Differential Effects of Prenatal Exposure to Bisphenol-A or ...

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Endocrinology 147(12):5956 –5966 Copyright © 2006 by The Endocrine Society doi: 10.1210/en.2006-0805

Developmental Programming: Differential Effects of Prenatal Exposure to Bisphenol-A or Methoxychlor on Reproductive Function Mozhgan Savabieasfahani, Kurunthachalam Kannan, Olga Astapova, Neil P. Evans, and Vasantha Padmanabhan Departments of Pediatrics and the Reproductive Sciences Program (M.S., O.A., V.P.), University of Michigan, Ann Arbor, Michigan 48109; Wadsworth Center (K.K.), New York State Department of Health, and Department of Environmental Health Sciences, State University of New York at Albany, Albany, New York 12201; and Division of Cell Sciences (N.P.E.), University of Glasgow Veterinary School, Glasgow G61 1QH, United Kingdom Increased occurrence of reproductive disorders has raised concerns regarding the impact of endocrine-disrupting chemicals on reproductive health, especially when such exposure occurs during fetal life. Prenatal testosterone (T) treatment leads to growth retardation, postnatal hypergonadotropism, compromised estradiol-positive feedback, polycystic ovaries, and infertility in the adult. Prenatal dihydrotestosterone treatment failed to affect ovarian morphology or estradiolpositive feedback, suggesting that effects of prenatal T may be facilitated via conversion of T to estradiol, thus raising concerns regarding fetal exposure to estrogenic endocrine-disrupting chemicals. This study tested whether fetal exposure to methoxychlor (MXC) or bisphenol A (BPA) would disrupt cyclicity in the ewe. Suffolk ewes were administered MXC (n ⴝ 10), BPA (n ⴝ 10) (5 mg/kg䡠d sc in cotton seed oil) or the vehicle (C; n ⴝ 16) from d 30 to 90 of gestation. On d 60 of treatment,

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OCIETAL CONCERN HAS been mounting over the potential deleterious effects on animal/human health of environmental exposure to endocrine-disrupting compounds (EDCs). EDCs are hormonally active, synthetic, or natural compounds that are present within our environment and food sources at concentrations that can interfere with the normal activity of endocrine systems/tissues, most notably the reproductive endocrine axis (1, 2). The controversy regarding the deleterious effects of EDC exposure has mainly been fueled by studies that point to likely effects on human health, including the recent dramatic increases in the incidence of estrogen sensitive cancers (breast, prostate and testicular) (3, 4), the decline in human sperm quality and quantity (5), a notable rise in endometriosis (2), an increase in genital abnormality in boys (6), and early puberty (7) in girls. EDCs that can interact with estrogen receptors have received considerable attention because they can modulate signaling by native estrogen, a key regulator of several physiologic functions including reproduction (8). In addition, it is First Published Online August 31, 2006 Abbreviations: BPA, Bisphenol A; C, control; CV, coefficient of variation; MXC, methoxychlor; P, progesterone; PG, prostaglandin; T, testosterone. Endocrinology is published monthly by The Endocrine Society (http:// www.endo-society.org), the foremost professional society serving the endocrine community.

maternal MXC concentrations in fat tissue and BPA in blood averaged approximately 200 ␮g/g fat and 37.4 ⴞ 3.3 ng/ml, respectively. Birth weights of BPA offspring were lower (P < 0.05) relative to C. There was no difference in the time of puberty between groups. BPA females were hypergonadotropic during early postnatal life and ended their breeding season later, compared with C. Characterization of cyclic changes after synchronization with prostaglandin F2␣ in five C, six MXC, and six BPA females found that the onset of the LH surge was delayed in MXC (P < 0.05) and the LH surge magnitude severely dampened (P < 0.05) in BPA sheep. These findings suggest that prenatal BPA and MXC exposure have long-term differential effects on a variety of reproductive endocrine parameters that could impact fertility. (Endocrinology 147: 5956 –5966, 2006)

well established in human medicine that fetal exposure to the synthetic estrogen, diethylstilbesterol, has resulted in a wide variety of problems in the daughters of mothers prescribed diethylstilbesterol during pregnancy, including increased risk of cancers and infertility (9, 10). Exposure to estrogenic EDCs such as dicofol and DDT (dichloro-diphenyl-trichlorethane), has also been reported to disrupt sexual differentiation in other species including turtles (11) and birds (12). Exposure to methoxychlor (MXC) or bisphenol A (BPA), EDCs that do interact with estrogen receptors, was found to masculinize the female brain (13), advance puberty (14), and cause sex reversal (15). In addition to these specific effects of EDCs on the reproductive system, EDCs have the potential to alter the endocrine status of the developing fetus and thus lead to adaptations that may predispose the fetus to obesity and other metabolic/endocrine diseases in adulthood (16 – 19). Based on current evidence, the U.S. Environmental Protection Agency (EPA) has thus advocated that although exposure to single estrogenic compounds, at current environmental levels, is insufficient to cause adverse effects in adult humans, more information is needed to determine whether the same holds true for the human fetus and neonate, which lack some of the protective mechanisms found in the adult (20). Another deficiency of the current EDC literature is the scarcity of information relative to levels of EDCs achieved in human. Low-dose studies carried out in

