Molecular biology of channel catfish brain cytochrome P450 aromatase (CYP19A2): cloning, preovulatory induction of gene expression, hormonal gene regulation and analysis of promoter region.

Cytochrome P450 aromatase (CYP19) converts androgens to estrogens. Unlike mammals, teleosts have two CYP19 genes, expressed differentially in ovary (CYP19A1) and neuronal tissues (CYP19A2). The primary purpose of this study was to demonstrate the potential involvement of CYP19A2 in the reproductive endocrinology of teleosts. Channel catfish CYP19A2 (ccCYP19A2) cDNAs were isolated from the brain using a PCR-based strategy. The ccCYP19A2 cDNA putatively encodes 500 amino acids which conferred aromatase activity in transfected COS-7 cells. Additionally, an alternatively spliced transcript was isolated which lacks the first 122 amino acids and is catalytically inactive. The brain and the pituitary were predominant sources of ccCYP19A2 transcript and the abundance in both tissues acutely increased prior to spawning. This preovulatory induction of ccCYP19A2 gene in the pituitary is remarkably similar to the pattern of gene expression for luteinizing hormone-beta (LHbeta). Estradiol-17beta (E(2)) and testosterone enhanced the transcript abundance of ccCYP19A2 and LHbeta in catfish pituitary cells cultured in vitro but the stimulatory effects of testosterone were abolished by an aromatase inhibitor, indicating an important role of E(2), the product of CYP19A2 activity, in the regulation of CYP19A2 and LHbeta. Structural and functional analysis of the 5'-flanking region of the gene suggested that the sequence from -1076 to - 435 bp is critical for the basal promoter activity in the pituitary. This report demonstrates that CYP19A2 functions as an important factor in the reproductive endocrinology of teleosts through the brain-pituitary-gonadal axis.

The 5'-flanking regions of CYP19A1 and CYP19A2 in zebrafish.

This report describes the structure of the 5'-flanking regions of both the CYP19A1 and A2 genes that were isolated from the genome of the zebrafish (Danio rerio). Consensus sequences of three cAMP-responsive elements (CRE), an aryl hydrocarbon-responsive element (AhR/Arnt), a steroidogenic factor 1 (SF-1) site, and a TATA box were observed in the 5'-flanking region of CYP19A1. In contrast, the 5'-flanking region of CYP19A2 was located upstream of an untranslated exon and possessed consensus sequences of a single CRE, an estrogen-responsive element (ERE), a peroxisome proliferator-activated receptor alpha/retinoid X receptor alpha heterodimer-responsive element (PPARalpha/RXRalpha), and a TATA box. Primer extension analysis revealed that the predominant transcription initiation sites for CYP19A1 and A2 transcripts were 28 and 91 bp upstream from the putative translation initiation codon, respectively. These analyses indicate that substantially different regulators, including a variety of environmental xenobiotics, control the expression the two CYP19 genes.

Developmental expression of cytochrome P450 aromatase genes (CYP19a and CYP19b) in zebrafish fry (Danio rerio).

Cytochrome P450 aromatase (CYP19) is the terminal enzyme in the steroidogenic pathway that converts androgens (e.g., testosterone) into estrogens (e.g., estradiol). Regulation of this gene dictates the ratio of androgens to estrogens; therefore, appropriate expression of this enzyme is critical for reproduction as well as being pivotal in sex differentiation for most vertebrates. It is assumed that most vertebrates have a single CYP19 gene that is regulated by multiple tissue-specific promoter regions. However, the zebrafish (Danio rerio) has two genes (CYP19a and CYP19b), each encoding a significantly different protein and possessing its own regulatory mechanism. The primary purpose of this study was to determine the pattern of expression of each of the CYP19 genes in the developing zebrafish. A fluorescent-based method of real-time, quantitative RT-PCR provided the sensitivity and specificity to determine transcript abundance in single embryos/juveniles harvested at days 0 through 41 days post-fertilization (dpf), which encompasses the developmental events of sex determination and gonadal differentiation. CYP19 transcripts could be detected as early as 3 or 4 dpf, (CYP19a and CYP19b, respectively) and peak abundance was detected on day five. In general, the CYP19 genes differed significantly in the ontogeny of their expression. In most cases, the gonadal form of CYP19 (CYP19a) was more abundant than the brain form (CYP19b); however, unlike CYP19a, the pattern of CYP19b expression could be clearly segregated into two populations, suggesting an association with sex differentiation. Pharmacological steroids (ethinylestradiol and 17 alpha-methyltestosterone) enhanced the expression of the CYP19b gene at all three days examined (4, 6, and 10 dpf). These data suggest that the timely and appropriate expression of CYP19 is important in development and that the expression of CYP19b (the "extra-gonadal" form) may be associated with sexual differentiation if not sexual determination. J. Exp. Zool. 290:475-483, 2001.

