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Endocrinology, doi:10.1210/en.2003-1174
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Endocrinology Vol. 145, No. 2 592-603
Copyright © 2004 by The Endocrine Society

Inhibition of Testicular Steroidogenesis by the Xenoestrogen Bisphenol A Is Associated with Reduced Pituitary Luteinizing Hormone Secretion and Decreased Steroidogenic Enzyme Gene Expression in Rat Leydig Cells

Benson T. Akingbemi, Chantal M. Sottas, Anna I. Koulova, Gary R. Klinefelter and Matthew P. Hardy

Center for Biomedical Research, The Population Council (B.T.A., C.M.S., A.I.K., M.P.H.), New York, New York 10021; and Reproductive Toxicology Division, National Health and Environmental Effects Research Laboratory, United States Environmental Protection Agency (G.R.K.), Research Triangle Park, North Carolina 27711

Address all correspondence and requests for reprints to: Dr. Matthew P. Hardy, The Population Council, 1230 York Avenue, New York, New York 10021. E-mail: m-hardy{at}popcbr.rockefeller.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Exposure of humans to bisphenol A (BPA), a monomer in polycarbonate plastics and a constituent of resins used in food packaging and dentistry, is significant. In this report exposure of rats to 2.4 µg/kg·d (a dose that approximates BPA levels in the environment) from postnatal d 21–35 suppressed serum LH (0.21 ± 0.05 ng/ml; vs. control, 0.52 ± 0.04; P < 0.01) and testosterone (T) levels (1.62 ± 0.16 ng/ml; vs. control, 2.52 ± 0.21; P < 0.05), in association with decreased LHß and increased estrogen receptor ß pituitary mRNA levels as measured by RT-PCR. Treatment of adult Leydig cells with 0.01 nM BPA decreased T biosynthesis by 25% as a result of decreased expression of the steroidogenic enzyme 17{alpha}-hydroxylase/17–20 lyase. BPA decreased serum 17ß-estradiol levels from 0.31 ± 0.02 ng/ml (control) to 0.22 ± 0.02, 0.19 ± 0.02, and 0.23 ± 0.03 ng/ml in rats exposed to 2.4 µg, 10 µg, or 100 mg/kg·d BPA, respectively, from 21–35 d of age (P < 0.05) due to its ability to inhibit Leydig cell aromatase activity. Exposures of pregnant and nursing dams, i.e. from gestation d 12 to postnatal d 21, decreased T levels in the testicular interstitial fluid from 420 ± 34 (control) to 261 ± 22 (P < 0.05) ng/ml in adulthood, implying that the perinatal period is a sensitive window of exposure to BPA. As BPA has been measured in several human populations, further studies are warranted to assess the effects of BPA on male fertility.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
HIGHER TRENDS in the incidence of several endocrine-related diseases, such as hypospadias and testicular, breast, and prostate cancer, are thought to be associated with exposures to environmental chemicals with estrogenic activity (1). Synthetic estrogens, also called xenoestrogens, are a diverse group of compounds in the environment that mimic the action of the natural hormone 17ß-estradiol (E2) in estrogen-dependent tissues (Fig. 1Go). Agents that cause adverse effects in target organs by interfering with the interaction of endogenous hormones and their receptors have been designated endocrine disruptors (EDs). The reproductive toxicity of EDs is mediated primarily by steroid hormone receptors, estrogen (ER) and androgen receptors. The ER is a member of the nuclear receptor superfamily of proteins that modulate gene expression typically as a consequence of ligand binding. Two subtypes of ER have been cloned to date, ER{alpha} and ERß. Although bisphenol A (BPA) preferentially binds ERß, it is capable of inducing ER{alpha}- and ERß-mediated gene expression with comparable efficacy (2). Binding of ER by a ligand induces conformational changes in the receptor, enabling the bound receptor complex to interact with specific sites on DNA. Once bound to DNA, the ligand-receptor complex alters the expression of estrogen-responsive genes that alter cell growth and differentiation. Although the effects of exposures to environmental chemicals in adulthood are typically transient, chemical exposures that alter gene activity during development disrupt differentiated function in hormone-responsive tissues of the adult (3). The possibility that xenoestrogens may cause adverse effects in the reproductive tract was first highlighted by reports on adolescent sons born to pregnant women who had taken the highly potent synthetic estrogen diethylstilbestrol (DES). These individuals developed a variety of testicular and epididymal abnormalities in adulthood (4).



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  		FIG. 1. The chemical structure of ER agonists, BPA, E2, DES, and HPTE, and the antiestrogen ICI 182,780.

 
BPA [2,2-(4,4-dihydroxydiphenol)propane] is an estrogenic compound that is widely used in the manufacture of polycarbonate plastics, which serve as containers for foods and beverages and as a constituent of dental sealants. The core structure of BPA resembles that of the natural estrogen E2, consisting of two phenolic rings joined by a bridging carbon (Fig. 1Go). Exposure of the human population to BPA is significant given its widespread use in consumer products. Indeed, BPA and its metabolites have been measured in the blood of normal men (1.49 ± 0.11 ng/ml) and women (0.64 ± 0.10 ng/ml), in food items (4–23 µg/can) and beverages (7–58 µg/g), and in saliva immediately after the application of certain dental sealants (90–913 µg/saliva·1 h); these concentrations are similar to those that induce proliferation of estrogen-sensitive tissues in vitro (5, 6). In rodents, developmental exposure to BPA increased prostate weight (7) and decreased epididymal weight (3) and daily sperm production (8). Thus, exposure to BPA exerts deleterious effects in male reproductive tissues. However, there is ongoing debate about whether BPA poses a hazard to human health at exposure levels typical of its occurrence in the environment, because reports of adverse reproductive effects at low doses in laboratory species were hard to duplicate in other studies (9, 10). These discrepancies were serious enough to make the Environmental Protection Agency constitute an expert panel to review the available data (11). The panel concluded that some ER agonists exert biological effects at levels lower than those presently considered safe. Thus, continued investigation of the antifertility effects of this agent at low dose exposure levels is warranted.

