Plasticizer bis(2-ethylhexyl) phthalate causes meiosis defects and

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Food Safety and Toxicology

Plasticizer bis(2-ethylhexyl) phthalate causes meiosis defects and decreases fertilization ability of mouse oocytes in vivo Zhenzhen Lu, Chengtu Zhang, Chengquan Han, Quanli An, Yuyao Cheng, Yongzhong Chen, Ru Meng, Yong Zhang, and Jianmin Su J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b00121 • Publication Date (Web): 28 Feb 2019 Downloaded from http://pubs.acs.org on March 1, 2019

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Journal of Agricultural and Food Chemistry

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Plasticizer bis(2-ethylhexyl) phthalate causes meiosis defects and

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decreases fertilization ability of mouse oocytes in vivo

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Running title: DEHP impairs mouse oocyte quality

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Zhenzhen Lu † , § , Chengtu Zhang ‡ , § , Chengquan Han † , Quanli An † , Yuyao

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Cheng†, Yongzhong Chen‡, Ru Meng‡, Yong Zhang* †, and Jianmin Su*

†

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

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Province, 712100, PR China.

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‡ Xining animal husbandry and veterinary station, Xining, Qinghai Province, 810003,

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PR China.

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§The first two authors contributed equally to this manuscript.

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* Corresponding author:

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[email protected]; [email protected]

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ACS Paragon Plus Environment


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Abstract

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Bis(2-ethylhexyl) phthalate (DEHP) is a widely used plasticizer in polyvinyl

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chloride (PVC) plastics. Humans and animals are widely and continuously exposed to

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DEHP, especially in dietary respect, which is associated with reproductive diseases.

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Nevertheless, the effects and underlying mechanisms of DEHP exposure on oocytes

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in vivo remain ambiguous. In this study, we found that oral administration of DEHP

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(40 µg/kg body weight per day for 14 days) markedly reduced the maturation and

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fertilization of oocytes in vivo. In addition, DEHP caused oxidative stress, increased

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reactive oxygen species generation, promoted early apoptosis, and resulted in DNA

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damage in mouse oocytes. Moreover, DEHP exposure caused mitochondrial damage,

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reduced ATP content, down-regulated actin expression, and disturbed the spindle

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assembly and chromosome alignment in mouse oocytes. Furthermore, DEHP

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exposure remarkably impaired the localization and protein level of Juno, the sperm

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receptor on the membrane of oocytes. The levels of DNA methylation, H3K9me3,

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and H3K9ac were aslo altered in the DEHP-exposed mouse oocytes. Thus, our results

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indicated that DEHP exposure reduced the maturation and fertilization capabilities of

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mouse oocytes by affecting cytoskeletal dynamics, oxidative stress, early apoptosis,

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meiotic spindle morphology, mitochondria, ATP content, Juno expression, DNA

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damage, and epigenetic modifications in mouse oocytes.

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Keywords: DEHP, Oocyte, Oxidative stress, Apoptosis, Epigenetic modification

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Introduction

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Bis(2-ethylhexyl) phthalate (DEHP) is a widely used plasticizer in polyvinyl

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chloride (PVC) plastics for daily necessities and medical devices. More than 8.3

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billion metric tons of virgin plastics have been produced, and about 6.3 billion metric

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tons of them have turned into plastic waste over the world 1. In China, the estimated

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daily dietary intake of DEHP for adults was 1.60 µg/kg/day 2. Given its weak

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molecular linkages with the PVC polymers, DEHP can easily be released from

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industrial settings and plastic wastes to enter and pollute the air, soil, and water

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Therefore, humans and animals are widely and continuously exposed to high levels of

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DEHP through ingestion, inhalation, or skin absorption 6. In general, the Agency for

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Toxic Substances and Disease Registry reported that humans are only allowed 3-30

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µg/kg/day exposure to DEHP, but this level can reach 700 mg/kg in household dust 7-8.

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Consequently, more than 95% of human blood samples and 100% of urine samples

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contain DEHP or DHEP metabolites. Moreover, more than 90% of amniotic fluid,

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newborn blood, human breast milk, and follicular fluid samples contain DEHP residue

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6-7, 9-11.

