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The Journal of Clinical Endocrinology & Metabolism Vol. 87, No. 8 3745-3753
Copyright © 2002 by The Endocrine Society

Original Article

Spatially Heterogenous Expression of Aromatase P450 through Promoter II Is Closely Correlated with the Level of Steroidogenic Factor-1 Transcript in Endometrioma Tissues

Hui-Juan Yang, Makio Shozu, Kouichi Murakami, Hiroshi Sumitani, Tomoya Segawa, Tadayuki Kasai and Masaki Inoue

Department of Obstetrics and Gynecology, School of Medicine, Kanazawa University, Kanazawa 920-0934, Japan

Address all correspondence and requests for reprints to: Makio Shozu, M.D., Ph.D., Department of Obstetrics and Gynecology, School of Medicine, Kanazawa University, Kanazawa 920-0934, Japan. E-mail: . shozu{at}med.kanazawa-u.ac.jp

Abstract

Endometriosis is an estrogen-dependent disease of women of reproductive age. Recent studies demonstrate that endometriosis per se express high levels of estrogen synthetase (aromatase P450). The resulting estrogen synthesized in situ may play a role in the development and exacerbation of the disease. For ovarian endometrioma, previous studies have been conducted ex vivo using cells obtained from endometrioma and have demonstrated that steroidogenic factor-1 is involved in the expression of aromatase. The aim of the present study was to provide in vivo evidence that steroidogenic factor-1 plays an important role in the regulation and overexpression of aromatase P450 in situ. First, promoter use of aromatase P450 in endometrioma tissue was determined using quantitative methods. Ovarian endometrioma tissue was chopped into small pieces, and two exon 1-specific transcripts of aromatase P450 (PII-specific and I.4-specific transcripts) were quantified using competitive RT-PCR. PII-specific transcript was more abundant than the I.4-specific transcript in 13 of the 15 endometriomas and less abundant in the remaining two. Spatial distribution of aromatase P450 transcripts in these endometrioma tissues revealed heterogeneous expression in the cyst wall, demonstrating wide variability even in the same endometrioma. Two possible regulators of aromatase expression (steroidogenic factor-1 and IL-1ß) were then measured in all endometrioma samples and the correlation between aromatase P450 transcripts and these possible regulators in the endometrioma samples were tested using Spearman’s rank order correlation test. Levels of steroidogenic factor-1 transcript were found to correlate closely with levels of PIIspecific transcript in eight of nine endometriomas examined. On the other hand, the level of IL-1ß weakly correlated with I.4-specific transcripts in three of the nine endometriomas. We next histologically examined samples of four endometriomas in which complete sets of tissue samples corresponded to the RNA samples. We could not identify any specific pathology to explain the heterogenous expression of PII-specific transcripts of aromatase P450, although the number of CD-68 positive macrophages in the tissue sections weakly correlated with the level of I.4-specific transcript in two of four endometriomas. These results provide strong evidence that promoter II is the predominant promoter of aromatase P450 in endometrioma tissues in vivo and that steroidogenic factor-1 in situ is a major determinant of aromatase P450 overexpression in endometrioma tissues in vivo.

ENDOMETRIOSIS IS A GYNECOLOGICAL disorder of women of reproductive age, which often requires surgical or medical management because of pelvic pain, dyspareunia, and impaired fecundity. Histology defines endometriosis as an extrauterine growth of endometrial tissues or endometrium-like tissues. Several theories have been proposed to explain the histogenesis of endometrial tissues or endometrium-like tissues outside the uterine cavity. The most widely accepted explanation, particularly for peritoneal endometriosis, is Sampson’s transplantation theory (1). Viable cells of eutopic endometrium are regurgitated, contact the pelvic surface via retrograde menstruation, and become implanted in it (1, 2, 3). Endometrial cells are frequently regurgitated through the fallopian tubes during menstruation, even in women who do not develop endometriosis (4, 5). Thus, retrograde menstruation could be necessary but not the only condition under which endometriosis develops (3). Other important factor(s) must differentiate women who develop endometriosis from those who do not. To date, several causative factors have been implicated, including environmental factors such as dioxin exposure, immunological abnormalities leading to dysfunction in peritoneal clearance of regurgitant cells, and genetic background (3, 6, 7, 8, 9). The actual pathogenesis, however, remains to be determined (3, 9).