Leydig cells produce the primary male steroid hormone testosterone (T), which stimulates virilization of the male urogenital system in fetal life and supports spermatogenesis and fertility in adulthood. Therefore, EDs that affect Leydig cell function potentially affect male fertility. Localization of ERs and aromatase activity at all levels of the hypothalamus-pituitary-gonadal axis suggests a role for ER-mediated activity in reproductive function. Leydig cells express ERs and are subject to estrogen action (12). The aromatase enzyme, which is encoded by the cyp19 gene and catalyzes the conversion of androgens to E2, is expressed more highly in the male reproductive tract than in other tissues in rodents (13). In the rat, prepubertal Sertoli cells are a source of estrogen, but testicular aromatase activity primarily resides in Leydig cells from 21 d of age onward, although aromatase has also been detected within mature germ cells and spermatozoa (14). Leydig cell T secretion is under the control of the gonadotropin LH, and LH release from the pituitary is regulated by hypothalamic GnRH, which signals through its receptors in pituitary gonadotropes. The locations of BPA-associated lesions within the hypothalamus-pituitary-gonadal axis have not been determined. Therefore, the objectives of the present study were 2-fold: 1) to determine whether exposure to environmentally relevant BPA levels affects testicular steroidogenesis, and if so, to identify the mechanisms associated with observed effects; and 2) to determine the outcome of perinatal and chronic BPA exposures on androgen biosynthesis by Leydig cells. We show that BPA has an inhibitory effect on testicular steroidogenesis at low dose exposure levels, presumably acting via the ER, while also suppressing aromatase gene expression and E2 biosynthesis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Experimental design
The Long-Evans strain of rat (Charles River, Wilmington, MA) was used in these studies because an extensive toxicological database is available from this strain in studies of EDs and testicular function (15). In all experiments, feed intake and body weight were measured for control and BPA-treated rats to continually adjust BPA doses and to monitor toxicity. BPA was dissolved in corn oil, and control rats received only the corn oil vehicle. Feed and water were provided ad libitum, and animals were kept on a 12-h light, 12-h dark cycle. Animals were fed Purina chow (Ralston-Purina Co., St. Louis, MO). Although this diet contains soybean meal, all animals were exposed to the same levels of phytoestrogen because feed intake was equivalent for control and BPA-treated rats (our unpublished observations). Pregnant and nursing dams (plus or minus pups) were housed one per plastic cage lined with wood bedding, whereas weanling rats were kept in groups of three. It has been suggested that prolonged use of polycarbonate cages may result in the leaching of BPA into the environment (16). The cages used in this study were washed, rinsed, and dried at least two times per week and were discarded once they began to get cloudy, and water bottles were cleaned daily. Repeated washing and rinsing are known to decrease the release of BPA from polycarbonate cages and water bottles (16). Therefore, exposure of experimental animals to phytoestrogens and BPA from these sources (feed, cages and water bottles) was minimal and equal for all groups. Assignment of rats to groups was made by body weight randomization to ensure equal weight distribution. In general, animals were killed within 24 h of the last BPA administration according to a protocol approved by the institutional animal care and use committee of Rockefeller University. At the end of each experiment and immediately following decapitation, trunk blood was obtained for measurement of serum steroid hormone concentrations, and Leydig cells were isolated for measurement of T production ex vivo. For experiments in which rats were allowed to attain adulthood, i.e. at 90 d of age, the seminal vesicles and whole prostate were collected, and wet weights were recorded. Testicular interstitial fluid (IF) was also harvested using the method described by Turner et al. (17). Briefly, the caudal pole of the testis was punctured with a 23-gauge needle, taking care to avoid damage to blood vessels and seminiferous tubules. Testes were then centrifuged at 50 x g for 15 min. IF samples were stored at -20 C until T levels were measured by RIA. For in vitro studies, BPA was dissolved in ethanol with the level of ethanol in the incubation medium not exceeding 0.001% (1 µl/ml); equivalent ethanol amounts were added to control cultures. This level of ethanol did not affect Leydig cell T production in vitro (our unpublished observations). A series of experiments was conducted to test the following hypotheses (Fig. 2Go).



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  		FIG. 2. Schema of the experimental designs. The exposure paradigms used in in vivo studies involved prepubertal (PND 21–35), perinatal (GD 12-PND 21), or chronic BPA (PND 21–90) exposures (A). Analyses included measurement of serum steroid hormone concentrations and the amounts of T produced by Leydig cells ex vivo (A). The effects of ER agonists on androgen biosynthesis were investigated by conducting dose-response studies in which adult Leydig cells were incubated with BPA, DES, and HPTE for 18 h (B). Identification of BPA-induced lesions in the steroidogenic pathway was performed by analysis of steroidogenic enzyme gene expression in Leydig cells after treatment in vitro (B). PND, Postnatal day; GD, gestation day.

 
Experiment 1: does BPA affect testicular steroidogenesis?
Environmental levels of BPA have been estimated at 2.4 µg/kg (18). Therefore, initial experiments were designed to evaluate the effect of this and higher doses of BPA on steroidogenesis. Higher doses were included in the experimental design to assess the effect of low vs. high BPA doses on the same end point, i.e. Leydig cell T production, given the current interest in the ability of xenoestrogens to cause opposing effects at different doses (19). Therefore, weanling Long-Evans rats were gavaged with low (2.4 or 10 µg/kg·d BPA) and high (100 or 200 mg/kg·d BPA) doses for 15 d, from 21–35 d of age. This age group of rats was used for these experiments because the prepubertal period is a time of active reproductive tract development, and hormonally active chemicals are known to exhibit greater potency during sexual differentiation in rodents and humans (20). At the end of treatment, animals were killed, and blood was collected for determination of serum hormone (LH and T) concentrations. Purified immature Leydig cells were also obtained for measurement of T production ex vivo after incubation in the absence (basal) and presence of a maximally stimulating dose of LH (100 ng/ml) or T precursors: 50 µM 22R-hydroxycholesterol (22R-CHO) or 20 µM pregnenolone, progesterone, and androstenedione. These steroid substrates are known to diffuse readily into Leydig cells and were used to measure enzyme reaction rates (21): cytochrome P450 cholesterol side-chain cleavage enzyme (P450scc; 22R-CHO), 3ß-hydroxysteroid dehydrogenase (3ßHSD; pregnenolone), cytochrome P450 17{alpha}-hydroxylase/17–20 lyase (P45017{alpha}; progesterone), and 17ßHSD (androstenedione). Pilot experiments indicated that the concentrations of steroids used were substrate saturating in immature Leydig cells. Initial observations indicated that BPA suppression of T production was associated with reduced pituitary LH secretion. As both ER subtypes are present in the rat pituitary, and estrogenic compounds are known to modulate expression of the ERs in target tissues (22), we conducted further experiments to identify the mechanisms of BPA action in the pituitary. Therefore, weanling rats were assigned to one of two groups: control or BPA treated (2.4 µg/kg·d, postnatal d 21–35). At the end of treatment we analyzed gene expression in the pituitary by measuring steady state mRNA levels for LHß, ERß, and ER{alpha} using RT-PCR.