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Savabieasfahani et al. • Prenatal EDC Exposure and Reproductive Dysfunction

recent years (21–23) target intake rather than levels achieved in circulation or tissue load. Considering that animals may metabolize, store, or respond to similar intake levels differently, it is essential to also have other reference points such as circulating and tissue concentrations of EDCs. Whereas a link has been suspected between EDC exposure and adverse effects on human and animal health, it is important to note that health risks have primarily been derived from epidemiological data and/or studies conducted in rodent models. Because the sensitivity to EDCs is likely to vary between species, studies in rodents need to be cross-validated using sensitive animal models with established periods of developmental susceptibility. Sheep provide a powerful model system to investigate reproductive consequences of in utero estrogenic/androgenic EDC exposures because a large body of literature already exists documenting critical periods of fetal susceptibility to native steroids (24 –35) in the sheep. For instance, prenatal exposure of female sheep to testosterone (T), an estrogen precursor, has been reported to lead to growth retardation (25, 26, 32) and neuroendocrine (24, 27–29) and ovarian (31–33) defects that culminate in a progressive loss of ovarian cyclicity (33–35). To assess the effects of EDC exposure, we selected two environmentally relevant endocrine disrupting compounds, MXC and BPA. The organochlorine insecticide, MXC, has been used extensively to control pests in agricultural, dairy, and domestic settings and is known to be moderately persistent in the environment (36, 37). Indeed, measurable concentrations of MXC can be found in the body fat of humans and rats (38, 39). BPA is a plasticizer, the production of which is estimated to be about 1.7 billion kg/yr (40), and again studies have found measurable concentrations of BPA in the maternal/fetal circulation, amniotic fluid, and placental tissue of pregnant women (40 – 42). In this study, we aimed to address the threat EDCs may pose to reproductive well-being. The study was designed bearing in mind the shortcomings of some of the current EDC literature and as such used the following: 1) a robust sheep model with known critical developmental windows and defined end points (24 –35), 2) EDC exposure based on EPApublished lowest observed effect level (21), 3) measures to quantify EDC burden in the circulation and tissues, and 4) appropriate controls. We hypothesized that fetal exposure to MXC and BPA, at levels approaching human exposure levels, would disrupt reproductive cyclicity in the ewe. Materials and Methods Breeding and prenatal EDC treatment Adult Suffolk ewes were maintained at a U.S. Department of Agriculture-inspected and University of Michigan Laboratory Animal Medicine-approved farm for breeding. Starting 2–3 wk before breeding, ewes were group fed daily with 0.5 kg shelled corn and 1.0 –1.5 kg alfalfa hay per ewe to increase energy balance. Ewes were mated to fertility-proven rams and the day of mating was noted. After breeding, all ewes were housed under natural photoperiod in the same pasture and group fed with a daily maintenance diet of 1.25 kg alfalfa/brome mix hay per ewe. Pregnant ewes (average weight 87.2 ⫾ 2.3 kg) received sc injections of MXC (purity 95⫹%, catalog no. M-1501; Sigma, St. Louis, MO) (n ⫽ 10) or BPA (purity 99⫹%, catalog no. 239658 –250G; Aldrich Chemical Co., Milwaukee, WI) (n ⫽ 10) at 5 mg/kg䡠d in cottonseed oil (Sigma) from d 30 through 90 of gestation (term 147 d). Critical time points relative to timing of the EDC exposure include initiation of gonadal

Endocrinology, December 2006, 147(12):5956 –5966

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differentiation at d 30 (43, 44), development of hypophyseal portal vasculature around d 50 (43), detection of LH and FSH in circulation around d 55 (45), and completion of primordial follicular differentiation by d 90 (44). EDC doses were selected on the basis of the lowest observed effect level established in the EPA National Toxicology Program’s Report of the Endocrine Disruptors (20). Recent rodent studies have achieved effects with much lower intake levels (21–23). Controls (C; n ⫽ 16) received vehicle. To determine concentrations of BPA achieved using this treatment regimen, blood samples were collected from both C and prenatal BPAtreated animals (n ⫽ 6/treatment group) at three different time points, 50, 70, and 90 d of gestation (20, 40 and 60 d of BPA treatment). To avoid the stress associated with collection of biopsies from experimental ewes, a preliminary estimate of MXC load was obtained after the collection of fat biopsies from one pregnant ewe and her twin lambs (a female and a male) after the animals were killed on d 90 of gestation (d 60 of MXC treatment). Fat biopsies were also obtained on d 30 and 60 of MXC treatment from two nonpregnant sheep. Fat biopsies were obtained from three untreated C. Choice of measurement of BPA levels in maternal blood and MXC in maternal fat was based on previous studies, which found that BPA accumulates in liver, blood, or proteinaceous tissues (46), and MXC is lipophilic and accumulates in fatty tissues (47).

Maternal care, neonatal measures, and postnatal care Six weeks before lambing, when maximal fetal growth was underway, pregnant ewes were group fed an additional 0.5–1 kg alfalfa hay and 250 mg Aureomycin crumbles (chlortetracycline) per ewe daily. All lambs, the majority twins, were born between March 15 and April 20, 2002. Birth dates, the number of offspring, and offspring gender were recorded. At birth each lamb was given oral vitamin E and selenium and injections for Clostridium perfringens types C and D and tetanus. Newborn weight and body dimensions were recorded from all male and female lambs the day after birth to allow adequate time for maternal bonding. These measures included weight, height (determined with the lambs standing), chest circumference, and anourethral, anonavel, and anoscrotal (males) distances. A blood sample was collected to measure circulating levels of insulin and IGF-I. Each ewe and its lamb(s) were housed together for the first 3 d and later group housed with other mothers and offspring, in a barn, under natural photoperiod. Lactating ewes were fed 1 kg shelled corn and 2–2.5 kg alfalfa hay while they were suckling lambs. A 60-W bulb in the lamb creep feed area was lit during the nights. Group-housed lambs had access to feed pellets (Shur-Gain, Elma, NY) containing 18% crude protein and alfalfa hay. At 8 wk of age, all lambs were weaned and the females transferred to the Sheep Research Facility (Ann Arbor, MI) where they were maintained in pens, outside, under natural photoperiod. Once weaned all lambs were provided ad libitum access to commercial feed pellets (as above). When they reached a weight of approximately 40 kg, to avoid fat deposition during the period of reduced growth, lambs were switched to a pelleted feed with 15% crude protein. Trace mineralized salt with selenium and vitamins A, D, and E (Armada Grain Co., Armada, MI) was freely accessible throughout the study. Postnatal weight gain was monitored weekly for 8 months in all female lambs.

