Aromatase P450arom Research Articles

Neurochemical characterization of the hypothalamus of the early fetal and newborn alpaca Vicugna pacos.

In this study we performed a neurochemical characterization of the hypothalamus in the developing alpaca (Vicugna pacos) with the aim of revealing the distributions of immunoreactive (-ir) cells containing parvalbumin (PV), calbindin (CB), calretinin (CR), the somatostatin (SOM), the enzyme aromatase P450 (P450Arom), the estrogen receptor α (ER-α), and estrogen receptor β (ER-β) in embryonal stages, early fetal age, and in the newborn. This analysis has been carried out on embryos at 20, 30, 45 days, fetuses at 90 days, and newborn alpaca. Our immunohistochemical results revealed no cells-ir throughout the embryonic hypothalami of 20, 30, and 45 days. On the fetal stage of 90 days, SOM-ir cells were observed in the lateral hypothalamus and the ventromedial nuclei of the tuberal region. We checked for the presence of P450Arom-ir cells in the periventricular area and dorsomedial hypothalamic nucleus of the tuberal region. In these fetal stages, no PV-ir, CB-ir, CR-ir or ERs-ir cells were identified. In the newborn, the PV-ir, CB-ir, CR-ir, and SOM-ir cells were detected in both the anterior and tuberal hypothalamic area. The P450Arom-ir cells the ER-α-ir and ER-β-ir cells were found in the anterior hypothalamus. Our results offer a contribution in the future purpose to obtain a time-expression pattern of the considered markers in alpaca during gestation and represents a foundation for future investigations on the alpaca brain to define the cross talk between PV, CB, CR, P450Arom, SOM, and ERs in the hypothalamus, the strategic region for the control of the reproductive behavior.

Effect of electroacupuncture on anti-Mullerian hormone expression in rats with polycystic ovarian syndrome

To observe whether the effect of electroacupuncture (EA) on improving sex hormone disorders and follicle development is by decreasing the expression of anti-Mullerian hormone (AMH) in rats with experimental polycystic ovarian syndrome (PCOS). Forty rats were randomly divided into four groups, a normal group (NG), a model group (MG), an EA at acupoints group (EAAG), and an EA at non-acupoints group (EANAG), with 10 rats in each group. The rats in the EAAG and EANAG were intervened by EA treatment for consecutive 14 d. Zhongji (CV 3) and Guanyuan (CV 4) were selected as the acupoints in the EAAG, and the tip of the tail and 1 cm up from the tail tip were selected as the non-acupoints in the EANAG. After treatment, the histomorphological changes of the ovary, the levels of aromatase P450 (P450arom), testosterone and estradiol in the ovarian tissues, and the expressions of follicle stimulating hormone (FSH) and AMH were observed. After treatment, compared with the MG and EANAG, the expression of AMH decreased (P<0.05), the levels of P450arom and estradiol increased significantly, and the level of testosterone decreased significantly (all P<0.01) in the EAAG. Additionally, several normal follicles were present and the number of cystically dilated follicles decreased in the EAAG. Compared with the MG and EANAG, the EAAG obviously had more follicular granulosa cells. EA can down-regulate the abnormally increased expression of AMH to improve sex hormone disorders and follicle development in PCOS rats.

Effects of cisplatin on surgically induced endometriosis in a rat model

Research has strongly suggested that the features of endometriosis serve as a precursor lesion of ovarian cancer. Cisplatin (CDDP) is the preferred drug against these cancer types. The present study investigated the effects of CDDP on surgically induced endometriosis in a rat model. Endometriosis was surgically induced by the autologous transplantation of endometrial tissue. A total of 36 model rats were randomly divided into three groups. The rats in Group 1 (control group, n=12) received no medication. The rats in Group 2 (n=12) and Group 3 (n=12) were administered 35 mg/m2 CDDP and 70 mg/m2 CDDP, respectively, every four days. All rats were treated for a total of 24 days. The growth and histologic scores of the implants were calculated. The expression of protein markers, including vascular endothelial growth factor (VEGF), aromatase P450 (P450arom), transforming growth factor-β (TGF-β) and matrix metalloproteinase (MMP)-2, were assessed using immunohistochemistry, an enzyme-linked immunosorbent assay and western blot analysis. Following CDDP treatment, the mean implant sizes were significantly reduced in Groups 2 and 3 compared with the control group (P=0.01). The mean histologic scores were also significantly lower in Groups 2 and 3. Furthermore, the protein expression of VEGF, P450arom, TGF-β and MMP-2 was significantly lower in Groups 2 and 3 when compared with the control group. A loss of hair was observed in 4 rats, which only occurred in Group 3. A dose-dependent effect was observed in the two CDDP-treated groups. In conclusion, the expression of proliferation- and angiogenesis-associated proteins was significantly lower following treatment with CDDP. CDDP caused a significant regression in the size of the endometriotic implants and induced atrophy of these lesions in rats.