Experiment 2: are the effects of perinatal BPA exposure on androgen biosynthesis, if any, reversible or do they persist into sexual maturity?
Pharmacokinetic studies in the rat demonstrated rapid transfer of BPA from dam to fetuses (23), and BPA concentrations ranging from 0.2–9.2 ng/ml were measured in human fetal plasma (24). Because critical events occur in the development of the male reproductive tract that are subject to interference by estrogenic chemicals (20), we conducted experiments to test the hypothesis that exposure to BPA in the perinatal period affect testicular function in adulthood, i.e. at 90 d of age. Pregnant Long-Evans rats were gavaged with 2.4 µg/kg·d BPA from gestation d 12 through nursing to weaning on postnatal d 21. Subsequently, male offspring received no further BPA treatment and were pooled by random selection from each dam and analyzed in adulthood at 90 d of age. Analysis included measurement of serum LH and T levels, T levels in testicular IF, Leydig cell T production ex vivo, and accessory sex organ weights (seminal vesicles and prostate).

Experiment 3: what are the effects of long-term (chronic) BPA exposures on androgen biosynthesis?
Given the use of BPA in several consumer products, it is conceivable that exposure of humans to this agent occurs over prolonged periods of time, as evidenced by the presence of measurable amounts of blood BPA levels in human populations (5, 24). Therefore, to determine the effects of chronic BPA exposure on Leydig cell androgen biosynthesis, weanling rats were gavaged with 2.4 µg/kg·d BPA from 21–90 d of age (a total of 70 d). Within 24 h of the last BPA administration, animals were killed, and blood was collected for measurement of serum LH and T concentrations. Leydig cells were isolated for measurement of T production ex vivo, and testicular IF was collected for assay of T concentrations. The weights of accessory sex organs (seminal vesicles and prostate) were also recorded.

Experiment 4: does BPA act directly in Leydig cells to inhibit androgen biosynthesis?
Given that BPA suppresses pituitary LH secretion in vivo, we asked whether BPA acts directly in Leydig cells to disrupt androgen biosynthesis in the absence of the confounding effect on LH secretion. The dose-dependent effects of BPA on steroidogenesis were evaluated by incubation of adult Leydig cells obtained from 90-d-old rats with 0, 0.01, 0.1, 1, 10, 100, and 1000 nM BPA for 18 h in medium containing 10 ng/ml LH, and T production was assayed in aliquots of the spent medium. We used adult Leydig cells in these experiments because of the inconsistent sensitivity of immature Leydig cells to ER agonists in vitro (21). To determine whether BPA action in Leydig cells is an estrogenic effect, Leydig cells were incubated with two other ER agonists as positive controls (Fig. 1Go). DES is a synthetic estrogen that has a high binding affinity for both ER subtypes and a potency greater than that of E2 (25). 2,2-Bis(p-hydroxyphenyl)-1,1,1-trichloroethane (HPTE) is the biologically active metabolite derived from the estrogenic pesticide methoxychlor after hydroxylation in the liver. This compound is known to compete with E2 for binding to ER (26), and we previously demonstrated that HPTE acts via the ER to decrease T production by rat Leydig cells in vitro (21). Therefore, Leydig cells were incubated with DES and HPTE at the same doses as BPA. The viability of Leydig cells after treatment with BPA, DES, and HPTE was assessed by the trypan blue exclusion test and did not differ from the control value. At the end of treatment, T production was measured in aliquots of spent medium by RIA. These experiments were conducted three times.

After determining that 0.01 nM BPA, but not higher doses, decreased T production, we conducted further analyses of the effects of BPA on androgen biosynthesis. Adult Leydig cells were incubated with 0.01 nM BPA for 18 h in medium containing 10 ng/ml LH. Cells were harvested after a 5-min incubation in a solution of 0.05% collagenase and 0.05% dispase in medium 199 buffered with 8.45 mM NaHCO3 and 8.8 mM HEPES, containing 0.1% BSA and 0.0025% trypsin inhibitor, at pH 7.1–7.2 (Sigma-Aldrich Corp., St. Louis, MO). To confirm the effects on androgen biosynthesis, we measured T production after aliquots of harvested cells (0.1 x 106 cells) were incubated in fresh medium without (basal) or with LH (100 ng/ml) for 3 h at 34 C. To establish that BPA-induced inhibition of androgen biosynthesis is ER mediated, we coincubated Leydig cells with 0.01 nM BPA and 0.1 nM of the synthetic antiestrogen ICI 182,780 (7{alpha}-[9-(4,4,5,5-pentafluoropentylsulfinyl)estra-1,3,5-(10)-triene-3,17ß-diol]) for 18 h in medium containing 10 ng/ml LH. ICI 182,780 binds to the two ER subtypes and does not exert partial ER agonist activity in rodent tissues (22). Rates of T production were assessed by RIA using aliquots of spent medium at the end of the 18-h treatment period. The doses of the antiestrogen were selected after pilot experiments indicated that coincubation of Leydig cells with 0.1 nM ICI 182,780 completely blocked BPA-induced (0.01 nM) inhibition of T biosynthesis. The steroidogenic acute regulatory protein (StAR) transports cholesterol, the steroid substrate used in T biosynthesis, into the inner mitochondrial membrane, whereas the steroidogenic enzymes catalyze the consecutive reactions that convert cholesterol into T in Leydig cells (27): P450scc, 3ß-HSD, P45017{alpha}, and 17ß-HSD. Therefore, to identify the site of the BPA-induced lesion in the steroidogenic pathway, steady state mRNA levels for StAR and androgen biosynthetic enzymes were measured by RT-PCR in total RNA obtained from adult Leydig cells harvested at the end of the 18-h treatment period. Because xenoestrogens are known to regulate ER expression in target tissues (22), we also measured ER steady state mRNA levels.