Dietary control To ensure that there is no confounding effect from feed differences across treatments, food was purchased from a single supplier in bulk and stored for use. All ewes, before and after breeding as well as all lambs after weaning therefore received the same standardized food. Whereas there was a potential for interaction between experimental EDC exposures with phytoestrogens from food, this feeding regimen ensured that exposure of phytoestrogens was similar across all treatment groups and thus allowed observation of effects of added experimental BPA and MXC exposure. The feeding/EDC exposure paradigm used in this study also had the added benefit that it reflects a true-life situation because humans and animals are likely to be exposed to industrial pollutants and phytoestrogens in parallel.

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Puberty and cycle characterization

RIAs

To establish the time of puberty (onset of progestogenic cycles), biweekly blood samples were collected from all female lambs starting at approximately 6 wk of age. Males were castrated by banding within first week of birth and not studied. At 40 wk of age, after achievement of puberty, C (n ⫽ 5) and prenatal EDC-treated females (n ⫽ 6) received two im injections of prostaglandin (PG) F2␣ (20 mg of 5 mg/ml Lutalyse; Pfizer Animal Health, Kalamazoo, MI) 11 d apart to synchronize their estrous cycles. To characterize cyclic changes in basal gonadotropin and steroid concentrations during the follicular phase, blood samples were obtained from all C, prenatal MXC-, and prenatal BPA-exposed females at 2-h intervals for 120 h, starting at the time of the second PGF2␣ injection. Only one female from any given dam was used in this study to ensure dam is the experimental unit. To monitor temporal changes in LH secretion, blood samples were collected frequently (12 min intervals for 8 h) twice during the presumptive follicular phase (24 –32 and 46 –54 h after administration of PGF2␣) and once during the luteal phase (d 8 after second PGF2␣ injection). To characterize the pattern of luteal progesterone (P) secretion, daily samples were collected for 2 wk starting from completion of 2-h samples. All animal use procedures were approved by the University of Michigan Committee for the Use and Care of Animals.

Circulating levels of LH were measured in twice-weekly samples collected during the first month of life and all samples in the cycle characterization study (except the daily samples) using a well-validated assay (50). Assay sensitivity, 50% displacement point, and intraassay coefficient of variation (CV) at 80 and 20% displacement points of the LH assay averaged 0.13 ⫾ 0.02 ng, 0.68 ⫾ 0.02 ng, 6.03 ⫾ 0.71%, and 3.70 ⫾ 0.42%, respectively (n ⫽ 17 assays). Interassay CV based on three quality control pools averaging 0.91 ⫾ 0.40, 13.29 ⫾ 0.23, and 22.40 ⫾ 0.55 ng/ml averaged 17.40, 8.40, and 9.56%, respectively. Circulating levels of FSH were measured in two-hourly samples collected during the synchronized estrus cycle using a validated RIA (51). Assay sensitivity, 50% displacement point, and intraassay CV at 80 and 20% displacement points of the FSH assay averaged 0.08 ⫾ 0.01 ng, 0.58 ⫾ 0.01 ng, 10.59 ⫾ 2.03%, and 5.30 ⫾ 1.02%, respectively (n ⫽ 5 assays). Interassay CV based on two quality control pools averaging 3.50 ⫾ 0.25 and 30.13 ⫾ 1.63 ng/ml averaged 14.17 and 10.80%, respectively. Plasma concentrations of P were measured using a commercial RIA kit (Coat-A-Count P; Diagnostic Products Corp., Los Angeles, CA) validated for use with sheep samples (52) in all twice-weekly samples to assess timing of puberty and the length of first breeding season and all daily samples to assess luteal phase length. Assay sensitivity, 50% displacement point, and intraassay CV at 80 and 20% displacement points of the P assay averaged 0.03 ⫾ 0.004 ng, 0.17 ⫾ 0.001 ng, 11.43 ⫾ 0.62%, and 5.71 ⫾ 0.32%, respectively (n ⫽ 7 assays). Interassay CV based on three quality control pools averaging 0.92 ⫾ 0.04, 1.82 ⫾ 0.08, and 13.07 ⫾ 0.46 ng/ml averaged 11.69, 12.32, and 9.34%, respectively. Plasma insulin concentrations were measured using ImmuChem-coated tube Insulin 125I RIA kit (ICN Pharmaceuticals, Costa Mesa, CA) in samples collected the day after birth. The sensitivity of the assay was 3.86 ␮U/ml and intraassay CV was 5%. Circulating IGF-I levels were measured using a validated assay (53) after an acid-ethanol extraction solution using a recombinant human IGF-I (R & D Systems, Minneapolis, MN) as the assay standard. The sensitivity of the assay was 2.9 ng/ml and intraassay CV and recovery were 10 and 96%, respectively. For all assays, all values below assay sensitivity were assigned the detection limit of the assay.

