Di-(2-ethylhexyl) phthalate and mono-(2-ethylhexyl) phthalate attenuate early embryonic development and induce blastocyst apoptosis via PERK/ATF4/CHOP pathway in mice

Di-(2-ethylhexyl) phthalate and mono-(2-ethylhexyl) phthalate attenuate early embryonic development and induce blastocyst apoptosis via PERK/ATF4/CHOP pathway in mice | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Di-(2-ethylhexyl) phthalate and mono-(2-ethylhexyl) phthalate attenuate early embryonic development and induce blastocyst apoptosis via PERK/ATF4/CHOP pathway in mice XueMei Teng, Nian Liu, ZhenZhen Song, XueHui Zeng, NianHua Yi, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6650176/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Di-2-ethylhexyl phthalate (DEHP) is widely used as a plasticizer, and mono-(2-ethylhexyl) phthalate (MEHP) is its primary metabolite. To investigate the effects of DEHP on mice ovaries, embryo development, and endoplasmic reticulum (ER) stress, adult female mice were daily exposed to DEHP (0, 0.05, 5, and 500 mg/kg/d) for five weeks, and ovaries and embryos were collected for examinations. The mRNA and protein levels of ER stress molecules and the blastocyst apoptosis were determined using real-time reverse-transcription PCR (qRT-PCR), Western Blotting and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL), respectively. For examining the effects of MEHP on embryo development and ER stress, two-cell embryos were randomly divided and cultured in KSOM containing MEHP (0, 10 -5 , 10 -4 , and 10 -3 M). We found that the mRNA expression of XBP-1 was decreased in the ovaries, with BiP, XBP-1S and CHOP increasing, and BiP and CHOP were consistent with the results in embryos. Besides, the protein expressions of p-PERK, eIF2α and ATF4 were increased in embryos of DEHP-treatment groups than that of the control. Furthermore, 500 mg/kg/d DEHP reduced the rates of fertilization and blastocyst formation and increased blastocyst apoptosis, which was stronger than that in 5 mg/kg/d DEHP group. Moreover, 10 -3 M MEHP significantly increased the mRNA expressions of BiP and CHOP in embryos, and retarded embryo development at the two-cell stage and decreased blastocyst formation rate, as well as induced blastocysts apoptosis. In summary, we found that DEHP and MEHP affected early embryo development by ER stress and induce blastocyst apoptosis via PERK/ATF4/CHOP pathway. Earth and environmental sciences/Environmental sciences/Environmental impact Health sciences/Health care Health sciences/Endocrinology/Endocrine system and metabolic diseases di-2-ethylhexyl phthalate mono-(2-ethylhexyl) phthalate ovarian toxicity early embryo development endoplasmic reticulum stress Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1 Introduction Over the past decade, the global use of assisted reproductive technology (ART) has triggered a constantly increasing research investigating whether lifestyle-related risk factors, such as smoking, caffeine, alcohol, or endocrine-disrupting chemicals (EDCs), affect ART outcomes. EDCs are a variety of exogenous compounds that interfere with any type of hormonal effects responsible for maintaining homeostasis and regulating developmental processes. EDCs have shown serious adverse effects on human reproductive health, 1 , 2 which has caused widespread concerns in recent years. Phthalic acid esters (PAEs) as the most prevalent EDC type are very common synthetic organic compounds, 3 which are widely used as solvents and plasticizers in cosmetics, automotive glass, printing and paint, and other chemical industries. 4 , 5 PAEs enter the human body via the digestive and the respiratory tract as well as through the skin by absorption, 6 which results in a ubiquitous exposure of the entire population. Clinical studies have shown that in addition to detecting PAEs and their metabolites in traditional biological samples (such as serum and urine), detectable levels of certain PAEs have been found in amniotic fluid, milk, semen, follicular fluid, and other body fluids closely related to reproduction, especially di-2-ethylhexyl phthalate (DEHP) and its metabolite, mono-(2-ethylhexyl) phthalate (MEHP), 7 indicating that DEHP and MEHP may affect the reproductive outcomes. MEHP as the primary DEHP metabolite is more toxic than DEHP. To date, many toxicological studies have been conducted assessing the effects on various systems and organs of DEHP and MEHP, including the reproductive toxicity and the early embryonic developmental toxicity. Zhao et al investigated that DEHP could significantly reduce the quality of sperm in mice, which was reflected in the damage of sperm DNA and the decline of sperm motility, and different degrees of endoplasmic reticulum (ER) swelling could also be observed under electron microscope, all of which ultimately lead to testicular spermatogenesis dysfunction. 8 DEHP mediated oxidative stress and ER stress by regulating the expression levels of Nrf2 and HSR -related genes, and exerted renal and splenic toxicity, which was manifested by decreased glomerular filtration function and changes in the structure of renal tubular epithelial cells, and structural damage to the splenic body. 9 – 11 Moreover, the study also found that the same dose of DEHP could regulate the balance of redox reactions. Once the balance was broken, structural changes in the liver occurred, including swelling of mitochondria, disorder of liver plates, degeneration of fat cells, chromatin bordering, deep staining of nuclei, and abnormally elevated liver enzyme levels. 12 , 13 In male, DEHP could also cause the damage of testicular stromal cells and pathological changes in quail heart, such as cardiomyocyte swelling and muscle fiber dilation, through Nrf2 signaling pathway and its downstream target genes. 14 , 15 Animal studies have shown that DEHP exerts female reproductive toxicity by interfering with the normal menstrual cycle, reducing the ovarian reserve function, and interfering with the oocyte meiosis. 16 – 20 MEHP reduces the developmental capacity of oocytes by increasing the number of GV-phase oocytes and decreasing the number of MII-phase oocytes; it also exerts embryonic developmental toxicity by reducing the proportion of two-cell embryos and the rate of blastocyst formation. 21 – 24 A previous report also indicated that the mouse two-cell embryos were blocked by MEHP. 25 In addition, related studies explored the effect of DEHP doses. Specifically, a high intrauterine MEHP exposure accelerates ovarian aging by accelerating primordial follicle activation, resulting in reproductive defects in F1/F2 adult females. 26 In general, exposure to low doses of DEHP or MEHP is not associated with reproductive toxicity. However, it has been demonstrated that equivalent exposure to a low DEHP dose in the population accelerated the rate of follicle recruitment, reduced or delayed the methylation level of the imprinted genes in oocytes, and increased the abnormalities of the mature oocyte MII spindle. 17 Thus, these studies indicated that environmental exposure to DEHP in daily life could also adversely affect reproductive outcomes (follicle formation, oocyte maturation, etc.). Therefore, studying the effects of environmental-level DEHP or MEHP exposure on early embryo development has substantial clinical value. The endoplasmic reticulum (ER) is critically involved in the correct folding and processing of newly synthesized proteins. Any damage to the ER and the resulting ER stress can lead to abnormalities in transcriptional regulation and gene expression, metabolism, signaling, or ion channel function. 27 – 29 Under ER stress, unfolded or misfolded proteins accumulated in the ER lumen, 30 activating the signaling pathways that are involved in the unfolded protein response (UPR). 31 There are three major signaling pathways for stimulating the UPR: (I) via the inositol-requiring enzyme 1 ( IRE1 ) involved in recruiting several signaling molecules, splicing, and generating activated transcription factor X-box binding protein 1 ( XBP-1 ), ER chaperones, such as the binding immunoglobulin protein ( BiP ; also known as glucose-regulated protein GRP78 ), along with C/EBP homologous protein ( CHOP ); (II) via the PKR-like eukaryotic initiation factor 2A kinase ( PERK ), which phosphorylates eukaryotic initiation factor-2α ( eIF2α ) to suppress protein translation; and (III) via the activating transcription factor 6 ( ATF6 ), which is translocated to the Golgi apparatus where proteolytic processing generates its activated form ATF6α , a transcription factor that stimulates the expression of BiP and XBP-1 , which act together to limit ER stress by reducing the protein synthesis, facilitating the protein degradation, and increasing the folding capacity. 27 – 29 However, the apoptotic pathway is activated when the level of misfolded or unfolded protein exceeds the processing capacity of the ER, leading to cell injury and death induced by XBP-1 , activating transcription factor 4 ( ATF4 ), and CHOP . 27 – 29 Several recent studies on the reproductive system found that ER stress fulfills a critical function in mouse oocyte maturation, 32 , 33 and functional pathways of ER stress and UPR signaling are essential for oocyte maturation and quality. 34 Multiple recent studies with a main focus on the XBP-1 pathway demonstrated that the ER stress affects preimplantation and post-implantation development of embryos. 35 – 38 In this study, we performed animal experiments for simulating DEHP exposure in the population. We explored the toxicity of DEHP on the ovaries and early embryo development, and we investigated the ER stress mechanism induced by DEHP and MEHP. Our work has a clinical value because it is designed to increase the people’s vigilance against the use of plasticizers in daily life, which could prevent the adverse effects that high exposure to environmental DEHP has on the ovaries and the early embryonic development. 2 Materials and Methods 2.1 Chemicals DEHP (99% purity) was purchased from Sigma-Aldrich (St. Louis, MO) and stored at 20-25°C until used. MEHP (99% purity) was purchased from AccuStandard (New Haven, CT). Stock solutions of MEHP were prepared using dimethylsulfoxide (DMSO) (Sigma- Aldrich) and stored at -20°C. The solution was then diluted to prepare KSOM medium containing 0, 10 − 5 , 10 − 4 , or 10 − 3 M MEHP. DMSO was added to the medium at the same concentration as compounds treatments (< 0.1%) to serve as vehicle control. Pregnant mare serum gonadotropin (PMSG) was purchased from Solarbio (Beijing Science & Technology Co., Ltd.). Human chorionic gonadotropin (HCG) was purchased from Livzon (Shanghai, China). HTF (fertilization medium) was purchased from Sigma (Millipore Corp. USA). KSOM was purchased from EasyCheck (Nanjing AIBI Biology Co., Ltd.). 2.2 Experimental animals and DEHP/MEHP treatment 2.2.1 Mice exposed to DEHP in vivo and mice used to recover embryos for MEHP exposure in vitro Healthy, sexually mature female (30–35 g) and male (35–40 g) ICR mice and 4–5 weeks old, healthy female and male mice were purchased from the Laboratory Animal Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology (Wuhan, China). Mice were housed in a temperature- and humidity-controlled room with a 12 h light/dark cycle. DEHP stock solution aliquots of 2 µL, 200 µL, 20 mL were added to 10 mL, 10 mL, and 30 mL corn oil, respectively, which were each added to 10 kg mouse feed, formulating the respective doses of 0.05 mg/kg/d, 5 mg/kg/d, and 500 mg/kg/d DEHP feed to simulate DEHP exposure in the human population. The female mice, 4–5 weeks old, were randomly divided into four groups for a 5-week DEHP exposure of 0, 0.05 mg/kg/d, 5 mg/kg/d, and 500 mg/kg/d (n = 13/group for qRT-PCR and n = 10/group for Western Blotting), which was repeated three times. Sexually mature, healthy female and male ICR mice, which did not receive any intervention, were sacrificed to generate embryos for exposure to MEHP. All mcie were sacrificed by cervical dislocation. All animal procedures were approved by the Animal Ethics Committee of Tongji Hospital. All methods were carried out in accordance with the ARRIVE guidelines 2.0. 2.3 Embryo collection and culture The female ICR mice were superovulated with PMSG, and cumulus-oocyte complexes (COCs) were collected in HTF medium from the ampullae of oviducts 15–17 h after injection of HCG. Spermatozoa were collected from the caudal epididymis of adult male ICR mice (35–40 g) and capacitated by preincubation for 1 h in HTF. The COCs were inseminated with capacitated spermatozoa in HTF embryo medium in a humidified atmosphere with 5% CO 2 at 37°C. After an insemination period of 4–5 h, the zygotes were washed and cultured in KSOM droplets. Then, 24 h after insemination, two-cell embryos were transferred to new KSOM droplets. 2.4 Experimental design This study is performed in accordance with relevant guidelines and regulations. All methods are reported in accordance with ARRIVE guidelines. The study included two sets of experiments to explore the developmental effect on early embryonic development and the mechanism of ER stress in mice ovaries and early embryos induced by DEHP or MEHP. The first and second set experiments consist of four parts and three parts respectively. The dosage regimen was selected to establish a dose range that would not be lethal, but may cause identification of other possible target organ effects. The lethal dose 50 (LD50) of DEHP was 30 g/kg. It was known that DEHP at dose of 1000 mg/kg was able to cause adverse effects in female rats without causing systemic toxicity. 39 In the DEHP in vivo exposure experiment, the female mice were randomly divided into four groups for a 5-week exposure with different DEHP doses (0, 0.05 mg/kg/d, 5 mg/kg/d and 500 mg/kg/d), while the adult males received no intervention. In the MEHP in vitro exposure experiment, all embryos generated by in vitro fertilization (IVF) were randomly divided into four groups and cultured in KSOM medium containing different concentrations of MEHP (0, 10 − 5 , 10 − 4 , and 10 − 3 M). The first part of the experiments was initiated by the DEHP in vivo exposure. There were 13 female and 3 male mice in each group for qRT-PCR and 10 female and 2 male mice in each group for Western Blotting, respectively, and each experiment was independently repeated three times. Female mice exposed to DEHP were superovulated using the conventional PMSG-HCG method and sacrificed to collect ovaries from all the groups for real-time reverse-transcription PCR (qRT-PCR) to detect ER stress-associated indicators ( BiP , XBP-1 , XBP-1S , CHOP , and ATF6 ). Simultaneously, the fertilized eggs obtained from mice of all groups were cultured in KSOM droplets and grouped on the culture dishes. The embryos of each group were divided into three samples to perform qRT-PCR, Western Blotting and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) analysis. Embryos were cultured in KSOM medium until the eight-cell stage. Then, the RNA of 150 and 100 eight-cell embryos was extracted for qRT-PCR and Western Blotting to assess the mRNA and protein expressions of ER stress-associated indicators, respectively. The remaining embryos were continuously cultured in KSOM droplets until they reached the blastocyst stage. Then, at least 10 blastocysts per group were collected for TUNEL analysis to detect apoptosis signals. Throughout the entire developmental period, the embryos were monitored continuously, and the rates for cleavage, as well as for morula and blastocyst formation, were recorded for each group. In the second part of the experiment, no intervention was performed on the mice. The female mice were superovulated, and the fertilized eggs were cultured in a humidified atmosphere with 5% CO 2 at 37°C. After an insemination period of 24 h, all well-developed two-cell embryos were randomly divided into four groups, with at least 180 embryos from 8 female mice in each group, and transferred to KSOM droplets containing different concentrations of MEHP (0, 10 − 5 , 10 − 4 , and 10 − 3 M) prepared in advance. The droplet cell cultures were performed in a humidified atmosphere with 5% CO 2 at 37°C. Then, RNA of 150 embryos at the eight-cell stage was extracted for qRT-PCR, and at least 10 blastocysts were collected per group for TUNEL. The rates for 4-cell embryo, cleavage, as well as for blastocyst formation, were recorded. These experiments were repeated five times. 2.5 RNA extraction and qRT-PCR analysis To analyze the expression of the target genes, we collected the ovaries and embryos (eight-cell stage) of each group after exposure to DEHP or MEHP. Total RNA was extracted using RNeasy Mini kits (Qiagen, Germany) according to the manufacturer’s instructions, and the first strand was synthesized using the PrimeScript™ RT Master Mix (TAKARA, Code No. RR036A). Then, qRT-PCR was performed with the TB Green Premix Ex Taq™ kit (Tli RNaseH Plus, TAKARA, Code No. RR420A) using the LightCycler 480 System (Roche, China). The data was normalized to the GAPDH values using the 2 −∆∆CT method. The PCR primers were designed and synthesized by PEPTBIO (Wuhan, China). The sequence numbers of primers are as follows: GAPDH - MQP027158, BiP -MQP074318, XBP-1 -MQP096646, XBP-1S -MQP077568, CHOP -MQP026921, and ATF6 -MQP026163. The tests were performed in triplicate, and the mRNA level of each sample was normalized to that of the GAPDH mRNA. 2.6 Western Blotting Relative protein levels of IRE1, PERK, eIF2α and ATF4 were detected by Western Blotting. Embryo protein extracts were separated on SAS-polyacrylamide gel electrophoresis. Separated proteins were electrophoretically transferred to a PVDF membrane (Servicebio, China) transferred for 50 min at 90V in Trisglycine buffer containing 20% methanol. The membranes were incubated with diluted antibody IRE1 (Abcam, 1:1000), PERK (Abcam, 1:1000), eIF2α (Bioswamp, 1:1000), ATF4 (Abcam, 1:1000) and β-actin (Bioswamp, 1:1000). The primary antibodies were localized with goat anti-rabbit IgG conjugated with horseradish peroxidase (Bioswamp, China). After protein bands were visualized with an enhanced chemiluminescence system (GeneGnome5, American), the intensities of the bands were quantified and analyzed by TANONGIS. 