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a Center for Biomedical Research, Population Council, New York, New York 10021
b Reproductive Toxicology Division, National Health and Environmental Effects Laboratory, United States Environmental Protection Agency, Research Triangle Park, North Carolina 27711
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ABSTRACT |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Environmental toxicants associated with infertility in wildlife and laboratory species include organophosphate and polychlorinated pesticides, as well as industrial chemicals. While DDT [1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane]) itself was banned in the United States in 1972, methoxychlor remains in agricultural use because of its diminished persistence in the atmosphere, rapid excretion from the body, and lower toxicity [13]. However, this compound was associated with a decrease in the weight of the testis, seminal vesicles, and prostate in the adult rat [14] and the testis in rabbits [15]. When administered to prepubertal rats, methoxychlor reduced testicular steroidogenesis, interstitial fluid T content, and spermatogenesis, suggesting a direct action on Leydig cells [16]. It is now known that endocrine disrupters cause demasculinization in rodents through altered androgen biosynthesis [17]. The estrogenic activities of methoxychlor were attributed to demethylated phenolic derivatives that result from its biotransformation in the liver. Furthermore, these metabolites, especially HPTE [2,2-bis(p-hydroxyphenyl)-1,1,1-trichloroethane], are known to bind the ER to a greater degree than methoxychlor itself [18, 19].
In the rat, Leydig cell development begins with proliferation and commitment of mesenchymal cells to the Leydig cell lineage by Day 14 postpartum; these are designated progenitor Leydig cells. Progenitor Leydig cells become immature Leydig cells between Days 21 and 35. Finally, immature Leydig cells differentiate into adult Leydig cells by Day 56 [20]. Since these Leydig cell stages differ in their capacities for T production, T levels provide an index for assessing Leydig cell differentiation. In the prepubertal period, T promotes the development of male secondary sex characteristics and hormonal imprinting of the liver, prostate, and hypothalamus. In the adult, T supports spermatogenesis, sperm maturation, and sexual function [21]. Therefore, disruption of T biosynthesis has implications for the masculinization process and fertility. This study was designed to characterize the effects of HPTE on male reproductive function through evaluation of T production capacity in differentiating Leydig cells.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Sodium bicarbonate (NaHCO3), Hepes, trypsin inhibitor, heparin, EDTA, BSA, bovine lipoprotein, Dulbecco's modified Eagle's medium nutrient mixture:Ham's F-12 (DMEM/F-12; 1:1 mixture without phenol red), E, diethylstilbestrol (DES), 5-pregnen-3ß-ol-20-one (pregnenolone), dibutyryl cAMP (dbcAMP: N6,2'-O-dibutyryl adenosine 3',5'-cyclic monophosphate), 22R-hydroxycholesterol (22R-CHO), albumin, Percoll, etiocholan-3ß-ol-17-one, nicotinamide adenine dinucleotide (NAD), nitro blue tetrazolium, and gentamycin were purchased from Sigma Chemical Company (St. Louis, MO). Dulbecco's PBS, Medium 199, and 10-strength Hanks' Balanced Salt Solution were obtained from Life Technologies (New York, NY); and collagenase, dispase, and DNase from Boehringer Mannheim GmbH (Mannheim, Germany). The antiestrogen ICI 182,780 [7
-[9-(4,4,5,5,-pentafluoropentylsulphinyl)estra-1,3,5(10)-triene-3,17ß-diol]] was a gift from Zeneca Pharmaceuticals (Cheshire, UK). Ovine LH was generously provided by the National Hormone and Pituitary Program (National Institute of Diabetes and Digestive and Kidney Diseases, Rockville, MD). HPTE was synthesized by Dr. W.R. Kelce (Monsanto Company, St. Louis, MO).
