Aromatase Isoforms Research Articles

Porcine-specific expression ofthe three functional CYP19paralogs in early conceptus, placenta, and gonads.

Although three porcine aromatase isoforms have been identified, their gene expression profiles in reproduction are still poorly understood. Here, we identified by Sanger sequencing unique nucleotide signatures for the three paralogous copies of Cyp19 and analyzed by RT-PCR the occurrence of the Cyp19 and Cyp17a1 transcripts at different tissues and stages of conceptus and fetal-placental development. Cyp19a1 and Cyp19a3 expression was detected in conceptuses and gonads, respectively. Cyp19a2 transcripts were identified on both conceptuses and placenta samples. Transcripts for Cyp17a1 were detected predominantly in conceptus and gonads. In the endometrium of Day 21 pregnant, as well as Days 12 and 17 pseudopregnant females, we did not detect expression of Cyp19a1, Cyp19a2 or Cyp19a3. In our study we have demonstrated distinct transcriptional regulation for the three functional Cyp19 paralogs and a potential role for Cyp17a1 in controlling secretion of estrogen from the conceptus and the placenta.

Plastic originated bisphenol A: A potential endocrine disruptor for fish reproduction

Bisphenol A (BPA) is an organic synthetic compound, abundantly used worldwide for the production of polycarbonate plastic and epoxy resins. It is an endocrine disruptor that can mimic estradiol by binding to and activating the same estrogen receptor as the natural hormone. It is not only hazardous to human population but also found to be acutely toxic to aquatic organisms in the range 1000–10,000μg/L for freshwater and marine species. In 2010, the U.S. Environmental Protection Agency reported that over one million pounds of BPA are released into the environment annually. BPA can enter the environment either directly from chemical, plastics, coat and staining manufacturers, from paper or material recycling companies, or indirectly leaching from plastic, paper and metal waste in landfills or ocean-borne plastic trash. BPA in anaerobic or semi aerobic sediment environments can persist for a prolonged period of time, leading to higher BPA levels in sediments than in surface water. Interestingly BPA can persist longer in seawater than in fresh water without any degradation (about 30 day) and the possibility of BPA contamination is higher marine than freshwater organisms. BPA possess endocrine disruption in different types of fishes by vitellogenin induction, upregulation of brain aromatase isoform mRNA, reduction of total sperm counts and induction of testis ova and poor somatic growth of male. It was also observed that environmentally relevant low level of BPA increased the expression of genes related to reproduction axis such as kiss1, kiss1r, Gnrh3, LH β, FSHβ, and ERα and dmrt1. BPA is also assumed to involve THR, in increasing the rate of early embryonic development in several fish species. Further, BPA exhibit very high estrogenic activity on the cyp19a1b gene, and increase concentration of vitellogenin in swim-up fry of freshwater fish species. Keeping in view of these, the present study is aimed to elucidate a baseline information about contamination routes of BPA in the aquatic environment and its endocrine-disruptive effects on aquatic organisms.

Brain Aromatase Modulates Serotonergic Neuron by Regulating Serotonin Levels in Zebrafish Embryos and Larvae.

Teleost fish are known to express two isoforms of P450 aromatase, a key enzyme for estrogen synthesis. One of the isoforms, brain aromatase (AroB), cyp19a1b, is highly expressed during early development of zebrafish, thereby suggesting its role in brain development. On the other hand, early development of serotonergic neuron, one of the major monoamine neurons, is considered to play an important role in neurogenesis. Therefore, in this study, we investigated the role of AroB in development of serotonergic neuron by testing the effects of (1) estradiol (E2) exposure and (2) morpholino (MO)-mediated AroB knockdown. When embryos were exposed to E2, the effects were biphasic. The low dose of E2 (0.005 µM) significantly increased serotonin (5-HT) positive area at 48 hour post-fertilization (hpf) detected by immunohistochemistry and relative mRNA levels of tryptophan hydroxylase isoforms (tph1a, tph1b, and tph2) at 96 hpf measured by semi-quantitative PCR. To test the effects on serotonin transmission, heart rate and thigmotaxis, an indicator of anxiety, were analyzed. The low dose also significantly increased heart rate at 48 hpf and decreased thigmotaxis. The high dose of E2 (1 µM) exhibited opposite effects in all parameters. The effects of both low and high doses were reversed by addition of estrogen receptor (ER) blocker, ICI 182,780, thereby suggesting that the effects were mediated through ER. When AroB MO was injected to fertilized eggs, 5-HT-positive area was significantly decreased, while the significant decrease in relative tph mRNA levels was found only with tph2 but not with two other isoforms. AroB MO also decreased heart rate and increased thigmotaxis. All the effects were rescued by co-injection with AroB mRNA and by exposure to E2. Taken together, this study demonstrates the role of brain aromatase in development of serotonergic neuron in zebrafish embryos and larvae, implying that brain-formed estrogen is an important factor to sustain early development of serotonergic neuron.