EDC measurements BPA levels in plasma samples were quantified by reverse phase HPLC with fluorescence detection, as described previously (48). Briefly, a 1-ml aliquot of plasma was transferred to a tube and 10 ␮l of 1-ppm butylphenol added as an internal standard. Samples were extracted twice with 5 ml ethyl ether. The solvent (2 ⫻ 5 ml) was pooled and evaporated to dryness under nitrogen. The sample extract was then reconstituted with 1 ml of acetonitrile and analyzed by HPLC. BPA recovery was determined by fortifying charcoal-stripped fetal bovine serum (1 ml) with 50 and 100 ng of BPA and it averaged 77 ⫾ 12%. Standards of BPA were prepared in acetonitrile at concentrations of 0.1, 0.25, 0.5, and 1 ␮g/ml for calibration. Samples and standards were injected onto an analytical column (Prodigy ODS, 250 ⫻ 4.6 mm column; Phenomenex, Torrance, CA), which was connected to a guard column Prodigy ODS, 30 ⫻ 4.6 mm using a series 200 autosampler (PerkinElmer, Norwalk, CT) and eluted with a flow of acetonitrile and water at a gradient from 50% acetonitrile in water to 98% acetonitrile in water for 20 min delivered by a PerkinElmer series 200 pump (flow rate was at 1 ml/min). Each sequence began with a blank and calibration standards. High-purity analytical grade solvents were used throughout. Detection was accomplished using a Hewlett Packard 1046A fluorescence detector (Hewlett-Packard Co., Wilmington, DE) with an excitation wavelength of 229 nm and an emission wavelength of 310 nm. Florescence detector settings were: photomultiplier tube gain 12, lamp time -1, response time 2 sec, stop time 27 min, and gate and delay at zero. Blanks were analyzed concurrently to check for interfering peaks. Detection limit of BPA was 10 ng/ml. For measurement of MXC accumulation, 3 g of fat were weighed and extracted with Soxhlet apparatus for 16 h using dichloromethane and hexane (400 ml). The extract was rotary evaporated to 6 ml. Two milliliters of the extract was diluted in 2 ml dichloromethane and passed through a gel permeation chromatography column to remove fat, as described elsewhere (49). One milliliter of the extract was used for lipid determination. The extract from the gel permeation chromatography column was concentrated to 1 ml and injected into an Agilent 6890 N gas chromatograph equipped with an electron capture detector (Agilent Technologies, Palo Alto, CA). A capillary column coated with DB-5 (5% phenyl methylpolysiloxane, 30 m ⫻ 0.25 mm inner diameter ⫻ 0.25 ␮m film thickness; Agilent Technologies) was used for the separation of MXC. The column oven temperature was programmed to change from 120 C (1 min) to 180 C (2 min) at a rate of 10 C/min and then to 240 C at 3 C/min, with a final hold time of 5 min. Inlet and detector temperatures were held at 250 and 300 C, respectively. Concentrations were calculated from the peak area of the sample to that of the corresponding external standard, MXC. Detection limit of MXC was 10 ng/g on a wet weight basis. Recovery of MXC through the analytical method was 82 ⫾ 7%.

Statistical analysis In terms of newborn measures, the primary outcome measures were body weight, height, chest circumference, anogenital ratio (the ratio of anourethral to anonavel distance), anoscrotal to anonavel ratio (males), and circulating insulin and IGF-I levels. Each outcome measure was compared between groups using only the mean value from siblings from multiple gestations by a general linear model that included treatment and number of males in the multiple gestations as a covariate. This is similar to a repeated-measures ANOVA in which the siblings are repeated measures with gender and treatment as grouping variables (covariates). To assess whether prenatal BPA exposure had an effect on postnatal growth rate, a growth curve analysis was performed using a linear mixed model in which a separate regression line (random intercept and random slope) was calculated for each ewe. The overall effect of age, treatment, and the age by treatment interaction was examined. Age at puberty was defined as the first increase of the first progestogenic cycle. A progestogenic cycle was defined as a plasma P concentration that remained 0.5 ng/ml or greater for at least two consecutive twice-weekly time points. The duration of each progestogenic cycle was calculated from the day of P rise greater than 0.5 ng/ml to the day when P concentration fell below this value. The onset of LH and FSH surges was determined based on modification of previously established criteria (54). Briefly, the onset of an LH/FSH surge was defined as elevation of circulating LH/FSH above baseline by 2⫻ assay sensitivity lasting at least 8 h and peak concentration of LH/FSH exceeding at least twice the average levels of LH/FSH during baseline periods. The end of the surge was defined as the time when LH/FSH levels fell below the established criteria of surge onset. For secondary FSH surge, the sustained increase in FSH that followed termination of primary FSH surge was used and included same criteria as defined for primary surge. If secondary FSH levels did not fall back to baseline before end of the bleed, the duration of surge was computed as interval between start of secondary FSH surge and time of termination of bleed. Data from first follicular bleed (24 –32 h after PGF2␣) for control and


BPA (the second follicular bleed for most animals occurred during or after LH surge) and both the first and second follicular phase bleed (46 –54 h) for MXC animals and luteal period of all animals were subjected to pulse analysis using the Cluster algorithm (55). The Cluster algorithm identifies pulses using criteria that define a pulse such that the peak of the pulse differs significantly from both the preceding and following nadirs according to two-sample t tests. For analysis with Cluster, the minimum number of data points in a peak and nadir were set at 2 and 2, respectively and the t statistic values used to identify a significant increase from preceding nadir and a decrease to following nadir were both at 2.0. Timing of puberty, duration and end of breeding season, number/ duration of progestogenic cycles and peak P concentrations achieved, timing and attributes of gonadotropin surges, LH pulse frequency and amplitudes, and duration/peak concentration of luteal P increase achieved in the synchronized cycle were analyzed by ANOVA after appropriate transformations to account for heterogeneity of variance.

Results BPA or MXC levels achieved

Representative chromatographic profiles of BPA and MXC from maternal plasma of a BPA-treated and abdominal fat of MXC-treated sheep are shown in Fig. 1 (top panels). Maternal concentrations of BPA in blood were significantly higher in BPA-treated females, compared with controls (below detection limit) at all three time points studied (50, 70, and 90 d of gestation corresponding to d 20, 40, and 60 of treatment) (Fig. 1, bottom left). Thus, 20 d appeared sufficient to achieve a steady-state concentration of BPA. MXC was found in fat samples collected from nonpregnant female sheep treated with MXC with levels being higher 60 d after MXC treatment,


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compared with levels achieved after 30 d of treatment (Fig. 1, bottom right). The MXC concentrations in the pregnant ewe and her male and female fetus killed on d 90 of gestation (d 60 of MXC treatment) were comparable with levels achieved in nonpregnant ewes that received the same MXC treatment for the same duration. Overall MXC levels achieved in treated ewes were markedly higher compared with controls. Gestational length, birth measures, and growth rate