Molecular biology of the channel catfish gonadotropin receptors: 2. Complementary DNA cloning, functional expression, and seasonal gene expression of the follicle-stimulating hormone receptor.

Molecular cloning of the channel catfish FSH receptor is reported together with temporal changes in the gene expression throughout a reproductive cycle. A cDNA encoding the receptor was isolated from the testis using reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) procedures. The cDNA coded for a 662-amino acid protein that was most identical (51%-59%) to salmon gonadotropin receptor I and the FSH receptors of higher vertebrates, and less identical to LH receptors and thyrotropin receptors (45%-49% and 46%-47%, respectively). In addition, PCR analysis of the genomic DNA showed the absence of the LH receptor-specific intron. Expression of the channel catfish FSH receptor gene was highly restricted to the testis and ovary, except for a low-level expression in the spleen. Transfected COS cells expressed an active recombinant receptor as determined by the ligand-specific activation of a cAMP-responsive reporter gene (luciferase). The recombinant receptor was activated by human FSH and, to a small extent, hCG. Seasonal changes in the ovarian expression of the FSH receptor gene, examined by measuring the transcript abundance by quantitative real-time RT-PCR, showed a rise around the time of onset of ovarian recrudescence and a decrease prior to spawning. This pattern of seasonal expression of FSH receptor differs significantly from that of the LH receptor, which we reported recently. The differential expression of the two gonadotropin receptor genes, in addition to the differential secretion of the gonadotropic hormones, seem to be critical for the regulation of steroidogenesis and other gonadal physiological processes.

Piscine glycoprotein hormone (gonadotropin and thyrotropin) receptors: a review of recent developments.

Similar to the higher vertebrates, the pituitary in bony fishes express three glycoprotein hormones: thyroid-stimulating hormone (TSH), follicle-stimulating hormone (FSH) and luteinizing hormone (LH). In addition to the appropriate secretion of these hormones, the timely and quantitative expression of their specific receptors (TSHR, FSHR and LHR) in the target tissues is an essential requirement for their physiological action. In fishes that constitute a very diverse group of vertebrates, there are only a few published reports of primary structure of these receptors although other examples have been communicated briefly. This review will summarize these reports as well as to describe the insights gained from what is known about the mammalian receptors. The structural organization of the fish receptors (as deduced from the encoding cDNAs) is highly homologous to the higher vertebrate receptors in that there is a 7-pass transmembrane region and an N-terminal extracellular domain, which contributes to ligand specificity. In mammals, the FSHR and the TSHR genes are composed of 10 exons whereas the LHR gene is composed of 11 exons. The position of the 'extra intron' is conserved in the catfish LHR gene. In the mammals, the transmembrane domain of each of the three glycoprotein hormone receptors is encoded by a single exon, however, in the salmon genes and homologous invertebrate genes, this portion of the receptor is encoded by multiple exons. In general, the tissue-specific expression of these receptors is similar to that seen in mammals, however, the gonadal expression of TSHR in the striped bass and sunrise sculpin and the renal expression of LHR in the channel catfish are unique.

Recombinant perciform GnRH-R activates different signaling pathways in fish and mammalian heterologous cell lines.

