Alterations in oocytes and early zygotes following oral exposure to di(2-ethylhexyl) phthalate in young adult female mice

Reproductive Toxicology 90 (2019) 53–61

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Alterations in oocytes and early zygotes following oral exposure to di(2ethylhexyl) phthalate in young adult female mice

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Lyda Yuliana Parra-Foreroa, Arlet Veloz-Contrerasa, Shirley Vargas-Marína, María Angelica Mojica-Villegasb, Elim Alfaro-Pedrazaa, Mayrut Urióstegui-Acostac, ⁎ Isabel Hernández-Ochoaa, a

Departamento de Toxicología, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional (Cinvestav), Av. Instituto Politécnico Nacional 2508, Col. San Pedro Zacatenco, Ciudad de México, 07360, Mexico b Laboratorio de Toxicología de la Reproducción-Fertilidad, Departamento de Farmacia, Escuela Nacional de Ciencias Biológicas-IPN, Col. San Pedro Zacatenco, Ciudad de México, 2508, Mexico c Escuela Superior de Ciencias Naturales, Universidad Autónoma de Guerrero, Mexico

A R T I C LE I N FO

A B S T R A C T

Keywords: DEHP Zygote Oocyte In vivo fertilization Zygote fragmentation

Because di(2-ethylhexyl) phthalate (DEHP) toxicity on ovarian function is incomplete, effects of DEHP oocyte fertilization and the resulting zygotes were investigated. Further, an analysis characterizing the stage of zygote arrest was performed. Female CD1 mice were dosed orally with DEHP (0, 20, 200 and 2000 μg/kg/day) for 30 days. Following an in vivo mating post-dosing, DEHP-treated females exhibited fewer oocytes/zygotes, fewer oocytes displaying the polar body extrusion, fewer 1-cell zygotes having 2-pronuclei, more unfertilized oocytes, and decreased number of zygotes at every stage of development. DEHP induced blastomere fragmentation in zygotes. DNA replication in zygotes directly assessed by the 5-Ethynyl-2′-deoxyuridine (5-EdU) incorporation assay and indirectly by dosing mice with 5-fluorouracil (5-FU) suggested that DEHP inhibits DNA replication. Our data suggest that DEHP at doses found in ‘every-day’ (200 μg/Kg/day) or occupational (2000 μg/Kg/day) environments induces zygote fragmentation and arrests its development from the 2-cell stage potentially impairing DNA replication.

1. Introduction Di(2-ethylhexyl) phthalate (DEHP) is the most widely used phthalate in the plastics industry. DEHP is incorporated into plastics to confer transparency, flexibility and durability to polyvinyl chloride (PVC) based polymers. As DEHP is not covalently bound to the plastic matrix, it readily leaches from plastics into the environment. The main routes of human exposure are orally or through inhalation [1]. Since DEHP metabolites have been measured in up to 100% of analyzed human urine samples [2–4], concerns about the potential detrimental effects of this compound have been raised. DEHP metabolites were previously detected in several reproductive fluids, including amniotic [5,6], and follicular fluid [7,8], suggesting incorporation from DEHP or metabolites circulating in plasma. DEHP exposure is associated with several reproductive failures, including, poor sperm quality [9,10] and decreased production of steroid sex hormones such as testosterone [11–13]. In the ovary, DEHP-related

detrimental effects include decreased ovulation [14,15], decreased size of the follicular granulosa cells [16,17], increased apoptosis [18,19], suppressed synthesis of follicular-derived estradiol [20,21], delayed follicular growth [22], altered follicular steroidogenic enzymes [16], prolonged estrous cyclicity [23,24], decreased folliculogenesis [17], delayed onset of puberty [25,26], and endometriosis [27]. Furthermore, implantation failure and fetal loss have also been described as a result of contact with this compound [5,6]. Previous studies have suggested that DEHP and/or its main metabolite mono (2-ethylhexyl) phthalate (MEHP) reduce the ability of oocytes to complete meiosis and to develop to the blastocyst stage. Specifically, in vivo and in vitro studies using DEHP or MEHP have shown detrimental effects on meiotic maturation, including abnormalities in the metaphase II spindle and/or negative modulation of the prophase I to metaphase II transition [18,19,28]. Furthermore, other in vitro studies and/or mouse models have demonstrated that DEHP/ MEHP can delay development of 2-cell zygotes or cumulus cell-oocyte

⁎ Corresponding author at: Departamento de Toxicología, Cinvestav, Av. Instituto Politécnico Nacional 2508, Col. San Pedro Zacatenco, Ciudad de México, 07360, Mexico. E-mail address: [email protected] (I. Hernández-Ochoa).

https://doi.org/10.1016/j.reprotox.2019.08.012 Received 15 April 2019; Received in revised form 30 July 2019; Accepted 16 August 2019 Available online 20 August 2019 0890-6238/ © 2019 Elsevier Inc. All rights reserved.

