Expression Of P450arom Research Articles

Ontogenesis of gonadal aromatase gene expression in atlantic silverside (Menidia menidia) Populations with genetic and temperature‐dependent sex determination

Cytochrome P450 aromatase (P450arom), an enzyme that converts testosterone to 17beta-estradiol, is an important mediator of sex determination in teleosts with genetic sex determination (GSD) and temperature-dependent sex determination (TSD). We compared the ontogenetic expression of P450arom in two populations of Atlantic silversides, Menidia menidia, which exhibit TSD (South Carolina) or GSD (Nova Scotia, Canada) using quantitative, real-time polymerase chain reaction (qRT-PCR). Embryos and newly hatched larvae were reared at an intermediate sex ratio-producing temperature (21 degrees C), and older larvae and juveniles were reared at temperatures that feminize (15 degrees C) and masculinize (28 degrees C) to assess the temperature response of P450arom during development. Before sex determination, embryos and newly-hatched larvae displayed negligible P450arom expression, indicating minimal upregulation of this gene before sex determination. Gene expression increased in both populations during sex differentiation. Nova Scotia fish with GSD exhibited presumptive male- and female-like expression levels during early sex differentiation that were not influenced by temperature. South Carolina fish displayed low levels of expression at 28 degrees C with significantly heightened expression in some individuals at 15 degrees C, indicating that P450arom is temperature sensitive in the population with TSD. Populations also differed in the timing and maximal levels of P450arom expression, with fish from Nova Scotia exhibiting both the highest and earliest increase in expression in presumptive females. Our results support the hypothesis that P450arom is involved in female sex differentiation in this species, but is only responsive to temperature in M. menidia populations that exhibit TSD.

Inducible Mis- and Over-Expression of PKCIW Regulates the Expression of AMH and P450arom in Chicken Embryo Fibroblast

Sex in birds is determined genetically as in mammals and a Z-linked gene DMRT1 is required for testis development. But it remains possible that the W chromosome contains a similarly critical factor for female development. The W-linked gene PKCIW was put forward as the seductive candidate for avian sex determination and/or sex differentiation. Mis-expression and over-expression of PKCIW were performed respectively in male and female chicken embryo fibroblast with tet-on inducible expression system in this study. The results of RT-PCR showed that with the PKCIW recombinant vector introducing, PKCIW mRNA expression was significantly improved in female chicken embryo fibroblast and was close to that of the female controls and in male chicken embryo fibroblast, in which normally there was no expression. Both misand over-expression of PKCIW led to up-regulated expression of P450arom and down-regulated expression of AMH, while large increase of P450arom in female and large decrease of AMH in male appeared. With misand over-expression of PKCIW, there was no significant change of FOXL2, SOX9, DMRT1 and ATP5A1W mRNA expression. These results showed the possible regulatory effect of PKCIW on AMH and P450arom in transcription level.

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

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

Regulation of synthesis function of porcine ovarian granulose cells by mitogen-activated protein kinase signaling pathway

To investigate the role of mitogen-activated protein kinase (MAPK) signaling pathway regulating synthesis function of steroid hormone of porcine ovarian granulose cells. Porcine ovarian granulose cells were cultured in medium added with dimethyl sulfoxide (DMSO) at concentration of 50 micromol/L or with MAPK inhibitor PD98059 (PD) for 48 hours. Then, granulose cells in DMSO medium were added with activator of adenylate cyclase (20 micromol/L) or blank agent for next 48 hours incubation, which were defined as observation group 1 and control group 1, similarly, granulose cells in PD medium were also added with activator of adenylate cyclase (20 micromol/L) or blank agent for next 48 hours, which were defined as observation group 2 and control group 2. The level of estradiol (E(2)), progesterone (P) and testosterone (T) were detected by chemoluminescence. The mRNA expression of 17alpha-hydroxylase/17,20-lyase (CYP17) and aromatase P450 (P450 arom) were assessed by RT-PCR. (1) Hormone expression: the level of T, P and E(2) were (29.5 +/- 2.5) nmol/L, (80 +/- 5) nmol/L, (49 +/- 4) pmol/L in control group 1 and (42.3 +/- 3.4) nmol/L, (170 +/- 15) nmol/L, (75 +/- 6) pmol/L in control group 2, which showed significant difference (P < 0.05). In the mean time, the level of T, P and E(2) were (106.2 +/- 7.6) nmol/L, (210 +/- 16) nmol/L, (130 +/- 11) pmol/L in observation group 2 and (47.2 +/- 3.5) nmol/L, (130 +/- 6) nmol/L, (81 +/- 6) pmol/L in observation group 1, which also reach statistical difference (P < 0.05). (2) The expression of enzyme: the expression of CYP17 mRNA was increased by 50% and P450 arom mRNA was decreased by 20% between control group 1 and 2. However, the mRNA expression of P450 arom and CYP17 were upregulated remarkably, especially, the expression of CYP17 mRNA were increased by 125%. MAPK signaling pathway plays an inhibitory role in regulating synthesis of steroid hormone of ovarian granulose cell.

