Reproductive Alterations in Chronically Exposed Female Mice to Environmentally Relevant Doses of a Mixture of Phthalates and Alkylphenols

Reproductive Alterations in Chronically Exposed Female Mice to Environmentally Relevant Doses of... Abstract Endocrine-disrupting chemicals (EDCs) are exogenous compounds that modify hormone biosynthesis, causing adverse effects to human health. Among them, phthalates and alkylphenols are important due to their wide use in plastics, detergents, personal care products, cosmetics, and food packaging. However, their conjoint effects over reproductive female health have not been addressed. The aim of this work was to test the effect of chronically exposed female mice to a mixture of three phthalates [bis (2-ethylhexyl), dibutyl, and benzyl butyl] and two alkylphenols (4-nonylphenol and 4-tert-octylphenol) from conception to adulthood at environmentally relevant doses. These EDCs were administered in two doses: one below the minimal risk dose to cause adverse effects on human development and reproduction [1 mg/kg body weight (BW)/d of the total mixture] and the other one based on the reference value close to occupational exposure in humans (10 mg/kg BW/d of the total mixture). Our results show that both doses had similar effects regarding the uterus and ovary relative weight, estrous cyclicity, serum levels of progesterone and 17β-estradiol, and expression of key elements in the steroidogenesis pathway (acute steroidogenic regulatory protein and CYP19A1). However, only the 1-mg/kg BW/d dose delayed the onset of puberty and the transition from preantral to antral follicles, whereas the 10-mg/kg BW/d dose decreased the number of antral follicles and gonadotropin receptor expression. In addition, we observed changes in several fertility parameters in exposed females and in their progeny (F2 generation). In conclusion, our results indicate that chronic exposure to a complex EDC mixture, at environmentally relevant doses, modifies reproductive parameters in female mice. The absolute number of couples affected by infertility increased from 42 million in 1990 to 48.5 million in 2010 (1). The causes of infertility linked to the female factor include ovulatory disorders (33%), idiopathic disorders (40%), and tubal factor disorders/endometriosis (27%) (2). Multiple factors such as the exposure to environmental contaminants can contribute to the origin of unexplained infertility in females (3). Genome-wide association has shown that numerous diseases are associated with specific genes or gene clusters, several of which have been shown to be expressed in the presence of certain environmental factors such as pesticides or tobacco smoke, stretching the notion that environmental conditions are important to trigger genes that are at the onset of different diseases (4, 5). In this context, it has been calculated that humans are exposed to ∼85,000 chemicals classified as toxic, widely present in commercial use, which pose great risk to the development of many unanticipated diseases (6, 7). Several studies have shown diverse association of unique environmental factors to some diseases (8, 9), but most have been assessed in animal models focused on individual compounds or exposure at specific developmental stages. However, these experimental designs do not take into account that humans and animals are exposed to these chemicals throughout life (i.e., from conception to adulthood). Endocrine-disrupting chemicals (EDCs) are exogenous compounds that alter hormone biosynthesis, causing adverse effects to human health and intact organisms, their progenitors, or offspring (10). Among the EDCs, phthalates [bis (2-ethylhexyl) phthalate (DEHP), dibutyl phthalate (DBP), and benzyl butyl phthalate (BBP)] and alkylphenols [4-nonylphenol (NP) and 4-tert-octylphenol (OP)] are important due to their wide use in plastics, detergents, personal care products, cosmetics, and food packaging (11). In females, the presence of these compounds has been verified in fetal samples such as placenta, cord blood, amniotic fluid, meconium, and urine (12) and in biological fluids like serum, milk, and follicular fluid in adult samples (13, 14). Epidemiological studies have linked infertility problems in women with single exposure to phthalates or alkylphenols (13–15), especially due to a diminished ovarian reserve (16). Nevertheless, there are insufficient data to reveal a clear association between single phthalates and alkylphenol exposure and adverse effects on human female reproductive health, raising a need for studies focused on mixtures of EDCs (17). At the reproductive age, in the ovary, it is possible to find follicles at different stages of maturation, but only those with a cavity adjacent to the oocyte—antral follicles—are responsible for the production of steroid hormones (18). Luteinizing hormone binds to its cognate receptor [luteinizing hormone cognate receptor (LHCGR)] present in theca cells of antral follicles and increases the transcription of genes encoding for enzymes responsible for progesterone and androgen (androstenedione and testosterone) biosynthesis, which play an important role in the follicular progression from the preantral to antral follicle (19). Furthermore, on one hand, the correct expression of the acute steroidogenic regulatory protein (STAR), which transports cholesterol into the mitochondria, is a limiting step. On the other hand, the binding of the follicle-stimulating hormone (FSH) to its receptor (FSHR), expressed by granulosa cells, increases the transcription of the gene that encodes P450 aromatase (CYP19A1). The former is an enzyme responsible for irreversibly converting androgens from theca cells into estrogens [(17β-estradiol and estrone)] (20). Finally, estrogens stimulate follicle growth and ovulation, and its production triggers growth and vascularization of the endometrium, initiating the menarche at puberty and maintaining the menstrual cycles in human and estral cycles in mice (21). In vitro and in vivo studies using single exposure to phthalates or alkylphenols have shown that these compounds interfere with the biosynthesis of sex steroids (androgens, estrogens, and progestins), their receptors, and the expression of the enzymes involved in steroidogenesis (22, 23). Previous studies have shown that prenatal or perinatal exposure to a complex mixture of EDCs containing only phthalates or alkylphenols, along with other chemicals, accelerated the onset of puberty, impaired folliculogenesis, and decreased fertility in female mice (24–27). However, some of these data have been contradictory, and the exposure to EDCs occurred during a specific developmental time, which is not comparable to human exposure. Therefore, in this study, we wanted to evaluate a chronic exposure to a mixture of three phthalates (DEHP, DBP, and BBP) and two alkylphenols (NP and OP) in female mice, from conception to adulthood, at two different doses: (1) one below the minimal risk dose to cause adverse effects on human development and reproduction (28) and (2) one based on the reference value close to occupational exposure in humans (29, 30). The mixture defined in our work was at least ∼100- and ∼1000-fold lower than the no observed adverse effect level and low observed adverse effect level values for DEHP to cause adverse effects in humans (31). In these models, we fully studied the changes in the reproductive health of exposed mice, including the progesterone, testosterone, and 17β-estradiol levels, along with the quantification of messenger RNA (mRNA)/protein levels implicated in the steroidogenesis pathway. Materials and Methods Animals Adult female C57BL/6J of 60 days of age were obtained from the Animal Facility of Pontificia Universidad Católica de Chile and maintained in bisphenol A–free H-TEMP polysulfone cages (TECNIPLAST). The mice were housed under a 12-hour light:12-hour dark cycle, and temperature was maintained at 22 ± 1°C. Food and water were provided for ad libitum consumption. Experiments were conducted in accordance with the rules laid down by the Consortium for Developing a Guide for the Care and Use of Agricultural Animals in Agricultural Research and Teaching and by the Guide for the Care and Use of Laboratory Animals of National Research Council, and they were approved by the Ethical Scientific Committee for the Care of Animals and the Environment at the Pontificia Universidad Católica de Chile No. 141222004. All animal protocols were endorsed by the Chilean National Fund of Science and Technology (FONDECYT). Chemicals DEHP, DBP, BBP, NP, OP, and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (St. Louis, MO). Ethanol was acquired from Winkler (Santiago, Chile). Experimental design The compounds of the mixture used in this study were chosen on the basis that they represent the most abundant phthalates and alkylphenols in everyday products (32, 33). Doses were chosen based on the nonoccupational and occupational exposure to these compounds between the range of 0.3 mg/kg/d and 143 mg/kg/d (29, 30, 34–37). In this way, we generated two doses of this mixture, resembling nonoccupational (1 mg/kg/d) and occupational (10 mg/kg/d) human exposure. It should be noted that these doses are, in relation to the no observed adverse effect level and low observed adverse effect level of DEHP and NP, ∼1000 and ∼100 times lower, respectively, for endocrine-reproductive adverse effects in humans, rats, and mice (31, 38, 39) (Supplemental Table 1). A bulk stock solution containing three phthalates (DEHP, DBP, and BBP) diluted in DMSO and two alkylphenols (NP and OP) diluted in ethanol was used to prepare the final mixture. The doses were freshly made and administered in the drinking water every other day according to the animal body weight (BW) and throughout the experimental period. The bottle was covered with foil to prevent the photolysis of the compounds. For both vehicle and treatments, the final DMSO and ethanol doses were 2.5 g/kg BW/d and 0.06 g/kg BW/d. In mice, orally tolerable doses have been established at 7.9 g/kg for DMSO and 2.5 g/kg for ethanol (40). To emulate chronic human exposure to an environmental mixture of EDCs, we administered the mixture of EDCs or vehicles (control) to pregnant mice from 0.5 postcoital day (biological n) during pregnancy and lactation. At weaning, only females were selected and maintained in a group of three to four individuals per cage. We continued the administration of the mixture of EDCs or control until adulthood (end point: postnatal day 60) (Fig. 1). Figure 1. View largeDownload slide Chronic mixture exposure model (exposome) on F1 female mice from conception to adulthood. Pregnant female mice (F0) and female F1 descendants were exposed to a mixture of three phthalates and two alkylphenols (DEHP, DBP, BBP, NP, and OP) or vehicle (DMSO and ethanol) in drinking water during all gestation periods and lactation (middle). Furthermore, F1 females were exposed from weaning to adulthood (60 days of age) when, at this point, treatment was suspended, fertility was assessed, and samples were collected for analyses. Figure 1. View largeDownload slide Chronic mixture exposure model (exposome) on F1 female mice from conception to adulthood. Pregnant female mice (F0) and female F1 descendants were exposed to a mixture of three phthalates and two alkylphenols (DEHP, DBP, BBP, NP, and OP) or vehicle (DMSO and ethanol) in drinking water during all gestation periods and lactation (middle). Furthermore, F1 females were exposed from weaning to adulthood (60 days of age) when, at this point, treatment was suspended, fertility was assessed, and samples were collected for analyses. Reproductive end points Reproductive end points assessed included age at vaginal opening, age of first estrous cycle, and estrous cycle length. Estrous cycles were determined via vaginal cytology followed by crystal violet staining (41) and recorded daily for 16-day spans since vaginal opening day. All cycles were measured daily between 08:00 and 10:00 hours. Fertility assay:estrous cycle was checked in adult females (60 days old), and those in proestrus were placed in a cage with an adult male (>90 days old) of proven fertility (i.e., it had fathered offspring at least once). To evaluate mating, vaginal plugs were checked the next day early in the morning (08:00 to 09:00 hours). All females with vaginal plugs were set apart and placed in a cage (four animals of the same experimental group) for 15 to 18 days. Then, females were separated in individual cages until delivery. Fertility in mice was assayed four times in the same animal, and the delivery rate was obtained by dividing the number of female mice that gave birth by the number of females that presented vaginal plugs. Litter size and sex ratio were recorded. Furthermore, pups were weighed at birth and 21 days old, and anogenital distance (AGD) and serum 17β-estradiol levels were measured at 21 days old. AGD was normalized to cubic root of BW to account for body size effects (42). Plasma hormonal levels At the end of the treatment, females (60 days old) in estrous were weighed and anesthetized by intraperitoneal administration of ketamine/xylazine (80 and 8 mg/kg, respectively). Then, blood samples were collected via cardiac puncture, clotted, and then centrifuged at 2300 g at 4°C for 10 minutes to collect serum. Finally, samples were stored at −80°C until measured. Serum progesterone, testosterone, and 17β-estradiol were measured in duplicate at the Pontificia Universidad Católica de Chile radioimmunoassay service. The sensitivity and intra- or interassay coefficients of variation (CVs) for the progesterone assay were 154 pg/mL and CVs <5.2 and <8.3, respectively; for the testosterone assay, they were 128 pg/mL and CVs <5.8 and <10.6, respectively; and those for the 17β-estradiol assay were 34 pg/mL and CVs <8.4 and <14.4, respectively. Tissue collection and body and organ weights After blood collection, females were euthanized by cervical dislocation, and uterus and ovaries were aseptically removed, cleaned of interstitial tissue, and weighed. Relative organ weights were calculated by dividing the weight of the uterus or ovaries by the body weight at the euthanasia time. Body and organ weights were recorded in grams. Histological evaluation of the number of preantral and antral follicles For this evaluation, collected ovaries were fixed in Bouin’s solution (Sigma-Aldrich). Then, samples were dehydrated and embedded in paraffin, and serial sections of each ovary were cut (8 μm) using a microtome followed by mounting onto glass slides and stained with the hematoxylin/eosin protocol. Finally, samples were rehydrated and mounted. For follicle counting, from each set of serial sections, four sections were counted for each ovary (the interval between sections was approximately 100 μm apart to avoid counting follicles twice) (43). Pictures were taken under a CX31 microscope (Olympus, Japan) with a Mshot camera (Guangzhou, China). The count of follicles and corpora lutea was based on previously defined criteria (44, 45). Briefly, preantral follicles contain an oocyte surrounded by at least two layers of cuboidal granulosa and theca cells, whereas antral follicles contain an oocyte surrounded by multiple layers of cuboidal granulosa cells with fluid-filled antral space and theca cells. Corpora lutea were identified by the hypertrophic aspect and large volume of cells. RNA extraction and real-time polymerase chain reaction Total RNA was isolated from ovaries that were collected in estrous using TRIzol Reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s recommendations. The quantity and integrity of total RNA were determined by a Thermo Scientific Nanodrop 2000 spectrophotometer. Real-time reverse transcription was used to verify gene expression using an Applied Biosystems 7500 thermal cycler. Complementary DNA was generated from 1 μg RNA using random primers and M-MLV Reverse transcription (Promega, WI), as well as after 1 μL complementary DNA was used for a 10-μL polymerase chain reaction reaction mixture containing 1 μM of primers (see sequences in Supplemental Table 2) and 2× SyBR Green Fast qPCR Master Mix (Biotool, TX). Polymerase chain reaction was carried out over 40 cycles at 95°C for 3 seconds and 60°C for 15 seconds. Expression data were normalized using the 2−∆∆Ct method (46) with Gapdh and β-actin as endogenous reference genes. Protein extraction and Western