3-5.

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DEHP exposure of humans and animals is associated with reproductive diseases,

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including reproductive hormone alteration, anovulation, suppressed or delayed

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ovulation, irregular estrogen secretion, small preovulatory follicles, sterility, and

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damaged sperm DNA

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DEHP could impair estrous cycle and inhibit ovulation in female rats 16. Hannon et al.

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demonstrated that low doses of DEHP disrupt the estrous cycle and accelerate

6, 12-15.

An earlier report showed that oral administration of

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primordial follicle recruitment in adult mice; they hypothesized that DEHP interferes

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with normal reproductive function and disrupts reproductive senescence

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research has shown that DEHP exposure could impair the meiotic progression of

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oocytes in vitro in mares, bovines, and mice

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DEHP exposure on the meiotic progression of mouse oocytes in vitro and suggested

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that DEHP could perturb the meiosis of oocytes and disturb the expression of genes

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associated with ovarian development

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DEHP negatively affects bovine oocyte maturation and influences oocyte

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developmental competence in vitro

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mechanisms of DEHP in vivo exposure on oocytes remain unclear.

22.

18-22.

17.

Recent

Liu et al. evaluated the effects of

In addition, some studies suggested that

19-21.

Nevertheless, the effects and underlying

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In the present study, we established a mouse model to explore the toxic effects and

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underlie mechanisms of DEHP on mammalian oocytes in vivo. We found that

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maturation of oocytes and in vitro fertilization decreased in mice orally administered

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with DEHP. Our results indicated that DEHP can affect the cytoskeletal dynamics,

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reactive oxygen species (ROS) levels, apoptosis, energy, and epigenetic modifications

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of mouse oocytes.

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Materials and methods

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Animals

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All animal experiments were carried out in accordance with the Animal Care

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Commission of the College of Veterinary Medicine, Northwest A&F University.

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(certificate no.: SCXK [SHAAN] 2017-003). Six to eight-week-old ICR female mice

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were kept under stable temperature (24°C–26°C) and illumination (12 h light–dark

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cycle) with free food and water available.

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Chemicals Anti-DNA/RNA damage, anti-H3K9ac, and anti-H3K9me3 were purchased from

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Abcam

(Cambridge,

UK).

Annexin

V-FITC

Apoptosis

Detection

Kit,

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Mito-Tracker@Green, 4,6-diamidino-2-phenylindole (DAPI), anti-α-tubulin were

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purchased from Beyotime (Haimen, China). Folr4-FITC was purchased from

93

BioLegend (CA, USA), CellROX® Green Reagent was purchased from life

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technologies (CA, USA), BODIPY FL ATP was obtained from Thermo Fisher

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Scientific (MA, USA). G-IVF medium was from Vitrolife (Goteborg, Sweden). All

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other reagents and chemicals used in this study were obtained by Sigma Chemical

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Company unless otherwise indicated.

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Feeding treatment

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Oocytes were harvested from 6–8-week-old female ICR mice treated with oral

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doses of DEHP. In brief, the mice orally received 0, 10, 40, or 80 µg/kg body weight

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daily dose of DEHP with a gavage needle (Engineering 360, NY, USA) in a corn oil

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carrier at 6:00 pm every day for 14 days before oocyte collection. In the control and

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the 40 µg/kg DEHP-treated group, there were 30 mice were used respectively. In the

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10 µg/kg and 80µg/kg DEHP-treated group, there were 7 and 6 mice were analyzed,

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respectively.

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107 108

Hematoxylin and eosin staining

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Ovaries were collected from mice and immediately fixed in 4% paraformaldehyde

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for 24 h, then the ovaries were embedded by paraffin. Serially ovaries were

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frozen-sectioned at 5 μm thick along the longitudinal direction. Ovary sections were

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stained with hematoxylin for a minute and washed with dripping water. The sections

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were then stained with eosin for 5–10 s and washed with dripping water. Finally, the

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stained sections were analyzed for the pathological changes of the ovaries under a

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microscope (Carl Zeiss LSM 700 META, Jena, Germany).

