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Toxicology |
a Population Health and Reproduction,
b Center for Health and the Environment,
c California National 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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-hydroxylase/17,20-lyase (P450c17) in the presence or absence of TCDD. Human LGCs were plated directly on plastic culture dishes in medium supplemented with 2 IU/ml of hCG. TCDD (10 nM) or its solvent was added directly to the cells at the time of medium change, every 48 h for 8 days. The objective of the experiment was to test the hypothesis that exogenous steroid, substrate for P450c17, would reduce the TCDD effects on E2 synthesis. With dehydroepiandrosterone (DHEA) (a P450c17 product), a dose-related increase in E2 production was observed and the effect of TCDD on lowering E2 production disappeared. In contrast, with increasing doses, up to 10 µM, of pregnenolone (P5), no change in E2 production was observed. However, 17-hydroxypregnenolone (17P5) at 10 µM produced a modest but significant increase in the E2 production. Treatments with P5 and 17P5 did not alter the effect of TCDD on E2 production. Radiolabeled substrate utilization by hLGC suggests that the principal metabolic pathway for 
5 substrates is the conversion to a 4 product probably by a very active 3ß-hydroxysteroid dehydrogenase. We conclude that estrogen production by hLGC is limited at the level of lyase activity. Thus, these data suggest that the most likely target for the TCDD-induced inhibition of estrogen synthesis by hLGC is the 17,20-lyase activity of the P450c17 enzyme complex.
granulosa cells, steroid hormones, toxicology
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In this article, we use an approach that involves metabolism of steroid substrate for specific steroidogenic enzymes. The challenge of separating, identifying, and quantifying individual steroids was met using high-pressure liquid chromatography (HPLC) as previously reported [20–22]. Specifically, a chromatographic system using reverse phase HPLC was employed to separate steroid metabolites produced by hLGC in vitro after incubation for defined periods of time in the presence of specific radiolabeled substrates.
The primary working hypothesis is that TCDD targets the rate-limiting step of E2 synthesis by hLGC. Steroids such as pregnenolone (P5), 17-hydroxy pregnenolone (17P5), and dehydroepiandrosterone (DHEA) can be used as a substrate for identifying cytochrome P450 17-hydroxylase/17,20-lyase (P450c17) as a possible rate-limiting step. Similarly, the use of androgens such as DHEA that eventually can be substrate for P450arom as well as androstenedione (A4) [19] may well answer whether the aromatase enzyme is working under saturating conditions or is able to handle more substrate for estrogen conversion. Using these same steroid substrates in the presence or absence of TCDD, it would be possible to find which substrate is able to revert the TCDD effect on E2 production, providing in this way evidence for identifying the target for TCDD toxicity in this model. We already demonstrate that P450arom is neither rate limiting nor a target for TCDD in the production of E2 [19]. The next likely possibilities are 17-hydroxylation or 17,20-lyase (both activities of P450c17) or some earlier enzymatic step involved in the production of E2.
The goal of this experiment was to characterize the effect of TCDD on P450c17, the enzyme that produces androgen substrates for aromatization in hLGC. We tested the hypothesis that the addition of P450c17 products, but not the addition of P450c17 substrates, would reverse the adverse effect of TCDD on the in vitro production of estrogen by hLGC.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Dulbecco Modified Eagle Medium (DMEM), antibiotic-antimycotic (10 000 units/ml penicillin G sodium, 10 000 µg/ml streptomycin sulfate, and 25 µg/ml amphotericin B as a Fungizone), fetal bovine and calf sera were purchased from Gibco BRL (Grand Island, NY). Pregnyl, a commercial hCG, was purchased from Organon Inc. (West Orange, NJ). TCDD was a kind gift from Dr. Steven Safe (Texas A&M University). Dimethyl sulfoxide (DMSO) was used as the TCDD solvent and was purchased from Sigma Chemical Co. (St. Louis, MO). P5, 17P5, DHEA, and A4 were purchased from Steraloids Inc. (Newport, RI). [7-3H(N)]P5 (21.1 Ci/mmol), [7-3H(N)]17P5 (21.2 Ci/mmol), [1,2,6,7-3H(N)]A4 (86.4 Ci/mmol), and [1,2,6,7-3H(N)]DHEA (92.5 Ci/mmol) were purchased from New England Nuclear (Boston, MA). A 3ß-hydroxysteroid dehydrogenase (3ßHSD) inhibitor, Trilostane, was obtained from Sanofi, Sterling Winthrop Pharmaceuticals Research Division (Rensselaer, NY). HPLC-grade distilled water and acetonitrile (ACN) were purchased from Fisher Scientific (Santa Clara, CA). All other chemicals were purchased from Sigma Chemical Co.