Perciforms have three forms of gonadotropin-releasing hormone (GnRH) in their brain. All three GnRHs are potent secretogogues for luteinizing hormone (LH) from the pituitary. The pivotal role of GnRH-R-GnRH interactions in reproductive homeostasis is well established; however, there is a paucity of information on how a GnRH-R responds to the three endogenous GnRH forms in a perciform species. In this study, a recombinant pituitary GnRH-R from striped bass (stb) was expressed in a mammalian cell line (COS-7) and a fish cell line (CHSE-214). Activation of the signaling pathways was monitored by reporter gene (luciferase) based assays, which were specific for cAMP-PKA or Ca 2+/calmodulin kinase (activated via c-fos promoter) signaling pathways. The stbGnRH-R expressed in two different cell lines triggered different downstream signaling in response to the treatments with chicken (c) GnRH II. Interestingly, when endogenous GnRHs were used in combinations, the luciferase activity was significantly attenuated in transfected CHSE-214 cells.

Molecular biology of channel catfish gonadotropin receptors: 1. Cloning of a functional luteinizing hormone receptor and preovulatory induction of gene expression.

There is little known about the molecular biology of piscine gonadotropin receptors, and information about gene expression during reproductive development is particularly lacking. We have cloned the LH receptor (LHR) in the channel catfish (cc), and examined its gene expression throughout a reproductive cycle. A cDNA encoding the receptor was isolated from the testis using reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends procedures. It encoded a 696-amino acid protein that showed the greatest homology (46-50% identity) with the known LHRs and lesser similarity with FSH receptors and thyroid-stimulating hormone receptors (44-47% and 42-44% identity, respectively). In addition, two characteristics unique to the LHRs were conserved in the cloned receptor and the encoding gene: presence of an intron corresponding to intron 10 in mammals and turkey and occurrence of a double cysteine residue in the cytoplasmic tail for potential palmitoylation. The ccLHR gene was well expressed in the gonads and kidney and merely detectable in the gills, muscle, and spleen. The isolated cDNA encoded an active ccLHR protein, as the recombinant receptor expressed in COS7 cells activated a cAMP response element-driven reporter gene (luciferase) upon exposure to hCG in a dose-dependent manner. Seasonal changes in the ovarian expression of the ccLHR gene, as examined by measuring the transcript abundance by quantitative real-time RT-PCR, remained rather low during most of the reproductive cycle but was acutely induced around the time of spawning. This pattern of expression correlates well with the reported expression of its ligand (LH) in fishes and concurs with the notion that LH is a key regulator of the periovulatory maturational events.

Porcine gonadal and placental isozymes of aromatase cytochrome P450: sub-cellular distribution and support by NADPH-cytochrome P450 reductase.

Functional differences exist between the porcine gonadal and placental aromatase cytochrome P450 (P450arom) isozymes that are encoded by separate genes. The present experiments investigated the sub-cellular location of these isozymes, their dependence on the redox partner protein NADPH-cytochrome P450 reductase (P450 reductase) for catalytic activity and the release of steroid intermediates during the aromatization of androgens to estrogen. After differential centrifugation, similar levels of gonadal and placental porcine P450arom were found along with P450 reductase in the microsomal compartment using activity and immunoblot analyses. Activity was stimulated much more by recombinant P450 reductase addition, and higher levels of 19-hydroxy and 19-oxo intermediates accumulated during androstenedione and testosterone metabolism, in cells expressing the gonadal compared to the placental isozyme. No other steroid products were identified by HPLC. Thus, the porcine gonadal P450arom is more sensitive to P450 reductase deprivation than is the placental P450arom, accounting in part for catalytic differences between these isozymes.

Changes in the expression of genes encoding steroidogenic enzymes in the channel catfish (Ictalurus punctatus) ovary throughout a reproductive cycle.