Expression of estrogen, estrogen related and androgen receptors in adrenal cortex of intact adult male and female rats.

Adrenocortical activity in various species is sensitive to androgens and estrogens. They may affect adrenal cortex growth and functioning either via central pathways (CRH and ACTH) or directly, via specific receptors expressed in the cortex and/or by interfering with adrenocortical enzymes, among them those involved in steroidogenesis. Only limited data on expression of androgen and estrogen receptors in adrenal glands are available. Therefore the present study aimed to characterize, at the level of mRNA, expression of these receptors in specific components of adrenal cortex of intact adult male and female rats. Studies were performed on adult male and female (estrus) Wistar rats. Total RNA was isolated from adrenal zona glomerulosa (ZG) and fasciculate/reticularis (ZF/R). Expression of genes were evaluated by means of Affymetrix® Rat Gene 1.1 ST Array Strip and QPCR. By means of Affymetrix® Rat Gene 1.1 ST Array we examined adrenocortical sex differences in the expression of nearly 30,000 genes. All data were analyzed in relation to the adrenals of the male rats. 32 genes were differentially expressed in ZG, and 233 genes in ZF/R. In the ZG expression levels of 24 genes were lower and 8 higher in female rats. The more distinct sex differences were observed in the ZF/R, in which expression levels of 146 genes were lower and 87 genes higher in female rats. Performed analyses did not reveal sex differences in the expression levels of both androgen (AR) and estrogen (ER) receptor genes in the adrenal cortex of male and female rats. Therefore matrix data were validated by QPCR. QPCR revealed higher expression levels of AR gene both in ZG and ZF/R of male than female rats. On the other hand, QPCR did not reveal sex-related differences in the expression levels of ERα, ERβ and non-genomic GPR30 (GPER-1) receptor. Of those genes expression levels of ERα genes were the highest. In studied adrenal samples the relative expression of ERα mRNA was higher than ERβ mRNA. In adrenals of adult male and female rats expression levels of estrogen-related receptors ERRα and ERRβ were similar, and only in the ZF/R of female rats ERRγ expression levels were significantly higher than in males. We also analyzed expression profile of three isoforms of steroid 5α-reductase (Srd5a1, Srd5a2 and Srd5a3) and aromatase (Cyp19a1) and expression levels of all these genes were similar in ZG and ZF/R of male and female rats. In contrast to Affymetrix microarray data QPCR revealed higher expression levels of AR gene in adrenal glands of the male rats. In adrenals of both sexes expression levels of ERa, ERb, non-genomic GPR30 (GPER-1), ERR α and ERRβ receptors were comparable. The obtained results suggest that acute steroidogenic effect of estrogens on corticosteroid secretion may be mediated by non-genomic GPR30.

Differential expression patterns of three aromatase genes and of four estrogen receptors genes in the testes of trout (Oncorhynchus mykiss).