Smad3/4 Binding to Promoter II of P450arom So As to Regulate Aromatase Expression in Endometriosis.

Activin A can stimulate aromatase P450 (P450arom) expression in eutopic endometrial stromal cells (ESCs) of endometriosis by activin type I receptor-Smad pathway. In order to identify Smad3/4 binding to P450arom promoter II that mediates activin A response in ESCs, polymerase chain reaction (PCR) products of a serial truncated deletion of the P450arom promoter II region between -904 and +87 bp were inserted into the pGL3-basic vector to generate the promoter reporter plasmids. Luciferase reporter plasmids were cotransfected into cells with or without activin A (25 ng/mL). The pGL3 -705/+87 revealed a luciferase activity similar to pGL3 -904/+87, whereas progressive truncation to position -464/+87 and -192/+87, the luciferase activity was significant variation. Chromatin immunoprecipitation assay and Smad4-small interfering RNA (siRNA) testify that Smad3/4 binds to the activin A-responsive aromatase promoter in ESCs. Chromatin immunoprecipitation assay-PCR assay demonstrated anti-Smad3 antibody complexes could interact with the amplified DNA of the activin A-responsive P450arom promoter. Mutations of the binding site (-141/-138 bp, -165/-162 bp) in P450arom promoter II significantly reduced promoter activity of activin A fold-induction to 26% and 28%, respectively. We cotransfected pGL3 -705/+87 with control siRNA and Smad4-siRNA into ESCs in the presence of activin A. Luciferase analysis showed that Smad4-siRNA abolished increased promoter activity of activin A-induced P450arom expression. The effect of activin A on the p-Smad3 accumulation in the cytoplasm and nucleus was significantly abrogated following the pretreatment of ESCs with Smad4-siRNA. In conclusion, activated Smad3 proteins can bind to P450arom promoter -705/+87 bp region, responsive to activin A in ESCs, which can promote P450arom transcription.

Effects of cisplatin and letrozole on surgically induced endometriosis and comparison of the two medications in a rat model

This study was to investigate the effects of cisplatin (CDDP) and letrozole on surgically induced endometriosis and comparison of the two drugs in a rat model. Endometriosis was surgically induced by autologous transplantation of endometrial pieces. Thirty model rats were divided into three groups, randomly. Group 1 (n=10) served as control and received no medication. Group 2 (n=10) received 0.2mg/kg/day of oral letrozole. Group 3 (n=10) received 35mg/m2 CDDP via peritoneal perfusion every four days. All the rats were treated for 24days. The growth and histologic score of the implants were evaluated. The proliferation- and angiogenesis-associated proteins were assessed using immunohistochemistry and western blotting. The serum sex hormones were assayed using ELISA. After the medication, the growth and histologic score of the implants were significantly lower in the 2 and 3 groups than in the control group. The protein expressions of vascular endothelial growth factor (VEGF), aromatase P450 (P450arom), transforming growth factor-beta (TGF-β), and matrix metalloproteinase (MMP)-2, were significantly lower in groups 2 and 3 than in the control group. Further, the P450arom level was lower in the letrozole group than in the CDDP group. The TGF-β and MMP-2 levels were lower in the CDDP group than in the letrozole group. Serum T level was significantly higher in the letrozole group, and serum E2 level was lower in the letrozole group. In conclusion, cisplatin and letrozole caused similar regression of the implants in the endometriosis model rats. But their effects on the proliferation- and angiogenesis-associated protein expressions and the serum sex hormone levels were different. Cisplatin and letrozole might cause the effects in the endometriotic foci through different mechanism.