Human LGC Culture
Granulosa cells were processed according to a protocol previously described [16] with modifications. Briefly, hLGC were obtained from a total of 27 patients undergoing assisted reproduction treatments at the Northern California Fertility Medical Center (Roseville, CA). Cells were retrieved by ultrasound-guided follicle aspiration. After oocytes and cumulus masses were removed, red blood cells (RBC) and hLGC were separated by centrifugation. The collected hLGC from each patient separately were resuspended gently with 4.0 ml DMEM. Residual RBC were removed by 40% Percoll gradient centrifugation. Human LGC were then washed twice, resuspended, and plated at 5 x 105 cells in 3 ml/60-mm plate (17 667 cells/cm2) or at 50 000 in 0.5 ml/four-well plate (26 315 cells/cm2) in DMEM supplemented with antibiotic, antimycotic, 0.02 IU/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 IU/ml hCG) and culture continued for 48 h before the addition of treatment experiments. Plates were assigned randomly to every experimental group, being careful to match control and treated plates within each individual patient.
TCDD Treatment
TCDD was dissolved in DMSO and added directly to the plate in a ratio of 1 µl/ml of conditioned medium. A final concentration of 10 nM was chosen as a treatment based on our previous studies [17, 19]. Control cells received an equal volume of TCDD solvent (DMSO). TCDD-containing medium was changed every other day for 8 days (throughout culture Day 10). An aliquot of the conditioned medium from the last day of culture was stored frozen at -20°C until hormone analysis. In those experiments involving steroid substrate addition, both the TCDD and the steroids were added on the same day (starting on culture Day 2).
Substrate Addition
Starting on culture Day 2, hLGC from several patients were incubated in the presence of exogenous steroid substrate at various concentrations in the presence or absence of TCDD. P5 and 17P5 were prepared in 100% ethanol, and 1–5 µl were added to each milliliter of conditioned medium to obtain final concentrations of 0.1, 1, 5, and 10 µM. DHEA, prepared in the same way, was added at a final concentration of 0.001, 0.01, 0.1, and 1 µM. The steroid substrates were added every other day together with the fresh medium and were present until the end of the experiment on culture Day 10.
3ßHSD Inhibition
The Trilostane is capable of inhibiting 3ßHSD with an inhibitory constant (Ki) that ranged from 0.04 to 0.061 µM for human placenta [23], was 0.16 µM for pig testes [24], and was 0.23 µM for rat adrenal microsome [25]. For monkey granulosa cells in vitro, a dose of 250 ng/ml (0.75 µM) produced a 90% inhibition on P4 production [26]; therefore, a dose of 1 µM was chosen to block 3ßHSD in this experiment. Human LGC were plated at 50 000 cells per well on a four-well plate and treated with TCDD as explained above. On Day 10 of culture, the cells were treated with P5 or 17P5 (3 µM) in the presence or absence of Trilostane (1 µM) for an additional 24 h. At the end of the incubation time, the conditioned medium was prepared for HPLC analysis of the metabolites. Cells from three different patients were included in this experiment.
Substrate for HPLC Analysis
At the end of TCDD treatment on culture Day 10, both control and TCDD-treated hLGC were incubated in serum-free medium containing a mixture of radiolabeled and cold steroid. Approximately 100 000 cpm of each substrate was added in conjunction with the unlabeled substrate (3 µM for P5 and 17P5, 150 nM for A4). Incubations were made for limited periods of time, from 0.5 to 24 h, to determine the time when the substrate was metabolized and the radiolabeled products were best recognized. Two additional vials containing the medium plus the steroids were incubated at the same time as the cells to be used as controls for total count and recovery efficiency (usually 70%–80%). Culture medium was saved at -20°C until HPLC analysis.
