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Published as doi: 10.1096/fj.06-6681com.
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(The FASEB Journal. 2007;21:586-595.)
© 2007 FASEB

Estrogen receptor-{alpha} mediates an intraovarian negative feedback loop on thecal cell steroidogenesis via modulation of Cyp17a1 (cytochrome P450, steroid 17{alpha}-hydroxylase/17,20 lyase) expression

Fuminori Taniguchi*,1, John F. Couse{dagger},1, Karina F. Rodriguez{dagger}, Judith M. A. Emmen{dagger},2, Donald Poirier{ddagger} and Kenneth S. Korach{dagger},3

* Department of Obstetrics and Gynecology, Tottori University Hospital, Yonago, Japan;

{dagger} Receptor Biology Section, Laboratory of Reproductive and Developmental Toxicology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina, USA; and

{ddagger} Medicinal Chemistry Division, Oncology and Molecular Endocrinology Research Center, Centre Hospitalier Universitaire de Quebec (CHUQ), Pavillon CHUL, Quebec, Canada

3Correspondence: Receptor Biology Section, Laboratory of Reproductive and Developmental Toxicology, National Institute of Environmental Health Sciences, NIH, MD B3–02, P.O. Box 12233, Research Triangle Park, NC 27709, USA. E-mail: korach{at}niehs.nih.gov


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Excess androgen synthesis by thecal cells is invariably detrimental to preovulatory follicles in the ovary and is considered a fundamental characteristic of polycystic ovary syndrome in women. Investigators have long postulated that granulosa cell-derived estrogens modulate thecal cell steroidogenesis via a short negative-feedback loop within the follicle. To test this hypothesis, we assessed the steroidogenic capacity of individual wild-type (WT) and estrogen receptor-{alpha} (ER{alpha})-null follicles when cultured in vitro under comparable conditions. Late-stage ER{alpha}-null follicles exhibited markedly increased expression of the thecal cell enzyme CYP17A1 and secreted much greater amounts of its end product, androstenedione. This phenotype was reproduced in WT follicles when exposed to an aromatase inhibitor or ER-antagonist, and prevented when the former treatment was supplemented with an ER{alpha}-specific agonist. ER{alpha}-null follicles also exhibited increased testosterone synthesis due to ectopic expression of hydroxysteroid (17ß) dehydrogenase type 3 (HSD17B3), a testis-specific androgenic enzyme. These data indicate that ER{alpha} functions within thecal cells to negatively modulate the capacity for androgen synthesis by repressing Cyp17a1 expression, and the biological activity of androgens produced by inhibiting Hsd17b3 expression. Hence, these findings provide novel evidence of an intraovarian ER{alpha} function that may be critical to the latter stages of folliculogenesis and overall ovarian function.—Taniguchi, F., Couse, J. F., Rodriguez, K. F., Emmen, J. M. A., Poirier. D., Korach, K. S. Estrogen receptor-{alpha} mediates an intraovarian negative feedback loop on thecal cell steroidogenesis via modulation of CYP17A1 (cytochrome P450, steroid 17{alpha}-hydroxylase/17,20 lyase) expression


Key Words: hydroxysteroid (17ß) dehydrogenase • hyperandrogenemia • folliculogenesis • aromatase


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
EHRMANN ET AL. (1) once characterized androgens as a "necessary evil" in the ovary, referring to their obligatory role as intermediates in estradiol synthesis vs. their conspicuous atretogenic properties in late-stage follicles. During folliculogenesis, androgens are synthesized by the primary thecal cells of growing follicles in response to the pituitary gonadotropin LH. The androgens then diffuse across the basement membrane of the follicle and into the granulosa cells, where they act in dual roles, first as a hormone via the androgen receptor (AR) to augment FSH induction of the estrogenic enzymes, and second as the immediate substrates for conversion to estrogens by these same enzymes (2) . The subsequent rise in intrafollicular estradiol levels leads to activation of estrogen receptor (ER) signaling, which assumes the role of augmenting FSH actions and results in rapid follicle growth and differentiation (2) . Hence, follicle maturation from the preantral to preovulatory stage is marked by a shift in the role of androgens from hormone to substrate (2) . If this transition fails to occur, the intrafollicular androgen levels rise above the steroidogenic capacity of the granulosa cells and invariably cause atresia (3) . Therefore, progression of viable preovulatory follicles and, hence, female fertility depends on stringent regulation of thecal cell androgen synthesis during the later stages of folliculogenesis. In fact, strong evidence indicates that ovarian hyperandrogenism may be a leading cause of polycystic ovary syndrome (PCOS), which is estimated to account for 75% of anovulatory infertility in women (1) .

This need to limit androgen synthesis in preovulatory follicles implies the existence of specific mechanisms that modulate thecal cell function (4) . During the late follicular phase of the ovarian cycle, the endocrine actions of estradiol are well described to elicit negative-feedback on the hypothalamic–pituitary (H–P) axis and thereby decrease LH secretion and further stimulation of thecal cell steroidogenesis. However, studies more than 25 years ago demonstrated that estrogens can also directly inhibit androgen synthesis in rodent ovaries and isolated thecal cells, leading to speculation that granulosa cell-derived estrogens (e.g., estradiol) may also mediate a short, intrafollicular feedback loop to negatively modulate thecal cell steroidogenesis (4) . Supporting evidence indicates that estradiol specifically targets CYP17A1 (P45017{alpha}-hydroxylase:C17,20-lyase), the thecal cell-specific enzyme that converts C21- to C19-steroids [e.g., progesterone to androstenedione; pregnenelone to dehydroepiandrosterone (DHEA) (4 , 5) ], yet estradiol does not appear to directly inhibit substrate binding or CYP17A1 enzymatic activity (6) , nor the capacity of thecal cells to respond to LH (7 , 8) . Instead, descriptions that estradiol repression of CYP17A1 activity is blocked by an estrogen receptor (ER)-antagonist (8 , 9) and that estrogens reduce the level of gonadal CYP17A1 expression (10 , 11) indicate that regulation may occur via receptor-mediated mechanisms at the transcriptional level.