Endometriotic Implants Regress in Rat Models Treated With Puerarin by Decreasing Estradiol Level

Phytoestrogens, which have a weak estrogenic effect, bind to estrogen receptors (ERs), thereby competing with estradiol, have an antiestrogenic effect on women of reproductive age with high estrogenic level. Herein, we examined the ability of the phytoestrogen Puerarin to treat endometriosis in rat models of endometriosis. In total, 75 adult, mature female Sprague-Dawley rats in which endometriotic implants were successfully induced by transplanting autologous endometrial tissue to ectopic sites were used in this study. Oral gavage of Puerarin (at doses of 600, 200, or 60 mg/kg per day) or Danazol (80 mg/kg per day) started 4 weeks after implantation. Control model rats received vehicle alone. After administration for 4 weeks, the weight of the ectopic implants, estradiol concentration, as well as ER-α and Aromatase P450 (P450arom) expression in different groups of rat tissues were evaluated after treatment. The endometriotic tissue weight and serum estrogen levels were significantly lower in high, medium, low dose of Puerarin and Danazol treatment groups than that in control group (P < .05 or P < .01). Low-dose Puerarin inhibited P450arom expression and significantly reduced estrogen levels in endometriotic tissue (P < .01). Three doses of Puerarin had no adverse effects on liver, kidney, and ovary, whereas high-dose Puerarin administration caused thinner bone trabecula with distortion and breakage and Danazol administration caused mild or moderate hepatic cell damage. These data demonstrate that Puerarin was able to effectively suppress the growth and development of ectopic endometrium in the rat endometriosis model, even at low doses, suggesting it may be an effective treatment for endometriosis.

Effect of Yikun Neiyi Wan on the Expression of Aromatase P450, COX-2, and ER Related Receptor in Endometrial Cells in Vitro from Patients with Endometriosis

To investigate the effect of yikun neiyi wan (YKNYW) and gestrinone on the expression of aromatase P450 (P450arom), cyclo-oxygenase-2 (COX-2) and estrogen receptor (ER) in isolated ectopic and normal endometrial stroma cells in vitro. Digestion and serial filtration were used to isolate and culture the ectopic and eutopic endometrial cells from patients with chocolate cyst in virto. Transformation of the cell morphology was observed in a inverted microscope. The effect of YKNYW on the expression of aromatase P450, cyclo-oxygenase-2, estrogen receptor in cultured endometriosis cells were detected by immunohistochemical method. The expression levels of P450arom, COX-2 in glandular epithelium cells in vitro were decreased significantly by YKNYW compared with gestrinone (P < 0.05). ER expression in mesenchymal cells of endometriosis was increased by YKNYW in the large and medium dosage groups compared with gestrinone. The mechanism by which YKNYW alleviates endometriosis pain is possibly related to the decrease in ectopic endometrial P450 arom and COX-2 expression in glandular epithelium, contrary to gestrinone, and the increase in ER expression in mesenchymalis, consistent with gestrione in patients with endometriosis.

Mechanisms in the regulation of aromatase in developing ovary and placenta

During human gestation, the placental syncytiotrophoblast develops the capacity to synthesize large amounts of estrogen from C 19-steroids secreted by the fetal adrenals. The conversion of C 19-steroids to estrogens is catalyzed by aromatase P450 (P450arom), product of the CYP19 gene. The placenta-specific promoter of the hCYP19 gene lies ∼100,000 bp upstream of the translation initiation site in exon II. In studies using transgenic mice and transfected human trophoblast cells we have defined a 246-bp region upstream of placenta-specific exon I.1 that mediates placental cell-specific expression. Using transgenic mice, we also observed that as little as 278 bp of DNA flanking the 5′-end of ovary-specific hCYP19 exon IIa was sufficient to target ovary-specific expression. This ovary-specific promoter contains response elements that bind cAMP-response element-binding protein (CREB) and the orphan nuclear receptors SF-1 and LRH-1, which are required for cAMP-mediated stimulation of CYP19 expression in granulosa and luteal cells during the estrous cycle and pregnancy. In this article, we review our studies to define genomic regions and response elements that mediate placenta-specific expression of the hCYP19 gene. The temporal and spatial expression of LRH-1 versus SF-1 in the developing gonad during mouse embryogenesis and in the postnatal ovary also will be considered.