Experiment 5: is the action of BPA in Leydig cells dose dependent?
Discrepancies in reports of the effects of BPA from different laboratories have received considerable attention (9). In the present study we observed that 2.4 µg/kg·d BPA, but not higher doses, suppressed serum LH and T levels. This finding thus raises the question of whether doses other than 2.4 µg have an effect in Leydig cells. However, BPA has been credited with the ability to modulate aromatase activity in several tissues (28). Because estrogen biosynthesis is a function of rat Leydig cells from the pubertal period onward, we conducted experiments to test the hypothesis that BPA doses that cause no changes in pituitary LH secretion and Leydig cell T production affect aromatase gene expression and E2 biosynthesis in Leydig cells. Weanling rats were gavaged with 2.4 µg, 10 µg, 100 mg, or 200 mg/kg·d BPA for 15 d, from 21–35 d of age. At the end of treatment, rats were killed, and blood was collected for measurement of serum E2 levels. To determine whether the changes in estrogen biosynthesis were not simply due to reduced substrate (T) availability, but were associated with changes in aromatase activity, adult Leydig cells were incubated with 0.01 nM BPA for 18 h (this BPA dose was previously demonstrated to affect Leydig cells in pilot experiments). At the end of treatment, E2 levels were measured in aliquots of spent medium. Levels of aromatase gene expression in control vs. BPA-treated Leydig cells were analyzed after amplification by RT-PCR.

Hormone measurements
Serum steroid hormone concentrations were determined without extraction. Serum LH concentrations were measured using [125I]rat LH (Amersham Pharmacia Biotech, Piscataway, NJ) and materials acquired from the National Hormone and Pituitary Program, namely, rat antibody NIDDK anti-rLH-S11, and LH reference standards (NIDDK rLF-RP-3). Iodination was performed with the Bolton-Hunter reagent following the manufacturer’s instructions (Pierce Chemical Co., Rockford, IL). The secondary immunoglobin G was supplied by ICN Pharmaceuticals (Costa Mesa, CA). The lower limit of detection for this assay is 0.12 ng/ml, and LH values are expressed in relation to the RP-3 standards. The intra- and interassay coefficients of variation were 5% and 10%, respectively (29). Total T and E2 concentrations were determined by a previously described tritium-based RIA with an interassay variation of 7.8% (30). The lower limit of detection for this assay is 0.01 ng/ml.

Purification of Leydig cells
Purified Leydig cells were obtained from the testes of 35- and 90-d-old rats by collagenase digestion, followed by Percoll density centrifugation as described previously (31). In an initial purification step, Leydig cells from 90-d-old rats were subjected to centrifugal elutriation to remove germ cell and sperm contaminants. After centrifugation through a 55% continuous Percoll gradient, Leydig cells from 35-d-old rats were harvested at densities between 1.070 and 1.088 g/ml, whereas cells from 90-d-old rats were located at a density corresponding to 1.070 or greater, i.e. to the bottom of the tube. Cell yields were estimated with a hemocytometer, and purity was assessed by histochemical staining for 3ßHSD using 0.4 mM etiocholan-3ß-ol-17-one as the enzyme substrate (32). Leydig cells were 95–97% enriched for cells that stained intensely for this marker enzyme. To measure the rates of T production, aliquots of 0.1–0.2 x 106 Leydig cells were incubated in microcentrifuge tubes in 1 ml culture medium. The culture medium consisted of DMEM/F-12 buffered with 14 mM NaHCO3, containing 0.1 BSA and 0.5 mg/ml bovine lipoprotein (Sigma-Aldrich Corp.). Incubations were conducted at 34 C for 3 h using the maximally stimulating dose of 100 ng/ml ovine LH (provided by the National Hormone and Pituitary Program, NIDDK, Bethesda, MD). Incubations conducted in culture plates for longer than 3 h were performed with the lower dose of 10 ng/ml to maintain LH responsivity for the incubation period. Leydig cell T production values were normalized to nanograms per 106 cells.

RNA analysis by RT-PCR
Total RNA was isolated by a single-step method. Pituitaries collected after in vivo BPA exposure (Experiment 1) and Leydig cells harvested after incubation with 0.01 nM BPA for 18 h in medium containing 10 ng/ml LH (experiments 4 and 5) were lysed with phenol and guanidium thiocyanate (Tri-Reagent, Molecular Research Center, Cincinnati, OH) in accordance with the manufacturer’s instructions. First-strand cDNA synthesis from 400 ng total RNA was performed using avian myeloblastosis virus reverse transcriptase, random primers, and deoxy-NTPs at 37 C for 75 min. The reaction was ended by heating at 95 C for 5 min. Primers for target cDNAs were synthesized on an oligonucleotide synthesizer (Keystone Laboratories, Camarillo, CA) using their published sequences (Table 1Go). As determined from preliminary studies, cDNAs were amplified linearly between 15–35 cycles of PCR. PCR products were size-fractionated by gel electrophoresis (2% agarose) and stained with ethidium bromide, after which DNA bands from four or five reactions were quantified relative to ribosomal protein S16 that was used as the internal control. Quantitation was performed using an imaging system (Kodak Digital Sciences, Rochester, NY) after normalizing individual bands to the respective S16 density to correct for differences in gel loading.


