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The generation of attosecond pulse trains at free-electron lasers opens new opportunities in ultrafast science, as it gives access, for the first time, to reproducible, programmable, extreme ultraviolet (XUV) waveforms with high intensity. In this work, we present a detailed analysis of the theoretical model underlying the temporal characterization of the attosecond pulse trains recently generated at the free-electron laser FERMI. In particular, the validity of the approximations used for the correlated analysis of the photoelectron spectra generated in the two-color photoionization experiments are thoroughly discussed. The ranges of validity of the assumptions, in connection with the main experimental parameters, are derived.

Synopsis There is a profound, almost symbiotic relation between electrons and photons. When light interacts with matter the electrons in the material will start to move and oscillate. When a charged particle, such as an electron, oscillates it will act as a dipole and emit light. We utilize this symbiotic relation and extend the control of light to the extreme ultraviolet (XUV) region using the newly developed XUV opto-optical modulator.

Free-electron lasers providing ultra-short high-brightness pulses of X-ray radiation have great potential for a wide impact on science, and are a critical element for unravelling the structural dynamics of matter. To fully harness this potential, we must accurately know the X-ray properties: intensity, spectrum and temporal profile. Owing to the inherent fluctuations in free-electron lasers, this mandates a full characterization of the properties for each and every pulse. While diagnostics of these properties exist, they are often invasive and many cannot operate at a high-repetition rate. Here, we present a technique for circumventing this limitation. Employing a machine learning strategy, we can accurately predict X-ray properties for every shot using only parameters that are easily recorded at high-repetition rate, by training a model on a small set of fully diagnosed pulses. This opens the door to fully realizing the promise of next-generation high-repetition rate X-ray lasers., X-ray free-electron lasers, important light sources for materials research, suffer from shot-to-shot fluctuations that necessitate complex diagnostics. Here, the authors apply machine learning to accurately predict pulse properties, using parameters that can be acquired at high-repetition rates.

Wepresent the first measurement of the vectorial response of strongly dressed helium atoms probed by an attosecond pulse train (APT) polarised either parallel or perpendicular to the dressing field polarisation. The transient absorption is probed as a function of delay between the APT and the Linearly polarised 800 nmfield of peak intensity 1.3∗1014 W cm-2. The APT spans the photon energy range 1642 eV, covering the first ionisation energy of helium (24.59 eV). With parallel polarised dressing and probing fields, we observe modulations with periods of one half and one quarter of the dressing field period. When the polarisation of the dressing field is altered from parallel to perpendicular with respect to the APT polarisation we observe a large suppression in the Modulation depth of the above ionisation threshold absorption. In addition to this we present the intensity dependence of the harmonic modulation depth as a function of delay between the dressing and probe fields, with dressing field peak intensities ranging from 2∗1012 to 2∗1014 W cm-2.We compare our experimental results with a full-dimensional solution of the single-atom time-dependent (TD) Schrdinger equation obtained using the recently developed abinitio TDB-spline ADCmethod and find good qualitative agreement for the above threshold harmonics.

Rapid proton migration is a key process in hydrocarbon photochemistry. Charge migration and subsequent proton motion can mitigate radiation damage when heavier atoms absorb X-rays. If rapid enough, this can improve the fidelity of diffract-before-destroy measurements of biomolecular structure at X-ray-free electron lasers. Here we study X-ray-initiated isomerization of acetylene, a model for proton dynamics in hydrocarbons. Our time-resolved measurements capture the transient motion of protons following X-ray ionization of carbon K-shell electrons. We Coulomb-explode the molecule with a second precisely delayed X-ray pulse and then record all the fragment momenta. These snapshots at different delays are combined into a ‘molecular movie’ of the evolving molecule, which shows substantial proton redistribution within the first 12 fs. We conclude that significant proton motion occurs on a timescale comparable to the Auger relaxation that refills the K-shell vacancy., United States. Dept. of Energy. Office of Basic Energy Sciences

We present a technical review of a beamline built to perform pump-probe experiments with a temporal resolution of < 200 attosecond. This is designed specifically to use the technique of attosecond transient absorption spectroscopy (ATAS) to resolve ultra-fast electron dynamics in atoms and molecules. A non-collinear, interferometrically stable geometry is adopted to allow us to individually control the characteristics of each of the pump and probe arms independent from each other. With the use of an auxiliary interferometer to correct for long-term drifts between the pump and probe arms we measure better than 150as resolution for our time-corrected delay despite having separated beam paths of over 4m in length. In our first experiment we have focused on the time dependence of the electronic states of an atom in a strong laser field. An extreme ultra-violet (XUV) attosecond pulse train (APT) and a precisely synchronized 30fs IR pulse are used in this work. Delay-dependent absorption modulations are observed at even multiples (2 and 4) of the IR dressing field frequency as the pump-probe delay is scanned. We investigate the dependence of the 2ω order absorption modulation amplitude from the transient absorption of laser-dressed helium as the IR dressing field ellipticity is varied, and we discuss the issues in obtaining such results. We present qualitative data indicating a clear anisotropy in the response of the atom to an IR dressing field, and discuss how we will improve this measurement in future experiments.

The effect of circulating oestrogen deficiency on sleep regulation and locomotor activity was investigated in aromatase cytochrome P450 deficient mice (ArKO) and wild-type (WT) controls. Sleep was recorded in 3-month old mice during a 24-h baseline day, 6-h sleep deprivation (SD) and 18-h recovery, and activity was recorded at the age of 3, 9 and 12 months. In mice deficient of oestrogen, the total amount of sleep per 24 h was the same as in WT controls. However, in ArKO mice, sleep was enhanced in the dark period at the expense of sleep in the light phase, and was more fragmented than sleep in WT mice. This redistribution of sleep resulted in a damped amplitude of slow-wave activity (SWA; power between 0.75-4.0 Hz) in non-rapid eye movement sleep across 24 h. After SD, the rebound of sleep and SWA was similar between the genotypes, suggesting that oestrogen deficiency does not affect the mechanisms maintaining the homeostatic balance between the amount of sleep and its intensity. Motor activity decreased with age in both genotypes and was lower in ArKO mice compared to WT at all three ages. After SD, the amount of rest in 3-month old WT mice increased above baseline and was more consolidated. Both effects were less pronounced in ArKO mice, reflecting the baseline differences between the genotypes. The results indicate that despite the pronounced redistribution of sleep and motor activity in oestrogen deficient mice, the basic homeostatic mechanisms of sleep regulation in ArKO mice remain intact.