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In vitro maturation of oocytes

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Germinal vesicle oocytes were collected, washed with M2 medium, and then

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incubated in M2 medium with mineral oil covering the surface at 37 °C in 5% CO2

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for 16 h until maturation. The rate of the first polar body extrusion was calculated as

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the oocyte maturation rate.

122 123

Metaphase II (MII) oocyte collection

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Except calculating maturation rates, all oocytes used in other experiments were MII

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oocytes in vivo. MII-stage oocytes were collected from the oviductal ampullae. In

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brief, female mice were intraperitoneally injected with 10 IU pregnant mare serum

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gonadotrophin for the stimulation of follicle growth, followed by 10 IU human

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chorionic gonadotrophin (hCG) 48 h later. At 13-15 h after hCG injection, oocytes

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were collected in M2 medium.

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In vitro fertilization

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Sperms were collected from 12-week-old male mice. In brief, caudal epididymides

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and spermaduct were obtained by surgery, washed with G-IVF (Vitrolife, Goteborg,

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Sweden) medium, and then placed in a new dish of G-IVF to release the sperm with

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shears and tweezers. Sperms floated on the surface of 3 mL G-IVF in a 15 mL

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centrifuge tube for 30 min at 37 °C in 5% CO2. The sperm suspension was

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centrifuged at 735 × g for 5 min, transferred to 200 µL of G-IVF, and then incubated

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with MII oocytes for 6 h at 37 °C in 5% CO2. Zygotes were counted for fertilization

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rate statistics.

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Immunofluorescence staining of oocytes

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Oocytes were fixed with in Immunol Staining Fix Solution (Beyotime, Haimen,

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China) for 2 h, permeabilized with 0.5% Triton X-100 for 20 min at room

144

temperature, and then blocked with Immunol Staining Blocking Solution (Beyotime,

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Haimen, China) at 4°C overnight. The samples were stained with anti-DNA/RNA

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damage (1:500, Abcam, Cambridge, UK ), anti-α-tubulin (1:100, Beyotime),

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anti-vimentin (1:500, Sigma, St. Louis, MO, USA), Phalloidin-TRITC (1:200, sigma),

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Folr4-FITC (1:100, BioLegend, CA, USA), anti-H3K9ac (1:500, Abcam, Cambridge,

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UK), and anti-H3K9me3 (1:500, Abcam) antibodies for 12 h at 4 °C. After extensive

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washing, oocytes were incubated with Alexa Fluor 488/555-labeled IgG (Beyotime)

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at room temperature for 3 h. After washing three times, the oocytes were

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counterstained with 4,6-diamidino-2-phenylindole (DAPI, Beyotime) for 4-5 min at

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room temperature. Finally, the oocytes were mounted on glass slides and analyzed

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using a confocal laser scanning microscope (Ador REVOLUTION WD, Northern

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Ireland, England), Those oocytes with Anti-DNA/RNA Damage antibody were

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analyzed under a fluorescence microscope (Carl Zeiss LSM 700 META, Jena,

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Germany).

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For 5mC and 5hmC staining, permeabilized samples were treated with 4 N HCl for

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10 min and neutralized with Tris-HCl (pH 8.5) for 10 min at room temperature. Other

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procedures were in accordance with our previous work 23, except DAPI staining was

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performed for 10 min.

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The fluorescence intensity was measured and analyzed using Image-Pro Plus 6.0

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(Media Cybernetics, Silver Spring, MD, USA) based on previous reports of our

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laboratory

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were measured. The average normalized fluorescence intensity for oocytes was

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represented by ‘sum IOD / sum area’. To quantify fluorescence intensity, the intensity

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levels of DEHP-treated oocytes were presented relative to the mean intensity level of

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control oocytes.

. Using Image-Pro plus, the integrated optical density (IOD) and area

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169 170

ROS detection and Annexin-V staining

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CellROX® Green Reagent (1:500, life technologies, CA, USA) was used to

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examined the ROS level in living oocytes. In brief, MII oocytes were incubated in M2

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medium with 50 μL of CellROX® Green Reagent for 30 min at 37 °C in 5% CO2.

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After washing three times in PBS-PVA, the oocytes were placed on glass slides and

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examined immediately under the fluorescence microscope.

