Reproductive toxicity of acute Cd exposure in mouse: Resulting in oocyte defects and decreased female fertility

Accepted Manuscript Reproductive toxicity of acute Cd exposure in mouse: Resulting in oocyte defects and decreased female fertility

Yuyao Cheng, Jun Zhang, Teng Wu, Xianlei Jiang, Huiqun Jia, Suzhu Qing, Quanli An, Yong Zhang, Jianmin Su PII: DOI: Article Number: Reference:

S0041-008X(19)30292-3 https://doi.org/10.1016/j.taap.2019.114684 114684 YTAAP 114684

To appear in:

Toxicology and Applied Pharmacology

Received date: Revised date: Accepted date:

5 April 2019 13 July 2019 16 July 2019

Please cite this article as: Y. Cheng, J. Zhang, T. Wu, et al., Reproductive toxicity of acute Cd exposure in mouse: Resulting in oocyte defects and decreased female fertility, Toxicology and Applied Pharmacology, https://doi.org/10.1016/j.taap.2019.114684

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT Reproductive toxicity of acute Cd exposure in mouse: resulting in oocyte defects and decreased female fertility

RI PT

Running title: Cd impairs oocyte quality in mouse

Yuyao Chenga, Jun Zhangb, Teng Wua, Xianlei Jianga, Huiqun Jiaa, Suzhu Qinga,

College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi

Province, 712100, PR China.

Academy of Animal Science and Veterinary Medicine, Qinghai University, Xining,

ED

b

MA

a

NU

SC

Quanli Ana, Yong Zhanga,*, and Jianmin Sua,*

CE PT

Qinghai Province, 810003, PR China.

*Correspondence:

AC

Jianmin Su and Yong Zhang, College of Veterinary Medicine, Northwest A&F University, Yangling 712100, Shaanxi, China. Email: [email protected]; [email protected]

1

ACCEPTED MANUSCRIPT Abstract Cadmium (Cd), a known metal contaminant, is widespreadly used in industry, thereby human health is severely affected through the way of occupational and environmental exposure. The adverse effects of the exposure to Cd on the female

RI PT

reproductive system, especially oocyte maturation and fertility have not been clearly defined. In this study, we found the arrested development of ovaries and uteri after

SC

Cd exposure and determined oocyte quality via assessing the key regulators during

NU

meiotic maturation and fertilization. We found that Cd exposure impeded the mouse oocyte meiotic progression by disrupting the normal spindle assembly, chromosome

MA

alignment and actin cap formation. Besides, exposure to Cd induced oxidative stress with the increased reactive oxygen species and apoptosis levels, leading to abnormal

ED

mitochondrial distribution, insufficient energy supply, and DNA damage, which

CE PT

ultimately led to oocyte quality deterioration. We also analyzed the effects of cadmium on epigenetic modifications, and the levels of 5mC, H3K9me3 and H3K9ac decreased after acute exposure to cadmium. Further experiments showed

AC

that the litter size in Cd-exposed female mice reduced, thereby indicating increased reproductive Cd toxicity. In conclusion, Cd exposure impairs oocyte maturation and fertilization ability induced by oxidative stress, early apoptosis and epigenetic modifications, which lead to the decrease of female fertility. Keywords: heavy metal pollution, oocyte quality, oxidation stress, epigenetic modification

2

ACCEPTED MANUSCRIPT 1. INTRODUCTION With increasing human activities, especially mining and industrial processing, heavy metal pollution has become a worldwide environmental problem affecting organism metabolism in the ecosystem (Zhuang et al., 2009; Qing et al., 2015; Islam

RI PT

et al., 2018). Cadmium (Cd) is one of the most toxic heavy metals because of its typical toxicological properties, such as severe toxicity, universality, persistence, and

SC

transferability (Li et al., 2015). The atmospheric deposition of airborne Cd,

NU

contaminated fertilizers, and sewage sludge on agricultural soil causes the transfer of Cd compounds adsorbed by plants and crops of crucial importance in food chain and

MA

accumulated in the human body (Jarup and Akesson, 2009; Faroon et al., 2012; Rafati Rahimzadeh et al., 2017). Most notably, Cd has an extremely extended half-period

ED

(approximately 20–30 years in humans) and low excretion rate in various body organs,

CE PT

thereby causing many adverse effects on human health (Rani et al., 2014). Recent studies have shown that Cd has diverse toxic effects, including renal dysfunction, oncogenicity, teratogenicity, and endocrine and reproductive system

AC

toxicity (Bernard, 2008; Zhang et al., 2017a). Considering that the reproductive system is highly sensitive to toxin, Cd exists latent toxicity to reproductive system. Studies have shown that after long-term treatment of cadmium, cadmium in Xenopus laevis is accumulated in the reproductive system in addition to accumulating in the liver and kidney (Huang and Liu, 2018). Furthermore, Cd may interfere with ovarian endocrine functions, inhibit follicle growth and development, increase the chance of

3

ACCEPTED MANUSCRIPT

follicle atresia, leading to ovulation failure, defective implantation, spontaneous abortion, and birth defects (Thompson and Bannigan, 2008; Wan et al., 2010; Wang et al., 2015; Zhang et al., 2017b). Studies have measured the concentration of cadmium in follicular fluid is 6.73 ±

RI PT

0.31 μg/L (Zenzes et al., 1995). The follicular fluid component may reflect changes in the microenvironment of the oocyte, which may affect the quality of the developing

SC

oocyte and embryo. Therefore, it is very important and necessary to explore the

NU

effects and mechanisms of cadmium exposure on oocyte quality. Other than that, the impact of Cd exposure on offspring remains unclear. Thus, this study aimed to

MA

explore the in vivo effects of Cd on mouse oocyte maturation, fertilization and offspring development.

ED

In the present study, we administered CdCl2 intraperitoneally to female mice for 7

CE PT

days and explored the toxic impacts on the oocytes. We further examined the effects of Cd on the spindle assembly, cytoskeletal integrity, ROS levels, apoptosis, mitochondrial

distribution,

ATP

content,

DNA/RNA

damage,

epigenetic

AC

modifications and investigated the mechanism underlying the potential deleterious effects of Cd on oocyte quality and the subsequent development competence of the offspring.

2. MATERIALS AND METHODS 2.1 Ethics statement

4

ACCEPTED MANUSCRIPT

All experiments were approved by the Animal Care and Use Committee of Northwest A&F University and were performed in accordance with institutional guidelines. The Kunming strain mice were purchased from the Xi’an Jiaotong University Health Science Center (certificate no.: SCXK [SHAAN] 2017-003) and

RI PT

acclimated with free supplied food and water under a constant temperature (23 ± 2 °C) and a 12h light/12h dark cycle. During oocytes collection, mice were treated

NU

SC

humanely to alleviate the suffering.