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Fig. 1. Experimental design. Female mice exposed to DEHP (20, 200 and 2000 μg/kg/day) were euthanized at 24-, 48-, 72-, 84-, or 96- h post-mating for the evaluation of 1-, 2-, and 4-cell zygotes, morula, and blastocysts, respectively.

2.2. Exposure to DEHP

complexes to the blastocyst stage [29,30]. However, our understanding of the mechanism of DEHP toxicity on oocytes and zygote development is incomplete. Specifically, the effect of DEHP on the fertilization ability of oocytes, the quality of the resulting zygote, the stage at which zygote development is delayed, and the mechanism of this delay have not previously been described. Therefore, the present study evaluated the effect of subchronic DEHP exposure on zygote development by quantifying pre-implantation embryos at each stage, in treated and control animals; including 1-,2-,4- cell zygotes, morulas and blastocysts. A more detailed analysis was then performed to establish at what stage DEHP causes pre-implantation embryos to arrest. In addition, we examined the effect of chronic DEHP exposure on numbers of the following: retrieved oocytes/ zygotes, fertilized oocytes and oocytes having polar bodies. Since delayed pronuclear formation has previously been linked to alterations in zygotic development [31], the effects of DEHP exposure on the numbers of 1- or 2- pronuclei was evaluated, and the effect on DNA replication was explored in zygotes as a potential mechanism of toxicity.

Female and male CD-1 mice (28–32 days-old), provided by the animal facility (UPEAL, Cinvestav), were handled according to the International Guidelines for the Use and Care of Laboratory Animals. All procedures were approved by the Institutional Laboratory Animal Use and Care Committee at Cinvestav. Mice were housed in filtered polysulfonate cages and maintained in an animal room at the UPEAL under a controlled environment of 21 ± 1 °C, 12 h light/dark cycle, and a relative humidity of 50%. Mice were provided with standard food (Formulab Diet for rodents 5008; LabDiet, Brentwood, MO, USA) and high purity water ad libitum. Females were randomly assigned to four groups (n = 6 per group) as follows: vehicle control animals were fed corn-oil free tocopherol, whilst three further groups were exposed to DEHP (at levels of 20, 200 or 2000 μg/kg/day respectively). In a fifth group, 5 females were injected intraperitoneally with a single dose of 125 mg/kg 5-FU (equivalent to 375 mg/m2) [32]. Saline solution (0.9%) was used as vehicle for 5-FU in a final volume of 0.1 mL per 10 g of mouse body weight [33]. The 5-FU dose used was chosen such that it would not modify body weight gain, and was also within the range of doses used for antineoplastic therapies [34,35]. DEHP levels of 20 and 200 μg/kg/ day were previously shown to be attainable in ‘every-day’ environments [36], whereas the dose of 2000 μg/kg/day is realistic in occupational environment [37,38] (Do et al., 2012; Ginsberg et al., 2016, Testai 2016). Levels of DEHP chosen for this study were also based on previous work showing delayed zygotic progression [39–41]. The oral dosing technique and classification of mice into age groupings (e.g. ‘young adulthood’) were derived from a previous study performed by our research group [42]. Since female fertility in mice, from puberty onset to three months of age, is thought to involve waves of activated follicles and also slow-growing adult primordial follicles [43], the 1month-dosing period was selected to ensure that both dynamics of ovarian follicles were exposed to DEHP.

2. Material and methods 2.1. Reagents The di(2-ethylhexyl) phthalate (DEHP; 99% purity), 5′-fluorouracil (5-FU), hyaluronidase (HA), sodium hydroxide, Triton X-100, Hoechst 33342, paraformaldehyde, glycerol, pyruvic acid, streptomycin, sodium lactate, penicillin, D-glucose, magnesium chloride hexahydrate, pregnant mare serum gonadotropin (PMSG) (Folligon Intervet, Boxmeer, The Netherlands) and human chorionic gonadotrophin (hCG), aphidicolin were obtained from Sigma-Aldrich Co. (St. Louis, MO). Corn oil free tocopherol and bovine serum albumin were obtained from MP Biomedicals (Illkirch, France). VectaShield was obtained from (Vector Laboratories, Burlingame, CA, USA). FHM HEPES, and KSOMaa medium with D-Glucose were obtained from Millipore (Darmstadt, Germany). Sodium chloride, monobasic potassium phosphate, calcium chloride, sodium bicarbonate and potassium chloride were bought from J.T Baker, (México). CBZ culture medium was purchased from In vitro, (México). The HBSS media, Tyrode's, and DAPI, were obtained from Sigma-Aldrich Co. (St. Louis, MO).

