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Articles |
a Institute of Toxicology and Environmental Health,
b Population Health and Reproduction,
c California Regional Primate Research Center, University of California-Davis, Davis, California 95616
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-hydroxyprogesterone, the inhibition of E2 synthesis by TCDD would seem not to involve steps such as cholesterol transport. Furthermore, the TCDD effect on E2 concentration in these cells disappeared in the presence of excess androgens. We conclude that the inhibition of E2 secretion by TCDD involves intermediate steps, specifically, the provision of androgens for aromatization.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Minimum essential medium (MEM), Dulbecco's modified Eagle's medium (DMEM), and antibiotic-antimycotic (10 000 U/ml penicillin G sodium, 10 000 µg/ml streptomycin sulfate, and 25 µg/ml amphotericin B as Fungizone), as well as fetal bovine and calf serum, were purchased from GIBCO BRL (Grand Island, NY). Pregnyl, a commercial hCG, was obtained from Organon Inc. (W. Orange, NJ), and TCDD was from CIL (Woburn, MA). Cold steroids, 5-androsten-3ß-ol-17-one (DHEA), and 4-androsten-3,17-dione (androstenedione) were purchased from Steraloids (Wilton, NH). [1ß,2ß-3H]-Androstenedione (specific activity, 42.0 Ci/mmol), [14C]E2 (53.2 mCi/mmol), and 3H-water were obtained from New England Nuclear (Boston, MA). All other chemicals were from Sigma Chemical Co. (St. Louis, MO).
hLGC Culture
Granulosa cells were processed according to a protocol previously described [19] with modifications. Briefly, hLGC were obtained from 21 patients undergoing assisted reproduction treatments at the Pacific Fertility Center (Sacramento, CA). Cells were retrieved by ultrasound-guided follicle aspiration. After oocytes and cumulus masses were removed, the follicular fluid was transported on ice to the California Regional Primate Research Center. Red blood cells (RBC) and hLGC were separated by centrifugation. The collected hLGC were resuspended gently with 4.0 ml MEM. Residual RBC were removed by Percoll (40%) gradient centrifugation. hLGC were then washed twice, resuspended, and plated at 5 x 105 cells per 60-mm plate in MEM supplemented with antibiotic, antimycotic, 0.02 USP/ml of hCG, and 5% fetal calf serum at 37°C in an atmosphere of 5% CO2 in air. Medium was changed after 24 h of preincubation to DMEM (supplemented with antibiotic-antimycotic, 10% calf serum, and 2 USP/ml hCG) in a final volume of 3 ml per dish, and culture continued for 48 h before the addition of treatment (designated culture Day 2). Plates were assigned randomly to every experimental group, with care taken to match control and treated plates within each individual patient. Samples of medium were assayed for steroid hormone content and, when the number of available cells permitted, enzyme activity was determined and enzyme protein levels were evaluated by Western blot analysis. Experiments were also conducted to investigate the saturation of aromatase activity by addition of excess androgen substrate, and estrogen metabolism was examined after addition of labeled estradiol.
TCDD Treatment
Previous studies [8] determined that a dose of 10 nM TCDD was the lowest maximally effective level that altered hormone production in hLGC. Therefore, 3 µl of a 10-5 M TCDD stock was added directly to each plate drop-wise to avoid precipitation. Control cells received an equal volume of TCDD solvent (dioxane). Medium was changed with fresh TCDD every other day for 8 days (throughout culture Day 10) and stored frozen at -20°C until hormone analysis. In those experiments combining TCDD with steroid substrate addition, both the TCDD and the steroids were added on the same day (starting on culture Day 2). After a dose-response analysis for both androgens (data not shown), saturating concentrations of DHEA (1 µM) and androstenedione (56 µM) were used for these experiments.
Hormone Determination
Conditioned medium was saved every 48 h from culture Day 2 (baseline) through the end of the experiment (culture Day 10). Concentrations of estradiol (E2) and progesterone (P4) in this medium were determined using available commercial RIA kits (Diagnostic Products Corporation, Los Angeles, CA). 17-Hydroxyprogesterone (17OHP4) concentrations were measured in collaboration with Dr. Paul Terranova at the Kansas University Medical Center as previously reported [20].