Estrogens are implicated in male gonad function, although their physiological roles remain uncertain. In the present study, we take advantage of the original model of spatio-temporal organization of trout spermatogenesis to revisit the synthesis and action sites of estrogens in fish testis. Within this system, somatic cell and germ cell development are synchronized due to a strict seasonal spermatogenetic cycle and the cystic organization of gonads. We evaluated the expression patterns and regulation of three aromatase isoforms (cyp19a, cyp19b-I, and cyp19b-II) and four estrogen receptors (esr1a, esr1b, esr2a, and esr2b) by quantitative reverse-transcriptase PCR during testicular maturation and in isolated germ cell populations. Our data demonstrated a reciprocal relationship between cyp19a and cyp19b (I and II) expression during testicular development (cyp19a decreased while cyp19b increased with maturation). Furthermore, cyp19b is significantly expressed in late germ cells. At the protein level, aromatase was immunohistochemically identified in interstitial tissue and in germ cells. Remarkable elevation of esr1a and esr2a was observed during the final stage of spermiation, while esr1b was expressed in an early stage of spermatogenetic development. Estrogen implants reduced testicular cyp19a transcript abundance while up-regulating cyp19b levels, whereas androgens up-regulated testicular esr1a, esr2a, and esr2b. Together, the distinct spatio-temporal expression profiles and regulation of aromatases and estrogen receptors suggest that estrogens have discrete physiological functions during an early step of spermatogenesis and in the final stages of germ cell maturation and/or excretion.

Disruptions in aromatase expression in the brain, reproductive behavior, and secondary sexual characteristics in male guppies (Poecilia reticulata) induced by tributyltin

Although bioaccumulation of tributyltin (TBT) in fish has been confirmed, information on possible effects of TBT on reproductive system of fish is still relatively scarce, particularly at environmentally relevant levels. To evaluate the adverse effects and intrinsic toxicological properties of TBT in male fish, we studied aromatase gene expression in the brain, sex steroid contents, primary and secondary sexual characteristics, and reproductive behavior in male guppies (Poecilia reticulata) exposed to tributyltin chloride at the nominal concentrations of 5, 50, and 500ng/L for 28 days in a semi-static exposure system. Radioimmunoassay demonstrated that treatment with 50ng/L TBT caused an increase in systemic levels of testosterone of male guppies. Gonopodial index, which showed a positive correlation with testosterone levels, was elevated in the 5ng/L and 50ng/L TBT treated groups. Real-time PCR revealed that TBT exposure had inhibiting effects on expression of two isoforms of guppy aromatase in the brain, and these changes at the molecular levels were associated with a disturbance of reproductive behavior of the individuals, as measured by decreases in frequencies of posturing, sigmoid display, and chase activities when males were paired with females. This study provides the first evidence that TBT can cause abnormalities of secondary sexual characteristics in teleosts and that suppression of reproductive behavior in teleosts by TBT is due to its endocrine–disrupting action as an aromatase inhibitor targeting the nervous system.

Inhibition of the Brain Cytochrome P450 Aromatase Isoform Expression in Jenynsia multidentata Reflects Changes in Water Quality

Cytochrome P450 aromatase is the steroidogenic enzyme that catalyzes the conversion of androgens to estrogens. Teleosts express two structurally and functionally different P450 aromatase isoforms, termed Cytochrome 19a1a and Cytochrome 19a1b. The first is preferentially expressed in ovary while the second is constitutively expressed in brain. The aim of the present study was to evaluate changes in cyp19a1b expression in brain of Jenynsia multidentata collected in Suquía river (Córdoba, Argentina) in order to assess if this biological response reflects changes in water quality. For this purpose we first identified cyp19a1b parcial cDNA sequence and adjusted the method to quantify mRNA expression by qRT-PCR (quantitative RT-PCR). Then, water and fish samples were collected in five monitoring stations located along a pollution gradient in Suquía river. The qRT-PCR analysis of female brains reveals that cyp19a1b mRNA levels change as water pollution does, showing the highest levels at both unpolluted and recovered areas, with suppression as pollution increases. Even when cyp19a1b expression in J. multidentata will need further studies, the results here presented indicate that this biological response appear as a promising biomarker of water pollution that may also point out the probable presence of endocrine disruptors.

