Sequence analysis and expression of the P450 aromatase and estrogen receptor genes in the Xenopus ovary

doi:10.1016/S0960-0760(00)00164-3

The Journal of Steroid Biochemistry and Molecular Biology

Volume 75, Issues 2–3, 15 December 2000, Pages 101-107

https://doi.org/10.1016/S0960-0760(00)00164-3 Get rights and content

Abstract

Recent studies point to a key role for the estrogen synthesizing enzyme P450 aromatase (P450 arom) in ovary determination in fish, birds and reptiles. It is unclear whether estrogen synthesis is important in sex determination of Xenopus gonad. To determine whether the aromatase gene is transcribed in the gonads of Xenopus tadpoles during the sex determination, we cloned a P450 arom cDNA and examined the level of P450 arom and estrogen receptor (ER) gene expression in association with estrogen activity. cDNA clones for P450 arom were isolated from a Xenopus ovarian cDNA library. There was an open reading frame (ORF) of 1500 bp from the ATG start to TAA stop codons encoding 500 predicted amino acids. cDNAs for P450 arom have previously been cloned from various vertebrates. The homology between the Xenopus P450 aromatase and the human P450 arom was higher. The expression of the P450 arom gene was mainly limited to reproductive organs. To determine the beginning of estrogen activity in gonads of embryos, expression of the aromatase and ER gene was also examined by RQ-RT-PCR. Both Xenopus aromatase and ER mRNA was detected at stage 51 in gonads. These observations are consistent with estrogens having a key role in ovarian development in various other vertebrates.

Introduction

P450 aromatase catalyzes the conversion of androgen to estrogen, which is a rate-limiting step in estrogen biosynthesis [1]. cDNA clones encoding aromatase have been isolated from human [2], rat [3], bovine [4], chicken [5], rabbit [6], catfish [7] and tilapia [8]. The aromatase activity in cell is closely related to the level of the very labile aromatase mRNA species, suggesting that transcriptional control is the main mechanism of regulation of enzyme activity [9]. Recent studies have shown that the regulation of P450 arom gene expression is important because estrogens are considered to play a role in sex differentiation. Treatment of embryos or larvae with sex steroid hormones have led to sex reversal in fish [10], reptiles [11], [12] and birds [13], [14]. A major role of estrogen and aromatase in ovary differentiation has also been confirmed by the use of aromatase inhibitors [15], [16], [17], [18], [19]. In contrast to the well-defined physiological functions of P450 arom and estrogens of fish, birds and reptiles [20], [21], [22], very little is known about the molecular characteristics and mechanisms of the regulation of transcription of the P450 arom gene in Xenopus ovaries [23]. In the present study, we characterized a full-length cDNA of the Xenopus ovary aromatase gene and compared it with vertebrates.

Estrogen exerts its biological effects through a specific nuclear receptor protein, which functions as ligand activated transcription factor, and a Xenopus estrogen receptor (ER) cDNA has been cloned [24]. Therefore, we examined the level of P450 arom and ER mRNA during development of embryos by real-time quantitative polymerase chain reaction after reverse transcription (RQ-RT-PCR) [25].

Section snippets

Isolation of RNA and cDNA synthesis

Total RNA was isolated from adult ovary, testis, liver, kidney and heart of Xenopus and gonads with the attached mesonephros of Xenopus tadpoles at various stages with RNAzol B (Cinna/Biotecx Laboratories, Inc.). The total RNA of gonads was used for RQ-RT-PCR. Poly (A) RNA of the adult ovary was isolated using an oligo (dT) column. A total of 3 μg poly (A) RNA was used for first- and second-strand cDNA synthesis (Stratagene). The double-stranded cDNA was ligated into λZAP II and packaged with

Nucleotide and deduced amino acid sequence of P450 arom cDNA

The sequence of the 2191-bp containing complete open reading frame (ORF) of the P450 arom cDNA is shown in Fig. 1. Despite similarities in the coding regions of P450 arom among various species, the sizes of the transcripts differed markedly. Two transcripts of 3.4 and 2.9 kb in size were observed in human tissues [6] and there are three rat transcripts of 3.3, 2.6 and 1.9 kb [3]. The large size of transcripts appears to result from the presence of a long 3′-untranslated region, which could have

Discussion

Estrogen is implicated in sex differentiation of fish, avians and reptiles. We cloned a cDNA for aromatase, which is essential for estrogen synthesis, from Xenopus ovaries. The predicted amino acid sequence of the aromatase showed high identity with that of human [2].