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  		TABLE 1. Parameters of oligonucleotide primer pairs used for PCR amplification

 
Statistics
Data are presented as the mean ± SEM. For in vivo studies, data represent the averages from two different experiments, and data for in vitro studies were combined from at least three separate experiments. Data were analyzed by one-way ANOVA with multiple comparisons performed by the Duncan multiple range test to identify differences between groups. Differences were considered significant at P <= 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Low dose BPA exposure decreases pituitary LH secretion, induces pituitary ERß gene expression, and decreases Leydig cell T production
Exposure of weanling Long-Evans rats to 2.4 µg/kg·d BPA for 15 d, i.e. from 21–35 d of age, decreased both serum LH and T levels (Fig. 3Go, A and B). Measurement of T production ex vivo showed that although basal T production was similar in immature Leydig cells from control and BPA-exposed males, LH-stimulated T production and T production after incubation with 22R-CHO, pregnenolone, and progesterone were decreased by BPA treatment (Table 2Go; P < 0.05). Immature Leydig cells from rats exposed to 10 µg/kg·d BPA also produced lesser amounts of T after incubation with pregnenolone and progesterone compared with controls (Table 2Go; P < 0.05). BPA at milligram doses did not affect serum LH and T levels or Leydig cell T production (Fig. 3Go and Table 2Go; P > 0.05). Furthermore, BPA exposure (2.4 µg/kg·d from postnatal d 21–35), down-regulated pituitary LHß expression, but increased steady state ERß mRNA levels, whereas ER{alpha} expression was unchanged (Fig. 4Go). These observations indicate that decreases in serum LH levels are the result of BPA-induced declines in pituitary LH synthesis and secretion. Diminished LH stimulation of Leydig cells in vivo is presumably the cause of reduced serum T concentrations and the decrease in steroidogenic capacity seen at microgram doses (Table 2Go).



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  		FIG. 3. Effect of BPA treatment on serum LH and T levels. Exposure of rats to 2.4 µg/kg·d BPA (n = 10–12) from 21–35 d of age decreased serum LH (A) and T (B) levels compared with control values, indicating reduced pituitary LH secretion and Leydig cell steroidogenesis. This effect was not seen at higher doses (10 µg, and 100 and 200 mg). *, P < 0.01 compared with controls.

 

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  		TABLE 2. Measurements of T production (nanograms per 106 cells) after exposures of Long-Evans rats to BPA from 21–35 d of age and incubation of Leydig cells with LH or steroid substrates

 


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  		FIG. 4. Effect of BPA on pituitary gene expression. Exposure of rats to 2.4 µg/kg·d BPA from 21–35 d of age reduced steady state mRNA levels for LHß and ERß, whereas ER{alpha} levels were unchanged compared with control values. Total RNA was isolated from pituitaries obtained from two separate experiments (n = 7), and the bands shown in A are representative of the results of at least three independent RT-PCR analysis. *, P < 0.01 compared with controls.

 
Perinatal BPA exposures decrease testis weight, suppress Leydig cell T production, and reduce seminal vesicle weights in adulthood
Although body weights were similar at birth (8.7 ± 0.1 vs. 8.5 ± 0.2 g; P > 0.05), male rats maternally exposed to 2.4 µg/kg·d BPA from gestation d 12 to postnatal d 21 (through nursing) exhibited increased body weights but reduced testis weights at 90 d of age (Table 3Go). Serum LH and T levels were equivalent in control and BPA-treated rats at 90 d (Table 3Go), but the rate of T production by Leydig cells was decreased by BPA treatment (Fig. 5AGo; P < 0.01). Consistent with reduced Leydig cell steroidogenic capacity, T concentrations in the testicular IF of BPA-treated rats were lower than control values (Fig. 5BGo; P < 0.01). The weights of the seminal vesicles in BPA-treated rats were reduced compared with control values (P < 0.01), but prostate weights were unchanged (Table 3Go). These findings show that perinatal exposure of rats to low dose BPA levels decreased androgen biosynthesis by adult Leydig cells, as measured in the testicular interstitium and after incubation of Leydig cells ex vivo, and reduced seminal vesicle weight in adulthood.


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  		TABLE 3. Reproductive parameters in adult Long-Evans rats after perinatal and chronic postnatal BPA exposures

 


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  		FIG. 5. Effects of perinatal and chronic BPA exposures on Leydig cell androgen biosynthesis. Perinatal exposures of rats to BPA through their dams from gestation d 12 to postnatal d 21 suppressed T production (A), as evidenced by the presence of reduced T levels in the testicular interstitium of BPA-treated rats measured at 90 d of age (B). Similarly, postnatal exposures of rats to BPA for a prolonged period (postnatal d 21–90) decreased Leydig cell T biosynthesis (C) and reduced T levels in the testicular IF (D) measured at the end of BPA treatment. T production was measured in Leydig cells obtained after two separate experiments involving either perinatal or chronic BPA exposures. Each Leydig cell isolation utilized seven to eight rats/group. Testicular IF was measured in individual rats; n = 12–14 per group. *, P < 0.05 compared with controls.

 
Chronic postnatal exposure to BPA from 21–90 d of age suppresses Leydig cell T production and decreases seminal vesicle weights
Chronic exposure to 2.4 µg/kg·d BPA from weaning at 21–90 d of age did not affect body weight or paired testes weight (Table 3Go). Although serum T levels were equivalent in control and BPA-treated rats, BPA treatment caused elevations in serum LH levels (P < 0.05; Table 3Go). Leydig cell T production, measured ex vivo, was reduced by BPA treatment (Fig. 5CGo; P < 0.01), and T concentrations in the testicular interstitium of BPA-treated rats were lower than control values (Fig. 5DGo; P < 0.01). Seminal vesicle weight, but not that of the prostate, were reduced in BPA-treated rats compared with controls (Table 3Go; P < 0.01). These observations indicate that chronic and direct exposures to low BPA levels, from weaning at 21 d to adulthood at 90 d, suppressed androgen biosynthesis by adult Leydig cells and decreased seminal vesicle weights.