Our understanding of the direct actions of estradiol in the ovary has historically been impeded by the inherent difficulties of studying the effect of a hormone within the tissue it is synthesized. This is further complicated by the discovery of ERß and its extraordinarily high expression in the granulosa cells of mammalian ovaries, whereas ER{alpha}, the originally discovered isoform, is largely limited to thecal cells (2) . However, the development of ER-null animal models and isoform-specific ER-agonists present new opportunities to better study the contribution of each ER isoform to mediating the intraovarian functions of estradiol. We have previously shown that the ovaries and thecal cells of ER{alpha}-null ({alpha}ERKO) but not ERß-null (ßERKO) mice exhibit abnormally high Cyp17a1 expression and activity despite a milieu of elevated estradiol (12 , 13) . These data are consistent with a modulating action of estradiol on thecal cell steroidogenesis and suggest that ER{alpha} is primarily involved. However, further insight from these data is confounded by the endocrine effects that follow the systemic loss of ER{alpha} functions, more specifically the chronically high LH levels and subsequent hyperstimulation of the ovarian theca that invariably results from the loss of estradiol-mediated negative-feedback in the H–P axis of {alpha}ERKO females (12) . Therefore, to better investigate the putative intraovarian feedback loop of estradiol on thecal cell androgen synthesis in growing follicles, we compared the steroidogenic capacity of individual WT and {alpha}ERKO follicles when grown in vitro under normalized gonadotropin levels. Follicles of each genotype were exposed to an aromatase inhibitor (AI) to allow androgen accumulation and more accurate assessment of synthesis rates. The resulting data definitively show that {alpha}ERKO follicles possess an increased capacity for androgen synthesis that correlates with abnormally high Cyp17a1 expression and that this phenotype is innate to the loss of ER{alpha} within the follicle. Furthermore, this phenotype was reproduced in WT follicles when acutely treated with an aromatase inhibitor or ER-antagonist and abated by cotreatment with estradiol or an ER{alpha}-specific agonist. These data provide convincing support for the long-standing hypothesis that estradiol mediates a short feedback loop within the follicle to prevent overproduction of androgens and definitively demonstrates this mechanism is dependent on functional ER{alpha}.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals
The Animal Care and Use Committee of the NIEHS preapproved all protocols and procedures involving animals. Animals were maintained in plastic cages under a 12-h light:12-h dark schedule in a temperature-controlled room (21–22°C), fed NIH 31 mouse chow and fresh water ad libitum. The generation of Esr1–/– ({alpha}ERKO) mice has been described previously (14 , 15) . WT (Esr1+/+), and {alpha}ERKO female mice were generated via heterozygous (Esr1+/–) breeding pairs of C57BL/6 strain. A tail biopsy was collected from female offspring at 19 d of age for genotyping as described previously (12) .

Chemicals
The aromatase inhibitor (AI), 4-(imidazolylmethyl)-1-nitro-9H-9-xanthenone, was purchased from Calbiochem, Inc. (San Diego, CA, USA). The nonspecific ER antagonist ICI 182,780 was purchased from Zeneca Pharmaceuticals (Cheshire, UK). 17ß-Estradiol (E2) was purchased from Steraloids (Newport, RI, USA). The ER{alpha}-specific agonist, 4, 4', 4''-(propyl-[1H]-pyrazole-1,3,5-triyl) trisphenol (PPT) and ERß-specific agonist, 2,3-bis(4-hydroxyphenyl)-propionitrile (DPN) were purchased from Tocris Cookson, Inc. (Ellisville, MO, USA). The 17ß-HSD3 inhibitor, 3ß-propyl-androsterone (DP3–1), was generated and previously characterized by D.P. (16) .

In vitro follicle culture
Individual mouse follicles were isolated and cultured in vitro as described previously (17 , 18) . In brief, female mice of 21–25 d of age were killed by CO2 asphyxiation, and the ovaries were immediately dissected and removed to Leibovitz’s L-15 Medium (Invitrogen, Carlsbad, CA, USA) supplemented with insulin (5 µg/ml; Invitrogen), transferrin (10 µg/ml; Sigma, St. Louis, MO, USA), selenium (2 ng/ml; Sigma), ascorbic acid (50 µg/ml; Sigma), and 0.3% BSA (Sigma) that was prewarmed and maintained at 37°C. Individual preantral follicles of 190–210 µm in diameter were isolated by manual dissection using 25-gauge needles and then transferred to {alpha}-minimal essential medium ({alpha}-MEM; Invitrogen) supplemented with Pen/Strep (Invitrogen), insulin (5 µg/ml; Invitrogen), transferrin (10 µg/ml; Sigma), selenium (2 ng/ml; Sigma), ascorbic acid (50 µg/ml; Sigma), 5% FBS (containing 1.2 ng LH/ml according to supplier, Hyclone, Logan, UT) and 100 mIU recombinant human FSH (Serono Inc., Rockland, MA, USA). After harvesting, follicles were transferred to Millipore CM (Millipore Corp., Bedford, MA, USA) culture plate inserts prefilled with 0.25 ml {alpha}-MEM medium containing the above supplements and maintained in a humidified incubator with a 95% O2/5% CO2 atmosphere at 37°C. As shown in Fig. 1 , follicles were cultured for a total of 5 d, reevaluated daily and allowed to remain in culture only if they continued to exhibit an intact basement membrane, a dense complement of granulosa cells, a centrally located oocyte and attached thecal cells. The medium was replaced after 1 and 3 d of culture, and follicle diameter was measured and recorded daily. On the 4th day of culture, all or 60% of the medium was replaced with fresh media containing one or more of the chemical treatments. After an additional 24-h incubation period, the media and follicle were collected separately and stored at –70°C for later analysis of steroid content and gene expression, respectively.


Figure 1
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  		Figure 1. Scheme used for in vitro culture of WT and {alpha}ERKO follicles for the assessment of steroidogenesis and gene expression. Large preantral follicles of ~200 µm in diameter were isolated from immature (21–24 d) WT and {alpha}ERKO females and individually propagated in a 250 µl vol of medium for 5 d, during which they grow to ~340 µm in diameter. Media were changed and or collected on the indicated days. Follicles were inspected for integrity and health every 24 h and removed from the study if they did not satisfy the conditions described in the Materials and Methods. The 24-h period between days 4–5 was found to be the period of peak steroid synthesis by follicles and was therefore selected for all experimental treatments. Media were changed on day 4 of culture and replaced with media containing vehicle or a combination of the indicated treatments. The media were then collected after 24 h and stored for evaluation of androstenedione, testosterone, and estradiol content by EIA. The follicle was also collected for later evaluation of gene expression.

Steroid enzyme immunoassays (EIAs)
Estradiol, androstenedione, and testosterone content in collected media were assessed using the respective active enzyme immunoassay (EIA) kits (Diagnostics Systems Laboratories, Webster, TX, USA) according to the manufacturer’s protocol. Due to limited sample volume, samples were measured in singlicate for each steroid. The least-detectable concentration, intra-assay coefficient of variation and interassay coefficient of variation for each EIA were as follows: estradiol, 7 pg/ml, 7%, 15%; androstenedione, 0.03 ng/ml, 4%, 8%; and testosterone, 0.04 ng/ml, 2.5%, 12%. The level of each steroid in fresh {alpha}-MEM medium was below the level of detection.