Although the etiology remains unknown, estrogen plays a critical role in the development and/or worsening of endometriosis (10, 11). An excess of estrogen promotes, whereas ablation of estrogen by GnRH agonist therapy or oophorectomy prevents progression of the disease. Thus, ovarian estrogen is essential for endometriosis. In addition to the pivotal role of ovarian estrogen, recent studies suggest an additional role for in situ estrogen synthesized within endometriotic implants (12, 13, 14). Endometriotic tissues and cultured cells derived from endometriosis express high levels of estrogen synthetase (aromatase P450) but lack the enzyme (17-ß hydroxysteroid dehydrogenase, type 2) that converts E2 into estrone, an inactive form of estrogen (12, 13, 14). Therefore, endometrioma cells per se produce active estrogen in situ without inactivating it. Several groups demonstrated that the eutopic endometrium of women with endometriosis can synthesize estrogen, whereas that of disease-free women does not (12, 15, 16, 17). This finding suggests that the eutopic endometrium of women with endometriosis expresses aromatase P450 and produces in situ estrogen that favors successful implantation through the up-regulation of some growth factors (18, 19). If in situ estrogen plays a role in the implantation and development of endometriosis, ablation of in situ estrogen might represent a novel conservative therapy for endometriosis. The mechanism that maintains high levels of in situ aromatase expression must, therefore, be ascertained, and a strategy for targeting aromatase expression in endometriosis cells is required.

Studies of aromatase expression in endometriosis have been limited in terms of number of samples and type of endometriosis tissues analyzed. For ovarian endometriomas, the expression of aromatase P450 has been studied on primary cells obtained from endometrioma tissues (endometriosis-derived stroma cells) ex vivo instead endometrioma tissues in vivo (20). They concluded that, in these primary cells in culture, aromatase P450 is driven predominantly by promoter II, a proximal promoter of the aromatase P450 gene (CYP19), and steroidogenic factor-1 (SF-1) is involved in its transcription (13, 20). However, no evidence has shown that SF-1 actually determines the transcriptional level of aromatase in endometrioma tissues in vivo.

The present study measures the precise levels of aromatase p450 and SF-1 expression in endometrioma tissues. We found that in situ expression of aromatase P450 was spatially heterogenous even in the same endometrioma and that the expression levels of promoter II of aromatase and SF-1 were closely correlated. This provides strong evidence that SF-1 actually regulates the expression level of in situ aromatase in endometriosis tissues in vivo.

Materials and Methods

Tissue acquisition and preparation

Fifteen endometrioma tissues were obtained from 14 women at laparotomy or after laparoscopic surgery for endometrioma following approval from the Medical Ethics Committee of Kanazawa University. Written informed consent was obtained before surgery from all patients who were randomly selected for enrollment in this study. All endometrioma tissue specimens except one that was enucleated from an excised ovary immediately after surgery were enucleated from the ovary during surgery. Clinical parameters are listed in Table 1Go. None of the endometriomas was potentially malignant. Fresh samples of enucleated endometriomas were examined under a dissecting microscope to confirm complete removal of ovarian tissue, unfolded onto a prechilled plate, and chopped into 1-cm squares. In 11 of 15 endometriomas, three or more 1-cm2 pieces at the periphery of unfolded endometrioma tissues were randomly selected and assigned for histological diagnosis. Other 1-cm2 pieces were snap-frozen and stored at -74 C for RT-PCR assay. Of the remaining four endometriomas, all 1-cm2 pieces were further divided into two parts along an oblique line and a (lower) half was similarly snap-frozen and stored for RT-PCR assay. The other (upper) half was fixed in 10% buffered formalin for comparative histological examination. Formalin-fixed samples were sectioned and stained with hematoxylin and eosin. Endometrioma cysts were composed of fibrous outside tissues associated with inside endometrial stromalike tissues and sometimes columnar epithelium similar to that of eutopic endometrium at the innermost location. Contamination of ovarian cortex or medulla in endometrioma samples was histologically suspected on the basis of the following findings: primordial, growing, and atretic follicles; corpus luteum and corpus albicans; and loose connective tissues of ovarian medulla associated with a mass of contorted blood vessels characteristic of rete ovarii.