BPA acts directly in Leydig cells
Measurement of T production after incubation of Leydig cells with BPA, DES, or HPTE showed that dose-dependent suppression of Leydig cell androgen biosynthesis differed markedly among the three chemicals (Fig. 6Go; P < 0.01). For example, although only 0.01 nM BPA (but not higher doses) reduced androgen biosynthesis, DES decreased T production at all doses tested, and exposure to HPTE doses equal to or greater than 100 nM inhibited androgen biosynthesis. Leydig cells, purified from the testes of 90-d-old rats, were incubated with 0.01 nM BPA for 18 h in medium containing 10 ng/ml LH. At the end of treatment, cells were harvested and incubated further without (basal) or with 100 ng/ml LH for 3 h. Basal T production, measured in aliquots of the spent medium, was equivalent in control and BPA-treated Leydig cells, but LH-stimulated T production was decreased by BPA treatment (Fig. 7AGo; P < 0.01). When Leydig cells were coincubated with 0.1 nM ICI 182,780 for the 18-h treatment period, the inhibitory effect of 0.01 nM BPA on androgen biosynthesis was alleviated, and the antiestrogen did not by itself affect T production at 0.1 nM (Fig. 7BGo). Suppression of androgen biosynthesis in Leydig cells by BPA was associated with inhibition of steroidogenic enzyme activity because there was a decrease in steady state mRNA levels of the P45017{alpha} enzyme, measured at the end of the 18-h treatment period (Fig. 7CGo; P < 0.05). Expression of StAR and other biosynthetic enzymes was unaffected (data not shown). We did not detect ERß in Leydig cells by RT-PCR, and steady state mRNA levels for ER{alpha} were not affected by BPA (0.01 nM; 18 h; data not shown).



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  		FIG. 6. Effects of treatment with estrogenic compounds on Leydig cell T production. Leydig cells obtained from 90-d-old rats were incubated with BPA, DES, and HPTE for 18 h in medium containing 10 ng/ml LH. T production was measured in aliquots of the spent medium. Inhibition of Leydig cell T production was caused by exposure to 0.01 nM BPA (A), by DES at all concentrations tested (B), and by HPTE at 100 and 1000 nM (C). These experiments were conducted three times. *, P < 0.05 compared with controls.

 


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  		FIG. 7. Analysis of BPA effects on Leydig cell T production. Leydig cells from 90-d old rats were incubated with BPA for 18 h. At the end of treatment, cells were harvested and incubated for 3 h in the absence of (basal) and in the presence of maximally stimulating concentrations of ovine LH (100 ng/ml); T production was then measured in aliquots of the spent medium. Although basal T production was equivalent in control and BPA-treated Leydig cells, BPA treatment decreased LH-stimulated T production (A). Measurement of T production after coincubation of Leydig cells with BPA and the antiestrogen ICI 182,780 for 18 h indicated that BPA-induced inhibition was ER mediated in Leydig cells (B). The antiestrogen alone did not affect Leydig cell T production at 0.1 nM (B). Inhibition of androgen biosynthesis was associated with reduced expression of the steroidogenic enzyme P45017{alpha} (C). RT-PCR was conducted with total RNA obtained from three separate experiments, and the audiogram shown in C is representative of three to five independent RT-PCR analysis. *, P < 0.05 compared with controls.

 
BPA action in Leydig cells is dose dependent
After gavage of weanling rats with BPA at 2.4 or 10 µg, and 100 or 200 mg/kg·d BPA from 21–35 d of age, serum E2 levels were decreased (P < 0.05) in all but the 200-mg dose group, similar to controls (Fig. 8AGo). This effect was replicated in vitro with isolated adult Leydig cells exposed to 0.01 nM BPA for 18 h showing suppressed E2 production compared with controls (Fig. 8BGo; P < 0.05). The decrease in estrogen biosynthesis was associated with inhibition of aromatase gene expression, because steady state aromatase mRNA levels were reduced in BPA-treated Leydig cells compared with controls (Fig. 8CGo; P < 0.05). As there were no differences in serum T levels in controls and, for example, the 100-mg dose group in which serum E2 levels were decreased (Fig. 3Go), these observations confirm that BPA-induced decreases in E2 biosynthesis were not due to changes in substrate availability, but were related to inhibition of aromatase activity.



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  		FIG. 8. Effect of BPA on estrogen biosynthesis. Exposure of rats to 2.4 µg, 10 µg, or 100 mg/kg·d BPA (n = 12) from 21–35 d of age decreased serum E2 levels (A). This effect was replicated in vitro after incubation of adult Leydig cells with 0.01 nM BPA (B) in three separate experiments. Inhibition of estrogen biosynthesis was associated with decreased gene expression for the aromatase enzyme, which catalyzes the conversion of androgens to estrogen (C). RT-PCR was conducted with total RNA obtained from three separate experiments, and the bands shown in C are representative of three to five independent measurements. *, P < 0.05 compared with controls.

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study demonstrates that exposure to environmentally relevant BPA levels has adverse effects on testicular function by decreasing pituitary LH secretion and reducing Leydig cell steroidogenesis. For example, exposure of pubertal rats to 2.4 µg/kg·d for 15 d, i.e. from 21–35 d of age, decreased serum LH and T levels as well as Leydig cell androgen biosynthetic capacity. Although BPA suppresses T production via decreased LH secretion, there is also evidence that BPA interferes with LH receptor-ligand binding (42); uncoupling of LH from the LH receptor potentially contributes to diminished LH stimulation of steroidogenesis. BPA also suppressed aromatase gene expression and Leydig cell E2 biosynthesis. After perinatal and chronic postnatal exposures, T production by adult Leydig cells ex vivo and T levels in the testicular IF at 90 d of age were reduced by BPA treatment. The present data further show that the perinatal period of development (gestation d 12 to postnatal d 21) is a sensitive window of exposure to BPA, as evidenced by measurement of reduced Leydig cell T production and smaller testicular and seminal vesicle weights at 90 d of age, i.e. long after BPA exposure had been terminated on postnatal d 21. The ability to produce latent effects in adulthood subsequent to prepubertal exposure is commonly observed for xenoestrogens and other endocrine-active chemicals. Presumably, this results from increased sensitivity during reproductive tract development (43). The fact that reduced androgen biosynthesis by Leydig cells was not reflected in serum T levels after chronic BPA exposure may be due to compensatory mechanisms that were activated in vivo, e.g. increased pituitary LH secretion (1.3 ± 0.13 vs. 0.6 ± 0.05 ng/ml; Table 3Go). Higher serum LH levels in BPA-treated rats presumably result from decreased sensitivity to androgen negative feedback on the hypothalamus and pituitary and the consequent stimulation of LH secretion.

