Prematurely ruptured dominant follicles often retain competent oocytes in infertile women.

Ovulation consists of a follicle's rupture and subsequent oocyte extrusion, although there is a paucity of evidence regarding whether every follicle's rupture is associated with extrusion of its oocyte. We examined this issue in a large-scale window-of-opportunity study by attempting aspiration of single dominant follicles that were found to have ruptured before a scheduled oocyte retrieval during in vitro fertilisation and embryo transfer treatment of infertile women. We were able to aspirate 587 of 1,071 ultrasonographically confirmed post-rupture dominant follicles from 1,071 women (i.e. one dominant follicle per woman) and retrieved 225 oocytes (oocyte recovery ratio: 43.4% of aspirated follicles), which yielded 28 live births (live birth ratio: 11.0% of retrieved oocytes). Interestingly, the live birth ratio for post-rupture dominant follicles was not statistically different from that achieved using regular pre-rupture aspiration of dominant follicles (1,085/8,977, 12.1%). These findings suggest that oocyte extrusion frequently does not occur after follicle rupture in infertile women undergoing in vitro fertilisation treatment, although the oocyte retained in the follicle can remain competent for use during that treatment.

Metformin alters DNA methylation genome-wide via the H19/SAHH axis.

The molecular mechanisms underlying the antineoplastic properties of metformin, a first-line drug for type 2 diabetes, remain elusive. Here we report that metformin induces genome-wide alterations in DNA methylation by modulating the activity of S-adenosylhomocysteine hydrolase (SAHH). Exposing cancer cells to metformin leads to hypermethylation of tumor-promoting pathway genes and concomitant inhibition of cell proliferation. Metformin acts by upregulating microRNA let-7 through AMPK activation, leading to degradation of H19 long noncoding RNA, which normally binds to and inactivates SAHH. H19 knockdown activates SAHH, enabling DNA methyltransferase 3B to methylate a subset of genes. This metformin-induced H19 repression and alteration of gene methylation are recapitulated in endometrial cancer tissue samples obtained from patients treated with antidiabetic doses of metformin. Our findings unveil a novel mechanism of action for the drug metformin with implications for the molecular basis of epigenetic dysregulation in cancer. This novel mechanism of action also may be occurring in normal cells.

Phase II study of medroxyprogesterone acetate plus metformin as a fertility-sparing treatment for atypical endometrial hyperplasia and endometrial cancer.

BACKGROUND: Metformin, widely used in the treatment of type 2 diabetes mellitus, reduces the risk of cancer and relapse after treatment. Fertility-sparing treatment for endometrial cancer (EC) with progestin is associated with a high chance of disease regression, and the high relapse rate continues to be a problem. We assessed the efficacy of metformin in preventing recurrence after medroxyprogesterone acetate (MPA) as fertility-sparing treatment for atypical endometrial hyperplasia (AEH) and EC. PATIENTS AND METHODS: This phase II study enrolled 17 patients with AEH and 19 patients with EC limited to the endometrium (age, 20-40 years). MPA (400 mg/day) and metformin (750-2250 mg/day) were administered for 24-36 weeks to achieve a complete response (CR). Metformin was administered until conception, even after MPA discontinuation. The primary end point was relapse-free survival (RFS) after remission. We analyzed all efficacy end points in the full analysis set. RESULTS: The body mass index was ≥25 kg/m(2) in 27 patients (mean, 31 kg/m(2); range, 19-51 kg/m(2)), and the homeostasis model assessment for insulin resistance index was ≥2.5 in 24 patients (mean, 4.7; range, 0.7-21). Two patients showed progression at 12 weeks [6%; 95% confidence interval (CI) 2-18]. At 36 weeks, 29 (81%; 95% CI 65-90) patients achieved CR, and 5 (14%; 95% CI 6-29) patients achieved partial response. During a median follow-up of 38 months (range, 9-66 months) after remission, relapse was confirmed in three of the patients who had achieved CR (relapse rate, 10%). The 3-year estimated RFS rate was 89%. No patients experienced severe toxicity. CONCLUSIONS: Metformin inhibited disease relapse after MPA therapy. The combination of metformin and MPA in EC treatment should be studied further. TRIAL REGISTRATION NUMBER: UMIN 000002210.