P450arom Activity Assay
At the conclusion of the experiment, P450arom activity was assayed by measuring the incorporation of tritium from [1ß,2ß-3H]androstenedione (150 nM) into water as previously described [21,22]. Briefly, on culture Day 10, hLGC were incubated for 30 min or 2 h at 37°C in a 5% CO2/95% air atmosphere with a serum-free medium containing labeled substrate. Medium was removed from the cells, proteins in the medium were precipitated with trichloroacetic acid (10% final concentration), and steroid was then extracted with chloroform. Charcoal/dextran was used to extract all remaining steroids, and 3H-water was measured in an aliquot from the aqueous phase by scintillation counting. After substrate incubation, cells were removed from the culture dish in 0.1 N NaOH, then briefly sonicated to disrupt the cell membrane. Protein determinations were made in the cell lysate using the Bicinchoninic Acid Protein Assay Reagent (Pierce, Rockford, IL) and were used both as an index for cell viability and in the final determination of P450arom activity, which was expressed as picomoles per milligram of protein per hour. Tritium release during aromatization is fractionally less from [1ß,2ß-3H]androstenedione than from [1ß-3H]androstenedione [22], and correspondingly more label is retained on the steroid product. This allowed for analysis of the organic phase by HPLC, which confirmed the identity of product as estrone (data not shown).
Western Immunoblot
At the end of the experiment, cells from additional plates were homogenized in PBS containing 1% sodium cholate 0.1% SDS and sonicated for 3 sec. Protein concentrations were estimated as explained earlier. Cell homogenates (25 µg protein/lane) were fractionated by electrophoresis on an 8% polyacrylamide-SDS gel at 150 V for 1 h in buffer containing 50 mM Tris, 383 mM glycine, 10% SDS, and 0.4 mM EDTA. The separated proteins were electroblotted onto a polyvinylidene fluoride (PVDF) membrane (Immobilon-P; Millipore, Bedford, MA) and immunoblotted with rabbit antisera (1:400 dilution) raised against recombinant human P450arom (courtesy of Dr. N. Harada, Fujita Health University, Japan). Immunoblotting procedures were carried out at room temperature in PBS with 0.1% Tween 20 according to the manufacturer's instructions (ECL; Amersham, Arlington Heights, IL) using a donkey anti-rabbit horseradish peroxidase (HRP)-linked IgG whole antibody (Amersham) at 1:10 000 dilution. Immunoreactive bands were visualized by autoradiographic detection of the chemiluminescent signal (New England Nuclear). After detection, membranes were stripped (100 mM 2-ß mercaptoethanol, 2% SDS, and 62.5 mM Tris-HCl pH 6.7) for 30 min at 50°C and reblotted for P45017 hydroxylase/17, 20 lyase (P450c17) using antisera raised against purified porcine P450c17 (1:2000 dilution; gift of Dr. A. Payne, Stanford University Medical Center, Stanford, CA).
Estradiol Metabolism
On culture Day 8, both control and TCDD-treated hLGC were incubated for 24 h in serum-free medium containing radiolabeled estradiol (0.019 µM; 6750 cpm/plate). An aliquot (1 ml) of this conditioned medium was extracted with 4 vol of diethyl ether, reconstituted in a mixture of 36% acetonitrile in water (HPLC grade), and analyzed by HPLC (Altex 110A system; Altex, Santa Clara, CA) using a Vydac reverse-phase 201TP5415 C18 column (Vydac, Hesperia, CA) under isocratic conditions (36% acetonitrile, 64% H2O, 1 ml/min flow rate). Fractions were collected in an automated Micro Fractionator (Gilson Medical Electronics, Inc., Middleton, WI), added to scintillation cocktail (Ultima Gold Scintillation cocktail; Packard, Downers Grove, IL), and counted. The identity of radioactive peaks was confirmed with authentic standards.
