Cytochrome P450 Aromatase Research Articles

Estrogens, a Female Hormone Concerned in Spermatogenesis

In the mammalian testis, the irreversible conversion of androgens into estrogens is catalyzed by the cytochrome P450 aromatase, a microsomal enzymatic complex encoded by a unique gene (Cyp19) in humans which contained 18 exons, 9 of them being translated. In addition that gene includes 12 non-coding exons I, located in the 5' end and controlled by tissue-specific promoters that are spliced alternatively onto a common site in exon II. However, a unique protein of 55 kDa is produced in human beings. In most of mammals studied so far, at least in rodents, all testicular cells except peritubular cells contain aromatase which is therefore constitutively expressed and very precisely controlled (in terms of expression and enzyme activity) according to somatic and germ cell origin (both estrogen receptors ERα and ERβ) as well as GPER (or GPR30), a seven transmembrane receptor which was concerned in these rapid effect of estrogens. In man, our data obtained from both ejaculated spermatozoa and immature gem cells demonstrate the presence of a biologically active aromatase; moreover estrogen receptors (ER alpha and ER beta) are also present. All together according to the widespread localisation of aromatase and estrogen receptors in testicular cells, our review clearly shows that besides gonadotrophins and androgens, estrogens produced locally, should be considered as physiologically relevant hormones involved in the regulation of spermatogenesis and spermiogenesis. Keywords: Aromatase, estrogens, estrogen receptors (genomic effect), germ cells, mammals, rapid membrane effects of estrogens, spermatogenesis, reproduction, testis

Motion and Flexibility in Human Cytochrome P450 Aromatase

The crystal structures of human placental aromatase in complex with the substrate androstenedione and exemestane have revealed an androgen-specific active site and the structural basis for higher order organization. However, X-ray structures do not provide accounts of movements due to short-range fluctuations, ligand binding and protein-protein association. In this work, we conduct normal mode analysis (NMA) revealing the intrinsic fluctuations of aromatase, deduce the internal modes in membrane-free and membrane-integrated monomers as well as the intermolecular modes in oligomers, and propose a quaternary organization for the endoplasmic reticulum (ER) membrane integration. Dynamics of the crystallographic oligomers from NMA is found to be in agreement with the isotropic thermal factors from the X-ray analysis. Calculations of the root mean square fluctuations of the C-alpha atoms from their equilibrium positions confirm that the rigid-core structure of aromatase is intrinsic regardless of the changes in steroid binding interactions, and that aromatase self-association does not deteriorate the rigidity of the catalytic cleft. Furthermore, NMA on membrane-integrated aromatase shows that the internal modes in all likelihood contribute to breathing of the active site access channel. The collective intermolecular hinge bending and twisting modes provide the flexibility in the quaternary association necessary for membrane integration of the aromatase oligomers. Taken together, fluctuations of the active site, the access channel, and the heme-proximal cavity, and a dynamic quaternary organization could all be essential components of the functional aromatase in its role as an ER membrane-embedded steroidogenic enzyme.

Ontogeny of Cytochrome P450 Aromatase mRNA Expression in the Developing Sheep Brain

The intraneuronal conversion of testosterone to oestradiol constitutes a critical step in the development and sexual differentiation of the brain of many short gestation mammalian species and has been inferred to play a similar role in long gestation sheep. This conversion is catalysed by cytochrome P450 aromatase (CYP19), which is expressed in specific brain structures during foetal development. The present study was undertaken to examine the specific neuroanatomical distribution and relative expression of aromatase mRNA in the developing sheep hypothalamus. The foetal sheep is a highly tractable model system for localising the region-specific expression of aromatase in the brain during prenatal development that can help predict regions where oestrogen acts to shape neural development. Our results, obtained using real time quantitative reverse transcriptase-polymerase chain reaction, revealed that aromatase mRNA was expressed throughout mid to late gestation in the foetal preoptic area and amygdala. In the preoptic area, aromatase expression declined with advancing gestation, whereas, it increased in the amygdala. No sex differences were observed in either brain area. We next investigated the anatomical distribution of aromatase using in situ hybridisation histochemistry and found that the pattern of mRNA expression was largely established by midgestation. High expression was observed in the medial preoptic nucleus, bed nucleus of the stria terminalis and corticomedial amygdala. We also observed substantial expression in the dorsal striatum. These results extend our understanding of the developmental expression of aromatase in the foetal sheep brain and lend support to the view that it plays an essential role in sexual differentiation and maturation of the neuroendocrine, motor and reward control systems.

