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. 2015 Apr 8;156(7):2563–2570. doi: 10.1210/en.2014-1634

Acute Influences of Bisphenol A Exposure on Hypothalamic Release of Gonadotropin-Releasing Hormone and Kisspeptin in Female Rhesus Monkeys

Joseph R Kurian 1,✉, Kim L Keen 1, Brian P Kenealy 1, James P Garcia 1, Curtis J Hedman 1, Ei Terasawa 1
PMCID: PMC4475715  PMID: 25853665

Abstract

Bisphenol A (BPA) is an industrial compound with pervasive distribution in the environments of industrialized countries. The U.S. Centers for Disease Control recently found that greater than 90% of Americans carry detectable levels of BPA, raising concern over the direct influences of this compound on human physiology. Epidemiologic evidence links elevated BPA serum concentrations to human reproductive dysfunction, although controlled studies on the acute effect of BPA exposure on reproductive function are limited, particularly in primates. We evaluated the effect of direct BPA exposure on female primate hypothalamic peptide release. Specifically, using a microdialysis method, we examined the effects of BPA (0.1, 1, and 10nM) directly infused to the stalk-median eminence on the release of GnRH and kisspeptin (KP) in mid to late pubertal ovarian intact female rhesus monkeys. We found that the highest level of BPA exposure (10nM) suppressed both GnRH and KP release, whereas BPA at lower concentrations (0.1 and 1nM) had no apparent effects. In addition, we measured BPA in plasma and hypothalamic dialysates after an iv bolus injection of BPA (100 μg/kg). We found a relatively stable distribution of BPA between the blood and brain (plasma:brain ≅ 5:1) persists across a wide range of blood BPA concentrations (1–620 ng/mL). Findings of this study suggest that persistent, high-level exposures to BPA could impair female reproductive function by directly influencing hypothalamic neuroendocrine function.

Bisphenol A (BPA) is an inexpensive, synthetic base for industrial preparation of polycarbonate plastic; it is also a component of resins that line cans for consumable packaging, dental sealants, and thermal printed receipts. To meet these manufacturing needs, 6 billion pounds of BPA was produced worldwide in 2008 (1). Manufacturing is projected to top 12 billion pounds by 2015, with the majority produced in the United States and Europe (2). Consequently, whereas BPA is already pervasive in the environment, these levels and human exposures are likely to increase in coming years.

More than 90% of Americans were recently found to carry detectable levels of BPA (3). The biological effect of these exposures on human health remains unclear; however, epidemiologic studies suggest that BPA levels found in humans are related to female reproductive dysfunction including polycystic ovarian syndrome (4, 5) and recurrent miscarriage (6). In addition, serum concentrations of BPA were found to covary with steroid hormone levels in men and women (7, 8). These correlations suggest that BPA could have an acute effect on human reproductive function, specifically through manipulation of circulating steroid hormones. However, based on little evidence of direct BPA influences on primate physiology, a recent National Toxicology Program review could not conclude whether BPA causes reproductive toxicity in exposed human adults (4), and further implied the necessity for more experimental investigations. Consequently, the current studies were designed to determine how circulating BPA might have a direct effect on neuroendocrine function in nonhuman primates.

GnRH released from the hypothalamus, stimulates the synthesis and release of gonadotropins (LH and FSH) from the anterior pituitary gland. The release of GnRH and LH are pulsatile (9–11) and they regulate gamete maturation and gonadal steroid synthesis and release in both sexes, and ovulation and the maintenance of luteal function in females (12–13). Abnormalities in pulsatile patterns of LH secretion (and presumably GnRH secretion) are associated with reproductive disorders such as polycystic ovarian syndrome, anorexia nervosa, and amenorrhea (14–15).

GnRH/LH release is greatly influenced by gonadal steroids, particularly by estradiol in females. Although it is speculated that estradiol can directly influence primate GnRH neurons through membrane-associated receptors (16), more commonly estradiol modifies GnRH release through transsynaptic mechanisms because GnRH neurons do not express estrogen receptor alpha (17). In contrast, primate hypothalamic kisspeptin (KP) neurons do express estrogen receptor alpha (18, 19) and control positive and negative feedback actions of estradiol on LH release (20–22).

Based on the estrogenic potential of BPA (23), we hypothesized that BPA alters release of GnRH and KP in the hypothalamus of female rhesus monkeys. To date, there are no reports of the direct influence of BPA exposure on primate in vivo GnRH or KP release. Therefore, in the present study we examined the effect of direct BPA exposure on the primate hypothalamus, focusing on the release dynamics of GnRH and KP. We also measured BPA concentrations in peripheral blood and the hypothalamus after an iv bolus injection of BPA (100 μg/kg). Collectively, the data provide direct experimental evidence that circulating BPA readily crosses the primate blood-brain barrier (BBB) and influences hypothalamic release of peptides critical to reproductive function.