Sample Preparation for HPLC
One-milliliter aliquots of tissue culture medium were extracted once with 5 ml diethyl ether. The organic phases were dried under a N2 stream while in a 37°C water bath. One milliliter of 100% pure EtOH was then added to each dried sample and 0.5-ml aliquots of each sample were transferred into two vials (A and B), which were subsequently dried in a vacuum with a Speed Vac Concentrator (Savant Instruments, Farmingdale, NY). Each sample A vial received 50 µl 40%:60% (v:v) ACN:H2O and 50 µl of cocktail A (47.4 ng estrone, 57.6 ng 17ß-estradiol, and 200 dpm/µl each of [3H]-DHEA and [3H]-17P5 in 40%:60% [v:v] ACN:H2O). Each sample B vial received 50 µl 40%:60% (v:v) ACN:H2O and 50 µl of cocktail B (1.7 ng androstenedione and 7.9 ng testosterone in 40%:60% (v:v) ACN:H2O.
Equipment
All chromatography was performed with an Agilent model 1100 HPLC system (Agilent Technologies, Wilmington, DE) that included a model 1100 binary pump module, a thermal column compartment, and a variable wavelength detector. Radioactive peaks were detected and counted with a ß-Ram (Inus Systems, Inc., Tampa, FL) in-line detector. All peak data (UV and radioactivity) were collected and analyzed by microcomputer using Scintflow software (Scintco, Augusta, NJ). The data are the area under the curve for each detectable peak, and they are expressed as a percentage of the total for each HPLC analysis.
HPLC of Samples
Sample steroid analytes were analyzed by reverse-phase HPLC using an Adsorbosphere C18 (5-µm, 150 x 4.6-mm) column (Alltech Associates, Inc., Deerfield, IL) and were eluted isocratically with 40%:60% (v:v) ACN:H2O at a 1.5 ml/min flow rate and at 37°C.
All detectable metabolites from samples were identified by comparison with corresponding known standards in both the radioactive and UV detection profiles.
Hormone Determination
Concentrations of P4, E2, A4, and DHEA in the conditioned medium were determined on Day 10 using commercial RIA kits (Diagnostic Products Corporation, Los Angeles, CA). Interassay coefficients of variation for P4 were below 3% and for E2 were lower than 5%.
Data Analysis
Hormonal concentrations were represented as the mean ± SEM. Normality was determined by Kolmogorov-Smirnov test. For data that were not normally distributed, the values were log transformed. Comparisons between control and TCDD-treated groups were made by one-way ANOVA followed by a pairwise multiple comparison procedure using the Student-Newman-Keuls method. For the substrate addition and TCDD treatment of block design experiment, the data were analyzed by ANOVA with repeated measures and a significance level of alpha = 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Estradiol concentrations were significantly decreased (P = 0.01, n = 13) by treatment from 1.6 ± 0.6 to 0.8 ± 0.4 ng/ml (Fig. 1A). On the other hand, the mean P4 concentration in conditioned medium of hLGC (Fig. 1B) decreased slightly, from 4.8 ± 1.2 to 2.6 ± 0.8 µg/ml (P = 0.06, n = 6). There was no significant change between the conditioned medium (DMEM) alone and the medium plus DMSO vehicle controls; therefore, all subsequent comparisons were made using the DMSO vehicle control group. It is important to note that several attempts were made to measure A4 and DHEA in the conditioned media but levels for these androgens were below the limit of detection of the assay used, currently 8 pg/ml.
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Effect of Substrate Addition
The addition of exogenous P5, substrate for 17-hydroxylase, did not affect the production of E2 or P4 (Fig. 2, A and B), whereas the addition of 17P5, substrate for 17,20-lyase, significantly increased E2 production, almost twofold (from 1.6 ± 0.6 to 3.0 ± 0.9 ng/ml) at the highest dose (10 µM) (Fig. 2C). P4 concentration presented a trend to decrease with increasing doses of 17P5 (from 4.8 ± 1.2 without 17P5 to 2.7 ± 0.6 µg/ml at 10 µM) (Fig. 2D). As previously demonstrated, the addition of DHEA to the hLGC stimulated the production of E2, and the inhibitory effect of TCDD on E2 production disappeared following this treatment [19]. In the present study, we observed that DHEA exponentially stimulated E2 production in a concentration-dependent manner, to a maximum of 180 ± 30 ng/ml (Fig. 3), more than 100 times the levels of control without additional substrate (1.6 ± 0.6 ng/ml). Thus, addition of just 0.01 µM of DHEA increased E2 production to a level similar to that observed with addition of three orders of magnitude higher concentrations of 17P5 (10 µM) (Fig. 3). The inhibitory effect of TCDD on E2 production was not observed in the presence of added DHEA (data not shown), as previously reported [19], but was maintained when either P5 or 17P5 were added (Fig. 2).