The application of a newly developed linear stapler preloaded with tissue reinforcement for distal pancreatectomy in the management of ovarian cancer.

Advanced ovarian cancer may extend into the spleen, and even the pancreatic tail, in which a splenectomy associated with distal pancreatectomy is crucial for optimal cytoreduction. A new linear stapler preloaded with tissue reinforcement is currently introduced. We herein report the first three cases of successful application of this device for distal pancreatectomy performed during cytoreductive surgery for ovarian cancer.

mRNA induces RANTES production in trophoblast cells via TLR3 only when delivered intracellularly using lipid membrane encapsulation.

BACKGROUND: Trophoblasts express Toll-like receptor 3 (TLR3). The artificial TLR3 ligand, PolyI:C, induces an inflammatory response in trophoblasts but an endogenous ligand has not been identified. Notably, inflammatory disorders of pregnancy are associated with increased circulating placenta-derived mRNA. Endogenous degraded, uncapped mRNA is recognized by TLR3 in other cell lines. OBJECTIVE: We tested the hypothesis that plasma-derived mRNA induces an inflammatory response in a trophoblast cell line via TLR3. METHODS: Experiments were performed in the human first trimester extravillous trophoblast cell line HTR-8/SV neo. Plasma-derived mRNA was amplified using modified template switching and final in vitro transcription. We compared free mRNA (which favors cell surface interaction) to liposomally encapsulated mRNA (which favors intracellular mRNA delivery). We tested for the specific requirement of TLR3 signaling using siRNA. We tested for involvement of the canonical signaling pathway downstream of TLR3 by measuring NF-κB and IFN regulatory factor transcriptional activity using firefly-luciferase constructs. RESULTS: Free mRNA did not induce RANTES production. In contrast, liposomal mRNA resulted in marked induction of RANTES production (non-stimulated control 3.4 ± 0.6 pg/mL, liposomal mRNA 169.7 ± 26.2 pg/mL, p < 0.001), and this RANTES production was abolished by siRNA for TLR3. Downstream of TLR3, liposomal mRNA-induced dose-response NF-κB and IFN regulatory factor transcriptional activity, and IFN beta production. CONCLUSION: Plasma-derived 5' uncapped mRNA delivered intracellularly signals to induce NF-κB activation and increase RANTES production via TLR3.

The risk of post-molar gestational trophoblastic neoplasia is higher in heterozygous than in homozygous complete hydatidiform moles.

BACKGROUND: Complete hydatidiform mole (CHM) is a high-risk pregnancy for gestational trophoblastic neoplasia (GTN). Patients with CHM have a 10-30% chance of trophoblastic sequelae. CHM includes androgenic homozygous (monospermic) and androgenic heterozygous (dispermic) moles. It is controversial whether the risk of GTN is higher with heterozygous than with homozygous CHM. A prospective cohort study was conducted to assess risk of GTN in homozygous and heterozygous CHM using short tandem repeat (STR) polymorphisms, and a meta-analysis of previous reports. METHODS: Twenty-eight consecutive molar pregnancies were evacuated and followed by regular hCG measurements to detect GTN. Persistent GTN was diagnosed according to the International Federation of Gynecology and Obstetrics 2000 system. Cytogenesis of the mole was determined by STR polymorphisms of molar tissue and parental blood. A meta-analysis of the GTN rate from previous reports was conducted using Mantel-Haenszel methods. RESULTS: Of 28 molar pregnancies, 24 were homozygous and three were heterozygous CHM. The remaining mole was diandric triploidy (a partial hydatidiform mole). Of the 24 homozygous CHMs, six (25%) cases developed GTN and received chemotherapy. Meanwhile, all three cases (100%) of heterozygous mole developed GTN and needed chemotherapy. The GTN risk was higher in heterozygous (P = 0.029, Fisher's exact test) than homozygous moles. A systematic review revealed only five previous reports (with more than 15 cytogenetically diagnosed cases), and the pooled relative risk of persistent GTN for heterozygous mole was not significant (odds ratio, 2.0; 95% confidence interval, 0.98-4.07). CONCLUSIONS: Heterozygous CHM had a higher risk for GTN than homozygous CHM.

Serum YKL-40 as a marker for cervical adenocarcinoma.