Data Analysis
Hormonal concentration data were log-transformed for statistical analysis and represented as the mean ± SEM of each sample per day. Comparisons were made at all sample times by one-way repeated-measures ANOVA, followed by a pair-wise multiple-comparison procedure using the Student-Neuman-Keuls Method. Correlation analysis among the steroids and between steroids and P450arom activity were determined using the Pearson's product-moment correlation method. Differences with a P < 0.05 were considered statistically significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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P4 concentrations were higher than 17OHP4 at all times and increased with time in culture. However, P4 concentrations were seen to plateau by culture Day 4, whereas 17OHP4 concentrations continued to increase in both control and treated groups until culture Day 6 (Fig. 1, A and B). In control hLGC, P4 increased from 8.7 ± 1.5 µg/ml to 15.7 ± 2.2 µg/ml, and 17OHP4 from 0.49 ± 0.17 µg/ml to 2.57 ± 0.78 µg/ml, from Day 2 to Day 10. TCDD did not alter either P4 or 17OHP4 levels, which were similar to those of controls on all days of culture (P4, 8.9 ± 1.5 and 14.9 ± 2.0 µg/ml; 17OHP4, 0.54 ± 0.21 and 1.52 ± 0.32 µg/ml, on culture Days 2 and 10, respectively). Similarly, levels of E2 in medium (Fig. 1C) increased from culture Day 2 to 10 (0.94 ± 0.31 to 27.55 ± 10.87 ng/ml) in control hLGC. Even though obvious in cells from only some of the patients, TCDD significantly decreased estradiol, from culture Day 4 to 10 (P < 0.05), by which time levels were 4-fold lower (6.97 ± 1.64 ng/ml) in TCDD-treated cells than in controls. E2 concentrations varied greatly between individual patients (ranging from 0.034 to 6.19 ng/ml on Day 2, and from 0.086 to 162.7 ng on Day 10 for the control cells; and from 0.022 to 4.87 ng/ml on Day 2, and from 0.065 to 29.31 ng/ml on Day 10 for the treated cells). 17OHP4 and E2 were positively correlated in both control (R = 0.89) and the treated groups (R = 0.95). P4 was correlated positively with both the 17OHP4 and the E2 levels in treated (R from 0.85 to 0.98; P < 0.05) but not control cells.
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TCDD Effects on P450arom
Aromatase activity measured in hLGC from ten different patients on Day 10 of culture ranged from 138–640 pmol/mg per hour and was not correlated with either P4 or E2 accumulation in culture. TCDD did not alter the aromatase activity (Table 1). This was not due to a loss of cells because protein concentrations remained equal in control and TCDD-treated groups. Immunoblot analysis detected a major band at about 53 kDa that corresponds well with the previously reported size of this protein [23]. TCDD exposure appeared not to change the level of P450arom protein (Fig. 2). Furthermore, aromatase activity was not always closely correlated with the levels of immunodetectable P450arom in all patients. P450c17 protein levels were below the limits of detection using the anti-porcine P450c17 antisera.
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Effect of Androgen Substrate Addition
To determine whether or not substrate for P450arom was limiting, estradiol was measured in culture medium from cells provided with saturating levels of androgen. hLGC from three patients were treated with TCDD or dioxane in the presence or absence of 1 µM DHEA. The treatment with DHEA increased E2 concentration from 2.88 ± 1.6 ng/ml to 197.5 ± 55.7 ng/ml in 48 h for the control group, with a similar change in the TCDD-treated group. E2 remained elevated until the end of the experiment. Under these conditions, the effect of TCDD on E2 concentration disappeared (Fig. 3B). In a separate experiment, hLGC from three patients were treated with 56 µM androstenedione. This treatment significantly increased (from 12 ± 1.8 ng/ml to 351 ± 172 ng/ml) the production of E2 in both control and TCDD-treated groups. This E2 increase was evident after 48 h of androgen exposure and remained at this level throughout the rest of the experiment. Under this condition, TCDD treatment also failed to decrease E2 concentration (Fig. 3C).
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TCDD Effect on E2 Metabolism
HPLC analysis of medium following incubation of hLGC with estrogen identified a single peak that corresponded to the added substrate. No metabolism of labeled estradiol was detected during 24 h of incubation with hLGC from any of three different patients in either control or TCDD treatment conditions (Fig. 4).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Previous studies have established the utility of cultured hLGC as an appropriate model for the human luteal function. The rise and eventual fall of E2 and P4 production closely mimic the luteal phase of a menstrual cycle [19]. Although not examined specifically in these previous studies, 17OHP4 production by hLGC was observed in the current experiments and established a profile similar to that seen during the menstrual cycle of rhesus monkeys [26] and during early pregnancy in humans [27,28]. The production of P4, 17OHP4, and E2 by hLGC in culture strongly suggests that these cells express the full complement of steroidogenic enzymes necessary for androgen as well as estrogen production. In particular, the fact that 17OHP4 increased for an additional 48 h beyond the plateau reached by P4 on culture Day 4 strongly supports the idea that P450c17 is functional in this cellular model. Steroidogenic enzymes such as P450c17 and P450arom have been shown to be regulated during the normal menstrual cycle [29]; therefore, they may be susceptible to alterations by diverse xenobiotic exposure.