We present an apparatus for performing gas phase high-harmonic generation spectroscopy of molecules primarily found in the liquid phase. Liquid molecular samples are heated in a temperature controlled bath and their vapour is used to back a continuous ow gas jet, with vapour pressures of over 1 bar possible. In order to demonstrate the system, we perform high harmonic spectroscopy experiments in benzene with a 1.8 µm driving field. Using the unique capabilities of the system, we obtain spectra that are nearly free from the effects of longitudinal phase-matching, amenable to comparison with advanced numerical modelling., This work was supported by EPSRC programme Grant No. EP/I032517/1 and ERC ASTEX Project No. 290467. A.S.J. and D.A.W. also acknowledge support from Marie Curie ITN EC317232, A.S.J. acknowledges support from NSERC PGSD3-454096-2014, and D.R.A. acknowledges support from the Imperial College JRF scheme. M.C. thanks support from Project No. CTQ2013-43086-P (MINECO, Spain)

We present technical and experimental advances for performing HHG experiments in a range of substituted benzene molecules starting in the liquid phase. We demonstrate sub 3% fluctutaions in the harmonic signal generated in a benzene vapour backed continuous flow gas jet using a mid-IR laser source. The longer drive wavelength is necessary as the target molecules have low ionization potential and exhibit femtosecond timescale dynamics. We present the acquisition of stable and reproducible harmonic spectra from a range of substituted benzene molecules and the dependence of HHG upon the ellipticity of the laser beam was measured for a number of molecules.

Objective To report visual acuity during the first three years after iodine 125 (I125) brachytherapy for medium-sized choroidal melanoma and to identify important baseline and treatment factors associated with posttreatment visual acuity in a group of patients who were treated and observed prospectively as part of a large, randomized clinical trial. Design Observational case series within a randomized, multicenter study. Participants Patients enrolled in the Collaborative Ocular Melanoma Study randomized trial of I125 brachytherapy versus enucleation had choroidal melanoma of at least 2.5 mm but no more than 10.0 mm in apical height, and no more than 16.0 mm in largest basal dimension. One thousand three hundred seventeen patients enrolled from February 1987 through July 1998; 657 patients were assigned to I125 brachytherapy. Visual acuity data for 623 patients who received I125 brachytherapy as randomly assigned and who have been observed for at least 1 year were analyzed for this report. Methods Under study protocol, an ophthalmic evaluation, including best-corrected visual acuity measurement of each eye, was performed at baseline, every 6 months thereafter for 5 years, and once yearly thereafter. Two poor vision outcomes, visual acuity of 20/200 or worse that was confirmed at the next follow-up examination and loss of six lines or more of visual acuity from baseline that was confirmed at the next follow-up examination, were analyzed to identify baseline and treatment characteristics that were associated with posttreatment visual acuity. Results At baseline, median visual acuity in the eye with choroidal melanoma was 20/32, with 70% of eyes having 20/40 or better and 10% of eyes having 20/200 or worse visual acuity. Three years after I125 brachytherapy, median visual acuity was 20/125, with 34% having 20/40 or better and 45% having 20/200 or worse visual acuity, including eyes that were enucleated within 3 years of treatment. Life-table estimates of percentages of patients who lost six or more lines of visual acuity from baseline, a quadrupling of the minimum angle of resolution, with this finding confirmed at the next 6-month follow-up examination, were 18% by 1 year, 34% by 2 years, and 49% by 3 years after treatment. Life-table estimates of percentages of patients with baseline visual acuity better than 20/200 whose visual acuity decreased to 20/200 or worse, confirmed at the next follow-up examination, were 17% by 1 year, 33% by 2 years, and 43% by 3 years after treatment. As soon as a poor vision outcome was observed, improvement of visual acuity to a level that no longer met the definition for a poor vision outcome was rare. Greater baseline tumor apical height and shorter distance between the tumor and the foveal avascular zone (FAZ) were the factors most strongly associated with loss of six or more lines of visual acuity after treatment. These two factors and baseline visual acuity also were strongly associated with visual acuity 20/200 or worse after treatment. Patient history of diabetes, presence of tumor-associated retinal detachment, and tumors that were not dome shaped also were associated with greater risk for both of the poor vision outcomes. Conclusions Forty-three percent to 49% of treated eyes had substantial impairment in visual acuity by 3 years after I125 brachytherapy, defined as a loss of six or more lines of visual acuity from the pretreatment level (49% of eyes) or visual acuity of 20/200 or worse (43% of eyes) that was confirmed at the next 6-month examination. Patients with a history of diabetes and patients whose eyes had thicker tumors, tumors close to or beneath the FAZ, tumor-associated retinal detachment, or tumors that were not dome shaped were those most likely to have a poor visual acuity outcome within 3 years after I125 brachytherapy.

A radiolabelled cRNA was synthesized using a 1.4 kb cDNA complementary to mRNA encoding bovine basic fibroblast growth factor (bFGF) as a template, and used as a probe to investigate the expression of mRNA encoding bFGF in bovine ovarian tissue, and luteal cells in primary culture. Northern analysis of poly(A +)RNA prepared from follicles and corpora lutea of various stages revealed a major mRNA species of 7 kb in corpora lutea of all stages, the amount of which was higher late in the luteal phase. No hybridizable message was detectable in follicles of any size. When luteal cells were established in primary culture, expression of the 7 kb mRNA species was maintained. This expression was increased markedly when cells were treated with LH/hCG or Bt2cAMP. Prostaglandin F-2 alpha treatment caused a marked decrease in the basal content of this 7 kb mRNA, and also severely impaired the ability of LH to stimulate this expression.

Models of estrogen insufficiency have revealed new and unexpected roles for estrogens in males as well as females. These models include natural mutations in the aromatase gene in humans, as well as mouse knock-outs of aromatase and the estrogen receptors, and one man with a mutation in the ERa gene. These mutations, both natural and experimental, have revealed that estrogen deficiency results in a spectrum of symptoms. These include loss of fertility and libido in both males and females; loss of bone in both males and females; a cardiovascular and cerebrovascular phenotype; development of a metabolic syndrome in both males and females, with truncal adiposity and male-specific hepatic steatosis. Most of these symptoms can be reversed or attenuated by estradiol therapy. Thus estrogen is involved in the maintenance of general physiological homeostasis in both sexes.

Models of estrogen insufficiency have revealed new and unexpected roles for estrogens in males as well as females. These models include natural mutations in the aromatase gene in humans, as well as mouse knock-outs of aromatase and the estrogen receptors, and one man with a mutation in the ERα gene. These mutations, both natural and experimental, have revealed that estrogen deficiency results in a spectrum of symptoms. These include loss of fertility and libido in both males and females; loss of bone in both males and females; a cardiovascular and cerebrovascular phenotype; development of a metabolic syndrome in both males and females, with truncal adiposity and male-specific hepatic steatosis. Most of these symptoms can be reversed or attenuated by estradiol therapy. Thus estrogen is involved in the maintenance of general physiological homeostasis in both sexes.