2.7 Determination of apoptosis Blastocysts were fixed with 4% paraformaldehyde (PFA) in PBS for 30 min and then permeabilized in 0.5% Triton X-100 in PBS for 40 min at 20-25°C. After three washes in PBS containing 0.1% BSA (PBS/BSA), apoptosis was determined by TUNEL assay using the In-Situ Cell Detection kit (Beyotime, C1086). Briefly, embryos were incubated in the TUNEL mixture (deoxynucleotidyl transferase enzyme and fluorescein-dUTP in a 1:9 ratio) for 1 h at 37°C in the dark. Positive controls were preincubated with 5 IU DNase I for 30 min at 37°C. Negative controls were processed without enzyme. After three times washing in PBS/BSA, the embryos were stained with Hoechst 33342 (10 µg/mL) for 40 min at 37°C in the dark. After a quick wash in PBS/BSA, the embryos were transferred to a glass slide and covered with anti-fluorescence quencher. The embryos were immediately viewed with a scanning laser confocal microscope (Zeiss, Germany). Green fluorescence represented the apoptotic signals, and blue fluorescence represented the nucleus. Three and five independent experiments were performed in vivo and in vitro , respectively. In each experiment, at least 10 embryos per group were collected and examined, generating data sets from at least 30 and 50 embryos examined per in vivo and in vitro group, respectively. 2.7 Statistical analysis All experiments are presented as means ± SEMs, and all the studies used for quantification were repeated at least three times. Data analysis was performed using GraphPad Prism8. The differences between the means were evaluated by one-way ANOVA when appropriate, followed by the least significant difference (l.s.d.) post hoc test. Asterisks (*) indicated statistically significant differences to the control group, * P < 0.05 VS control, ** P < 0.01, *** P < 0.001, **** P < 0.0001. 3 Results 3.1 ER stress in mouse ovary induced by DEHP exposure in vivo To investigate whether DEHP induces ER stress and UPR signaling pathways in the mouse ovary, 4–5 weeks old female ICR mice were exposed by consuming food that contained different doses of DEHP (0.05, 5, and 500 mg/kg/d). The qRT-PCR analysis showed that the mRNA expression of BiP in the ovaries of the 0.05, 5, and 500 mg/kg/d DEHP groups was significantly increased (Figure. 1A), compared to that of the control. There was no significant difference of the mRNA expression of XBP-1 (Figure. 1B) in all groups. DEHP increased the mRNA expression of XBP-1S (Figure. 1C) and CHOP (Figure. 1D) in a dose-dependent manner. The ATF6 mRNA level (Figure. 1E) was significantly higher in the group with 500 mg/kg/d DEHP treatment than in the control group. All data were shown in Table 1 . These results indicated that DEHP induced ER stress in the mouse ovary, and the three UPR signaling pathways responded at different degrees. Table 1 The mRNA expression of ER stress in ovaries exposed to DEHP(X ± S) Control (n = 13) 0.05 mg/kg/d (n = 13) 5 mg/kg/d (n = 13) 500 mg/kg/d (n = 13) BiP 0.8016 ± 0.02114 1.461 ± 0.05610 *** 1.203 ± 0.09615 * 0.9545 ± 0.03135 * XBP-1 1.450 ± 0.1308 0.8493 ± 0.02159 * 0.7026 ± 0.01573 ** 0.6340 ± 0.09619 ** XBP-1S 0.5271 ± 0.03468 0.8029 ± 0.03852 ** 0.9937 ± 0.02506 *** 1.639 ± 0.2726 * CHOP 0.5358 ± 0.03789 0.8418 ± 0.04672 ** 1.203 ± 0.07647 ** 1.776 ± 0.06287 **** ATF6 0.5061 ± 0.06300 0.8874 ± 0.1364 0.7479 ± 0.06358 1.231 ± 0.1594 * * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001 All data were presented as means ± SEMs. D 0.05: 0.05 mg/kg/d DEHP, D 5: 5 mg/kg/d DEHP, D 500: 500 mg/kg/d DEHP; * P < 0.05 VS control, ** P < 0.01, *** P < 0.001, **** P < 0.0001. (A)-(E): The mRNA expression of ER stress-related molecules in ovaries exposed to DEHP. (F)-(J): The mRNA expression of ER stress-related molecules in embryos obtained from mice exposed to DEHP. The mRNA expression of BiP and CHOP was gradually increased in the DEHP-treatment groups than that in the control group. 3.2 Effects of DEHP on the rates for fertilization and blastocyst formation in mice The development from high-quality oocytes to high-quality embryos depends on a complex crosstalk network connecting the developing oocyte, the supporting cumulus, and other cells within the follicle. 20 In this study, embryos obtained from mice exposed to DEHP were cultured in KSOM until the blastocyst stage. There was no significant difference in the embryonic development (cell numbers and differentiation) between the groups treated with 0.05 mg/kg/d and 5 mg/kg/d DEHP and the control (Fig. 2A, Fig. 3A). However, the rates for fertilization and blastocyst formation in the group treated with 500 mg/kg/d DEHP was significantly lower than in the control group (Fig. 3A, Fig. 3B, Fig. 3C). Data were shown in Table 2 . Table 2 Fertilization rate and blastocyst formation rate exposed to DEHP (X ± S) Control (n = 13) 0.05 mg/kg/d (n = 13) 5 mg/kg/d (n = 13) 500 mg/kg/d (n = 13) Fertilization rate 74 ± 4 60.667 ± 5.372 68.533 ± 10.072 50.533 ± 5.082 * Blastocyst formation rate 43.533 ± 1.474 38.733 ± 2.150 32.167 ± 1.692 21.167 ± 2.699 ** * P < 0.05; ** P < 0.01 Figure 2 . Embryo development diagrams obtained from DEHP-exposed mice and embryo development diagrams after direct exposure to MEHP. ICR female mice were exposed to di-2-ethylhexyl phthalate (DEHP) (0.05, 5, and 500 mg/kg/d) for five weeks (n = 13 for female mice and n = 3 for male mice). In the part of DEHP experiment, embryos were cultured in KSOM medium until blastocyst stage. In the part of MEHP experiment, two-cell embryos were divided into four groups and culture in KSOM containing different concentrations of MEHP (0, 10 − 5 , 10 − 4 , and 10 − 3 M). All data were presented as means ± SEMs. D 0.05: 0.05 mg/kg/d DEHP, D 5: 5 mg/kg/d DEHP, D 500: 500 mg/kg/d DEHP. 2 cell: embryos in two-cell stage; 3–4 cell: embryos in three-four cell stage; 7–8 cell: embryos in seven-eight stage; morula: embryos in morula stage; blastocyst: embryos in blastocyst stage. (A) Developmental map from fertilized eggs to blastocysts. All the embryos were obtained from mice exposed to DEHP. (B) Developmental map of two-cell embryos exposed to MEHP for 96h. 3.3 ER stress in early embryos obtained from mice exposed to DEHP in vivo The qRT-PCR and Western Blotting analysis were performed to detect ER stress-associated indicators in eight-cell embryos to further explore whether DEHP could induce ER stress in early embryos. Figure 1F showed that the BiP mRNA was significantly higher in the group treated with 500 mg/kg/d DEHP than in the control group. CHOP was increased in a dose-dependent manner induced by DEHP (Fig. 1I) ( P < 0.05). However, there was no significant difference in the mRNA expression of XBP-1 , XBP-1S , and ATF6 among the embryos from all groups (Fig. 1G, Fig. 1H, Fig. 1J). Western Blotting showed that there was no significant difference in the protein expression levels of IRE1 and PERK in the embryos of the experimental group and the control group (Fig. 4A, Fig. 4B), however, the protein expression levels of p-PERK , eIF2α and ATF4 in embryos of the DEHP-treatment groups were significantly higher than those of the control ( P < 0.05) (Fig. 4C, Fig. 4D, Fig. 4E), and the ratio of p-PERK to PERK in the treatment groups also gradually increased (Fig. 4F). Thus, the data indicated that DEHP might induce ER stress during early embryogenesis via the PERK/ATF4/CHOP pathway. Data were shown in Table 3 and Table 4 , respectively. Table 3 The mRNA expression of ER stress in embryo exposed to DEHP(X ± S) Control (n = 13) 0.05 mg/kg/d (n = 13) 5 mg/kg/d (n = 13) 500 mg/kg/d (n = 13) BiP 0.6074 ± 0.09673 0.9553 ± 0.1307 1.225 ± 0.1088 * 1.676 ± 0.05986 *** XBP-1 1.139 ± 0.2158 0.9705 ± 0.09108 1.039 ± 0.1930 1.019 ± 0.3212 XBP-1S 0.9961 ± 0.3412 1.344 ± 0.3429 1.602 ± 0.3887 1.274 ± 0.4324 CHOP 0.7354 ± 0.09626 1.061 ± 0.06617 * 1.233 ± 0.07917 * 1.607 ± 0.1169 ** ATF6 1.361 ± 0.4422 1.439 ± 0.3200 0.8681 ± 0.03653 1.382 ± 0.2360 * P < 0.05; ** P < 0.01; *** P < 0.001 Table 4 The protein expression of ER stress in embryo exposed to DEHP(X ± S) Control (n = 10) 0.05 mg/kg/d (n = 10) 5 mg/kg/d (n = 10) 500 mg/kg/d (n = 10) IRE1 1.143 ± 0.0819 1.097 ± 0.08996 1.297 ± 0.06067 1.047 ± 0.07647 PERK 0.3877 ± 0.01302 0.4104 ± 0.01210 0.4229 ± 0.001425 0.4190 ± 0.01035 p-PERK 0.4700 ± 0.01114 0.5450 ± 0.01296* 0.7875 ± 0.01343**** 0.9247 ± 0.01410**** p-PERK /PERK 0.7869 ± 0.09628 1.068 ± 0.06623 1.443 ± 0.06317** 1.674 ± 0.1349** eIF2α 0.2379 ± 0.01299 0.3124 ± 0.02567 0.3960 ± 0.02012** 0.9705 ± 0.06526*** ATF4 0.7466 ± 0.01567 0.9505 ± 0.07455 1.321 ± 0.1116** 1.477 ± 0.720*** * P < 0.05 VS control; ** P < 0.01; *** P < 0.001; **** P < 0.0001 All data were presented as means ± SEMs. D 0.05: 0.05 mg/kg/d DEHP, D 5: 5 mg/kg/d DEHP, D 500: 500 mg/kg/d DEHP; * P < 0.05 VS control; ** P < 0.01; *** P < 0.001; **** P < 0.0001. (A), (B) There was no difference in the protein expression levels of IRE1 and PERK among all the DEHP-treatment groups. (C), (D), (E) The protein expression levels of p-PERK , eIF2α and ATF4 were significantly increased in DEHP-treatment groups than that of the control. (F) The ratio of p-PERK to PERK was also significantly increased in groups D 5 and D 500. 3.4 Effect of DEHP exposure on blastocyst apoptosis in vivo CHOP , which is encoded by the growth arrest- and DNA damage-inducible gene 153 ( GADD153 ), has been identified as a factor that responds to DNA damage. 40 We found that DEHP exposure increased the mRNA expression of CHOP in embryos. A TUNEL analysis was performed to investigate further the developmental potential of embryos that reached the blastocyst stage after exposure to DEHP and to explore whether DEHP-induced changes in CHOP expression were directly related to blastocyst apoptosis. The TUNEL results showed that apoptosis signals appeared in blastocysts of groups treated with 5 and 500 mg/kg/d DEHP, and the latter treatment group had stronger apoptotic signals than the former (Fig. 5A), indicating that CHOP might be directly linked to blastocyst apoptosis. Green fluorescence represents the apoptotic signal and blue fluorescence represents the nucleus. (A) After five weeks exposure to di-2-ethylhexyl phthalate (DEHP) (0.05, 5, and 500 mg/kg/d), embryos were obtained and cultured until blastocyst stage, then apoptosis was determined by TUNEL staining. Apoptosis signals were observed in groups of 5 mg/kg/d and 500 mg/kg/d DEHP treatment, and the latter had stronger apoptotic signals than the former (Fig. 5A). (B) Sexually mature, healthy female without intervention were superovulated and sacrificed to obtain embryos. Two-cell embryos obtained from were cultured in KSOM containing mono-(2-ethylhexyl) phthalate (MEHP) (0, 10 − 5 , 10 − 4 , and 10 − 3 M) until blastocysts, and 10 blastocysts at least per group were collected for TUNEL assay. As shown, apoptosis signal was observed in group of 10 − 3 M MEHP treatment (Fig. 5B). 3.5 Effect of MEHP on early embryo development DEHP exerts ovarian toxicity through its metabolite MEHP. 41 Two-cell embryos obtained from female mice with no intervention were cultured in KSOM containing different concentrations of MEHP (0, 10 − 5 , 10 − 4 , and 10 − 3 M) for 96 h. Figure 2B showed the effect of different concentrations of MEHP on the morphology of early embryo development. It showed that exposure to 10 − 3 M MEHP retarded early embryo development (Fig. 3D-3F) and significantly reduced the blastocyst formation rate (Fig. 3F). The rates of 4-cell, 8-cell and blastocyst formation of each group were shown in Fig. 3G-3I. Data were shown in Table 5 . Table 5 rate of 4-cell and 8-cell embryo and blastocyst formation exposed to MEHP (X ± S) Control (n = 8) 10 − 5 M MEHP (n = 8) 10 − 4 M MEHP (n = 8) 10 − 3 M MEHP (n = 8) 4-cell embryo rate 77.77 ± 3.998 76.67 ± 1.934 67.77 ± 2.936 10.00 ± 1.905 * 8-cell embryo rate 71.10 ± 1.100 73.33 ± 1.934 63.33 ± 3.839 10.03 ± 3.333 ** Blastocyst formation rate 56.67 ± 1.934 52.67 ± 4.421 52.10 ± 3.035 10.00 ± 1.905 **** * P < 0.05 VS control; ** P < 0.01; **** P < 0.0001 All data were presented as means ± SEMs. D 0.05: 0.05 mg/kg/d DEHP, D 5: 5 mg/kg/d DEHP, D 500: 500 mg/kg/d DEHP; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. (A) ICR female mice were exposed to di-2-ethylhexyl phthalate (DEHP) (0.05, 5, and 500 mg/kg/d) for five weeks, and then they were superovulated to obtain embryos, which were cultured in KSOM until blastocysts. (A) Histograms of embryos development at all stages exposed to DEHP. (B), (C) The rates of two-cell and blastocyst formation of each group of embryos obtained in mice exposed to DEHP. (D), (E), (F): Diagram showing the proportions of embryo development at all stages exposed to MEHP. (G), (H), (I): The rates of four-cell, eight-cell embryo and blastocyte formation in embryos exposed to MEHP. 3.6 ER stress in mouse embryos induced by MEHP exposure in vitro The RNA of 150 eight-cell embryos was extracted for qRT-PCR to assess the expression of ER stress-associated factors. The results indicated that there was a significant difference in the mRNA expression levels of BiP (Fig. 6A), CHOP (Fig. 6D), and ATF6 (Fig. 6E) in the group treated with 10 − 3 M MEHP, compared to those in the control ( P < 0.05), which confirmed that MEHP might be involved in the regulation of early embryogenesis and apoptosis mainly via the ER stress-related CHOP pathway. There was no correlation between the mRNA expression of XBP-1 , XBP-1S and the concentration of MEHP in embryos (Fig. 6B, Fig. 6C). Data were shown in Table 6 . Table 6 The mRNA expression of ER stress in embryo exposed to MEHP(X ± S) Control (n = 8) 10 − 5 M MEHP (n = 8) 10 − 4 M MEHP (n = 8) 10 − 3 M MEHP (n = 8) BiP 0.9009 ± 0.1040 0.9181 ± 0.06335 1.016 ± 0.04475 1.213 ± 0.04119 * XBP-1 1.011 ± 0.09296 1.044 ± 0.2092 1.023 ± 0.1486 1.034 ± 0.1595 XBP-1S 1.003 ± 0.05593 1.001 ± 0.03038 1.021 ± 0.1387 0.9994 ± 0.04095 CHOP 0.8567 ± 0.04273 0.9552 ± 0.04964 0.9828 ± 0.03349 1.706 ± 0.1068 ** ATF6 0.8721 ± 0.05552 1.068 ± 0.1130 1.004 ± 0.1866 1.273 ± 0.07181 * * P < 0.05; ** P < 0.01 All data were presented as means ± SEMs. 150 embryos in eight-cell stage were collected for qRT-PCR to detect ER stress-associated molecules ( BiP , XBP-1 , XBP-1S , CHOP , ATF6 ). * P < 0.05, ** P < 0.01. (A) There was a significant difference in group of 10 − 3 M MEHP treatment compared to that of the control ( P < 0.05). (B), (C) There was no correlation between the mRNA expression of XBP-1 , XBP-1S and the concentration of MEHP in embryos. (D) The mRNA expression of CHOP in group of 10 − 3 M MEHP treatment was increased ( P < 0.01) compared to that of the control group. (E) As with CHOP , the mRNA expression of ATF6 in the group of 10 − 3 M MEHP treatment was also significantly increased, and there was no statistical difference in other two groups. 3.7 Effect of MEHP exposure on blastocyst apoptosis in vitro We found that MEHP exposure increased the mRNA expression of CHOP in embryos. A TUNEL analysis was performed to further investigate the developmental potential of embryos that reached blastocyst stage after exposure to MEHP and to explore whether MEHP-induced changes in CHOP expression were directly related to blastocyst apoptosis. The TUNEL results showed that apoptosis signals only appeared in blastocysts treated with 10 − 3 M MEHP (Fig. 5B), indicating that CHOP was directly linked to blastocyst apoptosis. 4 Discussion In this mouse study on DEHP exposure, we observed that the high-dose exposure (500 mg/kg/d DEHP) significantly decreased the rates for fertilization and blastocyst formation, the medium-dose exposure (5 mg/kg/d DEHP) reduced the blastocyst formation rate, but not significantly, and the low-dose exposure (0.05 mg/kg/d DEHP) did not affect the rates for fertilization and blastocyst formation. In the second part of the experiment, we found that the high dose of MEHP (10 − 3 M) significantly retarded or even abrogated early embryonic development at the two-cell stage, and significantly reduced the rates of 8-cell embryo and blastocyst formation, whereas the medium and low-dose of MEHP (10 − 4 M and 10 − 5 M, respectively) barely affected embryonic development. Thus, because the in vitro and in vivo experiments detected certain effects of DEHP on early embryonic development, we further explored the specific molecular mechanisms. The qRT-PCR analysis showed that in mouse ovaries, the expression level of XBP-1 decreased with the increasing DEHP dose, while XBP-1S showed the opposite trend. In embryos, however, there was no difference in the expression level of XBP-1 among each group. In addition, whether in ovaries or embryos, the expression of CHOP mRNA increased with the increasing DEHP dose. Western Blotting results of embryos in vivo showed that there was no difference in the expression level of IRE1 in each group, which was consistent with the expression of XBP-1 in embryos in vitro . In addition, we could see from the figure that the expression of PERK protein in embryos was obviously showing a gradual decrease, and p-PERK protein was showing an increasing trend, although there was no statistical difference, the ratio of p-PERK to PERK was obviously showing a gradual rising trend. The TUNEL analysis indicated that DEHP-induced changes in the expression of CHOP were directly related to blastocyst apoptosis. We further confirmed in this mouse study the toxicity of environmental DEHP levels for the ovaries and the early embryonic development, and the effect of MEHP, the primary metabolite of DEHP, on early embryonic development and blastocyst apoptosis was revealed by in vitro experiments. Among the ER stress-related indicators detected in this study, the expression levels of BiP , CHOP and ATF6 mRNA in embryos intervened with 10 − 3 M MEHP were significantly increased, and there was no significant difference in the mRNA expression levels of XBP-1 and XBP-1S , which was consistent with the trend in embryos exposed to DEHP. In this study, we specifically found that DEHP and MEHP induced ER stress in the ovaries and early embryos, and we found a direct association between CHOP and blastocyst apoptosis. Due to a lack of self-stabilizing mechanism as in somatic cells, oocytes and early embryos are very susceptible to various exogenous stimulants, such as temperature, osmotic pressure, chemical exposure, or oxidative stress. 30 , 32 , 42 The ER is one of the largest organelles in eukaryotic cells. It plays a major role in the synthesis, folding, and maturation of at least 1/3 of all cellular proteins, 43 and many of those are involved in the normal development and differentiation of the embryo, which is therefore dependent on the normal ER function. 44 It has been shown that ER stress affects the early embryonic development, and the inhibition of ER stress may improve this developmental process. 33 It has been reported previously that ER stress promoted protein folding by up-regulating BiP , which might restore the normal ER function during the early stage of ER stress. 45 However, if the adaptive response could not adequately restore the protein-folding homeostasis, the UPR signaling would be sustained, indicating high or chronic ER stress induced by pro-apoptotic signaling, 46 of which CHOP is an important member. 