2.2 Administration schedule

MA

Since cadmium compounds are not easily digested and absorbed by the intestinal tract of organisms, and intraperitoneal injection can avoid the first-pass effect of

ED

intestinal administration and absorb into the blood circulation directly, we used

CE PT

intraperitoneal injection of cadmium chloride solution to establish acute poisoning model in mice. Cd solutions were prepared by dissolving CdCl2 in 0.9% saline (202908, Sigma, St. Louis, MO, USA). 6- to 8-week-old female mice received daily

AC

intraperitoneal (IP) injection of 0.5, 1, 1.5, 2, 3 or 5 mg Cd/kg body mass for 7 consecutive days (Cd group). For the control group, mice were injected with an equal volume of 0.9% saline (control group). 45 and 42 mice were used for the control group and the 1.5 mg/kg b.w. Cd treatment group, respectively. In the 0.5, 1, 2, 3 mg/kg b.w. and 5mg/kg b.w. Cd-treated group, there were 6 mice analyzed, respectively. The Cd treatment was carried out according to the previous report and

5

ACCEPTED MANUSCRIPT

our preliminary experiment (Acharya et al., 2008).

2.3 Collection and culture of oocytes and embryos in vivo Seven days after the administration, mice were superovulated by injecting 10 IU of

RI PT

pregnant mare serum gonadotropin (PMSG, SHU SHENG HORMONE, Cixi, China), then 48h later injected of 10 IU of human chorionic gonadotropin (hCG, SHU

SC

SHENG HORMONE). After 15 h, mice were sacrificed for cervical dislocation, and the cumulus oocyte complexes were flushed out by tearing the ampulla of fallopian

NU

tube. After the treatment with 300 μg/mL of hyaluronidase (H3506, Sigma) for 4 min

MA

at 37 °C and washing three times with a glass pipette in the M2 medium (M7167,

rates were calculated.

ED

Sigma) to remove the surrounding cumulus cells, the first polar body extrusion (PBE)

CE PT

Female mice were superovulated according to the above method and mated with male mice immediately after hCG injection. The zygotes were collected from the oviducts 0.5 days after seeing the vaginal plugs. After treatment with hyaluronidase,

AC

the embryos were cultured in KSOM+AA medium (IVL04, Caisson, Smithfield, UT, USA) at 37 °C under 5% carbon dioxide in humidified air. Finally, blastocysts were collected at 4.5 days and the blastocyst rate was calculated.

2.4 Breeding assay The 3 and 5 mg Cd/kg body mass exposed mice were mated with fertile adult male

6

ACCEPTED MANUSCRIPT

mice overnight. Next morning, if female mice with vaginal plugs were observed, then copulation was successful. The litter size, sex ratio, and postnatal weights of the neonatal mice were also counted.

RI PT

2.5 Histological examination

The estrus cycles of mice were observed by external observation, and 3 female

SC

mice with normal estrus cycle for each group were selected. Mice were treated with

NU

PMSG/hCG for simultaneous estrus and superovulation, the 1.5 mg Cd/kg b.w. group and control groups were administered for injection for 7 days and the uterus and

MA

ovaries were collected at the estrus of the subsequent estrus cycle. Ovaries and uteri were fixed in 4% paraformaldehyde, embedded in paraffin, dehydrated in ethanol,

ED

and then serially sectioned into 5μm thickness. Tissue sections were stained with

CE PT

hematoxylin and eosin (H&E) following routine procedures for histopathological assessment by light microscopy (Andrew H. Fischer, 2008; Lin et al., 2018)(Carl

AC

Zeiss LSM 700 META, Jena, Germany).

2.6 In vitro fertilization Spermatozoa were collected from the caudal epididymis of adult male mice, followed by capacitation in G-IVF (10136, Vitrolife, Goteborg, Sweden) for 1 hour at 37 °C in humidified atmosphere with 5% CO2 and added to ovulated oocytes at a concentration of 2×106/mL sperm in 200 μL of G-IVF microdrops under paraffin oil

7

ACCEPTED MANUSCRIPT

for 6 hour (37 °C, 5% CO2). The formation of two pronuclei was considered as successful fertilization.

2.7 Immunofluorescence staining in oocytes

RI PT

The staining of oocytes is mainly through fixation, permeabilization, blocking, primary antibody incubation, and secondary antibody incubation. Detailed procedures

SC

refer to the previous articles in the laboratory (Lu et al., 2019). The oocytes were

NU

incubated with anti-DNA/RNA damage (ab62623, AB_940049, 1:800, Abcam, Cambridge, UK), anti-alpha tubulin (AT819, 1:500, Beyotime, Haimen, China),

MA

anti-phalloidin TRITC antibody (P5282, 1:200, Sigma), anti‐H3K9me3 antibody (ab8898, AB_306848, 1:1000, Abcam), anti-H3K9ac (ab4441, AB_2118292, 1:500,

ED

Abcam) and anti-mouse FR4-FITC antibody (125005, AB_1134204, 1:100,

CE PT

BioLegend, CA, USA) for 12 h at 4 °C. Alexa Fluor 488-labeled goat anti-rabbit IgG and 555-labeled goat anti-mouse IgG were obtained from TransGen (Beijing, China). After

washing

by

PBS

extensively,

oocytes

were

counterstained

with

AC

4,6-diamidino-2-phenylindole (C1006, DAPI, Beyotime) for 4–5 min. Finally, oocytes were mounted onto glass slides and observed under a confocal laser scanning microscope (Revolution WD, Andor, Belfast, Northern Ireland ) (Su et al., 2012; Su et al., 2015b). Deoxyribonucleic acid 5mC (33D3, anti-5mC, 1:1000, Eurogentec, Angers,France) and 5hmC (39770, anti-5hmC, 1:1000, Active Motif, Carlsbad,CA,USA) staining

8

ACCEPTED MANUSCRIPT

were strictly conducted in accordance with our previous works (An et al., 2019). In short, before blocking, the oocytes were treated with 4 N HCl for 5 min and then neutralized with Tris-HCl (pH 8.5) for 5 min. Other steps were the same as our above work, except stained with DAPI 10 min.

RI PT

To quantify fluorescence intensity, we acquired oocytes both from control and Cd group mice by setting up the same scanning parameters of confocal microscope.

NU

used as described previously (Su et al., 2015a).

SC

Image-Pro Plus software (v6.0, Media Cybernetics, Silver Spring, MD, USA) was

MA

2.8 Measurement of intracellular ROS and glutathione (GSH) The intracellular ROS levels in the denuded oocytes were measured using

ED

CellROX™ Green Reagent (C10444, 1:500, Invitrogen, Eugene, OR, USA). In brief,

CE PT

oocytes were incubated with CellROX reagent for 30 min at 37 °C. Afterward, the oocytes were washed three times with PBS and placed on glass slides. Fluorescent intensity was assessed by the fluorescence microscope (Zeiss LSM 700 META).

AC

In like manner, the intracellular GSH level was measured using a 20 μM ThiolTracker Violet (T10096, Invitrogen).

2.9 Annexin-V staining The externalization of apoptotic cells phosphatidylserine was detected by Annexin V-FITC Apoptosis Detection Kit (C1062S, Beyotime). A total of 195 μL of Annexin

9

ACCEPTED MANUSCRIPT V-FITC binding solution and 5 μL of Annexin V-FITC were lightly mixed. Oocytes were incubated with dye solution for 15 minutes and then washed twice with PBS. The fluorescent intensity was measured by Andor Revolution WD confocal system.

RI PT

2.10 Staining of mitochondria in oocytes

The activities of mitochondria in the oocytes were evaluated with the Mito Tracker

SC

Green (C1048, Beyotime), which selectively labels live mitochondria. After washing

NU

twice, MII oocytes were incubated with 200 nM Mito Tracker Green in a M2 medium for 30 min at 37 °C in the dark. The images of each oocyte were captured by a

MA

confocal microscope (Zeiss LSM 700 META).