Materials and Methods

Animals

Eight mid to late pubertal (37.9 ± 0.3 months of age, 5.6 ± 0.4 kg) female rhesus monkeys (Macaca mulatta) were used in this study. All animals were born and raised at the Wisconsin National Primate Research Center and housed in pairs (cages: 172 × 86 × 86 cm) in a room with a lighting schedule of 12 hours of light and 12 hours of dark, at a controlled temperature (22°C). They were fed a standard diet of Teklad Primate Chow (No. 2050, Harlan, Madison, WI) twice per day and water was available ad libitum. Fresh fruit or other enrichment was also provided on a daily basis. To define the menstrual stage, all animals were examined daily for sex-skin development and menstrual status. All direct BPA exposure experiments were conducted during the follicular phase of the menstrual cycle. That is, experiments were performed 8.2 ± 1.5 (10nM; n = 6), 8.3 ± 2.6 (1nM; n = 3), 6.7 ± 3.3 (0.1nM; n = 3), and 7.0 ± 1.4 (vehicle; n = 5) days after menstruation (one-way ANOVA, F (3, 13) = 0.1377; P = .9357). Note that female monkeys at the age that we examined exhibit irregular menstrual cycles with prolonged follicular phase, which are not accompanied by ovulation (48). Although we did not directly measure estradiol levels, based on our previous studies circulating estradiol levels in animals used in this study are estimated to be 20–80 pg/mL.

The protocol of this study was approved by the Animal Care and Use Committee; University of Wisconsin–Madison, and all experiments were conducted under the National Institutes of Health and U.S. Department of Agriculture guidelines.

Experimental design

In Experiment 1, we evaluated the amount of BPA in the hypothalamus, when BPA was injected iv. This experiment is very important for implications of acute exposure in humans. BPA was administered through an indwelling catheter in the saphenous vein. For the dose of BPA injection, we chose 100 μg/kg, based on the pharmacokinetic study in rhesus monkey by Doerge et al (24). BPA levels in the hypothalamus were assessed by the measurement of BPA in microdialysates. Blood samples were obtained from a total of four animals (two late pubertal animals from the microdialysis experiments and two additional late pubertal female rhesus monkeys). Blood samples (1 mL) were taken from the saphenous vein through an indwelling catheter at 5 minutes prior to injection and 0 (immediately prior to injection) 5, 15, 30, 60, 120, 180, 240, and 300 minutes after bolus injection, whereas microdialysates were collected continuously 120 minutes before through 300 minutes after the iv injection at 20-minute intervals. Plasma was separated by centrifugation and stored at −80°C until extraction and HPLC tandem mass spectroscopy (MS/MS) analysis.

To examine the effect of direct hypothalamic BPA exposure on two peptides in the hypothalamus, we conducted Experiment 2 in a total of six mid to late pubertal female monkeys. After 240 minutes of baseline dialysate collection, BPA at 10, 1, or 0.1nM was infused into the stalk-median eminence (S-ME) for 240 minutes through a microdialysis probe while dialysates were continuously collected at 20-minute intervals. All six monkeys were examined for the effects of 10nM BPA and vehicle, whereas three of the six and the remaining three were examined for BPA 1 and 0.1nM, respectively. The effects of doses and vehicle were tested in random order. To determine whether peptide release patterns recovered after BPA exposure, microdialysate collection was continued for an additional 240 minutes. As a control, vehicle was similarly infused, whereas dialysates were similarly collected. In each animal a minimum of 4 weeks separated microdialysis sampling sessions.

Preparation of BPA solutions

For direct infusions, BPA (> 99% purity; Sigma Chemical Company, St. Louis, MO) was dissolved into solution with water/ethanol (50/50 by volume) to 10−2M then serially diluted to 10−8, 10−9, or 10−10M using artificial cerebrospinal fluid (aCSF) [aCSF: 147mM NaCl, 2.7mM KCl, 1.2mM CaCl2, 0.85mM MgCl2, and bacitracin (4U/mL)]. These concentrations equate to approximately 2, 0.2, and 0.02 ng/mL, which span reported baseline human blood levels [0.3–4 ng/mL (24)]. Control (vehicle) solutions were prepared similar to BPA solutions, beginning with water/ethanol and serially diluting with aCSF. For iv injections, BPA was dissolved into solution with water/ethanol (60/40 by volume) to 10−2M then diluted with heparinized saline to a final concentration of 100 μg/mL. This concentration and dosing protocol mimics the dosing of Doerge et al (24) pharmacokinetic study, which intended to generate blood levels relevant to human exposure and allow for detection of the parent compound in blood and brain after iv injection.

Microdialysis experiments

The effect of BPA exposure on GnRH and KP release were examined using an in vivo microdialysis method, previously described (25, 26). aCSF was perfused into the S-ME of the hypothalamus through a microdialysis probe at a speed of 2 μL/min, whereas dialysates were collected at 20-minute intervals for up to 12 hours for each sampling session. To accommodate the measurement of GnRH and KP in the same samples, microdialysate samples were split into two vials.

BPA measurements by HPLC-MS/MS

Bisphenol A concentrations were analyzed by the Wisconsin State Laboratory of Hygiene using methods based upon solid phase extraction (Strata-X, Phemomenex, Torrance, CA) [Phenomenex Application Note 14454 (27)] and isotope dilution HPLC (Agilent 1100, Waldbronn, Germany) with MS/MS (5500 QTRAP, AB/SCIEX, Frammingham, MA) with atmospheric-pressure chemical ionization-negative mode ionization (28). Aliquots of 7 μL were injected on an Agilent Zorbax SB-C18 Rapid Resolution Cartridge Column, 2.1 × 30 mm, 3.5 micron (Agilent Technologies, Santa Clara, CA) using a binary gradient elution program (solvent A = 18 Mohm·cm water; solvent B = methanol) at a 0.8 mL/min flow rate as follows: 0–0.6 minutes hold at 25% B; to 98% B at 4.0 minutes; hold at 98% B to 7.0 minutes; to 25% B at 7.1 minutes, and hold at 25% B to 10 minutes for column re-equilibration. Pertinent MS/MS analysis parameters follow: bisphenol A multiple reaction monitoring transitions = 227.1/133.2 (quantitative) and 227.1/211.1 (confirmatory); d16 bisphenol A internal standard multiple reaction monitoring transition = 241.1/223.2. Declustering potential settings of −70 V (bisphenol A) and −90 V (d16 bisphenol A), collision energy settings of −36 V (bisphenol A) and −26 V (d16 bisphenol A), and collision cell exit potential of −7 V (bisphenol A quantitative), −9 V (bisphenol A confirmatory), and −5 V(d16 bisphenol A) were used. Source conditions used were as follows: curtain gas = 30 psig; collision gas setting = medium; nebulizer current = −4 V; source temperature = 500 degrees C; and nebulizer gas = 20 psig.