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For Annexin-V staining, Annexin V-FITC Apoptosis Detection Kit (Beyotime) was

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used to identify phosphatidylserine exteriorization in early apoptotic cells. After

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washing three times, living oocytes were stained for 20 min in darkness with 195 µL

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of Annexin V-FITC binding buffer containing 5 µL of Annexin-V-FITC and 10 µL of

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propidium iodide at room temperature. After washing three times in PBS-PVA, the

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oocytes were placed on glass slides and examined immediately under the confocal

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laser scanning microscope.

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Staining of mitochondria in oocytes

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Mito-Tracker@Green (Beyotime) was used to evaluate mitochondrial activity in the

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oocytes. After washing three times, MII oocytes were stained with 200 nM of

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Mito-Tracker@Green in M2 medium for 30 min at 37 °C in 5% CO2. The oocytes

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were washed three times in PBS-PVA, mounted onto glass slides, and examined

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immediately under the fluorescence microscope.

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ATP content assay

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After washing three times in PBS-PVA, denuded oocytes were incubated in M2

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medium supplemented with BODIPY FL ATP (1:10000, Thermo Fisher Scientific,

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MA, USA) for 30 min at room temperature in darkness. The oocytes were washed

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three times in PBS-PVA, mounted onto glass slides, and examined immediately under

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the fluorescence microscope.

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Statistical analysis

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Statistical analysis was performed with the SPSS 19.0 software (SPSS Inc.,

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Chicago, IL, USA). Each experiment was repeated three times. When multiple

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comparisons were made, data were analyzed by one-way ANOVA and LSD tests.

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When only two comparisons were made, Student’s t test was used. Data were

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presented as means ± SEM. Differences were considered statistically significant when

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p < 0.05.

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Results

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DEHP causes primary follicle damage

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In the control group (Fig. 1A), at the center of the primary follicle is the oocyte

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which has clear nucleus and zona pellucida, and the oocyte is surrounded by

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granulosa cells of normal shape. This is the morphology of a normal primary follicle.

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However, in the DEHP-treated group, in the primary follicle, the oocyte nucleus

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disappeared (Fig. 1B), the granular cells around the oocyte were extremely loose and

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detached, and exhibited disorganized and disrupted morphologies (Fig. 1C). In

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addition, degraded oocyte was found in the DEHP-exposed follicle (Fig. 1D).

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DEHP interferes with mouse oocyte maturation and fertilization

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Considering that good oocyte quality is essential for female fertility, we analyzed

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the maturation and fertilization of mouse oocytes exposed to different doses of DEHP.

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After 16 h of culture, more than 76% oocytes reached the MII stage in the control

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group (76.75% ± 1.33%, n = 224; Fig. 2A, 2B). However, the maturation rate

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significantly decreased with DEHP treatment (10 µg/kg/day: 71.77% ± 0.87%, n =

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223, p < 0.05; 40 µg/kg/day: 50.86% ± 2.14%, n = 235, p < 0.01; 80 µg/kg/day:

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34.62% ± 0.55%, n = 228, p < 0.01; Fig. 2A, 2B). These phenomena indicate that

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DEHP can inhibit mouse oocyte maturation in a dose-dependent manner. Then, we

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detected the fertilization rates in DEHP-exposed oocytes. As shown in figure 2, more

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than 83% oocytes successfully fertilized and initiated cleavage into 2-cell embryos

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(83.61% ± 2.47%, n = 183; Fig. 2C, 2D). However, exposure to 40 and 80 µg/kg

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DEHP significantly decreased fertilization rates (40 µg/kg: 64.47% ± 2.78%, n = 104,

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p < 0.01; 80 µg/kg, 5.36% ± 1.06%, n = 97, p < 0.01; Fig. 2C, 2D). We demonstrated

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that 40 µg/kg and 80 µg/kg DEHP treatment could cause both oocyte maturation and

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fertilization defects. However, the toxicity of 80 µg/kg dose, which causes extremely

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low fertilization, is too high to explore the toxicity mechanism of DEHP in living

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oocytes. Therefore, we used 40 µg/kg DEHP treatment (DEHP group) for the

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following experiments.