ED

2.11 ATP content assay in oocytes

CE PT

After being fixed for 20 min, Cumulus-denuded oocytes were incubated in M2 medium supplemented with 500 nM BODIPY FL ATP (A12410, Invitrogen, Eugene, OR, USA) for one hour at 20 °C in the dark, then mounted on cover slips, and

AC

observed under a fluorescence microscope (Leica DMi8, Wetzlar, Germany).

2.12 Statistical analysis At least 30 oocytes from a Cd-exposed mouse were examined in one experiment. Values were mean ± SEM from triplicate experiments. The total number of oocytes participating in the experiment was denoted by n. When multiple comparisons were

10

ACCEPTED MANUSCRIPT performed, the data were analyzed by one-way ANOVA, followed by LSD’s post hoc test. Student's t-test was used when only two comparisons were made, which was provided by SPSS 19.0 software (SPSS Inc., Chicago, IL, USA). A p value of <0.05

RI PT

was considered significant.

SC

3. RESULTS

3.1 Histological analysis of cadmium caused ovarian and uterine decline in mice

NU

First of all, variation of follicle morphology and uterine development were

MA

observed under a microscope. During estrus, the endometriums were in a proliferative phase, and there were many mature follicles before ovulation in the ovary. Few

ED

primordial follicles and mature follicles were found, and more atresia follicles were

CE PT

observed in the Cd group, which showed oocyte atrophy and dissolution, and zona pellucida shrinkage. Morphologically, compared with the control group (Figure 1a), the oocyte nuclear deviation, shrinkage deformation, and zona pellucida collapse were

AC

observed; some granulosa cells loosed and fell into the follicular cavity in the atresia follicle shown by the arrow in Figure 1b. Moreover, uterus sections of the control group (Fig.1c) showed normal endometrial structure (E) with intact endometrial epithelium and developed uterine gland (UG) in the lamina propria. However, in the experimental group (Figure 1d), decreased plica of uterine formed conjunctions, embryonic connective tissue cells, lymphocytes increased, and myometrium (M) significantly thickened. 11

ACCEPTED MANUSCRIPT

3.2 CdCl2 compromised oocyte maturation and fertilization potential Given that oocyte quality is an essential factor of female fertility, we first compared the oocyte maturation and fertilization ability. Meiotic progression was assessed by

RI PT

the calculation of first polar body extrusion rate in oocytes isolated from mice exposed to different IP doses of CdCl2, that is 0.5, 1, 1.5, and 2 mg/kg body mass/day,

SC

and the average maturation rates were 71.65% ± 1.20% (n = 187), 67.46% ± 1.82% (n

NU

= 160), 51.24% ± 1.56% (n = 213), and 39.96% ± 1.18% (n = 193), respectively, compared with the control group (90.04% ± 1.07%, n = 235; Figure 2a). The PBE rate

MA

of oocytes exposed to Cd mice was obviously decreased, thereby suggesting that Cd led to meiotic arrest during maturation.

ED

Furthermore, the same doses were administered to evaluate the in vitro

CE PT

developmental rates at two-cell embryo. The Cd groups exhibited an increasingly decreased fertilization rate with increased CdCl2 dose, particularly in the high-dose group (1.5 mg/kg b.w.; 87.9 ± 1.74% [n = 134] vs. 50.0% ± 1.48% [n = 115]; Figure

AC

2b). The treatment with 1.5 mg Cd/kg/day was selected for subsequent investigations since this concentration not only led to meiotic arrest but also allowed a proportion of oocytes to develop to MII stage for other investigations.

3.3 Cd exposure impairs embryo development and reduces the female fertility Next, we examined the development of embryos from cadmium-treated female

12

ACCEPTED MANUSCRIPT

mice and the control female mice. After 4.5 days in culture, 51.85% of in vivo fertilized zygotes reached the blastocyst stage in the Cd group, while 82.61% did so in the control group. And more embryos were found dead or fragmented (Figure 2c) in the Cd group. Further, we isolated oocytes from the oviduct and uterus exposed to

RI PT

cadmium for in vitro fertilization. The blastocyst development rate was significantly decreased in the Cd group than that of the control group 55.17% vs 86.11%, p < 0.05).

SC

We postulated from these results that the embryo development of cadmium-treated

NU

female mouse oocytes was impaired.

We assessed the fecundity of CdCl2-exposed female mice, which were caged with

MA

male mice. Sure though, the number of litters produced by the female mice in 3 mg Cd/kg group was remarkably lower than the number produced by the controls (11.83

ED

± 0.71 vs. 15.17 ± 0.63; Figure 2d), thereby indicating that female fertility was

CE PT

impaired. In conclusion, Cd impairs female fertility by compromising the oocyte

AC

maturation and fertilization abilities.

3.4 Cd exposure disturbed meiotic spindle assembly and actin accumulation and distribution during polar body extrusion Spindle and chromosomes were visualized by immunofluorescence staining with anti-α-tubulin FITC antibody and DAPI respectively. The results showed that 85% of the oocytes showed abnormal spindles, and approximately 70% of the oocytes displayed misaligned chromosomes in the Cd group (p < 0.05; Figure 3b and c). 13

ACCEPTED MANUSCRIPT

In the process of oocyte maturation, chromosomes moved to the oocyte cortex and stimulate CGFD formation in which actin was polymerized to form an actin cap. In the later stages of meiosis, the actin-rich region protruded outward, while the contraction ring began to contract, and then the polar body was discharged. In general,

RI PT

F-actin is labeled with phalloidin. As shown in Figure 4, the actin caps formed normally in the CGFD region in the control group, and the aberrant actin cap

NU

SC

formation rate was increased in the Cd group (p < 0.05).

3.5 Cd reduces the protein level of Juno in the mouse egg membrane

MA

During fertilization, spermatozoon binds and penetrates the zona pellucida and then fuses with the oocyte membrane via Juno binding to IZUMO1, which is a sperm

ED

cell surface receptor, to complete the sperm–egg recognition (Bianchi et al., 2014; Dai

CE PT

et al., 2017). Juno distributed evenly on the control oocyte membrane but was rarely distributed in Cd group. The fluorescence intensity of Juno in the Cd-exposed eggs

AC

was obviously lower than control eggs (p < 0.05; Figure 5).

3.6 Cd exposure results in decreased the GSH, ROS generation, and early apoptosis To determine the mechanisms underlying the effect of Cd exposure on oocyte quality, we analyzed the ROS and GSH levels in mouse oocytes. The ROS level was markedly increased in the Cd group (p < 0.05; Figure 6a and b). By contrast, the GSH

14

ACCEPTED MANUSCRIPT

levels were markedly lower in the Cd groups than those in the control group (p < 0.05; Figure 6a and c). Since the high ROS level is prone to apoptosis, we next detected early apoptosis levels. A green circle fluorescent signal locating on the external cellular membrane of

RI PT

the oocyte was observed, defined as Annexin V positive, while the rest was defined as negative. The results showed that green fluorescence signal was clearly observed on

SC

the plasma membrane of the oocytes in Cd group but was hardly detected in the

NU

control group. (p < 0.05; Figure 6d and e). This result indicated that Cd increased the

MA

early apoptosis levels.

