly alters the epigenetic pattern of oocytes, resulting in transgenerational epigenetic inheritance [43,44]. Similar studies have reported reproductive consequences to NPsexposure including for gold nanoparticles which significantly changed the global DNA methylation pattern in human embryonic stem cells [45]. However, the specific mechanisms require further study to elucidate the role that epigenetic modifications have during oocyte development.
In conclusion, the present results indicate that nZnO can affect pre-and post-natal oogenesis related processes and result in increased DNA damage and decrease the ovarian follicle reserve. The need remains, however, for investigation into the effects of different types of NPs on the development and functioning of the female gonad and of the possible consequences that exposure to such inorganic materials during pregnancy has on fertility in the offspring.

Animals
CD1 mice were purchased from Beijing Vital River Laboratory Experimental Animal Technology Co. LTD (Beijing, China). Animal housing conditions were: 21 -22 °C, 12 h dark /12 h light cycles, free access to water and food. 6-week female mice were mated overnight and the presence of the vaginal plug the next morning was considered as 0.5 dpc. All procedures involved in this study were approved by the Ethical Committee of Qingdao Agricultural University. AGING Nanoparticles nZnO were purchased from Beijing DK Nano Technology Co. LTD (Beijing, China), supplied as a white powder, it has a purity of 99.9% and a specific surface area 50 cm 2 /g. bulk ZnO (bZnO; ~ 230 nm) particles were purchased from Beijing Sorlabio Life Science Co. LTD (YZ-111619, Beijing, China) and ZnSO 4 was purchased from Sigma-Aldrich, Inc (Sigma, Z0251, USA). nZnO characterization was performed using transmission electron microscope (TEM, JEM-2100F, JEOL Inc., Japan) ( Figure S1A) and scanning electron microscope (SEM, JSM-7500F, JEOL Inc.) ( Figure S1B). The average nZnO diameter was ~ 30 nm whereas that of bZnO particles were ~ 230 nm ( Figure  S1C) as we previously described [46,47]. Before addition to the culture medium or animal injection, 4 mg nZnO was dissolved in 1 ml of α-Minimal Essential Medium (α-MEM, Hyclone, SH30265.01B, Beijing, China) and ultrasonicated for 15 min. 100 µl nZnO suspension was injected via tail vein on two consecutive days (12.5 dpc and 13.5 dpc). The dosage of nZnO was used according to previous studies [5].

Ovary culture
Ovaries were isolated from 12.5 dpc mouse embryos and cultured according to previously described procedures [48]. The culture medium was α-MEM, supplemented with 10% fetal bovine serum (FBS, Gibco, 10099-141, USA), 0.23 mM sodium pyruvate (Hyclone, SH40003-12) and 100 IU/ml of penicillin and 100 mg/ml of streptomycin sulfate, 10 mIU/ml follicle stimulating hormone (FSH; RD, 5925-FS, MN, USA). Half ovaries were cultured in a 24 well plate for six days in a humidified incubator at 37 °C, 5% CO 2 in air and medium changed every other day.

Ovary immunostaining
For immunostaining, ovaries were fixed with 4% paraformaldehyde, processed following standard paraffin embedding and serially sectioned at 5 μm. Slides were deparaffinized in xylene, rehydrated in a gradient ethanol series before antigen retrieval with 0.01 M sodium citrate buffer at 96 °C for 10 min. For immunofluorescence (IF), after blocking procedure, the slides were incubated with first antibody MVH (rabbit polyclonal, Abcam, ab13840) and γH2AX (Abcam, ab26350) overnight at 4 °C; CY3-labeled goat antirabbit (Beyotime, A0516) or FITC-conjugated goat anti-mouse secondary antibody (Beyotime, A0568), were used in the next morning at a dilution of 1:200; Hoechst 33342 was used for nuclei staining. Oocytes in cysts or into primordial follicles were scored in five section as previous described [50,51]. Images were obtained using an Olympus fluorescence microscope (BX51) or a Leica Laser Scanning Confocal Microscope (Leica TCS SP5 II, Wetzlar, Germany). Reflection analyses of ovary tissue sections were performed according to a previously described procedure [52,53]; the signal intensity of nZnOs were compared using 3D intensity analysis tools provided by Leica Laser Scanning Confocal Microscope Software [54,55].
For immunohistochemistry (IHC), after blocking, sections were exposed to 3% H 2 O 2 for 10 min, blocked with BDT (3% BSA, 10% normal goat serum in TBS) for 30 min and incubated with first antibody against rabbit MVH (Abcam, ab13840) at 4 °C overnight. After careful washing, horseradish peroxidase (HRP)-AGING conjugated goat anti-Rabbit IgG (Beyotime, A0258) was added at room temperature for 45 min. DAB (ZSGB-BIO, Beijing, China) was added to the slides and incubated for 5 -10 min at room temperature. Hematoxylin was used for nuclei staining and the slides mounted with neutral balsam (Sinopharm, Shanghai, China).

TUNEL assay
TUNEL staining was performed using the "Bright Red Apoptosis Detect Kit" (Vazyme, A113-02, China). Briefly, ovary sections were heated at the 60 °C in an air oven for 2 h, washed in xylene and rehydrated through a series of ethanol and double distilled water. The samples were then treated with proteinase K for 15 min at room temperature, rinsed twice with PBS and incubated for 60 min at 37 °C in the dark in 100 µl of the TUNEL reaction mixture; nuclei were counterstained with Hoechst 33342.

TEM and SEM
For transmission electron microscope (TEM) analysis, ovary tissues were fixed with 2% glutaraldehyde in sodium phosphate buffer (pH 7.2) for 2 h at 4 °C and then processed following standard procedures. Samples were sectioned with a Leica ultramicrotome (Leica EM UC7, Wetzlar, Germany) at 50 nm, transferred to a copper grids and stained with uranyl acetate. Pictures were taken with a HITACHI HT7700 transmission electron microscope (HITACHI, Tokyo, Japan) at an accelerating voltage of 80 kV.
For scanning electron microscope (SEM) analysis, samples were mounted on a SEM-specific stubs and samples coated with platinum with an auto fine coater (JEOL). Pictures were taken using a SEM (JEOL) equipped with an EDX detector (Oxford Instruments, Oxford, UK).

Quantitative real-time PCR (qRT-PCR)
Total RNA was extracted from ovarian tissues using RNA prep pure Micro Kit (Aidlab, RN07, Beijing, China), according to the manufacturer's instructions. Reverse transcription was performed using TransScript ® One-Step gDNA Removal Kit and cDNA Synthesis Kit (TransGen, AT311-03, Beijing, China) following the manufacturer's instructions. All primers used in this were listed in Table S1 and qRT-PCR were performed with LightCycler 480 II (Roche, LC480) using the LightCycler ® 480 SYBR Green I Master Kit (Roche, 04887352001). Reaction condition of q-PCR was set at 95 °C for 10 min, followed by 55 cycles at 95 °C for 10 s, 60 °C for 30 s, and finally cooling at 4 °C. Relative gene expression was calculated using the 2 -CT method and normalized to β-actin or Mvh, each sample contained three technical repeats [57].

Statistical analysis
All statistical analysis data involved in this study were performed in triplicate and variance and statistical comparisons were determined with GraphPad Prism 5.0 software (GraphPad Software, San Diego, CA, USA). Student's two-tailed t-test was used to determine statistical significance between means and a significant difference was considered at P < 0.05 for all tests.

Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.