RNA isolation and gene expression assays
Total RNA was isolated from individual follicles using the PicoPure RNA isolation kit (Arcturus, Mountain View, CA, USA) according to the manufacturer’s protocol. All RNA preparations were rid of contaminating DNA using the DNA-free® reagents (Ambion, Austin, TX, USA), according to the manufacturer’s protocol and the concentration of each preparation was determined from an A260/280 reading using an undetermined (ND)-1000 spectrophotometer (NanoDrop, Wilmington, DE, USA). A cDNA preparation was generated from each sample using the whole preparation of RNA (a vol of 10 µl) in a 25 µl reaction using random hexamers and the Superscript cDNA synthesis system (Invitrogen), according to the manufacturer’s protocol. Traditional (semiquantitative) polymerase chain reaction (PCR) reactions were prepared from the equivalent of 1 µl cDNA per 15 µl reaction for each respective primer set using PCR reagents and Platinum Taq Polymerase (Invitrogen) as described previously (12). PCR was performed in a Thermo Hybaid Multiblock System (Thermo-Hybaid) as follows: 95°C/30 s (1X); 95°C/30 s, 58°C/45 s, 72°C/30 s (32X); 72°C/7 min. All samples were electrophoresed on an agarose gel (2% NuSieve/0.7% SeaKem, BMA Bioproducts, Rockland, ME, USA) in 1x Tris borate-EDTA buffer, stained with ethidium bromide and photographed using an EC3 Imaging System (UVP, Upland, CA, USA). Primers used for the detection of murine Cyp17a1, Cyp11a1, and Hsd17b3 transcripts have been described previously (19) ; primers for the detection of murine Actb transcripts were purchased from Clonetech (Mountain View, CA, USA).

Real-time RT-polymerase chain reaction (RT-PCR) assessment of Cyp17a1 and Hsd17b3 expression used primers described previously (13) . Each sample was assayed in duplicate using the equivalent of 1 µl cDNA, 10 pmoles primer, and 1x SYBR Green Master Mix (Applied Biosystems) in a total reaction vol of 25 µl. For normalization purposes, an identical set of reactions was prepared using primers specific for ribosomal 18S RNA (Rn18s) as described previously (13) . Amplification was performed in an ABI PRISM 7700 Sequence Detection System (Applied Biosystems) as follows: 50°C/2 min, 95°C/10 min (1x); 95°C/15 s, 60°C/30 s (40x). Quantitative differences in the cDNA target among samples were determined using the mathematical model of Pfaffl (20) in which an expression ratio was determined for each sample by calculating (Etarget){Delta}Ct(target)/(ERn18s){Delta}Ct(Rn18s), where E is the efficiency of the primer set and {Delta}Ct = Ct(Rn18s) – Ct(experimental cDNA). The amplification efficiency of each primer set was calculated from the slope of a standard amplification curve of log µl cDNA/reaction vs. Ct value over at least 4 orders of magnitude (E=10—(1/slope)); Hsd17b3 primers, E = 1.97 (vs. WT testis cDNA); Cyp17a1 primers, E = 2.16 (vs. WT ovary cDNA); Cyp11a1 primers, E = 2.13 (vs. WT ovary cDNA).

Statistics
All data sets were analyzed for statistical significance (P<0.05) using JMP software (SAS Institute, Cary, NC, USA). Data sets were first tested for homoscedasticity of variance using the Levene’s test and if failed were log-transformed prior tofurther statistical analysis. All data sets were then evaluated by a one-way ANOVA followed by the Tukey-Kramer HSD posthoc test when applicable.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
{alpha}ERKO follicles exhibit elevated androgen synthesis in culture
Steroid secretion by individual WT and {alpha}ERKO follicles was assessed over the course of two consecutive 24 h periods between days 3–5 of culture (Fig. 1) . Pilot studies indicated that the period between culture days 3–4 and 4–5 was marked by a 5-fold increase in steroid production by follicles of both genotypes (data not shown). Therefore, all subsequent experiments and treatments were conducted during the latter 24-h period. As shown in Fig. 2 , {alpha}ERKO follicles exhibited >5-fold increase in estradiol synthesis relative to WT follicles (P<0.05). These data are consistent with earlier descriptions of increased circulating estradiol levels exhibited by adult {alpha}ERKO female mice in vivo (12) and ER{alpha}-null follicles in vitro (17) . Given that estradiol synthesis is limited by the availability of androgen precursors, {alpha}ERKO follicles accordingly exhibited a 3- and 4.5-fold increase in androstenedione and testosterone secretion, respectively, relative to WT follicles grown under comparable conditions (Fig. 2) . To better compare the rates of androstenedione and testosterone synthesis in WT and {alpha}ERKO follicles, in vitro cultures of each were exposed to an aromatase inhibitor (AI) to block CYP19A1-mediated aromatization of C19-steroids, thereby allowing the precursors to accumulate in the medium. The AI used is reported to specifically inhibit CYP19A1 with minimal effect on CYP17A1 enzymatic activity (21) . The lowest concentration of AI (0.5 µM) used eliminated all detectable estradiol synthesis in WT follicles and reduced estradiol synthesis in {alpha}ERKO follicles by ~95% (Fig. 2) . A 10-fold increase in AI (5 µM) totally inhibited all detectable estradiol synthesis in {alpha}ERKO follicles (Fig. 2) . As expected, inhibition of CYP19A1 activity led to a dose-dependent increase in androgen accumulation in follicles of both genotypes but {alpha}ERKO follicles continued to exhibit a 3- to 5-fold higher rate of androstenedione and testosterone synthesis relative to WT at all three AI concentrations (Fig. 2) . These data indicate that {alpha}ERKO follicles possess a marked increase in their capacity for androgen synthesis and that this phenotype is innate to the follicle rather than a consequence of increased gonadotropin stimulation.


Figure 2
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  		Figure 2. {alpha}ERKO follicles exhibit elevated steroidogenesis in vitro. Shown are the average (±SEM) levels of estradiol, androstenedione, and testosterone synthesized by WT (open bar) and {alpha}ERKO (filled bar) follicles during the 24-h culture period between days 4–5. Follicles were exposed to either vehicle (Veh) or increasing amounts of an aromatase inhibitor to allow thecal cell-derived androgens to accumulate and be measured. Individual {alpha}ERKO follicles clearly synthesize greater amounts of all three steroids when cultured under a controlled hormonal milieu. Furthermore, by inhibiting aromatization of androstenedione to estrone, or testosterone to estradiol, the increased level of androgen synthesis in {alpha}ERKO follicles becomes even more apparent. Bars that do not share a letter are significantly different (P<0.05). The data shown represent the pooled values of four independent experiments, and a total of 14–47 follicles per genotype, per treatment.