RIA

To accommodate measurements of GnRH and KP release in single samples, dialysates were divided into two vials (20 μL each) and samples were subjected to the GnRH and KP RIA. For GnRH, R42 antiserum was used as previously described (25). Assay sensitivity was 0.02 pg/tube. Intra- and interassay coefficients of variation were 8.9 and 11.6%, respectively. For KP, GQ2 antiserum was used as previously described (25). Assay sensitivity was 0.05 pg/tube. Intra- and interassay coefficients of variation were 8.0% and 8.9%, respectively. Peptide levels in dialysates were expressed in pg/mL/20 min.

Statistical analyses

We analyzed three critical parameters of peptide release for a 4-hour period after starting the BPA exposures: 1) mean peptide release, 2) pulse amplitude, and 3) interpulse interval (IPI). This 4-hour time frame focused analyses on the period of direct BPA exposure through the microdialysis probe. Changes in GnRH and KP release by BPA were evaluated with one-way ANOVA with respect to each parameter followed by Tukey post-hoc analyses. Peaks (pulses) of peptide release were identified using the PULSAR algorithm (29). The cutoff criteria for pulse determination, G1, G2, G3, G4, and G5 were 3.1, 2.26, 1.56, 1.13, and 0.83, respectively. Parameters of peptide release were calculated as follows: 1) mean release was calculated as an average value in pg/mL/20 min across all time points in the 4-hour range for each experiment; 2) pulse amplitude, defined as the difference between peak and trough was determined by the PULSAR program; 3) interpulse interval, defined as the intervals between peptide release peaks was also determined by the PULSAR program. Differences were considered significant at P ≤ .05.

Results

BPA concentration and distribution between blood and brain after bolus iv injection

BPA concentrations reached maximal levels in serum within 5 minutes after a bolus iv injection (Figure 1A). After reaching maximal concentrations (n = 4, 366.0 ± 95.6 ng/mL; mean ± SEM), plasma levels of BPA quickly declined, and returned to baseline levels between 180–240 minutes after the injection (Figure 1A). Elevated BPA was also detected in the S-ME within 20 minutes after iv injection (Figure 1A). BPA levels in the brain were approximately 12–23% of plasma BPA concentrations across the exposure period (Figure 1B). Note, that comparisons of blood and brain BPA concentrations were made between specific blood draw time points and the closest 20-minute dialysate collection period (ie, −5 refers to a blood draw taken 5 min prior to the iv BPA injection, which was compared with the −20 through 0 minutes brain dialysate collection period). The remaining comparisons were between the following time points: 15 to 0–20, 60 to 40–60, 120 to 100–120, and 240 to 220–240. The average percent plasma [BPA] detected in the brain across all time points was 17.42 ± 1.79. This percent distribution persisted across a wide range of blood BPA concentrations (1–620 ng/mL); the percent plasma [BPA] detected in the brain was not statistically different between any time point before or after iv BPA challenge (one-way ANOVA F(5, 9) = 1.206; P = .3795).

Figure 1.

Figure 1.

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Coordinate BPA measurements in the blood and brain after a bolus iv injection of BPA. A, Representative case of BPA levels in plasma (open square) and the hypothalamus (closed circle) before and after a BPA injection (100 μg/mL) obtained from the same experiment is shown. Note that BPA levels in the brain were assessed by collecting microdialysates from the stalk median eminence at 20-min intervals, whereas circulating plasma BPA levels were assessed by serial blood sampling at 5–60-min intervals. B, Percent plasma [BPA] detected in the brain. BPA levels in the brain were approximately 12–23% of plasma BPA concentrations across the exposure period. Note that comparisons of blood and brain BPA concentrations were made between specific blood draw time points and the closest 20-min dialysate collection period; ie, −5 refers to a blood draw taken 5 min prior to the iv BPA injection, which was compared with the −20- through 0-min brain dialysate collection period. The remaining comparisons were between the following time points: 15 to 0–20, 60 to 40–60, 120 to 100–120, and 240 to 220–240. The percent plasma [BPA] detected in the brain was not statistically different between any time point [one-way ANOVA F(5, 9) = 1.206; P = .3795].

Effects of acute BPA exposures on GnRH release

Mean GnRH release was suppressed during direct hypothalamic exposure to the highest dose of BPA (Table 1; control: 2.34 ± 0.23; BPA: 10nM, 1.08 ± 0.26; 1nM, 2.07 ± 0.22; 0.1nM, 2.57 ± 0.19 pg/mL; one-way ANOVA F(3, 13) = 7.759; P = .0032; Tukey post hoc at α = 0.05; 10nM BPA < control, 10nM BPA < 0.1nM BPA). BPA exposure also suppressed the average GnRH pulse amplitude (Table 1; control: 4.65 ± 0.47; BPA: 10nM, 2.17 ± 0.60; 1nM, 3.00 ± 0.55; 0.1nM, 4.28 ± 0.22 pg/mL; one-way ANOVA F(3, 13) = 4.925; P = .0168; Tukey post hoc at α = 0.05; 10nM BPA < control). In general, GnRH pulses detected during BPA exposure were low and prolonged resulting in low pulse amplitude. The IPI was not significantly changed by any BPA exposure, although in general the IPI became more variable compared with vehicle control (Figure 2 and Table 1; control: 55.0 ± 9.57; BPA: 10nM, 90.0 ± 23.8; 1nM, 80.0 ± 11.6; 0.1nM, 73.3 ± 24.0 min; one-way ANOVA F(3, 13) = 0.6445; P = .6001).