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Steroid Metabolism
Human LGC from 11 different patients were used for this experiment and several time points were analyzed for each patient. Human LGC metabolized P5 to E2 in approximately 1 h (see Fig. 4), with several additional products detected. The results indicated that P5 was principally converted to 17P4 (Fig. 4A), with only a fraction (from 2% to 4%) of radiolabel identified as E2 following 1–24 h of incubation (Fig. 4B). In only one experiment was it possible to detect androgens in the form of testosterone (T) after 6 h of P5 substrate incubation. Human LGC metabolized 17P5 preferentially to 17P4 (Fig. 5). The intermediary and product of P450c17, DHEA, was observed in only one experiment following 30 min and 1 h of 17P5 substrate incubation. It is interesting to note that the level of DHEA in TCDD-treated cells was reduced by 50% while no differences between control and TCDD-treated cells were observed for 17P4. The final product, E2, appears in relatively small amounts (less than 5%) only after 4 h of incubation with 17P5. At 1 h of 17P5 incubation, more than 90% of substrate was metabolized, with the majority of the products (more than 50%) being 17P4 (Fig. 5B); no differences in the metabolism of 17P5 were observed for control and TCDD-treated cells. The metabolism of A4 showed that almost all added substrate (80%) was converted to E2 (47%) and E1 (7.6%), with only traces of T (1%) detected in 30 min. No differences in the level of detected metabolites were observed with or without TCDD from 0.5 to 8 h of A4 incubation (Fig. 6). These data support the concept that 4 substrates tend to accumulate at 17P4, with only small amounts being converted to androgens and estrogens, and that the potential for aromatization is high. Further, the accumulation of 17P4 is also consistent with 3ßHSD activity being more efficient than that of P450c17.
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3ßHSD Inhibition
On Day 10 of culture, the cells were treated with P5 or 17P5 (3 µM) in the presence or absence of Trilostane (1 µM) for an additional 24 h. At the end of the incubation time, the conditioned medium was prepared for HPLC analysis following the same protocol explained above. The metabolism of 17P5 was primarily to 17P4, with 7% of the substrate remaining as 17P5 and 59% converted to 17P4 (Fig. 7). Trilostane decreased the metabolism of substrate; 33% of added 17P5 remained after incubation and 14% of substrate remained unmetabolized in cultures of cells treated with TCDD. The combination of TCDD and Trilostane further decreased 17P5 metabolism in comparison with either treatment alone, with 60% of substrate remaining at the end of incubation. At the same time, 17P4 product followed a similar but reversed pattern as compared with 17P5. Trilostane decreased the 17P4 to 47%; TCDD did not affect the 17P4 level, but the combination of TCDD and Trilostane decreased 17P4 to 15%. Despite the inhibition of 3ßHSD, there were no other metabolite products of 17P5. On the other hand, the metabolism of P5 was minimal, with only 2% converted to 17P4 and more than 70% of the radiolabel recovered as P5 substrate (Fig. 8).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The primary working hypothesis was that TCDD targets the rate-limiting step of E2 synthesis by hLGC. The experiments with substrates P5 and 17P5 plus the data from DHEA addition point to the 17,20-lyase as the rate-limiting step. Therefore, if P5 were able to alleviate the adverse effect of TCDD on E2 production, then the blockage and target for TCDD toxicity would be at a step earlier than 17-hydroxylation. The data presented here do not support this possibility. Alternatively, if the addition of 17P5 were able to alleviate the adverse effect of TCDD on E2 production, then the target for TCDD toxicity would be at least partially at the level of production of 17P5, i.e., the 17-hydroxylation activity of the P450c17. At the present time, it is not possible to directly measure any enzyme activity other than P450arom, as reported earlier [19]. We found that the addition of P5 and 17P5 substrates did not eliminate the effect of TCDD on E2 production at any concentration up to 10 µM. In contrast, DHEA completely overcame the TCDD-induced block in hLGC, and A4 does it on rat granulosa cells [30]; the P5 and 17P5 metabolism data support the concept that the flow of these steroids in hLGC is altered by TCDD at the point of P450c17. Therefore, the results from P5 metabolism studies do not support the hypothesis that TCDD is targeting at a step proximal to P450c17. This interpretation contrasts with that proposed for the effects of TCDD on testicular steroidogenesis in the rat, i.e., that TCDD alters early steps in steroid biosynthesis, specifically cholesterol transport to the inner mitochondria membrane [8]. It has also been suggested that the effect of the arylhydrocarbon