{alpha}ERKO follicles exhibit aberrantly increased Cyp17a1 expression
The above data strongly indicate that individual {alpha}ERKO follicles continue to possess increased CYP17A1 activities, even when maintained in conditions of controlled gonadotropin stimulation. Therefore, we sought to compare the level of Cyp17a1 expression in individual WT and {alpha}ERKO follicles following 5 d in culture. As shown in Fig. 3 , Cyp17a1 expression in {alpha}ERKO follicles was 3-fold higher than that of WT follicles, suggesting this phenotype is inherent to the loss of ER{alpha} functions within the follicle. To test this hypothesis, Cyp17a1 expression was evaluated in WT follicles following acute, in vitro exposure to an AI or ER-antagonist (ICI 182,780), both of which were expected to pharmacologically mimic the loss of ER{alpha} function. Interestingly, both treatments increased Cyp17a1 expression in WT follicles (P<0.05 vs. untreated WT) to levels that approximated those observed in untreated {alpha}ERKO follicles (Fig. 3) . Therefore, acute inhibition of ER-mediated actions via either removal of activating ligand or direct repression of receptor function leads to increased Cyp17a1 expression in WT follicles, hence reproducing the {alpha}ERKO phenotype. Similar in vitro exposure of {alpha}ERKO follicles to the AI or ER-antagonist had no additive effect on Cyp17a1 expression (Fig. 3) .


Figure 3
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  		Figure 3. Loss or inhibition of intrafollicular ER{alpha} functions leads to increased Cyp17a1 expression in individually cultured follicles. A) Shown is a representative ethidium bromide-stained agarose gel (inverted) of semiquantitative RT-PCR for Cyp17a1, Cyp11a1, and Actb transcripts in WT and {alpha}ERKO day 5 follicles following 24 h of treatment with either vehicle (V), an aromatase inhibitor (AI), or an ER-antagonist (ICI). B) Shown is quantitative data (average±SEM) from real-time RT-PCR for Cyp17a1 and Cyp11a1 expression from these same experiments. {alpha}ERKO follicles clearly exhibited increased Cyp17a1 expression relative to vehicle-treated WT follicles, and this phenotype was reproduced in the latter genotype following acute withdrawal of endogenous estradiol synthesis (via an AI) or direct repression of ER action (via ICI). In contrast, Cyp11a1 expression does not differ among genotypes and was not affected by the various treatments. Bars that do not share a letter are significantly different (P<0.05). The data shown are from one of two independent experiments that yielded comparable results. Sample sizes were 8–9 follicles per genotype, per treatment, per experiment.

Androgens are reported to down-regulate thecal cell androgen synthesis via an AR-mediated autoregulatory loop (22 , 23) . To determine whether increased androgen accumulation in the presence of the AI or loss of ER{alpha} may provide for some repression of Cyp17a1 expression, WT and {alpha}ERKO follicles were exposed to the AI plus an AR-antagonist (Flutamide, 10 µM). Cyp17a1 expression in AI-exposed WT follicles treated with Flutamide was actually reduced by 30%, a measurable but not statistically significant decline compared with WT follicles exposed to the AI alone (data not shown). A similar decrease in Cyp17a1 expression was observed in {alpha}ERKO follicles exposed to the AI plus Flutamide (data not shown).

Similar assays for Cyp11a1, another steroidogenic enzyme that is LH-regulated in thecal cells, indicated little difference in expression between WT and {alpha}ERKO follicles and minimal changes following all of the above treatments. Therefore, the inhibitory effect of ER{alpha} is specific to Cyp17a1 expression (Fig. 3) .

ER{alpha} mediates the inhibitory effect of estradiol on Cyp17a1
A phenotype of elevated ovarian Cyp17a1 expression and activity in {alpha}ERKO but not ßERKO females (12) , along with the predominance of ER{alpha} in thecal cells, strongly suggests that estradiol modulation of Cyp17a1 expression is ER{alpha}-mediated. To test this hypothesis, in vitro cultured WT follicles were exposed to an AI to eradicate endogenous synthesis of ER ligand (i.e., estradiol) while simultaneously exposed to either exogenous estradiol, an ER{alpha}-specific agonist (PPT) or an ERß-specific agonist (DPN) for 24 h. As shown in Fig. 4 , the increased Cyp17a1 expression elicited by removal of endogenous estradiol synthesis was completely abated by exogenous estradiol replacement at 0.2 nM, indicating the inhibitory effect is specific to estrogen action. Furthermore, this effect of estradiol was fully mimicked by the ER{alpha}-agonist but not the ERß-agonist, indicating that ER{alpha} solely mediates estradiol repression of Cyp17a1 expression (Fig. 4) . None of the treatments affected Cyp11a1 expression (Fig. 4) , demonstrating the specificity of ER{alpha}-mediated actions to Cyp17a1 regulation.


Figure 4
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  		Figure 4. Estradiol suppression of Cyp17a1 expression is mediated by ER{alpha}. Shown are quantitative data (average±SEM) from real-time RT-PCR for Cyp17a1 and Cyp11a1 transcripts in WT day 5 follicles following 24 h of treatment with either vehicle (Veh), an aromatase inhibitor (AI), an ER-antagonist (ICI), or an AI plus estradiol (E2), an ER{alpha}-agonist (PPT) or an ERß-agonist (DPN). As expected, acute withdrawal of endogenous estradiol synthesis (via an AI) or direct repression of ER action (via ICI) leads to increased Cyp17a1 expression (top). However, this can be prevented by cotreatment with exogenous E2 or an ER{alpha}-specific agonist (PPT), indicating that ER{alpha} mediates the estradiol suppression of Cyp17a1 expression in growing follicles. In contrast, Cyp11a1 expression (bottom) was not affected by the various treatments, indicating that the effects of ER{alpha}-mediated estradiol are specific to Cyp17a1. Bars that do not share a letter are significantly different (P<0.05). The data shown are from one of two independent experiments that yielded comparable results. Sample sizes were 8–9 follicles per genotype, per treatment, per experiment.

Elevated testosterone synthesis in {alpha}ERKO follicles is mediated by ectopic HSD17B3 activity
In addition to increased androstenedione synthesis, {alpha}ERKO follicles also exhibited remarkably high rates of testosterone secretion in vitro, exhibiting a T/A4 ratio of 3.4 (±0.7) vs. 1.6 (±0.1) in WT follicles (P<0.05). An overabundance of precursor, i.e., androstenedione, could provide the basis for increased testosterone synthesis in {alpha}ERKO follicles. However, we have described previously that adult {alpha}ERKO females exhibit male-like plasma testosterone levels in vivo due to ectopic ovarian expression of HSD17B3 (12 , 13) , a testis-specific enzyme that specifically reduces androstenedione to testosterone (24 , 25) . In the current study, Hsd17b3 transcripts continued to be detected in individual {alpha}ERKO follicles but not WT follicles following 5 d in culture (Fig. 5 ), which indicates that the in vivo ovarian phenotype is preserved in {alpha}ERKO follicles under in vitro conditions. In contrast to the effect on Cyp17a1 expression, however, acute exposure to the AI or ER-antagonist did not lead to an {alpha}ERKO-like induction of Hsd17b3 expression in WT follicles (Fig. 5) . Some WT follicles exposed to the AI exhibited a detectable rise inHsd17b3 expression, but this was neither reproducible nor comparable with the levels detected in {alpha}ERKO follicles. Therefore, ectopic Hsd17b3 expression is innate to {alpha}ERKO follicles and exists prior to in vitro culture.