2.3. Superovulation protocol Following 1-month-dosing period, animals at estrus were administered with a single dose of 5 IU of PMSG intraperitoneally (ip) and 48 h 54

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2.4. In vivo fertilization Following hCG injection in the superovulation protocol, females were mated at a ratio of 1:1 with fertile males. Males fertility was prescreened by at least one previous mating to a nonexposed female that resulted in the birth of live, healthy pups. Mating was performed for 24 h and the females were sacrificed at intervals as shown in Fig. 1 and Table 1 [44]. Zygotes and oocytes were collected by flushing oviducts through their respective infundibula using an insulin syringe with a 30 G blunt needle [45–47], and then they were reported. Collection was performed using supplemented Embryomax KSOMaa medium containing 4 mg/mL BSA and 5.6 mM D-Glucose. Unfertilized oocytes were considered as those having morphological characteristics, such as granulations in the cytoplasm, alterations in the pellucid zone, germinal vesicle breakdown, metaphase I or II, and evidence of a polar body extrusion without pronucleus formation. An important criterion for unfertilized oocytes was the absence of 1- or 2-pronuclei. Zygotes recently fertilized were considered as those having decondensed chromatin (1-cell stage), no granulations in the cytoplasm, and the presence of 1- or 2- pronuclei together with extrusion of at least one polar body when first observed in bright field and phase contrast microscopy, and then confirmed after 10 min staining with Hoechst 33342. Zygotes at the 2-, 4-cell, and morula stages were considered as those having two, four, and more than eight blastomeres (cells produced by cleavage), respectively. Blastocysts were considered as those having an inner cell mass and a thin trophoblast layer enclosing a cavity [48,49].

Morula

2 1 5 13

Recovered N Arrest phase

1.62 0.74 16.9** 5.08 1.3 0 0 1.7

Percentage (%)b

4-Cell

5.5 8.5 12.4** 35.1** 4.6 7.0 33.8** 21.0** 5.6 11.4 13.2** 29.8**

4-Cell

2 1 16 5 1 0 0 1

Recovered N Arrest phase Percentage (%)b

2.5. Assessment of pronuclei numbers and DNA replication

2-Cell

2-Cell

The 5-Ethynyl-2′-deoxyuridine (5-EdU) incorporation assay was performed with some modifications of the Click-It Kit manufacturer’s protocol (EdU Alexa Fluor 488 kit; from Invitrogen, Carlsbad, CA, USA, Cat. C10340). The fertilized oocytes were obtained 18 h after mating, and then stripped by incubating in 0.5% hyaluronidase solution for 2 min, followed by three consecutive washes using PBS. Oocytes were then incubated for 2 h in a working solution containing EdU and CBZ maturation media (1:30 v/v), and fixed in 3.7% paraformaldehyde for 15 min. A group of oocytes treated with aphidicolin (2 μg/mL of CBZ + EdU) was included as a positive control for the inhibition of DNA synthesis. The fixed oocytes were then permeabilized with Triton X-100 (0.5%) for 15 min at room temperature. Intermediate washes using PBS solution were performed between the staining and permeabilization steps. Then, oocytes were incubated with the ‘Click-it’ labelling cocktail for 30 min, washed with the buffer (Component F) for 5 min, and incubated with DAPI for 5 min (Salic and Mitchison, 2008; Wang et al., 2014). Finally, oocytes were mounted with VectaShield covered with glass coverslips and analyzed using a Leica Confocal Microscope TCS SP8 (Leica Microsystems, Wetzlar, Germany) and LAS AF Lite program (Leica Microsystems, Wetzlar, Germany). The transverse and vertical resolutions were 0.25 μm and 0.5 μm, respectively. Consecutive projections were made using photomultipliers with 0.5 −1 μm with gray level (1024 × 1024), the speed was 1 frame per 0.5–1 second.

Blastocyst (96 h)

Morula (84 h)

a.Expected stage at the specified time of euthanasia. b.Regarding the total recovery. N. Total recovered. ** Statistically different from control.