45 In the mouse ovaries obtained after DEHP exposure, we observed different expression patterns for BiP and CHOP mRNA under ER stress. The mRNA expression of BiP was elevated at the low DEHP dosage and reduced at the higher DEHP dose. However, at the same exposure time, the mRNA expression of CHOP was dose-dependently elevated among all the groups of DEHP. A dose of 0.05 mg/kg/d DEHP stimulated the mRNA expression of BiP and CHOP in the ovaries. At the 5 mg/kg/d dose, the mRNA expression of BiP was reduced, and it diminished at the 500 mg/kg/d dosage, although this BiP mRNA level was still higher than that in the control. However, the mRNA expression of CHOP showed a continuous upward trend within the DEHP exposure range. This suggested that the mRNA expression levels of the protective molecular chaperone BiP and the apoptosis-inducing CHOP were up-regulated during the ER stress in the ovaries. However, ER stress induced by DEHP was initially dominated by BiP -mediated protective effects; although cells in the ovarian tissue might have entered apoptosis, it was not significant. In the presence of strong, long-lasting stimulants (high exposure to DEHP), the apoptosis signal pathway in ER stress was dominant. At this time point, the mRNA expression of BiP with the protective effects was gradually reduced, and the mRNA expression of CHOP molecules had a major role in inducing apoptosis in ovarian tissue cells. To verify that high-dose DEHP-induced apoptosis through the BiP - CHOP signaling pathway, embryos from female mice exposed to DEHP were further examined. We found that the mRNA expression of CHOP in embryos was also gradually increased, depending on the increase of the DEHP dosage. But unlike in ovaries, the expression level of BiP mRNA in embryos showed a tendency to gradually increase with the increasing of DEHP dose. In this regard, we thought that under the same exposure time and dose of DEHP, the ovarian tissue first response to ER stress, and it reflected the comprehensive situation of all phrase of follicles in the ovaries, not only primordial follicles, 47 which could not exactly represent the actual situation of the follicles forming the embryo in this follicular cycle, but overall, they responded to each dose of DEHP. Some studies believed that this might be the continuous overexpression of BiP caused by UPR in embryos, which was a key feature of the adaptive response to mild chronic ER stress, leading to cell survival. 48 In the future, we will further study the effect of DEHP on all phrase of follicles. Besides, from the perspective of the effect of DEHP on ER stress in embryos, Western Blotting showed no changes of IRE1 protein, nor did it cause changes in the expression level of XBP-1 mRNA in embryos. In ER stress, XBP-1 was the downstream target of IRE1 activation, which sheared XBP-1 mRNA and form active XBP-1S . 40 In this study, there was no change in the expression of the IRE1 / XBP-1 signaling pathway. Therefore, we thought that under the exposure time and dose of DEHP performed in this study, the ER stress occurring in the embryo had nothing to do with the IRE1 signaling pathway, and this was consistent with the scientific statement that the three main signal pathways of ER stress might not be activated at the same time, but showed a dose effect and time sequence. 49 In a similar manner to IRE1 , dissociation of BiP from PERK ’ER luminal face leads to dimerization, interdimer trans-phosphorylation and activation of its cytosolic kinase domain. In the context of the UPR, PERK ’ main cellular target is eIF2α . PERK phosphorylates eIF2α , resulting in the reduction in global protein synthesis allowing the cell time to refold or degrade any unfolded protein in the ER. 50 eIF2α is an essential constituent of ternary complex, which is required for the initiation of translation of an mRNA into a polypeptide. 51 In response to eIF2α phosphorylation, ATF4 are upregulated, which further upregulates a number of key genes including CHOP . CHOP is downstream of ATF4 and is itself a transcription factor which is produced in high quantity after prolonged ER stress, promoting apoptosis as well as ER stress-induced cytokine production. 52 In this study, there was no significant difference in the protein expression levels of PERK in embryos among DEHP-treatment groups and the control. However, we found that there were significant statistical differences in each DEHP-treatment group compared with the control, and there were also statistical differences between the experimental groups ( P < 0.05), showing a dose-dependent manner. Besides, regarding phosphorylated protein, the effective indicator we need to observe should be the ratio of phosphorylated protein to total protein in order to effectively reflect the actual phosphorylation level. According to statistics, the ratio of p-PERK to PERK protein also showed a gradually increasing trend, which were statistically different in the medium and high DEHP-exposure groups, indicating that DEHP could indeed cause changes in the PERK signaling pathway in embryos. Under long-term high-dose exposure, PERK signaling pathway showed a sustained and enhanced response to promote embryos to cope with ER stress caused by the external environment. As a downstream molecule of PERK , eIF2α was activated in the manner of phosphorylation, resulting in the rapid downregulation of global protein synthesis and preferential translation of genes. 53 In this study, we found that the expression of eIF2α protein in the embryos of each DEHP-treatment group showed an increasing trend, and there were significant differences in the medium and high exposure groups. Elevated phosphorylation of eIF2α was very important in ER stress, which could enhance the transcription and translation levels of ATF4 and CHOP in cells after DEHP treatment. 54 Just like the results of our study, the protein expression of ATF4 in each DEHP-treatment group also showed a gradual increasing trend, which was consistent with eIF2α . Studies indicated that DEHP effectively triggered the ER stress as demonstrated by the increased phosphorylation PERK and its downstream substrate (eIF2α, as well as the increased levels of ATF4 and CHOP 54 . During long-term ER stress, ATF4 might also stimulate genes of CHOP, which was responsible for initiation of the apoptotic cascade. 52 In summary, our study showed that the protein expression PERK , p-PERK , eIF2α and ATF4 in embryos exposed to DEHP were consistent with the theoretical basis of ER stress. The TUNEL staining revealed that the apoptosis signal in blastocysts of the high-dose group (500 mg/kg/d DEHP) was significantly stronger than that in the medium-dose group (5 mg/kg/d DEHP), and the results were consistent with the significant increase of CHOP mRNA in embryos exposed to 5 and 500 mg/kg/d DEHP. Whereas, in the group of 0.05 mg/kg/d DEHP, the expression level of CHOP mRNA was significantly increased, but the blastocysts formed by its continued development did not show apoptosis signals. We thought that these were compatible with a transient UPR induction in which CHOP levels were insufficient to trigger massive apoptosis, likely due to concomitant overexpression of BiP and Bcl-2 that could prevent or markedly delay cell death to facilitate adaptation. In support of this assumption, it had been reported that cell fate was dependent not only on the severity or duration of ER stress but also on the balance between pro-death and pro-survival factors. 48 It has been repeatedly suggested that CHOP gene expression is strictly involved in cell death by apoptosis, but research has not confirmed existence of any relationship between induction of CHOP and apoptosis. The mechanisms that may explain how apoptotic cell death is induced by CHOP still remain unclear but, there is ample evidence that it is implicated in numerous human disease entities, including diabetes, neurodegenerative diseases, ischemic diseases, and tumor development. 55 Although ER stress and sustained UPR signaling have been well documented in affected tissues in diabetes, neurodegeneration, stroke, pulmonary fibrosis, viral infection, inflammatory disorders, cancer, and heart disease, 56 their contributions to preimplantation development have not been investigated to date. Several studies have investigated individual components of the UPR, and the results indicated that the UPR pathway constituents are expressed during early development. 57 – 59 In porcine embryos, blastocysts recovered from a tunicamycin-treated group exhibited a significantly increased apoptosis rate along with up-regulated mRNA levels of pro-apoptotic BAX and down-regulated mRNA levels of anti-apoptotic BCL2 . 60 CHOP was identified as a factor that responded to DNA damage. 32 Under conditions of severe or prolonged ER stress, BiP was released from PERK to trigger activating transcription factor ( ATF4 ), leading to the upregulation of CHOP expression. 34 CHOP mainly induced apoptosis by downregulation of the anti-apoptotic BCL2 gene, 30 , 32 which supported our findings to some extent. XBP-1S is considered an appropriate marker for the induction of the IRE1 pathway during the UPR, because XBP-1 is spliced exclusively under ER stress conditions. 40 The qRT-PCR analysis demonstrated that mRNAs for porcine XBP-1u and XBP-1S were clearly detected in GV-stage oocytes, but only the mRNA of XBP-1u was detected in MI and MII-stage oocytes in mice, 35 suggesting that XBP-1 might play a very important role in the maturation of oocytes. In our study, there was a significant difference in the mRNA expression of XBP-1S in the ovaries of mice exposed to DEHP. The mRNA expression of XBP-1S was gradually increased by the increasing DEHP dosage, indicating that XBP-1 might be associated with the development and maturation of oocytes. In mouse embryos, UPR inducers, such as TM and sorbitol, increase nuclear XBP-1S at the one- and two-cell stages and activate XBP-1 mRNA splicing at the eight-cell, morula, and blastocyst stages, 33 , 40 indicating that the IRE1 arm of the UPR was activated as an important coping response and adaption to ER stress in preimplantation embryos. In our study, however, there was no difference in the mRNA expression of XBP-1S in eight-cell stage embryos treated with DEHP and MEHP. A possible explanation for this result is that different ER stress signaling could be initiated in embryos when facing different stimulants, because the UPR pathways could be individually modulated instead of complete activation or suppression of all three signaling pathways. 49 Based on the DEHP/MEHP exposure duration and dose regimen implemented in this study, DEHP/MEHP might affect early embryo development through the BiP - CHOP signaling pathway under ER stress instead of the IRE1 / XBP-1 signaling pathway, which requires further investigation in the future. This study showed that there was a significant difference in the mRNA expression of ATF6 in the high-MEHP dose group compared to that of the control, which was consistent with the mRNA expression patterns of CHOP and BiP detected by qRT-PCR. These results further supported that under high-MEHP dose exposure, the embryos failed to maintain cell homeostasis through ER stress, but instead exerted pro-apoptosis effects by promoting the expression of apoptotic signaling molecules regulated by ATF6 and CHOP . In this study, we established a DEHP exposure model in female mice. In addition, embryos were exposed to MEHP in vitro to verify further that DEHP exerted early embryonic developmental toxicity through its main metabolite MEHP. This study has three major limitations. Firstly, the implantation and live birth outcomes of well-developed blastocysts exposed to DEHP or MEHP were not studied. This experiment would determine the implantation potential of DEHP-exposed blastocysts with normal appearance and morphology. Secondly, there was only one metabolite of PAEs tested in this study. More rigorous experiments should be conducted with PAE metabolite mixtures, which is more consistent with the actual exposure of the population, thus, generating more meaningful experimental results. Thirdly, further experiments in human embryos should be performed to collect DEHP/MEHP-exposed embryos for single-cell sequencing to detect changes in ER stress, thereby achieving the transformation of this basic research into a clinical research project. In conclusion, the current study demonstrated that DEHP and MEHP affected embryo fertilization and blastocyst apoptosis through ER stress. In addition, we further identified a direct correlation between CHOP stimulated under ER stress and blastocyst apoptosis induced by DEHP and MEHP. We hope that the results and interpretation of this study can enhance the people’s vigilance against the use of plasticizers in daily life, thus preventing the adverse effects caused by the exposure of the ovaries and the early-stage embryos to high levels of environmental DEHP. These efforts may contribute to reducing the abortion rate, increasing the clinical pregnancy rate, and improving the clinical pregnancy outcomes, which has an important clinical value. Abbreviations DEHP Di-(2-ethylhexyl) phthalate MEHP Mono-(2-ethylhexyl) phthalate ER Endoplasmic Reticulum qRT-PCR real-time reverse-transcription PCR TUNEL terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling ART Assisted Reproductive Technology EDCs Endocrine Disrupting Chemicals PAEs Phthalates Acid Esters GV Germinal Vesicle MI Metaphase I MII Metaphase II UPR Unfolded Protein Response BiP Immunoglobulin heavy chain binding protein GRP78 Glucose-regulated protein 78 IRE1 Inositol-requiring enzyme 1 XBP-1 X-box Binding Protein 1 PERK PKR-like eukaryotic initiation factor 2A kinase eIF2α eukaryotic Initiation Factor-2α CHOP C/EBP homologous protein ATF4 Activating transcription factor 4 ATF6 Activating transcription factor 6 DMSO Dimethylsulfoxide PMSG Pregnant Mare Serum Gonadotropin HCG Human Chorionic Gonadotrophin COCs Cumulus-oocyte complexes IVF in vitro Fertilization PFA Paraformaldehyde PBS Phosphate buffer saline BSA Bovine Serum Albumin TM Tunicamycin Declarations CONFLICT OF INTEREST Authors assure that there are no conflicts of interest. Author Contribution XueMei Teng performed the study, analyzed the data, and wrote the paper. All authors read, revised and approved the final manuscript. ACKNOWLEDGEMENTS This study was supported by the National Natural Science Foundation of China (grant No. 81571508) and the Natural Science Foundation of Hubei Province (grant No. 2014CFA069). Data Availability Data is provided within the manuscript. References Hertz-Picciotto, I. et al. A cohort study of in utero polychlorinated biphenyl (PCB) exposures in relation to secondary sex ratio. Environ. Health . 7 , 37 (2008). Sifakis, S., Androutsopoulos, V. P., Tsatsakis, A. M. & Spandidos, D. A. Human exposure to endocrine disrupting chemicals: effects on the male and female reproductive systems. Environ. Toxicol. Pharmacol. 51 , 56–70 (2017). Katsikantami, I. et al. A global assessment of phthalates burden and related links to health effects. Environ. Int. 97 , 212–236 (2016). Calafat, A. M. et al. 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T. et al. Adaptation to ER stress is mediated by differential stabilities of pro-survival and pro-apoptotic mRNAs and proteins. PLoS Biol. 4 (11), e374 (2006). Ma, Y., Shimizu, Y., Mann, M. J., Jin, Y. & Hendershot, L. M. Plasma cell differentiation initiates a limited ER stress response by specifically suppressing the PERK-dependent branch of the unfolded protein response. Cell. Stress Chaperones . 15 (3), 281–293 (2010). Hughes, D. & Mallucci, G. R. The unfolded protein response in neurodegenerative disorders - therapeutic modulation of the PERK pathway. FEBS J. 286 (2), 342–355 (2019). RKimball Aloop. Eukaryotic initiation factor eIF2. Int. J. Biochem. Cell Biol. 31 (1), 25–29 (1999). Nishitoh, H. CHOP is a multifunctional transcription factor in the ER stress response. J. BioChem. 151 (3), 217–219 (2011). Rozpędek DP, W., Mucha, B., Leszczyńska, H., Diehl, J. A. & Majsterek, I. The Role of the PERK-eIF2α-ATF4-CHOP Signaling Pathway in Tumor Progression During Endoplasmic Reticulum Stress. (2016). Sun, X. et al. Di(2-ethylhexyl) phthalate-induced apoptosis in rat INS-1 cells is dependent on activation of endoplasmic reticulum stress and suppression of antioxidant protection. J. Cell. Mol. Med. 19 (3), 581–594 (2015). Li, Y., Guo, Y., Tang, J., Jiang, J. & Chen, Z. New insights into the roles of CHOP-induced apoptosis in ER stress. Acta Biochim. Biophys. Sin (Shanghai) . 46 (8), 629–640 (2014). Wang, S. & Kaufman, R. J. The impact of the unfolded protein response on human disease. J. Cell. Biol. 197 (7), 857–867 (2012). Hao, L. et al. The unfolded protein response contributes to preimplantation mouse embryo death in the DDK syndrome. Biol. Reprod. 80 (5), 944–953 (2009). Takao, I. c,1, Ryoko Akaia,c, Shinya Yamanakad, and Kenji Kohnoc. Function of IRE1 alpha in the placenta is essential for placental development and embryonic viability. (2009). Luo, S., Mao, C., Lee, B. & Lee, A. S. GRP78/BiP is required for cell proliferation and protecting the inner cell mass from apoptosis during early mouse embryonic development. Mol. Cell. Biol. 26 (15), 5688–5697 (2006). Lin, T. et al. Tauroursodeoxycholic acid improves pre-implantation development of porcine SCNT embryo by endoplasmic reticulum stress inhibition. Reprod. Biol. 16 (4), 269–278 (2016). Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6650176","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":475901669,"identity":"37651454-8dc8-4eca-bd02-3600d53046de","order_by":0,"name":"XueMei Teng","email":"","orcid":"","institution":"Huazhong University of Science and Technology Tongji Medical College Maternal and Child Health Hospital of Hubei Province","correspondingAuthor":false,"prefix":"","firstName":"XueMei","middleName":"","lastName":"Teng","suffix":""},{"id":475901670,"identity":"5ad75e1c-38f4-41c4-81ec-974fb1044a6f","order_by":1,"name":"Nian Liu","email":"","orcid":"","institution":"Huazhong University of Science and Technology Tongji Medical College Maternal and Child Health Hospital of Hubei Province","correspondingAuthor":false,"prefix":"","firstName":"Nian","middleName":"","lastName":"Liu","suffix":""},{"id":475901671,"identity":"3df4b947-a7a4-434e-ac50-f9ec5970d863","order_by":2,"name":"ZhenZhen Song","email":"","orcid":"","institution":"Huazhong University of Science and Technology Tongji Medical College Maternal and Child Health Hospital of Hubei Province","correspondingAuthor":false,"prefix":"","firstName":"ZhenZhen","middleName":"","lastName":"Song","suffix":""},{"id":475901672,"identity":"ad4d0f72-44e3-487a-b598-13b9149baa97","order_by":3,"name":"XueHui Zeng","email":"","orcid":"","institution":"Huazhong University of Science and Technology Tongji Medical College Maternal and Child Health Hospital of Hubei Province","correspondingAuthor":false,"prefix":"","firstName":"XueHui","middleName":"","lastName":"Zeng","suffix":""},{"id":475901673,"identity":"1b12b1c3-0064-4886-9735-b42a293d5cf9","order_by":4,"name":"NianHua Yi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAxklEQVRIiWNgGAWjYBACefkHCYf/VNTI8bM3EKnFsCHh4QGeM8eMJXsOEGvNgcTHB3jbmBM3zEggUgdjw+GEA5JtbMYGko833mCosYkmqIWdsS3hgME5GTlz6bRiC4ZjabkNBG1p5kk4kFDGZmw5O8dMAmgnYS0Mx/g/HDjABvTLzTPEajnDkHCwAeT9GzxEajGcwZBwmAEcyEC/JBDjF3kJhuTPDOCoPLzxxocaGyIchgQMJBJIUQ7RQqqOUTAKRsEoGBkAANmERGjtr2bPAAAAAElFTkSuQmCC","orcid":"","institution":"Huazhong University of Science and Technology Tongji Medical College Maternal and Child Health Hospital of Hubei Province","correspondingAuthor":true,"prefix":"","firstName":"NianHua","middleName":"","lastName":"Yi","suffix":""},{"id":475901674,"identity":"e3550737-027d-44e6-b1e4-dccc9729fd89","order_by":5,"name":"YuFeng Li","email":"","orcid":"","institution":"Huazhong University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"YuFeng","middleName":"","lastName":"Li","suffix":""}],"badges":[],"createdAt":"2025-05-13 01:08:12","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6650176/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6650176/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":85483736,"identity":"ad2a0208-906e-4685-a656-813d8e84e195","added_by":"auto","created_at":"2025-06-26 11:32:23","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":860035,"visible":true,"origin":"","legend":"\u003cp\u003eThe mRNA expression levels of ER stress-related molecules in ovaries and embryos obtained from mice exposed to DEHP. ICR female mice were exposed to di-2-ethylhexyl phthalate (DEHP) (0.05, 5, and 500 mg/kg/d) for five weeks (n=13 for female mice and n=3 for male mice). Ovaries were collected for qRT-PCR to detect the mRNA expression of ER stress-associated factors (\u003cem\u003eBiP\u003c/em\u003e, \u003cem\u003eXBP-1\u003c/em\u003e, \u003cem\u003eXBP-1S\u003c/em\u003e, \u003cem\u003eCHOP\u003c/em\u003e, \u003cem\u003eATF6\u003c/em\u003e).\u003c/p\u003e\n\u003cp\u003eAll data were presented as means ± SEMs. D 0.05: 0.05 mg/kg/d DEHP, D 5: 5 mg/kg/d DEHP, D 500: 500 mg/kg/d DEHP; * \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05 VS control, ** \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001, ****\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.0001. (A)-(E): The mRNA expression of ER stress-related molecules in ovaries exposed to DEHP. (F)-(J): The mRNA expression of ER stress-related molecules in embryos obtained from mice exposed to DEHP. The mRNA expression of \u003cem\u003eBiP\u003c/em\u003e and \u003cem\u003eCHOP\u003c/em\u003e was gradually increased in the DEHP-treatment groups than that in the control group.\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-6650176/v1/13746be4a145dd323769844f.png"},{"id":85483753,"identity":"f5d074a6-c9d2-4116-ab54-b6a6e729cca3","added_by":"auto","created_at":"2025-06-26 11:32:24","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":45342940,"visible":true,"origin":"","legend":"\u003cp\u003eEmbryo development diagrams obtained from DEHP-exposed mice and embryo development diagrams after direct exposure to MEHP. ICR female mice were exposed to di-2-ethylhexyl phthalate (DEHP) (0.05, 5, and 500 mg/kg/d) for five weeks (n=13 for female mice and n=3 for male mice). In the part of DEHP experiment, embryos were cultured in KSOM medium until blastocyst stage. In the part of MEHP experiment, two-cell embryos were divided into four groups and culture in KSOM containing different concentrations of MEHP (0, 10\u003csup\u003e-5\u003c/sup\u003e, 10\u003csup\u003e-4\u003c/sup\u003e, and 10\u003csup\u003e-3\u003c/sup\u003eM).\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-6650176/v1/f159b842a605d2cb6843aeaf.png"},{"id":85484697,"identity":"e3b3f9d9-890c-418a-937c-78711bee4bb9","added_by":"auto","created_at":"2025-06-26 11:40:23","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1538776,"visible":true,"origin":"","legend":"\u003cp\u003eHistograms of embryos development at all stages exposed to DEHP and MEHP.\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-6650176/v1/c2c4b918312f055766f1a6a6.png"},{"id":85483737,"identity":"965964ea-14f0-454d-bee9-395379c5f48f","added_by":"auto","created_at":"2025-06-26 11:32:23","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1000865,"visible":true,"origin":"","legend":"\u003cp\u003eThe protein expression level of ER stress-related molecules in the embryos obtained from mice exposed to DEHP.\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-6650176/v1/f32be6a399b74f93d108664e.png"},{"id":85483739,"identity":"728d3c69-fa3e-46cf-812b-f364f8031897","added_by":"auto","created_at":"2025-06-26 11:32:23","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2221902,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of DEHP and MEHP exposure on blastocyst apoptosis \u003cem\u003ein vivo \u003c/em\u003eand\u003cem\u003e in vitro\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-6650176/v1/6a59c27d049e287a038e4975.png"},{"id":85484698,"identity":"d4efc61d-17e0-461f-9b44-e62dd0eaf279","added_by":"auto","created_at":"2025-06-26 11:40:23","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":443203,"visible":true,"origin":"","legend":"\u003cp\u003eThe mRNA expression levels of ER stress-related molecules in embryos exposed to MEHP. There were 8 female and 2 male mice in each experimental group and control group, and each experiment was independently repeated five times. Mice without intervention were sacrificed and embryos were obtained by IVF, and then two-cell embryos were divided into four groups randomly and cultured in KSOM containing mono-(2-ethylhexyl) phthalate (MEHP) (0, 10\u003csup\u003e-5\u003c/sup\u003e, 10\u003csup\u003e-4\u003c/sup\u003e, and 10\u003csup\u003e-3\u003c/sup\u003eM).\u003c/p\u003e","description":"","filename":"Fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-6650176/v1/8ef7726fea412ef339b2fa74.png"},{"id":101752555,"identity":"71bdf615-7062-4b23-bbad-47ae19aecc6d","added_by":"auto","created_at":"2026-02-03 10:28:08","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":46343736,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6650176/v1/879a4738-87cb-4b56-8355-c4fc9c8f92c9.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Di-(2-ethylhexyl) phthalate and mono-(2-ethylhexyl) phthalate attenuate early embryonic development and induce blastocyst apoptosis via PERK/ATF4/CHOP pathway in mice","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003eOver the past decade, the global use of assisted reproductive technology (ART) has triggered a constantly increasing research investigating whether lifestyle-related risk factors, such as smoking, caffeine, alcohol, or endocrine-disrupting chemicals (EDCs), affect ART outcomes.\u003c/p\u003e \u003cp\u003eEDCs are a variety of exogenous compounds that interfere with any type of hormonal effects responsible for maintaining homeostasis and regulating developmental processes. EDCs have shown serious adverse effects on human reproductive health,\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e which has caused widespread concerns in recent years. Phthalic acid esters (PAEs) as the most prevalent EDC type are very common synthetic organic compounds,\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e which are widely used as solvents and plasticizers in cosmetics, automotive glass, printing and paint, and other chemical industries.\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e PAEs enter the human body via the digestive and the respiratory tract as well as through the skin by absorption,\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e which results in a ubiquitous exposure of the entire population. Clinical studies have shown that in addition to detecting PAEs and their metabolites in traditional biological samples (such as serum and urine), detectable levels of certain PAEs have been found in amniotic fluid, milk, semen, follicular fluid, and other body fluids closely related to reproduction, especially di-2-ethylhexyl phthalate (DEHP) and its metabolite, mono-(2-ethylhexyl) phthalate (MEHP),\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e indicating that DEHP and MEHP may affect the reproductive outcomes.\u003c/p\u003e \u003cp\u003eMEHP as the primary DEHP metabolite is more toxic than DEHP. To date, many toxicological studies have been conducted assessing the effects on various systems and organs of DEHP and MEHP, including the reproductive toxicity and the early embryonic developmental toxicity. Zhao et al investigated that DEHP could significantly reduce the quality of sperm in mice, which was reflected in the damage of sperm DNA and the decline of sperm motility, and different degrees of endoplasmic reticulum (ER) swelling could also be observed under electron microscope, all of which ultimately lead to testicular spermatogenesis dysfunction.\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e DEHP mediated oxidative stress and ER stress by regulating the expression levels of \u003cem\u003eNrf2\u003c/em\u003e and \u003cem\u003eHSR\u003c/em\u003e-related genes, and exerted renal and splenic toxicity, which was manifested by decreased glomerular filtration function and changes in the structure of renal tubular epithelial cells, and structural damage to the splenic body.\u003csup\u003e\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e Moreover, the study also found that the same dose of DEHP could regulate the balance of redox reactions. Once the balance was broken, structural changes in the liver occurred, including swelling of mitochondria, disorder of liver plates, degeneration of fat cells, chromatin bordering, deep staining of nuclei, and abnormally elevated liver enzyme levels.\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e In male, DEHP could also cause the damage of testicular stromal cells and pathological changes in quail heart, such as cardiomyocyte swelling and muscle fiber dilation, through \u003cem\u003eNrf2\u003c/em\u003e signaling pathway and its downstream target genes.\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eAnimal studies have shown that DEHP exerts female reproductive toxicity by interfering with the normal menstrual cycle, reducing the ovarian reserve function, and interfering with the oocyte meiosis.\u003csup\u003e\u003cspan additionalcitationids=\"CR17 CR18 CR19\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e MEHP reduces the developmental capacity of oocytes by increasing the number of GV-phase oocytes and decreasing the number of MII-phase oocytes; it also exerts embryonic developmental toxicity by reducing the proportion of two-cell embryos and the rate of blastocyst formation.\u003csup\u003e\u003cspan additionalcitationids=\"CR22 CR23\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e A previous report also indicated that the mouse two-cell embryos were blocked by MEHP.\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e In addition, related studies explored the effect of DEHP doses. Specifically, a high intrauterine MEHP exposure accelerates ovarian aging by accelerating primordial follicle activation, resulting in reproductive defects in F1/F2 adult females.\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e In general, exposure to low doses of DEHP or MEHP is not associated with reproductive toxicity. However, it has been demonstrated that equivalent exposure to a low DEHP dose in the population accelerated the rate of follicle recruitment, reduced or delayed the methylation level of the imprinted genes in oocytes, and increased the abnormalities of the mature oocyte MII spindle.\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e Thus, these studies indicated that environmental exposure to DEHP in daily life could also adversely affect reproductive outcomes (follicle formation, oocyte maturation, etc.). Therefore, studying the effects of environmental-level DEHP or MEHP exposure on early embryo development has substantial clinical value.\u003c/p\u003e \u003cp\u003eThe endoplasmic reticulum (ER) is critically involved in the correct folding and processing of newly synthesized proteins. Any damage to the ER and the resulting ER stress can lead to abnormalities in transcriptional regulation and gene expression, metabolism, signaling, or ion channel function.\u003csup\u003e\u003cspan additionalcitationids=\"CR28\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e Under ER stress, unfolded or misfolded proteins accumulated in the ER lumen,\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e activating the signaling pathways that are involved in the unfolded protein response (UPR).\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e There are three major signaling pathways for stimulating the UPR: (I) via the inositol-requiring enzyme 1 (\u003cem\u003eIRE1\u003c/em\u003e) involved in recruiting several signaling molecules, splicing, and generating activated transcription factor X-box binding protein 1 (\u003cem\u003eXBP-1\u003c/em\u003e), ER chaperones, such as the binding immunoglobulin protein (\u003cem\u003eBiP\u003c/em\u003e; also known as glucose-regulated protein \u003cem\u003eGRP78\u003c/em\u003e), along with \u003cem\u003eC/EBP\u003c/em\u003e homologous protein (\u003cem\u003eCHOP\u003c/em\u003e); (II) via the PKR-like eukaryotic initiation factor 2A kinase (\u003cem\u003ePERK\u003c/em\u003e), which phosphorylates eukaryotic initiation factor-2α (\u003cem\u003eeIF2α\u003c/em\u003e) to suppress protein translation; and (III) via the activating transcription factor 6 (\u003cem\u003eATF6\u003c/em\u003e), which is translocated to the Golgi apparatus where proteolytic processing generates its activated form \u003cem\u003eATF6α\u003c/em\u003e, a transcription factor that stimulates the expression of \u003cem\u003eBiP\u003c/em\u003e and \u003cem\u003eXBP-1\u003c/em\u003e, which act together to limit ER stress by reducing the protein synthesis, facilitating the protein degradation, and increasing the folding capacity.\u003csup\u003e\u003cspan additionalcitationids=\"CR28\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e However, the apoptotic pathway is activated when the level of misfolded or unfolded protein exceeds the processing capacity of the ER, leading to cell injury and death induced by \u003cem\u003eXBP-1\u003c/em\u003e, activating transcription factor 4 (\u003cem\u003eATF4\u003c/em\u003e), and \u003cem\u003eCHOP\u003c/em\u003e.\u003csup\u003e\u003cspan additionalcitationids=\"CR28\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e Several recent studies on the reproductive system found that ER stress fulfills a critical function in mouse oocyte maturation,\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e and functional pathways of ER stress and UPR signaling are essential for oocyte maturation and quality.\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e Multiple recent studies with a main focus on the \u003cem\u003eXBP-1\u003c/em\u003e pathway demonstrated that the ER stress affects preimplantation and post-implantation development of embryos.\u003csup\u003e\u003cspan additionalcitationids=\"CR36 CR37\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eIn this study, we performed animal experiments for simulating DEHP exposure in the population. We explored the toxicity of DEHP on the ovaries and early embryo development, and we investigated the ER stress mechanism induced by DEHP and MEHP. Our work has a clinical value because it is designed to increase the people\u0026rsquo;s vigilance against the use of plasticizers in daily life, which could prevent the adverse effects that high exposure to environmental DEHP has on the ovaries and the early embryonic development.\u003c/p\u003e"},{"header":"2 Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Chemicals\u003c/h2\u003e \u003cp\u003eDEHP (99% purity) was purchased from Sigma-Aldrich (St. Louis, MO) and stored at 20-25\u0026deg;C until used. MEHP (99% purity) was purchased from AccuStandard (New Haven, CT). Stock solutions of MEHP were prepared using dimethylsulfoxide (DMSO) (Sigma- Aldrich) and stored at -20\u0026deg;C. The solution was then diluted to prepare KSOM medium containing 0, 10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e, 10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e, or 10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003eM MEHP. DMSO was added to the medium at the same concentration as compounds treatments (\u0026lt;\u0026thinsp;0.1%) to serve as vehicle control. Pregnant mare serum gonadotropin (PMSG) was purchased from Solarbio (Beijing Science \u0026amp; Technology Co., Ltd.). Human chorionic gonadotropin (HCG) was purchased from Livzon (Shanghai, China). HTF (fertilization medium) was purchased from Sigma (Millipore Corp. USA). KSOM was purchased from EasyCheck (Nanjing AIBI Biology Co., Ltd.).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Experimental animals and DEHP/MEHP treatment\u003c/h2\u003e \u003cp\u003e \u003cb\u003e2.2.1 Mice exposed to DEHP\u003c/b\u003e \u003cb\u003ein vivo\u003c/b\u003e \u003cb\u003eand mice used to recover embryos for MEHP exposure\u003c/b\u003e \u003cb\u003ein vitro\u003c/b\u003e\u003c/p\u003e \u003cp\u003eHealthy, sexually mature female (30\u0026ndash;35 g) and male (35\u0026ndash;40 g) ICR mice and 4\u0026ndash;5 weeks old, healthy female and male mice were purchased from the Laboratory Animal Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology (Wuhan, China). Mice were housed in a temperature- and humidity-controlled room with a 12 h light/dark cycle. DEHP stock solution aliquots of 2 \u0026micro;L, 200 \u0026micro;L, 20 mL were added to 10 mL, 10 mL, and 30 mL corn oil, respectively, which were each added to 10 kg mouse feed, formulating the respective doses of 0.05 mg/kg/d, 5 mg/kg/d, and 500 mg/kg/d DEHP feed to simulate DEHP exposure in the human population. The female mice, 4\u0026ndash;5 weeks old, were randomly divided into four groups for a 5-week DEHP exposure of 0, 0.05 mg/kg/d, 5 mg/kg/d, and 500 mg/kg/d (n\u0026thinsp;=\u0026thinsp;13/group for qRT-PCR and n\u0026thinsp;=\u0026thinsp;10/group for Western Blotting), which was repeated three times. Sexually mature, healthy female and male ICR mice, which did not receive any intervention, were sacrificed to generate embryos for exposure to MEHP. All mcie were sacrificed by cervical dislocation. All animal procedures were approved by the Animal Ethics Committee of Tongji Hospital. All methods were carried out in accordance with the ARRIVE guidelines 2.0.