Figure 5
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  		Figure 5. {alpha}ERKO follicles exhibit ectopic expression of the Leydig cell-specific gene Hsd17b3. A) Shown is a photograph of a representative ethidium bromide-stained agarose gel (inverted) of semiquantitative RT-PCR for Hsd17b3, Cyp11a1, and Actb transcripts in WT and {alpha}ERKO day 5 follicles following 24 h of treatment with either vehicle (V), an aromatase inhibitor (AI), or an ER-antagonist (ICI). B) Shown are quantitative data (average±SEM) from real-time RT-PCR for Hsd17b3 expression from these same experiments. {alpha}ERKO follicles clearly exhibit ectopic Hsd17b3 expression relative to WT follicles, and this phenotype cannot be reproduced in wild follicles following acute withdrawal of endogenous estradiol synthesis (via an AI) or direct repression of ER action (via ICI), indicating it is a fixed phenotype in {alpha}ERKO follicles prior to culture. Bars that do not share a letter are significantly different (P<0.05). The data shown are from one of two independent experiments that yielded comparable results. Sample sizes were 8–9 follicles per genotype, per treatment, per experiment.

The above findings indicate that increased testosterone synthesis in {alpha}ERKO follicles is due to ectopic HSD17B3 activity. However, a definitive conclusion is precluded by reports that HSD17B1, a related family member that functions to reduce estrone to estradiol and is highly expressed in granulosa cells, can also reduce androstenedione to testosterone in rodents (26 , 27) . Therefore, to discern the contributions of HSD17B type 1 and type 3 activities to the overall capacity for testosterone synthesis in {alpha}ERKO follicles, follicles of each genotype were exposed to an HSD17B3-specific inhibitor (DP3–1) in the presence or absence of the AI. In the absence of the AI, DP3–1 inhibited testosterone synthesis by >85% in {alpha}ERKO follicles (Fig. 6 ). When the AI was included to allow for the accumulation of androstenedione, the common substrate for HSD17B types 1 and 3, DP3–1 still inhibited testosterone synthesis in {alpha}ERKO follicles by >60% (P<0.05) (Fig. 6) . Furthermore, DP3–1 treatment led to a measurable accumulation in androstenedione (Fig. 6) , indicating that decreased testosterone synthesis was not due to parallel reductions in available precursor. The failure of DP3–1 to inhibit testosterone synthesis in WT follicles indicates this synthesis is likely mediated by the androgenic actions of HSD17B1 (Fig. 6) .


Figure 6
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  		Figure 6. Ectopic HSD17B3 activity accounts for the aberrantly elevated capacity for testosterone synthesis in {alpha}ERKO follicles. WT and {alpha}ERKO follicles were grown as described in Fig. 1 . During the 24-h culture period between days 4–5, follicles were exposed to an aromatase inhibitor (AI) and/or an HSD17B3-specific inhibitor (DP3–1). Shown is the average (±SEM) amount of testosterone (top) and androstenedione (bottom) synthesized during the 24 h period. As was first shown in Fig. 2 , both WT and {alpha}ERKO follicles synthesized increased amounts of androstenedione and testosterone when exposed to an AI; however, the levels of both androgens are much higher in the {alpha}ERKO follicles. Furthermore, testosterone synthesis in {alpha}ERKO follicles is inhibited by DP3–1, which indicated that it is mediated by HSD17B3, a Leydig cell-specific enzyme. In contrast, that DP3–1 had no effect on testosterone synthesis in WT follicles indicated that this is likely mediated by the androgenic properties of rodent HSD17B1. The data shown are from one of two independent experiments that yielded comparable results. Sample sizes were 8–9 follicles per genotype, per treatment, per experiment.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The two-cell, two-gonadotropin model of steroidogenesis in ovarian follicles states that androgens are synthesized solely by primary thecal cells in response to LH and then diffuse across the basement membrane to serve as immediate substrates for estradiol synthesis by granulosa cells in response to FSH (5) . Hormonal actions of androgens also promote estradiol synthesis in preantral follicles by enhancing FSH-induction of CYP19A1 (2) . This notwithstanding, elevated androgen synthesis and/or accumulation during the later stages of folliculogenesis is undoubtedly detrimental to the follicle (3 , 5) and is an invariable characteristic in women diagnosed with PCOS (28) . Therefore, follicle integrity relies on a delicate balance between the steroidogenic capacities of the theca and granulosa cells. It has long been speculated that this balance is achieved by granulosa cell-derived estradiol acting in a paracrine loop to negatively modulate thecal cell function (3 4 5) . Indeed, estradiol is known to inhibit androgen synthesis in thecal cells under experimental conditions (4 , 29) , and ER{alpha} is highly expressed in the primary thecal cells of growing follicles in multiple species (2) . However, the generation of definitive experimental evidence to support this hypothesis has been precluded by the lack of appropriate investigative tools. Herein, we used in vitro follicle culture and ER{alpha}-null mice to demonstrate that the loss of functional ER{alpha} within growing follicles leads to markedly elevated rates of androstenedione synthesis that can be attributed to increased expression of CYP17A1, the thecal cell-specific enzyme that is directly involved in androstenedione synthesis. We also provide evidence that ER{alpha} functions to repress testosterone synthesis in the ovary by inhibiting expression of HSD17B3, an enzyme that efficiently reduces androstenedione to testosterone but is normally testis-specific. These data indicate that ER{alpha} functions within thecal cells to maintain the proper steroidogenic environment of growing follicles by a) controlling the overall capacity for androgen and estrogen synthesis by negatively modulating Cyp17a1 expression, and b) inhibiting the synthesis of the more biological active androgen, testosterone, by repressing Hsd17b3 expression.

The current data are consistent with earlier our reports that adult {alpha}ERKO but not ßERKO female mice exhibit increased ovarian Cyp17a1 and ectopic Hsd17b3 expression and correlating levels of circulating androstenedione and testosterone (12 , 13) . However, gonadal Cyp17a1 and Hsd17b3 expression are highly dependent on LH stimulation (4 , 30) , and therefore any inference from in vivo observations must consider the effects of chronically increased LH secretion that results from the loss of ER{alpha}-mediated actions in the H–P axis (12) . Herein, we have overcome this caveat by comparing the phenotypes of individual WT and {alpha}ERKO follicles when propagated in vitro under a normalized gonadotropin milieu. The preservation of increased Cyp17a1 and ectopic Hsd17b3 expression, and increased rates of androstenedione and testosterone synthesis, in individually cultured {alpha}ERKO follicles indicates these traits are inherent to the loss of intrafollicular ER{alpha} functions and not the secondary effects of LH-hyperstimulation. Indeed, even when WT or ßERKO female mice are forced to possess comparably elevated LH levels via possession of the LH-CTP transgene, they do not exhibit comparable increases in ovarian Cyp17a1 expression, presumably because the inhibitory actions of ER{alpha} within the ovary remain intact (19) . Interestingly, Heikkilä et al. recently reported that ovaries of newborn Wnt4-null mice exhibit a more than 60-fold increase in Cyp17a1 expression that is concurrent with a 8-fold reduction in ER{alpha} expression but no change in ERß levels (31) . Therefore, increased Cyp17a1 expression in {alpha}ERKO ovaries is likely the compound effect of the loss of ER{alpha}-mediated functions in both the ovary and H–P axis. Recent reports that estradiol down-regulates Cyp17a1 expression in the testes of rats (10) and fish (11) , and that the testes of {alpha}ERKO males exhibit aberrantly high Cyp17a1 expression and activity (32) , indicate that ER{alpha} likely plays a comparable role in the male gonad.