receptor and its ligands (dioxins, such as TCDD) on the production of steroids may be a direct effect on the steroidogenic acute regulatory protein (StAR) [31]. The modest decrease we observed in P4 production would be consistent with this possibility. However, the decrease in progestagen synthesis cannot explain the decrease in estrogen production observed here. Specifically, the production of progesterone exceeded that of estrogen by three orders of magnitude, even after TCDD treatment, suggesting that substrate supply at the level of pregnanes, even if severely inhibited, is unlikely to be limiting and that another target of toxicity must exist. Our data demonstrate that enzyme activities leading to androgen synthesis, 17-hydroxylase and/or 17,20-lyase, are substrate saturated, apparently rate-limiting for estrogen production, and therefore the most likely target of relevance to estrogen synthesis in hLGC. Inhibition of progesterone production by TCDD has not been a consistent finding in studies using hLGC cultures. The results of some investigations revealed a decrease in progesterone production by hLGC that were cultured in the presence of TCDD as reported here [32]. Other reports, however, have not verified these findings [19, 27]. The trend of a dose-dependent decrease in P4 concentration in response to increasing concentrations of 17P5 suggests that exogenous 17P5 may compete with endogenous P5 for 3ß HSD conversions to their respective 4 products. If true, this competition would also explain how it would be possible to observe decreased P4 levels when 17,20-lyase inhibition resulted in a concomitant and critical increase in competition for 3ßHSD by the 17,20-lyase substrate.
In the present study, the duration of exposure of TCDD was 10 days, which is longer than many other studies investigating the effect of TCDD on steroidogenesis [27]. Early effects of TCDD may be different than those shown here and could be limited to processes upstream of P450c17. Because TCDD is a long-acting toxicant in vivo, we believe that the longer incubation times better simulate in vivo exposures, as evidenced by the similar effects of TCDD on estrogen production in vivo [11, 12, 14, 15]. Furthermore, it seems more likely that adverse toxic effects would be directed to a rate-limiting step rather than a process that does not regulate downstream events. In steroidogenic cells, the rate-limiting steps identified to date include StAR and P450 side chain cleavage [33, 34]. We propose therefore that the rate-limiting step in our model for the production of estrogens is P450c17 and this step is the target of TCDD toxicity. This conclusion is supported by the findings that high concentrations of P5 or 17P5 did not stimulate E2 production as did DHEA.
Questions remain regarding intracellular partitioning, i.e., events related to exogenous substrates reaching the cell, crossing the plasma membrane, and becoming available for metabolism. The results of our experiments with radiolabeled substrates and HPLC separation of the resulting products clearly indicate that the substrates were available for metabolism. From these data, we conclude that the majority of the substrates progress through the 4 pathway, supporting again the idea of a rate-limiting step at the P450c17 enzyme. In order to enhance the flux of steroid substrates toward DHEA and ultimately to E2, we included in the experimental protocol the use of a 3ßHSD inhibitor. This inhibitor, Trilostane, was able to maintain P5 and 17P5 on the pathway as 5 metabolites, a pathway that is more effective for the conversion of c21 to c19 steroids in humans [35]. Similar observations were made with luteinizing granulosa cells from the rhesus macaque, in which Trilostane decreased the production of P4 and at the same time increased E2, indicating an increased flux through the 5 pathway when 3ßHSD was inhibited [26]. However, the data presented here do not allow analysis of the flux through the 5 pathway, as no metabolism other than the production of 17P4 was achieved by the Trilostane in the 24-h experiment. These data support the concept of a very active 3ßHSD enzyme relative to a lower and less efficient 17,20-lyase enzyme activity that is probably the rate-limiting step in the E2 production by hLGC. In summary, the current data indicate that hLGC treated with TCDD in the presence of exogenous DHEA induced a dose-related increase in E2 production and the effect of TCDD on lowering E2 production disappeared. In contrast, when cells were incubated with even higher concentrations of P5 (up to 10 µM), no change in E2 production was observed. However, when cells were incubated with 10 µM 17P5, there was a twofold increase in the E2 production. Treatments with P5 and 17P5 did not alter the effect of TCDD on E2 production. Our data also suggest that the principal metabolic pathway for 5 substrates is the conversion to a 4 product, probably by a very active 3ßHSD.