3.7 ATP contents decreased due to mitochondria dysfunction in the Cd-treated

ED

oocytes

CE PT

We investigated the mitochondrial activity and distribution with Mito Tracker Green staining in MII oocytes. As shown in Figure 7a, the difference between the treatment and control groups as measured by the fluorescence intensity was

AC

significant (p < 0.05). Moreover, the mitochondria in the control group were uniformly distributed in the oocyte cytoplasm. However, the mitochondria in the experimental group only exhibited peripheral distribution with overall weak fluorescence signals. The normal distribution rate of mitochondria was also evidently lower (p < 0.05). Accordingly, the ATP contents in the Cd group were lower than those in the

15

ACCEPTED MANUSCRIPT

control group (p < 0.01; Figure 7d).

3.8 Cd increased the DNA damage level caused by oxidative stress It is known that excessive ROS production can cause damage to mitochondrial

RI PT

proteins and DNA. Oxo-8-dG is an excellent marker for DNA damage produced by oxidants. The 8-oxo-dG levels were apparently higher in the Cd group (p < 0.01;

NU

SC

Figure 8)

3.9 Effects of Cd on the epigenetic modifications in mouse MII oocytes

MA

The levels of the global histone lysine methylations (H3K9me3), acetylations (H3K9ac), the global DNA methylation (5mC) and hydroxymethylation (5hmC) were

ED

measured in Cd and control groups. As shown in Figure 9, the global H3K9me3 and

CE PT

H3K9ac levels were lower in the Cd oocytes than those in the control oocytes (p < 0.05). Compared with those in the control oocytes, the fluorescence intensities of 5mC in the treated oocytes were significantly decreased (p < 0.05; Figure 10). However,

AC

there was no significant difference in the 5hmC level between the control and Cd oocytes.

4. DISCUSSION In recent years, heavy metal pollution has become of great concern. In the general

16

ACCEPTED MANUSCRIPT

population, exposure to Cd mainly through food, water and smoking, the average dietary intake of cadmium in urban residents in China is estimated to be 15.5 μg/day, the mean blood level of cadmium is 0.74 μg/L, and the average cadmium content in urine is 0.34 μg/g (Wang et al., 2012; Ikeda et al., 2018). Recent advances on the

RI PT

basis of molecular cell biology have shown that Cd has multiplex influences on cells (Waisberg et al., 2003; Bertin and Averbeck, 2006). Cd affects cell cycle progression,

SC

proliferation, differentiation, DNA replication, and repair, as well as apoptotic

NU

pathways (Dong et al., 2001; Fang et al., 2002; Yang et al., 2004; Oh and Lim, 2006). In this study, we investigated the effects of cadmium treatment of female mice on

MA

meiotic maturation of their oocytes and sub-sequent embryo development, and the possible mechanisms responsible for the adverse effects.

ED

Acute exposure of cadmium is known to affect the hypothalamic-pituitary-ovarian

CE PT

axis and decrease FSH, LH and basal progesterone levels, which are responsible for ovarian development and ovulation (Paksy et al., 1989). Histologic and morphometric variations reflected the toxic effects of cadmium on mouse ovaries, and the arrested

AC

development of ovaries induced by Cd exposure, mainly manifested as the decrease of mature follicles and the increase of atresia follicles. Besides, cadmium is an environmental pollutant with a hormone-like effect that activates the estrogen receptor and increases the thickness of the mouse endometrium (Johnson et al., 2003; Kluxen et al., 2012). These findings demonstrated that adverse effects of cadmium exposure were found in female reproductive system. It also reminded us that changes in the

17

ACCEPTED MANUSCRIPT

oocyte microenvironment may affect the quality of developing oocytes and embryos. In this study, we found that cadmium exposure had a strong influence on oocyte maturation, fertilization ability and subsequent embryo development. The decreased PBE rate implied that oocyte meiosis arrest in the MI stage. The fertilization rate

RI PT

gradually decreased with the increase in the CdCl2 dose. Our results suggested that cadmium exposure had an effect on the development of fertilized embryos in vivo and

SC

in vitro. In particular, embryo development from in vitro fertilization was also

NU

impaired, despite the potential impacts of cadmium on sperm motility and fertilization through contact with oviduct and uterus (Zhao et al., 2017), suggesting that the

MA

negative effects of cadmium exposure sustained on oocytes. The litter sizes of female mice exposed to cadmium were further evidence of this. The difference in the number

ED

of newborn mice born at the 1.5 mg/kg dose was insignificant. However, at the 3

CE PT

mg/kg dose, the number of newborn mice decreased significantly, the body weight was lower, and increased slowly with the growth. This finding was even stronger as it suggested that Cd exposure has a significant negative effect on female fertility.

AC

To elucidate the mechanism underlying low maturation rate, we examined the spindle microtubules organization and actin filaments, two key cytoskeletal components in oocytes. Actin filaments regulate spindle movements, initiate polar body extrusion and cell division, and establish cortical polarity; meanwhile, microtubules form meiotic spindle to drive oocyte chromosome aggregation and separation (Yi and Li, 2012; Zhu et al., 2016; Laband et al., 2017). In this study, we

18

ACCEPTED MANUSCRIPT

found high frequencies of spindle and chromosome abnormalities, which suggested that cadmium exposure led to abnormal oocyte meiosis progression by disrupting spindle morphology and chromosome arrangement. The present results also showed that Cd exposure disrupted actin distribution, which may be partly responsible for the

RI PT

failure of polar body extrusion, resulting in low oocyte maturation rates. Fertilization is an essential biologic process in sexual reproduction and comprises a

SC

series of molecular interactions between the sperm cells and egg (Aydin et al., 2016).

NU

Fertilization ability is an important criterion for oocyte quality assessment. Thus, we investigated the possible mechanisms responsible for fertilization defects. The

MA

IZUMO1 oocyte receptor Juno is considered to be an important factor in the interaction and fusion of sperm and oocytes (Bianchi et al., 2014; Aydin et al., 2016;

ED

Miao et al., 2018). These results in this study revealed that the amount of Juno was

CE PT

reduced in the Cd group. Hence, we speculated that part of the reason for the failure of fertilization caused by Cd exposure is due to Juno deficiency. Several studies showed that Cd can induce the increase in ROS level in many

AC

kinds of cells (Koizumi et al., 1996; Szuster-Ciesielska et al., 2000; Shih et al., 2004). Likewise, our results showed that Cd exposure induced a prominently increased ROS level inside the oocytes. Similar to other studies, we found the intracellular GSH content in the Cd group decreased obviously. The mechanisms underlying acute Cd toxicity are involved in the decrease in GSH and protein-bound sulfhydryl groups, thereby leading to an increase in ROS (Dudley and Klaassen, 1984; Liu et al., 2009).

19

ACCEPTED MANUSCRIPT

To determine whether excessive reactive oxygen species affects the oocyte quality, we examined the ATP content, mitochondrial distribution, 8-oxodG level, and the early apoptosis rate. Cd toxicity brings about a decrease in intracellular antioxidants and the increase in

RI PT

mitochondrial ROS, which indirectly causes oxidative stress (Waisberg et al., 2003). Redundant ROS in turn causes mitochondrial dysfunction. In the present study, Cd

SC

exposure remarkably reduced the activities of mitochondria and ATP content in

NU

oocytes. Low ATP levels predict a decrease in mitochondrial function, thereby indicating insufficient energy supply in critical events such as spindle assembly, actin

MA

cap formation, and fertilization activity. This phenomenon can be part of the cause of oocyte meiosis arrest and failure to fertilize.