1-Cell

1-Cell

1-Cell 4-Cell (72 h)

211 188 162 153** 200 175 180 134** 208 189 160** 169** 189 147 138** 124** 2-Cell (48 h)

CONTROL 20 200 2000 CONTROL 20 200 2000 CONTROL 20 200 2000 CONTROL 20 200 2000

1-Cell

15 16 14 37 5 8 7 12 3 1 7 5 3 2 4 6

7.5 8.8 9.9 25.12** 4.3 7.8 6.3 14.6** 1.4 0.7 4.3** 3.1** 3.1 2.5 6.8** 13.7**

2-Cell

7 9 13 28 6 8 32 21 5 9 8 13

Recovered N Arrest phase Percentage (%)b Recovered N Arrest phase Recovered N Treatment (μg/ kg/day) Expected stage/ euthanasia timea

Table 1 The effect of DEHP on developmental stages of the zygote.

later, with 5 IU of human chorionic gonadotropin (hCG; also ip). Sixteen hours later, the mice were euthanized by cervical dislocation and oocytes collected from oviducts (Fig. 1).

2.38 1.15 10.28** 30.13**

Percentage (%)b

L.Y. Parra-Forero, et al.

2.6. Statistical analysis The results are presented as mean ± standard error of the mean (SEM). Comparisons were performed using one-way analysis of variance (ANOVA) followed by the Bonferroni’s post hoc multiple comparison test. P-values < 0.05 were considered statistically significant. 55

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Fig. 2. DEHP alters the numbers of retrieved oocytes/zygotes. A. Number of oocytes retrieved from the ampulla of the oviduct. Data are expressed as mean ± SEM from 6 mice per group. * p < 0.05 compared to control, according to one-way ANOVA- Bonferroni´s post hoc.

3. Results 3.1. Impact of oral dosing to DEHP on the numbers of retrieved oocytes/ zygotes The numbers of oocytes/zygotes recovered were significantly decreased from animals dosed at 2000 μg/kg/day DEHP but not at 20- and 200-μg/kg/day DEHP, compared to the vehicle control (Fig. 2).

3.2. Impact of oral dosing to DEHP on oocyte fertilization rate and polar body extrusion following mating The data show that unfertilized oocytes recovered from animals dosed at 200 μg/kg/day DEHP were significantly increased compared to the vehicle control (Fig. 3A). A common pattern in 1-cell zygotes, identified by the presence of one or two pronuclei, from animals dosed at 200 μg/kg/day DEHP was the absence of the polar body extrusion (Fig. 3C and B). The polar body is a cytoplasmic exclusion body enclosing the excess of DNA following the oocyte meiosis and sperm fertilization, indicating herein successful oocyte maturation and fertilization [50,51]. Further, unfertilized oocytes, exhibiting characteristic morphological features such as high granularity content in the cytoplasm and a perivitelline space, are shown in Fig. 3B. Representative zygotes recently fertilized displaying prototypical polar bodies are shown in Fig. 3D.

3.3. Morphology in zygotes from DEHP-treated female mice after mating The percentage of zygotes with abnormal morphology recovered from in vivo fertilized oocytes were significantly increased in animals dosed with 200 and 2000 μg/kg/day DEHP compared to the vehicle control. This group of aberrant zygotes was mainly composed of fragmented blastomeres (Fig. 4A). Micrographs in Fig. 4B (1–3) show typical fragmentation patterns observed during zygote development (2cell to morula). Since it has been reported that zygotes with fragmented blastomeres occupying more than 10% of the inner side are less likely to successfully implant in the uterus [52], this cut-off point was used to evaluate the effect of DEHP on zygote quality (Figs. 4C). The data show that 2000 μg/kg/day DEHP treatment significantly increased the percentage of zygotes with fragmented blastomeres occupying at least 10% of their inner sides. Zygotes in Fig. 4D (2–4) are representative examples of the severe damage caused by 2000 μg/kg/day DEHP to the cytoplasm and/or blastomeres.