Animals
Male Sprague-Dawley rats were purchased from Charles River Laboratories (Wilmington, MA). Progenitor, immature, and adult Leydig cells were isolated from rats that were, respectively, 21, 35, and 90 days of age on the day of isolation; these isolations used, respectively, 50 male pups, 18 pubertal, and 10 sexually mature rats. The animals were killed by CO2 asphyxiation, according to a protocol approved by the Institutional Animal Care and Use Committee of Rockefeller University (Protocol 91200-R2). Leydig cells were isolated from the testes by collagenase digestion, centrifugal elutriation, and Percoll density gradient centrifugation according to the method of Klinefelter et al. [22]. Cell yields were estimated with a hemocytometer, and purity was assessed by histochemical staining for 3ß-hydroxysteroid dehydrogenase (3ß-HSD) using 0.4 mM etiocholan-3ß-ol-17-one as the enzyme substrate [23]. Progenitor Leydig cells were 90% enriched for cells that stain weakly for the marker enzyme 3ß-HSD, and immature and adult Leydig cell fractions were typically 95–97% enriched for cells that stain intensely.
Culture of Leydig Cells
The culture medium consisted of DMEM/F-12 buffered with 14 mM NaHCO3, containing 0.1% BSA and 0.5 mg/ml bovine lipoprotein [24]. Progenitor and immature Leydig cells were cultured at a density of 0.5 to 1.0 x 106 cells per well in 6-well plates, and adult Leydig cells at 0.2 to 0.3 x 106 cells in 24-well plates (Corning-Costar Company, New York, NY), in an atmosphere containing 5% O2 and 5% CO2 and a temperature of 34°C. Leydig cells were allowed 2 h to attach in fresh medium alone that were thereafter replaced by test solutions (fresh media containing test compounds). Except for 22R-CHO, which was solubilized in dimethylsulfoxide (DMSO), test compounds were dissolved in 95% ethanol and kept as 10 mM stock solutions at -20°C. When required, stock solutions were serially diluted such that 1 µl in 1 ml of the culture media gave the desired concentration of test compound. Equivalent amounts (1 µl/ml) of the solvent were added to the media for the control cultures. This level (0.1%) of ethanol or DMSO in the media did not significantly alter T production by Leydig cells. Culture of Leydig cells with low amounts of LH maintains or increases the number of LH receptors in Leydig cells, but incubation with high doses for prolonged periods results in a decrease in the number of LH receptors and Leydig cell desensitization [4]. To avoid desensitization, incubation of Leydig cells for longer than 6 h was done using a low concentration of ovine LH (10 ng/ml), while incubations of shorter duration were conducted with a high dose (100 ng/ml). T concentrations were determined by RIA as previously described, with an interassay variation of 7.8% [25]. T production values were normalized to ng T/106 cells.
Dose Dependency of HPTE Effect
To establish that HPTE has an effect on T production, progenitor, immature, and adult Leydig cells were cultured for 18 h in the absence or presence of 0, 0.01, 0.05, 0.1, 0.2, 0.5, 1, 2, 5, and 10 µM HPTE. After treatment, cells were harvested following 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. To facilitate the process of detachment, culture plates were subsequently placed on an orbital shaker at 70 cycles/min for 10 min; Leydig cells were removed thereafter. The viability of harvested cells was assessed by the trypan blue exclusion test. Aliquots of harvested cells (0.25–0.5 x 106) were subsequently incubated in fresh media for 3 h at 34°C. The incubations were conducted in the absence or presence of a maximally stimulating (100 ng/ml) dose of ovine LH [26]. At the end of incubation, the tubes were centrifuged at 500 x g and stored at -20°C until RIA.
Onset and Reversibility of HPTE Effect
Dose-response data indicated that 1.11 ± 0.02 µM HPTE caused half-maximal inhibition (IC50) of T production by Leydig cells at all three stages of differentiation. Therefore, subsequent studies were conducted using HPTE at a concentration of 1 µM; cells remained attached throughout the incubation period. To ensure that this dose was not directly cytotoxic, a cell fluorescence assay (Promega, Madison, WI) was performed after incubation of Leydig cells in media containing 10 ng/ml LH for 18 h with and without 1 µM HPTE. After treatment, a premixed dye solution containing a tetrazolium salt was added to the cultures, and incubation was continued for another 4 h. Cell viability corresponded to cellular conversion of the tetrazolium salt into a colored formazan product and was quantified using an ELISA plate reader (Dynex Technologies, Chantilly, VA). The results were in agreement with those of the trypan blue exclusion test.