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Embryo collection and culture\u003c/h2\u003e \u003cp\u003eThe female ICR mice were superovulated with PMSG, and cumulus-oocyte complexes (COCs) were collected in HTF medium from the ampullae of oviducts 15\u0026ndash;17 h after injection of HCG. Spermatozoa were collected from the caudal epididymis of adult male ICR mice (35\u0026ndash;40 g) and capacitated by preincubation for 1 h in HTF. The COCs were inseminated with capacitated spermatozoa in HTF embryo medium in a humidified atmosphere with 5% CO\u003csub\u003e2\u003c/sub\u003e at 37\u0026deg;C. After an insemination period of 4\u0026ndash;5 h, the zygotes were washed and cultured in KSOM droplets. Then, 24 h after insemination, two-cell embryos were transferred to new KSOM droplets.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Experimental design\u003c/h2\u003e \u003cp\u003eThis study is performed in accordance with relevant guidelines and regulations. All methods are reported in accordance with ARRIVE guidelines. The study included two sets of experiments to explore the developmental effect on early embryonic development and the mechanism of ER stress in mice ovaries and early embryos induced by DEHP or MEHP. The first and second set experiments consist of four parts and three parts respectively. The dosage regimen was selected to establish a dose range that would not be lethal, but may cause identification of other possible target organ effects. The lethal dose 50 (LD50) of DEHP was 30 g/kg. It was known that DEHP at dose of 1000 mg/kg was able to cause adverse effects in female rats without causing systemic toxicity.\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e In the DEHP \u003cem\u003ein vivo\u003c/em\u003e exposure experiment, the female mice were randomly divided into four groups for a 5-week exposure with different DEHP doses (0, 0.05 mg/kg/d, 5 mg/kg/d and 500 mg/kg/d), while the adult males received no intervention. In the MEHP \u003cem\u003ein vitro\u003c/em\u003e exposure experiment, all embryos generated by \u003cem\u003ein vitro\u003c/em\u003e fertilization (IVF) were randomly divided into four groups and cultured in KSOM medium containing different concentrations of MEHP (0, 10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e, 10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e, and 10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003eM).\u003c/p\u003e \u003cp\u003eThe first part of the experiments was initiated by the DEHP \u003cem\u003ein vivo\u003c/em\u003e exposure. There were 13 female and 3 male mice in each group for qRT-PCR and 10 female and 2 male mice in each group for Western Blotting, respectively, and each experiment was independently repeated three times. Female mice exposed to DEHP were superovulated using the conventional PMSG-HCG method and sacrificed to collect ovaries from all the groups for real-time reverse-transcription PCR (qRT-PCR) to detect ER stress-associated indicators (\u003cem\u003eBiP\u003c/em\u003e, \u003cem\u003eXBP-1\u003c/em\u003e, \u003cem\u003eXBP-1S\u003c/em\u003e, \u003cem\u003eCHOP\u003c/em\u003e, and \u003cem\u003eATF6\u003c/em\u003e). Simultaneously, the fertilized eggs obtained from mice of all groups were cultured in KSOM droplets and grouped on the culture dishes. The embryos of each group were divided into three samples to perform qRT-PCR, Western Blotting and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) analysis. Embryos were cultured in KSOM medium until the eight-cell stage. Then, the RNA of 150 and 100 eight-cell embryos was extracted for qRT-PCR and Western Blotting to assess the mRNA and protein expressions of ER stress-associated indicators, respectively. The remaining embryos were continuously cultured in KSOM droplets until they reached the blastocyst stage. Then, at least 10 blastocysts per group were collected for TUNEL analysis to detect apoptosis signals. Throughout the entire developmental period, the embryos were monitored continuously, and the rates for cleavage, as well as for morula and blastocyst formation, were recorded for each group.\u003c/p\u003e \u003cp\u003eIn the second part of the experiment, no intervention was performed on the mice. The female mice were superovulated, and the fertilized eggs were cultured in a humidified atmosphere with 5% CO\u003csub\u003e2\u003c/sub\u003e at 37\u0026deg;C. After an insemination period of 24 h, all well-developed two-cell embryos were randomly divided into four groups, with at least 180 embryos from 8 female mice in each group, and transferred to KSOM droplets containing different concentrations of MEHP (0, 10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e, 10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e, and 10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003eM) prepared in advance. The droplet cell cultures were performed in a humidified atmosphere with 5% CO\u003csub\u003e2\u003c/sub\u003e at 37\u0026deg;C. Then, RNA of 150 embryos at the eight-cell stage was extracted for qRT-PCR, and at least 10 blastocysts were collected per group for TUNEL. The rates for 4-cell embryo, cleavage, as well as for blastocyst formation, were recorded. These experiments were repeated five times.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 RNA extraction and qRT-PCR analysis\u003c/h2\u003e \u003cp\u003eTo analyze the expression of the target genes, we collected the ovaries and embryos (eight-cell stage) of each group after exposure to DEHP or MEHP. Total RNA was extracted using RNeasy Mini kits (Qiagen, Germany) according to the manufacturer\u0026rsquo;s instructions, and the first strand was synthesized using the PrimeScript\u0026trade; RT Master Mix (TAKARA, Code No. RR036A). Then, qRT-PCR was performed with the TB Green Premix Ex Taq\u0026trade; kit (Tli RNaseH Plus, TAKARA, Code No. RR420A) using the LightCycler 480 System (Roche, China). The data was normalized to the \u003cem\u003eGAPDH\u003c/em\u003e values using the 2\u003csup\u003e\u0026minus;∆∆CT\u003c/sup\u003e method. The PCR primers were designed and synthesized by PEPTBIO (Wuhan, China). The sequence numbers of primers are as follows: \u003cem\u003eGAPDH\u003c/em\u003e- MQP027158, \u003cem\u003eBiP\u003c/em\u003e -MQP074318, \u003cem\u003eXBP-1\u003c/em\u003e-MQP096646, \u003cem\u003eXBP-1S\u003c/em\u003e-MQP077568, \u003cem\u003eCHOP\u003c/em\u003e-MQP026921, and \u003cem\u003eATF6\u003c/em\u003e-MQP026163. The tests were performed in triplicate, and the mRNA level of each sample was normalized to that of the \u003cem\u003eGAPDH\u003c/em\u003e mRNA.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Western Blotting\u003c/h2\u003e \u003cp\u003eRelative protein levels of IRE1, PERK, eIF2α and ATF4 were detected by Western Blotting. Embryo protein extracts were separated on SAS-polyacrylamide gel electrophoresis. Separated proteins were electrophoretically transferred to a PVDF membrane (Servicebio, China) transferred for 50 min at 90V in Trisglycine buffer containing 20% methanol. The membranes were incubated with diluted antibody IRE1 (Abcam, 1:1000), PERK (Abcam, 1:1000), eIF2α (Bioswamp, 1:1000), ATF4 (Abcam, 1:1000) and β-actin (Bioswamp, 1:1000). The primary antibodies were localized with goat anti-rabbit IgG conjugated with horseradish peroxidase (Bioswamp, China). After protein bands were visualized with an enhanced chemiluminescence system (GeneGnome5, American), the intensities of the bands were quantified and analyzed by TANONGIS.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Determination of apoptosis\u003c/h2\u003e \u003cp\u003eBlastocysts were fixed with 4% paraformaldehyde (PFA) in PBS for 30 min and then permeabilized in 0.5% Triton X-100 in PBS for 40 min at 20-25\u0026deg;C. After three washes in PBS containing 0.1% BSA (PBS/BSA), apoptosis was determined by TUNEL assay using the In-Situ Cell Detection kit (Beyotime, C1086). Briefly, embryos were incubated in the TUNEL mixture (deoxynucleotidyl transferase enzyme and fluorescein-dUTP in a 1:9 ratio) for 1 h at 37\u0026deg;C in the dark. Positive controls were preincubated with 5 IU DNase I for 30 min at 37\u0026deg;C. Negative controls were processed without enzyme. After three times washing in PBS/BSA, the embryos were stained with Hoechst 33342 (10 \u0026micro;g/mL) for 40 min at 37\u0026deg;C in the dark. After a quick wash in PBS/BSA, the embryos were transferred to a glass slide and covered with anti-fluorescence quencher. The embryos were immediately viewed with a scanning laser confocal microscope (Zeiss, Germany). Green fluorescence represented the apoptotic signals, and blue fluorescence represented the nucleus. Three and five independent experiments were performed \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e, respectively. In each experiment, at least 10 embryos per group were collected and examined, generating data sets from at least 30 and 50 embryos examined per \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e group, respectively.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Statistical analysis\u003c/h2\u003e \u003cp\u003eAll experiments are presented as means\u0026thinsp;\u0026plusmn;\u0026thinsp;SEMs, and all the studies used for quantification were repeated at least three times. Data analysis was performed using GraphPad Prism8. The differences between the means were evaluated by one-way ANOVA when appropriate, followed by the least significant difference (l.s.d.) post hoc test. Asterisks (*) indicated statistically significant differences to the control group, *\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 VS control, **\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, ***\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, ****\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001.\u003c/p\u003e \u003c/div\u003e"},{"header":"3 Results","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003e3.1 ER stress in mouse ovary induced by DEHP exposure \u003cem\u003ein vivo\u003c/em\u003e\u003c/h2\u003e\n \u003cp\u003eTo investigate whether DEHP induces ER stress and UPR signaling pathways in the mouse ovary, 4\u0026ndash;5 weeks old female ICR mice were exposed by consuming food that contained different doses of DEHP (0.05, 5, and 500 mg/kg/d). The qRT-PCR analysis showed that the mRNA expression of \u003cem\u003eBiP\u003c/em\u003e in the ovaries of the 0.05, 5, and 500 mg/kg/d DEHP groups was significantly increased (Figure. 1A), compared to that of the control. There was no significant difference of the mRNA expression of \u003cem\u003eXBP-1\u003c/em\u003e (Figure. 1B) in all groups. DEHP increased the mRNA expression of \u003cem\u003eXBP-1S\u003c/em\u003e (Figure. 1C) and \u003cem\u003eCHOP\u003c/em\u003e (Figure. 1D) in a dose-dependent manner. The \u003cem\u003eATF6\u003c/em\u003e mRNA level (Figure. 1E) was significantly higher in the group with 500 mg/kg/d DEHP treatment than in the control group. All data were shown in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e. These results indicated that DEHP induced ER stress in the mouse ovary, and the three UPR signaling pathways responded at different degrees.\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eThe mRNA expression of ER stress in ovaries exposed to DEHP(X\u0026thinsp;\u0026plusmn;\u0026thinsp;S)\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eControl (n\u0026thinsp;=\u0026thinsp;13)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e0.05 mg/kg/d (n\u0026thinsp;=\u0026thinsp;13)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e5 mg/kg/d (n\u0026thinsp;=\u0026thinsp;13)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e500 mg/kg/d (n\u0026thinsp;=\u0026thinsp;13)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBiP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.8016\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02114\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.461\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05610 ***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.203\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09615 *\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.9545\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03135 *\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eXBP-1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.450\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1308\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.8493\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02159 *\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.7026\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01573 **\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.6340\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09619 **\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eXBP-1S\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.5271\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03468\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.8029\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03852 **\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.9937\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02506 ***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.639\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2726 *\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCHOP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.5358\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03789\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.8418\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04672 **\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.203\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07647 **\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.776\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06287 ****\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eATF6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.5061\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06300\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.8874\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1364\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.7479\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06358\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.231\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1594 *\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"5\"\u003e* P\u0026thinsp;\u0026lt;\u0026thinsp;0.05; ** P\u0026thinsp;\u0026lt;\u0026thinsp;0.01; *** P\u0026thinsp;\u0026lt;\u0026thinsp;0.001; **** P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eAll data were presented as means\u0026thinsp;\u0026plusmn;\u0026thinsp;SEMs. D 0.05: 0.05 mg/kg/d DEHP, D 5: 5 mg/kg/d DEHP, D 500: 500 mg/kg/d DEHP; * \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 VS control, ** \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, ***\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, ****\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001. (A)-(E): The mRNA expression of ER stress-related molecules in ovaries exposed to DEHP. (F)-(J): The mRNA expression of ER stress-related molecules in embryos obtained from mice exposed to DEHP. The mRNA expression of \u003cem\u003eBiP\u003c/em\u003e and \u003cem\u003eCHOP\u003c/em\u003e was gradually increased in the DEHP-treatment groups than that in the control group.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003e3.2 Effects of DEHP on the rates for fertilization and blastocyst formation in mice\u003c/h2\u003e\n \u003cp\u003eThe development from high-quality oocytes to high-quality embryos depends on a complex crosstalk network connecting the developing oocyte, the supporting cumulus, and other cells within the follicle.\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e In this study, embryos obtained from mice exposed to DEHP were cultured in KSOM until the blastocyst stage. There was no significant difference in the embryonic development (cell numbers and differentiation) between the groups treated with 0.05 mg/kg/d and 5 mg/kg/d DEHP and the control (Fig.\u0026nbsp;2A, Fig.\u0026nbsp;3A). However, the rates for fertilization and blastocyst formation in the group treated with 500 mg/kg/d DEHP was significantly lower than in the control group (Fig.\u0026nbsp;3A, Fig.\u0026nbsp;3B, Fig.\u0026nbsp;3C). Data were shown in Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n \u003ctable id=\"Tab2\" border=\"1\"\u003e\n \u003ccaption\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eFertilization rate and blastocyst formation rate exposed to DEHP (X\u0026thinsp;\u0026plusmn;\u0026thinsp;S)\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eControl (n\u0026thinsp;=\u0026thinsp;13)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e0.05 mg/kg/d (n\u0026thinsp;=\u0026thinsp;13)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e5 mg/kg/d (n\u0026thinsp;=\u0026thinsp;13)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e500 mg/kg/d (n\u0026thinsp;=\u0026thinsp;13)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFertilization rate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e74\u0026thinsp;\u0026plusmn;\u0026thinsp;4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e60.667\u0026thinsp;\u0026plusmn;\u0026thinsp;5.372\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e68.533\u0026thinsp;\u0026plusmn;\u0026thinsp;10.072\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e50.533\u0026thinsp;\u0026plusmn;\u0026thinsp;5.082 *\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBlastocyst formation rate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e43.533\u0026thinsp;\u0026plusmn;\u0026thinsp;1.474\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e38.733\u0026thinsp;\u0026plusmn;\u0026thinsp;2.150\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e32.167\u0026thinsp;\u0026plusmn;\u0026thinsp;1.692\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e21.167\u0026thinsp;\u0026plusmn;\u0026thinsp;2.699 **\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"5\"\u003e* P\u0026thinsp;\u0026lt;\u0026thinsp;0.05; ** P\u0026thinsp;\u0026lt;\u0026thinsp;0.01\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003e\u003cstrong\u003eFigure 2\u003c/strong\u003e. Embryo development diagrams obtained from DEHP-exposed mice and embryo development diagrams after direct exposure to MEHP. ICR female mice were exposed to di-2-ethylhexyl phthalate (DEHP) (0.05, 5, and 500 mg/kg/d) for five weeks (n\u0026thinsp;=\u0026thinsp;13 for female mice and n\u0026thinsp;=\u0026thinsp;3 for male mice). In the part of DEHP experiment, embryos were cultured in KSOM medium until blastocyst stage. In the part of MEHP experiment, two-cell embryos were divided into four groups and culture in KSOM containing different concentrations of MEHP (0, 10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e, 10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e, and 10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003eM).