BACKGROUND: The current study examined the clinical usefulness of YKL-40 in detection and prognosis of uterine cervical cancer. PATIENTS AND METHODS: Serum levels of YKL-40, cancer antigen 125 (CA 125), carbohydrate antigen 19-9 (CA19-9), and squamous cell carcinoma (SCC) antigen were determined by enzyme-linked immunosorbent assay in women with benign gynecologic disease (n=24), cervical malignancy (SCC, n=104; adenocarcinoma, n=37), and age-matched healthy controls (n=45). Immunohistochemical analysis for local YKL-40 expression was carried out on 28 adenocarcinomas. RESULTS: Receiver operating characteristic curve analysis showed that YKL-40 [area under the curve (AUC)=0.882] was significantly better at discriminating adenocarcinoma from healthy control than SCC antigen, CA 125, and CA19-9. For SCC, YKL-40 (AUC=0.898) carried out similarly to SCC antigen and was better than CA 125 and CA19-9. Using a cut-off YKL-40 value of 92.2 ng/ml, sensitivity of YKL-40 in stage I adenocarcinoma (68%) was higher than that of the other three markers (11%-21%). Tumor-associated macrophages showed immunoreactivity for YKL-40 in 2 of 28 adenocarcinoma tissue samples, but adenocarcinoma cells themselves were nonimmunoreactive in all samples. Multivariate Cox regression analysis revealed that elevated pretreatment YKL-40 levels predicted unfavorable prognosis, independent of International Federation of Gynecology and Obstetrics stage and age at diagnosis. CONCLUSIONS: Pretreatment serum YKL-40 level is a possible prognosticator of cervical adenocarcinoma.

ADAMTS-1 is involved in normal follicular development, ovulatory process and organization of the medullary vascular network in the ovary.

To clarify the role of disintegrin-like and metalloproteinase with thrombospondin type I motifs-1 (ADAMTS-1) in ovarian function, we examined abnormalities in ovulatory processes, folliculogenesis and the vascular system of ADAMTS-1 null ovaries. First, when immature female mice were treated with pregnant mare serum gonadotropin (PMSG)/human chorionic gonadotropin (hCG), the number of ovulated oocytes was markedly decreased in ADAMTS-1 null mice in comparison to ADAMTS-1 (+/-) controls. The proportion of anovulated follicles to total mature follicles was significantly higher in ADAMTS-1 null females when compared with controls. The numbers of growing follicles at each stage were counted. The number of follicles at type 5b (late preantral) and later stages was markedly reduced in ADAMTS-1 null mice, irrespective of gonadotropin treatment (no gonadotropins, PMSG alone or PMSG/hCG). These data demonstrate that impairment of ovarian function to ovulate oocytes in ADAMTS-1 null mice occurs at two different levels: in the development of growing follicles and ovulatory processes. Furthermore, ADAMTS-1 null ovaries included a number of unusual atretic follicles that showed no sign of oocyte degeneration but lost the surrounding granulosa cell layers and were considered to be derived from type 4 or 5a follicles. These results suggest that ADAMTS-1 is important for follicular development beyond the type 4 and/or 5a and for maintaining normal granulosa cell layers in follicles. Finally, the number of large blood vessels in the medullar zone was significantly decreased in ADAMTS-1 null mice ovaries, suggesting that ADAMTS-1 is also involved in the organization of the medullary vascular network.

Temporary amniotic membrane patching for acute chemical burns.

PURPOSE: To describe the surgical technique, and its usefulness, of temporary amniotic membrane patching (AMP) in the acute phase of ocular chemical injury. METHODS: Temporary AMP with modification in suture placement was performed on five eyes of five consecutive patients inflicted with acute chemical injury having a greater than grade II injury by the Roper-Hall classification. RESULTS: All patients reported herein presented with a large epithelial defect on the cornea and conjunctiva. Case 3 was classified as grade III while the other four cases were classified as grade II. The causative chemical agents were anhydrous acetic acid in Case 1, calcium oxide in Case 2, sodium hydroxide in Case 3, sodium silicate in Case 4, and sulphuric acid in Case 5. All cases experienced rapid relief of pain after AMP. Epithelialization of the cornea with improvement of visual acuity was observed in all cases when the amniotic membrane was removed within 2 weeks after surgery. During the mean follow-up of 19.6 months, the ocular surface remained stable and no cicatricial complications were noted. CONCLUSIONS: These results suggest that immediate AMP is quite useful for managing moderately severe acute ocular chemical injury by facilitating rapid epithelialization and pain relief, and securing ocular surface integrity.