Large and small cells of the porcine corpus luteum: differential capacity to secrete estradiol and aromatize exogenous androgen during mid- and late luteal phase.

It has been demonstrated previously that aromatization capacity of luteal cells increased as the luteal phase progresses, suggesting that estradiol secretion by regressing corpora lutea could play an active role in regulation of the estrus cycle. To determine whether a specific luteal subpopulation is responsible for this process, luteal cells collected from mature (8-10 days after ovulation) corpora lutea were separated at unit gravity into small (14-26 microns) and large (26-38 microns) cell types. Aliquots of separated cells were incubated overnight in M199 medium alone (= control) or in medium containing: 10 M-5 T, 10(-6) M or 10(-7) M T or 0.1 x 10(-3) M, 0.5 x 10(-3) M or 1 x 10(-3) M aromatase inhibitor CGS 16949A. Both cell types were capable of estradiol production without the addition of substrates. The endogenous content of estradiol was always higher in large granulosa-derived luteal cells (LC) than in small theca-derived luteal cells (SC), and was higher in the late (LLP) than in the mid-luteal (MLP) phase. Both types of cells responded to testosterone by increasing estradiol secretion. Adding aromatase inhibitor to LC isolated from MLP and LLP decreased testosterone-stimulated estradiol secretion. On the other hand, aromatase inhibitor had no effect on testosterone-stimulated estradiol secretion by SC isolated during MLP, and inhibited testosterone-stimulated estradiol secretion by SC isolated during LLP. These data are the first to show the ability of both populations of porcine luteal cells to aromatize exogenous testosterone. The observed lack of aromatase inhibitor influence upon basal secretion of estradiol by SC separated from mature corpora lutea, and the decrease of testosterone-stimulated estradiol secretion by SC isolated from regressing corpora lutea, indicate that cytodifferentiation during luteinization could cause expression of P450arom in theca-derived luteal cells.

Porcine Hypothalamic Aromatase Cytochrome P450: Isoform Characterization, Sex-Dependent Activity, Regional Expression, and Regulation by Enzyme Inhibition in Neonatal Boars1

Domestic pigs have three CYP19 genes encoding functional paralogues of the enzyme aromatase cytochrome P450 (P450arom) that are expressed in the gonads, placenta, and preimplantation blastocyst. All catalyze estrogen synthesis, but the gonadal-type enzyme is unique in also synthesizing a nonaromatizable biopotent testosterone metabolite, 1OH-testosterone (1OH-T). P450arom is expressed in the vertebrate brain, is higher in males than females, but has not been investigated in pigs, to our knowledge. Therefore, these studies defined which of the porcine CYP19 genes was expressed, and at what level, in adult male and female hypothalamus. Regional expression was examined in mature boars, and regulation of P450arom expression in neonatal boars was investigated by inhibition of P450arom with letrozole, which is known to reprogram testicular expression. Pig hypothalami expressed the gonadal form of P450arom (redesignated the "gonadal/hypothalamic" porcine CYP19 gene and paralogue) based on functional analysis confirmed by cloning and sequencing transcripts. Hypothalamic tissue synthesized 1OH-T and was sensitive to the selective P450arom inhibitor etomidate. Levels were 4-fold higher in male than female hypothalami, with expression in the medial preoptic area and lateral borders of the ventromedial hypothalamus of boars. In vivo, letrozole-treated neonates had increased aromatase activity in hypothalami but decreased activity in testes. Therefore, although the same CYP19 gene is expressed in both tissues, expression is regulated differently in the hypothalamus than testis. These investigations, the first such studies in pig brain to our knowledge, demonstrate unusual aspects of P450arom expression and regulation in the hypothalamus, offering promise of gaining better insight into roles of P450arom in reproductive function.