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DEHP exposure increases ROS level and early apoptosis in mouse oocytes To determine the specific mechanism by which DEHP affects oocyte maturation 11



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and fertilization, we measured the ROS levels in mouse oocytes exposed to DEHP.

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Compared to control group (40.24 ± 6.95, n = 52), the fluorescence intensity of ROS

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in oocytes dramatically increased after DEHP exposure (92.11 ± 8.5, n=58, p < 0.01;

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Fig. 3A, 3B). Considering that high ROS levels can induce early apoptosis through

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cellular oxidative stress, we examined early apoptosis in DEHP-exposed oocytes. As

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refer to previously study 25, if the external cellular membrane of oocyte with a clearly

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green circle fluorescent signal, it was defined as Annexin V positive, otherwise it

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would be defined as negative. The immunofluorescence signals of early apoptosis

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increased in the DEHP-treated oocytes (Fig. 3C). The results indicated that the early

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apoptosis rate of mouse oocytes significantly increased after DEHP exposure (DEHP:

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72.14% ± 1.49%, n =65 vs. control: 34.91% ± 1.11%, n =43, p < 0.01; Fig. 3D). The

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following data was also shown as DEHP-treated group vs. control group.

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DEHP exposure causes DNA damage in mouse oocytes

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8-Oxo-7,8-dihydroguanine is one of the most common DNA damages resulting

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from ROS. Therefore, anti-DNA/RNA Damage antibody was used to investigate

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whether DEHP exposure could cause DNA damage in mouse oocytes. As shown in

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figure 4, DNA damage level significantly increased with DEHP exposure (1.93 ±

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0.11, n=26 vs. 1.00 n=29; p < 0.01; Fig. 4A, 4B).

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DEHP exposure causes mitochondrial damage and reduces ATP content in

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mouse oocytes 12


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Mitochondrion is a determinant factor in oocyte maturation and fertilization26-27.

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Thus, we detected the mitochondrial activation in DEHP-exposed oocytes. The

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quantification of fluorescence intensity showed that the signals of the mitochondria

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was lower in the DEHP-exposed groups than in the controls (0.68 ± 0.01, n=83 vs.

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1.00, n=102, p < 0.01; Fig. 5A, 5B) As produced in mitochondria, ATP content is

265

closely associated with mitochondrial activity, and ATP content could affect oocyte

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biological process by serving as an energy source. Further examination showed that

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the ATP content was significantly lower in the DEHP-exposed oocytes than in the

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control group (0.38 ± 0.03, n=67 vs. 1.00, n=66, p < 0.01; Fig. 6A, 6B).

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DEHP disturbs the spindle assembly and chromosome alignment during mouse

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oocyte meiotic maturation

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Aberrant spindle organization often activates the spindle assembly checkpoint,

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resulting in meiotic arrest. Therefore, we analyzed the morphologies of meiotic

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spindles in DEHP-treated and control oocytes through α-tubulin-FITC staining to

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observe the spindle apparatus and DAPI staining to show the chromosome alignment.

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As shown in figure 7, most oocytes in the control group showed typical barrel-shaped

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spindle morphologies and well-aligned chromosome nearby the equatorial plate

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(normal spindles). However, most of DEHP-exposed oocytes exhibited disorganized

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and disrupted spindle morphologies (abnormal spindles). (Fig. 7A). Statistically, the

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proportion of abnormal spindles significantly increased in the DEHP-exposed oocytes

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compared with the control oocytes (72.24% ± 0.83%, n=108 vs. 11.71% ± 1.07%, 13



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n=109; p < 0.01; Fig. 7B). Normal chromosome was a well-aligned chromosome on

283

the equatorial plate, otherwise it would be misaligned chromosomes. The rate of

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misaligned chromosomes also significantly increased in the DEHP-exposed oocytes

285

(66.81% ± 2.18%, n = 108 vs. 12.09% ± 0.62%, n =109; p < 0.01; Fig. 7C). Taken

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together, these phenomena indicate that DEHP impairs the meiotic maturation of

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mouse oocyte by disturbing spindle assembly and chromosome alignment.