The putative autoregulatory actions of androgens on thecal cell steroidogenesis (22 , 23) were not observed in the current studies using in vitro follicle culture. In fact, {alpha}ERKO follicles continued to exhibit elevated Cyp17a1 expression despite their self-generation of an environment rich in testosterone. Furthermore, treatment of WT and {alpha}ERKO follicles with an AR-antagonist actually led to a slight decrease in Cyp17a1 expression, an effect that is opposite that which could be expected if AR-mediated androgen actions repress Cyp17a1 expression. These data suggest that either ER{alpha} is involved in the postulated AR-mediated autoregulatory loop on thecal cell function or that ER{alpha} is the more predominant negative modulator of thecal cell steroidogenesis.

The {alpha}ERKO phenotype of increased Cyp17a1 expression could be reproduced in WT follicles during acute withdrawal of endogenous estrogenic ligand or inhibition of ER{alpha} action. Furthermore, only exogenous estradiol or the ER{alpha}-specific agonist (PPT) prevented the increase in Cyp17a1 expression in WT follicles following withdrawal of endogenous estrogen synthesis. These data indicate that estradiol repression of Cyp17a1 expression is clearly ER{alpha}-mediated as well as acute and reversible in nature. Interestingly, AI or ICI treatment did not elicit ectopic Hsd17b3 expression in WT follicles, suggesting that this phenotype is fixed in {alpha}ERKO follicles prior to culture. This divergence in CYP17A1 and HSD17B3 regulation in the ovary is consistent with the role of the former enzyme in the synthesis of androstenedione, which is obligatory for estradiol synthesis; whereas continuous repression of Hsd17b3 expression in the ovary is conducive to a) shunting the available thecal cell-derived androstenedione toward the path of estrogen rather than testosterone synthesis and b) preventing the generation of testosterone to potentially harmful levels.

Considerable divergence in CYP17A1 expression patterns occurs among different species and steroidogenic tissues (24) , which makes it difficult to speculate on the mechanism by which ER{alpha} represses expression in thecal cells. Tissue-specific CYP17A1 expression is at least partly achieved by differential receptor expression among the steroidogenic tissues. For example, LH is necessary to stimulate CYP17A1 expression in the gonads, whereas ACTH stimulates expression in the adrenal glands (24) . In contrast, mechanisms that actively repress CYP17A1 expression are gaining attention as another important regulatory mode of CYP17A1 expression, and several nuclear factors and signaling pathways have been implicated, including UBC9 (33) , RIP-140 (34) , protein kinase-c (PKC) (23 , 35) , Src-tyrosine kinases (36) , and transforming growth factor-ß (TGF-ß) (37 38 39 40) ; the latter of which is estrogen-regulated in thecal cells (41) . Indeed, isolated thecal cells from women with PCOS exhibit an aberrant increase in basal CYP17A1 activity in vitro (42 , 43) , which is currently attributed to a loss of mechanisms that normally repress CYP17A1 expression (44 45 46) . Although a comparison of ER{alpha} and ERß expression levels in normal vs. PCOS human ovaries showed that ER{alpha} levels are in fact increased in thecal cells from the diseased ovaries (47) , an intronic PvuII single-nucleotide polymorphism in the ESR1 (ER{alpha}) gene is associated with increased androstenedione levels in postmenopausal women (48) . Furthermore, we found marked levels of Cyp17a1 transcripts in the adrenal glands of {alpha}ERKO females (J.F. Couse and K.S. Korach, unpublished observations) despite reports that rodent adrenal glands are normally void of CYP17A1 (24) , which thereby provide further evidence that ER{alpha} functions are critical to the repression of CYP17A1 expression in steroidogenic tissues.

In summary, direct effects of estradiol on the ovary were first demonstrated more 60 years ago (49 , 50) , yet in-depth studies toward understanding the mechanisms of intraovarian estrogen actions are impeded by difficulties inherent to investigating hormone action within the source tissue. The discovery of ERß and its marked expression in the ovary, the generation of ER-null and CYP19A1-null mice, and the development of ER-specific agonists has led to a resurgence in the field of intraovarian estrogen actions. We used an in vitro follicle culture method using follicles from ER{alpha}-null mice along with recently developed ER-specific compounds to demonstrate an ER{alpha}-dependent paracrine loop within late-stage follicles that allows granulosa cell-derived estradiol to negatively modulate thecal cell androgen synthesis by specifically reducing Cyp17a1 expression, confirming a role for estradiol that was first postulated more than 25 years ago (4 , 29 , 51) .


   ACKNOWLEDGMENTS
 
We are grateful to numerous colleagues who have supported our efforts over the course of these studies, and Drs. William Schrader and Darryl Zeldin for reviewing the manuscript. This research was supported by the Intramural Research Program of the National Institutes of Health (NIH), National Institute of Environmental Health Sciences (NIEHS).


   FOOTNOTES
 
1 These authors contributed equally to this work.

2 Current address: Department of Pharmacology and Toxicology, Cardiovascular Research Institute, University of Maastricht, P.O. Box 616, 6200 MD Maastricht, The Netherlands.