International audience; We report the first measurement of the near oxygen K-edge auger spectrum of the glycine molecule. Our work employed an x-ray free electron laser as the photon source operated with input photon energies tunable between 527 and 547 eV. Complete electron spectra were recorded at each photon energy in the tuning range, revealing resonant and non-resonant auger structures. Finally ab initio theoretical predictions are compared with the measured above the edge auger spectrum and an assignment of auger decay channels is performed.

While the ovaries are the principal source of systemic estrogen in the premenopausal nonpregnant woman, other sites of estrogen biosynthesis are present throughout the body and these become the major sources of estrogen beyond menopause. These sites include the mesenchymal cells of the adipose tissue and skin, osteoblasts, and perhaps chondrocytes in bone, vascular endothelial and aortic smooth muscle cells, as well as a number of sites in the brain including the medial preoptic/anterior hypothalamus, the medial basal hypothalamus and the amygdala. These extragonadal sites of estrogen biosynthesis possess several fundamental features which differ from those of the ovaries. Principally, the estrogen synthesized within these compartments is probably only biologically active at a local tissue level in a paracrine or 'intracrine' fashion. Thus the total amount of estrogen synthesized by these extragonadal sites may be small, but the local tissue concentrations achieved are probably quite high, and exert significant biological influence locally. Thus these sources of estrogen play an important but hitherto largely unrecognized, physiological and pathophysiological role.

Estrogen has been suggested to play a neuromodulatory and neuroprotective role on the brain dopamine system. We used aromatase knockout (ArKO) mice that lack a functional aromatase enzyme and are unable to convert testosterone into estrogen, and assessed prepulse inhibition of acoustic startle, locomotor hyperactivity to amphetamine treatment and rotarod performance. Mice were tested at either 1 month, 4-5 months or 12-18 months of age. In male, but not female ArKO mice, there was an age-related reduction of prepulse inhibition. The 12-18 months old male ArKO mice also showed significantly greater amphetamine-induced hyperactivity. Mice heterozygous for the mutation showed no deficits or were in-between wildtype mice and ArKO mice. We postulate that these data indicate a neuroprotective role of estrogen, particularly in male mice, on ageing of brain mechanisms involved in pre-pulse inhibition and locomotor activity regulation. It is likely that these brain mechanisms are or include dopaminergic activity.

Previous studies employing the male aromatase knockout (ArKO) mouse have indicated that local expression of estrogens appears to be important for the progression of spermatogenesis. In the absence of estrogen biosynthesis round spermatids are observed to undergo apoptosis and thus fail to differentiate into mature, elongated spermatids. This lesion appears to arise between the ages of 18 weeks and 1 year. To ultimately determine if the disruption to spermatogenesis arises earlier than 18 weeks, we performed an intensive study to examine the fertility of younger male ArKO mice. This involved an analysis of their mating capacity together with an extensive stereological analysis, determination of the in vitro potential of mature sperm, and sexual behavior. ArKO and wild-type (w/t) males at 7 weeks of age were placed with w/t females for 7 weeks. At age 14 weeks, the males were killed and the testes removed. ArKO mice were observed to sire significantly fewer litters than the w/t mice; 5 out of the 10 sired no litters at all. Stereological analysis performed on the removed testes found a significant decrease in round spermatid numbers between w/t and ArKO mice at this age; however, there were no differences in all other germ cells and Sertoli cell numbers. When mature spermatozoa were analyzed, sperm from 15-week-old ArKO mice had a significant reduction in motility. This was further reduced by 1 year of age with a decrease in concentration. A preliminary examination of sexual behavior found that ArKO mice did not attempt to mount the females, in contrast to the w/t mice, which mounted consistently during the time period. In conclusion, we observed that ArKO mice have reduced fertility at age 14 weeks. This may be due in part to a disruption in spermatogenesis because the phenotype does appear to arise earlier than 18 weeks, possibly leading to abnormalities in the mature spermatozoa. Or, in part, this may be attributable to an impairment in the development of copulatory behavior, which is consistent with the available evidence that points to a crucial role for estrogens in the neural development and initiation of male sexual behavior.

Estrogen biosynthesis is catalyzed by aromatase cytochrome P-450 (the product of the CYP19 gene). Adipose tissue is the major site of estrogen biosynthesis in postmenopausal women, with the local production of estrogen in breast adipose tissue implicated in the development of breast cancer. In human adipose tissue, aromatase is primarily expressed in the mesenchymal stromal cells and is a marker of the undifferentiated preadipocyte phenotype. Aromatase expression in adipose tissue is regulated via the distal promoter I.4, under the control of glucocorticoids and class I cytokines such as oncostatin M, interleukin 6, and interleukin 11, as well as tumor necrosis factor alpha. These cytokines, which are expressed in adipose, also inhibit adipocyte differentiation. Therefore, we hypothesized that factors which stimulate adipocyte differentiation should inhibit aromatase expression. These factors include synthetic peroxisome proliferator-activated receptor gamma (PPARgamma) ligands such as thiazolidinediones, e.g., troglitazone and rosiglitazone (BRL49653) and the endogenous PPARgamma ligand 15-deoxy-delta12,14-prostaglandin J2. We have demonstrated by measurement of aromatase activity and by reverse transcription-PCR/Southern blotting that these PPARgamma ligands inhibit aromatase expression in cultured breast adipose stromal cells stimulated with oncostatin M or tumor necrosis factor alpha plus dexamethasone in a concentration-dependent manner, whereas a metabolite of troglitazone that does not activate PPARgamma has no effect. We have also shown that troglitazone inhibits luciferase activity of reporter constructs containing various lengths of the upstream region of promoter I.4 transfected into mouse 3T3-L1 preadipocyte mesenchymal cells, whereas the troglitazone metabolite does not. Because local estrogen production in breast fat is implicated in breast cancer development in postmenopausal women, the actions of PPARgamma ligands suggest that they may have potential therapeutic benefit in the treatment and management of breast cancer.

Aromatase enzyme is the product of the CYP19 gene. Human aromatase deficiency is a rare disorder and is usually caused by single base-pair changes resulting in amino acid substitution or premature stop codons. In most cases, the affected mother presents with virilization in the third trimester of pregnancy. Affected female newborns have pseudohemaphrodism with clitoromegaly and hypospadias. The cause of these presentations in pregnancy is the inability to convert fetal dehydroepiandrosterone to estrogen in the placenta and subsequent conversion to androgens in the periphery. Affected male newborns present with tall stature secondary to failed epiphyseal fusion. They also have delayed bone age, osteopenia, and undermineralization, which can be corrected with the addition of estrogen, highlighting estrogen's critical role in men as well as women. The aromatase knock-out male mouse (ArKO) has shortened femur length and bone undermineralization. Female ArKO mice at 10-12 weeks have multiple ovarian follicles arrested in the antral phase and stromal hyperplasia. By 1 year the ovaries become grossly dysmorphic with numerous cystic follicles and fibrous stroma. Male ArKO mice testes demonstrate arrest of spermatogenesis at the level of round spermatids and Leydig cell hyperplasia. ArKO mice also exhibit evidence of insulin resistance and visceral adiposity.