Effect of butyrate on aromatase cytochrome P450 levels in HT29, DLD-1 and LoVo colon cancer cells

Epidemiological studies suggest that colonic production of butyrate and estrogen may be involved in human susceptibility to colorectal cancer (CRC). Estrone (E1) can be produced by the aromatase pathway during the conversion of androstenedione (A) to E1. Therefore, we studied the effect of sodium butyrate (NaBu) on the CYP19A1 transcript and protein levels and on the conversion of A to E1 in HT29, DLD-1 and LoVo CRC cells. We found that NaBu significantly downregulated CYP19A1 transcript and protein levels, a phenomenon that was associated with reduced conversion of A to E1 in HT29, DLD-1 and LoVo cells. Our studies demonstrated that, although butyrate exhibited a protective role in CRC development, this compound may reduce aromatase activity and the production of E1 in colon cancer cells.

Effects of bisphenol A on the expression of cytochrome P450 aromatase (CYP19) in human fetal osteoblastic and granulosa cell-like cell lines

The effects of bisphenol A (BPA), an endocrine disruptor, on aromatase (CYP19) expression in human osteoblastic (SV-HFO) and ovarian granulosa-like (KGN) cell lines were examined. CYP19 enzyme activity was suppressed in the presence of BPA in a dose-dependent fashion in both cell lines. CYP19 gene transcript expression, as well as activities of promoter I.4 in SV-HFO and promoter II in KGN, was down-regulated by BPA, suggesting that BPA affects CYP19 at the gene-expression level. These data and the previous finding that BPA induced the down-regulation of promoter I.1 activity within the human placental cell line suggest that there may be a conserved signaling pathway that down-regulates CYP19 expression in response to BPA in both cell lines. Additionally, differences between promoter I.4 and II suggest that there may be cell- and promoter-specific down-regulating mechanisms downstream from the actions of BPA.

Estradiol Promotes Purkinje Dendritic Growth, Spinogenesis, and Synaptogenesis During Neonatal Life by Inducing the Expression of BDNF

Neurosteroids are synthesized de novo from cholesterol in the brain. In rodents, the Purkinje cell actively produces several kinds of neurosteroids including estradiol during neonatal life, when cerebellar neuronal circuit formation occurs. Estradiol may be involved in cerebellar neuronal circuit formation through promoting neuronal growth and synaptic contact, because the Purkinje cell expresses estrogen receptor-β. To test this hypothesis, in this study we examined the effect of estradiol on dendritic growth, spinogenesis, and synaptogenesis in the Purkinje cell using neonatal wild-type (WT) mice or cytochrome P450 aromatase knock-out (ArKO) mice. Administration of estradiol to neonatal WT or ArKO mice increased dendritic growth, spinogenesis, and synaptogenesis in the Purkinje cell. In contrast, WT mice treated with tamoxifen, an ER antagonist, or ArKO mice exhibited decreased Purkinje dendritic growth, spinogenesis, and synaptogenesis at the same neonatal period. Estrogen administration to neonatal WT or ArKO mice increased the expression of brain-derived neurotrophic factor (BDNF) in the cerebellum, whereas tamoxifen decreased the BDNF level in WT mice similar to ArKO mice. BDNF administration to tamoxifen-treated WT mice increased Purkinje dendritic growth. These results indicate that estradiol induces dendritic growth, spinogenesis, and synaptogenesis in the developing Purkinje cell via BDNF action during neonatal life.