\u003c/p\u003e\n \u003cp\u003eAll data were presented as means\u0026thinsp;\u0026plusmn;\u0026thinsp;SEMs. D 0.05: 0.05 mg/kg/d DEHP, D 5: 5 mg/kg/d DEHP, D 500: 500 mg/kg/d DEHP. 2 cell: embryos in two-cell stage; 3\u0026ndash;4 cell: embryos in three-four cell stage; 7\u0026ndash;8 cell: embryos in seven-eight stage; morula: embryos in morula stage; blastocyst: embryos in blastocyst stage. (A) Developmental map from fertilized eggs to blastocysts. All the embryos were obtained from mice exposed to DEHP. (B) Developmental map of two-cell embryos exposed to MEHP for 96h.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n \u003ch2\u003e3.3 ER stress in early embryos obtained from mice exposed to DEHP \u003cem\u003ein vivo\u003c/em\u003e\u003c/h2\u003e\n \u003cp\u003eThe qRT-PCR and Western Blotting analysis were performed to detect ER stress-associated indicators in eight-cell embryos to further explore whether DEHP could induce ER stress in early embryos. Figure\u0026nbsp;1F showed that the \u003cem\u003eBiP\u003c/em\u003e mRNA was significantly higher in the group treated with 500 mg/kg/d DEHP than in the control group. \u003cem\u003eCHOP\u003c/em\u003e was increased in a dose-dependent manner induced by DEHP (Fig.\u0026nbsp;1I) (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). However, there was no significant difference in the mRNA expression of \u003cem\u003eXBP-1\u003c/em\u003e, \u003cem\u003eXBP-1S\u003c/em\u003e, and \u003cem\u003eATF6\u003c/em\u003e among the embryos from all groups (Fig.\u0026nbsp;1G, Fig.\u0026nbsp;1H, Fig.\u0026nbsp;1J). Western Blotting showed that there was no significant difference in the protein expression levels of \u003cem\u003eIRE1\u003c/em\u003eand \u003cem\u003ePERK\u003c/em\u003e in the embryos of the experimental group and the control group (Fig.\u0026nbsp;4A, Fig.\u0026nbsp;4B), however, the protein expression levels of \u003cem\u003ep-PERK\u003c/em\u003e, \u003cem\u003eeIF2\u0026alpha;\u003c/em\u003e and \u003cem\u003eATF4\u003c/em\u003e in embryos of the DEHP-treatment groups were significantly higher than those of the control (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;4C, Fig.\u0026nbsp;4D, Fig.\u0026nbsp;4E), and the ratio of \u003cem\u003ep-PERK\u003c/em\u003e to \u003cem\u003ePERK\u003c/em\u003e in the treatment groups also gradually increased (Fig.\u0026nbsp;4F). Thus, the data indicated that DEHP might induce ER stress during early embryogenesis via the \u003cem\u003ePERK/ATF4/CHOP\u003c/em\u003e pathway. Data were shown in Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e and Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e, respectively.\u0026nbsp;\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tab3\" border=\"1\"\u003e\n \u003ccaption\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eThe mRNA expression of ER stress in embryo exposed to DEHP(X\u0026thinsp;\u0026plusmn;\u0026thinsp;S)\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eControl (n\u0026thinsp;=\u0026thinsp;13)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e0.05 mg/kg/d (n\u0026thinsp;=\u0026thinsp;13)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e5 mg/kg/d (n\u0026thinsp;=\u0026thinsp;13)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e500 mg/kg/d (n\u0026thinsp;=\u0026thinsp;13)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBiP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.6074\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09673\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.9553\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1307\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.225\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1088 *\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.676\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05986 ***\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eXBP-1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.139\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2158\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.9705\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09108\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.039\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1930\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.019\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3212\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eXBP-1S\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.9961\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3412\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.344\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3429\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.602\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3887\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.274\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4324\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCHOP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.7354\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09626\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.061\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06617 *\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.233\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07917 *\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.607\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1169 **\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eATF6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.361\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4422\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.439\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3200\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.8681\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03653\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.382\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2360\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"5\"\u003e* P\u0026thinsp;\u0026lt;\u0026thinsp;0.05; ** P\u0026thinsp;\u0026lt;\u0026thinsp;0.01; *** P\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n \u003cdiv class=\"colspec\" align=\"char\"\u003e\u0026nbsp;\u003c/div\u003e\n \u003cdiv class=\"colspec\" align=\"char\"\u003e\u0026nbsp;\u003c/div\u003e\n \u003cdiv class=\"colspec\" align=\"char\"\u003e\u0026nbsp;\u003c/div\u003e\n \u003cdiv class=\"colspec\" align=\"char\"\u003e\u0026nbsp;\u003c/div\u003e\n \u003ctable id=\"Tab4\" border=\"1\"\u003e\n \u003ccaption\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eThe protein expression of ER stress in embryo exposed to DEHP(X\u0026thinsp;\u0026plusmn;\u0026thinsp;S)\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eControl (n\u0026thinsp;=\u0026thinsp;10)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e0.05 mg/kg/d (n\u0026thinsp;=\u0026thinsp;10)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e5 mg/kg/d (n\u0026thinsp;=\u0026thinsp;10)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e500 mg/kg/d (n\u0026thinsp;=\u0026thinsp;10)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eIRE1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.143\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0819\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.097\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08996\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.297\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06067\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.047\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07647\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePERK\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.3877\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01302\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.4104\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01210\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.4229\u0026thinsp;\u0026plusmn;\u0026thinsp;0.001425\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.4190\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01035\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ep-PERK\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.4700\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01114\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.5450\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01296*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.7875\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01343****\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.9247\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01410****\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ep-PERK\u003c/p\u003e\n \u003cp\u003e/PERK\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.7869\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09628\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.068\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06623\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.443\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06317**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.674\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1349**\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eeIF2\u0026alpha;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.2379\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01299\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.3124\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02567\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.3960\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02012**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.9705\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06526***\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eATF4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.7466\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01567\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.9505\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07455\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.321\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1116**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.477\u0026thinsp;\u0026plusmn;\u0026thinsp;0.720***\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"5\"\u003e* P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 VS control; ** P\u0026thinsp;\u0026lt;\u0026thinsp;0.01; *** P\u0026thinsp;\u0026lt;\u0026thinsp;0.001; **** P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003eAll data were presented as means\u0026thinsp;\u0026plusmn;\u0026thinsp;SEMs. D 0.05: 0.05 mg/kg/d DEHP, D 5: 5 mg/kg/d DEHP, D 500: 500 mg/kg/d DEHP; * \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 VS control; ** \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01; *** \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; **** \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001. (A), (B) There was no difference in the protein expression levels of \u003cem\u003eIRE1\u003c/em\u003e and \u003cem\u003ePERK\u003c/em\u003e among all the DEHP-treatment groups. (C), (D), (E) The protein expression levels of \u003cem\u003ep-PERK\u003c/em\u003e, \u003cem\u003eeIF2\u0026alpha;\u003c/em\u003e and \u003cem\u003eATF4\u003c/em\u003e were significantly increased in DEHP-treatment groups than that of the control. (F) The ratio of \u003cem\u003ep-PERK\u003c/em\u003e to \u003cem\u003ePERK\u003c/em\u003e was also significantly increased in groups D 5 and D 500.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n \u003ch2\u003e3.4 Effect of DEHP exposure on blastocyst apoptosis \u003cem\u003ein vivo\u003c/em\u003e\u003c/h2\u003e\n \u003cp\u003e\u003cem\u003eCHOP\u003c/em\u003e, which is encoded by the growth arrest- and DNA damage-inducible gene 153 (\u003cem\u003eGADD153\u003c/em\u003e), has been identified as a factor that responds to DNA damage.\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e We found that DEHP exposure increased the mRNA expression of \u003cem\u003eCHOP\u003c/em\u003e in embryos. A TUNEL analysis was performed to investigate further the developmental potential of embryos that reached the blastocyst stage after exposure to DEHP and to explore whether DEHP-induced changes in \u003cem\u003eCHOP\u003c/em\u003e expression were directly related to blastocyst apoptosis. The TUNEL results showed that apoptosis signals appeared in blastocysts of groups treated with 5 and 500 mg/kg/d DEHP, and the latter treatment group had stronger apoptotic signals than the former (Fig.\u0026nbsp;5A), indicating that \u003cem\u003eCHOP\u003c/em\u003e might be directly linked to blastocyst apoptosis.\u003c/p\u003e\n \u003cp\u003eGreen fluorescence represents the apoptotic signal and blue fluorescence represents the nucleus. (A) After five weeks exposure to di-2-ethylhexyl phthalate (DEHP) (0.05, 5, and 500 mg/kg/d), embryos were obtained and cultured until blastocyst stage, then apoptosis was determined by TUNEL staining. Apoptosis signals were observed in groups of 5 mg/kg/d and 500 mg/kg/d DEHP treatment, and the latter had stronger apoptotic signals than the former (Fig.\u0026nbsp;5A). (B) Sexually mature, healthy female without intervention were superovulated and sacrificed to obtain embryos. Two-cell embryos obtained from were cultured in KSOM containing mono-(2-ethylhexyl) phthalate (MEHP) (0, 10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e, 10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e, and 10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003eM) until blastocysts, and 10 blastocysts at least per group were collected for TUNEL assay. As shown, apoptosis signal was observed in group of 10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003eM MEHP treatment (Fig.\u0026nbsp;5B).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n \u003ch2\u003e3.5 Effect of MEHP on early embryo development\u003c/h2\u003e\n \u003cp\u003eDEHP exerts ovarian toxicity through its metabolite MEHP.\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e Two-cell embryos obtained from female mice with no intervention were cultured in KSOM containing different concentrations of MEHP (0, 10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e, 10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e, and 10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003eM) for 96 h. Figure\u0026nbsp;2B showed the effect of different concentrations of MEHP on the morphology of early embryo development. It showed that exposure to 10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003eM MEHP retarded early embryo development (Fig.\u0026nbsp;3D-3F) and significantly reduced the blastocyst formation rate (Fig.\u0026nbsp;3F). The rates of 4-cell, 8-cell and blastocyst formation of each group were shown in Fig.\u0026nbsp;3G-3I. Data were shown in Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e.\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tab5\" border=\"1\"\u003e\n \u003ccaption\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003erate of 4-cell and 8-cell embryo and blastocyst formation exposed to MEHP (X\u0026thinsp;\u0026plusmn;\u0026thinsp;S)\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eControl (n\u0026thinsp;=\u0026thinsp;8)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003eM MEHP (n\u0026thinsp;=\u0026thinsp;8)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003eM MEHP (n\u0026thinsp;=\u0026thinsp;8)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003eM MEHP (n\u0026thinsp;=\u0026thinsp;8)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4-cell embryo rate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e77.77\u0026thinsp;\u0026plusmn;\u0026thinsp;3.998\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e76.67\u0026thinsp;\u0026plusmn;\u0026thinsp;1.934\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e67.77\u0026thinsp;\u0026plusmn;\u0026thinsp;2.936\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e10.00\u0026thinsp;\u0026plusmn;\u0026thinsp;1.905 *\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8-cell embryo rate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e71.10\u0026thinsp;\u0026plusmn;\u0026thinsp;1.100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e73.33\u0026thinsp;\u0026plusmn;\u0026thinsp;1.934\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e63.33\u0026thinsp;\u0026plusmn;\u0026thinsp;3.839\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e10.03\u0026thinsp;\u0026plusmn;\u0026thinsp;3.333 **\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBlastocyst formation rate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e56.67\u0026thinsp;\u0026plusmn;\u0026thinsp;1.934\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e52.67\u0026thinsp;\u0026plusmn;\u0026thinsp;4.421\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e52.10\u0026thinsp;\u0026plusmn;\u0026thinsp;3.035\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e10.00\u0026thinsp;\u0026plusmn;\u0026thinsp;1.905 ****\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"5\"\u003e* P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 VS control; ** P\u0026thinsp;\u0026lt;\u0026thinsp;0.01; **** P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003eAll data were presented as means\u0026thinsp;\u0026plusmn;\u0026thinsp;SEMs. D 0.05: 0.05 mg/kg/d DEHP, D 5: 5 mg/kg/d DEHP, D 500: 500 mg/kg/d DEHP; * \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, ** \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, ***\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, ****\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001. (A) ICR female mice were exposed to di-2-ethylhexyl phthalate (DEHP) (0.05, 5, and 500 mg/kg/d) for five weeks, and then they were superovulated to obtain embryos, which were cultured in KSOM until blastocysts. (A) Histograms of embryos development at all stages exposed to DEHP. (B), (C) The rates of two-cell and blastocyst formation of each group of embryos obtained in mice exposed to DEHP. (D), (E), (F): Diagram showing the proportions of embryo development at all stages exposed to MEHP. (G), (H), (I): The rates of four-cell, eight-cell embryo and blastocyte formation in embryos exposed to MEHP.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\n \u003ch2\u003e3.6 ER stress in mouse embryos induced by MEHP exposure \u003cem\u003ein vitro\u003c/em\u003e\u003c/h2\u003e\n \u003cp\u003eThe RNA of 150 eight-cell embryos was extracted for qRT-PCR to assess the expression of ER stress-associated factors. The results indicated that there was a significant difference in the mRNA expression levels of \u003cem\u003eBiP\u003c/em\u003e (Fig.\u0026nbsp;6A), \u003cem\u003eCHOP\u003c/em\u003e (Fig.\u0026nbsp;6D), and \u003cem\u003eATF6\u003c/em\u003e (Fig.