Gestational length of C and prenatal MXC- and BPAtreated females did not differ and averaged 148.3 ⫾ 0.1, 147.0 ⫾ 0.5, and 147.3 ⫾ 0.6 d, respectively. All animals were born between March 15 and April 19, 2002. Eleven female and 18 males were born in the C group. There were 11 females and six males in the prenatal BPA-treated group and nine females and nine males in the prenatal MXC-treated group. Prenatal BPA- but not MXC-treated female lambs weighed less (P ⬍ 0.01) than the C at birth (Fig. 2). This difference was associated with significant effects on height (P ⬍ 0.01) and chest circumferences (P ⬍ 0.05). There were no differences in anogenital ratio among the three groups of female lambs (C: 0.28 ⫾ 0.09; prenatal MXC: 0.27 ⫾ 0.08; prenatal BPA: 0.23 ⫾ 0.08). Circulating levels of IGF-I in female lambs at 1 d of age were similar across treatment groups and averaged 5.14 ⫾ 0.07, 4.85 ⫾ 0.26, and 5.23 ⫾ 0.14 ng/ml in C and prenatal MXC- and BPA-treated females, respectively. Circulating levels of insulin also did not differ significantly among these groups: 2.41 ⫾ 0.24, 2.00 ⫾ 0.24, 2.17 ⫾ 0.12 ng/ml in C and prenatal MXC- and BPA-treated females, respectively. In the males, prenatal MXC but not BPA treatment was associated with a significant (P ⬍ 0.05) increase in birth weight relative to the C (C: 5.0 ⫾ 0.18; prenatal MXC: 5.74 ⫾ 0.30; prenatal BPA: 4.96 ⫾ 0.59 kg). This increase was reflected in their chest circumference (C: 40.5 ⫾ 0.7; prenatal MXC: 42.4 ⫾ 0.9; prenatal BPA: 40.4 ⫾ 1.4 cm). Whereas there was no difference in the anogenital ratio between males of the three groups (C: 0.70 ⫾ 0.07; prenatal MXC: 0.75 ⫾ 0.09; prenatal BPA: 0.63 ⫾ 0.07 cm), the anoscrotal to anonavel ratio was increased in both the prenatal MXC- (P ⬍ 0.05) and BPA-treated (P ⬍ 0.01) males, compared with C (C: 0.38 ⫾ 0.01; prenatal MXC: 0.42 ⫾ 0.02; prenatal BPA: 0.46 ⫾ 0.004). Postnatal growth

As expected, there was a highly significant positive slope for age (P ⬍ 0.0001) across treatments. Growth rate tended to be higher in prenatal BPA- and not MXC-treated females (P ⫽ 0.08), compared with C between 2 and 4 months of age. Timing of puberty and reproductive cycles FIG. 1. Top panels, Chromatographic profiles of BPA and MXC of BPA- and MXC-treated pregnant sheep. Bottom left panel, Levels of circulating BPA in C (open circles) and BPA-treated (closed circles) pregnant ewes on d 50, 70, and 90 of gestation (d 20, 40, and 60 of treatment). Bottom right panel, Levels of MXC found in fat obtained from three control animals (open bar; C), two nonpregnant ewes on d 30 and 60 of MXC treatment (hatched bars) and one pregnant ewe and its male and female fetuses on d 60 of MXC treatment (d 90 of pregnancy; treatment began d 30 of pregnancy) (closed bars).

Age at puberty (start of first breeding season) was similar in C and prenatal MXC- and BPA-treated sheep (Fig. 2). All females irrespective of treatment had repetitive progestogenic cycles during the first breeding season (Fig. 3). Whereas the date of onset of the breeding season was similar across treatment groups, the breeding season ended later (P ⬍ 0.05) in the prenatal BPA-treated females (Fig. 3). Prenatal MXC(P ⬍ 0.05) and BPA-treated (P ⫽ 0.054) animals had more

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FIG. 2. Newborn body weight, height, and chest circumference adjusted for litter size (mean ⫾ SEM) and age at puberty of female lambs born to ewes treated with cottonseed oil (C, open bars), sheep prenatally treated during d 30 –90 of pregnancy with MXC (hatched bars) or BPA (closed bars). Asterisks, Significant differences from C (P ⬍ 0.05).

progestogenic cycles than the C during the first breeding season (Fig. 3). Peak P levels and length of progestogenic cycles did not differ among the three groups (Fig. 3). One C and one prenatal MXC-treated female were excluded from this analysis because of abnormal elevations in P that lasted several weeks, precluding calculating the number of cycles. Mean circulating concentrations of LH measured from biweekly samples during the first month of life were higher in prenatal BPA- but not the MXC-treated lambs (Fig. 4).

FIG. 3. Patterns of biweekly P from three C (top left) and three prenatal MXC- (top middle) and three prenatal BPA-treated (top right) females during the first breeding season. Bottom panels, Mean ⫾ SEM of end of first breeding season (bottom left) as well as the mean ⫾ SEM of peak level, number, and duration of the progestogenic cycles in C and prenatal MXC- and BPA-treated females. Asterisks, Significant differences from C females.

Effects of prenatal MXC and BPA treatment on estrous cycle characteristics

Patterns of circulating LH and FSH from three C, three prenatal MXC-, and three BPA-treated females beginning from the time of second PGF2␣ injection are shown in Fig. 5. An LH surge was seen in all C and MXC-exposed females but not all BPA-exposed females. Only 50% of BPA-treated animals exhibited increases in LH that met the criteria defined for an LH surge (three of six animals) as opposed to 100% of controls (P ⫽ 0.0637 by ␹2 analysis). Summary statistics of primary gonadotropin and secondary FSH surges are provided in Figs. 6 and 7 (excluding the three prenatal BPAtreated females that failed to show a definable LH surge). Relative to the second PGF2␣ injection, the time of onset of the LH surge and time to peak were both significantly delayed (P ⬍ 0.05) in prenatally MXC- but not BPA-treated sheep (Fig. 6, top two panels), compared with C. Timing of the primary FSH surge was similar to that of LH (not shown). The peak LH concentration and the total amount of LH released during the primary LH surge were significantly lower (P ⬍ 0.05) in prenatal BPA- but not MXC-treated sheep when compared with C (Fig. 6). There were no differences in the duration of the LH surge between C and the two EDCtreated groups. There were also no differences in the primary and secondary FSH surge magnitudes or total FSH secreted. Duration of the primary FSH surge in prenatal MXC-treated

FIG. 4. Mean ⫾ SEM of circulating LH during early postnatal period in C (open bar) and prenatal MXC-treated (hatched bar) and prenatal BPA-treated (closed bar) females. For analysis, serial values from twice weekly samples taken during the first 2 months of postnatal life were averaged. Asterisk, Significant difference from C (P ⬍ 0.05).