blotting Protein extraction was performed by homogenizing ovaries (collected in estrous) in radioimmunoprecipitation assay buffer, adding a protease inhibitor cocktail (Sigma) and a phosphatase inhibitor cocktail with 2 mM 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride, 0.3 μM aprotinin, 130 μM bestatin hydrochloride, 14 μM E-64, 1 mM EDTA, and 1 μM leupeptin hemisulfate. Proteins were purified by centrifugation at 12,000 × g at 4°C for 10 minutes and subsequently quantified. After that, 20 μg of proteins was separated by electrophoresis on a 10% polyacrylamide gel (sodium dodecyl sulfate–polyacrylamide gel electrophoresis) under denaturing and reducing conditions and then transferred to a nitrocellulose membrane (Thermo Scientific) at 350 mA for 2 hours. Next, membranes were blocked with a solution of 3% (w/v) nonfat milk or bovine serum albumin 0.1% (v/v) Tween in Tris-buffered saline, pH 7.4, and incubated overnight with the respective primary antibodies for CYP19A1, LHCGR, and STAR (Abbexa, Cambridge, UK); FSHR and androgen receptor (AR) (Santa Cruz Biotechnology, TX); and B-TUBULIN (Sigma) as a loading control (Table 1). Finally, a second incubation took place with their respective secondary antibodies conjugated with horseradish peroxidase (KPL, Gaithersburg, MD) diluted 1:5000 in a blocking solution for 1 hour at room temperature. Peroxidase activity was detected by enhanced chemiluminescence (Pierce Biotechnology, Rockford, IL). Table 1. Antibodies Protein Target  Antigen Sequence  Name of Antibody  Manufacturer, Catalog No., and RRID  Raised Species  Dilution Used (μg/mL)  FSHR  UniProt P35378  FSHR (N-20) antibody  Santa Cruz Biotechnology, sc-7798, RRID: AB_2294263  Goat; polyclonal  1  LHCGR  UniProt P30730  LHCGR antibody  Abbexa, abx113542, RRID: AB_2665489  Rabbit; polyclonal  0.063  STAR  UniProt P51557  STAR antibody  Abbexa, abx000970, RRID: AB_2665490  Rabbit; polyclonal  0.32  CYP19A1  UniProt P28649  CYP19A1 antibody  Abbexa, abx001773, RRID: AB_2665491  Rabbit; polyclonal  0.43  AR  UniProt P19091  AR (N-20) antibody  Santa Cruz Biotechnology, sc-816, RRID: AB_1563391  Rabbit; polyclonal  0.125  B-TUBULIN  UniProt Q9ERD7  BETA-TUBULIN antibody  Innovative Research, 32-2600, RRID: AB_86547  Mouse; monoclonal  0.1  Protein Target  Antigen Sequence  Name of Antibody  Manufacturer, Catalog No., and RRID  Raised Species  Dilution Used (μg/mL)  FSHR  UniProt P35378  FSHR (N-20) antibody  Santa Cruz Biotechnology, sc-7798, RRID: AB_2294263  Goat; polyclonal  1  LHCGR  UniProt P30730  LHCGR antibody  Abbexa, abx113542, RRID: AB_2665489  Rabbit; polyclonal  0.063  STAR  UniProt P51557  STAR antibody  Abbexa, abx000970, RRID: AB_2665490  Rabbit; polyclonal  0.32  CYP19A1  UniProt P28649  CYP19A1 antibody  Abbexa, abx001773, RRID: AB_2665491  Rabbit; polyclonal  0.43  AR  UniProt P19091  AR (N-20) antibody  Santa Cruz Biotechnology, sc-816, RRID: AB_1563391  Rabbit; polyclonal  0.125  B-TUBULIN  UniProt Q9ERD7  BETA-TUBULIN antibody  Innovative Research, 32-2600, RRID: AB_86547  Mouse; monoclonal  0.1  Abbreviation: RRID, Research Resource Identifier. View Large Statistical analysis To evaluate if the data were normally distributed, the D’Agostino and Pearson omnibus normality test of GraphPad Prism 5 tests (GraphPad Software, San Diego, CA) was used. For mean comparisons, we used analysis of variance to conduct multiple comparisons between normally distributed experimental groups, followed by Tukey posttest to discriminate groups. Kruskal-Wallis test followed by the Dunn posttest was used for comparison between groups if data were not normally distributed. Statistical significance was defined as P < 0.05. Results Exposure to a mixture of EDC changes in F1 female pubertal outcomes First, we determined if the chosen doses were lethal or had unwanted side effects during pregnancy. Data showed that the increase of BW during pregnancy was similar in females exposed to both doses of the mixture (1 or 10 mg/kg/d) compared with controls (females only exposed to vehicle) (Supplemental Fig. 1A). In addition, exposure to the mixture of EDCs did not change gestational length, the number of litter size, or the average of BWs at birth of the F1 generation compared with the control group (Supplemental Fig. 1B–1D). Therefore, exposure of pregnant dams to the 1- or 10-mg/kg mixture of EDCs did not affect general pregnancy parameters. Next, we evaluated F1 female parameters related to sexual development. We found that F1 females exposed to the 1-mg/kg/d mixture of EDCs showed a substantial average delay of 5 days in vaginal opening compared with control (Fig. 2B and 2E). Interestingly, in the same animals, we detected an increase of the presence of a vaginal thread, a thick cord of mesenchymal tissue surrounded by epithelial cells that cross the vaginal opening (47, 48) (Fig. 2A and 2C). On the contrary, females exposed to the 10-mg/kg/d dose did not show any representative delay in vaginal opening or a vaginal thread (Fig. 2A–2C and 2E). Moreover, female mice exposed to the 1-mg/kg/d mixture of EDCs showed an important delay of 6.5 days in the first estrous, but those exposed to the 10-mg/kg/d mixture were similar to control (Fig. 2D and 2F). Once they reached the first estrous, female mice exposed to vehicle showed a characteristic 4- to 5-day estrous cyclicity (Fig. 2G, top panel). However, those exposed to the mixture of EDCs, regardless of the dose, had a decrease in the number of cycles recorded over the studied period (16 days), along with an increase in the number of days expended at the estrous phase and decrease of the other phases (Fig. 2G–2I). Overall, these data suggest that chronic exposure to a mixture of phthalates and alkylphenols modifies, in a dose-dependent manner, female mouse sexual maturation (vaginal opening, age of first estrous, and estrous cyclicity), suggesting changes in ovarian function and probably fertility. Figure 2. View largeDownload slide The mixture of phthalates and alkylphenols delays the onset of puberty and induces lengthened and irregular estrous. (A) Vaginal thread phenotype (white arrow) was increased in females treated with the 1-mg/kg/d dose (C) but not in those with the 10-mg/kg/d dose. (B, E) Vaginal opening and (D, F) first estrous age delay were observed only in female mice exposed to 1 mg/kg/d compared with control. The estrous cycle was evaluated by vaginal smear for a period of 16 days (from vaginal opening day) in the offspring of females exposed to the mixture (1 mg/kg/d and 10 mg/kg/d) or control. (G) Representative estrous cycles were measured by vaginal smear of control (top) and mixture of 1 mg/kg/d (middle) and 10 mg/kg/d (bottom). Note persistence in estrous in both treatments compared with the control. (H) A decrease in the number of estrous cycles in treatment groups compared with control can be observed. (I) An alteration was detected in the number of days in each stage of the cycle with an increase in the number of days in estrous in exposed animals compared with control. The mean ± standard error of the mean values are shown. n ≥ 4, *P < 0.05. M + D, metestrus and diestrus. Figure 2. View largeDownload slide The mixture of phthalates and alkylphenols delays the onset of puberty and induces lengthened and irregular estrous. (A) Vaginal thread phenotype (white arrow) was increased in females treated with the 1-mg/kg/d dose (C) but not in those with the 10-mg/kg/d dose. (B, E) Vaginal opening and (D, F) first estrous age delay were observed only in female mice exposed to 1 mg/kg/d compared with control. The estrous cycle was evaluated by vaginal smear for a period of 16 days (from vaginal opening day) in the offspring of females exposed to the mixture (1 mg/kg/d and 10 mg/kg/d) or control. (G) Representative estrous cycles were measured by vaginal smear of control (top) and mixture of 1 mg/kg/d (middle) and 10 mg/kg/d (bottom). Note persistence in estrous in both treatments compared with the control. (H) A decrease in the number of estrous cycles in treatment groups compared with control can be observed. (I) An alteration was detected in the number of days in each stage of the cycle with an increase in the number of days in estrous in exposed animals compared with control. The mean ± standard error of the mean values are shown. n ≥ 4, *P < 0.05. M + D, metestrus and diestrus. Effect of exposure to a mixture of EDCs in ovarian function of adult females First, we recorded ovarian and uterus weight in adult females exposed to the mixture of EDCs (60 days). Results showed that the uterus relative weight was higher in exposed females compared with control, but the opposite (lower relative weight) was observed in ovaries (Fig. 3A–3D). On the one hand, the quantification of follicle types in the whole ovary showed that females treated with the 1-mg/kg/d dose had a substantial increase in the number of preantral follicles (Fig. 3E) and a decrease in the number of antral follicles and corpora lutea (Fig. 3F and 3G) compared with control. On the other hand, in females treated with the 10-mg/kg/d dose, a decrease in the number of antral follicles was detected (Fig. 3F), and there were no changes in the number of preantral follicles or corpora lutea compared with control (Fig. 3E and 3G). These data suggest that a low dose of mixture of EDCs (1 mg/kg/d) reduced the progression of follicles to the antral stage, whereas a high dose had an effect on the antral stage. Figure 3. View largeDownload slide The exposure to a mixture of EDCs alters the reproductive organs and ovary folliculogenesis in mice. Graphs represent the relative weight of ovaries and uterus from exposed adult females (age 60 days). (A) A representative picture of a control uterus (left) and mixture of 1 mg/kg/d (middle) and 10 mg/kg/d (right). Both doses of the mixture (A, B) increased the relative weight of the uterus and (C, D) decreased the ovary compared with the control. The number of ovarian follicles of exposed females and control was evaluated by histology. (D) A representative picture of a control ovary (left) and mixture of 1 mg/kg/d (middle) and 10 mg/kg/d (right), where preantral follicles (arrowhead), antral follicles (arrow), and corpora lutea (asterisk) can be seen. An increase was observed in the number of (E) preantral follicles and a decrease in the number of (F) antral follicles and (G) corpora lutea in exposed females compared with the control. The mean ± standard error of the mean values are shown. n ≥ 4, *P < 0.05. The bar corresponds to 500 μm at ×40 magnification. Figure 3. View largeDownload slide The exposure to a mixture of EDCs alters the reproductive organs and ovary folliculogenesis in mice. Graphs represent the relative weight of ovaries and uterus from exposed adult females (age 60 days). (A) A representative picture of a control uterus (left) and mixture of 1 mg/kg/d (middle) and 10 mg/kg/d (right). Both doses of the mixture (A, B) increased the relative weight of the uterus and (C, D) decreased the ovary compared with the control. The number of ovarian follicles of exposed females and control was evaluated by histology. (D) A representative picture of a control ovary (left) and mixture of 1 mg/kg/d (middle) and 10 mg/kg/d (right), where preantral follicles (arrowhead), antral follicles (arrow), and corpora lutea (asterisk) can be seen. An increase was observed in the number of (E) preantral follicles and a decrease in the number of (F) antral follicles and (G) corpora lutea in exposed females compared with the control. The mean ± standard error of the mean values are shown. n ≥ 4, *P < 0.05. The bar corresponds to 500 μm at ×40 magnification. Females exposed to a phthalate and alkylphenol mixture present changes in the levels of steroidal sex hormones Next, we investigated whether the impairment of reproductive parameters observed in exposed females was related to changes in reproductive hormones. Our data showed that plasma levels of progesterone and 17β-estradiol, but not testosterone, were significantly reduced in adult female mice exposed to both doses of the mixture of EDCs compared with control (Fig. 4A–4C). Figure 4. View largeDownload slide The mixture of phthalates and alkylphenols alters levels of steroid hormones. Levels of 17β-estradiol, progesterone, and testosterone measured by radioimmunoassay in blood of adult female mice exposed to the mixture and control. A decrease in the plasma levels of (A) progesterone and (C) 17β-estradiol in adult female treatment groups compared with control was observed. (B) No changes were observed in testosterone plasmatic levels. The mean ± standard error of the mean values are shown. n ≥ 4, *P < 0.05. Figure 4. View largeDownload slide The mixture of phthalates and alkylphenols alters levels of steroid hormones. Levels of 17β-estradiol, progesterone, and testosterone measured by radioimmunoassay in blood of adult female mice exposed to the mixture and control. A decrease in the plasma levels of (A) progesterone and (C) 17β-estradiol in adult female treatment groups compared with control was observed. (B) No changes were observed in testosterone plasmatic levels. The mean ± standard error of the mean values are shown. n ≥ 4, *P < 0.05. Later, to explain the changes in plasma progesterone and 17β-estradiol levels, we evaluated whether the mRNA or protein levels of the receptors and enzymes involved in biosynthesis of these steroids were altered in the ovaries of exposed females. Results showed that LHCGR and FSHR mRNAs and protein levels solely decreased in ovaries of females exposed to the 10-mg/kg/d dose compared with control (Fig. 5A, 5B, 5F, and 5G). In addition, we found that the mRNA (Fig. 5C) and protein levels (Fig. 5H) of STAR were significantly lower in ovaries of exposed mice to both doses of the mixture of EDCs compared with control. Furthermore, we evaluated Cyp17a1 mRNA implicated in 17α-hydroxylase and 17,20-lyase activities, as well as Hsd17b implicated in the reduction and dehydrogenation of 17-ketosteroids and 17β-hydroxysteroids, respectively. Results showed an increase of the mRNA levels of Cyp17a1 in ovaries of mice exposed to both doses, whereas mRNA levels of Hsd17b were similar to control (Supplemental Fig. 2A and 2B). Similarly, the mRNA and protein levels of CYP19A (aromatase) were found diminished in ovaries of mice exposed to both doses of the mixture (Fig. 5D and 5I). Interestingly, on one hand, the mRNA and protein levels of the AR significantly decreased only in those females treated with the 10-mg/kg/d dose (Fig. 5E and 5J). On the other hand, the mRNA levels of the estrogen receptor (Esr1) were significantly reduced in ovaries of mice exposed to both doses (Supplemental Fig. 2C). Therefore, changes in the plasma levels of 17β-estradiol and progesterone could be due to alterations in the mRNA that is responsible for encoding the enzymes involved in the steroidoigenic pathway and cholesterol transport. Figure 5. View largeDownload slide The mixture of phthalates and alkylphenols impairs the steroidogenic enzyme levels. LHCGR, FSHR, STAR, CYP19A1, and AR mRNA and protein levels were determined by real-time polymerase chain reaction and normalized to b-actin and Gapdh or by Western blot and normalized with B-tubulin in ovaries of exposed and control females. A decrease in the (C, H) STAR and (D, I) CYP19A1 levels in the exposed groups compared with control was observed. (A, F) LHCGR, (B, G) FSHR, and (E, J) AR were decreased only at the 10-mg/kg/d dose. mean ± standard error of the mean values are shown. n ≥ 4, *P < 0.05. Figure 5. View largeDownload slide The mixture of phthalates and alkylphenols impairs the steroidogenic enzyme levels. LHCGR, FSHR, STAR, CYP19A1, and AR mRNA and protein levels were determined by real-time polymerase chain reaction and normalized to b-actin and Gapdh or by Western blot and normalized with B-tubulin in ovaries of exposed and control females. A decrease in the (C, H) STAR and (D, I) CYP19A1 levels in the exposed groups compared with control was observed. (A, F) LHCGR, (B, G) FSHR, and (E, J) AR were decreased only at the 10-mg/kg/d dose. mean ± standard error of the mean values are shown. n ≥ 4, *P < 0.05. Phthalate and alkylphenol mixture altered fertility of exposed female mice Given the above data, we wondered whether the exposure to the mixture of EDCs induced fertility problems in adult female mice (60 days old) or alterations in their offspring. Control and exposed