Phytoestrogens are compounds found in a wide variety of plant foods that historically are said to exhibit estrogen-like activity and, more recently, have been reported to display both estrogenic and anti-estrogenic effects. Population-based studies have been interpreted to suggest that consumption of a phytoestrogen-rich diet is protective against breast, prostate, and bowel cancer and cardiovascular disease and ameliorates estrogen-deficiency symptoms in postmenopausal women. Consequently, there is a global movement towards increased consumption of phytoestrogen-rich foods and tabletized concentrated isoflavone extracts are being heavily promoted. Evaluating the effects and hence the potential benefits and risks of phytoestrogens is a complex task. The interindividual diversity and complexity in dietary phytoestrogen absorption and metabolism make the bioactivity of these compounds unpredictable. Epidemiological studies of relationships between phytoestrogens and cancer and cardiovascular disease that take into account confounding factors are scarce. Results of many of the in vitro and in vivo studies are conflicting and confusing. These compounds do not simply mimic the effects of human steroidal estrogen but rather demonstrate both similar and divergent actions. The ultimate actions of these compounds in specific cells are determined by many factors, including the relative levels of estrogen receptor (ER) alpha and ER beta and the diverse cocktail of co-activators and co-repressors present in any given cell type. Therefore, effects vary according to the phytoestrogen studied, cell line, tissue, species, and response being evaluated. Overall, it is naive to assume that exposure to these compounds is always good; inappropriate or excessive exposure may be detrimental. Extensive documentation of the specific intracellular effects of the various phytoestrogens in different tissues, the relationships between timing and duration of exposure and disease, and results from prospective randomized studies in humans of their clinical effects and potential side effects are essential. Only then can widespread recommendations regarding the dietary and pharmacological intake of these compounds be made.

This report represents the first study in the literature linking development of severe gynecomastia, in a 17 1/2-yr-old boy, to high levels of aromatase expression in a large fibrolamellar hepatocellular carcinoma, which gave rise to extremely elevated serum levels of estrone (1200 pg/mL) and estradiol-17 beta (312 pg/mL) that suppressed FSH and LH (1.3 and 2.8 IU/L, respectively), and consequently testosterone (1.53 ng/mL). After removal of a 1.5-kg hepatocellular carcinoma, gynecomastia partially regressed, and essentially, normal hormone levels were restored (estradiol-17 beta,50 pg/mL; estrone, 74 pg/mL; testosterone, 6.85 ng/mL; and FSH/LH, 6.3/3.7 mIU/mL). Conversion of C19 steroids to estrogens occurs in a number of human tissues and is catalyzed by aromatase P450 (P450arom), the product of the CYP19 gene in a number of human tissues. Tissue-specific promoters are used to regulate P450arom gene transcription in adult human tissues, e.g. promoters I.4 and I.3 in adipose fibroblasts, and promoter II in the gonads. Human fetal liver uses promoter I.4 to express markedly high levels of P450arom, whereas hepatic P450arom expression normally becomes undetectable in postnatal life. Using immunohistochemistry, diffuse intracytoplasmic aromatase expression was detected in the liver cancer cells from this severely feminized boy. Northern analysis indicated the presence of P450arom transcripts in total RNA from the hepatocellular cancer but not in the adjacent liver nor in disease-free adult liver samples. Promoter use for aromatase expression was determined by a specific RT-PCR method. Promoters I.3 and II were used for P450arom gene expression in the hepatocellular cancer tissue. Because aromatase is not expressed in the disease-free adult liver, the presence of extremely high levels of aromatase expression in this fibrolamellar hepatocellular carcinoma tissue is intriguing, particularly because there is preferential use of the proximally located P450arom promoters I.3 and II by the tumor, instead of the much more distally located fetal liver-type promoter I.4.

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.

It has been shown that sexual dimorphic morphology of certain hypothalamic and limbic areas underlie gender-specific sexual behavior and neuroendocrine mechanisms. The key role played by locally formed estrogen in these developmental events has been revealed during a critical perinatal period. In this study, we aimed to document the presence of estrogen-synthetase (aromatase)-immunoreactive elements in the involved limbic system and hypothalamus of the developing rat brain. On postnatal day 5, animals of both sexes were perfusion-fixed, and sections from the forebrain and hypothalamus were immunolabelled for aromatase using an antiserum that was generated against a 20 amino acid sequence of placental aromatase. Aromatase-immunoreactivity was present in neuronal perikarya and axonal processes in the following limbic structures: the central and medial nuclei of the amygdala, stria terminalis, bed nucleus of the stria terminalis (BNST), lateral septum, medial septum, diagonal band of Broca, lateral habenula and all areas of the limbic (cingulate) cortex. In the hypothalamus, the most robust labelling was observed in the medial preoptic area, periventricular regions, ventromedial and arcuate nuclei. The most striking feature of the immunostaining with this antiserum was its intracellular distribution. In contrast to the heavy perikaryal labelling that can be observed with most of the currently available aromatase antisera, in the present experiments, immunoperoxidase was predominantly localized to axons and axon terminals. All the regions with fiber staining corresponded to the projection fields of neuron populations that have previously been found to express perikaryal aromatase. Our results confirm the presence of aromatase-immunoreactivity in developing limbic and hypothalamic areas. The massive expression of aromatase in axonal processes raises the possibility that estrogen formed locally by aromatase may not only regulate the growth, pathfinding and target recognition of its host neuronal processes, but may also exert paracrine actions on structures in close proximity, including the target cells.

The conversion of C19 steroids to estrogens is catalysed by aromatase P450 (P450arom; product of the CYP19 gene). Tissue sites of expression include the gonads and brain; however, in a small subset of mammals, P450arom is also expressed in the placenta. In humans, gonadal expression employs a promoter proximal to the start site of translation, whereas expression in the placenta relies on a promoter which is distal to this site. We characterized the bovine CYP19 gene in both the ovary (OV) and placenta (PL), to determine if this method of regulation of tissue-specific expression is employed in other species. Both bovine and human species express P450arom in these tissues, however, the pattern of expression is very different. In both humans and cattle, P450arom is expressed in the granulosa cells of the ovary, however, after ovulation only the luteinized granulosa cells of the human continue to express P450arom. There is a high degree of sequence identity (70%) shared between humans and cattle in the OV-specific 5'-flanking DNA, whereas little identity (40%) was found between humans and cattle in the PL-specific DNA. Promoters and 5'-flanking regions of human and bovine CYP19 genes were subcloned into a luciferase (LUC) vector. Bovine PL-specific constructs transfected into JEG-3 human choriocarcinoma cells failed to express LUC activity. When OV-specific constructs were transfected into luteinized bovine granulosa cells, there was no change in LUC activity in cells transfected with the bovine constructs after treatment with forskolin, whereas all of the corresponding human constructs expressed LUC activity. The bovine 5'-flanking DNA lacks a cAMP-responsive element-like sequence (CLS) critical for cAMP-stimulated transcription of P450arom in the human ovary. With the absence of this CLS sequence in the bovine gene, there appears to be little enhancement of transcription by cAMP in the portion of the 5'-flanking region studied so far. When the analogous region of the bovine promoter was mutated to the human CLS, only a partial restoration of LUC activity was observed. An additional element therefore appears to be important in preventing the full expression of the bovine CYP19 gene in luteinized granulosa cells.