Table 1.

GnRH Release Dynamics in S-ME of Young Adult Female Rhesus Monkeys During BPA Exposure

GnRH Release, pg/mL Pulse Amplitude, pg/mL Interpulse Interval, min
Control
    (n = 5) 2.34 ± 0.23 4.65 ± 0.47 55.0 ± 9.57
BPA
    10nm (n = 6) 1.08 ± 0.26a 2.17 ± 0.60b 90.0 ± 23.8
    1nm (n = 3) 2.07 ± 0.22 3.00 ± 0.55 80.0 ± 11.6
    0.1nm (n = 3) 2.57 ± 0.19 4.28 ± 0.22 73.3 ± 24.0
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Results are shown as mean ± sem

a

Mean GnRH release is suppressed during direct BPA exposure compared with direct vehicle exposure; one-way ANOVA F(3,13) = 7.759; P = .0032; Tukey post hoc at α = 0.05; 10nm BPA < vehicle, 10nm BPA < 0.1.nm BPA.

b

GnRH pulse amplitude is decreased during direct BPA exposure compared with direct vehicle exposure; one-way ANOVA F(3,13) = 4.925; P = .0168; Tukey post hoc at α = 0.05; 10nm BPA < vehicle.

Figure 2.

Figure 2.

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Direct BPA exposure suppresses hypothalamic GnRH and KP release. Example cases for the effect of BPA (10, 1, and 0.1nM) or vehicle directly infused to the S-ME on GnRH (left panels) and KP (right panels) release in the female rhesus monkey hypothalamus. aCSF was continuously infused through a microdialysis probe while dialysates were collected at 20-min intervals over a 12-h period. Dashed lines define the periods of BPA or vehicle control infusions. Panels A–D, G, and H are data collected from one animal. Panels E, F, I, and J are data collected from another animal. BPA exposure at the highest dose significantly suppressed GnRH and KP release dynamics. Peptide release pulses detected with the PULSAR algorithm are indicated with an asterisk (*). Note that peptide pulses during BPA exposure were generally prolonged low peaks.

Effects of acute BPA exposures on KP release

Similar to GnRH, mean KP release was suppressed during direct hypothalamic exposure to the highest dose of BPA (Table 2; control: 2.95 ± 0.38; BPA: 10nM, 1.05 ± 0.12; 1nM, 2.27 ± 0.13; 0.1nM, 3.17 ± 0.23 pg/mL; one-way ANOVA F(3, 13) = 16.58, P < .0001; Tukey post hoc at α = 0.05, 10nM BPA < control, 10nM BPA < 1nM BPA, 10nM BPA < 0.1.nM BPA). BPA exposure also suppressed the average KP pulse amplitude (Table 2; control: 4.95 ± 0.29; BPA: 10nM, 1.75 ± 0.22; 1nM, 3.91 ± 0.52; 0.1nM, 5.07 ± 0.49 pg/mL; one-way ANOVA F(3, 13) = 25.43; P < .0001; Tukey post hoc at α = 0.05, 10nM BPA < control, 10nM BPA < 1nM BPA, 10nM BPA < 0.1.nM BPA). The IPI was not significantly changed by any BPA exposure (Table 2; Control: 73.3 ± 6.67, BPA: 10nM, 65.0 ± 5.0; 1nM, 86.7 ± 17.6; 0.1nM, 53.3 ± 6.67 min; one-way ANOVA F(3, 13) = 2.149; P = .1433).

Table 2.

KP Release Dynamics in Stalk Median Eminence of Young Adult Female Rhesus Monkeys During BPA Exposure

KP release, pg/mL Pulse amplitude, pg/mL Interpulse interval, min
Control
    (n = 5) 2.95 ± 0.38 4.95 ± 0.29 73.3 ± 6.67
BPA
    10nm (n = 6) 1.05 ± 0.12a 1.75 ± 0.22b 65.0 ± 5.0
    1nm (n = 3) 2.73 ± 0.13 3.91 ± 0.52 86.7 ± 17.6
    0.1nm (n = 3) 3.17 ± 0.23 5.07 ± 0.49 53.3 ± 6.67
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Results are shown as mean ± sem.

a

Mean KP release is suppressed during direct BPA exposure compared with direct vehicle exposure; one-way ANOVA F(3,13) = 16.58; P < .0001; Tukey post hoc at α = 0.05; 10nm BPA < control, 10nm BPA < 1nm BPA, 10nm BPA < 0.1.nm BPA.

KP pulse amplitude is decreased during direct BPA exposure compared with direct vehicle exposure; ANOVA F(3,13) = 25.43; P < .0001; Tukey post hoc at α = 0.05; 10 nm BPA < control, 10nm BPA < 1nm BPA, 10nm BPA < 0.1.nm BPA.