Effect of letrozole on the proliferation of uterine leiomyoma cells in vitro.

Objective To investigate the expression of aromatase cytochrome P450 (P450arom), the secretion of estradiol and the suppressive effect of aromatase inhibitor letrozole on the proliferation of uterine leiomyoma cells in vitro. Methods Uterine leiomyoma cells were cultured in vitro, after the action of letrozole with different concentration, MTT was used to examine the proliferative activity of uterine leiomyoma cells, RT-PCR and immunocytochemistry was used to determine the expression of the P450arom protein and mRNA, and the concentration of estradiol in media supernatant was measured by radioimmunity. Results Low dosage (0.06 μmol/L) of letrozole had no effect on the expression of P450arom protein and mRNA, and estradiol synthesis in uterine leiomyoma cell (P>0.05). However, high dosage(0,6-6 μmol/L) of letrozole could down-regulate the expression of P450arom, and to inhibit estradiol synthesis and the proliferation activity of uterine leiomyoma cells(P<0.05). Furthermore, the inhibition were shown to decline gradually along with the increase in the concentrations of letrozole. Conclusion The aromatase inhibitor letrozole could suppress the proliferation of uterine leiomyoma cells in the dosage-dependent manner by down-regulating the expression of the P450arom and decreasing the estradiol synthesis, suggesting that the drug may possibly be effective in the clinical treatment of uterine leiomyoma. Key words: Uterine neoplasms; Letrozole; Aromatase; Estradiol

Histone H3 acetylation of StAR and decrease in DAX-1 is involved in the luteinization of bovine granulosa cells during in vitro culture

We investigated the expression of genes and transcription factors associated with steroidogenesis during the luteinization of granulosa cells isolated from bovine small follicles. Granulosa cells produced progesterone when cultivated in a culture medium including serum and attached to the substrate and began to display an elongated or fibroblastic aspect within 24 h of culture. We observed an increase in the number of granulosa cells at the same time. P450arom expression in the cultured granulosa cells had decreased at 24 h compared with 0 h of culture, and afterward was maintained at a low level. This expression was consistent with the decline of E2 concentration in the medium. Expression of StAR and P450scc mRNAs in the cultured granulosa cells was significantly increased at 72 h compared with 0 h of culture. Although the expression of Ad4BP/SF-1 mRNA began to increase during period between 48 and 72 h of culture, protein expression of Ad4BP/SF-1 remained at a constant level throughout the culture period. DAX-1 mRNA expression had decreased at 24 h of culture and remained at a low level. In parallel with this expression, the protein expression of DAX-1 began to decrease between 24 and 48 h of culture and then remained at a low level. Histone H3 acetylation of the StAR promoter region was observed at 72 h of culture period. Our data suggested that the decrease of Dax-1 transcription factor and the increase in histone H3 acetylation may play important roles in progesterone synthesis in luteinizing granulosa cells.

Exogenous androstenedione induces formation of follicular cysts and premature luteinization of granulosa cells in the ovary

To investigate the effects of androstenedione on ovarian follicle development. Experimental study. University research laboratory. Female Wistar-Imamichi rats and BDF1 mice. Rats were injected with androstenedione. Ovarian follicles of mice were cultured in the presence of androstenedione. Ovarian morphology; ovarian cell types undergoing apoptosis; ovarian expression of cytochrome P450 aromatase (P450arom), cytochrome P450 side-chain cleavage (P450scc), and cyclin-dependent kinase inhibitor p27(kip1); serum levels of T, E(2), and P in rats; and ultrastructure of granulosa cells from cultured follicles of mice. In androstenedione-treated rat ovaries, follicular cysts were formed, and apoptotic cells were found in the inner part of granulosa cell layers of antral follicles. Androstenedione administration down-regulated expression of P450arom but up-regulated expression of P450scc and p27(Kip1) in the granulosa cells of antral follicles. Serum T levels were significantly increased in androstenedione-treated rats. In mouse follicles exposed to androstenedione, the granulosa cells contained abundant lipid droplets and mitochondria with complex tubular cristae. Excess androgen enhances apoptosis in the inner part of granulosa cell layers of antral follicles, resulting in the formation of follicular cysts. It is also demonstrated that androgen stimulates premature luteinization of granulosa cells.