Aromatase (CYP19) gene variants influence ovarian response to standard gonadotrophin stimulation

The association of cytochrome P450 aromatase gene CYP19(TTTA) ( n ) polymorphism with ovarian response to FSH stimulation was explored. Three hundred women undergoing medically assisted reproduction and 300 women with at least one spontaneous pregnancy participated in the study. CYP19(TTTA) ( n ) polymorphism was genotyped, while serum hormones were determined. During oocyte retrieval, the follicular size, the follicle and oocyte numbers were recorded. Six CYP19(TTTA) ( n ) alleles with 7 to 12 repeats were revealed. Women homozygous for long CYP19(TTTA) ( n ) alleles presented with lower serum FSH levels at the third day of the menstrual cycle (p < 0.001) and higher large follicle numbers (p < 0.01), compared to women homozygous for short CYP19(TTTA) ( n ) alleles. The CYP19(TTTA) ( 7 ) allele was associated with higher serum FSH levels (p < 0.003), with lower total follicle (p < 0.02) and large follicle numbers (p < 0.03), while CYP19(TTTA) ( 7 ) allele-carriers presented more frequently with small follicles than CYP19(TTTA) ( 7 ) allele-non carriers (p < 0.01). CYP19 genetic variants were associated with ovarian reserve and response to standard gonadotrophin stimulation of women undergoing in vitro fertilization.

Ontogenic and sexually dimorphic expression of cyp19 isoforms in the rainbowfish, Melanotaenia fluviatilis (Castelnau 1878)

To investigate the role of cytochrome P450 aromatase, we isolated cyp19 isoforms in the Murray River rainbowfish, M. fluviatilis. The cloned cDNA for cyp19a1a and cyp19a1b had an open reading frame (ORF) of 492 and 499 amino acid residues, with shared identity of up to 83% and 87% with the corresponding homologues of other teleosts respectively. In contrast, the cyp19a1a and cyp19a1b of the Murray River rainbowfish had a shared identity of only 61%. Not surprisingly, the phylogenetic analysis clustered the M. fluviatilis cyp19 isoforms with the corresponding isoforms of other teleosts, suggesting a shared evolutionary ancestry of the respective isoforms. We also studied the expression of cyp19 isoforms during ontogeny and in adult fish using quantitative Real-Time PCR (qPCR). Results suggest that uniquely only cyp19a1b transcripts are maternally inherited, suggesting its role in early development and growth in the species. In contrast to reports in many teleosts, the cyp19a1a was exclusively expressed in the ovarian tissue and completely absent in other tissues examined, including testis. The cyp19a1b like in most teleosts was predominantly expressed in the brain of both males and females with low level of expression in other tissues including gonads of both sexes.

Differential regulation of the expression of cytochrome P450 aromatase, estrogen and androgen receptor subtypes in the brain–pituitary–ovarian axis of the Japanese eel ( Anguilla japonica) reveals steroid dependent and independent mechanisms

This study aimed at investigating the role of sexual steroids in the regulation of the expression of the single aromatase gene and steroid receptor subtypes in the brain–pituitary–ovarian axis of the Japanese eel. Unlike other teleosts, which possess duplicated genes for aromatase, cyp19a1a and cyp19a1b, expressed in the gonads and in the brain, respectively, eel species possess a single cyp19a1. Phylogenetic analysis indicated that eel brain/gonadal cyp19a1 branches at the basis of both teleost gonadal cyp19a1a and brain cyp19a1b clades. Female eels treated with catfish pituitary homogenate (CPH) to induce sexual maturation showed an increase in the expression of cyp19a1 and aromatase enzymatic activity in the brain and in the ovaries. Treatments with sex steroids (estradiol-17 β, E 2 or testosterone, T) revealed that the increase in cyp19a1 expression in the brain may result from E 2-specific induction. In contrast, the increase in cyp19a1 expression in the ovaries of CPH-treated eels is a result of steroid-independent control, probably from a direct effect of gonadotropins contained in the pituitary extract. Analysis of the expression of estrogen and androgen receptor subtypes, esr-α, esr-β, ar-α and ar-β, in eels treated with CPH or sex steroids revealed differential regulations. In CPH-treated eels, the expression of esr-α and ar-α was significantly increased in the brain, while the expression of ar- α and ar- β was increased in the ovaries. No change was observed in esr- β in any organ. Steroid treatments induced an upregulation by E 2 of esr- α, but not esr- β expression, in the brain, pituitary and ovaries, while no autoregulation by T of its own receptors could be observed. These results reveal both steroid-dependent and -independent mechanisms in the regulation of cyp19a1 and steroid receptor subtype expression in the eel.