WT1 and DAX-1 regulate SF-1-mediated human P450arom gene expression in gonadal cells

Binding activity of steroidogenic factor-1 (SF-1) to promoters of the majority of steroidogenic genes in response to gonadotropins is a critical mechanism that regulates steroidogenesis in gonads. Thus, the modulation of SF-1 action may be essential for the differential regulation of formation of sex steroids in the ovary. Aromatase P450 (P450arom) is the rate-limiting enzyme for estrogen formation. In this study, we characterize another nuclear receptor half site in the gonadal aromatase promoter which we show to be important for aromatase regulation. We also show herein that the stimulation of P450arom promoter activity by SF-1 in ovarian granulosa, testicular Sertoli and JEG-3 choriocarcinoma cells is inhibited by two transcription factors, Wilms’ tumor suppressor gene (WT1) and dosage sensitive sex reversal adrenal hypoplasia congenita critical region on the X chromosome gene 1 (DAX-1). Given the characterized roles of these transcription factors in gonadal development and function, modulation of SF-1 action by WT1 and DAX-1 may represent an important key mechanism in steroidogenesis.

Cloning and characterization of a novel endothelial promoter of the human CYP19 (aromatase P450) gene that is up-regulated in breast cancer tissue.

Intratumoral expression of aromatase P450 (P450arom) promotes the growth of breast tumors via increased local estrogen concentration. We cloned a novel 101-bp untranslated first exon (I.7) that comprises the 5'-end of 29-54% of P450arom transcripts isolated from breast cancer tissues (n = 7). The levels of P450arom transcripts with exon I.7 were significantly increased in breast tumor tissues and adipose tissue adjacent to tumors. We identified a promoter immediately upstream of exon I.7 and mapped this to about 36 kb upstream of ATG translation start site of the CYP19 (aromatase cytochrome P450) gene. Sequence analysis of I.7 revealed a TATA-less promoter containing an initiator, two consensus GATA sites, and cis-regulatory elements found in megakaryocytes and endothelial type promoters. Luciferase activity directed by the promoter I.7 sequence (-299/+81 bp) was 4-fold greater than a minimum length promoter sequence (-35/+81 bp) in human microvascular endothelial cells (HMEC-1), but only 2-fold greater in MCF-7 breast malignant epithelial cells. There was no promoter activity in primary breast adipose fibroblasts. Site-directed mutations demonstrated that maximal basal promoter activity required two GATA motifs at -146/-141 bp and -196/-191 bp. Gel shift and deoxyribonuclease I footprinting assays demonstrated the binding of GATA-2 transcription factor but not GATA-1 to the -196/-191-bp region. Overexpression of GATA-2 in HMEC-1 cells increases promoter I.7 activity by 5-fold. In conclusion, promoter I.7 is a GATA-2-regulated endothelial promoter of the human CYP19 gene and may increase estrogen biosynthesis in vascular endothelial cells of breast cancer. The activity of this promoter may also be important for intracrine and paracrine effects of estrogen on blood vessels.

Wilms’ tumor 1 inhibits aromatase P450 expression in human cells and is downregulated in endometriosis vs. endometrium-derived stromal cells

Wilms’ tumor 1 inhibits aromatase P450 expression in human cells and is downregulated in endometriosis vs. endometrium-derived stromal cells

WT1 and DAX-1 inhibit aromatase P450 expression in human endometrial and endometriotic stromal cells.