(caption on next page)

3.4. Impact of oral dosing to DEHP on zygote development Exposure to DEHP has previously been related to alterations in 56

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Fig. 3. DEHP increases the rate of unfertilized oocytes and decreases the number of 1-cell zygotes with polar body extrusion. A. Number of unfertilized oocytes/numbers of retrieved oocytes. B. Representative micrographs in light field microscope showing the formation of 1- or 2- pronuclei (B1) and unfertilized oocyte patterns (arrows) (B2). C. Number of 1-cell zygotes with polar body extrusion. D. Representative micrographs of polar body extrusion in the vehicle control and the 2000 μg/kg/day DEHP-treated groups. Data are expressed as mean ± SEM from 6 mice per group. * p < 0.05 compared to control, according to one-way ANOVA- Bonferroni’s post hoc. Scale bar: 100 μm.

zygote development [28,29,53,54]. To evaluate the effect of DEHP on this oviductal process in detail, pre-implantation embryos were collected from control and DEHP treated females euthanized at the indicated times (Fig. 1). These embryos were then counted and grouped by developmental stage. The resulting data show that DEHP significantly decreased the numbers of 2-, 4-cell zygotes, morula, and blastocysts recovered at the dose of 2000 μg/kg/day, and that it significantly decreased the numbers of morula and blastocysts at the dose of 200 μg/kg/day compared to vehicle controls (Fig. 5). Some blastocysts from females dosed with 2000 μg/kg/day of DEHP reached late development, such as the late and hatching stages. However, morphological damage was evident even in these few cases (Fig. 5B). As these data suggest a delay in zygote development, the percentages of zygotes arrested at earlier developmental stages than the one expected at the time of euthanasia, were estimated and shown in Table 1. The data showed that when 2-cell zygotes were expected, a dose of 2000 μg/kg/ day DEHP significantly increased the percentage of 1-cell zygotes (25.1%) compared to vehicle control (7.5%), suggesting that zygotes were arrested at the 1-cell stage. When 4-cell zygotes were expected, a dose of 2000 μg/kg/day DEHP significantly increased the percentages of 1-cell (14.6%) and 2-cell zygotes (35.1%), compared to vehicle controls (4.3 and 5.5%, respectively). Notably, when morula or blastocysts were expected, a dose of 200 μg/kg/day DEHP significantly increased zygotes at stages ranging from 1-cell to morula. 3.5. Potential mechanisms of DEHP toxicity on zygote arrest After fertilization, both male and female DNA decondense asynchronously to form the corresponding pronucleus [55]. Since data herein (see above) indicate that DEHP treatments can cause developmental arrest of the zygote from the 1-cell stage, these zygotes were assessed for the numbers of 1- or 2-pronuclei to evaluate whether DEHP alters the first steps of zygote formation. Notably, the numbers of zygotes having 1-pronucleus were not modified by DEHP treatments, compared to the vehicle control group. However, 200 and 2000 μg/kg/ day DEHP treatments significantly decreased the numbers of 1-cell zygotes having 2-pronuclei, compared to the vehicle control group. In addition, significant differences in the numbers of zygotes possessing 2pronuclei were recovered from DEHP treated animals at 200 versus 2000 μg/kg/day dosages (Fig. 6A). DNA replication is critical in mitotic cycles, including those occurring in zygotes throughout development to the blastocyst stage [56,57]. Thus, as a potential mechanism of toxicity on zygote arrest from the 1cell stage, DNA replication was assessed in 1-cell zygotes from DEHPtreated and control mice. The data showed that 200 and 2000 μg/kg/ day DEHP treatments significantly decreased the numbers of 1-cell zygotes that incorporated Edu into de novo DNA strands, compared to the control group. Significant differences were again observed between the 200 and 2000 μg/kg/day DEHP doses (Fig. 6B & C). To indirectly confirm that impaired DNA replication was sufficient to cause the developmental arrest of the zygotes we observed herein with DEHP treatment, a group of female mice was treated with 5-FU, a known inhibitor of de novo DNA synthesis [58]. 5-FU is a pyrimidine analog that inhibits the enzyme thymidylate synthase, therefore it blocks the synthesis of thymidine which is a nucleoside required for DNA replication [58]. The data showed that 5-FU treatment

(caption on next page)

significantly increased the percentage of unfertilized oocytes compared to vehicle control treated females (Fig. 7A). Furthermore, a significantly decreased number of 1-cell zygotes, that barely reached the 4-cell stage, 57

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Fig. 4. DEHP increases the number of zygotes having fragmented blastomeres. A. Percentage of fragmented zygotes at the 1-Cell stage. B. Representative micrographs of fragmented zygotes at different stages of development (2-cell to morula stages) in mice exposed to 200 or 2000 μg/kg/day DEHP. Scale bar: 100 μm. C. Percentage of fragmented zygotes (greater than 10%) at different stages of development (left). Representative micrographs and schematic drawings of zygotes that possess no fragmentation (normal), less than 10% fragmented blastomeres, more than 40% fragmented blastomeres, and more than 80% fragmented blastomeres. D. 2-cell zygotes with no evident damage to DNA (1). Scale bar (C–D): 20 μm. Representative fragmentation patterns observed in 2-cell zygotes with fragmentation greater than 50% and evident damage in the cytoplasm (2), with vacuoles in the perivitelline space and no apparent DNA damage (3), and with evident damage in the cytoplasm, and vacuoles in the perivitelline space accompanied by fragments of DNA (4). Data are expressed as mean ± SEM from 6 mice per group. * p < 0.05 compared to control, and **p < 0.05 between groups, according to one-way ANOVABonferroni’s post hoc.