Interactions between hypoxia and sewage-derived contaminants on gene expression in fish embryos

Fish embryos were used to evaluate the interaction among common environmental and chemical stressors found in urban coastal environments, namely hypoxia, aryl hydrocarbon receptor (AhR) agonists, and estrogenic compounds. At the molecular level, the systems responding to these stressors share common response factors, and evidence exists for cross-talk between them. Biomarkers of exposure to these stressors, cytochrome P4501a ( Cyp1a), estrogen receptor alpha ( ERα), brain cytochrome P450 aromatase ( Cyp19a2 or AromB), and hypoxia inducible factor 1 alpha ( Hif-1α) mRNA expression were examined using qRT-PCR simultaneously in embryos of two well studied species, the Atlantic killifish, Fundulus heteroclitus, and the zebrafish Danio rerio. Embryos of both species were exposed to the model Cyp1a inducer β-naphthoflavone (BNF) or 17-β estradiol (E2) under either normoxic or hypoxic (5% oxygen atmosphere) conditions and harvested prior to hatch at 9 days post fertilization (dpf) for the killifish, and 48 h post fertilization (hpf) for the zebrafish. BNF significantly induced Cyp1a expression in embryos of both species with killifish embryos being more responsive (700-fold > control) than zebrafish embryos (7-100-fold > control). AromB was also significantly influenced by treatment, but to a lesser extent, with mean expression levels increased by less than two-fold over control values in response to E2, and in one case upregulated by BNF. ERα and Hif-1α were constitutively expressed in embryos of both species, but expression was unaffected by exposure to either BNF or E2. Hypoxic conditions downregulated AromB expression strongly in killifish but not in zebrafish embryos. The impact of hypoxia on expression of other genes in either species was inconsistent, although an interactive effect between hypoxia and BNF on several of the genes evaluated was observed. These data are the first to examine expression patterns of these important environmental response genes together in embryos of two important model fish species. The results support the use of Cyp1a expression as a biomarker of AhR agonists in fish embryos, and indicate that AromB may be more responsive than ERα to estrogenic chemicals at this stage in development. Hif-1α expression was not found to be a good biomarker of hypoxic exposure in either killifish or zebrafish embryos. The interaction observed between BNF and co-exposure to hypoxia warrants further investigation. Finally killifish embryos are generally more sensitive than zebrafish embryos at this stage of development supporting their use in environmental assessments.

Endurance Exercise Training Enhances Local Sex Steroidogenesis in Skeletal Muscle