The orphan nuclear receptor steroidogenic factor-1 (SF-1) induces the expression of Müllerian inhibiting substance (MIS) and many steroidogenic genes, including aromatase P450 (P450arom). Dosage-sensitive sex reversal adrenal hypoplasia congenita critical region on the X chromosome gene 1 (DAX-1) inhibits SF-1-mediated induction of MIS and other steroidogenic genes, whereas Wilms' tumor suppressor gene (WT1) augments SF-1-mediated MIS expression. The effects of WT1 on steroidogenesis or P450arom expression have not been explored to date. In human endometriotic stromal cells, extremely high levels of P450arom mRNA and enzyme activity are present. Prostaglandin E(2) stimulates cAMP formation, SF-1 binding activity, P450arom mRNA levels, and estrogen synthesis in endometriotic stromal cells. Stromal cells of eutopic endometrium from disease-free women, on other hand, do not contain readily detectable levels of P450arom mRNA. Thus, we evaluated herein the possible roles of WT1 and DAX-1 in cAMP/SF-1-mediated regulation of P450arom expression in endometriotic and endometrial stromal cells. We also determined the cellular distribution and levels of these transcription factors in pathological endometriotic vs. normal eutopic endometrial tissues by immunohistochemistry to understand their in vivo roles. In vitro transcriptional regulation studies showed that both WT1 and DAX-1 inhibited cAMP and/or SF-1-induced P450arom promoter activity in a dose-dependent fashion in cultured human endometriotic and endometrial stromal cells. Site-directed disruption of the SF-1 binding site (-136/-124 bp) in the P450arom promoter abolished basal or cAMP/SF-1-induced promoter activity in the presence or absence of WT1 or DAX-1. Immunohistochemistry and H-scoring showed that DAX-1 was ubiquitously present in epithelial and stromal cells of both tissues. WT1, on the other hand, was preferentially expressed in stromal (vs. epithelial) cells. Moreover, WT1 levels in endometriotic stromal cells are significantly down-regulated compared with normal endometrial stromal cells. In summary, WT1 or DAX-1 inhibits cAMP-SF-1 pathway-dependent P450arom expression in cultured human endometriotic and endometrial stromal cells. In vivo down-regulation of WT1 in endometriotic stromal cells (vs. normal endometrial stromal cells) may in part be responsible for aberrantly increased P450arom expression and estrogen formation in this pathological tissue.

Genetic or Enzymatic Disruption of Aromatase Inhibits the Growth of Ectopic Uterine Tissue

Aromatase P450 (P450arom) is the key enzyme for the biosynthesis of estrogen that is essential for the growth of human endometriosis, a pathology characterized by endometrium-like tissue on the peritoneal surfaces of abdominal organs manifest by pelvic pain and infertility. Surgically transplanted autologous uterine tissue to ectopic sites on the peritoneum in mice has been used as an animal model to study endometriosis. Using this mouse model, we evaluated the roles of the P450arom gene and aromatase enzyme activity in the growth of endometriosis represented by ectopic uterine tissues in mice. Endometriosis was induced surgically in the following groups of mice: 1) untreated transgenic mice with disrupted P450arom gene (ArKO); 2) ArKO mice treated with systemic estrogen; 3) untreated wild-type (WT) mice; 4) WT mice treated with estrogen; 5) WT mice treated with the aromatase inhibitor, letrozole; and 6) WT mice treated with letrozole and estrogen. Each group contained eight mice; +/+ littermates of ArKO mice were used as WT controls. Treatment with estrogen increased the size of ectopic uterine tissues in ArKO and WT mice significantly. The ectopic uterine lesions in untreated and estrogen-treated ArKO mice were strikingly smaller than those in untreated and estrogen-treated WT controls, respectively. Systemic treatment of WT mice with letrozole significantly decreased the lesion size in a dose-dependent manner. The addition of estrogen to letrozole treatment increased the ectopic lesion size, although these lesions were significantly smaller than those in mice treated with estrogen only. As tissue controls, the effects of these conditions on normally located (eutopic) uterine tissue were evaluated. The effects of disruption of the P450arom gene and treatments with letrozole and estrogen seemed to be more profound on ectopic tissues, suggesting that ectopic tissues might be more sensitive to estrogen for growth. We conclude that both an intact P450arom gene and the presence of aromatase enzyme activity are essential for the growth of ectopic uterine tissue in a mouse model of endometriosis.

Regulation of aromatase P450 expression in endometriotic and endometrial stromal cells by CCAAT/enhancer binding proteins (C/EBPs): decreased C/EBPbeta in endometriosis is associated with overexpression of aromatase.