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FIG. 5. Circulating patterns of LH (closed circles) and FSH (open circles) in three C (ewes 299, 244, and 268) and three prenatal MXC- (ewes 233, 273, and 274) and three BPA-treated (ewes 265, 234, and 262) females taken at 2-h intervals for 120 h after induction of luteolysis with two injections of PGF2␣ 11 d apart. Note the onset of LH surge is delayed in the three prenatal MXCtreated animals relative to C. The prenatal BPA-treated females showed severely dampened LH surges (ewes 265 and 234). Ewe 262 showed a sustained but very small increase in LH, which fit the surge definition but not a clearly definable surge. The other three prenatal BPAtreated females failed to show LH increases that met the criteria established (see Materials and Methods).

females was, however, significantly (P ⬍ 0.05) longer than the other two groups (Fig. 7). The LH pulse characteristics during the follicular and luteal phase of the cycle in C and prenatal MXC- and BPAtreated females are summarized in Table 1. The first follicular phase LH pulse determination began 12.4 ⫾ 2.8, 29.0 ⫾ 3.0, and 14.0 ⫾ 2.7 h before the onset of LH surge in C and prenatal MXC- and BPA-treated females, respectively. The second follicular phase determination for MXC animals be-

FIG. 6. Mean (⫾ SEM) LH surge onset and peak relative to second PGF2␣ administration are shown in the top panels. Bottom panels shows LH surge characteristics (LH surge peak, total LH, and duration of the LH surge) from C (open bar) and prenatal MXC-treated (hatched bar) and prenatal BPA-treated (closed bar) females. Note measures in prenatal BPA-treated females are from only three animals. The other three animals did not show clearly definable LH surge. Significant differences between treatment and control groups are indicated by differing letters.

gan 9.2 ⫾ 1.6 h before the onset of LH surge. The second follicular phase determination occurred during or after LH surge in most of C and prenatal BPA-treated females. As expected, LH pulse frequency was higher in the follicular than luteal phase across treatment groups (P ⬍ 0.05). There

FIG. 7. Characteristics of primary and secondary FSH surge (peak height, total FSH, and duration) in C (open bar) and prenatal MXC(hatched bar) and prenatal BPA-treated (closed bars) females are shown in the top and bottom panels, respectively. Note measures in prenatal BPA-treated females are from three animals. The other three animals did not show clearly definable LH surges and hence other parameters could not be calculated. Asterisk, Significant difference from C (P ⬍ 0.05).

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TABLE 1. LH pulse dynamics during the follicular and luteal phase of the estrous cycle Groups

C (first) MXC (first) MXC (second) BPA (first)c

Folliculara

Lutealb

Mean

Frequency

Amplitude

Mean

Frequency

Amplitude

5.31 ⫾ 1.05 3.03 ⫾ 0.44 2.42 ⫾ 0.40 4.92 ⫾ 1.62

7.25 ⫾ 0.85 6.50 ⫾ 0.85 6.00 ⫾ 0.55 6.67 ⫾ 1.2

3.49 ⫾ 0.67 2.51 ⫾ 0.50 2.36 ⫾ 0.80 4.48 ⫾ 1.67

1.82 ⫾ 0.23

3.25 ⫾ 0.75

2.87 ⫾ 0.42

2.10 ⫾ 0.37 2.58 ⫾ 0.53

3.00 ⫾ 0.45 3.67 ⫾ 0.67

4.78 ⫾ 0.96 4.78 ⫾ 0.27d

a Blood samples were collected frequently at 12-min intervals for 8 h twice during the presumptive early and late follicular phase (24 –32 and 46 –54 h after administration of PGF2␣). Because second follicular bleed occurred during or after LH surge in majority of the C and BPA-treated females, data from only the first follicular bleed is provided. For prenatal MXC animals, in which onset of LH surge was delayed, mean LH, LH pulse frequency, and amplitude are provided from both follicular bleeds. b Luteal phase blood samples were taken at 12-min intervals for 8 h on d 8 after PGF2␣ injection. c Because three of the prenatal BPA-treated females failed to show definable LH surges, they were not included in these analyses. d P ⬍ 0.05.

were no between treatment differences in frequency or amplitude of LH pulses during the follicular phase. LH pulse amplitude, but not frequency, was significantly increased (P ⬍ 0.05) in prenatal BPA- but not MXC-treated females during the luteal phase. All animals in C and prenatal MXC- and BPA-treated groups had luteal P increases. There was no difference in peak or duration of luteal P increase between C and prenatal MXC- or BPA-treated groups (Fig. 8), despite the severely disrupted LH surges in the prenatal BPA-treated females. Discussion

It has been proposed that in utero exposure to environmental pollutants that can act as hormone agonists or antagonists (8) could pose a considerable threat to mankind. Findings from this study using sheep as a model provide evidence that prenatal exposure to BPA or MXC, two envi-

ronmentally relevant EDCs, at environmentally relevant doses, can disrupt reproductive function in different ways. Prenatal BPA exposure, at levels close to that seen in human maternal circulation, resulted in female offspring of low birth weight, which is a recognized risk factor for adult-onset disease. Prenatal BPA exposure also caused early postnatal hypergonadotropism and prolonged the first breeding season, which is suggestive of a reduced sensitivity to estradiolnegative feedback. Prenatal MXC exposure, on the other hand, had no effect on birth weight or changes in gonadotropin secretion that are associated with activation of the reproductive axis but did affect hormone secretory dynamics during the estrous cycle such that the onset of primary gonadotropin surges (after PGF2␣) was delayed. Robustness of the animal model used