The presence of P450c17 on hLGC is controversial and not completed established by this study alone. However, our group has mounting evidence that this enzyme is present in this model system. In this article, we demonstrate that E2 is produced without the addition of an androgen substrate, that P5 is converted to 17P5, and 17P5 is converted to DHEA. Previously, we reported high, and parallel to progesterone, production of 17P4 by these cells [19], and we have preliminary evidence that demonstrates the presence of P450c17 in hLGC lysates by immunoblot as well as 17,20-lyase activity by a radiometric assay (unpublished manuscript). While cell purity was not determined directly and theca cells could be a contaminant providing additional enzyme activities, we believe that these are standard follicular aspirates that are widely accepted as containing only granulosa cells. However, even if there is theca cell contamination, the use of hLGC in culture as a model for the primate corpus luteum was validated by Stewart and VandeVoort [16] and it has been used as an experimental model for several years [17, 19] with reliable and reproducible results in term of steroid production. In addition, there can be blood contamination (depending on patient), but most of the red blood cells (RBC) are removed during the Percoll wash. Furthermore, the RBC do not plate and do not survive in culture for the 10 days. Therefore, it is considered that these cells are relatively pure granulosa cells, which spontaneously luteinize.
We conclude that one of the rate-limiting steps on hLGC estrogen production is at the level of 17,20-lyase activity (the production of DHEA) and that the inhibition of E2 secretion by TCDD is most likely at this rate-limiting step. While these data do not allow us to clearly identify the specific target for TCDD toxicity, the accumulated evidence suggests that TCDD most likely is targeting the proposed rate-limiting step, i.e., the 17,20-lyase activity of the P450c17 enzyme complex. At this time, we are working on specific ways to make more direct measurements of both protein amounts and enzyme activity of P450c17 in hLGC to provide further support for this hypothesis.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Bill L. Lasley, Center for Health and the Environment, University of California, Davis, One Shields Avenue, Davis, CA 95616-8615. FAX: 530 752 5300; bllasley{at}ucdavis.edu
Received: 3 May 2002.
First decision: 28 May 2002.
Accepted: 7 August 2002.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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4-ene-isomerase activity. J Steroid Biochem Mol Biol 1990 37:231-236[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]This article has been cited by other articles:
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  A. Guzeloglu, T. R. Bilby, A. Meikle, S. Kamimura, A. Kowalski, F. Michel, L. A. MacLaren, and W. W. Thatcher Pregnancy and Bovine Somatotropin in Nonlactating Dairy Cows: II. Endometrial Gene Expression Related to Maintenance of Pregnancy J Dairy Sci, October 1, 2004; 87(10): 3268 - 3279. [Abstract] [Full Text] [PDF]
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 A. Guzeloglu, P. Subramaniam, F. Michel, and W. W. Thatcher Interferon-{tau} Induces Degradation of Prostaglandin H Synthase-2 Messenger RNA in Bovine Endometrial Cells Through a Transcription-Dependent Mechanism Biol Reprod, July 1, 2004; 71(1): 170 - 176. [Abstract] [Full Text] [PDF]
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B.-M. Huang, K.-Y. Hsiao, P.-C. Chuang, M.-H. Wu, H.-A. Pan, and S.-J. Tsai Upregulation of Steroidogenic Enzymes and Ovarian 17{beta}-Estradiol in Human Granulosa-Lutein Cells by Cordyceps sinensis Mycelium Biol Reprod, May 1, 2004; 70(5): 1358 - 1364. [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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