ED

Apoptotic cell death, which is another criterion for oocyte quality evaluation, is an

CE PT

important factor that threatens the subsequent embryonic development. Our results showed that the apoptotic oocyte rate was dramatically higher in the Cd group. This finding indicated that Cd can cause mitochondrial dysfunction through ROS

AC

production, thereby leading to apoptosis. To explore whether oxidative stress can cause DNA damage further, we examined the levels of 8-oxo-dG, which is one of the major products of DNA oxidation in oocytes (de Souza-Pinto et al., 2001). The 8-oxodG contents were significantly higher in the Cd group. Thus, we speculated that Cd can cause DNA damage through oxidative stress, thereby affecting the oocyte genome stability and ultimately affecting

20

ACCEPTED MANUSCRIPT

oocyte nuclear maturation. Increasing evidence demonstrated that epigenetic modifications could be changed after exposure to environmental chemicals (Baccarelli and Bollati, 2009), and identified several classes of toxicants that modify epigenetic marks, including metals,

RI PT

peroxisome proliferators, air pollutants and endocrine-disrupting/reproductive toxicants (Mass and Wang, 1997; Li et al., 2003; Dolinoy et al., 2007). Several

SC

epigenetic mechanisms, including DNA methylation, histone modifications, and

NU

microRNA (miRNA) expression can regulate gene expression and are essential for the developmental competence of oocytes (Reik et al., 2001; Liu et al., 2004; Racedo et In growing oocytes, trimethylation of histone H3K9 colocalizes with the

MA

al., 2009).

concentrated DNA domain and is involved in the suppression of gene expression in

ED

the euchromatin and the formation of heterochromatin in the oocyte genome, which

CE PT

would lead to the silencing of the entire genome during oocyte growth (Erhardt et al., 2003; Kageyama et al., 2007). Acetylation of histone 3 lysine 9 (H3K9ac) has an important role in the regulation of gene expression and the establishment of an open

AC

chromatin configuration (Huang et al., 2012). Defective deacetylation of H3K9 in the oocytes is correlated with misaligned chromosomes, which results in a high incidence of aneuploidy and embryo death (Akiyama et al., 2006). Previously, it has been reported in the context of cadmium carcinogenesis that cadmium-induced cell proliferation in K562 cells was accompanied by global DNA hypomethylation (Huang et al., 2008). Our results showed that acute Cd exposure decreased the H3K9me3 and

21

ACCEPTED MANUSCRIPT

H3K9ac level, which may result in late DNA replication and aneuploidy after fertilization. Therefore, Cd can affect epigenetic modifications, which were shown by the altered DNA and histone methylation levels for mammalian oocyte maturation. This may be partly responsible for the failure of embryo development when the

RI PT

oocytes were from cadmium-treated female mice. In addition, changes in epigenetic modifications can cause changes in the transcriptional pattern of the gene, which is

SC

likely to be related to other cellular activities such as oocyte oxidation and apoptosis.

NU

In this study, mice were injected intraperitoneally with 1.5 mg Cd/kg/bw, which was equivalent to human body 122 μg/kg/bw according to the dose conversion

MA

between mice and humans (Nair and Jacob, 2016). This dose is close to the human environment exposure in the contaminated area,140-260 µg/day ((IPCS), 1992).

ED

Collectively, our findings provide an implication that Cd exposure has a

CE PT

detrimental effect on mouse oocyte maturation, fertilization, embryo development, and fertility by affecting meiosis spindle dynamics, actin cap formation, anti-oxidative stress, early apoptosis and epigenetic modifications. In conclusion, cadmium exposure

AC

reduces female fertility by impairing oxidative stress and early apoptosis-induced oocyte maturation and fertilization.

ACKNOWLEDGEMENTS This work was supported by the National Key R&D Program of China (2018YF D0502304),

National Natural Science Foundation of China (31873001) and Qinghai

22

ACCEPTED MANUSCRIPT

Science and Technology Project (2017-NK-111, 2018-NK-132). The authors wish to thank Dr. Bo Xiong and Dr. Shaochen Sun from Nanjing Agricultural University, Dr. Yiliang Miao from Huazhong Agricultural University for their generous technical

RI PT

assistance.

CONFLICT OF INTERESTS

NU

SC

The authors declare that there are no conflicts of interest.

AUTHOR CONTRIBUTIONS

MA

J. S. and Y. Z. designed the research; Y. C., J. Z., T. W., X. J., and H. J. performed the experiments; and Y. C., Y. Z., and J. S. analyzed the data and wrote the

CE PT

ED

manuscript. Y. C. and J. Z. contributed equally to this work.

Data Availability Statement

Data sharing is not applicable to this article as no new data were created or

AC

analyzed in this study.

23

ACCEPTED MANUSCRIPT References

AC

CE PT

ED

MA

NU

SC

RI PT

(IPCS), I.P.o.C.S., 1992. Environmental Health Criteria 134. Cadmium. In Organization, W.H., (Ed.), 3. Sources of Human and Environmental Exposure, Geneva, pp. Acharya, U.R., Mishra, M., Patro, J., Panda, M.K., 2008. Effect of vitamins C and E on spermatogenesis in mice exposed to cadmium. Reproductive toxicology 25, 84-88. Akiyama, T., Nagata, M., Aoki, F., 2006. Inadequate histone deacetylation during oocyte meiosis causes aneuploidy and embryo death in mice. Proceedings of the National Academy of Sciences of the United States of America 103, 7339-7344. An, Q., Peng, W., Cheng, Y., Lu, Z., Zhou, C., Zhang, Y., Su, J., 2019. Melatonin supplementation during in vitro maturation of oocyte enhances subsequent development of bovine cloned embryos. Journal of cellular physiology 234, 17370-17381. Andrew H. Fischer, K.A.J., Jack Rose, Rolf Zeller, 2008. Hematoxylin and Eosin Staining of Tissue and Cell Sections., Cold Spring Harbor Laboratory Protocols, pp. Aydin, H., Sultana, A., Li, S., Thavalingam, A., Lee, J.E., 2016. Molecular architecture of the human sperm IZUMO1 and egg JUNO fertilization complex. Nature 534, 562-565. Baccarelli, A., Bollati, V., 2009. Epigenetics and environmental chemicals. Current opinion in pediatrics 21, 243-251. Bernard, A., 2008. Cadmium & its adverse effects on human health. The Indian journal of medical research 128, 557-564. Bertin, G., Averbeck, D., 2006. Cadmium: cellular effects, modifications of biomolecules, modulation of DNA repair and genotoxic consequences (a review). Biochimie 88, 1549-1559. Bianchi, E., Doe, B., Goulding, D., Wright, G.J., 2014. Juno is the egg Izumo receptor and is essential for mammalian fertilization. Nature 508, 483-487. Dai, X., Lu, Y., Zhang, M., Miao, Y., Zhou, C., Cui, Z., Xiong, B., 2017. Melatonin improves the fertilization ability of post-ovulatory aged mouse oocytes by stabilizing ovastacin and Juno to promote sperm binding and fusion. Human reproduction 32, 598-606. de Souza-Pinto, N.C., Eide, L., Hogue, B.A., Thybo, T., Stevnsner, T., Seeberg, E., Klungland, A., Bohr, V.A., 2001. Repair of 8-oxodeoxyguanosine lesions in mitochondrial dna depends on the oxoguanine dna glycosylase (OGG1) gene and 8-oxoguanine accumulates in the mitochondrial dna of OGG1-defective mice. Cancer research 61, 5378-5381. Dolinoy, D.C., Huang, D., Jirtle, R.L., 2007. Maternal nutrient supplementation counteracts bisphenol A-induced DNA hypomethylation in early development. 24