adult females were left in a cage with a nonexposed adult male (>90 days old) of proven fertility. Data showed that control and exposed females had evidence of copulation (vaginal plug), but regardless the dose, a slight decrease in the delivery rate was observed in exposed females (Table 2). The gestational length was shortened at both doses, and there was a decrease in litter size and an increase in pups’ mortality (F2 generation) of F1 female mice exposed to 1 mg/kg/d but not to 10 mg/kg/d. Furthermore, BW of pups (F2 offspring) was significantly reduced in both treatments (∼10%), and this reduction in weight was even more pronounced at 21 days old (∼17%) only in F2 offspring of female mice exposed to 1 mg/kg/d (Table 2). To predict neonatal and adult reproductive disorder in F2 offspring, we determined the AGD. Males aged 21 days exposed to 1 mg/kg/d showed a decrease in AGD compared with control mice. However, the female AGD was significantly reduced in pups born from females exposed to both doses of the mixture of EDCs (Table 2). In the same manner, a decrease of 17β-estradiol plasma levels was observed in females at 21 days of age (Table 2). All these data indicate that fertility in mice exposed to the mixture of EDCs is compromised, and there is a generational effect in the offspring. Table 2. Reproductive Outcome of F1 Female Mice Treated With the Mixture of EDCs and Morphological Indices of Offspring (F2) Exposed Female Mice/Parameter  Control  1 mg/kg/d  10 mg/kg/d  F1         No. of females (4 repeats)  5  5  5   Positive plug (%)  100  100  100   Gestation length (d)  20.1 ± 0.24  19.3 ± 0.21a  19.3 ± 0.13a   Delivery (%)  100  80  90   Litter size (n)  8.22 ± 0.49  6.44 ± 0.38a  6.56 ± 0.56  F2         Viability index (%)  91.54  72.62a  82.45   Body weight at birth (g)  1.33 ± 0.02  1.19 ± 0.01a  1.20 ± 0.04a   Female body weight at PND 21 (g)  8.35 ± 0.20  6.95 ± 0.38a  7.21 ± 0.42   Female AGD at PND 21 (mm/g1/3)  4.33 ± 0.14  3.28 ± 0.24a  3.39 ± 0.21a   Male AGD at PND 21 (mm/g1/3)  6.79 ± 0.34  5.48 ± 0.33a  6.36 ± 0.47   Female 17β-estradiol levels at PND 21 (pg/mL)  91.03 ± 6.03  55.70 ± 4.79a  58.47 ± 6.33a  Exposed Female Mice/Parameter  Control  1 mg/kg/d  10 mg/kg/d  F1         No. of females (4 repeats)  5  5  5   Positive plug (%)  100  100  100   Gestation length (d)  20.1 ± 0.24  19.3 ± 0.21a  19.3 ± 0.13a   Delivery (%)  100  80  90   Litter size (n)  8.22 ± 0.49  6.44 ± 0.38a  6.56 ± 0.56  F2         Viability index (%)  91.54  72.62a  82.45   Body weight at birth (g)  1.33 ± 0.02  1.19 ± 0.01a  1.20 ± 0.04a   Female body weight at PND 21 (g)  8.35 ± 0.20  6.95 ± 0.38a  7.21 ± 0.42   Female AGD at PND 21 (mm/g1/3)  4.33 ± 0.14  3.28 ± 0.24a  3.39 ± 0.21a   Male AGD at PND 21 (mm/g1/3)  6.79 ± 0.34  5.48 ± 0.33a  6.36 ± 0.47   Female 17β-estradiol levels at PND 21 (pg/mL)  91.03 ± 6.03  55.70 ± 4.79a  58.47 ± 6.33a  Values are mean ± standard error of the mean, n ≥ 4. Each female mated four times with an unexposed fertile male. Delivery: (number of females with litter/number of females with vaginal plug) × 100. Viability index: (number of pups at weaning/number of pups alive on PND 4) × 100. Bold text indicates values significantly different from control. Abbreviations: PND, postnatal day; SE, standard error. a P < 0.05. View Large Discussion In this study, we show that chronic exposure to a mixture of phthalates and alkylphenols at low doses relative to levels of human exposure deregulates levels of progesterone and 17β-estradiol in female mice by changing the expression of enzymes involved in their synthesis and modifying follicle progression and estrous cyclicity. Interestingly, exposure to the lowest mixture of EDCs (1 mg/kg/d) resulted in a more deleterious phenotype, showing additional effects in the delay of puberty and decreased fertility. Data presented in this work indicate that the mixture containing phthalates and alkylphenols did not cause gestational difficulties in F0 females because exposed dams successfully gave birth to live litters comparable to controls. This indicates that the phthalate and alkylphenol mixture exposure did not cause gestational damage or fetal toxicity. Epidemiological data of different cohorts have shown changes in the timing of puberty, thelarche, and menarche associated with the exposure to phthalates and phenols (49, 50). A precocious puberty or a delay in puberty can result in diseases such as infertility, obesity, and cancer. However, the epidemiological studies fail to demonstrate a direct association between EDC exposure and changes in the reproductive parameters. Data of the present work show that chronic exposure to 1 mg/kg/d of phthalates and alkylphenols delays the vaginal opening and first estrous, which are external signs of puberty in female mice (51). The onset of puberty is related to the beginning of the secretion of gonadotropin-releasing hormone (GnRH) and the activation of the hypothalamus-pituitary-gonadal axis (52), and previous studies have shown that exposure to single phthalates altered the levels of GnRH in the hypothalamus and its receptor in the pituitary (53, 54), thus changing the production of gonadotropins and puberty onset. Further studies should be done to determine the levels of GnRH, FSH, and luteinizing hormone in mice exposed to both doses of the mixture of EDCs to decipher the participation of hypothalamus-pituitary-ovarian axis in this process and regulate puberty in humans and animal models. In addition, we observed prolonged estrous cycles in female mice exposed to both doses of the mixture, which is another sign of infertility problems in the adult. This alteration in the estrous cycle may be due to either an indirect consequence of hormonal alterations observed in the exposed mice or a direct action of the mixture on the uterus and/or the vagina. Previous works have shown that exposure to a low or high dose of single phthalates and alkylphenols is related to disruption in the estrous cyclicity (55–58). In this sense, our work shows the same results as a previous one where females were exposed to a mixture of six phthalates from gestational day 10 until birth (27). Because the desquamation of the cells observed during the estrous phase is induced by an increase of 17β-estradiol levels, and our data indicate that in treated females, there is a decrease of this hormone in plasma, it is probable that what is observed as persistence in estrous in the treated females is no more than an estrogenic and direct effect of the mixture on the vaginal epithelium and not a consequence of the disruption of the hypothalamic-pituitary-ovary axis. This same hypothesis could also explain the observed increase in uterine size, which was also seen in a previous study with a mixture of phthalates at a dose similar to that of the current study (27). In addition, both NP and OP, two of the compounds contained in the mixture, induce uterine growth in vivo (22, 59). In the current study, we observed a reduction in the ovary weight, which could be explained as due to a reduction in the number of total antral follicles in mice exposed to both doses. One possible explanation, on one hand, is that as a result of lower levels of Esr1 mRNA (and probably lower levels at the protein level), the proliferation signaling cascade elicited by this receptor is not turned on in these mice, and therefore the granulosa cells in the follicle do not increase in number and block the antrum formation. On the other hand, because androgen signaling is crucial to follicle development (60), and phthalates and alkylphenols have been reported to bind to AR (61), it is possible that they also block the signaling event elicited by this receptor, hampering follicle development and decreasing ovary size. Interestingly, exposure to the 10-mg/kg/d dose did not alter preantral follicle numbers but decreased antral follicle numbers, which differs from previous studies where single exposure to DEHP or NP modified the number of preantral follicles (56, 62, 63). This difference could be due to the exposure time or that previous works have been done with single compounds and not a mixture of phthalates and alkylphenols like the present work. The results of the current study are similar to those reported in female mice lacking the AR (ARKO), with a decrease in the antral follicle numbers but without changes in the preantral follicle numbers (64). In ovary, AR signaling induces the activation of the insulin pathway via PI3K, promoting the expression of the steroidogenic enzymes such as CYP19A1 (65), which acts cooperatively with gonadotropins to induce steroidogenesis by an increase in the number of FSHR and LHCGR (66, 67). Here we observed a decrease in the expression of the AR at the mRNA and protein levels, which could account for the observed effects of these mice and explain the similarity with the ARKO model. In this way, the reduced Ar mRNA expression could explain the observed decrease in FSHR, LHCGR, and CYP19A1 at the mRNA and protein levels. One possible mechanism, on one hand, is that this mixture induces alterations in insulin signaling upstream of the steroidogenesis due to an Ar mRNA decrease, which mimics the phenotype of the ARKO mouse (64). Thus, a reduction in Ar mRNA results in decreased levels of the AR, insulin signaling, steroidogenesis, and loss of antral follicle probably due to an increase in apoptosis (60). Also, it is feasible that the reduction of the Ar mRNA levels is due to a disruption in the expression levels of the promoter because genomic and epigenomic mutations have been found in both mice and rats exposed to phthalates (25, 68). On the other hand, the 1-mg/kg/d mixture inhibits and/or reduces the progression of the preantral to antral follicle, which is consistent with a previous report in pregnant mice exposed to 5 mg/kg/d DEHP (69). Furthermore, in mice exposed to the 1-mg/kg/d mixture, we observed a decrease in corpora lutea, suggesting a failure in the progression of folliculogenesis. Previous studies have shown that DEHP exposure decreases the number of antral follicles by increasing atresia and apoptosis of granulosa cells (23). However, in the present work, this decline in antral follicles could be explained by a reduction in the preantral-to-antral transition rather than increasing atresia or apoptosis. On one hand, female mice exposed to the 1-mg/kg/d mixture did not show a decrease in mRNA or protein levels of FSHR and LHCGR, suggesting that preantral follicles are fully responding to gonadotropin stimulus. On the other hand, we observed a decrease in progesterone and 17β-estradiol plasma levels and also the expression of mRNAs and proteins levels of STAR and CYP19A1. Interestingly, we also observed an increment in the levels on Cyp17a1, which could be a kind of compensatory mechanism to maintain the levels of androstenedione while not affecting testosterone levels. Because 17β-estradiol is key to promote follicle growth, it is possible that the decrease in the levels of this hormone is due to a reduction in the Cyp19a1 mRNA expression levels. This finding is in agreement with previous studies showing a decrease in the levels of Cyp19a1 mRNA in female mice exposed to DEHP (58, 69, 70). Alternatively, it is possible that the mixture impairs LHCGR signaling and/or the expression of cell cycle cyclins, thus impairing follicle growth, which is similar to the effect of elevated concentrations of FSH in the prepuberal ovary (71). Therefore, further studies are required to determine the mechanism by which this mixture of phthalates and alkylphenols reduces the levels of CYP19A1 and in this manner possibly controls the preantral-to-antral follicle transition. In the present work, we reveal a decrease in fertility in females exposed to a mixture of phthalates and alkylphenols (F1-exposed generations), which agrees with previous reports showing that chronic occupational exposure in women to single phthalates is associated with decreased rates of pregnancy and high rates of miscarriage (72, 73). In exposed female mice, a decrease in the fertility (delivery percent) was more noticeable with the lowest dose (Table 2), and more interestingly, female offspring (F2 generations) exposed to both doses of the mixture of EDCs presented a decrease in anogenital distance and in the 17β-estradiol serum levels, which is a marker of endocrine disruption in the offspring and multigenerational effects (24). Therefore, these data suggest that a mixture of EDCs can alter germline epigenetic programming in a similar way reported by single exposure to phthalates and alkylphenols (25, 69). In addition, it shows that chronic exposure to EDCs induces changes in fertility that may a pose risk to the development of other pathologies such as cancer. In summary, our data demonstrate that chronic exposure to a mixture of phthalates and alkylphenols at an environmentally relevant dose, from prenatal to adult life in female mice (the exposome paradigm), induces changes in the steroidogenesis pathway and estrous cyclicity and is dose dependent of the levels of the gonadotropin receptor, follicle development, puberty onset, and fertility. These results indicate that not only exposure but also its level is relevant to assess the effective contribution of EDCs in the development of diseases. Abbreviations: AGD anogenital distance AR androgen receptor ARKO female mice lacking the androgen receptor BBP benzyl butyl phthalate BW body weight CV coefficient of variation DBP dibutyl phthalate DEHP bis (2-ethylhexyl) phthalate DMSO dimethyl sulfoxide EDC endocrine-disrupting chemical FSH follicle-stimulating hormone FSHR follicle-stimulating hormone receptor GnRH gonadotropin-releasing hormone LHCGR luteinizing hormone cognate receptor mRNA messenger RNA NP 4-nonylphenol OP 4-tert-octylphenol STAR acute steroidogenic regulatory protein. Acknowledgments Financial Support: This work was supported by FONDECYT (Grant 1150352 to R.D.M.) and CONICYT (Grant 63140090 to D.P.-G.), Chile. Disclosure Summary: The authors have nothing to disclose. References 1. Mascarenhas MN, Flaxman SR, Boerma T, Vanderpoel S, Stevens GA. National, regional, and global trends in infertility prevalence since 1990: a systematic analysis of 277 health surveys. PLoS Med . 2012; 9( 12): e1001356. Google Scholar CrossRef Search ADS PubMed  2. Healy DL, Trounson AO, Andersen AN. Female infertility: causes and treatment. Lancet . 1994; 343( 8912): 1539– 1544. Google Scholar CrossRef Search ADS PubMed  3. Marques-Pinto A, Carvalho D. Human infertility: are endocrine disruptors to blame? Endocr Connect . 2013; 2( 3): R15– R29. Google Scholar CrossRef Search ADS PubMed  4. Penning TM. Human aldo-keto reductases and the metabolic activation of polycyclic aromatic hydrocarbons. Chem Res Toxicol . 2014; 27( 11): 1901– 1917. Google Scholar CrossRef Search ADS PubMed  5. Chin-Chan M, Navarro-Yepes J, Quintanilla-Vega B. Environmental pollutants as risk factors for neurodegenerative disorders: Alzheimer and Parkinson diseases. Front Cell Neurosci . 2015; 9: 124. Google Scholar CrossRef Search ADS PubMed  6. Fenner-Crisp PA, Maciorowski AF, Timm GE. The Endocrine Disruptor Screening Program developed by the U.S. Environmental Protection Agency. Ecotoxicology . 2000; 9( 1/2): 85– 91. Google Scholar CrossRef Search ADS   7. World Health Organization. United Nations Environment Programme (WHO-UNEP). In: Bergman A, Heindel JJ, Jobling S, Kidd KA, Zoeller RT, eds. State of the Science of Endocrine Disrupting Chemicals—2012. Geneva, Switzerland: World Health Organization and United Nations Environment Programme. Available at: http://www.who.int/ceh/publications/endocrine/en/index.html. Accessed 23 June 2017. 8. De Coster S, van Larebeke N. Endocrine-disrupting chemicals: associated disorders and mechanisms of action. J Environ Public Health . 2012; 2012: 713696. Google Scholar CrossRef Search ADS PubMed  9. Sathyanarayana S, Butts S, Wang C, Barrett E, Nguyen R, Schwartz SM, Haaland W, Swan SH; TIDES Team. Early prenatal phthalate exposure, sex steroid hormones, and birth outcomes. J Clin Endocrinol Metab . 2017; 102( 6): 1870– 1878. Google Scholar CrossRef Search ADS PubMed  10. Diamanti-Kandarakis E, Bourguignon JP, Giudice LC, Hauser R, Prins GS, Soto AM, Zoeller RT, Gore AC. Endocrine-disrupting chemicals: an Endocrine Society scientific statement. Endocr Rev . 