Decreased expression of aromatase in the Ishikawa and RL95-2 cells by the isoflavone, puerarin, is associated with inhibition of c-jun expression and AP-1 activity

Aromatase P450 (P450 arom) is overexpressed in endometriosis, endometrial cancers and uterine fibroids. With weak estrogen agonists/antagonists and some other enzymatic activities, isoflavones are increasingly advocated as a natural alternative to estrogen replacement therapy (ERT) and are available as dietary supplements. Puerarin is major isoflavonoid compound isolated from Pueraria lobata, a Chinese medicine known as Gegen. Our clinical study shows that puerarin can be used in the treatment of endometriosis, which improves pain and infertility. Assuming that the effect of puerarin on endometriosis may result from the regulation of aromatase expression or activity, we carried out this study to test the effects of puerarin on aromatase in Ishikawa and RL95-2 cell lines. Our data have demonstrated a significant decrease of P450 arom expression at both mRNA and protein levels by low dose puerarin treatment in both cell lines. Besides, we found that the −410/−401 bp and −565/−559 bp regions of aromatase promoter II contained the critical cis-acting elements, binding AP-1 and c-jun. We also found that puerarin exerted a time-course effect on the inhibition of c-jun mRNA, which parallelled that of P450 arom. To further confirm if c-jun is responsible for the P450 arom regulation by puerarin, we knocked down c-jun expression using siRNA and it indicates that c-jun acts as a considerable transcription factor in regulating P450 arom expression and activity. Accordingly, the suppression of P450 arom expression and activity by puerarin treatment may associate with the downregulation of transcription factor AP-1 or c-jun.

Cloning, Structure, and Expression Pattern of the P-450 Aromatase Gene in Rice Field Eel (Monopterus albus)

We report the cloning, tissue expression, and structural analysis of the aromatase gene in the rice field eel (Monopterus albus). The ovary-derived cDNA (1,802 bp) has a 49 bp 5'-untranslated region (UTR), a 202 bp 3'-UTR, and a 1,551 bp open-reading frame, which encodes a protein of 517 amino acid residues with a predicted molecular weight of 58.2 kDa. The amino acid sequence alignment suggests that the rice field eel ovarian P-450 aromatase shares 63-80% identity with that of other fish species, reduced to 59-60% with brain-derived aromatases of other fishes and to 50% with human placenta aromatases. Between the 5' and 3' untranslated terminal regions, the rice field eel CYP19 gene contained seven introns at the same sites as in medaka and human but lacked an intron between the I-helix and the aromatase-specific conserved region. All introns conformed to the GT/AG rule. Sequence analysis of the 1,065 bp upstream of the translation start site revealed that the transcription initiation site was 51 bp upstream from the translation start site. This region had one estrogen receptor recognition half site (nt -62), five copies of an SRY/iSRY binding motif, a C/EBP (CCAAT enhancer binding protein) binding site (nt -751), chicken ovalbumin upstream promoter-transcription factor (nt -986) and GATA-2 (nt -186, -249) recognition sequences, but no binding sequence for steroidogenic factor-1 and the cAMP response element binding protein activating transcription factor family. In females, levels of relative expression were, in descending order, hypothalamus, pituitary, forebrain, ovary, and liver. In males, P450arom was detected only in the pituitary and the liver, with half the expression found in females. In fry, the P450arom expression level increased during development and was significantly higher in the brain than in the gonad.