In vertebrates, the growth and maturation of the ovarian follicle is dependent on the appropriate dynamics of sex steroid secretion, which is dictated by gene expression of the steroidogenic enzymes. The molecular aspects of steroid regulation are poorly understood in fishes, so as a first step we determined the pattern of expression of four key steroidogenic genes throughout the ovarian cycle in an annually spawning teleost, the channel catfish (Ictalurus punctatus). The abundance of transcripts encoding 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) and cholesterol side chain cleavage (P450(scc)), 17 alpha-hydroxylase/lyase (P450(c17)), and aromatase (P450(arom)) were determined by rtqRT-PCR or ribonuclease protection assay and correlated to ovarian growth and plasma titers of estradiol (E(2)) and testosterone (T) in two populations of catfish. Elevations in transcript abundance for P450(c17), P450(scc), and P450(arom) were observed at the onset of ovarian recrudescence and during early vitellogenic growth of the oocytes; however, all three decreased precipitously with the completion of vitellogenesis. Changes in the expression of these genes strongly suggest a direct correlation to E(2) and T titers. Alternatively 3 beta-HSD transcript abundance was relatively stable throughout the year. This study suggests that the genes encoding the three steroidogenic cytochrome P450s have a similar regulatory mechanism.

Characterization of a pituitary GnRH-receptor from a perciform fish, Morone saxatilis: functional expression in a fish cell line.

Gonadotropin-releasing hormones (GnRHs) bind to the specific receptor present on the gonadotrophs to activate the synthesis and release of gonadotropins (follicle stimulating hormone or FSH and luteinizing hormone or LH), which in turn control gonadal maturation, gametogenesis and gamete release. Perciform species have three endogenous GnRHs. The main objective of this study was to characterize the gonadotropin-releasing hormone receptor (GnRH-R) present in the pituitary of a perciform species, striped bass (Morone saxatilis) and demonstrate how it interacts with its potential ligand. In this study, a cDNA for GnRH-R from the pituitaries of striped bass was cloned. The cloned cDNA has an open reading frame (ORF) that codes for a 419 amino acids peptide. Like other G-protein coupled receptors including the non-mammalian GnRH-Rs, the peptide has seven putative transmembrane domains and a C-terminal tail. Comparative analysis of the amino acid sequence of striped bass (stb) GnRH-R shows 38-87% similarity with the known GnRH-Rs. A Northern blot analysis revealed a single GnRH-R transcript in the pituitary; however, its expression in various extrapituitary tissues was demonstrated by a reverse-transcription-PCR (RT-PCR). Functionally, upon induction by endogenous forms of GnRHs (seabream, chicken II and salmon GnRHs) and a mammalian GnRH-agonist, the recombinant stbGnRH-R mediated a reporter gene (luciferase) activity in a fish cell line (CHSE-214). A real-time relative quantitation method established that significantly higher (P<0.05) levels of stbGnRH-R mRNA were present in the pituitaries of striped bass with advanced stages of ovarian development, compared to the pituitaries of fish with less developed ovaries.

Characterization of gonadal and extra-gonadal forms of the cDNA encoding the Atlantic stingray (Dasyatis sabina) cytochrome P450 aromatase (CYP19).

Cytochrome P450 aromatase (P450arom; CYP19) mediates the conversion of androgens to estrogens and its activity has been found in all vertebrates studied to date. This study describes the full-length cDNA encoding the ovarian form of P450arom and the differences in the 5'-untranslated region (5'-UTR) of the extra-gonadal P450arom transcript expressed by the Atlantic stingray (Dasyatis sabina). Elasmobranchs (cartilaginous fishes such as sharks, rays and skates) diverged from the other vertebrates more than 350 million years ago, therefore the stingray P450arom cDNA may represent an ancient form of this gene. Northern blot analysis showed that the ovarian follicle expressed transcripts of 3.1 and 1.7 kb in size which correspond to the clones isolated from a stingray ovarian follicle cDNA library. Both transcripts consisted of an identical 1.5 kb coding region and a 41 bp 5'-UTR, however the 3'-UTRs differed in the use of the most proximate and the most distal of four polyadenylation signals. COS cells transfected with the 1.7 kb cDNA had twice the aromatase activity as cells transfected with the 3.1 kb cDNA. The coding region of the cDNA predicted a 58.5 kDa protein which consisted of 511 residues. Alignment of the stingray protein indicates that the P450arom is equally identical (53-59%) to all other vertebrate forms of P450arom characterized to date, thus indicating a common ancestry. The evolutionary relationship of the stingray form of P450arom clearly predates the other forms and belongs to a unique lineage. Transcripts of P450arom were expressed in ovarian follicles (of all sizes), the testis, the pituitary, in all sections of the brain, and in the kidney. The extra-gonadal transcripts appear to encode a protein identical to the ovarian form, however, the 5'-UTR was 657 bp longer presumably due to the transcription of an untranslated 'first exon' as seen in the mammalian form of this gene.