2009; 30( 4): 293– 342. Google Scholar CrossRef Search ADS PubMed  11. Lagos-Cabré R, Moreno RD. Contribution of environmental pollutants to male infertily: a working model of germ cell apoptosis induced by plasticizers. Biol Res . 2012; 45( 1): 5– 14. Google Scholar CrossRef Search ADS PubMed  12. Li LX, Chen L, Meng XZ, Chen BH, Chen SQ, Zhao Y, Zhao LF, Liang Y, Zhang YH. Exposure levels of environmental endocrine disruptors in mother-newborn pairs in China and their placental transfer characteristics. PLoS One . 2013; 8( 5): e62526. Google Scholar CrossRef Search ADS PubMed  13. Du YY, Fang YL, Wang YX, Zeng Q, Guo N, Zhao H, Li YF. Follicular fluid and urinary concentrations of phthalate metabolites among infertile women and associations with in vitro fertilization parameters. Reprod Toxicol . 2016; 61: 142– 150. Google Scholar CrossRef Search ADS PubMed  14. Dieterle S. Analysis of toxins in follicle fluid from women with unfulfilled pregnancy. 2017. Available at: https://clinicaltrials.gov/show/NCT01385605. Accessed 23 June 2017. 15. Tranfo G, Caporossi L, Paci E, Aragona C, Romanzi D, De Carolis C, De Rosa M, Capanna S, Papaleo B, Pera A. Urinary phthalate monoesters concentration in couples with infertility problems. Toxicol Lett . 2012; 213( 1): 15– 20. Google Scholar CrossRef Search ADS PubMed  16. Messerlian C, Souter I, Gaskins AJ, Williams PL, Ford JB, Chiu YH, Calafat AM, Hauser R; Earth Study Team. Urinary phthalate metabolites and ovarian reserve among women seeking infertility care. Hum Reprod . 2015; 31( 1): 75– 83. Google Scholar CrossRef Search ADS PubMed  17. Mínguez-Alarcón L, Gaskins AJ. Female exposure to endocrine disrupting chemicals and fecundity: a review. Curr Opin Obstet Gynecol . 2017; 29( 4): 202– 211. Google Scholar CrossRef Search ADS PubMed  18. Drummond AE. The role of steroids in follicular growth. Reprod Biol Endocrinol . 2006; 4( 1): 16. Google Scholar CrossRef Search ADS PubMed  19. Huang Z, Wells D. Molecular aspects of follicular development. In: Donnez J, Kim SS, eds. Principles and Practice of Fertility Preservation . Cambridge, UK: Cambridge University Press; 2011: 114– 128. Google Scholar CrossRef Search ADS   20. Senthilkumaran B, Yoshikuni M, Nagahama Y. A shift in steroidogenesis occurring in ovarian follicles prior to oocyte maturation. Mol Cell Endocrinol . 2004; 215( 1–2): 11– 18. Google Scholar CrossRef Search ADS PubMed  21. Caras ML. Estrogenic modulation of auditory processing: a vertebrate comparison. Front Neuroendocrinol . 2013; 34( 4): 285– 299. Google Scholar CrossRef Search ADS PubMed  22. Yoshida M, Takenaka A, Katsuda S, Kurokawa Y, Maekawa A. Neonatal exposure to p-tert-octylphenol causes abnormal expression of estrogen receptor alpha and subsequent alteration of cell proliferating activity in the developing Donryu rat uterus. Toxicol Pathol . 2002; 30( 3): 357– 364. Google Scholar CrossRef Search ADS PubMed  23. Hannon PR, Flaws JA. The effects of phthalates on the ovary. Front Endocrinol (Lausanne) . 2015; 6: 8. Google Scholar PubMed  24. Zhou C, Gao L, Flaws JA. Exposure to an environmentally relevant phthalate mixture causes transgenerational effects on female reproduction in mice. Endocrinology . 2017; 158( 6): 1739– 1754. Google Scholar CrossRef Search ADS PubMed  25. Manikkam M, Tracey R, Guerrero-Bosagna C, Skinner MK. Plastics derived endocrine disruptors (BPA, DEHP and DBP) induce epigenetic transgenerational inheritance of obesity, reproductive disease and sperm epimutations. PLoS One . 2013; 8( 1): e55387. Google Scholar CrossRef Search ADS PubMed  26. Johansson HK, Jacobsen PR, Hass U, Svingen T, Vinggaard AM, Isling LK, Axelstad M, Christiansen S, Boberg J. Perinatal exposure to mixtures of endocrine disrupting chemicals reduces female rat follicle reserves and accelerates reproductive aging. Reprod Toxicol . 2016; 61: 186– 194. Google Scholar CrossRef Search ADS PubMed  27. Zhou C, Gao L, Flaws JA. Prenatal exposure to an environmentally relevant phthalate mixture disrupts reproduction in F1 female mice. Toxicol Appl Pharmacol . 2017; 318: 49– 57. Google Scholar CrossRef Search ADS PubMed  28. Agency for Toxic Substances and Disease Registry. Minimal risk levels (MRLs): US Department of Health and Human Services, Public Health Service, Atlanta, GA; 2017. Available at: https://www.atsdr.cdc.gov/mrls/index.asp. Accessed 23 June 2017. 29. Jonsson B. Risk assessment on butylphenol, octylphenol and nonylphenol, and estimated human exposure of alkylphenols from Swedish fish. Uppsala, Sweden: Ecological Department, Uppsala University; 2006: 1– 52. Available at: http://www.uu.se/digitalAssets/177/c_177024-l_3-k_jonsson-beatrice-report.pdf. 30. Somasundaram DB, Manokaran K, Selvanesan BC, Bhaskaran RS. Impact of di-(2-ethylhexyl) phthalate on the uterus of adult Wistar rats. Hum Exp Toxicol . 2016; 36( 6): 565– 572. Google Scholar CrossRef Search ADS PubMed  31. Agency for Toxic Substances and Disease Registry. Toxicological profile for di(2-ethylhexyl)phthalate. Department of Health and Human Services, Public Health Service, Atlanta, GA; 2002. Available at: https://www.atsdr.cdc.gov/toxprofiles/tp9.pdf. Accessed 23 June 2017. 32. Snedeker SM, Hay AG. The alkylphenols nonylphenol and octylphenol in food contact materials and household items: exposure and health risk considerations. In: Snedeker SM, ed. Toxicants in Food Packaging and Household Plastics . London, UK: Springer-Verlag; 2014: 125– 150. Google Scholar CrossRef Search ADS   33. Zero Breast Cancer. Phthalates: the everywhere chemical. San Rafael, CA: Zero Breast Cancer; 2014. Available at: https://www.niehs.nih.gov/research/supported/assets/docs/j_q/phthalates_the_everywhere_chemical_handout_508.pdf. Accessed 23 June 2017. 34. Food and Drug Administration. Safety assessment of di(2-Ethylhexyl)phthalate (DEHP) released from PVC medical devices. Rockville, MD: Center for Devices and Radiological Health; 2001. Available at: https://www.fda.gov/downloads/MedicalDevices/.../UCM080457.pdf. Accessed 23 June 2017. 35. National Toxicology Program. NTP-CERHR monograph on the potential human reproductive and developmental effects of butyl benzyl phthalate (BBP). NTP CERHR MON . 2003;( 5): i– III90. 36. Kavlock R, Barr D, Boekelheide K, Breslin W, Breysse P, Chapin R, Gaido K, Hodgson E, Marcus M, Shea K, Williams P. NTP-CERHR expert panel update on the reproductive and developmental toxicity of di(2-ethylhexyl) phthalate. Reprod Toxicol . 2006; 22( 3): 291– 399. Google Scholar CrossRef Search ADS PubMed  37. Hines CJ, Hopf NB, Deddens JA, Silva MJ, Calafat AM. Estimated daily intake of phthalates in occupationally exposed groups. J Expo Sci Environ Epidemiol . 2009; 21( 2): 133– 141. Google Scholar CrossRef Search ADS PubMed  38. Chapin RE, Delaney J, Wang Y, Lenning L, Davis B, Collins B, Mintz N, Wolfe G. The effects of 4-nonylphenol in rats: a multigeneration reproduction study. Toxicol Sci . 1999; 52( 1): 80– 91. Google Scholar CrossRef Search ADS PubMed  39. Osimitz TG, Droege W, Driver JH. Human risk assessment for nonylphenol. Hum Ecol Risk Assess . 2015; 21( 7): 1903– 1919. Google Scholar CrossRef Search ADS   40. Gad SC, Spainhour CB, Shoemake C, Pallman DR, Stricker-Krongrad A, Downing PA, Seals RE, Eagle LA, Polhamus K, Daly J. Tolerable levels of nonclinical vehicles and formulations used in studies by multiple routes in multiple species with notes on methods to improve utility. Int J Toxicol . 2016; 35( 2): 95– 178. Google Scholar CrossRef Search ADS PubMed  41. McLean AC, Valenzuela N, Fai S, Bennett SA. Performing vaginal lavage, crystal violet staining, and vaginal cytological evaluation for mouse estrous cycle staging identification. J Vis Exp . 2012;( 67): e4389. 42. Gallavan RH Jr, Holson JF, Stump DG, Knapp JF, Reynolds VL. Interpreting the toxicologic significance of alterations in anogenital distance: potential for confounding effects of progeny body weights. Reprod Toxicol . 1999; 13( 5): 383– 390. Google Scholar CrossRef Search ADS PubMed  43. Chen ZG, Luo LL, Xu JJ, Zhuang XL, Kong XX, Fu YC. Effects of plant polyphenols on ovarian follicular reserve in aging rats. Biochem Cell Biol . 2010; 88( 4): 737– 745. Google Scholar CrossRef Search ADS PubMed  44. Flaws JA, Doerr JK, Sipes IG, Hoyer PB. Destruction of preantral follicles in adult rats by 4-vinyl-1-cyclohexene diepoxide. Reprod Toxicol . 1994; 8( 6): 509– 514. Google Scholar CrossRef Search ADS PubMed  45. Murphy BD. Models of luteinization. Biol Reprod . 2000; 63( 1): 2– 11. Google Scholar CrossRef Search ADS PubMed  46. Rao X, Huang X, Zhou Z, Lin X. An improvement of the 2ˆ(−delta delta CT) method for quantitative real-time polymerase chain reaction data analysis. Biostat Bioinforma Biomath . 2013; 3( 3): 71– 85. Google Scholar PubMed  47. Flaws JA, Sommer RJ, Silbergeld EK, Peterson RE, Hirshfield AN. In utero and lactational exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induces genital dysmorphogenesis in the female rat. Toxicol Appl Pharmacol . 1997; 147( 2): 351– 362. Google Scholar CrossRef Search ADS PubMed  48. Hotchkiss AK, Lambright CS, Ostby JS, Parks-Saldutti L, Vandenbergh JG, Gray LE Jr. Prenatal testosterone exposure permanently masculinizes anogenital distance, nipple development, and reproductive tract morphology in female Sprague-Dawley rats. Toxicol Sci . 2007; 96( 2): 335– 345. Google Scholar CrossRef Search ADS PubMed  49. Wolff MS, Teitelbaum SL, Pinney SM, Windham G, Liao L, Biro F, Kushi LH, Erdmann C, Hiatt RA, Rybak ME, Calafat AM; Breast Cancer and Environment Research Centers. Investigation of relationships between urinary biomarkers of phytoestrogens, phthalates, and phenols and pubertal stages in girls. Environ Health Perspect . 2010; 118( 7): 1039– 1046. Google Scholar CrossRef Search ADS PubMed  50. Zhang Y, Cao Y, Shi H, Jiang X, Zhao Y, Fang X, Xie C. Could exposure to phthalates speed up or delay pubertal onset and development? A 1.5-year follow-up of a school-based population. Environ Int . 2015; 83: 41– 49. Google Scholar CrossRef Search ADS PubMed  51. Nelson JF, Karelus K, Felicio LS, Johnson TE. Genetic influences on the timing of puberty in mice. Biol Reprod . 1990; 42( 4): 649– 655. Google Scholar CrossRef Search ADS PubMed  52. Ojeda SR, Urbanski HF, Ahmed CE. The onset of female puberty: studies in the rat. Recent Prog Horm Res . 1986; 42: 385– 442. Google Scholar PubMed  53. Liu T, Li N, Zhu J, Yu G, Guo K, Zhou L, Zheng D, Qu X, Huang J, Chen X, Wang S, Ye L. Effects of di-(2-ethylhexyl) phthalate on the hypothalamus-pituitary-ovarian axis in adult female rats. Reprod Toxicol . 2014; 46: 141– 147. Google Scholar CrossRef Search ADS PubMed  54. Liu T, Jia Y, Zhou L, Wang Q, Sun D, Xu J, Wu J, Chen H, Xu F, Ye L. Effects of di-(2-ethylhexyl) phthalate on the hypothalamus-uterus in pubertal female rats. Int J Environ Res Public Health . 2016; 13( 11): E1130. Google Scholar CrossRef Search ADS PubMed  55. Davis BJ, Maronpot RR, Heindel JJ. Di-(2-ethylhexyl) phthalate suppresses estradiol and ovulation in cycling rats. Toxicol Appl Pharmacol . 1994; 128( 2): 216– 223. Google Scholar CrossRef Search ADS PubMed  56. Willoughby KN, Sarkar AJ, Boyadjieva NI, Sarkar DK. Neonatally administered tert-octylphenol affects onset of puberty and reproductive development in female rats. Endocrine . 2005; 26( 2): 161– 168. Google Scholar CrossRef Search ADS PubMed  57. Hannon PR, Peretz J, Flaws JA. Daily exposure to di(2-ethylhexyl) phthalate alters estrous cyclicity and accelerates primordial follicle recruitment potentially via dysregulation of the phosphatidylinositol 3-kinase signaling pathway in adult mice. Biol Reprod . 2014; 90( 6): 136. Google Scholar CrossRef Search ADS PubMed  58. Moyer B, Hixon ML. Reproductive effects in F1 adult females exposed in utero to moderate to high doses of mono-2-ethylhexylphthalate (MEHP). Reprod Toxicol . 2012; 34( 1): 43– 50. Google Scholar CrossRef Search ADS PubMed  59. Zhang W, Yang J, Wang J, Xia P, Xu Y, Jia H, Chen Y. Comparative studies on the increase of uterine weight and related mechanisms of cadmium and p-nonylphenol. Toxicology . 2007; 241( 1–2): 84– 91. Google Scholar CrossRef Search ADS PubMed  60. Sen A, Prizant H, Light A, Biswas A, Hayes E, Lee HJ, Barad D, Gleicher N, Hammes SR. Androgens regulate ovarian follicular development by increasing follicle stimulating hormone receptor and microRNA-125b expression. Proc Natl Acad Sci USA . 2014; 111( 8): 3008– 3013. Google Scholar CrossRef Search ADS PubMed  61. Luccio-Camelo DC, Prins GS. Disruption of androgen receptor signaling in males by environmental chemicals. J Steroid Biochem Mol Biol . 2011; 127( 1–2): 74– 82. Google Scholar CrossRef Search ADS PubMed  62. Zhang XF, Zhang LJ, Li L, Feng YN, Chen B, Ma JM, Huynh E, Shi QH, De Felici M, Shen W. Diethylhexyl phthalate exposure impairs follicular development and affects oocyte maturation in the mouse. Environ Mol Mutagen . 2013; 54( 5): 354– 361. Google Scholar CrossRef Search ADS PubMed  63. Xu C, Chen JA, Qiu Z, Zhao Q, Luo J, Yang L, Zeng H, Huang Y, Zhang L, Cao J, Shu W. Ovotoxicity and PPAR-mediated aromatase downregulation in female Sprague-Dawley rats following combined oral exposure to benzo[a]pyrene and di-(2-ethylhexyl) phthalate. Toxicol Lett . 2010; 199( 3): 323– 332. Google Scholar CrossRef Search ADS PubMed  64. Wang RS, Chang HY, Kao SH, Kao CH, Wu YC, Yeh S, Tzeng CR, Chang C. Abnormal mitochondrial function and impaired granulosa cell differentiation in androgen receptor knockout mice. Int J Mol Sci . 2015; 16( 5): 9831– 9849. Google Scholar CrossRef Search ADS PubMed  65. Luo W, Wiltbank MC. Distinct regulation by steroids of messenger RNAs for FSHR and CYP19A1 in bovine granulosa cells. Biol Reprod . 2006; 75( 2): 217– 225. Google Scholar CrossRef Search ADS PubMed  66. Zhou J, Kumar TR, Matzuk MM, Bondy C. Insulin-like growth factor I regulates gonadotropin responsiveness in the murine ovary. Mol Endocrinol . 1997; 11( 13): 1924– 1933. Google Scholar CrossRef Search ADS PubMed  67. Poretsky L, Cataldo NA, Rosenwaks Z, Giudice LC. The insulin-related ovarian regulatory system in health and disease. Endocr Rev . 1999; 20( 4): 535– 582. Google Scholar CrossRef Search ADS PubMed  68. Huang XF, Li Y, Gu YH, Liu M, Xu Y, Yuan Y, Sun F, Zhang HQ, Shi HJ. The effects of di-(2-ethylhexyl)-phthalate exposure on fertilization and embryonic development in vitro and testicular genomic mutation in vivo. PLoS One . 2012; 7( 11): e50465. Google Scholar CrossRef Search ADS PubMed  69. Pocar P, Fiandanese N, Berrini A, Secchi C, Borromeo V. Maternal exposure to di(2-ethylhexyl)phthalate (DEHP) promotes the transgenerational inheritance of adult-onset reproductive dysfunctions through the female germline in mice. Toxicol Appl Pharmacol . 2017; 322: 113– 121. Google Scholar CrossRef Search ADS PubMed  70. Pocar P, Fiandanese N, Secchi C, Berrini A, Fischer B, Schmidt JS, Schaedlich K, Borromeo V. Exposure to di(2-ethyl-hexyl) phthalate (DEHP) in utero and during lactation causes long-term pituitary-gonadal axis disruption in male and female mouse offspring. Endocrinology . 2012; 153( 2): 937– 948. Google Scholar CrossRef Search ADS PubMed  71. François CM, Petit F, Giton F, Gougeon A, Ravel C, Magre S, Cohen-Tannoudji J, Guigon CJ. A novel action of follicle-stimulating hormone in the ovary promotes estradiol production without inducing excessive follicular growth before puberty. Sci Rep . 2017; 7: 46222. Google Scholar CrossRef Search ADS PubMed  72. Thomsen AM, Riis AH, Olsen J, Jönsson BA, Lindh CH, Hjollund NH, Jensen TK, Bonde JP, Toft G. Female exposure to phthalates and time to pregnancy: a first pregnancy planner study. Hum Reprod . 2017; 32( 1): 232– 238. Google Scholar PubMed  73. Peng F, Ji W, Zhu F, Peng D, Yang M, Liu R, Pu Y, Yin L. A study on phthalate metabolites, bisphenol A and nonylphenol in the urine of Chinese women with unexplained recurrent spontaneous abortion. Environ Res . 2016; 150: 622– 628. Google Scholar CrossRef Search ADS PubMed  Copyright © 2018 Endocrine Society http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Endocrinology Oxford University Press

Reproductive Alterations in Chronically Exposed Female Mice to Environmentally Relevant Doses of a Mixture of Phthalates and Alkylphenols

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Endocrine Society
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Copyright © 2018 Endocrine Society
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0013-7227
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1945-7170
D.O.I.