Aromatase P450 (P450arom) is responsible for conversion of C19 steroids to estrogens in a number of human tissues, such as the placenta, gonads, adipose tissue, skin and the brain. Aromatase expression in human tissues is regulated by use of alternative promoters in the placenta (promoter I.1), adipose tissue (promoters I.4, I.3 and II) and gonads (promoter II). Aromatase expression is absent in the disease-free adult liver, adrenal and uterine tissues. Excessive or inappropriate aromatase expression in adipose fibroblasts and endometriosis-derived stromal cells, as well as in testicular, hepatic, adrenal and uterine tumors, is associated with abnormally high circulating estrogen levels and/or with increased local estrogen concentrations in these tissues. Whether systemically delivered or locally produced, elevated estrogen levels will in turn promote the growth of hormone-responsive tissues. We recently studied aromatase expression in testicular tumor and adipose tissue samples from prepubertal boys with gynecomastia, in hepatocellular cancer and adrenocortical tumor samples from adult men with gynecomastia, in breast adipose tissue samples proximal to breast tumors, and in endometrial cancer, leiomyoma and endometriosis tissues. Excessive aromatase activity and P450arom transcript levels were found in these tissue samples or in cultured cells derived from these tissues. In these neoplastic or non-neoplastic tissues or cells, the regulation of aromatase expression was studied in terms of alternative promoter use, both in vivo and in response to various hormonal stimuli. Our results were suggestive of a common metabolic abnormality associated with activation of a cyclic AMP-dependent signalling pathway that gives rise to transcriptional transactivation of aromatase expression via promoters I.3 and II in all of the above tissues. This article describes the common pathophysiological and molecular features of excessive aromatase expression in these disease states.

Estrogen biosynthesis in adipose tissue has assumed great significance in terms of a number of estrogen-related diseases. The biosynthesis of estrogens from C19 steroids is catalyzed by a specific form of cytochrome P450, namely aromatase cytochrome P450 (P450arom; the product of the CYP19 gene). The human CYP19 gene comprises nine coding exons, II-X, and its transcripts are expressed in the ovary, placenta, testes, adipose tissue, and brain. Tissue-specific expression of the CYP19 gene is determined at least in part by the use of tissue-specific promoters, which give rise to transcripts with unique 5'-noncoding termini. Thus, the distal promoter I.1 is responsible for expression uniquely in placenta. On the other hand, the proximal promoter II, which regulates expression via a cAMP-dependent signaling pathway, is responsible for expression in the gonads. Transcripts in breast adipose tissue contain 5'-termini corresponding to expression derived from promoters I.4, II, and I.3, with I.4-specific termini predominating. The latter are derived from promoter I.4, which contains a glucocorticoid response element and an interferon-gamma activation site element and is responsible for expression in the presence of glucocorticoids and members of the class I cytokine family. The object of the present study was to determine the distribution of these various transcripts in adipose tissue from abdomen, buttocks, and thighs of women, as this would provide important clues to the factors regulating aromatase expression in these sites. To achieve this, we employed competitive reverse transcription-PCR to amplify unique 5'-ends of each of the transcripts of the CYP19 gene that are expressed in adipose tissue as well as for the coding region to evaluate total CYP19 gene (P450arom) transcript levels. We observed that exon I.4-specific transcripts were predominantly present in adipose tissue samples obtained from women regardless of the tissue site or the age of the individual. In these tissues, promoter II- and exon I.3-specific transcripts were present in lower copy numbers. We also demonstrated that in these sites total or exon-specific P450arom transcripts levels increased in direct proportion to advancing age and that transcript levels were the highest in buttocks, followed by thighs, and lowest in the abdomen. These results suggest that in normal human adipose tissue, aromatase expression is mainly under local control by a number of cytokines via paracrine and autocrine mechanisms in the presence of systemic glucocorticoids.

Family 19 of the P450 superfamily is responsible for the conversion of C19 androgenic steroids to the corresponding estrogens, a reaction known as aromatization, since it involves conversion of the delta 4-3-one A-ring of the androgens to the corresponding phenolic A-ring characteristic of estrogens. Its members occur throughout the entire vertebrate phylum. The reaction mechanism of aromatase is very interesting from a chemical point of view and has been studied extensively; however, a detailed examination of structure-function relationships has not been possible due to lack of a crystal structure. Recent attempts to model the three-dimensional structure of aromatase have permitted a model that accounts for the reaction mechanism and predicts the location of aromatase inhibitors. The gene encoding human aromatase has been cloned and characterized and shown to be unusual compared to genes encoding other P450 enzymes, since there are a number of untranslated first exons that occur in aromatase transcripts in a tissue-specific fashion, due to differential splicing as a consequence of the use of tissue-specific promoters. Thus, expression in ovary utilizes a proximal promoter that is regulated primarily by cAMP. On the other hand, expression in placenta utilizes a distal promoter that is located at least 40 kb upstream of the start of transcription and that is regulated by retinoids. Other promoters are employed in brain and adipose tissue. In the latter case, class I cytokines such as IL-6 and IL-11 as well as TNF alpha are important regulatory factors. PGE2 is also an important regulator of aromatase expression in adipose mesenchymal cells via cAMP and PGE2 appears to be a major factor produced by breast tumors that stimulates estrogen biosynthesis in local mesenchymal sites. In all of the splicing events involved in the use of these various promoters, a common 3'-splice junction is employed that is located upstream of the start of translation; thus, the coding regions of the transcripts- and hence the protein-are identical regardless of the tissue site of expression; what differ in a tissue-specific fashion are the 5'-ends of the transcripts. This pattern of expression has great significance both from a phylogenetic and ontogenetic standpoint as well as for the physiology and pathophysiology of estrogen formation. Recently, a number of mutations of the aromatase gene have been described, which give rise to complete estrogen deficiency. In females this results in virilization in utero and primary amenorrhea with hypergonadotropic hypogonadism at the time of puberty. In men the most striking feature is continued linear bone growth beyond the time of puberty, delayed bone age, and failure of epiphyseal closure, thus indicating an important role of estrogens in bone metabolism in men. In both sexes the symptoms can be alleviated by estrogen administration.