Neuroendocrine Effects of Endocrine Disruptors in Teleost Fish

Because a large proportion of potential endocrine disruptors (EDC) end up in surface waters, aquatic species are particularly vulnerable to their potential adverse effects. Recent studies identified a number of brain targets for EDC commonly present in environmentally relevant concentrations in surface waters. Among those neuronal systems disrupted by EDC are the gonadotropin-releasing hormone (GnRH) neurons, the dopaminergic and serotoninergic circuits, and more recently the Kiss/GPR54 system, which regulates gonadotropin release. However, one of the most striking effects of EDC, notably estrogen mimics, is their impact on the cyp19a1b gene that encodes the brain aromatase isoform in fish. Moreover, this is the only example in which the molecular basis of endocrine disruption is fully understood. The aims of this review were to (1) synthesize the most recent discoveries concerning the EDC effects upon neuroendocrine systems of fish and (2) provide, when possible, the underlying molecular basis of disruption for each system concerned. The potential adverse effects of EDC on neurogenesis, puberty, and brain sexualization are also described. It is important to point out the future environmental, social, and economical issues arising from endocrine disruption studies in the context of risk assessment.

Cloning of <I>CYP19a</I> gene and expression analysis during the reproductive cycle in males of <I>Verasper moseri</I>

Estrogens are key players in sexual differentiation and development and their production is essential for normal sexual development in both females and males. Cytochrome P450 aromatase is the terminal enzyme in steroidogenic pathway that converts androgens into estrogens. In most of vertebrates,P450arom is the product of the CYP19 gene,which exists as a single copy per haploid genome. In contrast,there are two isoforms of aromatase in teleosts,which are encoded by two different genes,CYP19a and CYP19b,as well as have different distribution. This work describes the molecular cloning of the cDNA encoding the barfin flounder(Verasper moseri) gonad cytochrome P450 aromatase(CYP19a) by means of reverse transcriptase polymerase chain reaction(RT-PCR),degenerate primers PCR amplification and 5'-and 3'-rapid amplification of cDNA ends(RACE) analyses. We cloned the complete cDNA sequence of CYP19a gene and the length is 2 167 bp,encoding a protein of 523 amino acids. Via Blastn,the CYP19a cDNA displayed high identity with Atlantic halibut(Hippoglossus hippoglossus),Japanese flounder(Paralichthys olivaceus) and southern flounder(Paralichthys lethostigma). Alignment analysis using barfin flounder CYP19a gene deduced animo acid sequence and animo acid sequences of other species showed that there were transmembrane helix region,I-helix region,Ozol's peptide region,aromatase-specific conserved region and heme-binding region in the deduced animo acid sequence. Phylogenetic analysis suggested the occurrence of a gene duplication for cytochrome P450 aromatase in the teleost lineage after its divergence from the tetrapods. Furthermore,the higher identity between barfin flounder P450aromA and P450aromAs of other teleosts suggested that they have the same origin. A semiquantitative PCR was developed to measure mRNA expression levels of CYP19a gene of barfin flounder. Expression analysis in different tissues demonstrated that P450aromA transcripts were highly abundant in brain,ovary and testis,less abundant in intestine,liver and kidney,showing that not only gonads can produce steroids but also other tissues have steroidogenic capability. And we analyzed the relative mRNA expression level of CYP19a in testes at different stages of reproductive cycle,there were the highest level at stageⅡ and the lowest level at stageⅤ. These changes in expression profiles may imply differences in the functionality of the enzyme between different stages,suggesting an important role for P450aromA in barfin flounder testes development.

Peroxisome proliferator-activated receptors, estrogenic responses and biotransformation system in the liver of salmon exposed to tributyltin and second messenger activator