Endurance training improves skeletal muscular function including energy metabolism and structure. Sex steroid hormones partly contribute to the exercise-induced muscular adaptations. Recently, we demonstrated that skeletal muscle contains steroidogenic converting enzymes to synthesize sex steroid hormones and an acute endurance exercise activates local steroidogenesis in skeletal muscle. However, whether chronic endurance training leads to enhanced steroidogenesis in skeletal muscle is unknown. Here, we examined changes in steroidogenic enzymes and sex steroid hormones in the skeletal muscle after chronic endurance exercise training. Eleven male rats were divided into two groups: sedentary (n = 6) and trained (n = 5). Endurance training was performed on a treadmill (30 m·min(-1), 30 min) for 5 d·wk(-1) for 12 wk. The posttraining harvesting was performed 48 h after the last exercise training. The mRNA expressions of 3β-HSD, aromatase cytochrome P450, and 5α-reductase in the skeletal muscle of trained rats were significantly higher than those of sedentary rats (P < 0.05). The protein expressions of aromatase cytochrome P450 and 5α-reductase in the skeletal muscle of trained rats were also significantly higher than those of sedentary rats (P < 0.05). The muscular dihydrotestosterone (DHT) concentrations in the skeletal muscle of trained rats were significantly higher than those of sedentary rats (P < 0.01), but there was no change in dehydroepiandrosterone, total testosterone, free testosterone, and estradiol. Furthermore, muscle weight corrected for body weight of trained rats was moderately correlated with the level of muscular DHT concentration in trained rats (r = 0.41, P < 0.05). Endurance exercise training enhances the muscular DHT concentration through 5α-reductase in the skeletal muscle of rats, suggesting that local bioactive androgen metabolism may participate in exercise training-induced skeletal muscular adaptation.

Differentiation of Steroid-Producing Cells and Folliculogenesis in the Developing Ovary of the Nile TilapiaOreochromis niloticus

Differentiation and development of steroid-producing cells (SPCs) and folliculogenesis during ovarian differentiation in the Nile tilapia Oreochromis niloticus were immunohistochemically and ultrastructurally examined. Clusters of immunopositive cells (IPCs) against antibodies (ABs) of cholesterol side-chain cleavage cytochrome P450 (P450scc), 3β-hydroxysteroid dehydrogenase (3βHSD), and cytochrome P450aromatase (P450arom) only appeared in the area near blood vessels in the fish ovaries at 50-60 days after hatching (dah). Ultrastructural results showed that differentiation and development of SPCs from undifferentiated to maturation occurred in the area near blood vessels, indicating that it would be the original site of SPCs. At 70-80 dah, IPC clusters invaded the interstices among oocytes at the perinucleolar stage from the area near the blood vessels. IPCs increased in number in the interstices among the previtellogenic oocytes, and some clusters began to enclose the outer thecal layer of the previtellogenic oocytes at 90 dah. The process of folliculogenesis was ultrastructurally observed. SPCs enclosed by fibroblastic cells invaded the interstitial areas among oocytes and some reached the surfaces of oocytes. The upper portions of these elongations opened and began to enclose the outer surfaces of developed oocytes to become thecal layer. Later, newly migrated SPCs reach the thecal layer to become thecal cells. These results indicate that steroid-producing thecal cells originate from the SPCs in the area near blood vessels. After thecal layer formation, an immunopositive reaction against P450arom AB, but not against P450scc or 3β-HSD ABs, appeared first in the granulosa cells enclosing the vitellogenic oocytes at 100 dah. At this time, estrogen production in serum levels rapidly increased. Thus, folliculogenesis could be essential for active production of estrogen in the ovary.

Modulation of estrogen synthesis through activation of protein kinase A in H295R cells by extracts of estuary sediments

Sediments from two estuaries within Liaodong Bay, China, were examined for the effects on steroidogenesis using H295R human adrenocortical carcinoma cells. Total extracts (TE) isolated from sediments by Soxhlet extraction were separated into three fractions (F1, F2, and F3) using Florisil columns. After exposing H295R cells to each TE and fractions, the expressions of six steroidogenic genes (cytochrome P450 cholesterol side-chain cleavage [CYP11A], 3β-hydroxysteroid dehydrogenase type 1 [3β-HSD1], 3β-hydroxysteroid dehydrogenase type 2 [3β-HSD2], cytochrome P450 17-hydroxylase/17-20 lyase [CYP17], cytochrome P450 aromatase [CYP19], 17β-hydroxysteroid dehydrogenase [17β-HSD]), and the production of six steroid hormones (progesterone [PGT], 17-hydroxyprogesterone [17-HPT], testosterone [TTR], androstenedione [ADD], estrone [E1], and 17β-estradiol [17β-E2]) were measured. The gene expressions of CYP11A, CYP17, 3β-HSD2, and CYP19, and hormone productions of PGT, 17-HPT, TTR, ADD, E1, and 17β-E2 were significantly increased after exposure to F3 extracts from the Daliao River. In particular, greater concentrations of E1 (up to 48-fold) and 17β-E2 (up to 20-fold), as well as up-regulation of CYP19 gene expression (up to tenfold), were caused by exposure to the F3 fraction from the Daliao River, but not from the Daling River. Insight into the mechanism of action was obtained by use of principal component analysis (PCA), the results of which were consistent with unidentified constituents in F3 from the Daliao River activating the protein kinase A (PKA) pathway. This hypothesis was confirmed by reversal of the effects caused by F3 through a co-exposure of a PKA inhibitor (H89) and F3 extract. The H89 down-regulated CYP19 messenger RNA (mRNA) expression with concomitant lesser production of E1 and 17β-E2 in the co-exposure group, indicating unidentified constituents that could modulate estrogen synthesis, primarily through a mechanism of PKA activation.