Xenopus tadpoles are capable of steroidogenic activity in the interrenal region after stage 47, but not in the gonads between stages 50 and 52 [28], [29]. Steroid metabolism and production in the gonads of Rana catesbeiana also

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Genes encoding aromatases in teleosts: Evolution and expression regulation
2014, General and Comparative Endocrinology
Citation Excerpt :
The present paper is intended to briefly summarize the current advancements in the study of cyp19a1 in teleosts, including the origin, evolution, and regulation of cyp19a1 genes. To date, studies have shown that there is only one copy of cyp19a1 in most tetrapods, including mammals (Simpson et al., 1997) except pigs and peccaries (Corbin et al., 2007), chicken (McPhaul et al., 1988), Xenopus (Miyashita et al., 2000), and alligator (Gabriel et al., 2001). In addition, only one copy of cyp19a1 was identified in Atlantic stingray (Ijiri et al., 2000), a cartilaginous fish.
Cytochrome P450 aromatases, encoded by cyp19a1 genes, catalyzes the conversion of androgens to estrogens and plays important roles in the reproduction of vertebrates. Vertebrate cyp19a1 genes showed high synteny in chromosomal locations and conservation in sequences during evolution. However, amphioxus cyp19a1 does not show synteny to vertebrate cyp19a1. Teleost fish possess two copies of the cyp19a1 gene, which were postulated to result from a fish-specific genome duplication. The duplicated copies of fish cyp19a1 genes evolved into the brain and ovarian forms of cytochrome P450 aromatase genes, cyp19a1a and cyp19a1b, respectively, with different regulatory mechanisms of expression, through subfunctionalization under long-term selective pressure. In addition to the estradiol (E2) auto-regulatory loop, there may be other mechanisms responsible for the high expression of aromatase in the teleost brain. The study of the two cyp19a1 copies in teleost fish will shed light on the general evolution, function, and regulation of vertebrate cyp19a1.
Molecular cloning and characterization of brain and ovarian cytochrome P450 aromatase genes in the catfish Heteropneustes fossilis: Sex, tissue and seasonal variation in, and effects of gonadotropin on gene expression
2014, General and Comparative Endocrinology
Citation Excerpt :
The placenta and ovary are the main sources of P450 arom and circulating estrogens, lesser amounts are formed in brain, fat, bone, testis and other tissues. Previous studies have characterized a single gene coding for P450 arom (Cyp19a1) in tetrapods including human and cartilaginous fish (Gabriel et al., 2001; Ijiri et al., 2000; McPhaul et al., 1988; Miyashita et al., 2000), and the tissue-specific expression is regulated by tissue-specific promoters of the gene (Simpson et al., 2002). In pigs and peccaries among mammals, on the other hand, two or more genes are characterized, which express in a tissue – specific manner (Corbin et al., 2007; Graddy et al., 2000).
Cytochrome P450 aromatase (Cyp19arom) is the rate-limiting enzyme controlling estrogen biosynthesis, coded by Cyp19a1 in most gnathostomes. Most teleosts have two forms expressed differentially in ovary (cyp19a1a) and neural tissue (cyp19a1b). In this study, full length cDNAs of 2006 bp and 1913 bp with ORFs of 1575 bp and 1488 bp were isolated from the brain and ovary, respectively, of the catfish Heteropneustes fossilis, an air-breathing species with high aquaculture potential. The ORFs encode predicted proteins of 495 and 524 amino acid residues, respectively. The proteins show 62% identity with each other and cluster in two distinct clades (the brain type and ovary type) in the teleost taxon, separated from the tetrapod type. In the in situ localization study, both cyp19a1a and cyp19a1b transcripts were localized in the brain but the signal intensity was higher for the brain type paralog. The transcript signals were observed in the radial glial cells and in neuronal populations of the dorso-lateral region of the telencephalon, pre-tectum, hypothalamus and medulla oblongata. In the ovary, both paralogs were expressed in the follicular layer with a high signal intensity of the ovarian type (cyp19a1a). The differential expression of the gene paralogs was evident from qPCR analysis. Cyp19a1b has relatively a high abundance in the female brain, followed by other peripheral tissues (gonads, liver, gill, kidney and muscle). On the other hand, cyp19a1a has relatively a high transcript abundance in the ovary and female brain, followed by the testis and male brain, and female liver and muscle. The expression was low in male liver and muscle, and the lowest in the gill and kidney. The expression of the two paralogs exhibit brain regional differences; both types have relatively a high transcript abundance in telencephalon-preoptic area with the cyp19a1b expression higher in females than males. In hypothalamus, the expression of both types is higher in males than females. In medulla, the expression of the cyp19a1b is higher than cyp19a1a, and the transcript abundance of the ovarian type is higher in females than males. The expression of the gene paralogs elicits significant seasonal variations in the ovary and brain. In both tissues, the expression increases from the resting to preparatory phases, and decreases through the prespawning phase to low levels in spawning phase. In vivo and/or in vitro treatments with human chorionic gonadotropin (hCG) stimulated the expression of the gene paralogs in the brain and ovary, time-dependently. In conclusion, both paralogs have an overlapping distribution at different levels of the brain–pituitary–gonad axis and may function as a single functional unit as far as the estrogen synthesis is concerned.
Clotrimazole exposure modulates aromatase activity in gonads and brain during gonadal differentiation in Xenopus tropicalis frogs
2009, Aquatic Toxicology