10.1210/en.2017-00614
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Abstract

Abstract Endocrine-disrupting chemicals (EDCs) are exogenous compounds that modify hormone biosynthesis, causing adverse effects to human health. Among them, phthalates and alkylphenols are important due to their wide use in plastics, detergents, personal care products, cosmetics, and food packaging. However, their conjoint effects over reproductive female health have not been addressed. The aim of this work was to test the effect of chronically exposed female mice to a mixture of three phthalates [bis (2-ethylhexyl), dibutyl, and benzyl butyl] and two alkylphenols (4-nonylphenol and 4-tert-octylphenol) from conception to adulthood at environmentally relevant doses. These EDCs were administered in two doses: one below the minimal risk dose to cause adverse effects on human development and reproduction [1 mg/kg body weight (BW)/d of the total mixture] and the other one based on the reference value close to occupational exposure in humans (10 mg/kg BW/d of the total mixture). Our results show that both doses had similar effects regarding the uterus and ovary relative weight, estrous cyclicity, serum levels of progesterone and 17β-estradiol, and expression of key elements in the steroidogenesis pathway (acute steroidogenic regulatory protein and CYP19A1). However, only the 1-mg/kg BW/d dose delayed the onset of puberty and the transition from preantral to antral follicles, whereas the 10-mg/kg BW/d dose decreased the number of antral follicles and gonadotropin receptor expression. In addition, we observed changes in several fertility parameters in exposed females and in their progeny (F2 generation). In conclusion, our results indicate that chronic exposure to a complex EDC mixture, at environmentally relevant doses, modifies reproductive parameters in female mice. The absolute number of couples affected by infertility increased from 42 million in 1990 to 48.5 million in 2010 (1). The causes of infertility linked to the female factor include ovulatory disorders (33%), idiopathic disorders (40%), and tubal factor disorders/endometriosis (27%) (2). Multiple factors such as the exposure to environmental contaminants can contribute to the origin of unexplained infertility in females (3). Genome-wide association has shown that numerous diseases are associated with specific genes or gene clusters, several of which have been shown to be expressed in the presence of certain environmental factors such as pesticides or tobacco smoke, stretching the notion that environmental conditions are important to trigger genes that are at the onset of different diseases (4, 5). In this context, it has been calculated that humans are exposed to ∼85,000 chemicals classified as toxic, widely present in commercial use, which pose great risk to the development of many unanticipated diseases (6, 7). Several studies have shown diverse association of unique environmental factors to some diseases (8, 9), but most have been assessed in animal models focused on individual compounds or exposure at specific developmental stages. However, these experimental designs do not take into account that humans and animals are exposed to these chemicals throughout life (i.e., from conception to adulthood). Endocrine-disrupting chemicals (EDCs) are exogenous compounds that alter hormone biosynthesis, causing adverse effects to human health and intact organisms, their progenitors, or offspring (10). Among the EDCs, phthalates [bis (2-ethylhexyl) phthalate (DEHP), dibutyl phthalate (DBP), and benzyl butyl phthalate (BBP)] and alkylphenols [4-nonylphenol (NP) and 4-tert-octylphenol (OP)] are important due to their wide use in plastics, detergents, personal care products, cosmetics, and food packaging (11). In females, the presence of these compounds has been verified in fetal samples such as placenta, cord blood, amniotic fluid, meconium, and urine (12) and in biological fluids like serum, milk, and follicular fluid in adult samples (13, 14). Epidemiological studies have linked infertility problems in women with single exposure to phthalates or alkylphenols (13–15), especially due to a diminished ovarian reserve (16). Nevertheless, there are insufficient data to reveal a clear association between single phthalates and alkylphenol exposure and adverse effects on human female reproductive health, raising a need for studies focused on mixtures of EDCs (17). At the reproductive age, in the ovary, it is possible to find follicles at different stages of maturation, but only those with a cavity adjacent to the oocyte—antral follicles—are responsible for the production of steroid hormones (18). Luteinizing hormone binds to its cognate receptor [luteinizing hormone cognate receptor (LHCGR)] present in theca cells of antral follicles and increases the transcription of genes encoding for enzymes responsible for progesterone and androgen (androstenedione and testosterone) biosynthesis, which play an important role in the follicular progression from the preantral to antral follicle (19). Furthermore, on one hand, the correct expression of the acute steroidogenic regulatory protein (STAR), which transports cholesterol into the mitochondria, is a limiting step. On the other hand, the binding of the follicle-stimulating hormone (FSH) to its receptor (FSHR), expressed by granulosa cells, increases the transcription of the gene that encodes P450 aromatase (CYP19A1). The former is an enzyme responsible for irreversibly converting androgens from theca cells into estrogens [(17β-estradiol and estrone)] (20). Finally, estrogens stimulate follicle growth and ovulation, and its production triggers growth and vascularization of the endometrium, initiating the menarche at puberty and maintaining the menstrual cycles in human and estral cycles in mice (21). In vitro and in vivo studies using single exposure to phthalates or alkylphenols have shown that these compounds interfere with the biosynthesis of sex steroids (androgens, estrogens, and progestins), their receptors, and the expression of the enzymes involved in steroidogenesis (22, 23). Previous studies have shown that prenatal or perinatal exposure to a complex mixture of EDCs containing only phthalates or alkylphenols, along with other chemicals, accelerated the onset of puberty, impaired folliculogenesis, and decreased fertility in female mice (24–27). However, some of these data have been contradictory, and the exposure to EDCs occurred during a specific developmental time, which is not comparable to human exposure. Therefore, in this study, we wanted to evaluate a chronic exposure to a mixture of three phthalates (DEHP, DBP, and BBP) and two alkylphenols (NP and OP) in female mice, from conception to adulthood, at two different doses: (1) one below the minimal risk dose to cause adverse effects on human development and reproduction (28) and (2) one based on the reference value close to occupational exposure in humans (29, 30). The mixture defined in our work was at least ∼100- and ∼1000-fold lower than the no observed adverse effect level and low observed adverse effect level values for DEHP to cause adverse effects in humans (31). In these models, we fully studied the changes in the reproductive health of exposed mice, including the progesterone, testosterone, and 17β-estradiol levels, along with the quantification of messenger RNA (mRNA)/protein levels implicated in the steroidogenesis pathway. Materials and Methods Animals Adult female C57BL/6J of 60 days of age were obtained from the Animal Facility of Pontificia Universidad Católica de Chile and maintained in bisphenol A–free H-TEMP polysulfone cages (TECNIPLAST). The mice were housed under a 12-hour light:12-hour dark cycle, and temperature was maintained at 22 ± 1°C. Food and water were provided for ad libitum consumption. Experiments were conducted in accordance with the rules laid down by the Consortium for Developing a Guide for the Care and Use of Agricultural Animals in Agricultural Research and Teaching and by the Guide for the Care and Use of Laboratory Animals of National Research Council, and they were approved by the Ethical Scientific Committee for the Care of Animals and the Environment at the Pontificia Universidad Católica de Chile No. 141222004. All animal protocols were endorsed by the Chilean National Fund of Science and Technology (FONDECYT). Chemicals DEHP, DBP, BBP, NP, OP, and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (St. Louis, MO). Ethanol was acquired from Winkler (Santiago, Chile). Experimental design The compounds of the mixture used in this study were chosen on the basis that they represent the most abundant phthalates and alkylphenols in everyday products (32, 33). Doses were chosen based on the nonoccupational and occupational exposure to these compounds between the range of 0.3 mg/kg/d and 143 mg/kg/d (29, 30, 34–37). In this way, we generated two doses of this mixture, resembling nonoccupational (1 mg/kg/d) and occupational (10 mg/kg/d) human exposure. It should be noted that these doses are, in relation to the no observed adverse effect level and low observed adverse effect level of DEHP and NP, ∼1000 and ∼100 times lower, respectively, for endocrine-reproductive adverse effects in humans, rats, and mice (31, 38, 39) (Supplemental Table 1). A bulk stock solution containing three phthalates (DEHP, DBP, and BBP) diluted in DMSO and two alkylphenols (NP and OP) diluted in ethanol was used to prepare the final mixture. The doses were freshly made and administered in the drinking water every other day according to the animal body weight (BW) and throughout the experimental period. The bottle was covered with foil to prevent the photolysis of the compounds. For both vehicle and treatments, the final DMSO and ethanol doses were 2.5 g/kg BW/d and 0.06 g/kg BW/d. In mice, orally tolerable doses have been established at 7.9 g/kg for DMSO and 2.5 g/kg for ethanol (40). To emulate chronic human exposure to an environmental mixture of EDCs, we administered the mixture of EDCs or vehicles (control) to pregnant mice from 0.5 postcoital day (biological n) during pregnancy and lactation. At weaning, only females were selected and maintained in a group of three to four individuals per cage. We continued the administration of the mixture of EDCs or control until adulthood (end point: postnatal day 60) (Fig. 1). Figure 1. View largeDownload slide Chronic mixture exposure model (exposome) on F1 female mice from conception to adulthood. Pregnant female mice (F0) and female F1 descendants were exposed to a mixture of three phthalates and two alkylphenols (DEHP, DBP, BBP, NP, and OP) or vehicle (DMSO and ethanol) in drinking water during all gestation periods and lactation (middle). Furthermore, F1 females were exposed from weaning to adulthood (60 days of age) when, at this point, treatment was suspended, fertility was assessed, and samples were collected for analyses. Figure 1. View largeDownload slide Chronic mixture exposure model (exposome) on F1 female mice from conception to adulthood. Pregnant female mice (F0) and female F1 descendants were exposed to a mixture of three phthalates and two alkylphenols (DEHP, DBP, BBP, NP, and OP) or vehicle (DMSO and ethanol) in drinking water during all gestation periods and lactation (middle). Furthermore, F1 females were exposed from weaning to adulthood (60 days of age) when, at this point, treatment was suspended, fertility was assessed, and samples were collected for analyses. Reproductive end points Reproductive end points assessed included age at vaginal opening, age of first estrous cycle, and estrous cycle length. Estrous cycles were determined via vaginal cytology followed by crystal violet staining (41) and recorded daily for 16-day spans since vaginal opening day. All cycles were measured daily between 08:00 and 10:00 hours. Fertility assay:estrous cycle was checked in adult females (60 days old), and those in proestrus were placed in a cage with an adult male (>90 days old) of proven fertility (i.e., it had fathered offspring at least once). To evaluate mating, vaginal plugs were checked the next day early in the morning (08:00 to 09:00 hours). All females with vaginal plugs were set apart and placed in a cage (four animals of the same experimental group) for 15 to 18 days. Then, females were separated in individual cages until delivery. Fertility in mice was assayed four times in the same animal, and the delivery rate was obtained by dividing the number of female mice that gave birth by the number of females that presented vaginal plugs. Litter size and sex ratio were recorded. Furthermore, pups were weighed at birth and 21 days old, and anogenital distance (AGD) and serum 17β-estradiol levels were measured at 21 days old. AGD was normalized to cubic root of BW to account for body size effects (42). Plasma hormonal levels At the end of the treatment, females (60 days old) in estrous were weighed and anesthetized by intraperitoneal administration of ketamine/xylazine (80 and 8 mg/kg, respectively). Then, blood samples were collected via cardiac puncture, clotted, and then centrifuged at 2300 g at 4°C for 10 minutes to collect serum. Finally, samples were stored at −80°C until measured. Serum progesterone, testosterone, and 17β-estradiol were measured in duplicate at the Pontificia Universidad Católica de Chile radioimmunoassay service. The sensitivity and intra- or interassay coefficients of variation (CVs) for the progesterone assay were 154 pg/mL and CVs <5.2 and <8.3, respectively; for the testosterone assay, they were 128 pg/mL and CVs <5.8 and <10.6, respectively; and those for the 17β-estradiol assay were 34 pg/mL and CVs <8.4 and <14.4, respectively. Tissue collection and body and organ weights After blood collection, females were euthanized by cervical dislocation, and uterus and ovaries were aseptically removed, cleaned of interstitial tissue, and weighed. Relative organ weights were calculated by dividing the weight of the uterus or ovaries by the body weight at the euthanasia time. Body and organ weights were recorded in grams. Histological evaluation of the number of preantral and antral follicles For this evaluation, collected ovaries were fixed in Bouin’s solution (Sigma-Aldrich). Then, samples were dehydrated and embedded in paraffin, and serial sections of each ovary were cut (8 μm) using a microtome followed by mounting onto glass slides and stained with the hematoxylin/eosin protocol. Finally, samples were rehydrated and mounted. For follicle counting, from each set of serial sections, four sections were counted for each ovary (the interval between sections was approximately 100 μm apart to avoid counting follicles twice) (43). Pictures were taken under a CX31 microscope (Olympus, Japan) with a Mshot camera (Guangzhou, China). The count of follicles and corpora lutea was based on previously defined criteria (44, 45). Briefly, preantral follicles contain an oocyte surrounded by at least two layers of cuboidal granulosa and theca cells, whereas antral follicles contain an oocyte surrounded by multiple layers of cuboidal granulosa cells with fluid-filled antral space and theca cells. Corpora lutea were identified by the hypertrophic aspect and large volume of cells. RNA extraction and real-time polymerase chain reaction Total RNA was isolated from ovaries that were collected in estrous using TRIzol Reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s recommendations. The quantity and integrity of total RNA were determined by a Thermo Scientific Nanodrop 2000 spectrophotometer. Real-time reverse transcription was used to verify gene expression using an Applied Biosystems 7500 thermal cycler. Complementary DNA was generated from 1 μg RNA using random primers and M-MLV Reverse transcription (Promega, WI), as well as after 1 μL complementary DNA was used for a 10-μL polymerase chain reaction reaction mixture containing 1 μM of primers (see sequences in Supplemental Table 2) and 2× SyBR Green Fast qPCR Master Mix (Biotool, TX). Polymerase chain reaction was carried out over 40 cycles at 95°C for 3 seconds and 60°C for 15 seconds. Expression data were normalized using the 2−∆∆Ct method (46) with Gapdh and β-actin as endogenous reference genes. Protein extraction and Western blotting Protein extraction