FTZ-F1 and FOXL2 up-regulate catfish brain aromatase gene transcription by specific binding to the promoter motifs

Cytochrome P450 aromatase (cyp19) catalyzes the conversion of androgens into estrogens. Teleosts have distinct, ovarian specific ( cyp19a1a) and brain specific ( cyp19a1b) cyp19 genes. Previous studies in teleosts demonstrated regulation of cyp19a1a expression by the NR5A nuclear receptor subfamily as well as a fork head transcription factor, FOXL2. In the present study, we investigated the involvement of fushi tarazu factor 1, FTZ-F1, a NR5A subfamily member, and FOXL2 in the regulation of cyp19a1b expression in brain of the air-breathing catfish, Clarias gariepinus. Based on the synchronous expression pattern of cyp19a1b, FTZ-F1 and FOXL2 in the brain, we isolated the 5′ upstream region of cyp19a1b to analyse regulatory motifs. Promoter motif analysis revealed FTZ-F1/NR5A1 and FOXL2 binding nucleotide sequences. Transient transfection studies showed that FTZ-F1 and FOXL2 together enhanced the transcriptional activity of cyp19a1b gene in mammalian cell lines. Mutation in either of their putative binding sites within the cyp19a1b promoter abolished this effect. Electrophoretic gel mobility shift experiments indicated that FTZ-F1 and FOXL2 proteins bind to the synthesized radio-labelled oligomers used as probes and mobility shifted upon addition of their respective antibodies. Chromatin immunoprecipitation assay confirmed the binding of both these transcription factors to their corresponding cis-acting elements in the upstream region of cyp19a1b. To our knowledge, this study is the first report on the transcriptional regulation of cyp19a1b by FTZ-F1 and FOXL2 in a teleost fish.

Expression patterns of cytochrome P450 aromatase genes during ovary development and their responses to temperature stress in female yellow catfish (Pelteobagrus fulvidraco)

Cytochrome P450 aromatase (P450arom) plays a pivotal role in ovary development. In this study, we used semi-quantitative reverse transcription PCR (RT-PCR) to analyze spatiotemporal expressions of two P450arom genes (CYP19A and CYP19B) and their responses to temperature stress in female yellow catfish (Pelteobagrus fulvidraco). Tissue distribution pattern of CYP19 showed that CYP19B was abundantly expressed in fish brain and ovary (brain > ovary), but weakly in intestines, whereas CYP19A was exclusively expressed in ovary. Semi-quantitative RT-PCR analyses showed high transcript abundance of both CYP19A and CYP19B in the ovarian reproductive cycle, corresponding with serum estradiol-17β (E2) levels. Increases in aromatases, serum E2 and testosterone (T) levels in fish exposed to higher temperature indicated stimulation of ovarian maturation and recrudescence by heat stress in stages II and V during the ovarian cycle, whereas associated decreases in stage III suggested vitellogenesis inhibition by heat stress. Gene expression of CYP19 was closely related to levels of serum E2. Results demonstrated CYP19 played a crucial role in the reproductive cycle of female yellow catfish. Different temperature stress affected CYP19 gene expression in the fish ovarian reproductive cycle. Associated P450arom genes could be useful for studying physiological aspects of yellow catfish.