\u0026nbsp;6E) in the group treated with 10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003eM MEHP, compared to those in the control (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), which confirmed that MEHP might be involved in the regulation of early embryogenesis and apoptosis mainly via the ER stress-related \u003cem\u003eCHOP\u003c/em\u003e pathway. There was no correlation between the mRNA expression of \u003cem\u003eXBP-1\u003c/em\u003e, \u003cem\u003eXBP-1S\u003c/em\u003e and the concentration of MEHP in embryos (Fig.\u0026nbsp;6B, Fig.\u0026nbsp;6C). Data were shown in Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e.\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n \u003ctable id=\"Tab6\" border=\"1\"\u003e\n \u003ccaption\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 6\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eThe mRNA expression of ER stress in embryo exposed to MEHP(X\u0026thinsp;\u0026plusmn;\u0026thinsp;S)\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eControl (n\u0026thinsp;=\u0026thinsp;8)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003eM MEHP (n\u0026thinsp;=\u0026thinsp;8)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003eM MEHP (n\u0026thinsp;=\u0026thinsp;8)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003eM MEHP (n\u0026thinsp;=\u0026thinsp;8)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBiP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.9009\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1040\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.9181\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06335\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.016\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04475\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.213\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04119 *\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eXBP-1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.011\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09296\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.044\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2092\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.023\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1486\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.034\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1595\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eXBP-1S\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.003\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05593\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.001\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03038\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.021\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1387\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.9994\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04095\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCHOP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.8567\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04273\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.9552\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04964\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.9828\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03349\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.706\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1068 **\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eATF6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.8721\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05552\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.068\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1130\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.004\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1866\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.273\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07181 *\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"5\"\u003e* P\u0026thinsp;\u0026lt;\u0026thinsp;0.05; ** P\u0026thinsp;\u0026lt;\u0026thinsp;0.01\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003eAll data were presented as means\u0026thinsp;\u0026plusmn;\u0026thinsp;SEMs. 150 embryos in eight-cell stage were collected for qRT-PCR to detect ER stress-associated molecules (\u003cem\u003eBiP\u003c/em\u003e, \u003cem\u003eXBP-1\u003c/em\u003e, \u003cem\u003eXBP-1S\u003c/em\u003e, \u003cem\u003eCHOP\u003c/em\u003e, \u003cem\u003eATF6\u003c/em\u003e). * \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, ** \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01. (A) There was a significant difference in group of 10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003eM MEHP treatment compared to that of the control (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). (B), (C) There was no correlation between the mRNA expression of \u003cem\u003eXBP-1\u003c/em\u003e, \u003cem\u003eXBP-1S\u003c/em\u003e and the concentration of MEHP in embryos. (D) The mRNA expression of \u003cem\u003eCHOP\u003c/em\u003e in group of 10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003eM MEHP treatment was increased (\u003cem\u003eP\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.01) compared to that of the control group. (E) As with \u003cem\u003eCHOP\u003c/em\u003e, the mRNA expression of \u003cem\u003eATF6\u003c/em\u003e in the group of 10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003eM MEHP treatment was also significantly increased, and there was no statistical difference in other two groups.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\n \u003ch2\u003e3.7 Effect of MEHP exposure on blastocyst apoptosis \u003cem\u003ein vitro\u003c/em\u003e\u003c/h2\u003e\n \u003cp\u003eWe found that MEHP exposure increased the mRNA expression of \u003cem\u003eCHOP\u003c/em\u003e in embryos. A TUNEL analysis was performed to further investigate the developmental potential of embryos that reached blastocyst stage after exposure to MEHP and to explore whether MEHP-induced changes in \u003cem\u003eCHOP\u003c/em\u003e expression were directly related to blastocyst apoptosis. The TUNEL results showed that apoptosis signals only appeared in blastocysts treated with 10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003eM MEHP (Fig.\u0026nbsp;5B), indicating that \u003cem\u003eCHOP\u003c/em\u003e was directly linked to blastocyst apoptosis.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"4 Discussion","content":"\u003cp\u003eIn this mouse study on DEHP exposure, we observed that the high-dose exposure (500 mg/kg/d DEHP) significantly decreased the rates for fertilization and blastocyst formation, the medium-dose exposure (5 mg/kg/d DEHP) reduced the blastocyst formation rate, but not significantly, and the low-dose exposure (0.05 mg/kg/d DEHP) did not affect the rates for fertilization and blastocyst formation. In the second part of the experiment, we found that the high dose of MEHP (10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003eM) significantly retarded or even abrogated early embryonic development at the two-cell stage, and significantly reduced the rates of 8-cell embryo and blastocyst formation, whereas the medium and low-dose of MEHP (10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003eM and 10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003eM, respectively) barely affected embryonic development. Thus, because the \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e experiments detected certain effects of DEHP on early embryonic development, we further explored the specific molecular mechanisms. The qRT-PCR analysis showed that in mouse ovaries, the expression level of \u003cem\u003eXBP-1\u003c/em\u003e decreased with the increasing DEHP dose, while \u003cem\u003eXBP-1S\u003c/em\u003e showed the opposite trend. In embryos, however, there was no difference in the expression level of \u003cem\u003eXBP-1\u003c/em\u003e among each group. In addition, whether in ovaries or embryos, the expression of \u003cem\u003eCHOP\u003c/em\u003e mRNA increased with the increasing DEHP dose. Western Blotting results of embryos \u003cem\u003ein vivo\u003c/em\u003e showed that there was no difference in the expression level of \u003cem\u003eIRE1\u003c/em\u003e in each group, which was consistent with the expression of \u003cem\u003eXBP-1\u003c/em\u003e in embryos \u003cem\u003ein vitro\u003c/em\u003e. In addition, we could see from the figure that the expression of \u003cem\u003ePERK\u003c/em\u003e protein in embryos was obviously showing a gradual decrease, and \u003cem\u003ep-PERK\u003c/em\u003e protein was showing an increasing trend, although there was no statistical difference, the ratio of \u003cem\u003ep-PERK\u003c/em\u003e to \u003cem\u003ePERK\u003c/em\u003e was obviously showing a gradual rising trend. The TUNEL analysis indicated that DEHP-induced changes in the expression of \u003cem\u003eCHOP\u003c/em\u003e were directly related to blastocyst apoptosis. We further confirmed in this mouse study the toxicity of environmental DEHP levels for the ovaries and the early embryonic development, and the effect of MEHP, the primary metabolite of DEHP, on early embryonic development and blastocyst apoptosis was revealed by \u003cem\u003ein vitro\u003c/em\u003e experiments. Among the ER stress-related indicators detected in this study, the expression levels of \u003cem\u003eBiP\u003c/em\u003e, \u003cem\u003eCHOP\u003c/em\u003e and \u003cem\u003eATF6\u003c/em\u003e mRNA in embryos intervened with 10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003eM MEHP were significantly increased, and there was no significant difference in the mRNA expression levels of \u003cem\u003eXBP-1\u003c/em\u003e and \u003cem\u003eXBP-1S\u003c/em\u003e, which was consistent with the trend in embryos exposed to DEHP. In this study, we specifically found that DEHP and MEHP induced ER stress in the ovaries and early embryos, and we found a direct association between \u003cem\u003eCHOP\u003c/em\u003e and blastocyst apoptosis.\u003c/p\u003e \u003cp\u003eDue to a lack of self-stabilizing mechanism as in somatic cells, oocytes and early embryos are very susceptible to various exogenous stimulants, such as temperature, osmotic pressure, chemical exposure, or oxidative stress.\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e The ER is one of the largest organelles in eukaryotic cells. It plays a major role in the synthesis, folding, and maturation of at least 1/3 of all cellular proteins,\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e and many of those are involved in the normal development and differentiation of the embryo, which is therefore dependent on the normal ER function.\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eIt has been shown that ER stress affects the early embryonic development, and the inhibition of ER stress may improve this developmental process.\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e It has been reported previously that ER stress promoted protein folding by up-regulating \u003cem\u003eBiP\u003c/em\u003e, which might restore the normal ER function during the early stage of ER stress.\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e However, if the adaptive response could not adequately restore the protein-folding homeostasis, the UPR signaling would be sustained, indicating high or chronic ER stress induced by pro-apoptotic signaling,\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e of which \u003cem\u003eCHOP\u003c/em\u003e is an important member.\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e In the mouse ovaries obtained after DEHP exposure, we observed different expression patterns for \u003cem\u003eBiP\u003c/em\u003e and \u003cem\u003eCHOP\u003c/em\u003e mRNA under ER stress. The mRNA expression of \u003cem\u003eBiP\u003c/em\u003e was elevated at the low DEHP dosage and reduced at the higher DEHP dose. However, at the same exposure time, the mRNA expression of \u003cem\u003eCHOP\u003c/em\u003e was dose-dependently elevated among all the groups of DEHP. A dose of 0.05 mg/kg/d DEHP stimulated the mRNA expression of \u003cem\u003eBiP\u003c/em\u003e and \u003cem\u003eCHOP\u003c/em\u003e in the ovaries. At the 5 mg/kg/d dose, the mRNA expression of \u003cem\u003eBiP\u003c/em\u003e was reduced, and it diminished at the 500 mg/kg/d dosage, although this \u003cem\u003eBiP\u003c/em\u003e mRNA level was still higher than that in the control. However, the mRNA expression of \u003cem\u003eCHOP\u003c/em\u003e showed a continuous upward trend within the DEHP exposure range. This suggested that the mRNA expression levels of the protective molecular chaperone \u003cem\u003eBiP\u003c/em\u003e and the apoptosis-inducing \u003cem\u003eCHOP\u003c/em\u003e were up-regulated during the ER stress in the ovaries. However, ER stress induced by DEHP was initially dominated by \u003cem\u003eBiP\u003c/em\u003e -mediated protective effects; although cells in the ovarian tissue might have entered apoptosis, it was not significant. In the presence of strong, long-lasting stimulants (high exposure to DEHP), the apoptosis signal pathway in ER stress was dominant. At this time point, the mRNA expression of \u003cem\u003eBiP\u003c/em\u003e with the protective effects was gradually reduced, and the mRNA expression of \u003cem\u003eCHOP\u003c/em\u003e molecules had a major role in inducing apoptosis in ovarian tissue cells. To verify that high-dose DEHP-induced apoptosis through the \u003cem\u003eBiP\u003c/em\u003e-\u003cem\u003eCHOP\u003c/em\u003e signaling pathway, embryos from female mice exposed to DEHP were further examined. We found that the mRNA expression of \u003cem\u003eCHOP\u003c/em\u003e in embryos was also gradually increased, depending on the increase of the DEHP dosage. But unlike in ovaries, the expression level of \u003cem\u003eBiP\u003c/em\u003e mRNA in embryos showed a tendency to gradually increase with the increasing of DEHP dose. In this regard, we thought that under the same exposure time and dose of DEHP, the ovarian tissue first response to ER stress, and it reflected the comprehensive situation of all phrase of follicles in the ovaries, not only primordial follicles,\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e which could not exactly represent the actual situation of the follicles forming the embryo in this follicular cycle, but overall, they responded to each dose of DEHP. Some studies believed that this might be the continuous overexpression of \u003cem\u003eBiP\u003c/em\u003e caused by UPR in embryos, which was a key feature of the adaptive response to mild chronic ER stress, leading to cell survival.\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e In the future, we will further study the effect of DEHP on all phrase of follicles. Besides, from the perspective of the effect of DEHP on ER stress in embryos, Western Blotting showed no changes of \u003cem\u003eIRE1\u003c/em\u003e protein, nor did it cause changes in the expression level of \u003cem\u003eXBP-1\u003c/em\u003e mRNA in embryos. In ER stress, \u003cem\u003eXBP-1\u003c/em\u003e was the downstream target of \u003cem\u003eIRE1\u003c/em\u003e activation, which sheared \u003cem\u003eXBP-1\u003c/em\u003e mRNA and form active \u003cem\u003eXBP-1S\u003c/em\u003e.\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e In this study, there was no change in the expression of the \u003cem\u003eIRE1\u003c/em\u003e/\u003cem\u003eXBP-1\u003c/em\u003e signaling pathway. Therefore, we thought that under the exposure time and dose of DEHP performed in this study, the ER stress occurring in the embryo had nothing to do with the \u003cem\u003eIRE1\u003c/em\u003e signaling pathway, and this was consistent with the scientific statement that the three main signal pathways of ER stress might not be activated at the same time, but showed a dose effect and time sequence.\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eIn a similar manner to \u003cem\u003eIRE1\u003c/em\u003e, dissociation of \u003cem\u003eBiP\u003c/em\u003e from \u003cem\u003ePERK\u003c/em\u003e\u0026rsquo;ER luminal face leads to dimerization, interdimer trans-phosphorylation and activation of its cytosolic kinase domain. In the context of the UPR, \u003cem\u003ePERK\u003c/em\u003e\u0026rsquo; main cellular target is \u003cem\u003eeIF2α\u003c/em\u003e. \u003cem\u003ePERK\u003c/em\u003e phosphorylates \u003cem\u003eeIF2α\u003c/em\u003e, resulting in the reduction in global protein synthesis allowing the cell time to refold or degrade any unfolded protein in the ER.\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e \u003cem\u003eeIF2α\u003c/em\u003e is an essential constituent of ternary complex, which is required for the initiation of translation of an mRNA into a polypeptide.\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e In response to \u003cem\u003eeIF2α\u003c/em\u003e phosphorylation, \u003cem\u003eATF4\u003c/em\u003e are upregulated, which further upregulates a number of key genes including \u003cem\u003eCHOP\u003c/em\u003e. \u003cem\u003eCHOP\u003c/em\u003e is downstream of \u003cem\u003eATF4\u003c/em\u003e and is itself a transcription factor which is produced in high quantity after prolonged ER stress, promoting apoptosis as well as ER stress-induced cytokine production.\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e In this study, there was no significant difference in the protein expression levels of PERK in embryos among DEHP-treatment groups and the control. However, we found that there were significant statistical differences in each DEHP-treatment group compared with the control, and there were also statistical differences between the experimental groups (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), showing a dose-dependent manner. Besides, regarding phosphorylated protein, the effective indicator we need to observe should be the ratio of phosphorylated protein to total protein in order to effectively reflect the actual phosphorylation level. According to statistics, the ratio of p-PERK to PERK protein also showed a gradually increasing trend, which were statistically different in the medium and high DEHP-exposure groups, indicating that DEHP could indeed cause changes in the \u003cem\u003ePERK\u003c/em\u003e signaling pathway in embryos. Under long-term high-dose exposure, \u003cem\u003ePERK\u003c/em\u003e signaling pathway showed a sustained and enhanced response to promote embryos to cope with ER stress caused by the external environment. As a downstream molecule of \u003cem\u003ePERK\u003c/em\u003e, \u003cem\u003eeIF2α\u003c/em\u003e was activated in the manner of phosphorylation, resulting in the rapid downregulation of global protein synthesis and preferential translation of genes.