The mechanisms by which organotin compounds produce modulations of the endocrine systems and other biological responses are not fully understood. In this study, juvenile salmon were force-fed diet containing TBT (0: solvent control, 0.1, 1 and 10 mg/kg fish) for 72 h. Subsequently, fish exposed to solvent control and 10 mg TBT were exposed to waterborne concentration (200 μg/l) of the adenylate cyclase (AC) stimulator, forskolin for 2 and 4 h. The overall aim of the study was to explore whether TBT endocrine disruptive effects involve second messenger activation. Liver was sampled from individual fish ( n = 8) at the end of the exposures. The transcription patterns of peroxisome proliferator-activated receptor (PPAR) isotype and acyl-coenzyme A oxidase 1 (ACOX1), aromatase isoform, estrogen receptor-α (ERα), pregnane X receptor (PXR), CYP3A and glutathione S-transferase (GST) genes were measured by quantitative polymerase chain reaction (qPCR). Our data showed a consistent increase in PPARα, PPARβ and PPARγ mRNA and protein expression after TBT exposure that were inversely correlated with ACOX1 mRNA levels. Forskolin produced PPAR isotype-specific mRNA and protein effects that were modulated by TBT. ACOX1 expression was decreased (at 2 h) and increased (at 4 h) by forskolin and the presence of TBT potentiated these effects. TBT apparently increased mRNA and protein levels of cyp19a, compared to the solvent control, whereas cyp19b mRNA levels were unaffected by TBT treatment. Combined TBT and forskolin exposure produced respective decrease and increase of mRNA levels of cyp19a and cyp19b, compared with control. TBT decreased ERα mRNA at low dose (1 mg/kg) and forskolin exposure alone produced a consistent decrease of ERα mRNA levels that were not affected by the presence of TBT. Interestingly, PXR and CYP3A mRNA levels were differentially affected, either decreased or increased, after exposure to TBT and forskolin, singly and also in combination. GST mRNA was increased by TBT exposure. Exposure to forskolin alone increased GST expression with time, and combined exposure with TBT potentiated these respective effects. Overall, the present study demonstrates multiple biological effects of TBT given singly or in combination with cAMP activator. There are no studies known to us that have evaluated the endocrine disruptive effects of TBT in the presence of a second messenger activator, and our data suggest that TBT may exert endocrine, biotransformation and lipid peroxidative effects through modulation of cAMP/PKA second messenger signaling with overt physiological consequences.

Regulation, expression and characterization of aromatase (cyp19b1) transcripts in ovary and testis of rainbow trout (Oncorhynchus mykiss)

Cytochrome P450 aromatase is the key enzyme in the pathway that converts androgens to estrogens. The enzyme functions in the smooth endoplasmic reticulum in a complex with NADPH-cytochrome P450 reductase. In teleost fish, at least two separate loci, cyp19a and cyp19b, encode distinct aromatase isoforms. The activity of cyp19a and cyp19b are predominantly associated with the ovary and brain, respectively, although their expression is not confined solely to these tissues. We found that at least five cyp19b1 transcripts with different 5'-UTRs are generated in the ovary and testis of rainbow trout. Regulation for selection of these variants may be through signals present in exon 2 that recruit alternative splicing factors. Also, binding elements for FOXL2 and SF-1 located within the cyp19b1 intron 1 may influence formation of transcripts that contain the 3'-end of the intron. Another transcript devoid of the exon 2 methionine initiator codon may utilize other downstream in-frame start codons. Less developed stages of ovarian and testicular tissues express only the intron-containing transcripts whereas precocious and more mature gonads express all five cyp19b1 messages. The function of these different 5'-UTRs may be for regulation of cyp19b1 at particular developmental stages or to specify control in distinct gonadal cell-types.

Brain aromatase mRNA expression in two populations of the peacock blenny Salaria pavo with divergent mating systems

Aromatase, the key enzyme in the conversion of androgens to estrogens, regulates the availability of these hormones in tissues and controls many physiological and behavioral processes. In fish and other vertebrates, the regulation of aromatase expression in the brain has been implicated in the modulation of male sexual and aggressive behaviors. Here, the pattern of mRNA expression of the brain aromatase isoform (encoded by the CYP19A2 gene also referred as CYP19b) was quantified at the peak of spawning season in brain macroareas from males and females of the blenny Salaria pavo originated from two populations displaying male alternative reproductive tactics but differing in their mating systems. In Trieste (Adriatic) nesting males aggressively defend nests and take the initiative in courtship and perform sexual displays more often than females while in Ria Formosa (Southern Portugal) the pattern is reversed as a result of shortage of appropriate nesting sites. Nesting males from Ria Formosa had overall higher levels of brain aromatase mRNA expression than nesting males from Trieste, suggesting a higher brain estrogen synthesis in these males. Since in some fish species exogenous estradiol administration has been shown to decrease sexual and agonistic behaviors, the higher levels of brain aromatase in Ria Formosa nesting males may explain their reduced expression of sexual and aggressive displays when compared with nesting males from Trieste. Alternatively, the higher brain aromatase levels in nesting males from Ria Formosa could be a mechanism to decrease the putative androgen-induced activation of aggressive and sexual displays by reducing the local availability of androgens through their metabolization into estrogens. Although females and parasitic female-like males also differ in their displays between populations, the interpopulational pattern of brain aromatase mRNA expression was similar, suggesting that other neuroendocrine agents mediate the expression of female and female-like behaviors. In conclusion, brain aromatase availability seems like a probable mechanism to regulate the effects of steroids on the brain circuits underlying the expression of sexual and agonistic displays in S. pavo.