Family 19 of the P450 super family is responsible for the conversion of C19 androgenic steroids to the corresponding estrogens, a reaction known as aromatization because it involves conversion of the delta4-3-one A-ring of the androgens to the corresponding phenolic A-ring characteristic of estrogens. The gene encoding human aromatase has been cloned and characterized and shown to be unusual compared to genes encoding other P450 enzymes, because there are numerous untranslated first exons that occur in aromatase transcripts in a tissue-specific fashion due to differential splicing as a consequence of the use of tissue-specific promoters. Thus, expression in the ovary uses a proximal promoter that is regulated primarily by cAMP. On the other hand, expression in the placenta uses a distal promoter located at least 40 kb upstream of the start of transcription that is regulated by retinoids. Other promoters are used in brain and adipose tissue. In the latter case, class I cytokines such as IL-6 and IL-11, as well as TNF-alpha, are important regulatory factors. A common 3'-splice junction located upstream of the start of translation is used in all of the splicing events involved in the use of these various promoters. Thus, the coding region of the transcripts, and hence the protein, are identical regardless of the tissue site of expression; what differs in a tissue-specific fashion is the 5'-end of the transcripts. This pattern of expression has great significance both from a phylogenetic and ontogenetic standpoint, as well as for the physiology and pathophysiology of estrogen formation, as will be discussed in this review.

The expression of aromatase cytochrome P450 (P450arom) in the adrenal glands, testes, and placentas of fetal and newborn pigs was investigated. Western immunoblot analysis detected a single 48-50-kDa protein band in these tissues as well as in other porcine tissues known to express P450arom including Day 12 tubular conceptuses, theca interna, and granulosa. Slight differences in migration suggested that the P450arom protein expressed in the testis was larger than that in the adrenal gland, which was, in turn, larger than that in placenta, theca, and granulosa. Consistent with P450arom expression in these tissues, a cDNA encoding porcine P450arom hybridized to a 2.3-kb transcripts in Northern analyses of porcine blastocysts, placentas, and fetal and newborn adrenal glands and testes, as well as in theca and granulosa tissues from preovulatory follicles. No differences in transcript size were detectable among tissues. The identity of P450arom transcripts was confirmed by sequence analysis of partial cDNA clones amplified from porcine fetal adrenal glands, testes, and placentas according to the RACE procedure. The sequences of the adrenal and testis clones were identical but differed from the placental sequence, which represented the first 85 amino acids of porcine P450arom. Specifically, the adrenal and testis clones expressed transcripts that resembled the ovarian isoform of porcine P450arom, rather than the porcine placental isoform, predicting a two-amino acid deletion and 12 predicted amino acid substitutions. P450arom activity was examined to further define expression in these tissues. Activity in adrenal, testis, and placental homogenates was inhibited by 4-hydroxyandrostenedione (4OH-A4) whereas inhibition by etomidate was demonstrated in the adrenal and testis homogenates but not in the placental homogenates. The sensitivity of activity in the newborn porcine adrenal glands and testes to inhibition by etomidate was similar to that of ovarian P450arom activity. The level of P450arom activity was highest in the placenta and lowest in the adrenal gland, and no effect of fetal sex was noted in either tissue. Immunocytochemical studies localized the expression of P450arom in the adrenal gland of newborns to cells at the corticomedullary junction and, with greater intensity, to cells around the developing medullary lobules. The same cells expressed cytochrome P450 17 alpha-hydroxylase, the expression of which also extended throughout the zona fasciculata. The interstitial cells were the site of P450arom expression in the testis, but no expression could be detected in any cells with the spermatic tubules. These data demonstrate that fetal and newborn porcine adrenal glands and testes express an active P450arom that resembles the isoform expressed in the ovary. The localization of adrenal expression suggests a possible role in medullary maturation and function in the fetal and newborn pig.

The aromatase enzyme complex catalyzes the conversion of androgens to estrogens in a wide variety of tissues, including the ovary, testis, placenta, brain, and adipose tissue. Only a single human gene encoding aromatase P450 (CYP19) has been isolated; tissue-specific regulation is controlled in part by alternative promoters in a tissue-specific manner. We report a novel mutation in the CYP19 gene in a sister and brother. The 28-yr-old XX proband, followed since infancy, exhibited the cardinal features of the aromatase deficiency syndrome as recently defined. She had nonadrenal female pseudohermaphrodism at birth and underwent repair of the external genitalia, including a clitorectomy. At the age of puberty, she developed progressive signs of virilization, pubertal failure with no signs of estrogen action, hypergonadotropic hypogonadism, polycystic ovaries on pelvic sonography, and tall stature. The basal concentrations of plasma testosterone, androstenedione, and 17-hydroxyprogesterone were elevated, whereas plasma estradiol was low. Cyst fluid from the polycystic ovaries had a strikingly abnormal ratio of androstenedione and testosterone to estradiol and estrone. Hormone replacement therapy led to breast development, menses, resolution of ovarian cysts, and suppression of the elevated FSH and LH values. Her adult height is 177.6 cm (+2.5 SD). Her only sibling, an XY male, was studied at 24 yr of age. During both pregnancies, the mother exhibited signs of progressive virilization that regressed postpartum. The height of the brother was 204 cm (+3.7 SD) with eunuchoid skeletal proportions, and the weight was 135.1 kg (+2.1 SD). He was sexually fully mature and had macroorchidism. The plasma concentrations of testosterone (2015 ng/dL), 5 alpha-dihydrotestosterone (125 ng/dL), and androstenedione (335 ng/dL) were elevated; estradiol and estrone levels were less than 7 pg/mL. Plasma FSH and LH concentrations were more than 3 times the mean value. Plasma PRL was low; serum insulin-like growth factor I and GH-binding protein were normal. The bone age was 14 yr at a chronological age of 24 3/12 yr. Striking osteopenia was noted at the wrist. Bone mineral densitometric indexes of the lumbar spine (cancellous bone) and distal radius (cortical bone) were consistent with osteoporosis; the distal radius was -4.7 SD below the mean value for age- and sex-matched normal men; indexes of bone turnover were increased. Hyperinsulinemia, increased serum total and low density lipoprotein cholesterol, and triglycerides and decreased high density lipoprotein cholesterol were detected.(ABSTRACT TRUNCATED AT 400 WORDS)

Conversion of androgens to estrogens is catalyzed by aromatase P450 (P450arom; the product of the CYP19 gene). Regulation of tissue-specific expression of P450arom in humans is due, in part, to alternative transcriptional start sites that arise as a consequence of the use of granulosa cells and placental tissue from cows, horses, and pigs (ungulates) in order to determine whether these species, like the human, utilize tissue-specific promoters to drive P450arom expression. The majority of transcripts in the placenta have 5'-termini that differ from those in the ovary upstream of a common site of divergence, indicative of a splice junction. The use of tissue-specific promoters by the bovine CYP19 gene would produce these results, as it does in the case of the human CYP19 gene. A bovine genomic library was then screened with probes that hybridize to ovary- or placenta-specific transcripts. Two clones of approximately 15 kb each in length were isolated; one hybridized with the ovary-specific sequence and the other hybridized with the placenta-specific sequence. Whereas the former sequence was contiguous with the downstream sequence containing the translational start site, the latter was identical only with the sequence of the placental transcripts upstream of the putative splice junction, indicating that this was the distal sequence. Bovine and human ovary-specific genomic sequences share 77% bp identity, while bovine and human placenta-specific sequences demonstrated only 39% bp identity. These results mirror those obtained in comparisons of human, bovine, equine, and porcine ovarian and placental RACE cDNA 5'-termini.(ABSTRACT TRUNCATED AT 250 WORDS)