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DEHP exposure reduces actin expression in mouse oocytes

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Actin and vimentin, together with microtubule, constitute the cytoskeleton and

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function in cell morphology maintenance and cell movement. In this study, exposure

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to DEHP significantly reduced actin level (0.53 ± 0.08, n=36 vs. 1.00, n=27, p < 0.01;

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Fig. 8A, 8B). However, exposure to DEHP did not significantly affect vimentin

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expression (1.07 ± 0.02, n=22 vs. 1.00, n = 30, p > 0.05; Fig. 8C, 8D).

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DEHP exposure reduces the protein level of Juno in the membrane of mouse

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oocytes

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The lack of Juno protein could disrupt sperm–egg fusion. Therefore, we analyzed

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the localization and quantification of Juno on the oocyte membrane using

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immunofluorescence. The Juno protein was equably distributed on the oocyte

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membrane in the control group, but it was partly or entirely lost in the membrane of

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the DEHP-exposed oocytes (Fig. 9A). The proportion of mislocalized Juno was

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considerably higher in the DEHP-exposed oocytes than in the controls (48.10% ± 14


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0.99%, n=86 vs. 17.33% ± 1.01%, n=82, p < 0.01; Fig. 9B). Interestingly, the

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fluorescence signals of Juno were remarkably lower in the DEHP-treated oocytes than

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in the control group ( 28.34 ± 0.70, n=86 vs.78.99 ±3.29, n=82, p < 0.05; Fig. 9C).

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Collectively, we speculated that the reduction of Juno protein on DEHP-exposed

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oocyte membrane is partly responsible for the low fertilization rate.

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DEHP exposure results in epigenetic alterations in mouse oocytes

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Correct and precise epigenetic modifications in oocytes are essential for

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fertilization and zygote cleavage. We examined DNA methylation, histone

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methylation, and acetylation levels in DEHP-exposed oocytes. As shown in figure 10,

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5mC level significantly increased in the DEHP-exposed oocytes (1.36 ± 0.08, n = 54

315

vs. 1.00, n = 33, p < 0.05; Fig. 10A, 10B), whereas 5hmC level significantly

316

decreased with DEHP exposure (0.58 ± 0.01, n = 54 vs. 1.00, n = 33, p < 0.05; Fig.

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10A, 10C). Moreover, DEHP treatment could significantly reduce H3K9me3 level

318

(0.62 ± 0.04, n = 52 vs. 1.00, n = 28, p < 0.05; Fig. 11A, 11B). By contrast, the

319

signals of H3K9ac were significantly higher in the DEHP-exposed oocytes than in the

320

control group (1.49 ± 0.03, n = 24 vs. 1.00, n = 33, p < 0.01; Fig. 11C, 11 D). Our

321

findings suggest that DEHP exposure could alter DNA methylation, histone

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methylation, and acetylation modifications in mouse oocytes.

323 324

Discussion

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325

Increasing evidence suggests that environmental agents cause reproductive

326

disorders in human populations and animals. DEHP is the most bounteous phthalate

327

in the environment

328

from the U.S. National Toxicology Program’s Center, thought that DEHP has the

329

potential to cause adverse effects in human reproduction. Phthalates exist in our daily

330

life as plasticizers. These substances are present in many personal-care necessities,

331

such as nail polish, hair spray, shampoo, soap, deodorants, lotions, and fragrances.

332

They are also used in some medical devices and pharmaceuticals, plastic products

333

(e.g., plastic raincoats and automotive plastics), adhesives, detergents, solvents, vinyl

334

tiles, and flooring. Humans are exposed to DEHP daily

335

the speciﬁc contribution of DEHP to oocyte development failure is important.

28.

Panel, an expert of evaluation of risks to human reproduction

28.

Therefore, understanding

336

In the present study, we performed a series of experiments on mouse models in

337

oocytes to investigate the negative effects of DEHP exposure on oocyte development.

338

We investigated cytoskeleton, apoptosis, ROS levels, epigenetic modifications, and

339

receptor protein Juno in mouse oocytes exposed to DEHP. We found that DEHP

340

could decline the maturation and fertilization abilities of oocytes by disturbing the

341

structures or biological processes in mouse oocytes.