Received for publication August 29, 2006. Accepted for publication September 6, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Ehrmann, D. A., Barnes, R. B., Rosenfield, R. L. (1995) Polycystic ovary syndrome as a form of functional ovarian hyperandrogenism due to dysregulation of androgen secretion. Endocr. Rev. 16,322-353[CrossRef][Medline]
  2. Couse, J. F., Hewitt, S. C., Korach, K. S. (2006) Steroid receptors in the ovary and uterus. Neill, J. D. eds. Knobil and Neill’s Physiology of Reproduction ,593-678 Elsevier Science San Diego, California, USA.
  3. Greenwald, G. S., Roy, S. K. (1994) Follicular development and its control. Knobil, E. Neill, J. D. eds. The Physiology of Reproduction ,629-724 Raven Press, Ltd. New York, New York, USA.
  4. Erickson, G. F., Magoffin, D. A., Dyer, C. A., Hofeditz, C. (1985) The ovarian androgen producing cells: a review of structure/function relationships. Endocr. Rev. 6,371-399[Medline]
  5. Gore-Langton, R. E., Armstrong, D. T. (1994) Follicular steroidogenesis and its control. Knobil, E. Neill, J. D. eds. The Physiology of Reproduction ,571-627 Raven Press, Ltd. New York, New York, USA.
  6. Johnson, D. C., Martin, H., Tsai-Morris, C. H. (1984) The in vitro and in vivo effect of estradiol upon the 17 alpha-hydroxylase and C17,20-lyase activity in the ovaries of immature hypophysectomized rats. Mol. Cell. Endocrinol. 35,199-204[CrossRef][Medline]
  7. Magoffin, D. A., Erickson, G. F. (1981) Mechanism by which 17ß-estradiol inhibits ovarian androgen production in the rat. Endocrinology 108,962-969[Abstract]
  8. Magoffin, D. A., Erickson, G. F. (1982) Direct inhibitory effect of estrogen on LH-stimulated androgen synthesis by ovarian cells cultured in defined medium. Mol. Cell. Endocrinol. 28,81-89[CrossRef][Medline]
  9. Banks, P. K., Meyer, K., Brodie, A. M. (1991) Regulation of ovarian steroid biosynthesis by estrogen during proestrus in the rat. Endocrinology 129,1295-1304[Abstract]
  10. Sakaue, M., Ishimura, R., Kurosawa, S., Fukuzawa, N. H., Kurohmaru, M., Hayashi, Y., Tohyama, C., Ohsako, S. (2002) Administration of estradiol-3-benzoate down-regulates the expression of testicular steroidogenic enzyme genes for testosterone production in the adult rat. J. Vet. Med. Sci. 64,107-113[CrossRef][Medline]
  11. Govoroun, M., McMeel, O. M., Mecherouki, H., Smith, T. J., Guiguen, Y. (2001) 17ß-estradiol treatment decreases steroidogenic enzyme messenger ribonucleic acid levels in the rainbow trout testis. Endocrinology 142,1841-1848[Abstract/Free Full Text]
  12. Couse, J. F., Yates, M. M., Walker, V. R., Korach, K. S. (2003) Characterization of the hypothalamic-pituitary-gonadal axis in estrogen receptor (ER) Null mice reveals hypergonadism and endocrine sex reversal in females lacking ER{alpha} but not ERß. Mol. Endocrinol. 17,1039-1053[Abstract/Free Full Text]
  13. Couse, J. F., Yates, M. M., Rodriguez, K. F., Johnson, J. A., Poirier, D., Korach, K. S. (2006) The intraovarian actions of estrogen receptor-{alpha} (ER{alpha}) are necessary to repress the formation of morphological and functional Leydig-like cells in the female gonad. Endocrinology 147,3666-3678[Abstract/Free Full Text]
  14. Lubahn, D. B., Moyer, J. S., Golding, T. S., Couse, J. F., Korach, K. S., Smithies, O. (1993) Alteration of reproductive function but not prenatal sexual development after insertional disruption of the mouse estrogen receptor gene. Proc. Natl. Acad. Sci. U. S. A. 90,11162-11166[Abstract/Free Full Text]
  15. Couse, J. F., Curtis, S. W., Washburn, T. F., Lindzey, J., Golding, T. S., Lubahn, D. B., Smithies, O., Korach, K. S. (1995) Analysis of transcription and estrogen insensitivity in the female mouse after targeted disruption of the estrogen receptor gene. Mol. Endocrinol. 9,1441-1454[Abstract]
  16. Tchedam Ngatcha, B., Luu-The, V., Labrie, F., Poirier, D. (2005) Androsterone 3{alpha}-ether-3ß-substituted and androsterone 3ß-substituted derivatives as inhibitors of type 3 17ß-hydroxysteroid dehydrogenase: chemical synthesis and structure-activity relationship. J. Med. Chem. 48,5257-5268[CrossRef][Medline]
  17. Emmen, J. M., Couse, J. F., Elmore, S. A., Yates, M. M., Kissling, G. E., Korach, K. S. (2005) In vitro growth and ovulation of follicles from ovaries of estrogen receptor (ER){alpha} and ERß null mice indicate a role for ERß in follicular maturation. Endocrinology 146,2817-2826[Abstract/Free Full Text]
  18. Nayudu, P. L., Osborn, S. M. (1992) Factors influencing the rate of preantral and antral growth of mouse ovarian follicles in vitro. J. Reprod. Fertil. 95,349-362[Abstract]
  19. Couse, J. F., Yates, M. M., Sanford, R., Nyska, A., Nilson, J. H., Korach, K. S. (2004) Formation of cystic ovarian follicles associated with elevated luteinizing hormone requires estrogen receptor-ß. Endocrinology 145,4693-4702[Abstract/Free Full Text]
  20. Pfaffl, M. W. (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res. 29,e45[Abstract/Free Full Text]
  21. Recanatini, M., Bisi, A., Cavalli, A., Belluti, F., Gobbi, S., Rampa, A., Valenti, P., Palzer, M., Palusczak, A., Hartmann, R. W. (2001) A new class of nonsteroidal aromatase inhibitors: design and synthesis of chromone and xanthone derivatives and inhibition of the P450 enzymes aromatase and 17{alpha}-hydroxylase/C17,20-lyase. J. Med. Chem. 44,672-680[CrossRef][Medline]
  22. Simone, D. A., Mahesh, V. B. (1993) An autoregulatory process for androgen production in rat thecal-interstitial cells. Biol. Reprod. 48,46-56[Abstract]
  23. Simone, D. A., Chorich, L. P., Mahesh, V. B. (1993) Mechanisms of action for an androgen-mediated autoregulatory process in rat thecal-interstitial cells. Biol. Reprod. 49,1190-1201[Abstract]
  24. Payne, A. H., Hales, D. B. (2004) Overview of steroidogenic enzymes in the pathway from cholesterol to active steroid hormones. Endocr. Rev. 25,947-970[Abstract/Free Full Text]
  25. Penning, T. M. (1997) Molecular endocrinology of hydroxysteroid dehydrogenases. Endocr. Rev. 18,281-305[Abstract/Free Full Text]
  26. Nokelainen, P., Puranen, T., Peltoketo, H., Orava, M., Vihko, P., Vihko, R. (1996) Molecular cloning of mouse 17ß-hydroxysteroid dehydrogenase type 1 and characterization of enzyme activity. Eur. J. Biochem. 236,482-490[Medline]