ACCEPTED MANUSCRIPT

AC

CE PT

ED

MA

NU

SC

RI PT

Proceedings of the National Academy of Sciences of the United States of America 104, 13056-13061. Dong, S.Y., Shen, H.M., Ong, C.N., 2001. Cadmium-induced apoptosis and phenotypic changes in mouse thymocytes. Mol Cell Biochem 222, 11-20. Dudley, R.E., Klaassen, C.D., 1984. Changes in hepatic glutathione concentration modify cadmium-induced hepatotoxicity. Toxicology and applied pharmacology 72, 530-538. Erhardt, S., Su, I.H., Schneider, R., Barton, S., Bannister, A.J., Perez-Burgos, L., Jenuwein, T., Kouzarides, T., Tarakhovsky, A., Surani, M.A., 2003. Consequences of the depletion of zygotic and embryonic enhancer of zeste 2 during preimplantation mouse development. Development 130, 4235-4248. Fang, M.Z., Mar, W., Cho, M.H., 2002. Cadmium affects genes involved in growth regulation during two-stage transformation of Balb/3T3 cells. Toxicology 177, 253-265. Faroon, O., Ashizawa, A., Wright, S., Tucker, P., Jenkins, K., Ingerman, L., Rudisill, C., 2012. Toxicological Profile for Cadmium. Agency for Toxic Substances and Disease Registry (US), Atlanta (GA), pp. Huang, D., Zhang, Y., Qi, Y., Chen, C., Ji, W., 2008. Global DNA hypomethylation, rather than reactive oxygen species (ROS), a potential facilitator of cadmium-stimulated K562 cell proliferation. Toxicology letters 179, 43-47. Huang, J., Li, T., Ding, C.H., Brosens, J., Zhou, C.Q., Wang, H.H., Xu, Y.W., 2012. Insufficient histone-3 lysine-9 deacetylation in human oocytes matured in vitro is associated with aberrant meiosis. Fertil Steril 97, 178-184 e173. Huang, M., Liu, Z., 2018. Cadmium Accumulation and Distribution in Xenopus Laevis after Long-Term Cadmium Treatment. IOP Conference Series: Materials Science and Engineering 452, 022159. Ikeda, M., Nakatsuka, H., Watanabe, T., Shimbo, S., 2018. Estimation of dietary intake of cadmium from cadmium in blood or urine in East Asia. Journal of trace elements in medicine and biology : organ of the Society for Minerals and Trace Elements 50, 24-27. Islam, M.A., Romic, D., Akber, M.A., Romic, M., 2018. Trace metals accumulation in soil irrigated with polluted water and assessment of human health risk from vegetable consumption in Bangladesh. Environmental geochemistry and health 40, 59-85. Jarup, L., Akesson, A., 2009. Current status of cadmium as an environmental health problem. Toxicol Appl Pharm 238, 201-208. Johnson, M.D., Kenney, N., Stoica, A., Hilakivi-Clarke, L., Singh, B., Chepko, G., Clarke, R., Sholler, P.F., Lirio, A.A., Foss, C., Reiter, R., Trock, B., Paik, S., Martin, M.B., 2003. Cadmium mimics the in vivo effects of estrogen in the uterus and mammary gland. Nature medicine 9, 1081-1084. Kageyama, S., Liu, H., Kaneko, N., Ooga, M., Nagata, M., Aoki, F., 2007. Alterations in epigenetic modifications during oocyte growth in mice. Reproduction 133, 25

ACCEPTED MANUSCRIPT

AC

CE PT

ED

MA

NU

SC

RI PT

85-94. Kluxen, F.M., Hofer, N., Kretzschmar, G., Degen, G.H., Diel, P., 2012. Cadmium modulates expression of aryl hydrocarbon receptor-associated genes in rat uterus by interaction with the estrogen receptor. Archives of toxicology 86, 591-601. Koizumi, T., Shirakura, H., Kumagai, H., Tatsumoto, H., Suzuki, K.T., 1996. Mechanism of cadmium-induced cytotoxicity in rat hepatocytes: cadmium-induced active oxygen-related permeability changes of the plasma membrane. Toxicology 114, 125-134. Laband, K., Le Borgne, R., Edwards, F., Stefanutti, M., Canman, J.C., Verbavatz, J.M., Dumont, J., 2017. Chromosome segregation occurs by microtubule pushing in oocytes. Nature communications 8, 1499. Li, P., Lin, C., Cheng, H., Duan, X., Lei, K., 2015. Contamination and health risks of soil heavy metals around a lead/zinc smelter in southwestern China. Ecotoxicology and environmental safety 113, 391-399. Li, S., Hursting, S.D., Davis, B.J., McLachlan, J.A., Barrett, J.C., 2003. Environmental exposure, DNA methylation, and gene regulation: lessons from diethylstilbesterol-induced cancers. Annals of the New York Academy of Sciences 983, 161-169. Lin, J., Zhao, H.S., Qin, L., Li, X.N., Zhang, C., Xia, J., Li, J.L., 2018. Atrazine Triggers Mitochondrial Dysfunction and Oxidative Stress in Quail ( Coturnix C. coturnix) Cerebrum via Activating Xenobiotic-Sensing Nuclear Receptors and Modulating Cytochrome P450 Systems. Journal of agricultural and food chemistry 66, 6402-6413. Liu, H., Kim, J.M., Aoki, F., 2004. Regulation of histone H3 lysine 9 methylation in oocytes and early pre-implantation embryos. Development 131, 2269-2280. Liu, J., Qu, W., Kadiiska, M.B., 2009. Role of oxidative stress in cadmium toxicity and carcinogenesis. Toxicology and applied pharmacology 238, 209-214. Lu, Z., Zhang, C., Han, C., An, Q., Cheng, Y., Chen, Y., Meng, R., Zhang, Y., Su, J., 2019. Plasticizer Bis(2-ethylhexyl) Phthalate Causes Meiosis Defects and Decreases Fertilization Ability of Mouse Oocytes in Vivo. Journal of agricultural and food chemistry. Mass, M.J., Wang, L., 1997. Arsenic alters cytosine methylation patterns of the promoter of the tumor suppressor gene p53 in human lung cells: a model for a mechanism of carcinogenesis. Mutation research 386, 263-277. Miao, Y., Zhou, C., Bai, Q., Cui, Z., ShiYang, X., Lu, Y., Zhang, M., Dai, X., Xiong, B., 2018. The protective role of melatonin in porcine oocyte meiotic failure caused by the exposure to benzo(a)pyrene. Human reproduction 33, 116-127. Nair, A.B., Jacob, S., 2016. A simple practice guide for dose conversion between animals and human. Journal of basic and clinical pharmacy 7, 27-31. Oh, S.H., Lim, S.C., 2006. A rapid and transient ROS generation by cadmium triggers apoptosis via caspase-dependent pathway in HepG2 cells and this is inhibited 26