Fig. 5. DEHP decreases the number of pre-implantation embryos. Following the DEHP or vehicle dosing period, the females were euthanized at specific times and pre-implantation embryos classified and counted as either 1-, 2- and 4-cell zygotes, morulas or blastocysts (A). Micrographs of blastocysts at late and hatching stages (B). Blastocysts with normal morphology obtained from the vehicle control group (B1). Zygotes obtained at stage-specific time of blastocyst from the DEHP-treated group (B2). The 100% corresponds to zygotes of the previous stages of development. Data are expressed as mean ± SEM from 6 mice per group. * p < 0.05 compared to control, and **p < 0.05 between groups, according to one-way ANOVA- Bonferroni’s post hoc. Scale bar: 100 μm. Fig. 6. DEHP decreases the numbers of 2-pronuclei and DNA replication. 1-cell zygotes were incubated with DAPI and 5-Edu-Alexa fluor 488 (an analog of thymidine which incorporates into the newly synthesized DNA chain), and then counted and analyzed for DNA replication. A) Numbers of 1- or 2-pronuclei in 1-cell zygotes. B) Representative micrographs from the pattern of no fertilized zygotes, and those fertilized possessing 1- or 2-pronuclei (PN) either 5-Edu (-) or (+). The graph represents the percentage of zygotes having 5-Edu (+) pronuclei and the control group of DNA synthesis inhibition (Aphidicolin) (C). Data in graphs are expressed as mean ± SEM from 6 mice per group. * p < 0.05 compared to control, and **p < 0.05 between groups, according to one-way ANOVA- Bonferroni’s post hoc. Scale bar: 20 μm.

were recovered from 5-FU treated females, compared to vehicle controls (Fig. 7A). Surprisingly, significantly increased percentage of fragmented zygotes were also recovered from 5-FU -treated females compared to vehicle controls (Fig. 7B). 4. Discussion Since fertility starts upon onset of puberty, we assessed the potential detrimental effects caused by DEHP exposure in a 30-day window after first estrus in mice. We used doses levels of DEHP below those 58