was performed by homogenizing ovaries (collected in estrous) in radioimmunoprecipitation assay buffer, adding a protease inhibitor cocktail (Sigma) and a phosphatase inhibitor cocktail with 2 mM 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride, 0.3 μM aprotinin, 130 μM bestatin hydrochloride, 14 μM E-64, 1 mM EDTA, and 1 μM leupeptin hemisulfate. Proteins were purified by centrifugation at 12,000 × g at 4°C for 10 minutes and subsequently quantified. After that, 20 μg of proteins was separated by electrophoresis on a 10% polyacrylamide gel (sodium dodecyl sulfate–polyacrylamide gel electrophoresis) under denaturing and reducing conditions and then transferred to a nitrocellulose membrane (Thermo Scientific) at 350 mA for 2 hours. Next, membranes were blocked with a solution of 3% (w/v) nonfat milk or bovine serum albumin 0.1% (v/v) Tween in Tris-buffered saline, pH 7.4, and incubated overnight with the respective primary antibodies for CYP19A1, LHCGR, and STAR (Abbexa, Cambridge, UK); FSHR and androgen receptor (AR) (Santa Cruz Biotechnology, TX); and B-TUBULIN (Sigma) as a loading control (Table 1). Finally, a second incubation took place with their respective secondary antibodies conjugated with horseradish peroxidase (KPL, Gaithersburg, MD) diluted 1:5000 in a blocking solution for 1 hour at room temperature. Peroxidase activity was detected by enhanced chemiluminescence (Pierce Biotechnology, Rockford, IL). Table 1. Antibodies Protein Target  Antigen Sequence  Name of Antibody  Manufacturer, Catalog No., and RRID  Raised Species  Dilution Used (μg/mL)  FSHR  UniProt P35378  FSHR (N-20) antibody  Santa Cruz Biotechnology, sc-7798, RRID: AB_2294263  Goat; polyclonal  1  LHCGR  UniProt P30730  LHCGR antibody  Abbexa, abx113542, RRID: AB_2665489  Rabbit; polyclonal  0.063  STAR  UniProt P51557  STAR antibody  Abbexa, abx000970, RRID: AB_2665490  Rabbit; polyclonal  0.32  CYP19A1  UniProt P28649  CYP19A1 antibody  Abbexa, abx001773, RRID: AB_2665491  Rabbit; polyclonal  0.43  AR  UniProt P19091  AR (N-20) antibody  Santa Cruz Biotechnology, sc-816, RRID: AB_1563391  Rabbit; polyclonal  0.125  B-TUBULIN  UniProt Q9ERD7  BETA-TUBULIN antibody  Innovative Research, 32-2600, RRID: AB_86547  Mouse; monoclonal  0.1  Protein Target  Antigen Sequence  Name of Antibody  Manufacturer, Catalog No., and RRID  Raised Species  Dilution Used (μg/mL)  FSHR  UniProt P35378  FSHR (N-20) antibody  Santa Cruz Biotechnology, sc-7798, RRID: AB_2294263  Goat; polyclonal  1  LHCGR  UniProt P30730  LHCGR antibody  Abbexa, abx113542, RRID: AB_2665489  Rabbit; polyclonal  0.063  STAR  UniProt P51557  STAR antibody  Abbexa, abx000970, RRID: AB_2665490  Rabbit; polyclonal  0.32  CYP19A1  UniProt P28649  CYP19A1 antibody  Abbexa, abx001773, RRID: AB_2665491  Rabbit; polyclonal  0.43  AR  UniProt P19091  AR (N-20) antibody  Santa Cruz Biotechnology, sc-816, RRID: AB_1563391  Rabbit; polyclonal  0.125  B-TUBULIN  UniProt Q9ERD7  BETA-TUBULIN antibody  Innovative Research, 32-2600, RRID: AB_86547  Mouse; monoclonal  0.1  Abbreviation: RRID, Research Resource Identifier. View Large Statistical analysis To evaluate if the data were normally distributed, the D’Agostino and Pearson omnibus normality test of GraphPad Prism 5 tests (GraphPad Software, San Diego, CA) was used. For mean comparisons, we used analysis of variance to conduct multiple comparisons between normally distributed experimental groups, followed by Tukey posttest to discriminate groups. Kruskal-Wallis test followed by the Dunn posttest was used for comparison between groups if data were not normally distributed. Statistical significance was defined as P < 0.05. Results Exposure to a mixture of EDC changes in F1 female pubertal outcomes First, we determined if the chosen doses were lethal or had unwanted side effects during pregnancy. Data showed that the increase of BW during pregnancy was similar in females exposed to both doses of the mixture (1 or 10 mg/kg/d) compared with controls (females only exposed to vehicle) (Supplemental Fig. 1A). In addition, exposure to the mixture of EDCs did not change gestational length, the number of litter size, or the average of BWs at birth of the F1 generation compared with the control group (Supplemental Fig. 1B–1D). Therefore, exposure of pregnant dams to the 1- or 10-mg/kg mixture of EDCs did not affect general pregnancy parameters. Next, we evaluated F1 female parameters related to sexual development. We found that F1 females exposed to the 1-mg/kg/d mixture of EDCs showed a substantial average delay of 5 days in vaginal opening compared with control (Fig. 2B and 2E). Interestingly, in the same animals, we detected an increase of the presence of a vaginal thread, a thick cord of mesenchymal tissue surrounded by epithelial cells that cross the vaginal opening (47, 48) (Fig. 2A and 2C). On the contrary, females exposed to the 10-mg/kg/d dose did not show any representative delay in vaginal opening or a vaginal thread (Fig. 2A–2C and 2E). Moreover, female mice exposed to the 1-mg/kg/d mixture of EDCs showed an important delay of 6.5 days in the first estrous, but those exposed to the 10-mg/kg/d mixture were similar to control (Fig. 2D and 2F). Once they reached the first estrous, female mice exposed to vehicle showed a characteristic 4- to 5-day estrous cyclicity (Fig. 2G, top panel). However, those exposed to the mixture of EDCs, regardless of the dose, had a decrease in the number of cycles recorded over the studied period (16 days), along with an increase in the number of days expended at the estrous phase and decrease of the other phases (Fig. 2G–2I). Overall, these data suggest that chronic exposure to a mixture of phthalates and alkylphenols modifies, in a dose-dependent manner, female mouse sexual maturation (vaginal opening, age of first estrous, and estrous cyclicity), suggesting changes in ovarian function and probably fertility. Figure 2. View largeDownload slide The mixture of phthalates and alkylphenols delays the onset of puberty and induces lengthened and irregular estrous. (A) Vaginal thread phenotype (white arrow) was increased in females treated with the 1-mg/kg/d dose (C) but not in those with the 10-mg/kg/d dose. (B, E) Vaginal opening and (D, F) first estrous age delay were observed only in female mice exposed to 1 mg/kg/d compared with control. The estrous cycle was evaluated by vaginal smear for a period of 16 days (from vaginal opening day) in the offspring of females exposed to the mixture (1 mg/kg/d and 10 mg/kg/d) or control. (G) Representative estrous cycles were measured by vaginal smear of control (top) and mixture of 1 mg/kg/d (middle) and 10 mg/kg/d (bottom). Note persistence in estrous in both treatments compared with the control. (H) A decrease in the number of estrous cycles in treatment groups compared with control can be observed. (I) An alteration was detected in the number of days in each stage of the cycle with an increase in the number of days in estrous in exposed animals compared with control. The mean ± standard error of the mean values are shown. n ≥ 4, *P < 0.05. M + D, metestrus and diestrus. Figure 2. View largeDownload slide The mixture of phthalates and alkylphenols delays the onset of puberty and induces lengthened and irregular estrous. (A) Vaginal thread phenotype (white arrow) was increased in females treated with the 1-mg/kg/d dose (C) but not in those with the 10-mg/kg/d dose. (B, E) Vaginal opening and (D, F) first estrous age delay were observed only in female mice exposed to 1 mg/kg/d compared with control. The estrous cycle was evaluated by vaginal smear for a period of 16 days (from vaginal opening day) in the offspring of females exposed to the mixture (1 mg/kg/d and 10 mg/kg/d) or control. (G) Representative estrous cycles were measured by vaginal smear of control (top) and mixture of 1 mg/kg/d (middle) and 10 mg/kg/d (bottom). Note persistence in estrous in both treatments compared with the control. (H) A decrease in the number of estrous cycles in treatment groups compared with control can be observed. (I) An alteration was detected in the number of days in each stage of the cycle with an increase in the number of days in estrous in exposed animals compared with control. The mean ± standard error of the mean values are shown. n ≥ 4, *P < 0.05. M + D, metestrus and diestrus. Effect of exposure to a mixture of EDCs in ovarian function of adult females First, we recorded ovarian and uterus weight in adult females exposed to the mixture of EDCs (60 days). Results showed that the uterus relative weight was higher in exposed females compared with control, but the opposite (lower relative weight) was observed in ovaries (Fig. 3A–3D). On the one hand, the quantification of follicle types in the whole ovary showed that females treated with the 1-mg/kg/d dose had a substantial increase in the number of preantral follicles (Fig. 3E) and a decrease in the number of antral follicles and corpora lutea (Fig. 3F and 3G) compared with control. On the other hand, in females treated with the 10-mg/kg/d dose, a decrease in the number of antral follicles was detected (Fig. 3F), and there were no changes in the number of preantral follicles or corpora lutea compared with control (Fig. 3E and 3G). These data suggest that a low dose of mixture of EDCs (1 mg/kg/d) reduced the progression of follicles to the antral stage, whereas a high dose had an effect on the antral stage. Figure 3. View largeDownload slide The exposure to a mixture of EDCs alters the reproductive organs and ovary folliculogenesis in mice. Graphs represent the relative weight of ovaries and uterus from exposed adult females (age 60 days). (A) A representative picture of a control uterus (left) and mixture of 1 mg/kg/d (middle) and 10 mg/kg/d (right). Both doses of the mixture (A, B) increased the relative weight of the uterus and (C, D) decreased the ovary compared with the control. The number of ovarian follicles of exposed females and control was evaluated by histology. (D) A representative picture of a control ovary (left) and mixture of 1 mg/kg/d (middle) and 10 mg/kg/d (right), where preantral follicles (arrowhead), antral follicles (arrow), and corpora lutea (asterisk) can be seen. An increase was observed in the number of (E) preantral follicles and a decrease in the number of (F) antral follicles and (G) corpora lutea in exposed females compared with the control. The mean ± standard error of the mean values are shown. n ≥ 4, *P < 0.05. The bar corresponds to 500 μm at ×40 magnification. Figure 3. View largeDownload slide The exposure to a mixture of EDCs alters the reproductive organs and ovary folliculogenesis in mice. Graphs represent the relative weight of ovaries and uterus from exposed adult females (age 60 days). (A) A representative picture of a control uterus (left) and mixture of 1 mg/kg/d (middle) and 10 mg/kg/d (right). Both doses of the mixture (A, B) increased the relative weight of the uterus and (C, D) decreased the ovary compared with the control. The number of ovarian follicles of exposed females and control was evaluated by histology. (D) A representative picture of a control ovary (left) and mixture of 1 mg/kg/d (middle) and 10 mg/kg/d (right), where preantral follicles (arrowhead), antral follicles (arrow), and corpora lutea (asterisk) can be seen. An increase was observed in the number of (E) preantral follicles and a decrease in the number of (F) antral follicles and (G) corpora lutea in exposed females compared with the control. The mean ± standard error of the mean values are shown. n ≥ 4, *P < 0.05. The bar corresponds to 500 μm at ×40 magnification. Females exposed to a phthalate and alkylphenol mixture present changes in the levels of steroidal sex hormones Next, we investigated whether the impairment of reproductive parameters observed in exposed females was related to changes in reproductive hormones. Our data showed that plasma levels of progesterone and 17β-estradiol, but not testosterone, were significantly reduced in adult female mice exposed to both doses of the mixture of EDCs compared with control (Fig. 4A–4C). Figure 4. View largeDownload slide The mixture of phthalates and alkylphenols alters levels of steroid hormones. Levels of 17β-estradiol, progesterone, and testosterone measured by radioimmunoassay in blood of adult female mice exposed to the mixture and control. A decrease in the plasma levels of (A) progesterone and (C) 17β-estradiol in adult female treatment groups compared with control was observed. (B) No changes were observed in testosterone plasmatic levels. The mean ± standard error of the mean values are shown. n ≥ 4, *P < 0.05. Figure 4. View largeDownload slide The mixture of phthalates and alkylphenols alters levels of steroid hormones. Levels of 17β-estradiol, progesterone, and testosterone measured by radioimmunoassay in blood of adult female mice exposed to the mixture and control. A decrease in the plasma levels of (A) progesterone and (C) 17β-estradiol in adult female treatment groups compared with control was observed. (B) No changes were observed in testosterone plasmatic levels. The mean ± standard error of the mean values are shown. n ≥ 4, *P < 0.05. Later, to explain the changes in plasma progesterone and 17β-estradiol levels, we evaluated whether the mRNA or protein levels of the receptors and enzymes involved in biosynthesis of these steroids were altered in the ovaries of exposed females. Results showed that LHCGR and FSHR mRNAs and protein levels solely decreased in ovaries of females exposed to the 10-mg/kg/d dose compared with control (Fig. 5A, 5B, 5F, and 5G). In addition, we found that the mRNA (Fig. 5C) and protein levels (Fig. 5H) of STAR were significantly lower in ovaries of exposed mice to both doses of the mixture of EDCs compared with control. Furthermore, we evaluated Cyp17a1 mRNA implicated in 17α-hydroxylase and 17,20-lyase activities, as well as Hsd17b implicated in the reduction and dehydrogenation of 17-ketosteroids and 17β-hydroxysteroids, respectively. Results showed an increase of the mRNA levels of Cyp17a1 in ovaries of mice exposed to both doses, whereas mRNA levels of Hsd17b were similar to control (Supplemental Fig. 2A and 2B). Similarly, the mRNA and protein levels of CYP19A (aromatase) were found diminished in ovaries of mice exposed to both doses of the mixture (Fig. 5D and 5I). Interestingly, on one hand, the mRNA and protein levels of the AR significantly decreased only in those females treated with the 10-mg/kg/d dose (Fig. 5E and 5J). On the other hand, the mRNA levels of the estrogen receptor (Esr1) were significantly reduced in ovaries of mice exposed to both doses (Supplemental Fig. 2C). Therefore, changes in the plasma levels of 17β-estradiol and progesterone could be due to alterations in the mRNA that is responsible for encoding the enzymes involved in the steroidoigenic pathway and cholesterol transport. Figure 5. View largeDownload slide The mixture of phthalates and alkylphenols impairs the steroidogenic enzyme levels. LHCGR, FSHR, STAR, CYP19A1, and AR mRNA and protein levels were determined by real-time polymerase chain reaction and normalized to b-actin and Gapdh or by Western blot and normalized with B-tubulin in ovaries of exposed and control females. A decrease in the (C, H) STAR and (D, I) CYP19A1 levels in the exposed groups compared with control was observed. (A, F) LHCGR, (B, G) FSHR, and (E, J) AR were decreased only at the 10-mg/kg/d dose. mean ± standard error of the mean values are shown. n ≥ 4, *P < 0.05. Figure 5. View largeDownload slide The mixture of phthalates and alkylphenols impairs the steroidogenic enzyme levels. LHCGR, FSHR, STAR, CYP19A1, and AR mRNA and protein levels were determined by real-time polymerase chain reaction and normalized to b-actin and Gapdh or by Western blot and normalized with B-tubulin in ovaries of exposed and control females. A decrease in the (C, H) STAR and (D, I) CYP19A1 levels in the exposed groups compared with control was observed. (A, F) LHCGR, (B, G) FSHR, and (E, J) AR were decreased only at the 10-mg/kg/d dose. mean ± standard error of the mean values are shown. n ≥ 4, *P < 0.05. Phthalate and alkylphenol mixture altered fertility of exposed female mice Given the above data, we wondered whether the exposure to the mixture of EDCs induced fertility