342

ROS participate not only in signal transduction, initiating mitosis, and pathogen

343

defense but also in follicular development, ovulation, corpus luteum function,

344

follicular atresia, and oocyte maturation

345

could increase oxidative stress levels in different cells. Consistent with their results,

346

our findings indicated that ROS accumulated in mouse oocytes exposed to DEHP.

29.

Previous studies have shown that DEHP

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Many previous reports have claimed that ROS could lead to apoptosis 30-32 in different

348

cells. ROS, such as H2O2, could induce apoptosis in neutrophils

349

increasing evidence showed that ROS could cause apoptosis in other cell types 33. In

350

addition, excess ROS can cause DNA damage and can induce damage to cell

351

structure, such as lipids, cell membranes, and proteins

352

showed that DEHP cause ROS accumulation and DNA damage in mouse oocytes.

353

29, 33.

32.

Moreover,

The results of this study

Mitochondria are the source and target of ROS, and ROS could induce oxidative 34.

354

damage to mitochondria

355

associated with ATP production because mitochondrial clustering may promote ATP

356

production

357

disrupted

358

and electron transport chain

359

ROS, apoptosis, and DNA damage based on our results. In brief, high ROS levels

360

may cause early apoptosis and DNA damage in mouse oocytes. After DEHP

361

treatment, the mitochondrial ROS levels increased, which may cause mitochondrial

362

oxidative damage in turn. In addition, mitochondria are the source of ATP. Therefore,

363

ATP levels decreased in the mouse oocytes treated with 40 µg/kg DEHP. As the

364

energy source for cellular biological processes, ATP provides energy for centrosome,

365

cytoskeleton, and chromosomal activity during meiosis and mitosis. ATP content

366

decrease can interfere with the polymerization and depolymerization of tubulin on the

367

spindle, thereby affecting chromosome segregation and leading to aneuploidy

368

Thus, we further explored the changes in cytoskeleton on DEHP-exposed mouse

35-37,

37.

Earlier studies showed that mitochondrial state is

and ATP production decreases when mitochondrial clusters are

ROS could reduce ATP levels by affecting the tricarboxylic acid cycle 38.

We summarize the underlying connections among

17


39.


369

Page 18 of 44

oocytes.

370

In general, the cytoskeleton mainly contains microtubules, microfilaments, and

371

intermediate fibers present in the cytoplasm. In addition, the actin filament regulates

372

the spindle movement, activates cell meiosis, causes polar body extrusion, and

373

regulates the microtubules forming meiotic spindles, which promote the aggregation

374

and separation of chromosomes in oocytes. Mycotoxins and zearalenone reduce the

375

maturation rate of mouse oocytes by altering the expression and localization of actin

376

filaments and α-tubulin, the basic components of microtubules

377

showed that DEHP exposure reduced cytoskeletal actin expression and increased

378

spindle abnormalities and chromosome misalignment. This result suggests that DEHP

379

exposure causes the arrest of oocyte meiosis possibly by damaging spindle

380

organization and chromosome alignment. Among the three cytoskeletal types, the

381

intermediate filaments are the most complex. When treated with a high-salt solution

382

or nonionic detergent, most of the cytoskeleton components in the cells are destroyed,

383

leaving only the intermediate fibers. Vimentin is the composition protein of the

384

intermediate filaments. In the present study, vimentin expression showed no

385

significant difference between the DEHP-exposed and control groups.

386

40-41.

Our experiments

Previous reports demonstrated that epigenetic modifications could be changed 42.

387

after exposure to environmental chemicals in various cell types

388

cytoskeleton could be affected by epigenetic modification states, especially DNA

389

methylation and histone methylation and acetylation. Aberrant epigenetic

390

modifications usually indicate slim survivability in cells

18


43.

Cell cycle and

Epigenetic modification

Page 19 of 44


44-45.