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  		Table 1. Clinical parameters and levels of aromatase P450 transcripts in ovarian endometriomas

 
Construction of plasmids carrying deletions for internal standards

Two plasmids (pCR{Delta}aromI.4 and pCR{Delta}aromPII) containing one copy each of exon I (exon I.4- and PII-specific sequence) and exon II through IV (with an internal deletion of 107 bp between exons II and III) were constructed by two-step PCR amplification (20, 21).

Another PCR-based strategy was used to introduce internal deletions into the wild sequence of IL-1ß, SF-1, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). For IL-1ß, two partial sequences of IL-1ß cDNA were PCR amplified by primer pairs: IL-1b175F (forward primer) and IL-1b/i (reverse i primer) for the region upstream of an internal deletion, and IL-1b/x (forward x primer) and IL-1ß 675R (reverse primer) for the region downstream of the internal deletion (Fig. 1Go). Equal amounts of these two PCR products were mixed and incubated at 37 C for 4 h to remove A-overhangs of 3'-ends. An aliquot (1 µl) of the mixture was then ligated using 10 µl ligation buffer containing 1 U T4-ligase (Invitrogen, Carlsbad, CA) at 16 C for 16 h. A target sequence carrying an internal deletion was PCR amplified from the ligation mixture using a primer pair (IL-1b175F and IL-1b675R). The resulting PCR product was subcloned into a pCR2.1 vector using a TA-cloning kit (Invitrogen). Plasmids carrying an internal deletion of GAPDH and SF-1 were constructed similarly using i and x primers for each gene. Primer sequences and the size of the internal deletions introduced are summarized in Table 2Go.



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  		Figure 1. Schematic representation of PCR strategy to construct deletion mutants (pCR2.1{Delta}IL-1ß, pCR2.1 {Delta}SF-1, and pCR2.1{Delta}GAPDH). *, A portion of ligation mixture was used as a template to amplify the target sequence carrying an internal deletion. This mixture contained various species of DNA fragments that were randomly ligated.

 

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  		Table 2. Primer sequences and PCR conditions

 
To introduce an internal deletion to an IGF-I sequence, wild-type sequence was digested by MSP I to remove a 143-bp internal sequence and religated. After PCR amplification, the target sequence was subcloned into a pCR2.1 vector as described above.

Fidelity and the direction of the sequence were confirmed using an automatic DNA sequencer, the ABI Prism 310 genetic analyzer (PE Applied Biosystems, Foster City, CA).

In vitro transcription of the internal standard

Internal standard RNAs were synthesized in vitro with T7-RNA polymerase (Invitrogen) using Hind III digests of pCR2.1 vectors carrying internal deletions (20). The synthetic RNAs were extracted twice with acid-phenol and chloroform, digested twice with DNase I (Invitrogen), and then purified by anion-exchange column chromatography (RNeasy kit, QIAGEN). RNA was spectrophotometrically quantified and purity and size were confirmed by polyacrylamide gel electrophoresis.

RNA extraction

Frozen tissue samples were triturated in liquid nitrogen and total RNA was extracted using an Ultraspec RNA isolation kit (Biotecx, Houston, TX) according to the manufacturer’s instructions. The RNA concentration was determined at OD260.

RT-PCR and quantitation of transcripts

We could not isolate a sufficient amount of RNA from all pieces of tissue for standard quantitative competitive RT-PCR assays. We, therefore, performed semiquantitative competitive RT-PCR assays in which levels of transcript were determined by a single RT-PCR reaction using a fixed amount of competitors. To optimize this analysis, appropriate amounts of internal standards are essential. Thus, we determined the average amount of the transcript by the standard quantitative competitive RT-PCR assay (first RT-PCR) for every gene of all endometriomas and used these values as ideal amounts of competitors for subsequent semiquantitative competitive RT-PCR assays (second RT-PCR).