In human endometriotic stromal cells, markedly high levels of aromatase P450 (P450arom) mRNA and promoter II activity are present and can be vigorously stimulated by PGE(2) via a cAMP-dependent pathway to give rise to physiologically significant estrogen biosynthesis. Stromal cells of eutopic endometrium, on the other hand, do not express sufficient levels of P450arom for detectable enzyme activity. Because P450arom is up-regulated in the ovaries of CCAAT/enhancer binding protein (C/EBP) beta knockout mice and activation of the ovarian-type P450arom promoter (II) is responsible for aberrant P450arom expression in endometriosis, we sought here to evaluate the possible roles of C/EBP isoforms in the regulation of P450arom expression in endometriotic vs. eutopic endometrial stromal cells. We previously found that the -517-bp flanking region of promoter II contained the critical cis-acting elements for baseline and cAMP (analog)-induced activity. In this study, we disrupted several potential sequences and found that mutations of a -211/-197-bp cAMP-response element (CRE) and a -317/-304-bp C/EBP binding site abolished both baseline and cAMP-induced promoter II activity. Ectopic expression of C/EBPalpha increased both baseline and cAMP-dependent promoter II activity significantly in endometriotic cells, whereas ectopic expression of C/EBPbeta or C/EBPdelta abolished promoter II activity in both untreated and cAMP-treated endometriotic stromal cells. Comparable changes in promoter II activity were observed using endometrial stromal cells, which showed, however, seemingly diminished levels of baseline and cAMP-induced promoter II activity in comparison with endometriotic cells. EMSA using a probe containing the critical -317/-304-bp C/EBP site upstream of promoter II demonstrated a distinct DNA-protein complex in endometriotic, but not in endometrial stromal cells. This specific complex, however, could not be altered using antibodies against C/EBPalpha, -beta, or -delta. Because CRE is another potential DNA motif that can bind C/EBP isoforms, we next used EMSA using a probe containing the -211/-197-bp CRE and demonstrated that specific DNA-protein complexes contained C/EBPalpha but not C/EBPbeta or C/EBPdelta in endometriotic stromal cells. In contrast, C/EBPbeta and C/EBPdelta but not C/EBPalpha were detected in DNA-protein complexes using nuclear extracts from endometrial stromal cells. Western blotting and immunohistochemistry demonstrated expression of C/EBPalpha, -beta, and -delta in human endometriotic and endometrial stroma and epithelium. Intriguingly, C/EBPbeta was expressed at increased levels in stromal cells of human eutopic endometrium compared with simultaneously biopsied endometriotic tissues. We conclude that both -317/-304 and -211/-197-bp elements in promoter II are critical for the robust cAMP-dependent induction in endometriosis. C/EBPalpha up-regulates, whereas C/EBPbeta and C/EBPdelta inhibit P450arom promoter activity via binding primarily to the -211/-197-bp CRE under in vitro conditions. In vivo down-regulation of C/EBPbeta in endometriotic stromal cells and its up-regulation in endometrial stromal cells may in part account for the induction of P450arom expression in endometriosis and its inhibition in endometrium.

Regulation of aromatase P450 promoter (II) activity in endometriotic stromal cells by CCAAT/enhancer binding proteins (C/EBPs).

Objective: Aromatase P450 (P450arom), the key enzyme in the biosynthesis of estrogen, is expressed aberrantly in endometriosis tissue. In human ectopic endometriotic stromal cells, markedly high levels of P450arom mRNA and activity are present and can be vigorously stimulated by dibutyryl (Bt2) cAMP or PGE2 to give rise to physiologically significant estrogen biosynthesis. On the other hand, eutopic endometrial tissue or stomal cells lack P450arom expression. We sought to determine herein the molecular basis for the presence of P450arom expression in endometriosis and its absence in eutopic endometrium.

Treatment with the aromatase inhibitor letrozole significantly reduced the size of ectopic uterine implants.

Objective: Surgically transplanted autologous uterine tissue to the peritoneum in rodents has been used as a model to study endometriosis. Estrogen was found to play an important role in the pathogenesis of endometriosis. Aromatase P450 (P450arom) is the key enzyme for the synthesis of estrogen. Significant levels of P450arom mRNA, protein and enzyme activity have been demonstrated in endometriotic tissue. Moreover, an unusually severe case of persistent postmenopausal endometriosis resolved after treatment with an aromatase inhibitor. The objective of this study is to determine the effects of an aromatase inhibitor on the growth of ectopic and eutopic uterine tissues in mice.

Localization of androgen receptor and cytochrome P450 aromatase in the follicle and corpus luteum of the porcine ovary

The following study was undertaken to localize androgen receptors (AR) and aromatase cytochrome P450 (P450arom) in porcine ovarian tissue because ovarian androgens may act locally to modulate follicular and luteal function in various species. Androgen receptor was detected immunohistochemically in granulosa and theca cells of preantral as well as in growing antral follicles. The most intensive staining was observed in the antral granulosa layer. Luteinizing granulosa cells of preovulatory follicles, and luteal cells from the early and midluteal phases stained weakly for the androgen receptor. Fully regressed corpora lutea in the early follicular phase of the next cycle did not stain for androgen receptor. In contrast, granulosa cells were very weakly stained for aromatase in early stages of follicular development. The P450arom was maximally expressed with the same intensity in mural and antral layers in large ovulatory follicles. Corpora lutea from the early luteal phase showed positive staining, whereas those from midluteal phase did not stain for aromatase, some cells of regressed corpora lutea unexpectedly exhibited aromatase staining.