The time course for HPTE inhibition was analyzed in order to determine whether HPTE action results from an acute effect that occurs in a short period of time, e.g., less than 1 h, or is delayed as would be expected with changes in gene expression, e.g., requiring several hours to manifest. Progenitor, immature, and adult Leydig cells were cultured in media containing 10 ng/ml LH with and without 1 µM HPTE for 3, 6, 10, 14, or 18 h. At the end of each time point, incubation was stopped, and spent media were collected for assay of T concentrations by RIA.
The reversibility of HPTE effect, as indicated by the ability of Leydig cells to restore T production capacity after cessation of treatment, was evaluated by incubating progenitor, immature, and adult Leydig cells in media containing 10 ng/ml LH with and without 1 µM HPTE for 3, 6, 10, 14, or 18 h. At the end of treatment, spent media were replaced by fresh media containing 10 ng/ml LH, and the incubation was continued for another 18 h regardless of the initial treatment period. At the end of culture, media were collected and stored at -20°C until RIA.
Antagonism of HPTE Effect by Coincubation with Antiestrogen
ICI 182,780 is a pure antiestrogen that binds the two isoforms of the ER [8]. This compound was used to determine whether HPTE inhibition of T production could be prevented by blockade of the Leydig cell ER. Immature Leydig cells were used for this determination and in subsequent studies because their sensitivity to HPTE action was intermediate between that of progenitor and that of adult Leydig cells. Leydig cells were incubated for 18 h with and without 1 µM HPTE, ICI 182,780 (5 µM) plus HPTE, or ICI 182,780 in medium containing 10 ng/ml LH. The 5 µM dose of ICI 182,780 was identified from pilot experiments as the concentration that prevented inhibition of T production by 1 µM HPTE in vitro. To test for the inhibitory effect of E and DES on Leydig cell steroidogenic function, immature Leydig cells were incubated for 18 h with graded doses (ranging from 10-11 to 10-6 M) of the two compounds. After the spent media were discarded and cells gently rinsed, incubation was continued in fresh media with and without 100 ng/ml LH for a 6-h period. Basal and LH-stimulated T production was measured in aliquots of the media collected at the end of this posttreatment period.
Evaluation of the T Biosynthetic Pathway
Immature Leydig cells were first incubated in culture media for 18 h with and without 1 µM HPTE. At the end of treatment, cells were gently rinsed without detachment, and incubation was continued for another 6 h in fresh media containing 100 ng/ml LH or 100 µM dbcAMP. These concentrations are known to maximally stimulate T production by Leydig cells in vitro [26, 27]. Previously exposed Leydig cells (HPTE, 18 h) were also incubated for 6 h in the presence of T precursors: 50 µM 22R-CHO or 20 µM of pregnenolone, progesterone, and androstenedione. These steroid substrates are known to diffuse readily into Leydig cells and are used at high doses to determine the capacity of their respective enzymes [28]: cytochrome P450 cholesterol side-chain cleavage (P450scc; 22R-CHO), 3ß-HSD (pregnenolone), cytochrome P450 17-hydroxylase/17-20 lyase (progesterone), and 17ß-hydroxysteroid dehydrogenase (androstenedione). Pilot studies confirmed that the concentrations of steroids used were substrate saturating for these enzymes in immature Leydig cells.