Cloning and functional expression of a thyrotropin receptor from the gonads of a vertebrate (bony fish): potential thyroid-independent role for thyrotropin in reproduction.

The thyroid stimulating hormone receptor (TSHR) mediates the pituitary control of the development, growth and function of the thyroid. The expression of the gene encoding this receptor is known only in the thyroid, lymphocytes, fibroblasts, retro-orbital tissue and fat cells. We have cloned a TSHR from the gonads of a non-mammalian vertebrate, a bony fish (striped bass, Morone saxatilis) in the course of our search for gonadotropin receptors (follicle stimulating hormone receptor, FSHR and luteinizing hormone receptor, LHR). RT-PCR analysis demonstrated that the striped bass TSHR (stbTSHR) transcripts were abundant in both the thyroid and gonads and detectable in skeletal muscle, heart and brain tissues. The stbTSHR cDNA encoded a 779-amino acid glycoprotein hormone receptor with much higher homology (57-59%) to the mammalian TSH receptors than the mammalian LH receptors (47-49%) and FSH receptors (47%), and salmon and catfish gonadotropin receptors (42-45%). There was a TSHR-specific insertion in the extracellular domain as seen in mammalian receptors. Moreover, PCR analysis of genomic DNA indicated the absence of the LHR-specific intron in the striped bass TSHR gene. Recombinant stbTSHR expressed in COS1 cells activated reporter genes (luciferase) driven by either a cAMP response element or the c-fos promoter in response to bovine TSH, stbLH or hCG, but not human FSH. In situ hybridization studies revealed the presence of stbTSHR transcripts in the gametes but not in the follicular cells. This pattern of expression is unique and suggests a direct, albeit unknown, role for TSH in gamete physiology.

Changes in testosterone metabolism associated with the evolution of placental and gonadal isozymes of porcine aromatase cytochrome P450.

Differences in the catalytic activity of the placental and gonadal isozymes of porcine aromatase cytochrome P450 (P450arom) were examined in cell lines exhibiting stable expression of recombinant enzyme. Cell lines were selected that expressed high, but similar, immunodetectable levels of each isozyme based on Western analysis. Aromatase activity varied with growth in culture, decreasing at confluence from a peak reached between 50-80% cell density. Cells expressing the placental isozyme had 3-5 times higher catalytic activity (per mg protein) than those expressing the gonadal isozyme. The P450arom inhibitor fadrazole (1 microM) inhibited more than 97% of this activity, whereas another imidazole, etomidate (1 microM), selectively inhibited gonadal P450arom activity by 92%. Estrogen synthesis from androstenedione and testosterone was determined by RIA and confirmed by HPLC analysis, which also identified the accumulation of the 19-hydroxy and 19-oxo intermediates of the respective substrates. There was no evidence of other steroid metabolites accumulating in the media of cell lines expressing either isozyme. Tritiated water formed during aromatization of substrates 3H labeled at the C1 and C2 positions was stereo-selective for the beta orientation, but less so for testosterone than androstenedione during metabolism by the porcine placental (and human) isozyme than the gonadal isozyme. Testosterone showed a higher affinity for the porcine placental P450arom than the gonadal P450arom, but both isozymes had similar affinities for androstenedione. Testosterone was also aromatized more slowly than androstenedione by the porcine gonadal P450arom. These data suggest that catalytic differences have arisen in the substrate binding pocket during the evolution of isozymes of porcine P450arom that affect androgen metabolism, particularly the aromatization of testosterone. The physiological significance of these differences to the reproductive biology of the pig remains to be determined.