Inhibition of in situ expression of aromatase P450 in leiomyoma of the uterus by leuprorelin acetate.

We have shown that in situ estrogen synthesized in leiomyoma of the uterus plays a possible role in the promotion of leiomyoma cell growth via an autocrine/paracrine mechanism. In the present study, we demonstrated that leuprorelin acetate, a GnRH agonist widely used for treatment of uterine leiomyoma by down-regulation of pituitary-ovarian function, suppressed the expression of aromatase P450 (an estrogen synthetase) in leiomyoma cells. Given the role of in situ estrogen in leiomyoma cell growth, the inhibition of in situ estrogen synthesis may play a role in GnRH agonist-induced rapid regression of leiomyomas. Quantitative RT-PCR revealed that in women receiving no medication uterine leiomyomas express aromatase P450 mRNA at levels 20 times higher than that in the surrounding myometrium. Leuprorelin acetate treatment (1.88 mg every 4 wk, sc injection) for 12-24 wk reduced the expression of aromatase P450 mRNA in leiomyoma tissue as well as in the myometrium, to approximately one tenth of that in the myometrium of untreated women. Suppression of aromatase P450 expression was also demonstrated by Western blot analysis and aromatase activity assay of microsomal fractions prepared from leiomyomas. On the other hand, no differences in the levels of activity and mRNA of aromatase P450 were observed between leiomyoma cells obtained from women treated with and without leuprorelin acetate injections when cells were cultured ex vivo and stimulated by various combinations of stimulants such as dexamethasone + IL-1beta. The addition of various concentrations of E2 did not affect the aromatase activity of leiomyoma cells, suggesting that deprivation of circulating (ovarian) estrogen is not a cause of decreased expression of aromatase during leuprorelin acetate therapy. On the other hand, 8-d treatment with leuprorelin acetate (100 nmol/liter) reduced dexamethasone + IL-1beta-induced activity and a mRNA level of aromatase by 28% and 42%, respectively. These results indicated that leuprorelin acetate inhibits the expression of aromatase P450 in leiomyoma cells, which contributes to the rapid regression of leiomyoma during leuprorelin acetate therapy.

Leak-proof puncture of ovarian cysts: instant mounting of plastic bag using cyanoacrylate adhesive.

BACKGROUND: Reliable leak-proof aspiration of cyst contents is required for treatment of large ovarian cysts by minilaparotomy. TECHNIQUE: Through a small abdominal wound a transparent plastic bag was instantly mounted onto the cyst surface using an ethyl-2-cyanoacrylate adhesive. A 1-2-cm-wide cut was made in the consolidated cyst wall through the inside of the bag and the contents directly aspirated. The fluid was trapped inside the bag without leaking into the abdominal cavity. This method can also be applied to relatively small cysts by holding the cyst just beneath the wound. EXPERIENCE: We used this method in 30 patients with unilateral ovarian cysts and in one patient with an ovarian cyst associated with an ipsilateral paraovarian cyst. All patients were successfully treated without spillage, although in one case a large mucinous ovarian cyst ruptured before surgery. CONCLUSION: Minilaparotomy using the instant adhesive is cost-effective, safe, reliable, and easily implemented. This procedure is also applicable to relatively small cysts and is a viable alternative to laparoscopic surgery for treatment of dermoid cysts showing considerable calcification.

Regulation of aromatase activity in bone-derived cells: possible role of mitogen-activated protein kinase.