Discussion

BPA has been implicated as an endocrine active agent with potential to impair reproductive function. To date, the vast majority of reproductive function studies have focused on the effect of pre or perinatal BPA exposures, particularly in rodents, and less attention has been made toward the direct effect of BPA on human or primate physiology. Our work focused on the direct effect of BPA exposure on the release of two neuropeptides, at doses (0.02–2 ng/mL) consistent with presumed baseline human serum concentrations (0.3–4 ng/mL) (30). In addition, by using the rhesus monkey model, findings of this study are more directly translatable to humans. We found that BPA infusion directly into the S-ME through a microdialysis probe suppressed the release of both GnRH and KP in the hypothalamus of intact mid to late pubertal female rhesus monkeys. GnRH and KP are known to play a key role in mammalian reproductive function. The minimum effective dose was 10nM (∼2 ng/mL). In addition, after a bolus iv BPA injection (100 μg/kg), we found that BPA readily crossed the BBB within 20 minutes. More importantly, we found that a relatively stable distribution of BPA between the blood and brain (plasma:brain ≅ 5:1) persists across a wide range of blood BPA concentrations (1–620 ng/mL). Altogether, these results suggest that BPA exposures could modify reproductive function by altering patterns of hypothalamic peptide release.

It seems that the effect of BPA on neuropeptide release was restricted to the period of exposure. We did not perform statistical analyses on data obtained after cessation of BPA exposure, as we were unable to collect microdialysate samples for another full 4 hours for every animal, the period necessary for meaningful pulse pattern analysis. Nonetheless, the findings of this study clearly suggest that GnRH and KP release patterns were influenced during direct exposure of the hypothalamus to BPA at 10nM or ∼2 ng/mL.

Relevance to human exposures and neuropeptide release

The direct exposure of BPA at the dose of 10nM or 2 ng/mL has relevance to proposed human exposure levels [blood concentrations between 0.3–4 ng/mL (30)]. Therefore, whereas we found that neuropeptide release patterns returned to pre-exposure levels when BPA levels decreased to baseline in rhesus monkeys, human screening studies suggest it is feasible that humans could continuously carry blood concentrations of BPA we have shown to affect neuropeptide release in these studies. Specifically, with the clear effect on neuropeptide release during direct hypothalamic exposure to BPA at 2 ng/mL, and apparent BPA distribution between blood and brain (∼5:1), it is plausible that humans with continuous serum concentrations of ∼10 ng/mL would have a significant, sustained effect on GnRH and KP release and consequently gonadotropin release, serum steroid hormone profiles, and reproductive function. It is important to note that our microdialysis probe targeted the S-ME, a region not protected by the BBB. As a result, the distribution of BPA between the blood and brain reported in this study may not be applicable to regions protected by the BBB. In addition, only a limited number of animals were available for analysis of BPA in both the blood and brain (N = 2 female animals). It is possible that inclusion of more animals would influence the measured blood:brain ratio. It is also possible that the blood:brain BPA ratio may be different between sexes and ages. Consequently, it is possible that the data presented in this study may not represent a typical BPA blood:brain ratio in males or sexually matured adult females.

The present study did not determine the effect of hypothalamic BPA exposure on serum gonadotropin levels. However, in general, an increased level of GnRH release reflects an increased level of LH, whereas a decreased level of GnRH release is parallel to a decreased LH level. Moreover, previous work in sheep has addressed the effect of continuous iv exposure to BPA on ovine plasma LH release dynamics. Collet and colleagues (31) found that stable BPA plasma concentrations of 38 ng/mL reduced LH pulse frequency. Importantly, LH release is closely coupled to hypothalamic GnRH release dynamics in female rhesus monkeys. Consequently, based on our current observation that BPA (10nM) directly suppressed GnRH pulse amplitude and mean release, it is likely that sustained exposures to BPA would impair LH pulsatility, which is critical to ovulation and luteal function in females (13).

Mechanism of BPA influence on GnRH and KP release

In the present study we initially suspected that any BPA influence on KP or GnRH release would be due to an estrogenic influence. We did not directly compare the effects of BPA on release of GnRH and KP with those of estradiol in female rhesus monkeys, as this laboratory has already reported (32, 34) the effects of estradiol benzoate (EB) infusion into the S-ME on GnRH and KP release. Given the well-documented ovarian estrogen-negative feedback actions on the hypothalamic-pituitary-gonadal axis, we had speculated that BPA might suppress GnRH release. However, surprising recent evidence from our laboratory suggests EB, directly infused into the ovariectomized female primate S-ME, actually stimulates GnRH release (32) and KP release (34). Surprisingly, continuous infusion of EB into the S-ME for 4 hours (similar to the present study) or 7 hours also stimulated release of GnRH and KP (33, 34). Moreover, local inhibition of aromatase, a terminal enzyme in estradiol synthesis, rapidly suppressed basal GnRH release (32). Consequently, we speculate that direct EB stimulation of GnRH release is indicative of estradiol acting as a neuromodulator. Specifically, estradiol may serve to increase basal excitability at the GnRH neuroterminals. Importantly, the stimulatory effect of EB (32) is in contrast with the findings of this study with direct exposure of the S-ME to BPA, which substantially suppressed GnRH release. It is possible that BPA directly interferes with neuroestradiol modulation of GnRH neurons, though this is less likely as the effective dose of BPA is similar to estradiol levels in the primate hypothalamus (32) and BPA affinity for estrogen receptors is substantially lower than estradiol (35). Alternatively, BPA may limit the local production of neuroestradiol, thereby suppressing GnRH release. This speculation is based on the report showing that prolonged exposure to BPA in adult rats impaired ovarian production of estradiol, apparently through degradation of aromatase (36). In addition, exposure of a human adenocarcinoma cell line (H295R) to BPA inhibited 17,20 lyase activity, an enzyme upstream of aromatase in estradiol synthesis (37). It is also possible that BPA quickly diffuses into the arcuate nucleus, where KP neurons are concentrated (38), resulting in the suppression of KP release and subsequent GnRH release, as KP neurons express estrogen receptor alpha. Deeper investigations of 1) the effect of BPA on brain steroidogenesis, 2) the differential mechanisms of estradiol and BPA action over KP/GnRH release, and 3) diffusion pattern of BPA within the hypothalamus are imperative; the results will help us understand the novel effect of neurosteroids and BPA on reproductive physiology.