ACCEPTED MANUSCRIPT

AC

CE PT

ED

MA

NU

SC

RI PT

through N-acetylcysteine-mediated catalase upregulation. Toxicol Appl Pharm 212, 212-223. Paksy, K., Varga, B., Horvath, E., Tatrai, E., Ungvary, G., 1989. Acute effects of cadmium on preovulatory serum FSH, LH, and prolactin levels and on ovulation and ovarian hormone secretion in estrous rats. Reproductive toxicology 3, 241-247. Qing, X., Yutong, Z., Shenggao, L., 2015. Assessment of heavy metal pollution and human health risk in urban soils of steel industrial city (Anshan), Liaoning, Northeast China. Ecotoxicology and environmental safety 120, 377-385. Racedo, S.E., Wrenzycki, C., Lepikhov, K., Salamone, D., Walter, J., Niemann, H., 2009. Epigenetic modifications and related mRNA expression during bovine oocyte in vitro maturation. Reproduction, fertility, and development 21, 738-748. Rafati Rahimzadeh, M., Rafati Rahimzadeh, M., Kazemi, S., Moghadamnia, A.A., 2017. Cadmium toxicity and treatment: An update. Caspian journal of internal medicine 8, 135-145. Rani, A., Kumar, A., Lal, A., Pant, M., 2014. Cellular mechanisms of cadmium-induced toxicity: a review. Int J Environ Heal R 24, 378-399. Reik, W., Dean, W., Walter, J., 2001. Epigenetic reprogramming in mammalian development. Science 293, 1089-1093. Shih, C.M., Ko, W.C., Wu, J.S., Wei, Y.H., Wang, L.F., Chang, E.E., Lo, T.Y., Cheng, H.H., Chen, C.T., 2004. Mediating of caspase‐independent apoptosis by cadmium through the mitochondria‐ROS pathway in MRC‐5 fibroblasts. J Cell Biochem 91, 384-397. Su, J., Wang, Y., Li, R., Peng, H., Hua, S., Li, Q., Quan, F., Guo, Z., Zhang, Y., 2012. Oocytes selected using BCB staining enhance nuclear reprogramming and the in vivo development of SCNT embryos in cattle. Plos One 7, e36181. Su, J., Wang, Y., Xing, X., Zhang, L., Sun, H., Zhang, Y., 2015a. Melatonin significantly improves the developmental competence of bovine somatic cell nuclear transfer embryos. Journal of pineal research 59, 455-468. Su, J.M., Wang, Y.S., Xing, X.P., Zhang, L., Sun, H.Z., Zhang, Y., 2015b. Melatonin significantly improves the developmental competence of bovine somatic cell nuclear transfer embryos. Journal of Pineal Research 59, 455-468. Szuster-Ciesielska, A., Stachura, A., Slotwinska, M., Kaminska, T., Sniezko, R., Paduch, R., Abramczyk, D., Filar, J., Kandefer-Szerszen, M., 2000. The inhibitory effect of zinc on cadmium-induced cell apoptosis and reactive oxygen species (ROS) production in cell cultures. Toxicology 145, 159-171. Thompson, J., Bannigan, J., 2008. Cadmium: toxic effects on the reproductive system and the embryo. Reproductive toxicology 25, 304-315. Waisberg, M., Joseph, P., Hale, B., Beyersmann, D., 2003. Molecular and cellular mechanisms of cadmium carcinogenesis. Toxicology 192, 95-117. Wan, X., Zhu, J., Zhu, Y., Zhu, Y., Ma, X., Zheng, Y., Wang, F., Liu, Z., Zhang, T., 27

ACCEPTED MANUSCRIPT

AC

CE PT

ED

MA

NU

SC

RI PT

2010. Rat ovarian follicle bioassay reveals adverse effects of cadmium chloride (CdCl2) exposure on follicle development and oocyte maturation. Toxicology and industrial health 26, 609-618. Wang, Y., Ou, Y.L., Liu, Y.Q., Xie, Q., Liu, Q.F., Wu, Q., Fan, T.Q., Yan, L.L., Wang, J.Y., 2012. Correlations of trace element levels in the diet, blood, urine, and feces in the Chinese male. Biological trace element research 145, 127-135. Wang, Y., Wang, X., Wang, Y., Fan, R., Qiu, C., Zhong, S., Wei, L., Luo, D., 2015. Effect of Cadmium on Cellular Ultrastructure in Mouse Ovary. Ultrastructural pathology 39, 324-328. Yang, P.M., Chiu, S.J., Lin, K.A., Lin, L.Y., 2004. Effect of cadmium on cell cycle progression in Chinese hamster ovary cells. Chemico-biological interactions 149, 125-136. Yi, K., Li, R., 2012. Actin cytoskeleton in cell polarity and asymmetric division during mouse oocyte maturation. Cytoskeleton 69, 727-737. Zenzes, M.T., Krishnan, S., Krishnan, B., Zhang, H., Casper, R.F., 1995. Cadmium accumulation in follicular fluid of women in in vitro fertilization-embryo transfer is higher in smokers**Supported by a grant of the Medical Research Council of Canada (MA-10428) (R.F.C.) and the Royal Bank of Canada. Fertility and Sterility 64, 599-603. Zhang, C., Lin, J., Ge, J., Wang, L.L., Li, N., Sun, X.T., Cao, H.B., Li, J.L., 2017a. Selenium triggers Nrf2-mediated protection against cadmium-induced chicken hepatocyte autophagy and apoptosis. Toxicology in vitro : an international journal published in association with BIBRA 44, 349-356. Zhang, W., Wu, T., Zhang, C., Luo, L., Xie, M., Huang, H., 2017b. Cadmium exposure in newborn rats ovary induces developmental disorders of primordial follicles and the differential expression of SCF/c-kit gene. Toxicology letters 280, 20-28. Zhao, L.L., Ru, Y.F., Liu, M., Tang, J.N., Zheng, J.F., Wu, B., Gu, Y.H., Shi, H.J., 2017. Reproductive effects of cadmium on sperm function and early embryonic development in vitro. PloS one 12, e0186727. Zhu, C.C., Zhang, Y., Duan, X., Han, J., Sun, S.C., 2016. Toxic effects of HT-2 toxin on mouse oocytes and its possible mechanisms. Archives of toxicology 90, 1495-1505. Zhuang, P., McBride, M.B., Xia, H., Li, N., Li, Z., 2009. Health risk from heavy metals via consumption of food crops in the vicinity of Dabaoshan mine, South China. The Science of the total environment 407, 1551-1561.

28

ACCEPTED MANUSCRIPT Figure legends Figure1. Histological changes of ovaries and uteri development in female mice exposed to Cd. (a, b) Suppression of ovary development. PF: primary follicle, SF: secondary follicle,

SC

endometrium, UG: uterine gland. Scale bar, 200 μm.