In the first RT-PCR assay, the levels of each transcript were determined for all endometriomas using pooled RNA samples from the same endometrioma. Graded amounts of synthetic RNAs (internal standards) were mixed with pooled RNA and then reverse transcribed and amplified by PCR. The amounts of added synthetic RNA were 10, 1, 0.1, and 0.05 amol for PII-specific transcript; 1, 0.1, 0.01, and 0.001 amol for exon I.4-specific transcript; 100, 10, 1, and 0.1 amol for IL-1ß; 100, 25, 10, and 1 amol for SF-1, 10, 10, 1, and 0.5 amol for IGF-I; 10, 5, 1, and 0.2 fmol for GAPDH. The PCR products were separated on agarose gels, stained with ethidium bromide, and photographed using a CCD camera system (Epi-light UV FA1100 system, AIC, Tokyo, Japan). Photographs were scanned (GT9500, Epson, Tokyo, Japan), and quantitative analyses were performed on a Macintosh Power PC G3 (Apple Japan, Toko, Japan) using the NIH Image (version 1.61) program. Densitometric values were normalized to the length of the band and the ratios of the densitometric values of the target to that of the internal standard were used to calculate the amount of transcripts in the pooled RNA samples. The logarithmically transformed ratio of the competitor intensity to the target intensity was plotted against the log amount of initially added competitor RNAs (Fig. 2AGo). The regression lines were linear, and the amount of transcripts in a sample (pooled RNA) was calculated from the equivalence point (log ratio = 0) on the regression lines.



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  		Figure 2. Representative quantitative RT-PCR to assay PII-specific transcripts. Levels of PII-specific transcript were determined on individual tissue pieces of all endometrioma samples by two-step sequential competitive RT-PCR assays. A (first RT-PCR), RNA samples obtained from the same endometrioma were pooled, and the levels of PII-specific transcript were estimated on this mixture by standard quantitative competitive RT-PCR, in which four concentrations of competitor RNA were mixed with a fixed (1 µg) amount of sample RNA. B (second RT-PCR), Amounts of PII-specific transcript were determined on individual tissue pieces of the same endometrioma by single semiquantitative RT-PCR. Scheme to indicate relative locations of sample pieces (A1-C3). *, Sample pieces assigned for histological examination. Amounts of PII-specific transcript were calculated based on the ratio of the target to the competitor as described in Materials and Methods. C (standard curve), The assay was validated by adding known amounts (0.05 to 12 amol) of target RNA to a fixed amount of competitor (0.25 attomol) before RT-PCR.

 
We then conducted the second RT-PCR to determine the level of each transcript in individual RNA samples. RNA samples from the same endometriomas were assayed in one reverse transcription and in one PCR reaction using fixed amounts of internal standards as determined by the first RT-PCR assays (Fig. 2BGo). A mixture of internal standard RNAs (PII, I.4, IL-1ß, SF-1, IGF-I, and GAPDH) was added to the RNA sample and reverse transcribed using 50 pmol random hexamers (PE Applied Biosystems) and 1 µl reverse transcriptase (RevatraAce, Toyobo, Tokyo, Japan). An aliquot of the resultant cDNA was used as the template for PCR amplification of each gene. Intensity was measured and values were normalized to the length of bands as described above. The level of transcripts in individual RNA samples was calculated from the ratio of the intensity of the target to that of the internal standard. The standard curve of this semiquantitative RT-PCR assay (logarithmically transformed ratios of target intensity to competitor intensity plotted against the log amount of initially added target) confirmed a significant linear regression over the two-log scale (Fig. 2CGo). Finally, levels of each transcript in individual samples were normalized by the level of GAPDH in each sample to eliminate the effect of RNA quality (amount of target transcripts was divided by amount of GAPDH transcripts and multiplied by 1000 for convenience).