Regulation of interrenal gland steroidogenesis in the Atlantic stingray (Dasyatis sabina).

The interrenal gland (the homologue of the mammalian adrenal cortex) of elasmobranchs (sharks, skates, and rays) produces 1alpha-hydroxycorticosterone (1alpha-B), which has been reported to function both as a gluco- and mineralocorticosteroid. In vitro synthesis of 1alpha-B by Atlantic stingray (Dasyatis sabina) interrenal glands was stimulated by short-term (2 hr) and long-term (24 hr) treatment with porcine adrenocorticotropic hormone (pACTH). Cycloheximide blocked the pACTH-induced effect on 1alpha-B synthesis, thus demonstrating that the mechanism for the short-term induction of steroidogenesis involved protein synthesis. However, gene transcription did not play a role in the short-term induction of 1alpha-B synthesis, as indicated by the lack of an effect with actinomycin D treatment. Long-term in vitro exposure to pACTH (but not short-term exposure) stimulated the synthesis of another steroid, 11-dehydrocorticosterone (A). This induction was partially blocked by cycloheximide and actinomycin D, which suggests enhanced expression of the 11beta-hydroxysteroid dehydrogenase gene. In addition, the 24-hr treatment with pACTH enhanced the activity of cytochrome P450 side chain cleavage several fold and doubled the activity of 3beta-hydroxysteroid dehydrogenase and cytochrome P450 21-hydroxylase in D. sabina interrenals, again suggesting the induction of steroidogenic genes. In contrast to other elasmobranch species, the salmon and human forms of angiotensin II had no effect on D. sabina interrenal steroidogenesis. J. Exp. Zool. 284:517-525, 1999.

Seasonal changes in the production of two novel and abundant ovarian steroids in the channel catfish (Ictalurus punctatus).

Timely and appropriate changes in steroid plasma titers are necessary for successful reproduction in all vertebrates. Gonadal steroidogenesis of the most intensively cultured teleost species in North America, the channel catfish (Ictalurus punctatus), is poorly understood so a year-long study was conducted to investigate seasonal changes in ovarian steroidogenesis. Incubations of ovarian tissue were conducted monthly with [3H]pregnenolone and the medium was analyzed by high-performance liquid chromatography (HPLC) with radioactivity detection. The suite of steroids produced by the catfish ovary included the expected sex steroids (estradiol and testosterone) and 18 additional ovarian metabolites, including five steroids that have yet to be identified. Androstenedione, 20beta-dihydroprogesterone, 5|P-dihydrotestosterone, estriol, 11beta-hydroxyandrostenedione, 17-hydroxypregnenolone, 17-hydroxyprogesterone, 11beta-hydroxytestosterone, and progesterone were characterized by a combination of HPLC and thin-layer chromatography. Two of the most abundant steroids were isolated and analyzed by gas chromatography coupled with mass spectrometry (GC-MS). One of the steroids, 7alpha-hydroxypregnenolone (7P5), is a novel steroid in teleosts and was produced late in vitellogenic growth of the oocyte. Evidence suggests that the enzyme responsible for converting pregnenolone to 7P5, 7alpha-hydroxylase, is a cytochrome P450. The second abundant steroid metabolite was partially characterized by GC-MS as an hydroxylated form of 17-hydroxy-pregnenolone (chi,17P5). This steroid was most abundant when the ovary was regressed and during early vitellogenesis and rapidly decreased prior to spawning. In mammals, 7P5 has been identified as an important neurosteroid; however, the reproductive significance of 7P5 and chi,17P5 in catfish is unknown.

Expression of cytochrome P450 aromatase in the channel catfish, Ictalurus punctatus.