Modulation of salmon ovarian steroidogenesis and growth factor responses by the xenoestrogen, 4-nonylphenol

Endocrine-disrupting chemicals are known to influence organismal reproductive processes, including the production and regulation of gonadal steroids. This study evaluated the effects of a xenoestrogen (nonylphenol: NP) on salmon ovarian steroidogenesis and growth factors using an in vitro organ culture system. Ovarian tissues were cultivated for 3 and 7 d with different concentrations of NP (0 (control), 1, 10 and 50 μM) dissolved in ethanol (0.1%). The mRNA expressions of steroidogenic acute regulatory (StAR) protein, P450-mediated cholesterol side-chain cleavage (P450 scc), aromatase isoforms, 3β-hydroxysteroid dehydrogenase (3β-HSD), Cyp11β-, Cyp17 and 21-hydroxylase, and insulin-like growth factors (IGF-1 and IGF-2) and IGF1-receptor (IGF1-R) were quantified by real-time PCR. Tissue levels of estradiol-17β (E2), testosterone (T) and 11-ketotestosterone (11-KT) were quantified using enzyme immunoassays. Our data show that nominal NP levels produced time- and concentration-specific effects on the expression of steroidogenesis- and IGF-related transcripts in salmon ovarian tissues. Tissue levels of ovarian E2, T and 11-KT were significantly modulated after NP exposure. Interestingly, elevated ovarian E2 levels after 10 μM NP exposure at day 3 paralleled P450 Arom isoforms mRNA expression at the same time interval. The expression patterns of other steroidogenic protein and enzyme genes, such as StAR, P450 scc, 3β-HSD and Cyp17 inversely paralleled this pattern, displaying consistent decreased transcript levels. These findings show that NP (an ubiquitous environmental pollutant) can produce variations in gonadal steroidogenesis and growth regulating responses with potential consequences for overt fecundity in teleosts.

Temporal expression of two Cytochrome P450 Aromatase isoforms during development in Oreochromis mossambicus, in association with histological development

Oreochromis mossambicus is targeted as a sentinel species for assay development to detect disruption of estrogen-dependent pathways in Southern Africa. Aromatase — an enzyme encoded by either of two cytochrome P450 19 ( cyp19) genes in O. mossambicus is the only enzyme able to catalyze the aromatization of androgens to estrogens. As part of background studies relating to evaluating the use of early life stages of O. mossambicus as an endocrine disruptor screen, we investigated the tissue specificity and temporal expression of cyp19 transcripts, cyp19a (ovarian cyp19) and cyp19b (brain cyp19) during development and estrogen exposure. The cyp19a transcript was detected only in the ovaries of adults, and expression during development reflected this result as induction of cyp19a coincides with histological development of putative ovaries. Using primers that only identify the ovarian transcript, cyp19a transcript levels demonstrated a typical reflection of puberty — after an initial surge in cyp19a, juveniles expressed very low levels, which increased again at the time histologically discernable vitellogenic ovaries were detected. Moreover, we found evidence of putative alternate transcript of cyp19a whose function is currently unknown. cyp19b transcripts were expressed in brain and muscle tissue of both male and female adults, in addition to ovaries in females. During development, cyp19b transcript levels were increased coincidental with cyp19a at 20 days post fertilization but the expression pattern was distinct from that observed for cyp19a. These studies set the foundation for utilizing this native species as a possible indicator of endocrine disruption and accentuates the importance of understanding “normal” basal levels of transcript levels and the nature of amplification of QPCR targets.