The majority of work testing EDC risk has relied on in vitro systems or rodent models. Because sensitivity to EDC may vary between species, comparative animal models using species other than rodents are also needed to establish safe exposure levels. The wealth of information available detailing the neuroendocrine and ovarian changes that regulate reproductive cyclicity in the sheep (56) and knowledge of the steroid sensitive developmental periods (24 –35) makes the sheep a superb model to assess the deleterious effects of EDCs on reproductive function. For example, our previous studies and those of others found that prenatal exposure of sheep to T from d 30 to 90 of gestation can lead to low birth weight (25, 26) and severe estrous cycle defects, which included neuroendocrine (24, 27–30) and ovarian (31–33) defects. Some of these reproductive defects such as disruption of estradiol feedback and development of polycystic ovarian morphology in prenatal T-treated females appear to be programmed by aromatization of T to estradiol as an exposure to a similar pattern of dihydrotestosterone, a nonaromatizable androgen, failed to induce the same abnormalities (24, 31). These findings raise the possibility that environmental EDCs that interact with estrogen and androgen receptors may also disrupt reproductive cyclicity. EDC exposure levels and relevance to human

FIG. 8. Mean ⫾ SEM circulating patterns of daily P in C and prenatal MXC- and prenatal BPA-treated females after synchronized estrus are shown on the left and mean ⫾ SEM of peak height and duration of luteal P increase are shown on the right.

It is highly debated whether EDC at current exposure levels are detrimental to human health. As reviewed in recent articles (20 –23), to assess human risks, animal studies should


include environmentally relevant doses as well as relevant and sensitive quantitative end points. Because uptake and metabolism are likely to vary from species to species and fetus to adult, it is also vitally important to establish a range of appropriate reference points for exposure assessment. For instance, species differences in the pharmacokinetics of a compound may alter the effective exposure such that low dose administration may lead to high exposure if absorption is high and metabolism is reduced, whereas a high exposure may provide a low exposure, if absorption is low and/or the compound is degraded quickly. Because different animals may store or metabolize EDCs quite differently from humans, blood/tissue levels need to be assessed in a variety of species in a variety of exposure paradigms to provide an initial reference point to relate experimental studies of EDC exposure to human exposure levels and assess potential human risk. Because the aim of this study was to investigate the effects of fetal exposure to EDCs, the model system targeted achievement of maternal levels of EDCs that can be compared with those reported in human. Subcutaneous injections were used to provide assurance that required levels are achieved. Whereas the method of administration used (sc injection) differs from that which would be encountered naturally, this does not detract from the result obtained using this model system. Maternal levels of BPA achieved in the present study averaged 37.4 ⫾ 3.3 ng/ml on d 60 of BPA treatment and were approximately 2-fold higher than the highest level reported in human maternal blood (41). Levels of unconjugated BPA in human maternal circulation ranged between 0.3 and 18.9 ng/ml and in fetal blood between 0.2 and 9.2 ng/ml (41), suggesting that BPA crosses the placental barrier. High levels of BPA have also been reported in human amniotic fluid and placental tissue (40). Considering the widespread use of BPA in industrial and consumer products (57– 61), our findings of reproductive disruptions after BPA exposure at concentrations not far from those reported in human maternal blood highlight the potential threat BPA poses to fetal and subsequent adult health. Our experiments with MXC exposure began before the use of MXC was banned in the United States (62). Nonetheless, studies focusing on disruptive effects of MXC are of relevance because the risks of exposure to MXC are likely to continue due to importation of many of the agricultural products from countries in which the use of MXC is not banned. Furthermore, adverse effects on developmental MXC exposure may not become evident for decades due to the long-term nature of the induced effects and the bioaccumulation of MXC in fat and its later release from people exposed to MXC in the recent past. Finally, there is also the potential for transgenerational transfer of altered phenotypes (63– 65). The levels of MXC achieved with daily administration of MXC for 60 d in this study are approximately 1000-fold higher than the reported level of 156 ng/g lipid in adipose fat of a human Spanish population (38). The increase in the concentration of MXC found in tissues of nonpregnant ewes after 60 d of exposure, compared with 30 d, substantiates the fact that MXC has the potential to bioaccumulate. Similar levels of MXC achieved in the one pregnant ewe and


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its fetuses, although very preliminary, suggest MXC also crosses the placental barrier and reaches the fetus. Effects of EDC on offspring measures

In previous studies, we reported that prenatal treatment with T leads to intrauterine growth retardation (32) and consequently low birth weight (26). Our finding that prenatal exposure to BPA, an estrogen mimic, also results in lowbirth-weight female offspring suggests that programming of low birth weight in prenatal T-treated females may be facilitated by aromatization of T to estrogen. This is further substantiated by the fact that offspring of dihydrotestosterone-treated females are not of lower birth weight (Steckler, T., and V. Padmanabhan, unpublished data). Low birth weight is viewed as an early marker of several adult disorders including infertility (66 – 69). Prenatal MXC treatment, on the other hand, had no effect on birth weight of female but increased body weight and size of the male offspring. Considering that MXC can interact with both estrogen and androgen receptors (70 –72), the growth-promoting effects on males may be facilitated by MXC acting as an estrogen antagonist or androgen agonist rather than as an estrogen agonist. The fact that both prenatal BPA and MXC treatments increased the anoscrotal to anonavel ratio in the males suggests that this effect is likely programmed by the two EDCs acting as an estrogen agonist. Whereas the results are discussed in the context of estrogen/androgen signaling, any number of mechanisms, many of which may have nothing to do with their ability to interact with gonadal steroid hormone receptor, may have contributed toward this disruption. The discussion is targeted to gonadal steroids due to the similarity of reproductive phenotype with T (androgenic ⫹ estrogenic) and not dihydrotestosterone (androgenic) animals. The subtle effects of prenatal MXC treatment on reproductive endocrine dynamics reflected as a delay in onset of LH surge, compared with prenatal BPA treatment, may relate to the levels achieved, which may be beyond the optimal response range. This is especially true because, as reviewed by Vom Saal et al. (73), current assumptions of monotonic dose-response curve (lack of risk below a certain threshold and similar or increased responses with increasing dose) do not appear to hold true. Alternatively, it is conceivable that in sheep MXC gets quickly stored in fat before being metabolized to its active metabolite, 2,2-bis (p-hydroxyphenyl)1,1,1-trichloroethane (74). A third possibility relates to the mixed effects of MXC at the level of estrogen and androgen receptors (70 –72). Effects of EDC on puberty