Fetal human osteoblast-like cells and the THP-1 cell line that differentiates into macrophage/osteoblast-like cells in the presence of Vitamin D3 and which possesses high aromatase activity, constitute a useful model with which to study the regulation of aromatase in bone. We showed that dexamethasone (DEX)-induced aromatase activity in the THP-1 cell line is completely suppressed by forskolin and by dibutyryl cAMP. We therefore investigated the contribution of mitogen-activated protein kinase (MAPK) to the regulation of aromatase, because cAMP inhibits MAPK in many cells. We examined the role of MAPK on aromatase activity using PD98059, a selective inhibitor of MEK-1. PD98059 (100 microM) reduced DEX+interleukin (IL)-1beta-induced aromatase activity in human osteoblast-like cells by more than 90%, whereas 50% of the aromatase mRNA concentration was retained compared with the control incubated with DEX+IL-1beta. PD98059 (50 microM) reduced the activity of aromatase in THP-1 cells by 80% without significantly affecting the mRNA level. These results indicated that MAPK plays an important role in aromatase activation at the post-transcriptional level.

In situ estrogen synthesized by aromatase P450 in uterine leiomyoma cells promotes cell growth probably via an autocrine/intracrine mechanism.

In the present study we characterized in detail the expression of aromatase P450 in leiomyomas to determine the role of in situ estrogen in the growth advantage of leiomyomas. The levels of aromatase P450 transcripts were determined by quantitative RT-PCR to be significantly higher in leiomyomas than in corresponding myometrium. The overexpression of aromatase P450 in leiomyomas was also confirmed by Western blot analysis. The estimated size of immunoreactive aromatase was 58 kDa, similar to that in placenta. To identify a cell type that express aromatase P450 in leiomyomas, histological specimens were stained for aromatase P450 using a polyclonal antibody. Strong immunoreactivity was detected in the cytoplasm of leiomyoma cells, whereas surrounding normal myometrium displayed weak or negative staining. Smooth muscle-like cells in culture obtained from leiomyomas, positive for actin D fiber, possessed immunoreactive granules of aromatase in the cytoplasm. Conversion of androgen to estrogen was effectively stimulated by phorbol myristate acetate and dexamethasone plus interleukin-1beta and was completely abolished by selective inhibitors of aromatase P450 (fadrozole and TZA-2209), but not by inhibitors of 5alpha-reductase (finasteride and flutamide). The apparent Km of androstenedione was 3 nM in the presence of dexamethasone and interleukin-1beta, corresponding to the plasma concentration of androstenedione in women of reproductive age. To determine whether endogenous aromatase P450 plays a role in the growth promotion of leiomyoma cells, we evaluated the cell growth of smooth muscle-like cells treated with various concentrations of estrogen and androgen using a WST-1 assay. Treatment with testosterone (10(-8) and 10(-7) M) and androstenedione (10(-8) and 10(-7) M) stimulated the growth of smooth muscle-like cells obtained from leiomyomas to the same extent as estradiol (10(-10)-10(-7) M), whereas dihydrotestosterone (10(-11)-10(-8) M) did not. The stimulatory effect of testosterone on cell growth was again abolished by cotreatment with fadrozole. The level of estradiol in the medium of testosterone (10(-8) M)-treated smooth muscle-like cells was 10(-11) M, which was 1 order lower than the minimum concentration of estradiol necessary to promote cell growth (10(-10) M). This indicates that estradiol synthesized in leiomyomas promotes their growth via an autocrine/intracrine mechanism. We conclude that myometrial cells of leiomyomas overexpress aromatase P450 and are able to synthesize sufficient estrogen to accelerate their own cell growth. Overexpression of aromatase P450 may play a role in the growth advantage of leiomyoma tissue over surrounding myometrium via an autocrine/intracrine mechanism.

TGF-beta1 stimulates expression of the aromatase (CYP19) gene in human osteoblast-like cells and THP-1 cells.