problems in adult female mice (60 days old) or alterations in their offspring. Control and exposed adult females were left in a cage with a nonexposed adult male (>90 days old) of proven fertility. Data showed that control and exposed females had evidence of copulation (vaginal plug), but regardless the dose, a slight decrease in the delivery rate was observed in exposed females (Table 2). The gestational length was shortened at both doses, and there was a decrease in litter size and an increase in pups’ mortality (F2 generation) of F1 female mice exposed to 1 mg/kg/d but not to 10 mg/kg/d. Furthermore, BW of pups (F2 offspring) was significantly reduced in both treatments (∼10%), and this reduction in weight was even more pronounced at 21 days old (∼17%) only in F2 offspring of female mice exposed to 1 mg/kg/d (Table 2). To predict neonatal and adult reproductive disorder in F2 offspring, we determined the AGD. Males aged 21 days exposed to 1 mg/kg/d showed a decrease in AGD compared with control mice. However, the female AGD was significantly reduced in pups born from females exposed to both doses of the mixture of EDCs (Table 2). In the same manner, a decrease of 17β-estradiol plasma levels was observed in females at 21 days of age (Table 2). All these data indicate that fertility in mice exposed to the mixture of EDCs is compromised, and there is a generational effect in the offspring. Table 2. Reproductive Outcome of F1 Female Mice Treated With the Mixture of EDCs and Morphological Indices of Offspring (F2) Exposed Female Mice/Parameter  Control  1 mg/kg/d  10 mg/kg/d  F1         No. of females (4 repeats)  5  5  5   Positive plug (%)  100  100  100   Gestation length (d)  20.1 ± 0.24  19.3 ± 0.21a  19.3 ± 0.13a   Delivery (%)  100  80  90   Litter size (n)  8.22 ± 0.49  6.44 ± 0.38a  6.56 ± 0.56  F2         Viability index (%)  91.54  72.62a  82.45   Body weight at birth (g)  1.33 ± 0.02  1.19 ± 0.01a  1.20 ± 0.04a   Female body weight at PND 21 (g)  8.35 ± 0.20  6.95 ± 0.38a  7.21 ± 0.42   Female AGD at PND 21 (mm/g1/3)  4.33 ± 0.14  3.28 ± 0.24a  3.39 ± 0.21a   Male AGD at PND 21 (mm/g1/3)  6.79 ± 0.34  5.48 ± 0.33a  6.36 ± 0.47   Female 17β-estradiol levels at PND 21 (pg/mL)  91.03 ± 6.03  55.70 ± 4.79a  58.47 ± 6.33a  Exposed Female Mice/Parameter  Control  1 mg/kg/d  10 mg/kg/d  F1         No. of females (4 repeats)  5  5  5   Positive plug (%)  100  100  100   Gestation length (d)  20.1 ± 0.24  19.3 ± 0.21a  19.3 ± 0.13a   Delivery (%)  100  80  90   Litter size (n)  8.22 ± 0.49  6.44 ± 0.38a  6.56 ± 0.56  F2         Viability index (%)  91.54  72.62a  82.45   Body weight at birth (g)  1.33 ± 0.02  1.19 ± 0.01a  1.20 ± 0.04a   Female body weight at PND 21 (g)  8.35 ± 0.20  6.95 ± 0.38a  7.21 ± 0.42   Female AGD at PND 21 (mm/g1/3)  4.33 ± 0.14  3.28 ± 0.24a  3.39 ± 0.21a   Male AGD at PND 21 (mm/g1/3)  6.79 ± 0.34  5.48 ± 0.33a  6.36 ± 0.47   Female 17β-estradiol levels at PND 21 (pg/mL)  91.03 ± 6.03  55.70 ± 4.79a  58.47 ± 6.33a  Values are mean ± standard error of the mean, n ≥ 4. Each female mated four times with an unexposed fertile male. Delivery: (number of females with litter/number of females with vaginal plug) × 100. Viability index: (number of pups at weaning/number of pups alive on PND 4) × 100. Bold text indicates values significantly different from control. Abbreviations: PND, postnatal day; SE, standard error. a P < 0.05. View Large Discussion In this study, we show that chronic exposure to a mixture of phthalates and alkylphenols at low doses relative to levels of human exposure deregulates levels of progesterone and 17β-estradiol in female mice by changing the expression of enzymes involved in their synthesis and modifying follicle progression and estrous cyclicity. Interestingly, exposure to the lowest mixture of EDCs (1 mg/kg/d) resulted in a more deleterious phenotype, showing additional effects in the delay of puberty and decreased fertility. Data presented in this work indicate that the mixture containing phthalates and alkylphenols did not cause gestational difficulties in F0 females because exposed dams successfully gave birth to live litters comparable to controls. This indicates that the phthalate and alkylphenol mixture exposure did not cause gestational damage or fetal toxicity. Epidemiological data of different cohorts have shown changes in the timing of puberty, thelarche, and menarche associated with the exposure to phthalates and phenols (49, 50). A precocious puberty or a delay in puberty can result in diseases such as infertility, obesity, and cancer. However, the epidemiological studies fail to demonstrate a direct association between EDC exposure and changes in the reproductive parameters. Data of the present work show that chronic exposure to 1 mg/kg/d of phthalates and alkylphenols delays the vaginal opening and first estrous, which are external signs of puberty in female mice (51). The onset of puberty is related to the beginning of the secretion of gonadotropin-releasing hormone (GnRH) and the activation of the hypothalamus-pituitary-gonadal axis (52), and previous studies have shown that exposure to single phthalates altered the levels of GnRH in the hypothalamus and its receptor in the pituitary (53, 54), thus changing the production of gonadotropins and puberty onset. Further studies should be done to determine the levels of GnRH, FSH, and luteinizing hormone in mice exposed to both doses of the mixture of EDCs to decipher the participation of hypothalamus-pituitary-ovarian axis in this process and regulate puberty in humans and animal models. In addition, we observed prolonged estrous cycles in female mice exposed to both doses of the mixture, which is another sign of infertility problems in the adult. This alteration in the estrous cycle may be due to either an indirect consequence of hormonal alterations observed in the exposed mice or a direct action of the mixture on the uterus and/or the vagina. Previous works have shown that exposure to a low or high dose of single phthalates and alkylphenols is related to disruption in the estrous cyclicity (55–58). In this sense, our work shows the same results as a previous one where females were exposed to a mixture of six phthalates from gestational day 10 until birth (27). Because the desquamation of the cells observed during the estrous phase is induced by an increase of 17β-estradiol levels, and our data indicate that in treated females, there is a decrease of this hormone in plasma, it is probable that what is observed as persistence in estrous in the treated females is no more than an estrogenic and direct effect of the mixture on the vaginal epithelium and not a consequence of the disruption of the hypothalamic-pituitary-ovary axis. This same hypothesis could also explain the observed increase in uterine size, which was also seen in a previous study with a mixture of phthalates at a dose similar to that of the current study (27). In addition, both NP and OP, two of the compounds contained in the mixture, induce uterine growth in vivo (22, 59). In the current study, we observed a reduction in the ovary weight, which could be explained as due to a reduction in the number of total antral follicles in mice exposed to both doses. One possible explanation, on one hand, is that as a result of lower levels of Esr1 mRNA (and probably lower levels at the protein level), the proliferation signaling cascade elicited by this receptor is not turned on in these mice, and therefore the granulosa cells in the follicle do not increase in number and block the antrum formation. On the other hand, because androgen signaling is crucial to follicle development (60), and phthalates and alkylphenols have been reported to bind to AR (61), it is possible that they also block the signaling event elicited by this receptor, hampering follicle development and decreasing ovary size. Interestingly, exposure to the 10-mg/kg/d dose did not alter preantral follicle numbers but decreased antral follicle numbers, which differs from previous studies where single exposure to DEHP or NP modified the number of preantral follicles (56, 62, 63). This difference could be due to the exposure time or that previous works have been done with single compounds and not a mixture of phthalates and alkylphenols like the present work. The results of the current study are similar to those reported in female mice lacking the AR (ARKO), with a decrease in the antral follicle numbers but without changes in the preantral follicle numbers (64). In ovary, AR signaling induces the activation of the insulin pathway via PI3K, promoting the expression of the steroidogenic enzymes such as CYP19A1 (65), which acts cooperatively with gonadotropins to induce steroidogenesis by an increase in the number of FSHR and LHCGR (66, 67). Here we observed a decrease in the expression of the AR at the mRNA and protein levels, which could account for the observed effects of these mice and explain the similarity with the ARKO model. In this way, the reduced Ar mRNA expression could explain the observed decrease in FSHR, LHCGR, and CYP19A1 at the mRNA and protein levels. One possible mechanism, on one hand, is that this mixture induces alterations in insulin signaling upstream of the steroidogenesis due to an Ar mRNA decrease, which mimics the phenotype of the ARKO mouse (64). Thus, a reduction in Ar mRNA results in decreased levels of the AR, insulin signaling, steroidogenesis, and loss of antral follicle probably due to an increase in apoptosis (60). Also, it is feasible that the reduction of the Ar mRNA levels is due to a disruption in the expression levels of the promoter because genomic and epigenomic mutations have been found in both mice and rats exposed to phthalates (25, 68). On the other hand, the 1-mg/kg/d mixture inhibits and/or reduces the progression of the preantral to antral follicle, which is consistent with a previous report in pregnant mice exposed to 5 mg/kg/d DEHP (69). Furthermore, in mice exposed to the 1-mg/kg/d mixture, we observed a decrease in corpora lutea, suggesting a failure in the progression of folliculogenesis. Previous studies have shown that DEHP exposure decreases the number of antral follicles by increasing atresia and apoptosis of granulosa cells (23). However, in the present work, this decline in antral follicles could be explained by a reduction in the preantral-to-antral transition rather than increasing atresia or apoptosis. On one hand, female mice exposed to the 1-mg/kg/d mixture did not show a decrease in mRNA or protein levels of FSHR and LHCGR, suggesting that preantral follicles are fully responding to gonadotropin stimulus. On the other hand, we observed a decrease in progesterone and 17β-estradiol plasma levels and also the expression of mRNAs and proteins levels of STAR and CYP19A1. Interestingly, we also observed an increment in the levels on Cyp17a1, which could be a kind of compensatory mechanism to maintain the levels of androstenedione while not affecting testosterone levels. Because 17β-estradiol is key to promote follicle growth, it is possible that the decrease in the levels of this hormone is due to a reduction in the Cyp19a1 mRNA expression levels. This finding is in agreement with previous studies showing a decrease in the levels of Cyp19a1 mRNA in female mice exposed to DEHP (58, 69, 70). Alternatively, it is possible that the mixture impairs LHCGR signaling and/or the expression of cell cycle cyclins, thus impairing follicle growth, which is similar to the effect of elevated concentrations of FSH in the prepuberal ovary (71). Therefore, further studies are required to determine the mechanism by which this mixture of phthalates and alkylphenols reduces the levels of CYP19A1 and in this manner possibly controls the preantral-to-antral follicle transition. In the present work, we reveal a decrease in fertility in females exposed to a mixture of phthalates and alkylphenols (F1-exposed generations), which agrees with previous reports showing that chronic occupational exposure in women to single phthalates is associated with decreased rates of pregnancy and high rates of miscarriage (72, 73). In exposed female mice, a decrease in the fertility (delivery percent) was more noticeable with the lowest dose (Table 2), and more interestingly, female offspring (F2 generations) exposed to both doses of the mixture of EDCs presented a decrease in anogenital distance and in the 17β-estradiol serum levels, which is a marker of endocrine disruption in the offspring and multigenerational effects (24). Therefore, these data suggest that a mixture of EDCs can alter germline epigenetic programming in a similar way reported by single exposure to phthalates and alkylphenols (25, 69). In addition, it shows that chronic exposure to EDCs induces changes in fertility that may a pose risk to the development of other pathologies such as cancer. In summary, our data demonstrate that chronic exposure to a mixture of phthalates and alkylphenols at an environmentally relevant dose, from prenatal to adult life in female mice (the exposome paradigm), induces changes in the steroidogenesis pathway and estrous cyclicity and is dose dependent of the levels of the gonadotropin receptor, follicle development, puberty onset, and fertility. These results indicate that not only exposure but also its level is relevant to assess the effective contribution of EDCs in the development of diseases. Abbreviations: AGD anogenital distance AR androgen receptor ARKO female mice lacking the androgen receptor BBP benzyl butyl phthalate BW body weight CV coefficient of variation DBP dibutyl phthalate DEHP bis (2-ethylhexyl) phthalate DMSO dimethyl sulfoxide EDC endocrine-disrupting chemical FSH follicle-stimulating hormone FSHR follicle-stimulating hormone receptor GnRH gonadotropin-releasing hormone LHCGR luteinizing hormone cognate receptor mRNA messenger RNA NP 4-nonylphenol OP 4-tert-octylphenol STAR acute steroidogenic regulatory protein. Acknowledgments Financial Support: This work was supported by FONDECYT (Grant 1150352 to R.D.M.) and CONICYT (Grant 63140090 to D.P.-G.), Chile. Disclosure Summary: The authors have nothing to disclose. References 1. Mascarenhas MN, Flaxman SR, Boerma T, Vanderpoel S, Stevens GA. National, regional, and global trends in infertility prevalence since 1990: a systematic analysis of 277 health surveys. PLoS Med . 2012; 9( 12): e1001356. Google Scholar CrossRef Search ADS PubMed  2. Healy DL, Trounson AO, Andersen AN. Female infertility: causes and treatment. Lancet . 1994; 343( 8912): 1539– 1544. Google Scholar CrossRef Search ADS PubMed  3. Marques-Pinto A, Carvalho D. Human infertility: are endocrine disruptors to blame? Endocr Connect . 2013; 2( 3): R15– R29. Google Scholar CrossRef Search ADS PubMed  4. Penning TM. Human aldo-keto reductases and the metabolic activation of polycyclic aromatic hydrocarbons. Chem Res Toxicol . 2014; 27( 11): 1901– 1917. Google Scholar CrossRef Search ADS PubMed  5. Chin-Chan M, Navarro-Yepes J, Quintanilla-Vega B. Environmental pollutants as risk factors for neurodegenerative disorders: Alzheimer and Parkinson diseases. Front Cell Neurosci . 2015; 9: 124. Google Scholar CrossRef Search ADS PubMed  6. Fenner-Crisp PA, Maciorowski AF, Timm GE. The Endocrine Disruptor Screening Program developed by the U.S. Environmental Protection Agency. Ecotoxicology . 2000; 9( 1/2): 85– 91. Google Scholar CrossRef Search ADS   7. World Health Organization. United Nations Environment Programme (WHO-UNEP). In: Bergman A, Heindel JJ, Jobling S, Kidd KA, Zoeller RT, eds. State of the Science of Endocrine Disrupting Chemicals—2012. Geneva, Switzerland: World Health Organization and United Nations Environment Programme. Available at: http://www.who.int/ceh/publications/endocrine/en/index.html. Accessed 23 June 2017. 8. De Coster S, van Larebeke N. Endocrine-disrupting chemicals: associated disorders and mechanisms of action. J Environ Public Health . 2012; 2012: 713696. Google Scholar CrossRef Search ADS PubMed  9. Sathyanarayana S, Butts S, Wang C, Barrett E, Nguyen R, Schwartz SM, Haaland W, Swan SH; TIDES Team. Early prenatal phthalate exposure, sex steroid hormones, and birth outcomes. J Clin Endocrinol Metab . 