Clotrimazole is a pharmaceutical used for treatment of fungal infections. It has been found in surface waters outside municipal wastewater treatment plants but data are scarce regarding its effects on aquatic organisms. It is known that clotrimazole and other imidazole fungicides are inhibitors of the enzyme aromatase (CYP 19). Aromatase converts androgens into estrogens and is suggested to be involved in the sex differentiation in amphibians. The aim of the present study was to evaluate effects of larval exposure to clotrimazole on aromatase activity in brain and gonads, and on gonadal differentiation in Xenopus tropicalis frogs. Another purpose was to determine if larval exposure to ethynylestradiol (EE₂), at a concentration known to cause male-to-female sex reversal, affects aromatase activity in brain and gonads during gonadal differentiation. Tadpoles were exposed from shortly after hatching (Nieuwkoop and Faber developmental stages 47–48) until complete metamorphosis (NF stage 66) to 6, 41, and 375 nM clotrimazole or 100 nM (nominal) EE₂. Aromatase activity was measured in the brain and gonad/kidney complex of tadpoles during gonadal differentiation (NF stage 56) and, in the clotrimazole experiment, also at metamorphosis. In clotrimazole-exposed tadpoles gonadal aromatase activity increased over exposure time in the 41 and 375 nM groups but did not differ significantly from the control group. Gonadal aromatase activity was increased in both sexes exposed to 41 and 375 nM clotrimazole at metamorphosis. Brain aromatase activity was decreased in tadpoles (NF stage 56) exposed to 375 nM clotrimazole, but at metamorphosis no differences were seen between groups or between sexes. No effects of clotrimazole on sex ratio or gonadal histology were noted at completed metamorphosis. EE₂-exposed tadpoles had a slightly decreased gonadal aromatase activity, though not significantly different from control group, and there was no effect of EE₂ on brain aromatase activity. All EE₂-exposed tadpoles developed ovaries. These findings indicate that estrogen-induced ovarian differentiation is not paralleled by increased gonadal aromatase activity in X. tropicalis. Further studies are needed, especially on developmental reproductive toxicity, to assess the risk for endocrine disruption in wild amphibians posed by clotrimazole and other imidazole fungicides.
Identification of single nucleotide polymorphism cytochrome P450-c19a and its relation to reproductive traits in Japanese flounder (Paralichthys olivaceus)
2008, Aquaculture
CYP19 is considered as an important factor affecting reproductive endocrinology in many fishes, and plays an important role in ovarian development, reproductive function and sexual differentiation. In this study, three single nucleotide polymorphisms (SNPs) within CDS of the CYP19a gene were tested and the associations between their genotypes and four reproductive traits were analyzed in 65 Japanese flounder individuals with Polymerase chain reaction and Single-stranded conformational polymorphism (PCR–SSCP). Results indicated that a SNP in the exon7 of CYP19a gene, SNP2, was significantly associated with 17β-estradiol (E₂) (P < 0.05) and gonadosomatic index (GSI) (P < 0.05). Individuals with genotype AB of SNP2 had significantly higher serum E₂ levels (P < 0.05) and GSI (P < 0.05) than those of genotype AA or BB. In addition, there was significant association between one diplotype based on three SNPs and reproductive trait. The genetic effects for both serum E₂ of diplotype D9 and GSI of diplotype D1 were respectively much higher than those of other diplotypes (P < 0.05). The evidence of the associations between genetic variants with serum E₂ and GSI may help explain effects of CYP19a gene in reproductive endocrinology of Japanese flounder.
Effect of atrazine on metamorphosis and sexual differentiation in Xenopus laevis
2008, Aquatic Toxicology
There is a growing international concern that commonly used environmental contaminants have the potential to disrupt the development and functioning of the reproductive system in amphibians. One such chemical of interests is the herbicide atrazine. Effects of atrazine on sex differentiation were studied using wild-type Xenopus laevis tadpoles and all-ZZ male cohorts of X. laevis tadpoles, produced by mating wild-type ZZ male to sex-reversed ZZ male (female phenotype). Stage 49 tadpoles were exposed to 0.1–100 ppb atrazine or 0.27 ppb (1 nM) 17β-estradiol (E₂) until all larvae completed metamorphosis (stage 66). Metamorphosis, gonadal morphology and histology, CYP19 (P450 aromatase) mRNA induction, and hepatic vitellogenin (VTG) induction were investigated. Effects of atrazine on VTG-induction were also assessed in vitro in primary-cultured X. laevis hepatocytes. Atrazine had no effect on metamorphosis of developing wild-type or all-male X. laevis larvae. Statistical increase in female ratios was observed in 10 and 100 ppb atrazine groups in comparison with control group. While no hermaphroditic froglet was observed in all atrazine groups. In ZZ males, sex reversal was induced by 0.27 ppb E₂, but not by atrazine at concentrations of 0.1 and 1.0 ppb. In addition, neither P450 aromatase mRNA in the gonad nor hepatic VTG were induced by atrazine. Furthermore, VTG was not induced by 1000 ppb atrazine in primary-cultured hepatocytes. Our results indicate that female ratios in developing X. laevis tadpoles were increased by 10 and 100 ppb atrazine under the present experimental conditions. While the other endpoints showed no effect in the range of 0.1–100 ppb atrazine. These results suggest that effect of atrazine on sexual differentiation was not caused by estrogenic action and has no induction ability of P450 aromatase gene in gonad.
Analysis of the p450 aromatase gene expression in the Xenopus brain and gonad
2007, Journal of Steroid Biochemistry and Molecular Biology
Analysis of 5′-RACE clones revealed two Cyp19 transcript variants (gonad p450 aroms 1 and 2) in the gonads and one Cyp transcript (brain p450 arom) in the brain that differed in their 5′-untranslated region (UTR). Since all cDNAs contained an identical open reading frame, it is considered that while Xenopus aromatase may be encoded by a single Cyp19 gene, it is transcribed by tissue-specific promoters, each of which may be regulated by a distinct set of transcriptional factors. While gonad p450 aroms 1 and 2 and brain p450 arom were expressed in the gonads, brain p450 arom was the predominantly expressed aromatase gene in the brain, with a low expression level of gonad p450 arom 1.