  27. Puranen, T., Poutanen, M., Ghosh, D., Vihko, R., Vihko, P. (1997) Origin of substrate specificity of human and rat 17ß-hydroxysteroid dehydrogenase type 1, using chimeric enzymes and site-directed substitutions. Endocrinology 138,3532-3539[Abstract/Free Full Text]
  28. Azziz, R. (2003) Androgen excess is the key element in polycystic ovary syndrome. Fertil. Steril. 80,252-254[Medline]
  29. Leung, P. C., Armstrong, D. T. (1980) Interactions of steroids and gonadotropins in the control of steroidogenesis in the ovarian follicle. Annu. Rev. Physiol. 42,71-82[CrossRef][Medline]
  30. Payne, A., Youngblood, G. (1995) Regulation of expression of steroidogenic enzymes in Leydig cells. Biol. Reprod. 52,217-225[Abstract]
  31. Heikkilä, M., Prunskaite, R., Naillat, F., Itaranta, P., Vuoristo, J., Leppaluoto, J., Peltoketo, H., Vainio, S. (2005) The partial female to male sex reversal in Wnt-4-deficient females involves induced expression of testosterone biosynthetic genes and testosterone production, and depends on androgen action. Endocrinology 146,4016-4023[Abstract/Free Full Text]
  32. Akingbemi, B. T., Ge, R., Rosenfeld, C. S., Newton, L. G., Hardy, D. O., Catterall, J. F., Lubahn, D. B., Korach, K. S., Hardy, M. P. (2003) Estrogen receptor-alpha gene deficiency enhances androgen biosynthesis in the mouse Leydig cell. Endocrinology 144,84-93[Abstract/Free Full Text]
  33. Kobayashi, S., Shibata, H., Kurihara, I., Yokota, K., Suda, N., Saito, I., Saruta, T. (2004) Ubc9 interacts with chicken ovalbumin upstream promoter-transcription factor I and represses receptor-dependent transcription. J. Mol. Endocrinol. 32,69-86[Abstract]
  34. Mellgren, G., Borud, B., Hoang, T., Yri, O. E., Fladeby, C., Lien, E. A., Lund, J. (2003) Characterization of receptor-interacting protein RIP140 in the regulation of SF-1 responsive target genes. Mol. Cell. Endocrinol. 203,91-103[CrossRef][Medline]
  35. Welsh, T. H., Jr, Jones, P. B., Hsueh, A. J. (1984) Phorbol ester inhibition of ovarian and testicular steroidogenesis in vitro. Cancer Res. 44,885-892[Abstract/Free Full Text]
  36. Chaturvedi, G., Arai, K., Limback, D., Roby, K. F., Terranova, P. F. (2004) Src tyrosine kinase regulates CYP17 expression and androstenedione secretion in theca-enriched mouse ovarian cells. Endocrine 25,147-154[CrossRef][Medline]
  37. Magoffin, D. A. (1991) Regulation of differentiated functions in ovarian theca cells. Sem. Reprod. Endocrinol. 9,321-331
  38. Hernandez, E. R., Hurwitz, A., Payne, D. W., Dharmarajan, A. M., Purchio, A. F., Adashi, E. Y. (1990) Transforming growth factor-ß1 inhibits ovarian androgen production: gene expression, cellular localization, mechanisms(s), and site(s) of action. Endocrinology 127,2804-2811[Abstract]
  39. Perrin, A., Pascal, O., Defaye, G., Feige, J. J., Chambaz, E. M. (1991) Transforming growth factor-ß1 is a negative regulator of steroid 17{alpha}-hydroxylase expression in bovine adrenocortical cells. Endocrinology 128,357-362[Abstract]
  40. Liakos, P., Lenz, D., Bernhardt, R., Feige, J. J., Defaye, G. (2003) Transforming growth factor-ß1 inhibits aldosterone and cortisol production in the human adrenocortical cell line NCI-H295R through inhibition of CYP11B1 and CYP11B2 expression. J. Endocrinol. 176,69-82[Abstract]
  41. Magoffin, D. A., Hubert-Leslie, D., Zachow, R. J. (1995) Estradiol-17ß, insulin-like growth factor-I, and luteinizing hormone inhibit secretion of transforming growth factor ß by rat ovarian theca-interstitial cells. Biol. Reprod. 53,627-635[Abstract]
  42. Nelson, V. L., Legro, R. S., Strauss, J. F., 3rd, McAllister, J. M. (1999) Augmented androgen production is a stable steroidogenic phenotype of propagated theca cells from polycystic ovaries. Mol. Endocrinol. 13,946-957[Abstract/Free Full Text]
  43. Wickenheisser, J. K., Quinn, P. G., Nelson, V. L., Legro, R. S., Strauss, J. F., 3rd, McAllister, J. M. (2000) Differential activity of the cytochrome P450 17{alpha}-hydroxylase and steroidogenic acute regulatory protein gene promoters in normal and polycystic ovary syndrome theca cells. J. Clin. Endocrinol. Metab. 85,2304-2311[Abstract/Free Full Text]
  44. Wickenheisser, J. K., Nelson-DeGrave, V. L., Quinn, P. G., McAllister, J. M. (2004) Increased cytochrome P450 17{alpha}-hydroxylase promoter function in theca cells isolated from patients with polycystic ovary syndrome involves nuclear factor-1. Mol. Endocrinol. 18,588-605[Abstract/Free Full Text]
  45. Nelson-Degrave, V. L., Wickenheisser, J. K., Hendricks, K. L., Asano, T., Fujishiro, M., Legro, R. S., Kimball, S. R., Strauss, J. F., 3rd, McAllister, J. M. (2005) Alterations in mitogen-activated protein kinase kinase and extracellular regulated kinase signaling in theca cells contribute to excessive androgen production in polycystic ovary syndrome. Mol. Endocrinol. 19,379-390[Abstract/Free Full Text]
  46. Wickenheisser, J. K., Nelson-Degrave, V. L., McAllister, J. M. (2005) Dysregulation of cytochrome P450 17{alpha}-hydroxylase messenger ribonucleic acid stability in theca cells isolated from women with polycystic ovary syndrome. J. Clin. Endocrinol. Metab. 90,1720-1727[Abstract/Free Full Text]
  47. Jakimiuk, A. J., Weitsman, S. R., Yen, H. W., Bogusiewicz, M., Magoffin, D. A. (2002) Estrogen receptor {alpha} and ß expression in theca and granulosa cells from women with polycystic ovary syndrome. J. Clin. Endocrinol. Metab. 87,5532-5538[Abstract/Free Full Text]
  48. Zofkova, I., Zajickova, K., Hill, M. (2002) The estrogen receptor alpha gene determines serum androstenedione levels in postmenopausal women. Steroids 67,815-819[CrossRef][Medline]
  49. Williams, P. C. (1940) Effect of stilbestrol on the ovaries of the hypophysectomized rat. Nature 145,388-389
  50. Pencharz, R. I. (1940) Effect of estrogens and androgens alone and in combination with chorionic gonadotropin on the ovary of the hypophysectomized rat. Science 91,554-555[Free Full Text]
  51. Evans, G., Leung, P. C., Brodie, A. M., Armstrong, D. T. (1981) Effect of an aromatase inhibitor (4-acetoxy-4-androstene-3,17-dione) on the stimulatory action of luteinizing hormone on estradiol-17ß synthesis by rat preovulatory follicles in vitro. Biol. Reprod. 25,290-294[Abstract]



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