Although ERß expression is thought to be significantly lower than ER{alpha} in gonadotrophs, estrogens are known to up-regulate both ER subtypes in the pituitary and cause transcriptional activation through both forms of the ER (44). In the present study BPA treatment increased steady state pituitary ERß mRNA levels, but not ER{alpha}. Whether increased ERß expression is a consequence of the higher binding affinity of BPA for this ER subtype [38-fold greater than for ER{alpha} (2)] or is the result of increased transcriptional activity mediated by this ER subtype is not clear. However, measurement of reduced LHß and increased ERß mRNA levels in the pituitaries of BPA-treated rats suggests that BPA-induced suppression of LHß expression is ER mediated. This interpretation is supported by the results of transfection analysis showing that the rat LHß is ER regulated, requiring estrogen response elements (45). A previous study showed that BPA increased the expression of both ER{alpha} and ERß in the anterior pituitary of pubertal Fischer 344 rats (40). The disparity between these and our observations is probably due to differences in the BPA doses used, 2.4 vs. 100 µg/kg·d (40).

The increase in body weights in adulthood after perinatal BPA exposure, i.e. at 90 d of age when BPA-treated rats were 10% heavier than controls, is consistent with a previous report that pups exposed to 1 nM BPA in utero were significantly heavier than controls at weaning on postnatal d 21 (46). The reasons for body weight gain are not known, but there were no differences in feed intake between control and BPA-treated rats (our unpublished observations). As male adipose tissue expresses ER, and the estrogen signaling pathway regulates adipocyte function and energy expenditure (47), the differences in body weights may be the consequence of BPA’s estrogenic action in nonreproductive tissues. The maintenance of accessory sex gland function is known to be androgen dependent, and BPA caused a decrease in seminal vesicle weights at 90 d of age after perinatal and chronic postnatal exposures. In this regard, the levels of T in the testicular interstitium were reduced on postnatal d 90 after both perinatal and chronic BPA exposures, although serum T levels were equivalent in control and BPA-treated rats. The concentrations of T in the testicular IF that directly bathes Leydig cells are approximately 25 times higher than those in the serum and may, therefore, be a more sensitive indicator of the androgen biosynthetic capacity of Leydig cells (17). Nevertheless, as there were no changes in serum T levels, decreases in the weights of the seminal vesicles probably result from direct BPA action as was previously reported (48). The reasons for the lack of a BPA effect on prostate weight, unlike the seminal vesicles, are not known, but species and strain differences in tissue-specific responsiveness to ER agonists have been described previously. For example, exposure to E2 suppressed spermatid maturation in C57BL/6J and C17/Jl strains of mice, whereas this effect was not seen in the CD-1 strain (49). It is also possible that the exposure paradigm used in the present study caused only biochemical effects in the prostate that did not result in changes in organ weight.

BPA was found to act directly in Leydig cells because it decreased T production after treatment of Leydig cells in vitro. Inhibition of steroidogenesis was ER mediated and was associated with inhibition of enzyme activity. BPA had been considered a weak estrogen because its binding affinity for the ER is about 5 orders of magnitude less than that of the natural ligand E2 (22). However, the present study shows that blockade of BPA-induced inhibition of Leydig cells required a higher (10-fold) concentration of ICI 182,780, an antiestrogen with high binding affinity for the ER. This observation supports the hypothesis that BPA has a greater potency for ER-mediated activity than was previously thought (22). Analysis of steroidogenic enzyme gene expression by RT-PCR indicated that BPA caused specific inhibition of the P45017{alpha} enzyme, which is known to be inhibited by estrogen (50). Although ER{alpha} was consistently demonstrated in rodent Leydig cells, localization of ERß has been variable, with reports of its absence (51) and its presence (52) in the mouse Leydig cell; although localized to fetal rat Leydig cells (53), it was not found in adult rat Leydig cells (54). As we did not detect ERß in Leydig cells in the present study, and BPA inhibition of T production was ER mediated, BPA suppression of the CYP17 gene, which encodes the P45017{alpha} enzyme, was presumably mediated by ER{alpha} as previously suggested (50). Furthermore, we observed that 0.01 nM BPA, and not higher doses (Fig. 6Go), suppressed Leydig cell T production, similar to findings from our in vivo studies (Fig. 3Go). The present data show that different effects may occur at varying dose levels, because 100 mg/kg·d BPA did not affect pituitary LH secretion and T production, but suppressed Leydig cell E2 biosynthesis. Therefore, the differential pattern of decreases observed in the serum levels of LH, T, and E2 at varying BPA doses implies that the effects of BPA on the male reproductive tract depend not only on the exposure paradigm employed, but also on the end points examined.

The present data show that BPA caused direct inhibition of aromatase gene expression and E2 biosynthesis that was not due to a decrease in substrate availability. It is not clear from the present data that this effect was ER mediated. However, inhibition of CYP19 gene expression of aromatase in Leydig cells was ER{alpha} mediated as indicated by the following: 1) estrogenic agents act via ER{alpha} to up-regulate the promoter region of the aromatase gene (55); and 2) ER{alpha}-mediated inhibition of E2 biosynthesis in breast cancer cells is blocked by antiestrogens such as tamoxifen and ICI 182,780 (55). However, BPA suppression of E2 biosynthesis has implications for male reproductive function. For example, estrogen is known to affect the development of immature germ cells, because incubation of differentiating gonocytes with E2 stimulated cell division in vitro (56). As gonocytes are the precursors of spermatogonia, it is reasonable to speculate that disturbances in endogenous estrogen biosynthesis during the early period of reproductive tissue differentiation could result in infertility at sexual maturity because the gonocyte population is established in the prepubertal period. Indeed, targeted disruption of the aromatase gene in mice was found to induce apoptosis in developing spermatids as well as cause disturbances in acrosome formation (57). Therefore, these observations support the hypothesis that exposure to environmentally relevant concentrations of xenoestrogens may result in low seminal quality. The effects of BPA modulation of estrogen biosynthesis are applicable to humans, because two isoforms of the ERß have been localized to the fetal and adult testis, implying that the human testis is subject to estrogen action (58). Moreover, sexual dimorphism is determined by the actions of androgen and estrogen during the critical period for perinatal differentiation of neural tissue (59). Thus, BPA-related suppression of E2 biosynthesis potentially affects male sexual behavior in adulthood.