Altered Sexual Development in Roach (Rutilus rutilus) Exposed to Environmental Concentrations of the Pharmaceutical 17α-Ethinylestradiol and Associated Expression Dynamics of Aromatases and Estrogen Receptors

Wild roach (Rutilus rutilus) inhabiting UK rivers contaminated with estrogenic effluents from wastewater treatment works show altered sexual development, including intersex, and this can impact negatively on their reproductive capabilities. The molecular events underlying these disruptions in gender assignment, however, are still poorly understood. In this study, two isoforms of aromatase (cyp19a1a and cyp19a1b) were cloned from the roach, and effects of exposure to 17alpha-ethinylestradiol (EE(2)) during early life were determined on the expression of both aromatases and on the estrogen receptors (ERs) (subtypes esr1 and esr2b) and analyzed against effects on the progression of gonadal sex differentiation. Exposure to environmentally relevant concentrations of EE(2) during the critical period of sex differentiation resulted in gonadal feminization and all roach exposed to 4 ng EE(2)/l were females. These effects on gonadal development were associated with alterations in the expression of both esr and cyp19a1 genes in bodies and heads of exposed fish with the most marked effects on the expression of esr1 and cyp19a1b. Our findings show that both aromatase isoforms and both ER subtypes are associated with sexual differentiation in roach, and alterations in their expression can signal for disruptions in sexual development.

Benzo( a)pyrene decreases brain and ovarian aromatase mRNA expression in Fundulus heteroclitus

The higher molecular weight polycyclic aromatic hydrocarbons (PAHs) such as benzo( a)pyrene (BaP) are typically associated with genotoxicity, however, newer evidence suggests that these compounds may also act as endocrine system disruptors. We hypothesized that altered expression of the P450 enzyme aromatase genes could be a target for reproductive or developmental dysfunction caused by BaP exposure. Aromatase is at least partially responsible for estrogen homeostasis by converting androgens into estrogens. In fish, there are two isoforms of aromatase, a predominantly ovarian form, CYP19A1, and a brain form, CYP19A2. CYP19 mRNA expression was measured following BaP exposure (0, 10, 100 μg/L waterborne for 10 or 15 days) in Fundulus adults, juveniles and embryos by in situ hybridization. The CYP19A1 expression was significantly decreased after BaP exposure in the 3-month-old Fundulus immature oocytes, but BaP did not affect CYP19A1 expression at any stage in adult oocytes. In embryo brains, BaP significantly decreased CYP19A2 compared to controls by 3.6-fold at 14 days post-fertilization. In adults, CYP19A2 expression was decreased significantly in the pituitary and hypothalamus (81% and 85% of controls, respectively). Promoter regions of Fundulus CYP19s were cloned, and putative response elements in the CYP19A1 and CYP19A2 promoters such as CRE, AhR and ERE may be involved in BaP-mediated changes in CYP19 expression. In order to compare the mechanism of BaP-mediated inhibition with that of a known aromatase inhibitor, fish were also exposed to fadrozole (20 and 100 μg/L). Fadrozole did not significantly decrease the mRNA expression in embryos or adult Fundulus. However, aromatase enzyme activity was significantly decreased in adult ovary and brain tissues. These studies provide a greater molecular understanding of the mechanisms of action of BaP and its potential to impact reproduction or development.

Distinct cytochrome P450 aromatase isoforms in the common carp (Cyprinus carpio): Sexual dimorphism and onset of ontogenic expression

Cytochrome P450 aromatase (CYP19) is a key enzyme in the steroidogenic pathway that catalyses the conversion of testosterone to estrogen, and therefore is thought to influence gonadal sex differentiation. In an effort to understand the role of this enzyme in ovarian differentiation, we isolated cDNA encoding the two distinct isoforms, ovarian and brain (termed cyp19a and cyp19b, respectively) of adult common carp, Cyprinus carpio. The cloned cDNA for cyp19a had an open reading frame (ORF) of 518 amino acid residues, in contrast to cyp19b with an ORF of 511 amino acids. Sequence and phylogenetic analysis showed that these CYP19 isoforms were orthologous with previously described cyp19a and cyp19b from other teleosts. Quantitative real-time PCR indicated that both isoforms are expressed in adult ovary and brain, with predominant expression of cyp19a in the ovary and cyp19b in the brain. The major aromatase expressing tissue was found to be the brain, with greatest cyp19b expression in the anterior quarter (telencephalon) in both sexes. The gonad showed sexually dimorphic expression of both genes and dimorphic expression of cyp19a was observed in the cerebellum and the liver. Ontogenic expression showed that only the ovarian aromatase transcript is inherited maternally, with lower expression observed through early larval development under warmer rearing conditions. The differential and overlapping expression suggests these two aromatase genes have different roles in reproductive physiology.