Recent evidence has shown that bone is not only a target of estrogen action but also a source of local estrogen production. Bone cells such as osteoblasts express aromatase (P450arom) and the expression of P450arom in osteoblasts is positively regulated in a tissue specific fashion, as in the case of other tissues which express P450arom. To clarify the physiological factors regulating expression of P450arom in bone, we tested TGF-beta1 using osteoblast-like cells obtained from human fetuses as well as THP-1 cells. TGF-beta1 increased IL-1beta+DEX- induced aromatase activity in osteoblast-like cells, while it inhibited activity in skin fibroblasts. Similar enhancement of aromatase activity by TGF-beta1 was found in DEX-stimulated THP-1 cells and this cell line was used for further experiments. In THP-1 cells, TGF-beta1 enhanced DEX-induced aromatase activity almost linearly by 12 h and thereafter. Increased levels of P450arom transcripts were also demonstrated by RT-PCR at 3 h of TGF-beta1 treatment and thereafter. Cyclohexamide abolished enhancement of activity but did not inhibit the accumulation of P450arom transcripts induced by TGF-beta1. Increase in P450arom expression by TGF-beta1 was attributable to expression driven by promoter I.4. TGF-beta1 did not change the half life of P450arom transcripts. To identify the cis-acting elements responsible for TGF-beta1 action on aromatase expression, transient transfection assays were performed using a series of deletion constructs for promoter I.4 (P450-I.4/Luc). Two constructs (-410/+14 and-340/+14) that contain a functional glucocorticoid response element (GRE) and downstream sequence showed significant increase of luciferase activity in response to TGF-beta1. Deletion and mutation of the GRE in P450-I.4/Luc (-340/+14) abolished the TGF-beta1. The luciferase activity of a (GRE)(1)-SV40/Luc construct was also stimulated by TGF-beta1. These results indicate that TGF-beta1 increases the expression of P450arom at the level of transcription through promoter I.4, at least in part via an enhancement of transactivation activity of the GR in THP-1 cells. TGF-beta1 is suggested to be one of the physiological up-regulatory factors of bone aromatase.

Changes with aging of steroidal levels in the cerebrospinal fluid of women.

OBJECTIVE: Age-related changes of steroid levels in the central nervous system (CNS) are not well understood. To investigate whether steroidal conditions in the CNS of women change with aging and menopause, steroid levels have been measured in serum and cerebrospinal fluid (CSF), and examined correlations with aging. METHODS: Serum and CSF concentrations of estradiol (E2), cortisol, dehydroepiandrosterone (DHEA), DHEA sulfate (DHEAS) and albumin were measured in 80 female patients who underwent operations for benign gynecological diseases. They had no endocrinological or neurological disorders and were aged 17-71 years; 62 patients were in premenopause and 18 were in postmenopause. RESULTS: Serum levels of E2 decreased markedly after menopause, while levels of DHEA and DHEAS decreased gradually with age. There was no significant change with age of serum cortisol levels. The CSF concentrations of E2 (0.2-3 pg/ml) decreased with age [correlation coefficient (r)= 0.31, P < 0.01]. The CSF DHEA levels (0.1-0.8 ng/ml) did not change with age although not significantly, but CSF cortisol levels (0.1-0.6 microg/dl) increased with age (r = 0.35, P < 0.01). The CSF DHEAS concentrations were below the sensitivity of the radioimmunoassay (RIA) (1 ng/ml). The CSF/serum ratios of cortisol increased with age (r = 0.30, P < 0.01), as did those of DHEA (r = 0.55, P < 0.01). Although serum albumin levels did not change throughout life, CSF albumin levels and CSF/serum albumin ratios increased gradually with age (r = 0.28, P = 0.052; r = 0.23, P = 0.114, respectively), but there was no significance. There were marked decreases of serum E2 and DHEA levels and CSF E2 levels in postmenopausal women (P < 0.05), but CSF cortisol levels increased (P < 0.05) and DHEA levels in CSF were maintained after menopause. CONCLUSION: These results indicate that steroids in CSF become cortisol dominated and deficient in estrogens with aging, especially after menopause.

Hydatidiform moles associated with multiple gestations after assisted reproduction: diagnosis by analysis of DNA fingerprint.

In spite of the widespread use of assisted reproductive technology, there have been, to our knowledge, only two reported cases of molar pregnancies after gamete intra-Fallopian transfer and five reported cases after in-vitro fertilization and embryo transfer. We report here a case of a complete hydatidiform mole in a twin pregnancy after gamete intra-Fallopian transfer, as well as a case of a complete hydatidiform mole in a triplet pregnancy after in-vitro fertilization and embryo transfer. The genetic constitution of each conceptus was determined by examination of the restriction fragment length polymorphism of the DNA with four different single-locus probes. This analysis revealed that both hydatidiform moles were of androgenetic origin and probably of monospermic origin. Moreover, the analysis confirmed that the pregnancies were dizygotic and trizygotic pregnancies respectively. The diagnostic utility of the analysis of DNA polymorphism is discussed in cases of a molar pregnancy with coexisting fetuses.