RT-PCR
Of the four substrates tested, only 22R-CHO failed to reverse HPTE inhibition, indicating that the decline in T production by Leydig cells is most likely due to reduced capacity for cholesterol side-chain cleavage rather than its transport. Steady-state mRNA levels for P450scc and steroidogenic acute regulatory protein (StAR), responsible for transporting cholesterol to the inner mitochondrial membrane location of P450scc [29], were measured after immature Leydig cells were incubated for 12 h in media containing 10 ng/ml LH with and without 1 µM HPTE. Total RNA was isolated by a single-step method after cells were lysed in culture plates without detachment using phenol and guanidinium thiocyanate (Trireagent; Molecular Research Center, Cincinnati, OH) in accordance with the manufacturer's instructions. First-strand cDNA synthesis from 400 ng of total RNA was done using avian myeloblastosis virus reverse transcriptase, random primers, and deoxyNTPs at 37°C for 75 min, and the reaction was terminated by heating at 95°C for 5 min. Target cDNA was coamplified with RPS16 as the internal control in an aliquot of the synthesized product. Primers for the target cDNAs were synthesized on an oligonucleotide synthesizer (Gene Assembler Special; LKB, Rockville, MD) using their published sequences. The sequences were 5'-AGGTGTAGCTCAGGACTTCA-3' (forward) and 5'-AGGAGGCTATAAAGGACACC-3' (reverse) for P450scc [30], 5'-TTGGGCATACTCAACAACCA-3' (forward) and 5'-ATGACACCGCTTTGCTCAG-3' (reverse) for StAR [31], and 5'-AAGTCTTCGGACGCAAGAAA-3' (forward), 5'-TTGCCCAGAAGCAGAACAG-3' (reverse) for RPS16 [32]. The expected product sizes were 399, 389, and 148 base pairs (bp) for P450scc, StAR, and RPS16, respectively. PCR was initiated by the addition of Taq DNA polymerase and continued for 35 cycles of 1 min each at 94°C, 57°C, and 72°C. Preliminary studies showed that the cDNAs of interest were amplified linearly between 15 and 35 cycles of PCR. PCR products were separated on 3% agarose, visualized by ethidium bromide staining, and quantified on a densitometer (Kodak Scientific Imaging Systems, New York, NY) using 0.65 µg of 100-bp DNA ladder as standard. The mass of PCR products for P450scc and StAR was normalized to RPS16. These experiments were conducted three times, and RT-PCR was replicated at least three times on each occasion.
Statistics
Data are presented as mean ± SEM and represent the average of three separate experiments for each determination (total of six determinations; Figs. 1–5, Table 1). The dose-response data (Fig. 1) were analyzed by a four-parameter logistic curve fitting using the SAAM II optimizer and IC50 values derived therefrom [33]. Other data were examined by one-way ANOVA with multiple comparisons performed by the Duncan multiple range test to identify differences between groups.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The viability of Leydig cells after treatment with 1 µM HPTE in vitro was also similar to the control value as determined by a cell fluorescence assay. It was necessary to establish that HPTE at 1 µM was not disruptive of Leydig cell membranes, as this dose was used for subsequent analysis of the HPTE effect in Leydig cells (Figs. 2–5, Table 1). Compared to observations in the control, inhibition of T production in culture was apparent after 10 h of HPTE treatment of progenitor and immature Leydig cells and 18 h for adult Leydig cells as described in Figure 2. This observation indicates that reduced T production is not the result of a rapidly acting cytotoxic effect. The reversibility of HPTE-induced inhibition was determined by the ability of Leydig cells to regain T production capacity after exposure to HPTE was terminated. In this regard, T production for the 18-h posttreatment period, after Leydig cells were treated with 1 µM HPTE for 3, 6, 10, 14, or 18 h, is shown in Figure 3. When treatment was for 3 h, T production by immature and adult Leydig cells was similar to the control value for the 18-h posttreatment period, but it was significantly lower for progenitor Leydig cells. However, when treatment was up to or longer than 6 h, immature and adult Leydig cells failed to restore T production capacity within the 18-h posttreatment period. Apparently, the steroidogenic lesion elicited by the action of HPTE continues beyond the period of direct exposure, because reduced T production by progenitor Leydig cells, for example, was evident after treatment for 10 h in the onset study (Fig. 2) and 3 h in the reversibility study (Fig. 3), while the total duration of culture was 10 and 21 h, respectively.