57-Isoform cytochromes P450 for bioconversions

There are likely to be up to 4500 isoforms of CYP in biota, including 57 in human tissues for isoform substrate specificities. These isoforms display differential inhibition by a wide range of biochemical drug-candidates. Drugs are targeted to CYP19 (aromatase) and CYP17 (17α-hydroxylase of sterols). The CYP19 isoform is rate limiting in oestrogen production in the body as it controls the biotransformation of testosterone to oestradiol by aromatisation of steroid ring A. A range of azoles is being developed for treatment of breast cancer by targeting the CYP19 (aromatase) isoform that catalyses the formation of an aromatic ring A as required for the conversion of testosterone to oestradiol. In addition, CYP17 (17α-hydroxylase of pregnenolone and of progesterone) is targeted by a range of drugs for the treatment of prostate cancer. Inhibitor-CYP comparisons are recommended therefore for CYP19 and CYP17 in silico and real-lab so as to continue the development for instance of therapeutic azole inhibitors of CYP19 (aromatase) such as anastrozole, by recognition in silico of quantitative structure/activity relationships (QSARs) between such therapeutic drugs and CYP-utilising pathways.

Real-time PCR analysis of cytochrome P450 aromatase expression in zebrafish: Gene specific tissue distribution, sex differences, developmental programming, and estrogen regulation

In teleost fish, the predominant brain form of cytochrome P450 aromatase (P450aromB) is a neural marker of estrogen effect, and an entry point for studying the role of hormonal and environmental estrogens on neurodevelopment and neuroplasticity. As part of a project using zebrafish to investigate these issues, we developed and validated a rapid, sensitive, and reproducible real-time polymerase chain reaction (PCR) assay for quantifying and comparing P450aromB and P450aromA expression in unfertilized eggs, embryos/larvae, and dissected tissues of adult fish. Results confirm that P450aromB and -A predominate in brain and ovary, respectively, and further show that the degree of overlapping expression (ratio, B:A) is 100:1 in brain, 1:50 in ovary, 1:1 in eye, and 2:1 in testis. Sex differences were observed in eye only (female > male). When compared to whole ovaries, unfertilized eggs had similar levels of P450aromA but enrichment of P450aromB, which suggests preferential synthesis or accumulation in mature oocytes. Both of the maternally derived aromatase isoforms were rapidly degraded post-fertilization, but the onset of embryonic P450aromB expression (5 hpf) was much earlier than P450aromA (48 hpf), and reached higher maximum levels (e.g., 10-fold at 72 hpf). Consistent with earlier reports, P450aromB but not -A was estrogen-inducible, but the estrogen response system in embryos was far more robust than in adults (>100- vs. <4-fold maximal induction, respectively). Application of this real-time PCR assay to measurement of P450aromB and -A in zebrafish embryos has utility for routine screening of chemicals and environmental samples for estrogen-like bioactivity and neural effects.

Aromatase in zebrafish: A potential target for endocrine disrupting chemicals

In zebrafish, two isoforms of the aromatase gene exist, namely cyp19a1 and cyp19a2, expressed predominantly in the gonads and brain, respectively. In this study, we focus on characterizing the specificity of antibodies against the aromatase isoforms, and on (xeno)estrogen-induced changes of individual cyp19a2 mRNA concentrations in the brains of adult male zebrafish. Among three polyclonal antibodies studied, the one against CYP19A2 was found to be specific in Western blots and immunohistochemistry. Real-time RT-PCR analyses revealed strong interindividual variation of cyp19a2 levels in the brains of adult male zebrafish. After a three-week-exposure to (xeno)estrogens, mean values of cyp19a2 mRNA levels tended to increase, with significant induction at 200 ng 17beta-estradiol/L, but interindividual variation of cyp19a2 expression was maintained.