To determine whether serum-free (SF) conditioned media (CM) from several human breast cancer cell lines and primary stromal cell cultures contain factor(s) that mimic the marked stimulatory effects of serum on aromatase activity and aromatase P450 (P450arom) gene expression in adipose stromal cells in culture (ASC) in the presence of dexamethasone (DEX).Adipose stromal cells, harvested from fresh adipose specimens, were grown to confluence, switched to SF media, and then incubated in the presence or absence of DEX with CM from T47-D breast cancer cells, pre-treated with or without 17 beta-estradiol (E2), and with CM from stromal cell cultures. Aromatase activity of the ASC was determined by the [3H] water release assay. Total RNA was isolated, and reverse transcription-polymerase chain reaction was performed to determine the expression of various 5'-termini.T47-D CM stimulated aromatase activity in a concentration-dependent manner, similar to that of serum, in ASC incubated with DEX. Estrogen potentiated this in a dose-dependent fashion. The ASC CM and endometrial stromal cell CM also markedly induced aromatase activity in ASC. Heat inactivation destroyed the stimulating ability of CM. The majority of P450arom 5'-termini expressed by ASC incubated with CM plus DEX contained the promoter I.4-specific sequence.Conditioned media from several breast cancer cell lines and primary stromal cell cultures can mimic the effects of serum in the presence of DEX to stimulate aromatase activity in ASC. These results suggest that undefined, heat-labile and proteinaceous factors are present in CM that stimulate P450arom expression in a fashion similar to that of serum.

Extraglandular conversion of C19 steroids to estrogens takes place primarily in the stromal cell component of adipose tissue and is catalyzed by aromatase cytochrome P450 (P450arom; the product of the CYP19 gene). CYP19 gene expression and aromatase activity in breast adipose stromal cells in culture are subject to complex hormonal regulation, which was recently found to be mediated in part by alternative use of tissue-specific promoters of the CYP19 gene. It has been proposed that increased local aromatase activity in breast adipose tissue may influence the growth of breast carcinomas. Using competitive RT-PCR, we quantified P450arom transcripts in breast adipose tissue from mastectomy specimens. In 10/15 patients, highest transcript levels were found in the quadrant where the tumor was located. We also found the highest proportions of adipose stromal cells versus adipocytes in those quadrants. Such findings suggest that regional differences in the relative proportions of the various histologic components give rise to locally elevated concentrations of estrogens. Although the initiating events are not known, once a neoplastic change has occurred, tumor growth may be promoted by the locally increased estrogen levels. We are currently investigating alternative promoter use for CYP19 gene transcription to explain this association. Our results underscore the importance of aromatase inhibitors as effective agents in treatment of hormone-responsive breast cancer, since aromatase inhibitors reduce local aromatase activity as well as blood estradiol levels.

The conversion of androgens to estrogens is catalyzed by an enzyme complex known as aromatase, whose activity results in aromatization of the A ring of androgens, to form the phenolic A ring characteristic of estrogens with concomitant loss of the C19 angular methyl group. This enzyme complex is localized to the endoplasmic reticulum of cells in which it is expressed and consists of two components. The first is a form of cytochrome P450 known as aromatase cytochrome P450 (P450arom) (Mendelson et al. 1985; Chen et al. 1986; Kellis and Vickery 1987), the product of the CYP19 gene (Nebert et al. 1991). This heme protein is responsible for binding the C19 steroid substrate and catalyzing the concerted series of reactions leading to formation of the phenolic A ring. The second component is a flavoprotein, NADPH-cytochrome P450 reductase, which is a ubiquitous protein in the endoplasmic reticulum of most cell types and is responsible for transferring reducing equivalents from NADPH to any microsomal cytochrome P450 species with which it comes into contact. The aromatase reaction utilizes 3mol oxygen and 3mol NADPH for every mole of C19 steroid metabolized (Thompson and Siiteri 1974) (Fig. 1). There is general agreement that the first two oxygen molecules are utilized in the oxidation of the C19 angular methyl group. A growing consensus of opinion considers that the third oxygen attack is also on the C19 methyl group resulting in its loss as formic acid.

A 56-year-old man presented with a left ciliary body mass, anterior vitreous hemorrhage and a subluxed, cataractous lens. The clinical course included rapid enlargement of the mass, anterior uveitis, cataract progression and secondary glaucoma. Investigation included fluorescein angiography, ultrasonography and computed tomography. The eye was enucleated because of progressive enlargement with poor vision and uncontrolled secondary glaucoma. Histopathological examination showed an adenoma of the nonpigmented ciliary epithelium with cystic areas of hyaluronidase-sensitive acid mucopolysaccharide. The mass distorted the iris, occluded the angle structures and produced a complete cataract. The basement membrane of the adjacent ciliary processes was extensively thickened. The authors discuss the clinical association with vitreous hemorrhage and the local damage caused by this benign tumour.

The conversion of androgens to estrogens is catalyzed by an enzyme complex named aromatase, which consists of a form of cytochrome P-450, aromatase cytochrome P-450 (cytochrome P-450AROM), and the flavoprotein, NADPH-cytochrome P-450 reductase. As a first step toward investigation of the structure-function relationships of cytochrome P-450AROM, we have used computer modeling to align the amino acid sequence of cytochrome P-450AROM with that of cytochrome P-450CAM from Pseudomonas putida and thus create a substrate pocket using the heme-binding region and the I-helix of cytochrome P-450CAM as the template. Site-directed mutagenesis was then carried out at two sites: one at a region that aligns with the bend in the I-helix of cytochrome P-450CAM and the other at a glutamate (Glu302) just N-terminal of this bend, which is predicted to be in close proximity to the C2-position of the androstenedione substrate. To determine the importance of the former region, three mutants were constructed: A307G (Ala307----Gly), P308V (Pro308----Val), and GAGV, which changed -Ile305-Ala306-Ala307-Pro308- to -Gly-Ala-Gly-Val- (the corresponding sequence found in 17 alpha-hydroxylase cytochrome P-450). When these proteins were expressed in COS-1 cells, it was found that the activity of P308V was approximately one-third that of the wild type. These observations are consistent with the concept that Pro308 causes a bend in the I-helix of cytochrome P-450AROM, similar to that observed in cytochrome P-450CAM, which is believed to be important in forming the substrate-binding pocket. The next set of mutants were designed to determine the importance of Glu302 in catalysis. Four mutants were prepared in which Glu302 was changed either to Ala, Val, Gln, or Asp, and the activities of the expressed proteins were examined. It was found that mutations in which the carboxylic acid was replaced were essentially devoid of activity. On the other hand, changing Glu302 to Asp resulted in a two-thirds reduction in the apparent Vmax. These results support the role of a carboxylic acid residue at position 302 in the catalytic activity of cytochrome P-450AROM.