Aromatase expression of human osteoblast-like cells.

Estrogen plays a major role in bone mineral homeostasis, maintaining a balance between bone formation and bone resorption not only in women but also in men. Extraglandular aromatization of circulating androgen is the major source of estrogen in post-menopausal women and men. In order to assess the capacity of bone cells as a local source of estrogen, osteoblast-like cells (OLCs) were obtained from human fetal bone in mid-trimester by the explant method and by mechanical disaggregation. The integrity of OLCs was confirmed by their ability to produce alkaline phosphatase and osteocalcin in response to vitamin D3 and also by their ability to deposit mineral. Aromatase activity was assessed by the formation of estrone from [1,2,6,7-3H]androstenedione and by the release of tritium from [1beta-3H]androstenedione into [3H]water. Formation of estrone was confirmed by thin layer chromatography (TLC) in OLCs stimulated with dexamethasone (DEX) + oncostatin M. The aromatase activity was 10 x higher in non-passaged OLCs than in passaged cells in the presence or absence of the stimulants (DEX + IL-1beta). The apparent Km and Vmax estimated by the release of [3H]water was 5.8+/-0.6 nM and 10.8+/-1.4 pmol/mg per 6 h in the presence of DEX + IL-1beta. The effects of several stimulants on aromatase activity in OLCs were examined: serum, IL-1beta, TNFalpha and type I cytokines stimulated activity in the presence of DEX, while PMA and PMA + dibutyryl cAMP did not. To confirm the expression of aromatase in OLCs, cells prepared from periosteal membranes were also examined: These cells in culture possessed aromatase activity corresponding to OLCs prepared from bone specimens. Moreover, the fresh periosteum expressed aromatase at higher levels than that of metaphyseal specimens. The aromatase gene employs several different promoters (I.1, 1.2, I.3, I.4, I.5, I.6, 2a, 1f and PII) and the usage of these promoters is known to be controlled in a tissue-specific fashion. Accordingly, promoter usage in OLCs and fetal long bone (tibia) tissue was examined using the 5' rapid amplification of cDNA ends (RACE) technique. The major promoter used was I.4, not only in stimulated and non-stimulated OLCs, but also in fetal tibia. Some minor transcripts were also found: 1f (brain-specific promoter), PII and I.6 in OLCs stimulated by DEX + IL-1beta, and PII and I.3 in OLCs stimulated by DEX + serum. Fetal tibia also expressed I.3 (15%) and I.6 (10%). Thus, regulation and promoter usage in OLCs was quite different from other tissues known as estrogen sources including adipose tissue, ovary and placenta. These results suggest that bone is an extraglandular source of local estrogen which plays an important role in bone mineral metabolism through autocrine and paracrine actions.

Multiple splicing events involved in regulation of human aromatase expression by a novel promoter, I.6.