Calculated values of intraassay and interassay variability were 8% and 24% for the PII-specific transcript, 12% and 34% for the I.4-specific transcript, 6% and 9% for the IL-1ß transcript, 7% and 19% for the SF-1 transcript, 15% and 36% for the IGF-I transcript, and 5% and 16% for the GAPDH transcript. Intraassay variability was calculated as the mean of variability from at least three samples assayed in triplicate and interassay variability was calculated from the results of three independent RT-PCR assays of the same RNA aliquot.

Immunohistochemistry

Fixed tissue samples were embedded and cut into 5-nm-thick sections. After blocking endogenous peroxidase with 0.3% hydrogen peroxide, sections were placed in 0.01 M sodium citrate buffer (pH 6.0) and boiled twice using a microwave oven set for antigen retrieval. The sections were then incubated for 15 min at room temperature in 10 mM PBS with anti-CD68 antibody (NeoMarkers, Fremont, CA). The sections were rinsed and sequentially incubated with a link antibody for 15 min, followed by streptavidin-biotin and streptavidin-peroxidase complexes using the catalyzed signal amplification system (DAKO Corp., Carpinteria, CA). Color was developed by incubation with 3,3-diaminobenzindine tetrahydrochloride for 3–6 min, followed by counterstaining with hematoxylin. A representative section of each sample piece was photographed, and two independent observers counted the number of CD-68-positive cells. The mean value of observed counts were used for statistical analysis.

Statistical analysis

Differences in levels of two species of aromatase P450 transcripts were evaluated using the Wilcoxon matched pairs signed-rank test. Differences in transcript levels between two groups were evaluated using the Mann-Whitney U test. Correlations among transcript levels were assessed by Spearman’s rank order correlation test for all endometrioma tissues. P values less than 0.05 were considered significant. Data are presented as means ±SEM of the mean. All analyses were performed using StatView software (version 5.0SAS Institute Inc., Cary, NC).

Results

Quantitative analysis of promoter use of aromatase P450

To determine which promoter is predominantly used for aromatase P450 expression, we measured the amount of mRNA containing I.4- and PII-specific sequences in endometrioma tissues. Table 1Go shows that 13 of 15 endometriomas contained much more PII-specific transcripts than exon I.4-specific transcripts. The P values of the difference were less than 0.05 in 10 samples and 0.06 in two. Statistical analysis was not performed on the remaining one endometrioma of which only one piece was obtained for quantitative RT-PCR analysis. In contrast, two endometriomas contained more I.4-specific than PII-specific transcript.

Endometriomas expressing a high level of mean PIIspecific transcripts also tended to possess high levels of mean I.4-specific transcripts (P < 0.06, Mann-Whitney U test).

Clinical parameters including age, preoperative GnRH agonist therapy, and revised AFS score did not show any correlation with the mean level of aromatase P450 transcripts.

Spatial distribution of aromatase P450 expression

To clarify the spatial distribution of aromatase P450 expression, we mapped aromatase P450 transcripts in the wall of each endometrioma. Figure 3Go shows two representative results in which I.4- and PII-specific transcripts predominated. The expression levels of each transcript varied considerably, even within the same endometrioma, and areas expressing high levels tended to lie in direct proximity to each other within the same endometrioma. The spatial distribution of I.4- and PII-specific transcript did not correlate. Because most endometriomas were obtained at the time of laparoscopic surgery without precise orientation, we could not localize areas expressing relatively high levels to the bony pelvis.



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  		Figure 3. Spatial distribution of aromatase P450 expression in ovarian endometriosis. Samples of the same endometrioma were assigned to squares based on relative positions. Squares are representational shapes. Height of pyramids on squares represents aromatase P450 transcripts normalized to GAPDH (divided by GAPDH transcript level and multiplied by 1000).

 
Correlation among expression levels of aromatase P450 (PII and I.4), IL-1ß, and SF-1

To clarify the possible regulatory role of IL-1ß and SF-1 on the regional expression of aromatase P450, the transcript levels in the endometriomas were assessed by Spearman’s rank order correlation test (Fig. 4Go, A and B). Nine endometriomas that consisted of six or more pieces and yielded sufficient RNAs for full analyses of the genes in all pieces were studied.