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Coincubation of immature Leydig cells with HPTE and the pure antiestrogen ICI 182,780 prevented HPTE-induced inhibition of Leydig cells as shown in Figure 4, indicating that HPTE inhibition was mediated through binding to ER. The ER antagonist by itself did not affect T production. However, we did not observe any consistent inhibition of basal and LH-stimulated T production by Leydig cells incubated with varying concentrations of E and DES (data not shown).
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Table 1 presents T production by immature Leydig cells treated with 1 µM HPTE for 18 h and subsequently incubated with LH, dbcAMP, 22R-CHO, pregnenolone, progesterone, and androstenedione for 6 h. After HPTE treatment, stimulation of Leydig cells with LH or dbcAMP did not restore T production to control levels. However, HPTE treatment did not alter Leydig cell responsiveness to LH, as indicated by a 4-fold increase in T production over basal levels (4.3 for control vs. 3.9 for HPTE-treated cells). T production by control and HPTE-treated immature Leydig cells was similar in the presence of pregnenolone, progesterone, and androstenedione; but HPTE-treated cells produced significantly lower amounts of T when incubations were conducted with 22R-CHO (P < 0.01). This finding indicated that HPTE inhibition is most likely due to a decrease in the activity of P450scc.
Steady-state mRNA levels for P450scc in immature Leydig cells were reduced by about half after treatment with 1 µM HPTE compared to control (P < 0.05). This observation shows that HPTE-induced inhibition is related to reduced gene expression for the cholesterol side-chain cleavage enzyme. On the other hand, StAR mRNA levels were similar in control and HPTE-treated cells (Fig. 5).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The present results show that the inhibitory action of HPTE was ER mediated. Early studies demonstrated that HPTE influences androgen formation by acting on ERs: it competitively inhibited binding of [3H]estradiol to testicular ER, even at concentrations as low as 0.1 µM [40]. The IC50 value obtained in the present study, about 1 µM, is similar to the concentration of HPTE, 0.75 µM, that displaced 50% of radiolabeled E from a human recombinant ER [41]. Together, these findings imply that HPTE has the ability to activate the ER and thereby regulate cellular function. The transfer of methoxychlor and its metabolites to male offspring in utero and during lactation, in association with developmental anomalies of the reproductive tract, has been reported in rats [16, 42]. As shown in Figures 2 and 3, sensitivity of Leydig cells to HPTE action was progenitor > immature > adult Leydig cells. This pattern is probably influenced by two factors. First, steady-state ER mRNA levels are greater in developing Leydig cells than in the adult [43], thus causing amplification of estrogen effects. Second, progenitor Leydig cells are known to possess androgen receptors (ARs) earlier in development than LH receptors, indicating a requirement for androgen during the process of differentiation [37]. Thus, ER-mediated reduction in T interferes with androgen stimulation of Leydig cell differentiation. For these reasons, developing Leydig cells are more susceptible to factors that hinder androgen biosynthesis and/or action. The present results are therefore in agreement with other studies showing that hormonally active chemicals, both xenoestrogens and antiandrogens, exhibit greater potency during sexual differentiation in rodents and humans [44–46].