The present study provides data indicating that TCDD targets specific steps in the steroidogenic pathway, ultimately inhibiting estrogen but not progesterone secretion. TCDD has been shown to inhibit steroidogenesis in a number of different tissues and species, but these effects vary depending on the major steroid products and the physiological state or experimental condition. Although steroidogenesis may be affected by a toxicant such as TCDD at any one, or several, of the steps leading to estrogen production, the major lesion most likely involves the rate-limiting enzyme or component of the pathway. In this study, P450arom was examined as a logical target for TCDD because it is the last committed step in estrogen synthesis [30]. We found that P450arom is neither a target for TCDD toxicity nor a rate-limiting step in the production of E2 in hLGC in vitro. This was clearly demonstrated by a lack of correlation between levels of aromatase activity and E2 synthesis in culture. Furthermore, there was no effect of TCDD on either activity or levels of aromatase enzyme as determined by the P450arom activity assay or the Western blot analysis, respectively. The levels of P450arom protein were not always correlated with enzyme activity, suggesting that other factors play a potentially important role in determining catalytic activity. Aromatase is active only upon formation of an enzyme complex with a redox partner protein, the flavoprotein NADPH-P450 reductase. Therefore, it is possible that hLGC lack sufficient flavoprotein reductase expression to fully support aromatase activity. Preliminary studies with hLGC have shown that the addition of flavoprotein reductase to lysates of hLGC stimulates aromatase activity, consistent with this possibility (data not shown). Regardless, our data suggest that the TCDD inhibitory effect on estrogen production involves steps in the pathway other than P450arom.
Previous reports support the possibility that TCDD may influence steroid production by effects on enzymes early in the biosynthetic pathway. Studies examining the inhibitory effects of TCDD on testicular steroidogenesis in the rat suggest the possibility that the mechanism involves interference of cholesterol transport to the inner mitochondria membrane, and cholesterol side-chain-cleavage P450 (P450scc) activity [31,32]. Contrary to these findings, one study has reported a similar depression of androgen secretion in male rats exposed to TCDD but concluded that it did not involve P450scc [33]. The reason for this discrepancy is not obvious. However, the data reported here are more consistent with those of Mebus et al. [33], suggesting that neither P450scc nor 3ß-hydroxysteroid dehydrogenase (3ß-HSD) is a likely target of TCDD for two reasons. First, TCDD had no effect on levels of P4, and it caused only a slight, nonsignificant depression of 17OHP4. Second, there was no effect of TCDD on the conversion of added DHEA to estradiol, the first step of which is catalyzed by 3ß-HSD. Therefore, although we did not directly measure the isolated activities of these enzymes, we believe our data indicate a block in the steroidogenic pathway between the synthesis of progestins and androgen production.
The possibility that TCDD may inhibit androgen secretion through effects on P450c17 was suggested in the above-mentioned studies by Mebus et al. [33], who reported a greater effect on 17-hydroxylase than on 17,20-lyase activity. However, there are significant differences in function between human and rat P450c17. Specifically, in humans, pregnenolone (Pe) is preferentially metabolized by P450c17 through the 
5 (i.e., Pe, 17-hydroxypregnenolone [17OHPe], and DHEA) rather than the 4 pathway (i.e., P4, 17OHP4, and A4) [34]. Regardless, the addition of androgen substrates eliminated the decrease in estrogen observed with TCDD exposure, consistent with inhibition at steps leading to androgen synthesis. A block in steroid metabolism at any step would be expected to result in accumulation of substrate. Unfortunately, none of the 5 metabolites were measured in the current studies to evaluate substrate levels. Still, substrates might not accumulate if alternative metabolic pathways were activated. Preliminary studies were conducted by incubating hLGC with both Pe and 17OHPe tracers, and the conditioned media were analyzed subsequently by HPLC. TCDD treatment did induce the appearance of additional steroid metabolites that we have not yet been able to either identify or quantify (data not shown).
Biochemical evidence supports the possibility that 17-hydroxylase and 17,20-lyase activities of P450c17 can be affected differentially [35]. Thus it is possible that lyase activity, but perhaps not hydroxylase activity, is a target for TCDD toxicity in hLGC. However, it was not possible to detect P450c17 protein by Western blot analysis. This may have been due to the lack of specificity of the antisera used or simply to low levels of P450c17 protein expression consistent with a previous report finding only low levels of P450c17 mRNA in human granulosa cells [36]. This lack of protein assessment precludes us from a more detailed interpretation of these results at this time. Clearly, further studies that involve specific antibody for the human protein, a P450c17 mRNA analysis, and an assay for the P450c17 enzyme activity will be necessary to properly evaluate the precise role of this enzyme in the inhibition of estrogen secretion by TCDD.