2017; 102( 6): 1870– 1878. Google Scholar CrossRef Search ADS PubMed  10. Diamanti-Kandarakis E, Bourguignon JP, Giudice LC, Hauser R, Prins GS, Soto AM, Zoeller RT, Gore AC. Endocrine-disrupting chemicals: an Endocrine Society scientific statement. Endocr Rev . 2009; 30( 4): 293– 342. Google Scholar CrossRef Search ADS PubMed  11. Lagos-Cabré R, Moreno RD. Contribution of environmental pollutants to male infertily: a working model of germ cell apoptosis induced by plasticizers. Biol Res . 2012; 45( 1): 5– 14. Google Scholar CrossRef Search ADS PubMed  12. Li LX, Chen L, Meng XZ, Chen BH, Chen SQ, Zhao Y, Zhao LF, Liang Y, Zhang YH. Exposure levels of environmental endocrine disruptors in mother-newborn pairs in China and their placental transfer characteristics. PLoS One . 2013; 8( 5): e62526. Google Scholar CrossRef Search ADS PubMed  13. Du YY, Fang YL, Wang YX, Zeng Q, Guo N, Zhao H, Li YF. Follicular fluid and urinary concentrations of phthalate metabolites among infertile women and associations with in vitro fertilization parameters. Reprod Toxicol . 2016; 61: 142– 150. Google Scholar CrossRef Search ADS PubMed  14. Dieterle S. Analysis of toxins in follicle fluid from women with unfulfilled pregnancy. 2017. Available at: https://clinicaltrials.gov/show/NCT01385605. Accessed 23 June 2017. 15. Tranfo G, Caporossi L, Paci E, Aragona C, Romanzi D, De Carolis C, De Rosa M, Capanna S, Papaleo B, Pera A. Urinary phthalate monoesters concentration in couples with infertility problems. Toxicol Lett . 2012; 213( 1): 15– 20. Google Scholar CrossRef Search ADS PubMed  16. Messerlian C, Souter I, Gaskins AJ, Williams PL, Ford JB, Chiu YH, Calafat AM, Hauser R; Earth Study Team. Urinary phthalate metabolites and ovarian reserve among women seeking infertility care. Hum Reprod . 2015; 31( 1): 75– 83. Google Scholar CrossRef Search ADS PubMed  17. Mínguez-Alarcón L, Gaskins AJ. Female exposure to endocrine disrupting chemicals and fecundity: a review. Curr Opin Obstet Gynecol . 2017; 29( 4): 202– 211. Google Scholar CrossRef Search ADS PubMed  18. Drummond AE. The role of steroids in follicular growth. Reprod Biol Endocrinol . 2006; 4( 1): 16. Google Scholar CrossRef Search ADS PubMed  19. Huang Z, Wells D. Molecular aspects of follicular development. In: Donnez J, Kim SS, eds. Principles and Practice of Fertility Preservation . Cambridge, UK: Cambridge University Press; 2011: 114– 128. Google Scholar CrossRef Search ADS   20. Senthilkumaran B, Yoshikuni M, Nagahama Y. A shift in steroidogenesis occurring in ovarian follicles prior to oocyte maturation. Mol Cell Endocrinol . 2004; 215( 1–2): 11– 18. Google Scholar CrossRef Search ADS PubMed  21. Caras ML. Estrogenic modulation of auditory processing: a vertebrate comparison. Front Neuroendocrinol . 2013; 34( 4): 285– 299. Google Scholar CrossRef Search ADS PubMed  22. Yoshida M, Takenaka A, Katsuda S, Kurokawa Y, Maekawa A. Neonatal exposure to p-tert-octylphenol causes abnormal expression of estrogen receptor alpha and subsequent alteration of cell proliferating activity in the developing Donryu rat uterus. Toxicol Pathol . 2002; 30( 3): 357– 364. Google Scholar CrossRef Search ADS PubMed  23. Hannon PR, Flaws JA. The effects of phthalates on the ovary. Front Endocrinol (Lausanne) . 2015; 6: 8. Google Scholar PubMed  24. Zhou C, Gao L, Flaws JA. Exposure to an environmentally relevant phthalate mixture causes transgenerational effects on female reproduction in mice. Endocrinology . 2017; 158( 6): 1739– 1754. Google Scholar CrossRef Search ADS PubMed  25. Manikkam M, Tracey R, Guerrero-Bosagna C, Skinner MK. Plastics derived endocrine disruptors (BPA, DEHP and DBP) induce epigenetic transgenerational inheritance of obesity, reproductive disease and sperm epimutations. PLoS One . 2013; 8( 1): e55387. Google Scholar CrossRef Search ADS PubMed  26. Johansson HK, Jacobsen PR, Hass U, Svingen T, Vinggaard AM, Isling LK, Axelstad M, Christiansen S, Boberg J. Perinatal exposure to mixtures of endocrine disrupting chemicals reduces female rat follicle reserves and accelerates reproductive aging. Reprod Toxicol . 2016; 61: 186– 194. Google Scholar CrossRef Search ADS PubMed  27. Zhou C, Gao L, Flaws JA. Prenatal exposure to an environmentally relevant phthalate mixture disrupts reproduction in F1 female mice. Toxicol Appl Pharmacol . 2017; 318: 49– 57. Google Scholar CrossRef Search ADS PubMed  28. Agency for Toxic Substances and Disease Registry. Minimal risk levels (MRLs): US Department of Health and Human Services, Public Health Service, Atlanta, GA; 2017. Available at: https://www.atsdr.cdc.gov/mrls/index.asp. Accessed 23 June 2017. 29. Jonsson B. Risk assessment on butylphenol, octylphenol and nonylphenol, and estimated human exposure of alkylphenols from Swedish fish. Uppsala, Sweden: Ecological Department, Uppsala University; 2006: 1– 52. Available at: http://www.uu.se/digitalAssets/177/c_177024-l_3-k_jonsson-beatrice-report.pdf. 30. Somasundaram DB, Manokaran K, Selvanesan BC, Bhaskaran RS. Impact of di-(2-ethylhexyl) phthalate on the uterus of adult Wistar rats. Hum Exp Toxicol . 2016; 36( 6): 565– 572. Google Scholar CrossRef Search ADS PubMed  31. Agency for Toxic Substances and Disease Registry. Toxicological profile for di(2-ethylhexyl)phthalate. Department of Health and Human Services, Public Health Service, Atlanta, GA; 2002. Available at: https://www.atsdr.cdc.gov/toxprofiles/tp9.pdf. Accessed 23 June 2017. 32. Snedeker SM, Hay AG. The alkylphenols nonylphenol and octylphenol in food contact materials and household items: exposure and health risk considerations. In: Snedeker SM, ed. Toxicants in Food Packaging and Household Plastics . London, UK: Springer-Verlag; 2014: 125– 150. Google Scholar CrossRef Search ADS   33. Zero Breast Cancer. Phthalates: the everywhere chemical. San Rafael, CA: Zero Breast Cancer; 2014. Available at: https://www.niehs.nih.gov/research/supported/assets/docs/j_q/phthalates_the_everywhere_chemical_handout_508.pdf. Accessed 23 June 2017. 34. Food and Drug Administration. Safety assessment of di(2-Ethylhexyl)phthalate (DEHP) released from PVC medical devices. Rockville, MD: Center for Devices and Radiological Health; 2001. Available at: https://www.fda.gov/downloads/MedicalDevices/.../UCM080457.pdf. Accessed 23 June 2017. 35. National Toxicology Program. NTP-CERHR monograph on the potential human reproductive and developmental effects of butyl benzyl phthalate (BBP). NTP CERHR MON . 2003;( 5): i– III90. 36. Kavlock R, Barr D, Boekelheide K, Breslin W, Breysse P, Chapin R, Gaido K, Hodgson E, Marcus M, Shea K, Williams P. NTP-CERHR expert panel update on the reproductive and developmental toxicity of di(2-ethylhexyl) phthalate. Reprod Toxicol . 2006; 22( 3): 291– 399. Google Scholar CrossRef Search ADS PubMed  37. Hines CJ, Hopf NB, Deddens JA, Silva MJ, Calafat AM. Estimated daily intake of phthalates in occupationally exposed groups. J Expo Sci Environ Epidemiol . 2009; 21( 2): 133– 141. Google Scholar CrossRef Search ADS PubMed  38. Chapin RE, Delaney J, Wang Y, Lenning L, Davis B, Collins B, Mintz N, Wolfe G. The effects of 4-nonylphenol in rats: a multigeneration reproduction study. Toxicol Sci . 1999; 52( 1): 80– 91. Google Scholar CrossRef Search ADS PubMed  39. Osimitz TG, Droege W, Driver JH. Human risk assessment for nonylphenol. Hum Ecol Risk Assess . 2015; 21( 7): 1903– 1919. Google Scholar CrossRef Search ADS   40. Gad SC, Spainhour CB, Shoemake C, Pallman DR, Stricker-Krongrad A, Downing PA, Seals RE, Eagle LA, Polhamus K, Daly J. Tolerable levels of nonclinical vehicles and formulations used in studies by multiple routes in multiple species with notes on methods to improve utility. Int J Toxicol . 2016; 35( 2): 95– 178. Google Scholar CrossRef Search ADS PubMed  41. McLean AC, Valenzuela N, Fai S, Bennett SA. Performing vaginal lavage, crystal violet staining, and vaginal cytological evaluation for mouse estrous cycle staging identification. J Vis Exp . 2012;( 67): e4389. 42. Gallavan RH Jr, Holson JF, Stump DG, Knapp JF, Reynolds VL. Interpreting the toxicologic significance of alterations in anogenital distance: potential for confounding effects of progeny body weights. Reprod Toxicol . 1999; 13( 5): 383– 390. Google Scholar CrossRef Search ADS PubMed  43. Chen ZG, Luo LL, Xu JJ, Zhuang XL, Kong XX, Fu YC. Effects of plant polyphenols on ovarian follicular reserve in aging rats. Biochem Cell Biol . 2010; 88( 4): 737– 745. Google Scholar CrossRef Search ADS PubMed  44. Flaws JA, Doerr JK, Sipes IG, Hoyer PB. Destruction of preantral follicles in adult rats by 4-vinyl-1-cyclohexene diepoxide. Reprod Toxicol . 1994; 8( 6): 509– 514. Google Scholar CrossRef Search ADS PubMed  45. Murphy BD. Models of luteinization. Biol Reprod . 2000; 63( 1): 2– 11. Google Scholar CrossRef Search ADS PubMed  46. Rao X, Huang X, Zhou Z, Lin X. An improvement of the 2ˆ(−delta delta CT) method for quantitative real-time polymerase chain reaction data analysis. Biostat Bioinforma Biomath . 2013; 3( 3): 71– 85. Google Scholar PubMed  47. Flaws JA, Sommer RJ, Silbergeld EK, Peterson RE, Hirshfield AN. In utero and lactational exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induces genital dysmorphogenesis in the female rat. Toxicol Appl Pharmacol . 1997; 147( 2): 351– 362. Google Scholar CrossRef Search ADS PubMed  48. Hotchkiss AK, Lambright CS, Ostby JS, Parks-Saldutti L, Vandenbergh JG, Gray LE Jr. Prenatal testosterone exposure permanently masculinizes anogenital distance, nipple development, and reproductive tract morphology in female Sprague-Dawley rats. Toxicol Sci . 2007; 96( 2): 335– 345. Google Scholar CrossRef Search ADS PubMed  49. Wolff MS, Teitelbaum SL, Pinney SM, Windham G, Liao L, Biro F, Kushi LH, Erdmann C, Hiatt RA, Rybak ME, Calafat AM; Breast Cancer and Environment Research Centers. Investigation of relationships between urinary biomarkers of phytoestrogens, phthalates, and phenols and pubertal stages in girls. Environ Health Perspect . 2010; 118( 7): 1039– 1046. Google Scholar CrossRef Search ADS PubMed  50. Zhang Y, Cao Y, Shi H, Jiang X, Zhao Y, Fang X, Xie C. Could exposure to phthalates speed up or delay pubertal onset and development? A 1.5-year follow-up of a school-based population. Environ Int . 2015; 83: 41– 49. Google Scholar CrossRef Search ADS PubMed  51. Nelson JF, Karelus K, Felicio LS, Johnson TE. Genetic influences on the timing of puberty in mice. Biol Reprod . 1990; 42( 4): 649– 655. Google Scholar CrossRef Search ADS PubMed  52. Ojeda SR, Urbanski HF, Ahmed CE. The onset of female puberty: studies in the rat. Recent Prog Horm Res . 1986; 42: 385– 442. Google Scholar PubMed  53. Liu T, Li N, Zhu J, Yu G, Guo K, Zhou L, Zheng D, Qu X, Huang J, Chen X, Wang S, Ye L. Effects of di-(2-ethylhexyl) phthalate on the hypothalamus-pituitary-ovarian axis in adult female rats. Reprod Toxicol . 2014; 46: 141– 147. Google Scholar CrossRef Search ADS PubMed  54. Liu T, Jia Y, Zhou L, Wang Q, Sun D, Xu J, Wu J, Chen H, Xu F, Ye L. Effects of di-(2-ethylhexyl) phthalate on the hypothalamus-uterus in pubertal female rats. Int J Environ Res Public Health . 2016; 13( 11): E1130. Google Scholar CrossRef Search ADS PubMed  55. Davis BJ, Maronpot RR, Heindel JJ. Di-(2-ethylhexyl) phthalate suppresses estradiol and ovulation in cycling rats. Toxicol Appl Pharmacol . 1994; 128( 2): 216– 223. Google Scholar CrossRef Search ADS PubMed  56. Willoughby KN, Sarkar AJ, Boyadjieva NI, Sarkar DK. Neonatally administered tert-octylphenol affects onset of puberty and reproductive development in female rats. Endocrine . 2005; 26( 2): 161– 168. Google Scholar CrossRef Search ADS PubMed  57. Hannon PR, Peretz J, Flaws JA. Daily exposure to di(2-ethylhexyl) phthalate alters estrous cyclicity and accelerates primordial follicle recruitment potentially via dysregulation of the phosphatidylinositol 3-kinase signaling pathway in adult mice. Biol Reprod . 2014; 90( 6): 136. Google Scholar CrossRef Search ADS PubMed  58. Moyer B, Hixon ML. Reproductive effects in F1 adult females exposed in utero to moderate to high doses of mono-2-ethylhexylphthalate (MEHP). Reprod Toxicol . 2012; 34( 1): 43– 50. Google Scholar CrossRef Search ADS PubMed  59. Zhang W, Yang J, Wang J, Xia P, Xu Y, Jia H, Chen Y. Comparative studies on the increase of uterine weight and related mechanisms of cadmium and p-nonylphenol. Toxicology . 2007; 241( 1–2): 84– 91. Google Scholar CrossRef Search ADS PubMed  60. Sen A, Prizant H, Light A, Biswas A, Hayes E, Lee HJ, Barad D, Gleicher N, Hammes SR. Androgens regulate ovarian follicular development by increasing follicle stimulating hormone receptor and microRNA-125b expression. Proc Natl Acad Sci USA . 2014; 111( 8): 3008– 3013. Google Scholar CrossRef Search ADS PubMed  61. Luccio-Camelo DC, Prins GS. Disruption of androgen receptor signaling in males by environmental chemicals. J Steroid Biochem Mol Biol . 2011; 127( 1–2): 74– 82. Google Scholar CrossRef Search ADS PubMed  62. Zhang XF, Zhang LJ, Li L, Feng YN, Chen B, Ma JM, Huynh E, Shi QH, De Felici M, Shen W. Diethylhexyl phthalate exposure impairs follicular development and affects oocyte maturation in the mouse. Environ Mol Mutagen . 2013; 54( 5): 354– 361. Google Scholar CrossRef Search ADS PubMed  63. Xu C, Chen JA, Qiu Z, Zhao Q, Luo J, Yang L, Zeng H, Huang Y, Zhang L, Cao J, Shu W. Ovotoxicity and PPAR-mediated aromatase downregulation in female Sprague-Dawley rats following combined oral exposure to benzo[a]pyrene and di-(2-ethylhexyl) phthalate. Toxicol Lett . 2010; 199( 3): 323– 332. Google Scholar CrossRef Search ADS PubMed  64. Wang RS, Chang HY, Kao SH, Kao CH, Wu YC, Yeh S, Tzeng CR, Chang C. Abnormal mitochondrial function and impaired granulosa cell differentiation in androgen receptor knockout mice. Int J Mol Sci . 2015; 16( 5): 9831– 9849. Google Scholar CrossRef Search ADS PubMed  65. Luo W, Wiltbank MC. Distinct regulation by steroids of messenger RNAs for FSHR and CYP19A1 in bovine granulosa cells. Biol Reprod . 2006; 75( 2): 217– 225. Google Scholar CrossRef Search ADS PubMed  66. Zhou J, Kumar TR, Matzuk MM, Bondy C. Insulin-like growth factor I regulates gonadotropin responsiveness in the murine ovary. Mol Endocrinol . 1997; 11( 13): 1924– 1933. Google Scholar CrossRef Search ADS PubMed  67. Poretsky L, Cataldo NA, Rosenwaks Z, Giudice LC. The insulin-related ovarian regulatory system in health and disease. Endocr Rev . 1999; 20( 4): 535– 582. Google Scholar CrossRef Search ADS PubMed  68. Huang XF, Li Y, Gu YH, Liu M, Xu Y, Yuan Y, Sun F, Zhang HQ, Shi HJ. The effects of di-(2-ethylhexyl)-phthalate exposure on fertilization and embryonic development in vitro and testicular genomic mutation in vivo. PLoS One . 2012; 7( 11): e50465. Google Scholar CrossRef Search ADS PubMed  69. Pocar P, Fiandanese N, Berrini A, Secchi C, Borromeo V. Maternal exposure to di(2-ethylhexyl)phthalate (DEHP) promotes the transgenerational inheritance of adult-onset reproductive dysfunctions through the female germline in mice. Toxicol Appl Pharmacol . 2017; 322: 113– 121. Google Scholar CrossRef Search ADS PubMed  70. Pocar P, Fiandanese N, Secchi C, Berrini A, Fischer B, Schmidt JS, Schaedlich K, Borromeo V. Exposure to di(2-ethyl-hexyl) phthalate (DEHP) in utero and during lactation causes long-term pituitary-gonadal axis disruption in male and female mouse offspring. Endocrinology . 2012; 153( 2): 937– 948. Google Scholar CrossRef Search ADS PubMed  71. François CM, Petit F, Giton F, Gougeon A, Ravel C, Magre S, Cohen-Tannoudji J, Guigon CJ. A novel action of follicle-stimulating hormone in the ovary promotes estradiol production without inducing excessive follicular growth before puberty. Sci Rep . 2017; 7: 46222. Google Scholar CrossRef Search ADS PubMed  72. Thomsen AM, Riis AH, Olsen J, Jönsson BA, Lindh CH, Hjollund NH, Jensen TK, Bonde JP, Toft G. Female exposure to phthalates and time to pregnancy: a first pregnancy planner study. Hum Reprod . 2017; 32( 1): 232– 238. Google Scholar PubMed  73. Peng F, Ji W, Zhu F, Peng D, Yang M, Liu R, Pu Y, Yin L. A study on phthalate metabolites, bisphenol A and nonylphenol in the urine of Chinese women with unexplained recurrent spontaneous abortion. Environ Res . 2016; 150: 622– 628. Google Scholar CrossRef Search ADS PubMed  Copyright © 2018 Endocrine Society

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EndocrinologyOxford University Press

Published: Feb 1, 2018

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