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  		Figure 4. Correlation of expression levels among aromatase P450 (I.4- and PII-specific), SF-1, IL-1ß, and IGF-I. A, Correlation coefficient matrix of Spearman’s correlation rank test. Coefficient was calculated among transcript levels of pieces from the same endometrioma. Bold type represents statistically significant correlation (P < 0.05). *, Identification of the patient. B, Summary of significant correlation found in above analysis. Numbers in table represent those with significant positive correlation for each pair among nine endometriomas tested. Parentheses indicate negative correlation.

 
The correlation of SF-1 with PII-specific transcripts was statistically significant in all endometriomas tested except one (patient 3), in which the correlation just failed to reach significance (P = 0.06), probably because of the low number of sample pieces (n = 6). The correlation between IL-1ß and I.4-specific transcripts was significant (P < 0.05) in three of nine endometriomas.

Correlation between aromatase P450 and IGF-I transcripts

The growth-promoting effect of ovarian estrogen on leiomyomas is mediated, at least in part, by the induction of IGF-I (22, 23, 24). We, therefore, surmised that locally synthesized estrogen plays a role in the growth of endometrioma cells through IGF-I expression, as does endocrine estrogen. To test this hypothesis, we similarly assessed the correlation between IGF-I and aromatase P450 transcripts. Figure 4Go shows that the level of IGF-I transcripts did not correlate with any aromatase P450 transcripts in all endometriomas except one in which the level of IGF-I transcripts significantly correlated with that of PII-specific transcripts (patient 9).

Histological examination

Contamination of the ovarian cortex may explain the localized overexpression of aromatase P450 in endometrioma tissues. To exclude this possibility, we histologically examined complete sets of tissue samples corresponding to the RNA samples in four endometriomas (patients 4 through 7). Two or three representative samples of other endometriomas were similarly examined. Among these, contamination with ovarian structures was not evident.

We examined the number of macrophages that migrated into endometriomas and found that it depended on the location of the sample pieces even within the same endometrioma (data not shown). Correlation between the number of macrophages and the expression level of IL-1ß, SF1, PII, and I.4 in the corresponding halves were assessed by Spearman’s rank order correlation test as described above. The number of macrophages and expression levels of tested genes did not significantly correlate, with an exception for I.4-specific transcripts. The number of infiltrated macrophages correlated with the level of the I.4-specific transcript in two of four endometriomas examined (patients 4 and 6).

Discussion

Aromatase P450 is a product of a single-copy gene (CYP19) on chromosome 15q21, and it is expressed in several tissues and cells, including gonads and extragonadal tissues. The expression of aromatase P450 is regulated in a tissue-dependent manner (25). Tissue-specific regulation is realized by the alternative use of seven promoters of CYP19, each of which has a unique cis-acting element function (25, 26, 27). Thus, identification of promoter use was the first step in understanding the mechanism of physiological and pathological expression of aromatase P450 in tissues (26). Promoter use in endometriosis was originally examined in endometriotic implants of the peritoneum. Noble et al. (12) examined two endometriotic implants by exon 1-specific RT-PCR and found that two aromatase P450 promoters, I.4 and PII, were used at a similar frequency. They later reexamined promoter use in three more endometriotic implants using the 5'-rapid amplification of cDNA ends procedure and found that PII was predominantly used, whereas both I.4 and I.3 were infrequently used (28). Although the methods that they used are essentially qualitative, these results suggest that the dominant promoter of aromatase P450 is PII or PII plus I.4 in endometriotic implants of the peritoneum.