In addition to GnRH, KP has an integral role in reproductive function. KP is released from neurons in the arcuate nucleus and regulates the release of GnRH (38), including preovulatory surges of GnRH in females (39). In fact, our laboratory previously reported that pulses of KP release were highly coordinated with GnRH pulses in the female primate hypothalamus (40), suggesting a very tight KP-mediated regulation of GnRH release in the female primate. Importantly, a recent study in our laboratory suggests that in pubertal monkeys ovarian estradiol is inhibitory to KP release, as KP release elevates after ovariectomy and systemic exposure to estradiol suppresses KP release (41). Moreover, ovariectomy increases, whereas estradiol replacement decreases KP mRNA expression (42).

We found that KP release was clearly suppressed during direct exposure of the S-ME to BPA (10nM). In fact, the suppression of KP closely resembled the BPA-mediated suppression of GnRH release. Consequently, it is tempting to suggest that BPA effects on GnRH release are the consequence of KP neuron suppression. Further, the current results suggest an estrogenic effect of BPA on KP release. However, it is important to mention that BPA may also act on KP neuron physiology through impairment of typical excitatory and inhibitory neurotransmission. For example, in male rats, developmental post natal exposure to BPA results in a reduction of hypothalamic GnRH peptide and glutamate content and an elevation of GABA (43, 44). Our laboratory has previously reported that GnRH release is suppressed by GABA neurotransmission in prepubertal female rhesus monkeys (45), a phenomenon that seems to involve KP signaling (25). In addition, the typical pubertal increase in GnRH release is accompanied by a decrease in GABA levels, followed by an elevation of glutamate levels in the female primate hypothalamus (46). Whether GABA and glutamate transmission are interrupted by BPA, and whether this is responsible for altered hypothalamic peptide release remain critical future questions.

Conclusion

Recent correlations have been drawn between adult reproductive physiology and elevated body burdens of BPA. For example, a recent study reports that females with high BPA burdens are potentially more susceptible to miscarriage (6) and males with high occupational exposure had lower sperm counts (46) and report reduced libido (47). The results of this study suggest that direct BPA exposure influences GnRH and KP release patterns in the nonhuman primate hypothalamus. Coordinated release of these peptides, particularly GnRH, is imperative for normal reproductive function. Most importantly, the effective BPA doses determined in this study (direct hypothalamic exposure to 10nM or ∼2 ng/mL) are close to baseline human unconjugated BPA serum levels [0.3–4 ng/mL (24)[. Altogether, these results imply that BPA could influence human reproductive function through modification of hypothalamic peptide release and emphasize the need to further examine the mechanisms of BPA action on neuroendocrine function.

Acknowledgments

This work was supported by National Institutes of Health (NIH) Grants K99/R00ES020878 (for J.R.K), R01HD15433, and R01HD11355 (for E.T.). This work also benefitted from NIH support (OD011106/RR00061) to the Wisconsin National Primate Research Center.

Disclosure Summary: The authors have nothing to disclose.

Footnotes

Abbreviations:
aCSF
artificial cerebro-spinal fluid
BBB
blood-brain barrier
BPA
Bisphenol A
EB
estradiol benzoate
IPI
interpulse interval
KP
kisspeptin
MS/MS
tandem mass spectroscopy
S-ME
stalk-median eminence.