Hypoxia Prevents Induction of Aromatase Expression in Human Trophoblast Cells in Culture: Potential Inhibitory Role of the Hypoxia-Inducible Transcription Factor Mash-2 (Mammalian Achaete-Scute Homologous Protein-2)

The human placenta has a remarkable capacity to aromatize C19-steroids, produced by the fetal adrenals, to estrogens. This reaction is catalyzed by aromatase P450 (P450arom), encoded by the CYP19 gene. In placenta, CYP19 gene expression is restricted to the syncytiotrophoblast layer. Cytotrophoblasts isolated from human placenta, when placed in monolayer culture in 20% O2, spontaneously fuse to form syncytiotrophoblast. These morphological changes are associated with a marked induction of aromatase activity and CYP19 gene expression. When cytotrophoblasts are cultured in an atmosphere containing 2% O2, they manifest increased rates of DNA synthesis and fail to fuse and form syncytiotrophoblast. The objective of the present study was to utilize cytotrophoblasts isolated from midterm human placenta to analyze the effects of O2 on CYP19 gene expression and the molecular mechanisms that mediate these effects. We observed that when trophoblast cells were maintained in 2% O2, there was only a modest induction of CYP19 expression as a function of time in culture, and aromatase activity was barely detectable. However, when cytotrophoblasts that had been maintained in 2% O2 for 3 days were placed in a 20% O2 environment, there was a rapid onset of cell fusion and induction of P450arom mRNA and aromatase activity. In addition, mRNAs for the helix-loop-helix factors Mash-2 (mammalian achaete-scute homologous protein-2) and Id1 (inhibitor of differentiation 1) were readily detectable in freshly isolated cytotrophoblasts and were markedly decreased upon differentiation to syncytiotrophoblast in 20% O2. By contrast, when cytotrophoblasts were cultured in 2% O2, mRNA levels for Mash-2 and Id1 remained elevated. Interestingly, overexpression of Mash-2 in primary cultures of human trophoblast cells markedly inhibited cell fusion and the spontaneous induction of P450arom mRNA levels and caused a marked decrease in expression of co-transfected fusion gene constructs containing either 125, 201, 246, or 501 bp of DNA flanking the 5'-end of the placenta-specific exon (exon I.1) of the human CYP19 gene linked to the human GH (hGH) structural gene, as reporter. In studies using BeWo, a human choriocarcinoma cell line, overexpression of Mash-2 also inhibited expression of cotransfected CYP19I.1:hGH fusion gene constructs. The findings that Mash-2 had no effect on the expression of a CYP19I.1(-42):hGH fusion gene in primary cultures of human trophoblast and BeWo cells suggest that Mash-2 exerts its inhibitory effects directly or indirectly though CYP19I.1 5'-flanking sequences that lie between -42 and -125 bp. By contrast, neither Id1 nor Id2 had an effect on CYP19I. 1 promoter activity in the transfected BeWo cells. These findings suggest that Mash-2 may serve as a hypoxia-induced transcription factor that prevents differentiation to syncytiotrophoblast and aromatase induction in human trophoblast cultured under low O2 conditions.

Stimulation of aromatase P450 promoter (II) activity in endometriosis and its inhibition in endometrium are regulated by competitive binding of steroidogenic factor-1 and chicken ovalbumin upstream promoter transcription factor to the same cis-acting element.