The same group subsequently studied the regulation of aromatase P450 in ovarian endometriomas (13, 28) They isolated stroma cells from the wall of ovarian endometriomas (endometrioma-derived cells) and compared the regulation of aromatase P450 in the cells with that in stromal cells obtained from eutopic endometrium (eutopic endometrial cells). They found that cells derived from endometrioma express high levels of aromatase P450 in response to several stimuli, including prostaglandin E2 (PGE2) and cAMP, whereas eutopic endometrial cells obtained from disease-free women did not express aromatase P450 (13). The 5'-rapid amplification of cDNA ends procedure followed by Southern blotting or sequencing demonstrated that both PGE2 and cAMP induce aromatase P450 expression through the promoter PII (13, 28). On the other hand, any combination such as IL-1ß + dexamethasone (DEX), TNF{alpha} + DEX, or IL-6 + DEX induced no or a minimal increase in aromatase activity in these endometrioma-derived cells (13). In contrast, these combinations are potent inducers of promoter I.4-driven transcription in many cell types. All of these ex vivo experiments suggest that PII is the most potent promoter functioning in cells derived from endometrioma.

The present study measured exon 1-specific transcripts of aromatase P450 in endometrioma tissues using quantitative RT-PCR and confirmed that usage of PII predominates in vivo, as indicated from ex vivo studies (12, 28). Our study also revealed that the expression of aromatase P450 is spatially heterogeneous within the same endometrioma. We, therefore, considered that the transcriptional level of aromatase P450 is locally determined in situ though a local regulatory factor(s). Moreover, our experimental model may provide a useful tool with which to identify this local factor(s) involved in the regulation of aromatase P450 in situ. We, therefore, quantified the expression levels of two possible regulatory factors (SF-1 and IL-1ß) for aromatase P450 and found that levels of SF-1 and PII transcripts were closely correlated in vivo. This provided evidence that SF-1 functions as a determinant of P450 aromatase expression in endometrioma tissues in vivo (28). SF-1 appears to up-regulate transcription through promoter PII according to ex vivo experiments. Cotransfection of an SF-1 expression vector potentiates the transcriptional activity of reporter vectors containing the promoter PII sequence in cells derived from endometrioma (28). EMSAs and transient transfection assays using cells derived from endometriosis have demonstrated that SF-1 binds to the nuclear half-site in promoter II and displaces chicken ovalbumin upstream promoter transcription factor (COUP-TF) from that site, which increases the amount of transcription (28). The transcriptional activity of SF-1 on promoter II of aromatase P450 has been established in granulosa or gonadal cells in many other animal species (29, 30). All of these ex vivo studies showed that SF-1 plays an essential role in the induction of aromatase P450. Our results provide powerful evidence that SF-1 actually functions as a key determinant in the transcriptional level of aromatase in endometrioma tissues in situ.

In addition to that between the SF-1 and PII-specific transcripts, we found a significant correlation between IL-1ß and I.4-specific transcripts in three of nine endometriomas. Other investigators and we have shown that IL-1ß profoundly stimulates the transcription of aromatase P450 through promoter I.4 in many primary cells including skin fibroblasts, aortic fibroblasts, adipose stromal cells, osteoblastlike cells, myometrial cells, and leiomyoma cells (31, 32, 33, 34). Therefore, IL-1ß probably up-regulates transcription through promoter I.4 of aromatase P450 in endometriomas. We initially considered that macrophages infiltrating endometriosis tissues secreted IL-1ß, which stimulated aromatase P450 expression through promoter I.4. The number of macrophages in endometriosis tissues did not, however, correlate with tissue levels of the IL-1ß transcript. Thus, IL-1ß seems to originate from another cell component(s) rather than from infiltrating macrophages. Stromal and epithelial cells in endometriosis tissues can actually synthesize IL-1ß (35, 36, 37, 38). On the other hand, the number of macrophages positively correlated with the I.4-specific transcript in two of four endometriomas tested despite the absence of correlation with the IL-1ß level. This means that macrophages contribute to the level of I.4 transcript possibly through cytokines other than IL-1ß. Both TNF-{alpha} and IL-6 can alternate with IL-1ß in terms of the promoter I.4-driven transcription of aromatase P450 in many of the cell types referred to above (20, 33, 39, 40, 41). The inducibility of aromatase P450 by cytokines including IL-1ß through promoter I.4 is currently under investigation using cells derived from endometrioma in culture.