\u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e In this study, we found that the expression of \u003cem\u003eeIF2α\u003c/em\u003e protein in the embryos of each DEHP-treatment group showed an increasing trend, and there were significant differences in the medium and high exposure groups. Elevated phosphorylation of \u003cem\u003eeIF2α\u003c/em\u003e was very important in ER stress, which could enhance the transcription and translation levels of \u003cem\u003eATF4\u003c/em\u003e and \u003cem\u003eCHOP\u003c/em\u003e in cells after DEHP treatment. \u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e Just like the results of our study, the protein expression of \u003cem\u003eATF4\u003c/em\u003e in each DEHP-treatment group also showed a gradual increasing trend, which was consistent with \u003cem\u003eeIF2α\u003c/em\u003e. Studies indicated that DEHP effectively triggered the ER stress as demonstrated by the increased phosphorylation PERK and its downstream substrate (eIF2α, as well as the increased levels of ATF4 and CHOP\u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e. During long-term ER stress, ATF4 might also stimulate genes of CHOP, which was responsible for initiation of the apoptotic cascade.\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e In summary, our study showed that the protein expression \u003cem\u003ePERK\u003c/em\u003e, \u003cem\u003ep-PERK\u003c/em\u003e, \u003cem\u003eeIF2α\u003c/em\u003e and \u003cem\u003eATF4\u003c/em\u003e in embryos exposed to DEHP were consistent with the theoretical basis of ER stress.\u003c/p\u003e \u003cp\u003eThe TUNEL staining revealed that the apoptosis signal in blastocysts of the high-dose group (500 mg/kg/d DEHP) was significantly stronger than that in the medium-dose group (5 mg/kg/d DEHP), and the results were consistent with the significant increase of \u003cem\u003eCHOP\u003c/em\u003e mRNA in embryos exposed to 5 and 500 mg/kg/d DEHP. Whereas, in the group of 0.05 mg/kg/d DEHP, the expression level of \u003cem\u003eCHOP\u003c/em\u003e mRNA was significantly increased, but the blastocysts formed by its continued development did not show apoptosis signals. We thought that these were compatible with a transient UPR induction in which \u003cem\u003eCHOP\u003c/em\u003e levels were insufficient to trigger massive apoptosis, likely due to concomitant overexpression of \u003cem\u003eBiP\u003c/em\u003e and \u003cem\u003eBcl-2\u003c/em\u003e that could prevent or markedly delay cell death to facilitate adaptation. In support of this assumption, it had been reported that cell fate was dependent not only on the severity or duration of ER stress but also on the balance between pro-death and pro-survival factors.\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e It has been repeatedly suggested that CHOP gene expression is strictly involved in cell death by apoptosis, but research has not confirmed existence of any relationship between induction of CHOP and apoptosis. The mechanisms that may explain how apoptotic cell death is induced by CHOP still remain unclear but, there is ample evidence that it is implicated in numerous human disease entities, including diabetes, neurodegenerative diseases, ischemic diseases, and tumor development.\u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eAlthough ER stress and sustained UPR signaling have been well documented in affected tissues in diabetes, neurodegeneration, stroke, pulmonary fibrosis, viral infection, inflammatory disorders, cancer, and heart disease,\u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e their contributions to preimplantation development have not been investigated to date. Several studies have investigated individual components of the UPR, and the results indicated that the UPR pathway constituents are expressed during early development.\u003csup\u003e\u003cspan additionalcitationids=\"CR58\" citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e In porcine embryos, blastocysts recovered from a tunicamycin-treated group exhibited a significantly increased apoptosis rate along with up-regulated mRNA levels of pro-apoptotic \u003cem\u003eBAX\u003c/em\u003e and down-regulated mRNA levels of anti-apoptotic \u003cem\u003eBCL2\u003c/em\u003e.\u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e \u003cem\u003eCHOP\u003c/em\u003e was identified as a factor that responded to DNA damage.\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e Under conditions of severe or prolonged ER stress, \u003cem\u003eBiP\u003c/em\u003e was released from \u003cem\u003ePERK\u003c/em\u003e to trigger activating transcription factor (\u003cem\u003eATF4\u003c/em\u003e), leading to the upregulation of \u003cem\u003eCHOP\u003c/em\u003e expression.\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e \u003cem\u003eCHOP\u003c/em\u003e mainly induced apoptosis by downregulation of the anti-apoptotic \u003cem\u003eBCL2\u003c/em\u003e gene,\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e which supported our findings to some extent.\u003c/p\u003e \u003cp\u003e \u003cem\u003eXBP-1S\u003c/em\u003e is considered an appropriate marker for the induction of the \u003cem\u003eIRE1\u003c/em\u003e pathway during the UPR, because \u003cem\u003eXBP-1\u003c/em\u003e is spliced exclusively under ER stress conditions.\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e The qRT-PCR analysis demonstrated that mRNAs for porcine \u003cem\u003eXBP-1u\u003c/em\u003e and \u003cem\u003eXBP-1S\u003c/em\u003e were clearly detected in GV-stage oocytes, but only the mRNA of \u003cem\u003eXBP-1u\u003c/em\u003e was detected in MI and MII-stage oocytes in mice,\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e suggesting that \u003cem\u003eXBP-1\u003c/em\u003e might play a very important role in the maturation of oocytes. In our study, there was a significant difference in the mRNA expression of \u003cem\u003eXBP-1S\u003c/em\u003e in the ovaries of mice exposed to DEHP. The mRNA expression of \u003cem\u003eXBP-1S\u003c/em\u003e was gradually increased by the increasing DEHP dosage, indicating that \u003cem\u003eXBP-1\u003c/em\u003e might be associated with the development and maturation of oocytes. In mouse embryos, UPR inducers, such as TM and sorbitol, increase nuclear \u003cem\u003eXBP-1S\u003c/em\u003e at the one- and two-cell stages and activate \u003cem\u003eXBP-1\u003c/em\u003e mRNA splicing at the eight-cell, morula, and blastocyst stages,\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e,\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e indicating that the \u003cem\u003eIRE1\u003c/em\u003e arm of the UPR was activated as an important coping response and adaption to ER stress in preimplantation embryos. In our study, however, there was no difference in the mRNA expression of \u003cem\u003eXBP-1S\u003c/em\u003e in eight-cell stage embryos treated with DEHP and MEHP. A possible explanation for this result is that different ER stress signaling could be initiated in embryos when facing different stimulants, because the UPR pathways could be individually modulated instead of complete activation or suppression of all three signaling pathways.\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e Based on the DEHP/MEHP exposure duration and dose regimen implemented in this study, DEHP/MEHP might affect early embryo development through the \u003cem\u003eBiP\u003c/em\u003e-\u003cem\u003eCHOP\u003c/em\u003e signaling pathway under ER stress instead of the \u003cem\u003eIRE1\u003c/em\u003e/\u003cem\u003eXBP-1\u003c/em\u003e signaling pathway, which requires further investigation in the future.\u003c/p\u003e \u003cp\u003eThis study showed that there was a significant difference in the mRNA expression of \u003cem\u003eATF6\u003c/em\u003e in the high-MEHP dose group compared to that of the control, which was consistent with the mRNA expression patterns of \u003cem\u003eCHOP\u003c/em\u003e and \u003cem\u003eBiP\u003c/em\u003e detected by qRT-PCR. These results further supported that under high-MEHP dose exposure, the embryos failed to maintain cell homeostasis through ER stress, but instead exerted pro-apoptosis effects by promoting the expression of apoptotic signaling molecules regulated by \u003cem\u003eATF6\u003c/em\u003e and \u003cem\u003eCHOP\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eIn this study, we established a DEHP exposure model in female mice. In addition, embryos were exposed to MEHP \u003cem\u003ein vitro\u003c/em\u003e to verify further that DEHP exerted early embryonic developmental toxicity through its main metabolite MEHP. This study has three major limitations. Firstly, the implantation and live birth outcomes of well-developed blastocysts exposed to DEHP or MEHP were not studied. This experiment would determine the implantation potential of DEHP-exposed blastocysts with normal appearance and morphology. Secondly, there was only one metabolite of PAEs tested in this study. More rigorous experiments should be conducted with PAE metabolite mixtures, which is more consistent with the actual exposure of the population, thus, generating more meaningful experimental results. Thirdly, further experiments in human embryos should be performed to collect DEHP/MEHP-exposed embryos for single-cell sequencing to detect changes in ER stress, thereby achieving the transformation of this basic research into a clinical research project.\u003c/p\u003e \u003cp\u003eIn conclusion, the current study demonstrated that DEHP and MEHP affected embryo fertilization and blastocyst apoptosis through ER stress. In addition, we further identified a direct correlation between \u003cem\u003eCHOP\u003c/em\u003e stimulated under ER stress and blastocyst apoptosis induced by DEHP and MEHP. We hope that the results and interpretation of this study can enhance the people\u0026rsquo;s vigilance against the use of plasticizers in daily life, thus preventing the adverse effects caused by the exposure of the ovaries and the early-stage embryos to high levels of environmental DEHP. These efforts may contribute to reducing the abortion rate, increasing the clinical pregnancy rate, and improving the clinical pregnancy outcomes, which has an important clinical value.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDEHP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eDi-(2-ethylhexyl) phthalate\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMEHP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMono-(2-ethylhexyl) phthalate\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eER\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eEndoplasmic Reticulum\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eqRT-PCR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ereal-time reverse-transcription PCR\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTUNEL\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eterminal deoxynucleotidyl transferase-mediated dUTP nick end labeling\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eART\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAssisted Reproductive Technology\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eEDCs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eEndocrine Disrupting Chemicals\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePAEs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePhthalates Acid Esters\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eGV\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eGerminal Vesicle\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMI\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMetaphase I\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMII\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMetaphase II\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eUPR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eUnfolded Protein Response\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cem\u003eBiP\u003c/em\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eImmunoglobulin heavy chain binding protein\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cem\u003eGRP78\u003c/em\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eGlucose-regulated protein 78\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cem\u003eIRE1\u003c/em\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eInositol-requiring enzyme 1\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cem\u003eXBP-1\u003c/em\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eX-box Binding Protein 1\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cem\u003ePERK\u003c/em\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePKR-like eukaryotic initiation factor 2A kinase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cem\u003eeIF2α\u003c/em\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eeukaryotic Initiation Factor-2α\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cem\u003eCHOP\u003c/em\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eC/EBP homologous protein\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cem\u003eATF4\u003c/em\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eActivating transcription factor 4\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cem\u003eATF6\u003c/em\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eActivating transcription factor 6\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDMSO\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eDimethylsulfoxide\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePMSG\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePregnant Mare Serum Gonadotropin\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHCG\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHuman Chorionic Gonadotrophin\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCOCs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eCumulus-oocyte complexes\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eIVF\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e \u003cem\u003ein vitro\u003c/em\u003e Fertilization\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePFA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eParaformaldehyde\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePBS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePhosphate buffer saline\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eBSA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eBovine Serum Albumin\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eTunicamycin\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eCONFLICT OF INTEREST\u003c/h2\u003e \u003cp\u003eAuthors assure that there are no conflicts of interest.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eXueMei Teng performed the study, analyzed the data, and wrote the paper. All authors read, revised and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eACKNOWLEDGEMENTS\u003c/h2\u003e \u003cp\u003eThis study was supported by the National Natural Science Foundation of China (grant No. 81571508) and the Natural Science Foundation of Hubei Province (grant No. 2014CFA069).\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eData is provided within the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eHertz-Picciotto, I. et al. A cohort study of in utero polychlorinated biphenyl (PCB) exposures in relation to secondary sex ratio. \u003cem\u003eEnviron. Health\u003c/em\u003e. \u003cb\u003e7\u003c/b\u003e, 37 (2008).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSifakis, S., Androutsopoulos, V. P., Tsatsakis, A. M. \u0026amp; Spandidos, D. A. 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Biol.\u003c/em\u003e \u003cb\u003e16\u003c/b\u003e (4), 269\u0026ndash;278 (2016).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"di-2-ethylhexyl phthalate, mono-(2-ethylhexyl) phthalate, ovarian toxicity, early embryo development, endoplasmic reticulum stress","lastPublishedDoi":"10.21203/rs.3.rs-6650176/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6650176/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eDi-2-ethylhexyl phthalate (DEHP) is widely used as a plasticizer, and mono-(2-ethylhexyl) phthalate (MEHP) is its primary metabolite. To investigate the effects of DEHP on mice ovaries, embryo development, and endoplasmic reticulum (ER) stress, adult female mice were daily exposed to DEHP (0, 0.05, 5, and 500 mg/kg/d) for five weeks, and ovaries and embryos were collected for examinations. The mRNA and protein levels of ER stress molecules and the blastocyst apoptosis were determined using real-time reverse-transcription PCR (qRT-PCR), Western Blotting and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL), respectively. For examining the effects of MEHP on embryo development and ER stress, two-cell embryos were randomly divided and cultured in KSOM containing MEHP (0, 10\u003csup\u003e-5\u003c/sup\u003e, 10\u003csup\u003e-4\u003c/sup\u003e, and 10\u003csup\u003e-3\u003c/sup\u003eM). We found that the mRNA expression of XBP-1 was decreased in the ovaries, with BiP, XBP-1S and CHOP increasing, and BiP and CHOP were consistent with the results in embryos. Besides, the protein expressions of p-PERK, eIF2α and ATF4 were increased in embryos of DEHP-treatment groups than that of the control. Furthermore, 500 mg/kg/d DEHP reduced the rates of fertilization and blastocyst formation and increased blastocyst apoptosis, which was stronger than that in 5 mg/kg/d DEHP group. Moreover, 10\u003csup\u003e-3\u003c/sup\u003eM MEHP significantly increased the mRNA expressions of BiP and CHOP in embryos, and retarded embryo development at the two-cell stage and decreased blastocyst formation rate, as well as induced blastocysts apoptosis. In summary, we found that DEHP and MEHP affected early embryo development by ER stress and induce blastocyst apoptosis via PERK/ATF4/CHOP pathway.\u003c/p\u003e","manuscriptTitle":"Di-(2-ethylhexyl) phthalate and mono-(2-ethylhexyl) phthalate attenuate early embryonic development and induce blastocyst apoptosis via PERK/ATF4/CHOP pathway in mice","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-26 11:32:18","doi":"10.21203/rs.3.rs-6650176/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f6abddd8-fb20-460d-a31b-2a6ac18006b4","owner":[],"postedDate":"June 26th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":50535445,"name":"Earth and environmental sciences/Environmental sciences/Environmental impact"},{"id":50535446,"name":"Health sciences/Health care"},{"id":50535447,"name":"Health sciences/Endocrinology/Endocrine system and metabolic diseases"}],"tags":[],"updatedAt":"2026-01-30T16:12:49+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-26 11:32:18","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6650176","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6650176","identity":"rs-6650176","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}