Of special interest is the variation observed on E2 levels on cells from different patients in the face of apparent abundant amounts of P450arom enzyme. This observation suggests that hLGC in vitro have a wide variation in the ability to produce substrate for aromatization and is in agreement with an observation made in early pregnancy in which different ovarian androgen levels were reported between women [28]. Such a difference in circulating androgen levels should directly affect E2 production if P450arom is generally not limiting. The difference in androgen concentration may be interpreted as reflecting differences in the P450c17 enzyme activity or different rates in metabolizing 17OHPe. Therefore, it is possible to hypothesize that the human ovary may have a different susceptibility in response to TCDD, which agrees with our postulate that TCDD is acting at the P450c17 step. Furthermore, we also observed that TCDD inhibited E2 in only some of the patients. Whether or not there is a correlation of the TCDD effect with the amount of E2 produced for hLGC in vitro is not evident from our current data.
The present data demonstrate a direct effect of TCDD on estrogen production by luteal cells and are consistent with some of the in vivo effects of TCDD on early pregnancy in monkeys [7]. Taken together, however, these data do permit the conclusion that a decrease in estrogen production is the primary adverse effect of TCDD on fertility in primates. Not only were the effects of TCDD on estrogen production subtle in both studies, but an essential role of estrogen is not clearly defined during the periimplantation period of human and monkeys [37,38]. It seems likely, however, that the effect of TCDD on ovarian estrogen production may be more important in the preovulatory period to block the ovarian follicle development, which is dependent upon estrogen production. This is consistent with the observations made with the animals in the study after fetal loss. Several months after this study was completed, the animals in the lower TCDD dose group had completely recovered and ovulated regularly whereas the animals in the highest dose group failed to ovulate (unpublished results). This failure to ovulate was associated with lowered estrogen and elevated FSH excretion, suggesting a toxicant-induced block on follicle development.
Finally, the present data do not exclude the possibility that TCDD may increase the metabolism of estradiol itself. The inter-conversion of estrone (E1) and E2 is catalyzed by 17ß-hydroxysteroid dehydrogenase (17ß-HSD) enzymes types I and II. Both isozymes are known to be expressed in the human ovary [39,40] as well as in isolated human granulosa cells [41]. While it is possible that an effect of TCDD on these 17ß-HSD enzymes could have altered the ratio of E2/E1 production, our data using an HPLC analysis for evaluating estrogen metabolites suggest that this hypothesis is also unlikely. Although TCDD has been shown to induce estrogen metabolism by MCF-7 breast cancer cells [42], results of the current study indicate that E2 is not further metabolized by hLGC, whether exposed to TCDD or not. While the results of this study do not support the concept that arylhydrocarbon receptor ligands such as TCDD induce changes in estrogen catabolism at the level of the granulosa cell, this cannot be generalized to include the circulating estrogens observed in vivo. The liver is not only a more likely site for steroid catabolism to take place but is known to respond to TCDD and related compounds with the induction of P450 enzyme activities [43,44]. It would seem most likely, therefore, that TCDD-like compounds may alter only the production rate of estradiol and estrone at the level of the granulosa cell and not change the nature of the kind of estrogens that are secreted. If such toxicants change the ratio of the 4- and 16-hydroxylated estrogens, then that most likely occurs postsecretion at some other organ and therefore we should not to expect to see such differences in our model. However, additional studies using radioactive labels and hLGC to identify all enzyme products will help in firmly establishing the molecular site of the TCDD-induced inhibition of E2 synthesis by hLGC.
In conclusion, we have clearly demonstrated that TCDD decreases E2 production without altering either the P450arom enzyme protein or activity. Since there was no change in either P4 or 17OHP4 secretion or in E2 metabolism by hLGC, we propose that TCDD toxicity is probably targeted to a step other than estrogen metabolism and upstream of aromatization but subsequent to progesterone production, which directly involves the production of androgen substrate for aromatization. This target for TCDD toxicity is most likely to be at the level of the P450c17 complex. We propose a predominant effect of TCDD on the 17–20 lyase activity with a lesser effect on 17-hydroxylase activity, a hypothesis that warrants further consideration.
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ACKNOWLEDGMENTS |
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OHP4. |
FOOTNOTES |
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1 Supported by NIH ESO 6198, RR 00169, and 1RO1HD36913. 