References

  • 1. Burridge E. Chemical profile: Bisphenol A. ICIS Chemical Business. 2008;274:48 http://www.icis.com/Articles/2008/01/14/9092025/chemical-profile-bisphenol-a.html. [Google Scholar]
  • 2. Global Industry Analysts, Inc. 2010. Bisphenol A: A global strategic business report. http://www.strategyr.com/Bisphenol_A_Market_Report.asp.
  • 3. Calafat AM, Ye X, Wong LY, Reidy JA, Needham LL. Exposure of the U.S. population to bisphenol A and 4-tertiary-octylphenol: 2003–2004. Environ Health Perspect. 2008;116:39–44. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4. National Toxicology Program U.S. Department of Health and Human Services; Center for the Evaluation of Risks to Human Exposure (2008) NTP brief on Bisphenol A [CAS No. 80–05-07]. http://ntp.niehs.nih.gov/ntp/ohat/bisphenol/bisphenol.pdf.
  • 5. Takeuchi T, Tsutsumi O, Ikezuki Y, Takai Y, Taketani Y. Positive relationship between androgen and the endocrine disruptor, bisphenol A, in normal women and women with ovarian dysfunction. Endocr J. 2004;51:165169. [DOI] [PubMed] [Google Scholar]
  • 6. Sugiura-Ogasawara M, Ozaki Y, Sonta S, Makino T, Suzumori K. Exposure to bisphenol A is associated with recurrent miscarriage. Hum Reprod. 2005;20:2325–2329. [DOI] [PubMed] [Google Scholar]
  • 7. Meeker JD, Calafat AM, Hauser R. Urinary bisphenol A concentrations in relation to serum thyroid and reproductive hormone levels in men from an infertility clinic. Environ Sci Technol. 2010;44:1458–1463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8. Takeuchi T, Tsutsumi O. Serum bisphenol A concentrations showed gender differences, possibly linked to androgen levels. Biochem Biophys Res Commun. 2002;291:76–78. [DOI] [PubMed] [Google Scholar]
  • 9. Levine JE, Pau KY, Ramirez VD, Jackson GL. Simultaneous measurement of luteinizing hormone-releasing hormone and luteinizing hormone release in unanesthetized, ovariectomized sheep. Endocrinology. 1982;111:1449–1455. [DOI] [PubMed] [Google Scholar]
  • 10. Gearing M, Terasawa E. Luteinizing hormone releasing hormone (LHRH) neuroterminals mapped using the push-pull perfusion method in the rhesus monkey. Brain Res Bull. 1988;21:117–121. [DOI] [PubMed] [Google Scholar]
  • 11. Dierschke DJ, Bhattacharya AN, Atkinson LE, Knobil E. Circhoral oscillations of plasma LH levels in the ovariectomized rhesus monkey. Endocrinology. 1970;87:850–853. [DOI] [PubMed] [Google Scholar]
  • 12. Crowley WF, Jr, Filicori M, Spratt DI, Santoro NF. The physiology of gonadotropin-releasing hormone (GnRH) secretion in men and women. Rec Prog Horm Res. 1985;41:473–531. [DOI] [PubMed] [Google Scholar]
  • 13. Knobil E, Hotchkiss J. 1988. The menstrual cycle and its neuroendocrine control. In: The physiology of reproduction. Knobil E, Neill J, eds. Raven Press: NY; 1971–1994. [Google Scholar]
  • 14. Yen SSC. 1980. Neuroendocrine regulation of the menstrual cycle. In: Neuroendocrinology: A hospital practice book. Krieger DT, Hughes JC, eds. Sinauer Associates, Inc: MA; 259–272. [Google Scholar]
  • 15. Marshall LA, Monroe SE, Jaffe RB. 1988. Physiologic and therapeutic aspects of GnRH and its analogs. In: Frontiers in neuroendocrinology. Martini L, Ganong WF, eds. Vol 10 Raven Press: NY; 239–278. [Google Scholar]
  • 16. Noel SD, Keen KL, Baumann DI, Filardo EJ, Terasawa E. Involvement of G protein–coupled receptor 30 (GPR30) in rapid action of estrogen in primate LHRH neurons. Mol Endocrinol. 2009;23:349–359. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17. Shivers BD, Harlan RE, Morrell JI, Pfaff DW. Absence of oestradiol concentration in cell nuclei of LHRH-immunoreactive neurones.. Nature. 1983;304:345–347. [DOI] [PubMed] [Google Scholar]
  • 18. Franceschini I, Lomet D, Cateau M, Delsol G, Tillet Y, Caraty A. Kisspeptin immunoreactive cells of the ovine preoptic area and arcuate nucleus co-express estrogen receptor alpha. Neurosci Lett. 2006;401:225–230. [DOI] [PubMed] [Google Scholar]
  • 19. Herbison AE. Multimodal influence of estrogen upon gonadotropin-releasing hormone neurons. Endocr Rev. 1998;19:302–330. [DOI] [PubMed] [Google Scholar]
  • 20. Smith JT. Sex steroid regulation of kisspeptin circuits. Adv Exp Med Biol. 2013;784:275–295. [DOI] [PubMed] [Google Scholar]
  • 21. Christian CA, Moenter SM. The neurobiology of preovulatory and estradiol-induced gonadotropin-releasing hormone surges. Endocr Rev. 2010;31:544–577. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22. Pielecka-Fortuna J, Chu Z, Moenter SM. Kisspeptin acts directly and indirectly to increase gonadotropin-releasing hormone neuron activity and its effects are modulated by estradiol. Endocrinology. 2008;149:1979–1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23. Dodds EC, Lawson W. Synthetic estrogenic agents without the phenanthrene nucleus. Nature. 1936;137:996. [Google Scholar]
  • 24. Doerge DR, Twaddle NC, Woodling KA, Fisher JW. Pharmacokinetics of bisphenol A in neonatal and adult rhesus monkeys. Toxicol Appl Pharmacol. 2010;248:1–11. [DOI] [PubMed] [Google Scholar]
  • 25. Kurian JR, Keen KL, Guerriero KA, Terasawa E. Tonic control of kisspeptin release in prepubertal monkeys: Implications to the mechanism of puberty onset. Endocrinology. 2012;153:3331–3336. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26. Frost SI, Keen KL, Levine JE, Terasawa E. Microdialysis methods for in vivo neuropeptide measurement in the stalk-median eminence in the rhesus monkey. J Neuorsci Methods. 2008;168:26–34. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27. Phenomenex Strata-X SPE Application Note 14454. http://www.phenomenex.com/Application/Detail/14454?alias=Strata Accessed on March 12, 2012.