The cDNA encoding the catfish ovarian aromatase has previously been isolated and described (accession number S75715). As demonstrated previously, the predicted amino acid sequence and enzymatic activity of the encoded protein share a significant degree of similarity to the forms of aromatase found in other vertebrates. Analyses utilizing reverse transcription coupled with the polymerase chain reaction (RT-PCR) demonstrate the expression of aromatase mRNA in catfish brain, testis and ovary. In spite of the evidence provided by Northern blot analysis for a single transcript encoding ovarian aromatase, RT-PCR analysis indicated transcript heterogeneity within the ovary, but not the testis or the brain. Although not characterized, PCR analysis indicated that the transcript complexity of ovarian aromatase was within the encoding region. Until this study, the expression of aromatase and its correlation with the reproductive physiology of fish had not been studied at the molecular level. In the catfish, significant changes in ovarian development were evident following elevation of plasma estradiol titers during October and again in February. Seasonal changes in the expression of ovarian aromatase and 3beta-hydroxysteroid dehydrogenase (3beta-HSD) mRNA was reflected in estradiol plasma titers. Ovarian expression of 3beta-HSD mRNA commenced a month before the message for aromatase was detected. Both transcripts were present from October to April. As the female approached the time of spawning (in May), the abundance of both aromatase and 3beta-HSD transcripts decreased. The aromatase message was not detected in post-spawning females but 3beta-HSD transcripts were evident. These data indicate that the timely synthesis of estradiol in catfish is caused by the regulation of both 3beta-HSD and aromatase.

Isolation of the putative cDNA encoding cholesterol side chain cleavage cytochrome P450 (CYP11A) of the southern stingray (Dasyatis americana).

Cholesterol side chain cleavage cytochrome P450 (P450scc; CYP11A) catalyzes the first step in the production of steroid hormones. By utilizing degenerate oligonucleotide primers in a reverse transcriptase-coupled polymerase chain reaction (RT-PCR), a specific 252 bp fragment of the putative P450scc was amplified from RNA of interrenal tissue (the adrenal cortex homolog) from the southern stingray (Dasyatis americana), blacktip shark (Carcharhinus limbatus), and the spiny dogfish shark (Squalus acanthias). The amino-acid sequences predicted by these PCR products were 73-90% identical to each other. Using the homologous PCR-generated probe, five positive clones were isolated from a cDNA library constructed from interrenal mRNA of the southern stingray. The longest clone (4619 bp) contained the 3'-untranslated region, including four putative polyadenylation signals. Northern blot analysis of stingray interrenal RNA revealed a single transcript of 4.2 kb in length. The incomplete amino-acid sequence predicted by the open reading frame of the cDNA (514 residues in length) is 48% homologous to the trout form and 39-40% homologous to mammalian forms. Even though the stingray P450scc contains an amino terminus longer than the other forms of P450scc, no translation initiation signal (ATG) was evident within the open reading frame. This report presents the first sequence of cytochrome P450scc from this evolutionary unique taxon of vertebrates.

Heterologous expression of the spiny dogfish shark (Squalus acanthias) cytochrome P450c17 (17 alpha-hydroxylase) in Escherichia coli.

Cytochrome P450c17 is a key steroidogenic enzyme for the production of sex steroids in gonadal tissue and for cortisol production in adrenal tissue. This protein possesses two enzymatic activities. The 17-alpha-hydroxylase activity introduces a hydroxyl group into the steroid nucleus. The resultant 17-alpha-hydroxylated, C(21) pregnenes can be converted to a C(19) androgen by the C(17,20)-lyase activity. The cDNA encoding the spiny dogfish shark (Squalus acanthias) testicular form of cytochrome P450c17 was used to direct heterologous expression in Escherichia coli. The wild-type P450c17 protein was not conducive to expression in E. coli using either a pET21 or a pCwori+ vector. However, modification of the amino terminus permitted overexpression of inactive shark protein with the pFT21 vector. This protein was fused with a hexahistidinyl peptide to facilitate purification by column chromatography using a Ni(2+)-chelated resin. Transformed bacteria yielded an average of 1.8 mg of purified, concentrated P450c17 protein per 100-ml culture. This modified protein was used to raise antisera in rabbits and the resultant antisera was used at a working titer of 1:10,000 for Western blot analysis. Culture conditions that result in the accumulation of bioactive recombinant bovine P450c17 failed to demonstrate expression of either the native or the modified form of shark P450c17 using the pCWori+ and pET21 vectors. These results suggest that the amino terminus of the native shark P450c17 was not conducive to synthesis in E. coli. However, modifications of the amino terminus permitted synthesis of an inactive protein that was protected from degradation within inclusion bodies.