Primary cell cultures from fetal bovine hypothalamus and cerebral cortex: A reliable model to study P450Arom and α and β estrogen receptors in vitro

Estrogens synthesized by neural P450 aromatase (P450Arom) are implicated in many aspects of mammalian brain development and particularly in sexual differentiation of the central nervous system (CNS). This study analyzes the usefulness of an in vitro model based on bovine primary cell cultures from the hypothalamus and frontal cortex to investigate the role of P450Arom and estrogen receptors (ERs) in the development of fetal neural structures. The mRNA expression of P450Arom, ERalpha and ERbeta was detected using RT-PCR analysis in both hypothalamic and cortical primary cell cultures. P450Arom was identified and localized by immunocytochemistry in both neurons and astrocytes. Our results indicate that, within our experimental settings, astrocytes do not express ERalpha. The experimental model that we propose may represent a standardized dynamic model to study cellular and molecular mechanisms involved in the complex process of brain sexual differentiation.

Expression of P450arom, AMH and ER.ALPHA. mRNA in Gonads of Turkey, Duck and Goose within One Week of Age

Although many studies have shown that P450aromatase (P450arom), anti-Mullerian hormone (AMH) and estrogen receptor α (ERα) play pivotal roles in sexual differentiation of the gonads during early embryonic development in chickens and quail, few studies have been reported for other domestic birds. Furthermore, little information is available in relation to the mRNA expression in gonads after sexual differentiation in posthatching birds. The present study was conducted to assay mRNA expression of P450arom, AMH and ERα in gonads at day of hatch in turkey and duck and 2 days after hatching in goose using the real-time PCR. The mRNA expression was also determined at one week of age in gonads of these birds. At the time of collection of the left gonads for total RNA extraction, the external appearance of the left and right gonads of male and female was documented by digital camera. Clear asymmetry was observed in the female ovary in which the right ovary was regressed completely by 1-2 days in posthatching turkey, duck and goose. Although gonadal asymmetry was as remarkable as in females, the left testis was larger than the right one in males. Remarkable expression of P450arom mRNA was observed only in females in all the 3 species but substantially no expression was detected in males. Significantly higher expression of AMH mRNA was detected in males than females only in goose but there was no sex difference in turkey and duck at 1-2 days posthatching and one week of age. Weak ERα mRNA expression without a sex difference was detected in the 3 birds. These results suggest that estrogen plays a key role for ovarian development via P450arom mRNA expression after hatching, whereas absence of its expression in males leads to testis development in turkey, duck and goose.

Sex differences in steroidogenesis in skeletal muscle following a single bout of exercise in rats

Sex steroid hormones, such as testosterone and estradiol, play important roles in developing both strength and mass of skeletal muscle. Recently, we demonstrated that skeletal muscle can synthesize sex steroid hormones. Whether there are sex differences in basal steroidogenesis or acute exercise-induced alterations of steroidogenesis in the skeletal muscle is unknown. We examined sex differences in the levels of testosterone, estradiol, and steroidogenesis-related enzymes, such as 17beta-hydroxysteroid dehydrogenase (HSD), 3beta-HSD, and aromatase cytochrome P-450 (P450arom), in the skeletal muscle at rest and after exercise. We studied the gastrocnemius muscles of resting rats (10 wk old) and exercised rats (10 wk old, treadmill running, 30 m/min, 30 min). Basal muscular testosterone levels were higher in males than females, whereas estradiol did not differ between sexes. Additionally, 17beta-HSD, 3beta-HSD, and P450arom transcript and protein expression were greater in females. After acute exercise, testosterone levels and 17beta-HSD expression increased in muscle in both sexes. By comparison, muscular estradiol levels increased in males following exercise but were unchanged in females. Expression of P450arom, which regulates estrogen synthesis, increased after acute exercise in males but decreased after exercise in females. Thus a single bout of exercise can influence the steroidogenic system in skeletal muscle, and these alterations differ between sexes. The acute exercise-induced alteration of steroidogenic enzymes may enhance the local steroidogenesis in the skeletal muscle in both sexes.