The two isoforms of the ER ( and ß) are present in Leydig cells, but the specific isoform mediating HPTE action in Leydig cells remains unclear because ICI 182,780 blocks both isoforms. ER is known to be present in the nuclei of fetal and adult Leydig cells but not in the seminiferous tubular compartment [9, 47]. In contrast, ERß is abundantly expressed in Sertoli cells and gonocytes of the fetal testis, as well as in the prostate, Sertoli and germ cells, and excurrent ducts of the adult [8, 10, 11]. However, to clearly distinguish between their functions in different tissues, the development of compounds capable of selective blockade of ER isoforms is required. Interestingly, there was no effect on T production when immature Leydig cells were incubated with various concentrations of E and DES. Data from previous studies have been variable and/or showed that relatively high doses of E are required to suppress T production by rat Leydig cells in vitro [4, 48–50]. On the other hand, the direct and inhibitory effects of E and DES on Leydig cell function in vivo have been clearly demonstrated. For example, a single injection of 2 mg aqueous E to young men reduced plasma T from a mean of 760 ng/dl to 295 ng/dl after 24 h without affecting serum LH levels [51]. When administered to neonatal mice, low doses of both E and methoxychlor decreased serum T [52]. These observations could be interpreted to mean that the effect of E and DES on Leydig cell T production probably requires potentiation by paracrine or other factors that are absent in vitro.
LH is known to regulate Leydig cells through cAMP-dependent and -independent pathways [53]. However, failure of LH and dbcAMP (Fig. 1, Table 1) to alleviate HPTE-induced inhibition of Leydig cells indicates that inadequate LH stimulation (e.g., from down-regulation of LH receptors) and disruption of the primary LH signaling pathway via phosphokinase A are not responsible for the decline in T production. Furthermore, HPTE-treated Leydig cells incubated with a high dose of 22R-CHO (50 µM), in spite of a 12-fold increase in T production over basal levels, produced significantly lower amounts of T compared to control. This finding suggests that cholesterol availability, even if contributory, is not the major factor causing the decrease in T production, because this steroid (22R-CHO) gains access to Leydig cells and does not require facilitated transport into the inner mitochondrial membrane. Incubation with T precursors other than 22R-CHO (Table 1) restored T production to control levels, suggesting that HPTE inhibition is related to a decrease in the activity of P450scc alone. RT-PCR confirmed that the loss of activity is due to reduced expression of this enzyme in HPTE-treated Leydig cells. Indeed, mRNA levels for StAR were equivalent for control and HPTE-treated cells, implying that expression of StAR in Leydig cells was unaffected by HPTE.
In conclusion, this study demonstrates that HPTE inhibits steroidogenic function in differentiating and mature Leydig cells, and that Leydig cells are more sensitive to this agent during pubertal differentiation versus adulthood. Since many DDT isomers continue to be used for agricultural purposes and/or have the ability to persist in the environment, this potential consequence for male reproductive function merits attention. Activation of the ER by endocrine disrupters is known to be moderated by estrogen-responsive elements, transcription factors, and the transcription-regulating domains of the target gene. Therefore, in vitro assays may underestimate or overestimate in vivo potency [54]. Endocrine disrupters may also cause their effects by more than one mechanism. For example, binding of [3H]5-dihydrotestosterone to androgen-binding protein is competitively inhibited by methoxychlor in the rat [55]. This effect further perturbs AR-mediated actions in androgen-dependent tissues. Other factors, such as long half-life and the tendency to remain in body tissues for prolonged periods [56], could facilitate the potency of environmental estrogens in vivo. The sources of human exposure to these compounds are largely dietary and therefore potentially chronic. Analysis of these issues will facilitate risk assessment regarding endocrine disrupters in the environment.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported in part by NIH Fogarty award, TW0-5350, to B.T.A. Preliminary data presented at the 31st Annual Meeting of the Society for the Study of Reproduction, August 8–11, 1998, College Station, TX. Although this study was funded in part and the data presented herein reviewed by the U.S. Environmental Protection Agency, this paper does not necessarily reflect the views and policies of the Agency. 
2 Correspondence: Matthew P. Hardy, Population Council, 1230 York Avenue, New York, NY 10021. FAX: 212 327 7678; m-hardy{at}popcbr.rockefeller.edu
Accepted: October 7, 1999.