RI PT

AF: atretic follicle. (c, d) Suppression of uterus development. M: myometrium, E:

Figure2. Effects of Cd on oocyte meiotic progression, in vitro fertilization, and

NU

fertility on female mice.

MA

(a) Representative photomicrographs of the first PBE in the control and Cd groups. Scale bar, 100 μm. (a′). The concentrations were 0.5, 1, 1.5, and 2 mg/kg. (b) The

ED

representative photomicrographs of fertilized oocytes in the control and Cd groups.

CE PT

Scale bar, 100 μm. (b′) The in vitro fertilization rate was recorded in the oocytes of the control mice and those of mice exposed to different Cd doses. (c) Representative photomicrographs of blastocyst development in the control and Cd groups. Scale bar,

AC

100 μm. (c′). The blastocyst rate was recorded. (d) Representative photomicrographs of the pups produced by the control and Cd-exposed female mice. (d′) Average litter sizes from the control and Cd female mice were recorded. The data represent the mean ± SEM of three independent experiments.

Figure3. Effects of Cd on the spindle assembly and chromosome arrangement of mouse oocytes 29

ACCEPTED MANUSCRIPT

(a) Representative photomicrographs of the spindle pattern and chromosome arrangement in the control (n = 114) and Cd groups (n = 123). Oocytes were stained with α-tubulin-FITC antibody to observe the spindles (green) and counterstained with DAPI to observe the DNA (blue). Scale bar, 20 μm. (b) Percentage of oocytes with

RI PT

aberrant spindles in the control and Cd groups. (c) Percentage of oocytes with dislocated chromosomes in the control and Cd groups. The data represent the mean ±

SC

SEM of three independent experiments. Values with different superscripts differ

NU

significantly (p < 0.05).

MA

Figure4. Effects of Cd on the actin cap formation in mouse oocytes (a) Representative photomicrographs of the actin caps in the control and Cd groups.

ED

Actin was immunostained with phalloidin-FITC, and each sample was snapped with

CE PT

light (DIC). Scale bar, 20 μm. (b) Percentage of oocytes with the abnormalof actin cap formation in the control (n = 174) and Cd groups (n = 148). The data representthe mean ± SEM of three independent experiments. Values with different superscripts

AC

differ significantly (p < 0.05).

Figure5. Effects of Cd on protein level of Juno in mouse oocytes (a) Representative photomicrographs of Juno in the control and Cd groups. Juno was immunostained with anti-mouse Folr4-FITC antibody, and DNA was counterstained with DAPI. Scale bar, 20 μm. (b) The fluorescence intensities of the Juno levels in the

30

ACCEPTED MANUSCRIPT

control (n = 114) and Cd groups (n = 92). The data represent the mean ± SEM of three independent experiments. Values with different superscripts differ significantly (p < 0.05).

RI PT

Figure6. Effects of Cd on ROS, GSH levels and Annexin-V in mouse oocytes (a) Representative photomicrographs of the ROS and GSH levels in the control and

SC

Cd groups. Scale bar, 100 μm. (b) The fluorescence strength of the ROS levels in the

NU

control (n = 128) and Cd groups (n = 110). (c) The fluorescence strength of the GSH levels in the control (n = 124) and Cd groups (n = 142). The data represent the mean ±

MA

SEM of three independent experiments. Values with different superscripts differ significantly (p < 0.05). (d) Representative photomicrographs of the apoptotic oocytes

ED

in the control and Cd groups. Oocytes were immunostained with Annexin-V-FITC,

CE PT

and each sample was snapped with light (DIC). Scale bar, 20 μm. (e) Percentage of Annexin-V oocytes in the control (n = 174) and Cd groups (n = 148). Data are represented as the mean ± SEM of three independent experiments. Values with

AC

different superscripts differ significantly (p < 0.05).

Figure7. Effects of Cd on the mitochondrial distribution and ATP contents in mouse oocytes (a) Representative photomicrographs of the mitochondria in the control and Cd groups. Scale bar, 20 μm. (b) Percentage of oocytes with aberrant mitochondrion

31

ACCEPTED MANUSCRIPT

distribution in the control and Cd groups. (c) The fluorescence intensities of the mitochondrion signals in the control (n = 126) and Cd groups (n = 113). The data represent the mean ± SEM of three independent experiments. Values with different superscripts differ significantly (p < 0.05). (d) Representative images of ATP contents

RI PT

in the control and Cd groups. ATP was immunostained with BODIPY-ATP, and each sample was snapped with light (DIC). Scale bar, 20 μm. (e) The fluorescence strength

SC

of the ATP contents in the control (n = 99) and Cd groups (n = 92). Data are

NU

represented as the mean ± SEM of three independent experiments. Values with

MA

different superscripts differ significantly (p < 0.05).

Figure8. Effects of Cd on DNA/RNA damage in mouse oocytes

ED

(a) Representative photomicrographs of DNA/RNA damage in the control and Cd

CE PT

groups. Oxo-8-dG was immunostained with anti-DNA/RNA damage antibody, and DNA was counterstained with DAPI. Scale bar, 20 μm. (b) The fluorescence intensities of the DNA/RNA damage in the control (n = 102) and Cd groups (n = 106).

AC

The data represent the mean ± SEM of three independent experiments. Values with different superscripts differ significantly (p < 0.05).

Figure9. Effects of Cd on global H3K9me3 and H3K9ac levels in mouse oocytes (a) Representative photomicrographs of the global H3K9me3 levels in the control and Cd groups. H3K9me3 was immunostained with anti-H3K9me3 antibody, and DNA

32

ACCEPTED MANUSCRIPT was counterstained with DAPI. Scale bar, 20 μm. (b) H3K9me3/DNA signal intensity quantification in the control (n = 112) and Cd groups (n = 92). (c) Representative photomicrographs of the global H3K9ac levels in the control and Cd groups. Scale bar, 20 μm. (b) H3K9ac/DNA signal intensity quantification in the control (n = 45) and Cd

RI PT

groups (n = 52). Data represent the mean ± SEM of three independent experiments.

SC

Values with different superscripts differ significantly (p < 0.05).

NU

Figure10. Effects of Cd on the DNA 5hmC and 5mC levels in mouse oocytes (a) Representative photomicrographs of the DNA 5hmC and 5mC levels in the control

MA

and Cd groups. 5hmC (red) and 5mC (green) staining in the control and Cd groups, and each sample was counterstained with DAPI to visualize DNA (blue). Scale bar, 10

ED

μm. (b) 5hmC and 5mC/DNA signal intensity quantification in the control (n = 57)

CE PT

and Cd groups (n = 63). Data represent the mean ± SEM of three independent

AC

experiments. Values with different superscripts differ significantly (p < 0.05).

33

ACCEPTED MANUSCRIPT

Highlights: 

Cd compromised the mouse oocyte meiotic progression, by disrupting spindle assembly and actin cap formation. Exposure to cadmium impaired fertilization ability, embryo development and

RI PT



reduced litter size. 

Cd induced oxidative stress with the increased oxygen species and apoptosis

NU

Cadmium exposure decreased the levels of DNA methylation, H3K9me3 and

CE PT

ED

MA

H3K9ac levels.

AC



SC

levels.

34

ACCEPTED MANUSCRIPT Conflicts of interest

AC

CE PT

ED

MA

NU

SC

RI PT

The authors have declared no conflicts of interest.

35