Effects of Daidzein on mRNA Expression of Gonadotropin Receptors and P450 Aromatase in Ovarian Follicles of White Silky Fowls

Effects of daidzein on expression of mRNAs of gonadotropin receptors (FSHR, LHR) and P450 aromatase (P450arom) were evaluated in ovarian follicles of white silky fowls. The hens were 13 months old in the post-peak period of egg laying and were randomly allocated as control and daidzein-treated groups, with daidzein supplemented to the basal diet at 10 mg/kg for 7 consecutive weeks. The mRNA expression of related genes was measured by semi-quantitative RT-PCR in the granulosa layers of the preovulatory follicle (PRF: Fl, F2 ...) and follicular layers of the small yellow follicle (SYF), large white follicle (LWF) and atretic follicle (ATF). Results showed that daidzein supplementation significantly increased the number of SYF and LWF (p<0.05). The relative abundance of the FSHR mRNA decreased in the granulosa layers from F3 to Fl, but LHR mRNA displayed opposite developmental changes. P450arom mRNA was highest in the SYF, but was very low in the granulosa layers after follicles finished selection. Treatment with daidzein resulted in increased mRNA expression of FSHR in F3 granulosa layer, LHR in granulosa layers of F3 to Fl and P450arom in LWF (p<0.05). These results indicated that dietary supplementation of daidzein up-regulated mRNA expression of gonadotropin receptors and P450arom to improve the development of preovulatory follicles in white silky fowls after the peak-laying period.

Evolution of Suiform Aromatases: Ancestral Duplication with Conservation of Tissue-Specific Expression in the Collared Peccary (Pecari tayassu)

Aromatase cytochrome P450 (P450arom), the enzyme that catalyzes estrogen synthesis, is required for successful reproduction and is encoded by a single copy gene (CYP19) in most mammals. However, pigs and their distant suiform relatives the peccaries experienced CYP19 duplication. Here, the evolutionary origin of CYP19 duplication, and the evolution of the gene paralogs, was explored further in collared peccaries (Pecari tayassu). Exons IV and V, and the intervening intron, representing duplicated CYP19 genes, were cloned and sequenced from collared peccary, pig, and hippopotamus. Sequence alignment and analysis identified a gene conversion in collared peccary with a breakpoint 102 base pairs (bp) upstream of exon V. Phylogenetic analyses of nucleotide and amino acid sequence upstream of the breakpoint supported a tree in which one peccary sequence was orthologous with the porcine gonadal gene. Cloning and sequencing of tissue transcripts, using reverse-transcriptase polymerase chain reaction techniques (RT-PCR), confirmed that the gonadal ortholog was expressed in collared peccary testis. Orthology of the other genomic sequence with the porcine placental gene was not resolved, but its placenta-specific expression in collared peccary was confirmed by similar transcript analysis. Immunoblot and enzyme activity in collared peccary testes demonstrated much lower levels of P450arom than in pig testis. Collared peccary placental P450arom expression also seemed much lower than pigs. Thus, suiform CYP19 genes arose from an ancestral duplication that has maintained gonad- and placenta-specific expression, but at lower levels in peccaries than pigs, perhaps facilitating the emergence of different reproductive strategies as Suiformes diverged and evolved.

Aromatase cytochrome P450 enzyme expression in human pituitary

Aromatase (P450AROM) converts testosterone to estrogen. This conversion could be important in normal physiology and estradiol-induced tumorigenesis in human pituitary. The objective of this study was to examine the expression of P450AROM in normal human pituitary and determine the gender difference. We examined aromatase expression in 19 normal human pituitary glands [13 males, 6 females, median age: 30 years (interquartile ranges, IQR: 23-63)] obtained from autopsy. We demonstrated aromatase gene expression levels by quantitative RT-PCR and aromatase protein with immunohistochemical staining in normal male and female human pituitary. Although median relative expression level of aromatase mRNA of male individuals [median DeltaCt = 42.6 (IQR: 7.6-93.9)] was higher than the female individuals [median DeltaCt = 3.9 (IQR:0-44.8)], we could not determine a significant gender difference in aromatase mRNA levels (p = 0.2). The difference between the aromatase protein density by immunohistochemistry was not significant between genders (p = 0.78). The aromatase levels were also not correlated with the age of the study subjects (p = 0.42 r = -0.21). The results indicate that aromatase enzyme is present in human pituitary. The amount and the density of the enzyme show a large variance among different individuals. Although higher mRNA expression was observed in male pituitary compared to female pituitary, there was no statistically significant difference for gender or age.