Received: June 11, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and ß) expression in the excurrent ducts of the adult male rat reproductive tract. J Androl 1997; 18:602–611. and ß. Endocrinology 1997; 138:863–870. within the testis and excurrent ducts of the rat and marmoset monkey from perinatal life to adulthood. J Endocrinol 1997; 153:485–495.[Abstract] (ER-) and ß (ER-ß) mRNA in the rat pituitary, gonad, and reproductive tract. Steroids 1998; 63:498–504.[CrossRef][Medline]-diol, 17ß-estradiol from male beagles with spontaneous or induced benign prostatic hyperplasia. Invest Urol 1981; 19:142–147.[Medline] mRNA levels and characterization of a partial length rat P45017 cDNA. Biochem Biophys Res Commun 1988; 154:151–158.[CrossRef][Medline]-hydroxylase/17–20 lyase in the fetal rat testis is reduced by maternal exposure to exogenous estrogens. Endocrinology 1996; 137:1063–1070.[Abstract]This article has been cited by other articles:
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  IN MEMORIAM Matthew P. Hardy, Ph.D. 1957-2007 Biol Reprod, March 1, 2008; 78(3): 563 - 564. [Full Text] [PDF]
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 T. M. Price, S. K. Murphy, and E. V. Younglai Perspectives: The Possible Influence of Assisted Reproductive Technologies on Transgenerational Reproductive Effects of Environmental Endocrine Disruptors Toxicol. Sci., April 1, 2007; 96(2): 218 - 226. [Abstract] [Full Text] [PDF]
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 K Svechnikov, V Supornsilchai, M-L Strand, A Wahlgren, D Seidlova-Wuttke, W Wuttke, and O Soder Influence of long-term dietary administration of procymidone, a fungicide with anti-androgenic effects, or the phytoestrogen genistein to rats on the pituitary-gonadal axis and Leydig cell steroidogenesis J. Endocrinol., October 1, 2005; 187(1): 117 - 124. [Abstract] [Full Text] [PDF]
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 |
|
 C. J. Thompson, S. M. Ross, and K. W. Gaido Di(n-Butyl) Phthalate Impairs Cholesterol Transport and Steroidogenesis in the Fetal Rat Testis through a Rapid and Reversible Mechanism Endocrinology, March 1, 2004; 145(3): 1227 - 1237. [Abstract] [Full Text] [PDF]
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B. T. Akingbemi, C. M. Sottas, A. I. Koulova, G. R. Klinefelter, and M. P. Hardy 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 Endocrinology, February 1, 2004; 145(2): 592 - 603. [Abstract] [Full Text] [PDF]
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B. T. Akingbemi, R. Ge, C. S. Rosenfeld, L. G. Newton, D. O. Hardy, J. F. Catterall, D. B. Lubahn, K. S. Korach, and M. P. Hardy Estrogen Receptor-{alpha} Gene Deficiency Enhances Androgen Biosynthesis in the Mouse Leydig Cell Endocrinology, January 1, 2003; 144(1): 84 - 93. [Abstract] [Full Text] [PDF]
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L. You, M. Casanova, E. J. Bartolucci, M. W. Fryczynski, D. C. Dorman, J. I. Everitt, K. W. Gaido, S. M. Ross, and H. d'A. Heck Combined Effects of Dietary Phytoestrogen and Synthetic Endocrine-Active Compound on Reproductive Development in Sprague-Dawley Rats: Genistein and Methoxychlor Toxicol. Sci., March 1, 2002; 66(1): 91 - 104. [Abstract] [Full Text] [PDF]
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 K B. Rhouma, O Tebourbi, R Krichah, and M Sakly Reproductive toxicity of DDT in adult male rats Human and Experimental Toxicology, August 1, 2001; 20(8): 393 - 397. [Abstract] [PDF]
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 |
|
 H. Nejaty, M. Lacey, and S. A. Whitehead Differing Effects of Endocrine-Disrupting Chemicals on Basal and FSH-Stimulated Progesterone Production in Rat Granulosa-Luteal Cells Experimental Biology and Medicine, June 1, 2001; 226(6): 570 - 576. [Abstract] [Full Text] [PDF]
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