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) increases the expression of cyclooxygenase-2 and aromatase cytochrome P450 in human endometrium

To investigate the role of 2,3,7,8-Tetrachlorodibenzo-p-dioxin(TCDD) on eutopic endometrium by stromal cell culture. Prospective experimental study. Human endometrial tissues obtained by hysterectomy specimens from 16 women with estrogen dependent diseases (endometriosis (n = 4), adenomyosis (n = 5), uterine fibroids (n = 7)) and 5 women who had undergone hysterectomy for carcinoma in situ of uterine cervix. Endometrial stromal cells were cultured under the following conditions: DMEM/F12 (10% FBS, 1 nM E2 and 100 nM P4). TCDD (10 nmol/l) and TNF-a (0.1ng/ml) were added according to experimental purposes. The protein expression of aromatase cytochrome P450 and COX-2 was examined by Western blot. In control group, the COX-2 protein in stromal cells cultured with TCDD was significantly increased (control; 0.63 ± 0.19 vs. TCDD; 0.94 ± 0.26, P<0.05) And the aromatase P450 protein in stromal cells cultured with TCDD and TCDD plus TNF-a was significantly increased. (control; 0.60 ± 0.26 vs. TCDD; 0.90 ± 0.36, TCDD plus TNF-a; 0.95 ± 0.43, P<0.05). In study group, the COX-2 protein in stromal cells cultured with TCDD and TCDD plus TNF-a was significantly increased. (control; 0.81 ± 0.26, TCDD; 1.04 ± 0.38, TCDD plus TNF-a; 1.30 ± 0.90, P<0.05). And the aromatase P450 protein in stromal cells cultured with TNF-a and TCDD and TCDD plus TNF-a was significantly increased (control; 0.76 ± 0.18 vs. TNF-a; 0.93 ± 0.34, TCDD; 0.97 ± 0.41, TCDD plus TNF-a; 1.16 ± 0.41 P<0.05). In the stromal cell cultured with TCDD plus TNF-a than TNF-a and TCDD alone, the aromatase protein was significantly increased (P<0.05). Treatment with the TCDD increased aromatase cytochrome P450 and COX-2 on the eutopic endometrium of patients with and without estrogen dependent diseases, by endometrial stromal cells in culture.

Molecular characterization and regulation of the angiotensin-converting enzyme type 2/Angiotensin-(1-7)/MAS receptor axis during the ovulation process in cattle

The objective of this study was to characterize the profiles of Ang-(1-7), MAS receptor, ACE(2), NEP and PEP during the ovulatory process in cattle. For this study, 40 synchronized cows with follicular diameter ≥ 12 mm were ovariectomized at different time-points (0, 3, 6, 12 and 24 h) after i.m. application of gonadotropin-releasing hormone (GnRH) to induce a luteinizing hormone surge. Follicular fluid was collected for measuring Ang-(1-7) by radioimmunoassay. Theca and granulosa cells were isolated from the preovulatory follicles to evaluate the gene expression of MAS receptor, ACE(2), NEP and PEP by qRT-PCR assay. Cross-contamination between theca and granulosa cells was tested by RT-PCR to detect cytochrome P450 aromatase (CYP19A1) and 17α-hydroxylase (CYP17A1) mRNA. Ang-(1-7) levels were constant until 12 h and then increased (p < 0.05) at 24 h after GnRH. Messenger RNA expression of MAS, ACE(2), NEP and PEP was detected in theca and granulosa cells at all time-points after GnRH. In granulosa cells, ACE(2), NEP and PEP were differentially expressed after GnRH treatment (p < 0.05). In conclusion, the Ang-(1-7), MAS receptor, ACE(2), NEP and PEP profiles in preovulatory follicles indicate that Ang-(1-7) plays a role in the regulation of the ovulatory process in cattle.