An in vitro transcription system has been developed to study the transcriptional control of steroid hydroxylase gene expression in steroidogenic tissues using upstream sequences of these genes in rabbit beta-globin promoter/reporter constructs. Analysis of deletion mutants of the upstream region of the bovine CYP17 (p45017 alpha) gene in whole cell extracts prepared from mouse Y1 adrenocortical cells revealed that sequences activating transcription in vitro are identical to those previously identified in vivo, namely the novel cAMP-responsive sequences crsI (-243 to -225 base pairs) and crsII (-80 to -40 base pairs). These sequences do not contain the known cAMP response elements, CRE or AP-2. In similar extracts from nonsteroidogenic mouse SVT2 cells, crsI was unable to activate transcription. Partially purified protein factor(s) binding to crsI were prepared from Y1 extracts by crsI-specific DNA affinity chromatography. This fraction activated transcription in vitro in SVT2 extracts only when crsI was present in the template. This in vitro transcription system will permit further investigation of crsI and its cognate factors and of the transcriptional regulation of the other steroid hydroxylase genes.

The biosynthesis of estrogens from androgens is catalyzed by a enzyme of the endoplasmic reticulum termed aromatase cytochrome P-450 (P-450AROM). The gene encoding P-450AROM was isolated in our laboratory utilizing a full-length P-450AROM cDNA and a primer-extended cDNA obtained from human placental libraries as probes. We have found that the P-450AROM gene spans at least 75 kilobases and the region encoding the P-450AROM protein is comprised of nine exons. In addition, there are at least two untranslated exons, I.1 and I.2, upstream of which are found putative promoter sequences thought to be responsible for expression of P-450AROM in placenta. To determine if these promoters are utilized to regulate P-450AROM expression in adipose tissue, we have used polymerase chain reaction technology in an attempt to amplify the untranslated exons out of human adipose total RNA. The untranslated exons could not be amplified out of adipose RNA although they could be amplified out of placental RNA. When oligonucleotides corresponding to these untranslated exons were used in Northern analysis of RNA from human adipose stromal cells, no hybridizable mRNA species was detectable. Putative promoter sequences 326 and 110 base pairs (bp) upstream of the 5' end of exon II were evaluated as adipose P-450AROM promoters by primer extension analysis and S1 nuclease protection assays. Both methods suggest a start site of transcription 26 bp down-stream of the TATAAA sequence located 110 bp from the placental intron-exon II junction. These results indicate that tissue-specific regulation of aromatase activity in the human is achieved in part by the use of alternative transcriptional start sites and tissue-specific promoters.

Cholesterol side chain cleavage cytochrome P450 (P450SCC) and 17 alpha-hydroxylase cytochrome P450 (P450(17) alpha) are key enzymes involved in steroid hormone biosynthesis. The 5'-flanking regions of the corresponding bovine genes have recently been characterized in the adrenal-derived Y1 cell line and specific DNA sequences, required for basal and cAMP-dependent gene expression, were identified. In order to investigate the molecular mechanisms controlling P450SCC and P450(17) alpha gene expression throughout the ovarian cycle, we devised an electroporation protocol to transfect bovine luteal cells in primary culture with specific chimeric DNA constructs. Transfection of such cells with reporter gene constructs containing 186 base pairs (bp) or more of the CYP11A (P450SCC) 5'-regulatory region resulted in cAMP-responsive reporter gene expression. Reporter gene constructs containing 101 bp or less of this regulatory region were expressed neither in the presence nor in the absence of forskolin. Thus, expression of the CYP11A gene in bovine luteal cells in primary culture appears to be controlled by a cis-acting element located between -186 to -101 bp. Transfection of bovine luteal cells in primary culture with reporter gene constructs containing increasing deletions of the bovine CYP17 (P450(17) alpha) 5'-regulatory region clearly demonstrated that none of the sequences were capable of promoting significant reporter gene expression, neither in the presence nor absence of forskolin. Since no 17 alpha-hydroxylase activity and no specific mRNA encoding P450(17) alpha have been detected in the bovine corpus luteum, the failure of expression of the P450(17) alpha reporter gene constructs mimics that of the endogenous gene. These results demonstrate that the regulation of CYP11A gene expression in bovine luteal cells is carried out by the same cis-acting element (-183/-101) which serves this role in the adrenal Y1 cell line. On the other hand, expression of the CYP17 gene in bovine luteal cells appears to be completely repressed, due to the absence of specific positive transcription factor(s) or the presence of negative regulatory factor(s). Thus, transfection experiments using bovine luteal cells in primary culture represent a first step towards the elucidation of molecular mechanisms underlying the regulation of cytochrome P450 gene expression throughout the ovarian cycle.

The promoter/regulatory region of the bovine CYP11A (P-450scc) gene was cloned from a bovine genomic library. One major start site of transcription was identified by primer extension analysis with a minor start site four nucleotides further upstream. A putative TATA box is located at position -31, and at position -68 resides a putative binding site for the transcription factor Sp1. Transient transfection of chimeric reporter gene constructs into mouse adrenal tumor Y1 cells was used to locate regions within the P-450scc 5'-flanking sequences that are important for basal and cAMP-dependent transcription of the reporter genes. While cAMP-dependent accumulation of mRNA derived from expression of the endogenous bovine P-450scc gene can be inhibited by protein synthesis inhibitors, transcription of reporter gene constructs containing the promoter/regulatory region of the P-450scc gene is not affected by cycloheximide following transient transfection of Y1 cells or primary bovine adrenocortical cells. Basal expression of these constructs as well as cAMP responsiveness is reduced upon deletion of sequences between -186 and -101, further deletion to -50 leading to loss of virtually all the remaining cAMP responsiveness. The sequence between -183 and -83 alone will direct both basal and cAMP-enhanced transcription when fused to a heterologous promoter and is equally active in either the correct or reverse orientation. No homology to the consensus cAMP-responsive element (CRE) or AP-2 binding site is found in this region whereas an activator protein 1-like sequence is found at position -116. It is concluded that the cAMP responsiveness of P-450scc gene expression is mediated by sequences different from canonical consensus regulatory elements. Whether or not there are sequences conferring cAMP responsiveness which are common both to P-450scc and the other steroidogenic P-450 genes remains to be established.