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from prophase I to metaphase II; a process termed oocyte maturation. This maturation is necessary for oocyte competence [63]. Thus, our data support those studies demonstrating that DEHP exposure impairs oocyte maturation. In our study, zygotes were collected from mice at defined time intervals following mating, ensuring that most zygotes from untreated mice were at one specific developmental stage [64]. This gave us a powerful tool to analyze the detrimental effects of DEHP exposure during zygote development and to identify potential stage-specific targets of toxicity. We found that DEHP exposure of > 200 μg/kg/day caused zygotes to arrest at every stage of development, starting from the 1-cell stage. Zygotic arrests or retardations beyond the 1-cell stage have been reported in previous studies. Dalman et al. (2008) showed that in vitro exposure of immature oocytes to MEHP (400 μM) prevented zygotes from developing beyond the 8-cell stage. Absalan et al. (2017) also observed that oral exposure to DEHP lowered the number of blastocysts. They further proposed that DEHP impairs the developmental ability of oocytes to progress to blastocysts by reducing transcription of genes involved in totipotency, genome activation, DNA damage repair, and cell cycle behavior, amongst others [28,65]. Furthermore, a failure to transition from maternal-to-embryonic genes is associated with delayed zygote development in DEHP-exposed experimental animals [29]. Following penetration of the sperm cell into the oocyte at fertilization, paternal and maternal pronuclei are assembled asynchronously to progress through S-phase [66]. Thus, in an attempt to evaluate a possible mechanism for the observed post-fertilization toxicity in zygotes from DEHP treated mice, we assessed whether DNA replication in the 1-cell was impaired. We found that DEHP treatment of > 200 μg/ kg/day impaired DNA replication in the 1-cell stage, suggesting that DEHP may impair the S-phase in the zygote. The detrimental effect of DEHP on DNA replication has previously been described in testicular tissue [67], Hep3B-liver derived cells [68], and FL83B-liver derived cells [69]. Further, DNA replication in murine zygotes has previously been modified by the inhibitor of histone deacetylase, Trichostatin A [70], and an excess of cholesterol [71]. Although the mechanism of toxicity to inhibit DNA replication in the zygote has not been described, the following are among the potential causes that warrant further investigation: 1) A failure in the exchange of cytosine to thymine, which is mediated by the enzymes translocation family of ten eleven (TET), 5-methylcytosine (5mC), and/or glycosylase thymine (TDG) [72]; 2) Histone modification (H3: H3K64ac, H3K122ac, H3K56ac) [73,74]; 3) Checkpoint activity of the cell cycle phases [75,76]; and 4) Alteration in the extrusion of the polar bodies [77,78]. The other dramatic effect we observed in treated mice exposed to > 200 μg/kg/day DEHP was that about ˜7% of zygotes were fragmented. This abnormality was characterized by the presence of small portions or fragments of cells separated from blastomeres [79]. This aberration is unusual in untreated murine zygotes collected from the oviduct [80], but it is evident in human zygotes that are fertilized and developed in vitro [81–83]. Degenerated blastocysts were previously reported in female mice exposed orally to DEHP (50 and 500 μg/kg/ day) for 8 weeks [84], though the exact nature of the morphological changes was not published. Since 5-FU is known to block DNA replication [58], a group of females was exposed to this compound to elucidate a possible mechanism for the impaired DNA replication in zygotes. Surprisingly, 5-FU treated females had significantly increased number of fragmented zygotes that barely reached the 4-cell stage, compared to vehicle control females. These data suggest that any defect in DNA replication can lead to zygote fragmentation and arrest in zygote development. Notably, the changes observed in fragmented zygotes in our study were similar to those changes reported during apoptosis. Indeed, a previous study showed that exposure to MEHP (10−3 M) in preimplantation zygotes induced apoptosis, which correlated with blocked development beyond the 2-

Fig. 7. 5-FU impairs fertilization rate and zygote development, and it induces blastomere fragmentation. Following tocopherol-stripped oil or 125 mg/kg 5FU treatments, the females were euthanized at a specific time to recover 2-cell zygotes. A) Percent of fertilized oocytes and early preimplantation zygotes. * p < 0.05 compared to control, and **p < 0.05 between groups, according to one-way ANOVA- Bonferroni’s post hoc. B) Percent of fragmented zygotes in vehicle control and 5-FU -treated female mice. Each bar represents the mean ± SEM from 5 mice per group. * p < 0.05 compared to control, according to the t-Student’s t-test.

estimated from exposures through medical devices (peak values up to 2200 μg/Kg/day) [38] and in occupational situations. Our main findings indicate that DEHP induces fragmentation in zygotes, and that it arrests zygote development potentially via interrupting DNA replication. In addition, our data show that exposure to DEHP reduces the numbers of retrieved oocytes/zygotes, oocyte fertilization, and the numbers of zygotes having a polar body. In our study, female mice treated with > 200 μg/kg/day DEHP produced less oocytes/zygotes, less oocytes possessing a polar body, and lower numbers of fertilized oocytes than the control females. Although our study was performed in an animal model, similar data have been reported in a human cohort study which found that the highest urinary DEHP levels group (> 0.22 μmol/L) had 2.9 fewer oocytes, 1.7 fewer mature oocytes, and 1.2 fewer fertilized oocytes than those with lower exposure to DEHP [59]. Other studies in adult female mice, however, have found that DEHP at similar doses to those used in our study did not impair the fertility index of treated mice, as indicated by the presence of pregnancy assessed immediately after a 10 day exposure [60]. In our study, the period of exposure to DEHP was 20 days longer than the one used by Chiang and Flaws (2019), suggesting that, for doses of ˜200 μg/kg/day, a 30-day-period-exposure to DEHP in female mice may be required to impair oocyte fertilization. On the other hand, our data on the reduced number of oocytes are also in agreement to those by Davis et al. [61] who observed that female rats exposed to a high dose of DEHP (2 g/kg/day) for 12 days had a decreased numbers of oocytes released into the oviduct. The reduced number of oocytes displaying the first polar body observed in our study is consistent with previous work showing that high doses of DEHP (10 nM - 1200 μM in vitro or 500–10000 μg/kg/d in vivo) or MEHP (50–400 μM) can impair the progression of meiosis in oocytes [15,41,49,62]. It is well known that the presence of the first polar body indicates that the oocyte has progressed through meiosis

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cell stage [29]. Other study showed that exposure to DEHP (0.12–1200 μM) in cumulus cell-oocyte complex induced apoptosis as well [18]. Thus, we cannot exclude the possibility that apoptosis may be also involved in DEHP induced zygote development arrest and/or fragmentation. In addition, chromosomal errors are among the other causes reported [83].