Our findings that prenatal MXC and BPA had no effect on EDC differ from rodent studies. Rodent studies (14, 75, 76) found that both MXC and BPA advance onset of puberty. Similar onset of progestogenic cycles in C and prenatal EDCtreated sheep is not surprising, considering the strict photoperiod requirement of female lambs for the synchronous timing of puberty (77). Postnatal but not prenatal estrogen exposure has been shown to delay onset of puberty in female lambs, possibly due to the profound postnatal hypogonadotropism it induces (78, 79). Whereas there were no differ-

5964


ences in onset of progestogenic cycles, the early increase in postnatal LH seen in prenatal BPA-treated females is consistent with advancement of neuroendocrine puberty and an early decline in estradiol-negative feedback, similar to that seen in prenatal T-treated females (24, 27, 28). Levels of LH are reported to be low in control females during the first 2 months of life (80). Differential effects of BPA and MXC on reproductive endocrine parameters

Whereas both prenatal MXC and BPA treatments resulted in altered reproductive endocrinological parameters in the female lambs after they had attained puberty, fertility outcomes were not assessed. Prenatal treatment with MXC or BPA had differential effects on LH surge dynamics with MXC treatment delaying the onset and BPA treatment reducing the magnitude of the LH surge. Rodent studies have found that BPA treatment disrupts estrous cyclicity (75, 81). The cycle defects seen in rodents (75, 81) and sheep (this study) are perhaps not surprising given the reports that BPA principally acts as an estrogen mimic (21–23), whereas MXC can interact with both estrogen and androgen receptors (70 – 72). From a mechanistic perspective, the reduced or absent LH surge magnitude such as that evidenced in prenatal BPAtreated females may be suggestive of impending loss of cycles. Prenatal T-treated females, the model prenatal BPAtreated females mimic in terms of low birth weight (25, 26), early LH excess (27, 28), and severely dampened LH surge (27), do show progressive loss of estrous cyclicity (33–35). Whereas it is possible that severe dampening of LH surge may be the result of reduced GnRH surge amplitude, earlier studies in sheep have found minimal increases in GnRH is sufficient to elicit a full amplitude LH surge (82). A second explanation would be a pituitary effect whereby exposure to the EDC has negative effects on LH production or release from the pituitary. A third possibility for compromised LH surge dynamics in prenatal BPA-treated females may relate to disrupted follicular function and a consequent reduction in the magnitude of preovulatory estradiol rise. These possibilities remain to be investigated. The delayed onset of LH surge in the prenatal MXCtreated sheep relative to the C cannot be explained by difference in magnitude and duration of luteal P. Previous reports have linked the timing of the LH surge to the levels of P experienced during the previous luteal phase (83), the LH surge being delayed in animals in which P levels in the previous cycle were high. The delayed LH surge onset relative to PGF2␣ administration may be a function of compromised follicular populations/development that induces a consequent delay in of the preovulatory estradiol rise. In this regard, MXC treatment has been found to disrupt ovarian folliculogenesis in rodents (84). Alternatively, preovulatory increase in estradiol may have occurred on time, but the required progression of changes in pulsatile GnRH secretion might be compromised (85). However, follicular phase LH pulse frequency was not different between prenatal MXCtreated and C females. Whereas no difference in pulse dynamics of LH was evident in the luteal phase of prenatal EDC-treated animals, compared with controls, LH pulse am-


plitude was increased in prenatal BPA-treated animals suggestive of reduced sensitivity to P-negative feedback. The increased postnatal LH and delayed ending of first breeding season in concert with reduced luteal P sensitivity in prenatal BPA-treated females are consistent with estradiol being the negative feedback regulator of LH during the prepubertal period and P assuming this role once cyclicity is established. The delayed/dampened LH surge in the prenatal MXCand BPA-treated animals did not disrupt luteal function, emphasizing the resiliency of the system to such cyclic perturbations. The dominant follicle, once differentiated and mature, appears to be capable of luteinizing and sustaining normal corpus luteum function, even when the ovulatory trigger is delayed or dampened. Our finding that very small but sustained increases in LH such as that seen in the prenatal BPA-treated female (ewe 262) was capable of eliciting a normal luteal P profile suggest a massive LH surge is not required. These findings also parallel what is seen in the prenatal T-treated sheep, in which repetitive P cycles are evident, despite severely compromised estradiol-positive feedback (27, 29). If the delayed or dampened LH surge in prenatal EDC-treated females has effects at other levels such as oocyte quality (86) remains to be determined. Dietary considerations

It should be recognized that, whereas care was taken to provide similar diet across all treatment groups, the effects of BPA or MXC described in this study might reflect synergistic or additive interactions with phytoestrogens in the feed. Nonetheless, because the diet was constant across treatments, any interaction with phytoestrogens does not reduce the value of the findings because in the context of developmental programming, this reflects a true-life situation with humans being exposed to EDCs in concert with other phytoestrogens. These studies are the first comprehensive study that relates the impact of prenatal exposure to MXC or BPA on programming of adult reproductive disruption to actual fetal exposure levels. Our findings draw attention to and validate public concern over human fetal exposure to synthetic exogenous steroids. Acknowledgments The authors are thankful to Mr. Douglas Doop and Gary R. McCalla for providing quality care and maintenance of the lambs used in this study; Dr. Mohan Manikkam, Mr. James Lee, and Ms. Carol Herkimer for their assistance with EDC treatment and blood sampling; and Dr. Teresa Steckler for assistance with the formatting of figures. Received June 15, 2006. Accepted August 21, 2006. Address all correspondence and requests for reprints to: Vasantha Padmanabhan, Department of Pediatrics, 300 North Ingalls Building, Room 1109 SW, Ann Arbor, Michigan 48109-0404. E-mail: [email protected]. This work was supported by U.S. Public Health Service Grants HD 41098, National Institutes of Health Grants F32 ES012366 and T32 ES07062, and the Office of the Vice President for Research, University of Michigan. Disclosure summary: None of the authors has anything to declare.



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