Functional expression of recombinant spiny dogfish shark (Squalus acanthias) cytochrome P450c17 (17 alpha-hydroxylase/C17,20-lyase) in yeast (Pichia pastoris).

The cDNA encoding the spiny dogfish shark (Squalus acanthias) testicular form of cytochrome P450c17 (CYP17) was used to direct the heterologous expression of a functional enzyme in yeast (Pichia pastoris). This protein possesses two enzymatic activities: 17 alpha-hydroxylase and C17,20-lyase reactions. Cytochrome P450c17 is a key steroidogenic enzyme for the production of sex steroids in gonadal tissue and for cortisol production in adrenal tissue. This study describes the culture conditions and the enzymatic activity of recombinant shark cytochrome P450c17. The shark enzyme was compatible with the endogenous yeast NADPH-cytochrome P450 reductase and was bioactive within the living yeast cell. Progesterone (at 15 microM) was metabolized (51 pmol/min/10(9) cells) faster than pregnenolone (36 pmol/min/10(9) cells). Both progesterone and pregnenolone were completely metabolized to their respective androgens (androstenedione and dehydroepiandrosterone). Although 11 beta-hydroxy-progesterone was readily 17 alpha-hydroxylated by the shark P450, the lyase reaction was not evident. Alterations to the 2-carbon sidechain of progesterone (21-hydroxylation or 20 beta-reduction) prevented metabolism. High-density cultures (> 1.5 x 10(9) cells/ml) yielded the greatest quantity of recombinant protein but cultures of lower density produced more recombinant protein per cell. This is the first report of heterologous expression in yeast of a steroidogenic cytochrome P450 from a lower vertebrate.

Isolation and characterization of the cDNA encoding the spiny dogfish shark (Squalus acanthias) form of cytochrome P450c17.

Cytochrome P450c17 is a key steroidogenic enzyme for the production of sex steroids in gonadal tissue and for cortisol production in adrenal tissue. This protein possesses two enzymatic activities. The 17 alpha-hydroxylase activity results in the introduction of a hydroxyl group at the 17 alpha-position. The resultant 17 alpha-hydroxylated, C21 pregnene is converted to a C19 androgen by the C17,20-lyase activity. A cDNA library was constructed from poly(A)-enriched mRNA isolated from spiny dogfish shark (Squalus acanthias) testis and ligated into EcoRI-cut lambda arms. The amplified library was screened using a bovine P450c17 cDNA probe and five positive clones were isolated. The described cDNA encompasses 23 bp of the 5'-untranslated region, a 1,527 bp open reading frame, and 414 bp of the 3'-untranslated region. A putative polyadenylation signal (AATAAA) is 18 bp from the poly(A) tail. Northern blot analysis showed a single transcript of 1.9 kb, thus indicating the isolated clone is a full-length cDNA. The deduced amino acid sequence of the shark form of P450c17 is 59% and 57% identical to the rainbow trout and chicken forms, respectively. The shark form is 43% to 46% identical to mammalian forms (rat, human, mouse, bovine, and porcine). There are large regions of extremely high identity shared among all the forms. The deduced shark 17 alpha-hydroxylase protein is 509 residues in length with a predicted weight of 57.2 kDa. Non-steroidogenic COS cells, transfected with the shark P450c17 cDNA, was capable of 17 alpha-hydroxylase and C17,20-lyase activities using both pregnenolone and progesterone as initial substrates.