Although the exact mechanism of estrogen action on the cellular growth of endometrial cells remains unknown, several reports indicate that estrogen induces the expression of growth stimulatory factors (such as IGF-I), which in turn stimulate cellular growth and implantation (23, 24, 42, 43, 44, 45). We examined the correlation between aromatase and IGF-I expression to demonstrate likely roles of in situ estrogen on cellular growth. We did not find a reproducible correlation between transcripts of aromatase P450 and IGF-I. This negated our hypothesis that in situ estrogen plays a role on the cellular growth of endometriomas through IGF-I mediated action. IGF-I may not be suitable for evaluating estrogen action on cellular growth. Further study is thus needed to address the possible role of in situ estrogen on this process.

We have demonstrated that uterine leiomyomas overexpress aromatase P450 through promoter I.4 and that preoperative GnRH analog therapy ablates the expression of aromatase P450 (20, 21). During GnRH agonist-induced suppression of the ovary, in situ estrogen would become the sole source of estrogen for leiomyoma cells. Given its growth stimulatory role, suppressing the in situ synthesis of estrogen would be necessary to achieve maximum shrinkage of leiomyomas during GnRH agonist therapy (21). The present study found that GnRH agonist did not suppress in situ aromatase in endometriomas. The levels of I.4- and PIIspecific transcripts of aromatase P450 in endometriomas from women with preoperative GnRH agonist therapy were not significantly different from those in women who did not undergo preoperative therapy. This means that the in situ production of estrogen persists during GnRH agonist therapy in endometrioma tissues, even after estrogen synthesis in the ovary ceases.

We assume that continuously synthesized in situ estrogen throughout GnRH agonist therapy favors the maintenance of endometrioma cells. We recently compared the levels of aromatase P450 transcripts in endometrioma with those in adipose tissues surrounding breast cancer, in which the growth-promoting role of in situ estrogen is established. The levels of aromatase P450 transcripts in the endometrioma were about one order higher than those in adipose tissues surrounding breast cancer tissues (in preparation). Therefore, the amount of estrogen synthesized in endometrioma tissues in situ may be sufficient to exert some growth stimulatory effects on their own cells. This in situ estrogen may become important, especially under conditions of hypoestrogenemia induced by ovarian ablation. We could not demonstrate a growth stimulatory role of in situ estrogen through a correlation with IGF-I expression. Further study using another approach is needed to define the role of in situ estrogen. This is important because if in situ estrogen functions in the cellular growth of endometriosis under hypoestrogenemic conditions induced by GnRH agonist therapy, an aromatase inhibitor would represent an alternate choice or an adjuvant to GnRH agonist therapy (18, 46). Unlike GnRH agonists, aromatase inhibitors decrease the amount of in situ estrogen produced by endometriosis in addition to endocrine estrogen from the ovary.

The present study, like others, used ovarian endometrioma tissues to study the regulation of aromatase P450 in endometriosis (13, 28). This is mainly because peritoneal implants are usually too small to be trimmed and release enough cells for reproducible experiments. As described earlier, histogenesis may, however, differ between these types of endometriosis (2). Ovarian endometrioma may arise from metaplasia of coelomic epithelium of the inclusion cyst in the ovary, unlike peritoneal implantation of endometrial reflux. Thus, the results of present study may be limited.

In summary, we identified PII as a major promoter used in aromatase P450 expression in ovarian endometriosis and provided evidence that the expression of aromatase is regulated through the expression level of SF-1 in situ. Even though the amount of in situ estrogen may be small, compared with the amount of estrogen supplied from the ovary, it would still play some role on growth under the hypoestrogenemic conditions induced by GnRH agonist or at menopause (46). To define the possible use of aromatase inhibitors, the regulation of aromatase P450 expression and the role of in situ synthesis of estrogen on cellular growth of endometrioma cells require further study.

Acknowledgments

Footnotes

This work was supported by Grants-in-Aid for Scientific Research B12557136 and B13470348 from the Ministry of Education, Science, Sports, and Culture, Japan.

Abbreviations: DEX, Dexamethasone; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; SF-1, steroidogenic factor-1.

Received January 31, 2002.

Accepted April 18, 2002.

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