In stromal cells of endometriosis, marked levels of aromatase P450 (P450arom) mRNA and activity are present and can be vigorously stimulated by (Bu)2cAMP or PGE2 to give rise to physiologically significant estrogen biosynthesis. Since eutopic endometrial tissue or stromal cells lack P450arom expression, we studied the molecular basis for differential P450arom expression in endometriosis and eutopic endometrium. First, we demonstrated by rapid amplification of cDNA 5'-ends that P450arom expression in pelvic endometriotic lesions is regulated almost exclusively via the alternative promoter II. Then, luciferase reporter plasmids containing deletion mutations of the 5'-flanking region of promoter II were transfected into endometriotic stromal cells. We identified two critical regulatory regions for cAMP induction of promoter II activity: 1) a-214/-100 bp proximal region responsible for a 3.7-fold induction, and 2) a -517/ -214 distal region responsible for potentiation of cAMP response up to 13-fold. In the -214/-100 region, we studied eutopic endometrial and endometriotic nuclear protein binding to a nuclear receptor half-site (NRHS, AGGTCA) and an imperfect cAMP response element (TGCACGTCA). Using electrophoretic mobility shift assay, cAMP response element-binding activity in nuclear proteins from both endometriotic and eutopic endometrial cells gave rise to formation of identical DNA-protein complexes. The NRHS probe, on the other hand, formed a distinct complex with nuclear proteins from endometriotic cells, which migrated at a much faster rate compared with the complex formed with nuclear proteins from eutopic endometrial cells. Employing recombinant proteins and antibodies against steroidogenic factor-1 (SF-1) and chicken ovalbumin upstream promoter transcription factor (COUP-TF), we demonstrated that COUP-TF but not SF-1 bound to NRHS in eutopic endometrial cells, whereas SF-1 was the primary NRHS-binding protein in endometriotic cells. In fact, COUP-TF transcripts were present in both eutopic endometrial (n = 12) and endometriotic tissues (n = 8), whereas SF-1 transcripts were detected in all endometriotic tissues (n = 12), but in only 3 of 15 eutopic endometrial tissues. Moreover, we demonstrated a dose-dependent direct competition between SF-1 and COUP-TF for occupancy of the NRHS, to which SF-1 bound with a higher affinity. Finally, overexpression of SF-1 in eutopic endometrial and endometriotic cells strikingly potentiated baseline and cAMP-induced activities of -517 promoter II construct, whereas overexpression of COUP-TF almost completely abolished these activities. In conclusion, COUP-TF might be one of the factors responsible for the inhibition of P450arom expression in eutopic endometrial stromal cells, which lack SF-1 expression in the majority (80%) of the samples; in contrast, aberrant SF-1 expression in endometriotic stromal cells can override this inhibition by competing for the same DNA-binding site, which is likely to account for high levels of baseline and cAMP-induced aromatase activity.

Maintenance of oestradiol production and expression of cytochrome P450 aromatase enzyme mRNA in long-term serum-free cultures of pig granulosa cells.

Studies were carried out to investigate the conditions required for maintenance of aromatase activity and expression in long-term cultures of pig granulosa cells. Cells from large (> 2 mm) and small (< or = 2 mm) follicles were cultured at 37 degrees C with 5% CO2 in McCoys 5a medium supplemented with 0.1% (w/v) BSA, testosterone (100 micrograms l-1), insulin (10 micrograms l-1) and long R3 insulin-like growth factor I (IGF-I) (100 micrograms l-1). Cells were cultured with five concentrations of USDA pFSH-I-2 (0-100 micrograms l-1) for 48, 96 or 144 h with or without fetal calf serum (FCS). The number of cells and oestradiol, progesterone and inhibin production were measured. In marked contrast to oestradiol production from cells cultured in plates precoated with FCS, 1 microgram FSH l-1 was optimal for the maintenance of high oestradiol production by granulosa cells from large follicles after 144 h of serum-free culture. Culture with FCS promoted cell proliferation, reduced oestradiol production, and supported FSH-dependent (P < 0.01) increased progesterone and inhibin production indicating cellular luteinization. Northern blot analysis of total RNA from cells cultured with 1 microgram FSH l-1 detected 2.5 and 1.8 kb transcripts encoding aromatase cytochrome P450 (P450arom) and cholesterol side-chain cleavage cytochrome P450 (P450scc), respectively. Transcript expression was hormone sensitive, irrespective of the presence of FCS. High concentrations of FSH (100 micrograms l-1) stimulated expression of P450scc, but inhibited P450arom expression as the cells luteinized after 144 h of culture. This serum-free system, which maintains the aromatase enzyme complex, is fundamental if physiologically relevant observations are to be made of the mechanisms regulating follicle hierarchy development from long-term cultures of pig cells.