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Sequence analysis and expression of the P450 aromatase and estrogen receptor genes in the Xenopus ovary

Abstract

Introduction

Section snippets

Isolation of RNA and cDNA synthesis

Nucleotide and deduced amino acid sequence of P450 arom cDNA

Discussion

J. Biol. Chem.

Gen. Comp. Endocrinol.

J. Biol. Chem.

Gen. Comp. Endocrinol.

Gen. Comp. Endocrinol.

Differentiation

J. Steroid. Biochem. Mol. Biol.

Gen. Comp. Endocrinol.

J. Steroid. Biochem. Mol. Biol.

Gen. Comp. Endocrinol.

Gen. Comp. Endocrinol.

Cell

The involvement of human placental microsomal cytochrome P-450 in aromatization

J. Biol. Chem.

Isolation of a full-length cDNA insert encoding human aromatase system cytochrome P-450 and its expression in nonsteroidgenic cells

Proc. Natl. Acad. Sci. USA

Aromatase cytochrom P450 in rat ovarian granulosa cells before and after luteinization: adenosin 3′, 5′-monophosphate-dependent and independent regulation. Cloning and sequencing of rat aromatase cDNA and 5′ genomic DNA

Mol. Endocrinol.

Isolation and characterization of a complementary deoxyribonucleic acid insert encoding bovine aromatase cytochrome P450

Endocrinology

Rapid sequencing of rabbit aromatase cDNA using RACE PCR

C. R. Acad. Sci. III

Isolation and characterization of the cDNA encoding the tilapia (Oreochromic niloticus) cytochrome P450 aromatase (P450arom): changes in P450arom mRNA, protein and enzyme activity in ovarian follicles during oogenesis

J. Mol. Endocrinol.