Ontogenesis of brain aromatase P450 expression in the bovine hypothalamus

Aromatase P450 (P450 AROM), converting testosterone (T) into estradiol (E), plays an important role in sexual differentiation of neural structures in the developing mammalian brain. The aim of the present study was to characterize the qualitative and quantitative profile of P450 AROM mRNA expression in the bovine hypothalamus (the region of the central nervous system in which the enzyme is mainly localized) using RT-PCR and quantitative real-time RT-PCR analysis, respectively. P450 AROM expression was examined in the developing hypothalamus in a series of experimental groups investigated at 10 weeks interval one from the other. Our data indicate that in the bovine fetal hypothalamus P450 AROM expression peaks at the second quarter of gestation. The presence of neural cells containing P450 AROM in the bovine fetal hypothalamus was confirmed by immunohistochemistry, and localized in the medial preoptic area. We conclude that second quarter of the gestation is the developmental stage which represents a critical period for hypothalamic differentiation in bovine ontogenesis, an important difference with the rat and mouse, short gestation species in which P450 AROM activity peaks around delivery.

Tamoxifen induces masculinization of genetic females and regulates P450 aromatase and müllerian inhibiting substance mRNA expression in Japanese flounder (Paralichthys olivaceus)

Japanese flounder, Paralichthys olivaceus, provides an excellent model to elucidate the roles of sex steroid hormones in gonadal sex differentiation because the sex is easily altered by sex steroid treatments or water temperature control during the sex differentiation. We have previously shown that high water temperature, an aromatase inhibitor (fadrozole), or 17alpha-methyltestosterone treatment causes the sex-reversal from genetic females to phenotypic males and suppression of mRNA expression of ovary-type P450 aromatase (P450arom), which is a steroidogenic enzyme responsible for the conversion of androgens to estrogens, in Japanese flounder. In the present study, we demonstrate that treatment of the genetic females with anti-estrogen (tamoxifen) leads to their masculinization, suppresses P450arom mRNA expression, and induces mRNA expression of Müllerian inhibiting substance (MIS), a member of the transforming growth factor-beta (TGF-beta) superfamily, while it has no effect on mRNAs expression of estrogen receptor-alpha (ERalpha) and ERbeta. In contrast, 17beta-estradiol counteracted masculinization of the genetic females by tamoxifen or high water temperature treatment, up-regulated P450arom mRNA expression, and down-regulated MIS mRNA expression. These results strongly suggest that estrogen signaling through ERs dramatically influences the gonadal sex differentiation by regulating P450arom and MIS mRNA expression.

DNA methylation and chromatin accessibility of the proximal Cyp19 promoter region 1.5/2 correlate with expression levels in sheep placentomes

Placental oestrogens play an important role as local regulators of placental growth and differentiation during gestation, and toward term they are also involved in the preparation of parturition. They are synthesized within the fetal cotyledons of placentomes by aromatase cytochrome P450 (P450arom; EC 1.14.14.1), the product of the Cyp 19 gene. The first step of regulation of P450arom expression, and hence enzyme activity and oestrogen production, takes place at the level of Cyp 19 transcription, which is driven by a proximal promoter region, P1.5/2, in the sheep placenta. The aim of the present study was to find out if different Cyp 19 expression levels, which previously had been observed in ovine placentome tissues, correlate with the tissue-specific chromatin structure of the promoter. To this end, we investigated the chromatin structure across the P1.5/2 region in caruncles and cotyledons from 100 and 125 days pregnant ewes, and in term placentae, respectively, by analyzing the DNA methylation and the accessibility to restriction digestion. Our data show that: (1) cotyledonal DNA was significantly lower methylated than caruncular DNA; (2) methylation of cotyledonal DNA was low at 100 and 125 days of pregnancy, and increased to a significant higher level in term placentae; and (3) concurrently, cotyledonal chromatin became inaccessible to restriction digestion at term of gestation. The results imply that DNA methylation and chromatin accessibility of the P1.5/2 promoter region correlate with expression levels of the Cyp 19 gene.