2 Correspondence: Bill L. Lasley, Division of Reproductive Biology, Institute of Toxicology and Environmental Health, University of California, Davis, Old Davis Road, One Shields Avenue, Davis, CA 95616-8615. FAX: 530 752 5300; bllasley{at}ucdavis.edu
Accepted: November 29, 1999.
Received: September 1, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-hydroxyprogesterone during the menstrual cycle of the rhesus monkey. Acta Endocrinol 1972; 71:755–764.-hydroxyprogesterone during human pregnancy. Acta Endocrinol 1972; 71:765–772.-hydroxylase and 17,20-lyase after administration of 2,3,7,8-tetra-chlorodibenzo-p-dioxin (TCDD). Biochem Pharmacol 1987; 36:727–731.[CrossRef][Medline]-hydroxylase and 3ß-hydroxysteroid dehydrogenase in the integration of gonadal and adrenal steroidogenesis via the 5 and 4 pathways of steroidogenesis in mammals. Biol Reprod 1997; 56:789–799.[CrossRef][Medline]-hydroxylase/17,20-lyase) in cultured human granulosa cells. J Clin Endocrinol Metab 1986; 63:202–207.This article has been cited by other articles:
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  Z. Shi, K. E. Valdez, A. Y. Ting, A. Franczak, S. L. Gum, and B. K. Petroff Ovarian Endocrine Disruption Underlies Premature Reproductive Senescence Following Environmentally Relevant Chronic Exposure to the Aryl Hydrocarbon Receptor Agonist 2,3,7,8-Tetrachlorodibenzo-p-Dioxin Biol Reprod, February 1, 2007; 76(2): 198 - 202. [Abstract] [Full Text] [PDF]
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 J. T. Sanderson The Steroid Hormone Biosynthesis Pathway as a Target for Endocrine-Disrupting Chemicals Toxicol. Sci., November 1, 2006; 94(1): 3 - 21. [Abstract] [Full Text] [PDF]
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S. A. Myllymaki, T. E. Haavisto, L. J. S. Brokken, M. Viluksela, J. Toppari, and J. Paranko In Utero and Lactational Exposure to TCDD; Steroidogenic Outcomes Differ in Male and Female Rat Pups Toxicol. Sci., December 1, 2005; 88(2): 534 - 544. [Abstract] [Full Text] [PDF]
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L.-A. Li and P.-W. Wang PCB126 Induces Differential Changes in Androgen, Cortisol, and Aldosterone Biosynthesis in Human Adrenocortical H295R Cells Toxicol. Sci., May 1, 2005; 85(1): 530 - 540. [Abstract] [Full Text] [PDF]
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 C. F. Skibola, J. D. Curry, C. VandeVoort, A. Conley, and M. T. Smith Brown Kelp Modulates Endocrine Hormones in Female Sprague-Dawley Rats and in Human Luteinized Granulosa Cells J. Nutr., February 1, 2005; 135(2): 296 - 300. [Abstract] [Full Text] [PDF]
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C.J. Corbin, F.M. Moran, J.D. Vidal, J.J. Ford, T. Wise, S.M. Mapes, V.C. Njar, A.M. Brodie, and A.J. Conley Biochemical Assessment of Limits to Estrogen Synthesis in Porcine Follicles Biol Reprod, August 1, 2003; 69(2): 390 - 397. [Abstract] [Full Text] [PDF]
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F. M. Moran, C. A. VandeVoort, J. W. Overstreet, B. L. Lasley, and A. J. Conley Molecular Target of Endocrine Disruption in Human Luteinizing Granulosa Cells by 2,3,7,8-Tetrachlorodibenzo-p-Dioxin: Inhibition of Estradiol Secretion Due to Decreased 17{alpha}-Hydroxylase/17,20-Lyase Cytochrome P450 Expression Endocrinology, February 1, 2003; 144(2): 467 - 473. [Abstract] [Full Text] [PDF]
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F.M. Moran, P. Lohstroh, C.A. VandeVoort, J. Chen, J.W. Overstreet, A.J. Conley, and B.L. Lasley Exogenous Steroid Substrate Modifies the Effect of 2,3,7,8-Tetrachlorodibenzo-p-Dioxin on Estradiol Production of Human Luteinized Granulosa Cells In Vitro Biol Reprod, January 1, 2003; 68(1): 244 - 251. [Abstract] [Full Text] [PDF]
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