  • 28. Ye X, Tao LJ, Needham LL, Calafat AM. Automated on-line column-switching HPLC-MS/MS method for measuring environmental phenols and parabens in serum. Talanta. 2008;76:865–871. [DOI] [PubMed] [Google Scholar]
  • 29. Merriam GR, Wachter KW. Algorithms for the study of episodic hormone secretion. Am J Physiol. 1982;243:E310–E318 [DOI] [PubMed] [Google Scholar]
  • 30. Vandenberg LN, Chahoud I, Heindel JJ, Padmanabhan V, Paumgartten FJ, Schoenfelder G. Urinary, circulating, and tissue biomonitoring studies indicate widespread exposure to bisphenol A. Environ Health Perspect. 2010;118:1055–1070. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31. Collet SH, Picard-Hagen N, Viguié C, Lacroix MZ, Toutain PL, Gayrard V. Estrogenicity of bisphenol A: A concentration-effect relationship on luteinizing hormone secretion in a sensitive model of prepubertal lamb. Toxicol Sci. 2010;117:54–62. [DOI] [PubMed] [Google Scholar]
  • 32. Kenealy BP, Kapoor A, Guerriero KA, et al. Neuroestradiol in the hypothalamus contributes to the regulation of gonadotropin releasing hormone release. J Neuroscience. 2013;33:19051–19059. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33. Kenealy BP, Keen KL, Garcia JP, Kurian JR, Terasawa E. Effects of dose and duration of estradiol benzoate infusion into the stalk median eminence on GnRH release in female rhesus macaques. Abstracts of the International Congress of Endocrinology and the 96th Annual Meeting of the Endocrine Society, Chicago, IL, 2014 (Abstract PP30–4). [Google Scholar]
  • 34. Kenealy BP, Keen KL, Garcia JP, Kurian JR, Terasawa E. Estradiol benzoate infusion into the stalk median eminence (S-ME) of ovariectomized female rhesus monkeys rapidly stimulates kisspeptin release. Abstracts of the 8th International Congress of Neuroendocrinology, Sydney, Australia, 2014 (Abstract 80). [Google Scholar]
  • 35. Blair RM, Fang H, Branham WS, et al. The estrogen receptor relative binding affinities of 188 natural and xenochemicals: Structural diversity of ligands. Toxicol Sci. 2000;54(1):138–53. [DOI] [PubMed] [Google Scholar]
  • 36. Zhang X, Chang H, Wiseman S, et al. Bisphenol A disrupts steroidogenesis in human H295R cells. Toxicol Sci. 2011;121:320–327. [DOI] [PubMed] [Google Scholar]
  • 37. Lee SG, Kim JY, Chung JY, et al. Bisphenol A exposure during adulthood causes augmentation of follicular atresia and luteal regression by decreasing 17β-estradiol synthesis via downregulation of aromatase in rat ovary. Environ Health Perspect. 2013;121:663–669. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38. Dungan HM, Clifton DK, Steiner RA. Minireview: Kisspeptin neurons as central processors in the regulation of gonadotropin-releasing hormone secretion. Endocrinology. 2006;147:1154–1158. [DOI] [PubMed] [Google Scholar]
  • 39. Guerriero KA, Keen KL, Millar RP, Terasawa E. Developmental changes in GnRH release in response to kisspeptin agonist and antagonist in female rhesus monkeys (Macaca mulatta): Implication for the mechanism of puberty. Endocrinology. 2012;153:825–836. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40. Keen KL, Wegner FH, Bloom SR, Ghatei MA, Terasawa E. An increase in kisspeptin-54 release occurs with the pubertal increase in luteinizing hormone-releasing hormone-1 release in the stalk-median eminence of female rhesus monkeys in vivo. Endocrinology. 2008;149:4151–4157. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41. Guerriero KA, Keen KL, Terasawa E. Developmental increase in kisspeptin-54 release in vivo is independent of the pubertal increase in estradiol in female rhesus monkeys (Macaca mulatta). Endocrinology. 2012;153:1887–1897. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42. Alçin E, Sahu A, Ramaswamy S, et al. Ovarian regulation of kisspeptin neurones in the arcuate nucleus of the rhesus monkey (macaca mulatta). J Neuroendocrinol. 2013;25:488–496. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43. Cardoso N, Pandolfi M, Lavalle J, et al. Probable gamma-aminobutyric acid involvement in bisphenol A effect at the hypothalamic level in adult male rats. J Physiol Biochem. 2011;67:559–567. [DOI] [PubMed] [Google Scholar]
  • 44. Cardoso N, Pandolfi M, Ponzo O, et al. Evidence to suggest glutamic acid involvement in Bisphenol A effect at the hypothalamic level in prepubertal male rats. Neuro Endocrinol Lett. 2010;31:512–516. [PubMed] [Google Scholar]
  • 45. Mitsushima D, Hei DL, Terasawa E. Gamma-aminobutyric acid is an inhibitory neurotransmitter restricting the release of luteinizing hormone-releasing hormone before the onset of puberty. Proc Natl Acad Sci U S A. 1994;91:395–399. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 46. Terasawa E, Luchansky LL, Kasuya E, Nyberg CL. An increase in glutamate release follows a decrease in gamma aminobutyric acid and the pubertal increase in luteinizing hormone releasing hormone release in the female rhesus monkeys. J Neuroendocrinol. 1999;11:275–282. [DOI] [PubMed] [Google Scholar]
  • 47. Li DK, Zhou Z, Miao M, et al. Relationship between urine bisphenol-A level and declining male sexual function. J Androl. 2010;31:500–506. [DOI] [PubMed] [Google Scholar]
  • 48. Terasawa E, Nass TE, Yeoman RR, Loose MD, Schultz NJ. 1983 Hypothalamic control of puberty in the female rhesus macaque. In: Neuroendocrine Aspects of Reproduction. Norman RL, ed. Academic Press: NY; 149–182. [Google Scholar]

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