Brain cytochrome P450 aromatase activity in roach ( Rutilus rutilus): Seasonal variations and impact of environmental contaminants

P450 aromatase catalyses the conversion of C19 androgens to C18 estrogens which is thought to be essential for the regulation of the reproductive function. In this study, brain aromatase activity (AA) was measured monthly over a reproductive cycle in wild roach ( Rutilus rutilus) sampled in a reference site in Normandy. AA peaked during the breeding season, reaching 35 fmol mg −1 min −1 in both male and female fish, and was low during the rest of the year except for a significant rise in October. AA was correlated with ovary maturation (measured either as gonado-somatic index or by histological analysis of the gonads) and plasma sex-steroid levels (11-ketotestosterone in males and 17-β-estradiol in females). Measurements of AA in polluted sites showed that activity was significantly upregulated in sites with fish showing high levels of plasma vitellogenin and large proportion of intersexuality (20–50%) thus suggesting the occurrence of estrogenic compounds and their involvement in AA modulation.

Effects of xenoestrogens on the expression of vitellogenin (vtg) and cytochrome P450 aromatase (cyp19a and b) genes in zebrafish (Danio rerio) larvae

In the present study, expression levels of vitellogenin (vtg) and cytochrome P450 aromatase genes (cyp19a and cyp19b) in zebrafish larvae during the early stages of development were investigated by quantitative real time-PCR assay. The results indicated that vtg gene transcription was induced seven days after zebrafish larvae fertilization, whereas the expression of cyp19a and cyp19b genes was detected as early as 3 and 4 days post-fertilization (dpf). Investigations into the effects of 17β-estradiol (E2) exposure on the expression of these genes showed that both vtg and cyp19b were upregulated by E2 in zebrafish larvae as early as four dpf, whereas no variation was observed in cyp19a gene expression. The estrogenic potential of pharmaceutical estrogen (DES), phenol estrogen (BPA) and the brominated flame retardants, TBBPA, DE-71 and 4-BP, were evaluated by analyzing the expression of these three genes in zebrafish larvae. The results demonstrated that natural estrogen, endocrine disrupting compounds and brominated flame retardants act as endocrine disrupters through different mechanisms. We have demonstrated for the first time that the polybrominated diphenyl ether mixture, DE-71, acts as an endocrine disrupter by upregulation of cyp19b gene expression at a relatively low concentration. These results indicate that analysis of vtg and cyp19b gene expression in zebrafish during early embryogenesis and organogenesis represents the basis of a sensitive and fast bioassay for the routine assessment of xenoestrogen effects.

The Bidder's organ of the toad Rhinella arenarum (Amphibia, Anura). Presence of steroidogenic enzymes

The Bidder's organ (BO) of male true toads of Bufonidae family is located in the anterior pole of the testis and it has been compared to a rudimentary ovary because of the presence of previtellogenic follicles. In some species, BO remains in both sexes, while in others only adult males preserve the structure. Several studies suggest that the development of BO is inhibited by the differentiation of the corresponding gonad. The purpose of this study is to describe morphological and histological variability of the BO of Rhinella arenarum and also analyze its steroidogenic capacity. Observations indicate that although most bidderian follicles are in pre vitellogenesis, there are others in early or late vitellogenesis. Moreover, we found that BOs weight was significantly lower in males during the pre-reproductive period and that there is no significant correlation between the weights of BO and the adjacent testis. We also analyzed the presence of steroidogenic enzymes using immunohistochemistry. Results indicate that all the follicles were immunoreactive with the antibody against aromatase, while only few of them were positive for the cytochrome P450 side-chain cleavage. Furthermore, activities of 3β-hydroxysteroid dehydrogenase/isomerase, cytochrome P450 17-hydroxylase, C17,20-lyase and aromatase were detected by the transformation of radioactive substrates into products. Taken together, these results confirm the steroidogenic capacity of the BO in adult males of R. arenarum.