In conclusion, data from the present study demonstrate that BPA has an inhibitory effect on testicular steroidogenesis at low dose exposure levels, presumably acting via the ER. BPA also suppressed aromatase gene expression and E2 biosynthesis. It is of interest that the BPA dose used in our studies, which corresponds to the levels of this agent in the environment, exerted inhibitory effects on Leydig cells. Although there is no evidence indicating that oral ingestion of BPA by humans at exposure levels typical of its presence in the environment has adverse effects, blood BPA levels in a group of pregnant mothers and their fetuses ranged from 0.3–18.9 and 0.2–9.2 ng/ml, respectively (24). These levels are higher than the concentration of 0.01 nM BPA used in our in vitro experiments, implying that human exposure to low BPA levels may exert adverse biological effects. As both T and E2 are required for male reproductive tract development and function, suppression of steroid hormone synthesis may be responsible for testicular anomalies associated with BPA in laboratory studies. The extensive use of BPA in consumer products to which humans are chronically exposed warrants the continued investigation of this compound at low dose exposure levels for the purpose of risk assessment.


   Acknowledgments
 
We thank Evan Read for help with manuscript preparation. Ovine LH and reagents for LH RIA were provided by the National Hormone and Pituitary Program (NIDDK, Bethesda, MD). HPTE was synthesized by Dr. W. R. Kelce (Pharmacia, Skokie, IL), and the antiestrogen ICI 182,780 was a gift from Zeneca Pharmaceuticals (Cheshire, UK).


   Footnotes
 
This work was supported in part by National Institute of Environmental Health Sciences Grant ES-10233. Access to the Cell Culture Core Facility was provided in part by the NICHHD, NIH, support through a cooperative agreement (U54-HD-13541) as part of the Specialized Cooperative Centers Program in Reproduction Research. Preliminary data were presented at the 81st Annual Meeting of The Endocrine Society, June 19–22, 2001, Denver, CO. Although this study was funded in part and the data presented herein were approved for publication by the U.S. Environmental Protection Agency, this paper does not necessarily reflect the views and policies of this agency.

Abbreviations: BPA, Bisphenol A; DES, diethylstilbestrol; E2, 17ß-estradiol; ED, endocrine disruptor; ER, estrogen receptor; HPTE, 2,2-bis(p-hydroxyphenyl)-1,1,1-trichloroethane; 3ßHSD, 3ß-hydroxysteroid dehydrogenase; IF, interstitial fluid; P45017{alpha}, cytochrome P450 17{alpha}-hydroxylase/17–20 lyase; P450scc, cytochrome P450 cholesterol side-chain cleavage enzyme; 22R-CHO, 22R-hydroxycholesterol; StAR, steroidogenic acute regulatory protein; T, testosterone.

Received September 5, 2003.

Accepted for publication October 28, 2003.


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Bisphenol A Exposure Induces Apoptosis and Upregulation of Fas/FasL and Caspase-3 Expression in the Testes of Mice
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L. N. Vandenberg, M. V. Maffini, C. Sonnenschein, B. S. Rubin, and A. M. Soto
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J. S. Brown Jr.
Effects of Bisphenol-A and Other Endocrine Disruptors Compared With Abnormalities of Schizophrenia: An Endocrine-Disruption Theory of Schizophrenia
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K. L. Howdeshell, J. Furr, C. R. Lambright, V. S. Wilson, B. C. Ryan, and L. E. Gray Jr
Gestational and Lactational Exposure to Ethinyl Estradiol, but not Bisphenol A, Decreases Androgen-Dependent Reproductive Organ Weights and Epididymal Sperm Abundance in the Male Long Evans Hooded Rat
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B. T. Akingbemi, T. D. Braden, B. W. Kemppainen, K. D. Hancock, J. D. Sherrill, S. J. Cook, X. He, and J. G. Supko
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G.-R. Chen, R.-S. Ge, H. Lin, L. Dong, C. M. Sottas, and M. P. Hardy
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P. Murugesan, M. Balaganesh, K. Balasubramanian, and J. Arunakaran
Effects of polychlorinated biphenyl (Aroclor 1254) on steroidogenesis and antioxidant system in cultured adult rat Leydig cells
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G. Delbes, C. Levacher, and R. Habert
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M Tanaka, S Nakaya, M Katayama, H Leffers, S Nozawa, R Nakazawa, T Iwamoto, and S Kobayashi
Effect of prenatal exposure to bisphenol A on the serum testosterone concentration of rats at birth
Human and Experimental Toxicology, July 1, 2006; 25(7): 369 - 373.
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W. V. Welshons, S. C. Nagel, and F. S. vom Saal
Large Effects from Small Exposures. III. Endocrine Mechanisms Mediating Effects of Bisphenol A at Levels of Human Exposure
Endocrinology, June 1, 2006; 147(6): s56 - s69.
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T. Hiroi, K. Okada, S. Imaoka, M. Osada, and Y. Funae
Bisphenol A Binds to Protein Disulfide Isomerase and Inhibits Its Enzymatic and Hormone-Binding Activities
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S. Ramaswamy
Pubertal Augmentation in Juvenile Rhesus Monkey Testosterone Production Induced by Invariant Gonadotropin Stimulation Is Inhibited by Estrogen
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R.-S. Ge, Q. Dong, E.-m. Niu, C. M. Sottas, D. O. Hardy, J. F. Catterall, S. A. Latif, D. J. Morris, and M. P. Hardy
11{beta}-Hydroxysteroid Dehydrogenase 2 in Rat Leydig Cells: Its Role in Blunting Glucocorticoid Action at Physiological Levels of Substrate
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