The expression of aromatase is regulated in a tissue-specific fashion through alternative use of multiple promoter-specific first exons. To date, eight different first exons have been reported in human aromatase, namely I.1., I.2, I.3. I.4, I.5, PII, 2a, and 1f. Recently, we have found a new putative exon I in a RACE-generated library of THP-1 cells and have conducted studies to characterize this new exon I. We confirmed that the constructs containing -1552/+17 or less flanking sequence of this exon function as a promoter in THP-1 cells, JEG-3 cells and osteoblast-like cells obtained from a human fetus. Results of transfection assays using a series of deletion constructs and mutation constructs indicate that a 1-bp mismatch of the consensus TATA-like box (TTTAAT) and the consensus sequence of the initiator site, which is located 45 bp downstream of the putative TATA box, were functioning cooperatively as a core promoter. The putative transcription site was confirmed by the results of RT-PCR southern blot analysis. We examined the regulation and the expression of this exon, I.6, in several human cells and tissues by RT-PCR Southern blot analysis. THP-1 cells (mononuclear leukemic origin) and JEG-3 cells (choriocarcinoma origin) expressed exon I.6 in serum-free media. The level of expression was increased by serum and phorbol myristyl acetate (PMA) in both cell lines. Adipose stromal cells also expressed exon I.6 in the presence of PMA. In fetal osteoblasts, the expression of exon I.6 was increased most effectively by serum and less so by dexamethasone (DEX) + IL-1beta and DEX + IL-11, whereas induction by serum was suppressed by the addition of DEX. The level of expression was low in granulosa cells in culture and did not change with forskolin. On the other hand, dibutyryl cAMP suppressed PMA-stimulated expression of exon I.6 in THP-1 cells and adipose stromal cells. This result supports the hypothesis that the expression of exon I.6 is regulated mainly via an AP-1 binding site that is found upstream of the initiator site of the promoter region. Expression of exon I.6-specific transcripts was examined in several human tissues. Testis and bone obtained from normal adults expressed exon I.6. Testicular tumor and hepatic carcinoma expressed high levels of exon I.6, whereas granulosa cell tumor did not. Fetal liver and bone also showed a significant level of exon I.6 expression, but not so much as testicular tumor and hepatic tumor. Several splicing variants of exon I.6 were detected especially in THP-1 and JEG-3 cells, and to a lesser extent in primary cultures and tissue samples. These variants were identified as an unspliced form, a form spliced at the end of exon I.4, a form spliced at the end of exon I.3 (truncated) and a form spliced 220 bp downstream of the 3' end of exon I.6. The last variant revealed a new splicing site. Because most of the splicing variants contain the sequence specific for exon I.3, RT-PCR specific for exon I.3 can coamplify these splicing variants of exon I.6 transcripts. These results suggests that it is necessary to examine the expression of I.6 in tissues that are known to express exon I.3 such as breast adipose tissue, in which promoter usage of exon I of the aromatase gene switches from exon I.4 to I.3 in the course of malignant transformation.

Estrogen biosynthesis in THP1 cells is regulated by promoter switching of the aromatase (CYP19) gene.

The expression of aromatase, the enzyme responsible for estrogen biosynthesis, has been studied in THP-1 cells of human mononuclear leukemic origin, which exhibit high rates of aromatase activity. These cells have the capacity to differentiate in the presence of vitamin D into cells with osteoclast-like properties. Differentiated cells displayed higher rates of aromatase than undifferentiated cells, and, in both cases, activity was stimulated 10- to 20-fold by dexamethasone. Phorbol esters also increased aromatase activity, but the effect was the same in differentiated as in undifferentiated cells. In a similar fashion to adipose stromal cells, serum potentiated the response to dexamethasone but had no effect on phorbol ester-stimulated activity. By contrast to its action in adipose stromal cells, (Bu)2cAMP markedly inhibited aromatase activity of THP-1 cells, as did factors whose actions are mediated by cAMP, such as PTH and PTH-related peptide. This was true of control cells, as well as of dexamethasone- and phorbol ester-stimulated cells. Previously we have shown that type 1 cytokines as well as tumor necrosis factor-alpha stimulate aromatase activity of adipose stromal cells in the presence of dexamethasone. By contrast, interleukin-6, interleukin-11, and leukemia-inhibitory factor had no effect on aromatase activity of THP-1 cells, whereas tumor necrosis factor-alpha, oncostatin M, and platelet-derived growth factor were slightly inhibitory of aromatase activity. Exon-specific Southern analysis of rapid amplification of cDNA ends-amplified transcripts was employed to examine the distribution of the various 5'-termini of aromatase transcripts. In the control group, most of the clones contained transcripts specific for the proximal promoter II, whereas in dexamethasone-treated cells, most transcripts contained exon I.4. In the phorbol ester-treated cells, a broader spectrum of transcripts was present, with equal proportions of I.4, II, and I.3-containing clones. Additionally, one clone containing a new sequence, exon I.6, was found. This was shown to be located about 1 kb upstream of exon II. By contrast, all clones from cells treated with (Bu)2cAMP contained promoter II-specific sequences. In addition to these transcripts, two clones in the library from the dexamethasone-treated cells contained the sequence previously defined as the brain-specific sequence, 1f. In one of these, the 1f sequence was fused downstream of exon I.4, indicative that its expression likely employed promoter I.4. These results point to similarities and important differences between aromatase expression in THP-1 cells and other cells such as adipose stromal cells, indicative of unique regulatory pathways governing aromatase expression in these cells.