391

states are also involved in meiosis and development during oocyte maturation

392

The epigenetic modification of cells is constantly adjusted through a range of

393

time-precise biological events, such as gamete development, fertilization, and fetal

394

development, which can be particularly vulnerably interfered by endocrine-disrupting

395

chemicals (EDCs) 46. These epigenetic modification changes could pass on to the next

396

generation

397

plastics and other EDC members could cause mammal oocytes meiosis defects and

398

mouse reproductive diseases in the F3 generation

399

DEHP exposure could alter DNA methylation, histone methylation, and acetylation

400

modifications in mouse oocytes, indicating that DEHP could affect epigenetic

401

modifications in mouse oocytes.

47-49.

Plasticizers are EDCs, and earlier reports showed that exposure to

50-53.

On the basis of our results,

402

Sperm-egg fusion is influenced by many factors, the most important of which is

403

Juno, a receptor protein on the oocyte, which binds to the Izumo1 protein on the

404

sperm. The lack of Juno protein causes a disorder in sperm-egg fusion. In the present

405

study, Juno was significantly reduced in the oocytes exposed to DEHP. This

406

phenomenon suggests that DEHP could impair oocyte fertilization.

407

According to the Food and Drug Administration, the oral tolerable intake range for

408

DEHP is 40 µg/kg/day 54, and the Environmental Protection Agency reported that 20

409

and 50 µg/kg/day of DEHP may cause hepatomegaly and testicular toxicity,

410

respectively 55. In addition, some previous studies found that 40 µg/kg/day of DEHP

411

could impair oocyte maturation through oral administration or injection

412

Therefore, we chose 0-80 µg/kg/day of DEHP as the oral dose to evaluate the effect of

19


56-57.


413

DEHP exposure on oocyte quality.

414

In conclusion, our results indicated that DEHP exposure affected actin and Juno

415

expression, meiotic spindle morphology, apoptosis, oxidative stress, ATP content,

416

mitochondria, DNA damage, and epigenetic modifications in mouse oocytes. These

417

alterations may be responsible for the decreased maturation and fertilization rates in

418

DEHP-exposed oocytes. The doses we used in this study are within the range of

419

environmental exposure levels in humans. Therefore, our findings provide some

420

supporting insights into the reproductive toxicity of DEHP in humans.

421 422

Acknowledgments

423

The authors wish to thank Dr. Bo Xiong and Dr. Shaochen Sun from Nanjing

424

Agricultural University for his generous technical assistance. This work was

425

supported by the National Natural Science Foundation of China (31873001),

426

National Key R&D Program of China (2018YFD0502304), and Qinghai Science and

427

Technology Project (2017-NK-111, 2018-NK-132).

428 429

Author contributions

430

JMS designed and conceived experiments. ZZL performed the experiments with

431

assistance from CTZ, CQH, QLA, YYC, YZC, and YM. ZZL analyzed data and

432

wrote the manuscript. JMS and YZ provided research guidance, critical revision of

433

the manuscript, and approval of the article.

434

20


Page 20 of 44

Page 21 of 44


435

Declaration of conflicting interests

436

The authors report that no conflict of interest exists to prejudice the impartiality of the

437

research reported.

438 439 440 441

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595

Figure legends

596

Figure 1. DEHP causes primary follicle damage

597

(A) Representative pictures of excellent primary follicles from the control group. The

598

black arrow indicates primary follicle. Scale bar=10 μm. (B, C, D) Representative

599

pictures of primary follicles from the DEHP-exposed group.

600 601

Figure 2. Effects of DEHP on mouse oocyte maturation and in vitro fertilization

602

(A) Representative images of the first polar body extrusion (PBE 1) in the control and

603

DEHP-exposed (10, 40, and 80 µg/kg) mouse oocytes. Scale bar=100 μm. (B) PBE

604

rates were recorded in the control and DEHP-exposed (10, 40, and 80 µg/kg) oocytes.

605

Data are presented as mean ± SEM (*p < 0.05, **p < 0.01). (C) Representative

606

images of fertilized oocytes in the control and DEHP-exposed (10, 40, and 80 µg/kg)

607

mouse oocytes. Scale bar=100 μm. (D) In vitro fertilization rates were analyzed in the

608

control and DEHP-exposed mouse oocytes. Data are presented as mean ± SEM (**p

Plasticizer bis(2-ethylhexyl) phthalate causes meiosis defects and

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