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5. Conclusions In conclusion, our data suggest that DEHP at doses found in ‘everyday’ environments (200 μg/Kg/day) or in occupational environments (2000 μg/Kg/day) induces zygote fragmentation and arrests its development from the 2-cell stage. Whether DEHP impairs DNA replication in the zygote warrants further investigation. Importantly, our study assessed alterations immediately after a 30-day DEHP treatment, which is roughly equivalent to the first stage of reproductive age. Since previous work has demonstrated that the detrimental effects of DEHP can persist into later life [22,40,60], future studies should examine whether the DEHP toxicity we describe herein represents a long-term or even permanent change in female reproductive capacity. Declaration of Competing Interest Nothing to disclose. Acknowledgments This study is derived from LYPF’s doctoral thesis. LYPF is enrolled in the PhD Toxicology Program at Cinvestav, and she was a recipient of a scholarship for graduate students from the National Council of Science and Technology-Mexico (CONACyT-Mexico). SVM, AVC, and EAP were enrolled in the MSc Toxicology Program, and they were also recipients of scholarships for graduate students from CONACyT-Mexico. The authors thank M. Sc. Elizabeth Estrada Muñiz for helping with the 5FU dosing group and Dr. Libia Vega Loyo for her advises on this dosing group. The authors also thank Ricardo Gaxiola, Benjamín Chavez, Rafael Leyva, and Rene Pánfilo Morales for their technical assistance with the animals. This study was funded by Cinvestav, Mexico. This paper was edited for English style by ScienceDocs Editor Dr. Stephen Dove. References [1] J.-P. Fong, F.-J. Lee, I.S. Lu, S.-N. Uang, C.-C. Lee, Estimating the contribution of inhalation exposure to di-2-ethylhexyl phthalate (DEHP) for PVC production workers, using personal air sampling and urinary metabolite monitoring, Int. J. Hyg. Environ. Health 217 (1) (2014) 102–109. [2] J.H. Kim, H.Y. Park, S. Bae, Y.-H. Lim, Y.-C. Hong, Diethylhexyl phthalates is associated with insulin resistance via oxidative stress in the elderly: a panel study, PLoS One 8 (8) (2013) e71392. [3] C. Philippat, D.H. Bennett, P. Krakowiak, M. Rose, H.-M. Hwang, I. Hertz-Picciotto, Phthalate concentrations in house dust in relation to autism spectrum disorder and developmental delay in the CHildhood Autism Risks from Genetics and the Environment (CHARGE) study, Environ. Health 14 (1) (2015) 56. [4] R. Hauser, A.J. Gaskins, I. Souter, K.W. Smith, L.E. Dodge, S. Ehrlich, J.D. Meeker, A.M. Calafat, P.L. Williams, E.S. Team, Urinary phthalate metabolite concentrations and reproductive outcomes among women undergoing in vitro fertilization: results from the EARTH study, Environ. Health Perspect. 124 (6) (2016) 831–839. [5] A.M. Calafat, J.W. Brock, M.J. Silva, L.E. Gray Jr, J.A. Reidy, D.B. Barr, L.L. Needham, Urinary and amniotic fluid levels of phthalate monoesters in rats after the oral administration of di(2-ethylhexyl) phthalate and di-n-butyl phthalate, Toxicology 217 (1) (2006) 22–30. [6] G. Latini, C. De Felice, G. Presta, A. Del Vecchio, I. Paris, F. Ruggieri, P. Mazzeo, In utero exposure to di-(2-ethylhexyl) phthalate and duration of human pregnancy, Environ. Health Perspect. 111 (14) (2003) 1783. [7] S.P. Krotz, S.A. Carson, C. Tomey, J.E. Buster, Phthalates and bisphenol do not accumulate in human follicular fluid, J. Assist. Reprod. Genet. 29 (8) (2012) 773–777. [8] Y.-Y. Du, Y.-L. Fang, Y.-X. Wang, Q. Zeng, N. Guo, H. Zhao, Y.-F. Li, Follicular fluid and urinary concentrations of phthalate metabolites among infertile women and associations with in vitro fertilization parameters, Reprod. Toxicol. 61 (2016) 142–150.

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