Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology
Ontogenic and sexually dimorphic expression of cyp19 isoforms in the rainbowfish, Melanotaenia fluviatilis (Castelnau 1878)
Introduction
Aromatase is a key steroidogenic enzyme that converts androgens to estrogens by aromatization of C19 androgens to C18 estrogens (Blázquez and Piferrer, 2004). This enzyme is membrane-bound and is located in the endoplasmic reticulum of estrogen-producing cells of ovaries, placenta, testes, adipose and brain tissues (Sundaray et al., 2005). It is expressed in different key tissues including brain, gonads, retina, spleen, kidney, liver and other tissues and is essential for gonad development and other physiological processes including growth, neurogenesis and reproductive behavior. Although, several putative multiple functions for both ovarian and brain aromatases of teleosts have been proposed (Barney et al., 2008, Penman and Piferrer, 2008, Guigen et al., 2010), a number these functions as well as the regulatory mechanisms largely remain unresolved.
In mammals, except in porcines, there is a single cyp19 gene which is expressed in different tissues. In contrast, two structurally and functionally different cyp19 isoforms have been found in teleosts which are products of different cyp19 gene loci, one preferentially expressed in the ovary and the other in the brain, designated cyp19a1a (CYP19a/P450AromA/CYP19A1) and cyp19a1b (CYP19b/P450AromB/CYP19A2) respectively (Tchoudakova and Callard, 1998, Blázquez and Piferrer, 2004, Chang et al., 2005, Guigen et al., 2010). The cDNAs encoding P450Arom isoforms have been isolated from several (over twenty) teleost species including the Japanese medaka (Fukuda et al., 1996), goldfish (Gelinas et al., 1998, Halm et al., 2001), zebrafish (Chiang et al., 2001), pejerrey (Strobl-Mazzulla et al., 2005) and common carp (Barney et al., 2008). This gene is duplicated in all the investigated teleosts except the Japanese eel, Anguilla japonica (Jeng et al., 2005).
Strikingly teleosts are peculiar in that their aromatase activity is 100–1000 times higher than that of mammals and other vertebrates (Pasmanik and Callard, 1985). The significance of elevated aromatase is not yet clear at the biological level even though several hypotheses on neuroprotection (Garcia-Segura et al., 2001) and neurogenesis (Callard et al., 2001) have been proposed. Among cyp19 genes, cyp19a1a is predominantly expressed in the ovary and believed to play a role in sex differentiation and ovarian development; whereas cyp19a1b is thought to be involved in neural development in brain, retina and pituitary as well as play a key role in sexual behavior (Kishida and Callard, 2001).
In teleosts, brain is the primary organ expressing aromatase with both isoforms exhibiting subtle species and or sex-specific differences in their spatial and temporal expression patterns. For example, the expression of cyp19a1a is strictly restricted to ovaries in some species, while in others albeit dominant in ovary, is also expressed in other tissues, including testes at low levels (See Table 1 for summary). Similar observations have been reported for cyp19a1b (Table 1), including within organ and between sex differences in the common carp (Barney et al., 2008). Further, cellular examination of cyp19a1b shows its expression to be restricted to radial glial cells in the brain of trout (Menuet et al., 2003) and zebrafish (Goto-Kazeto et al., 2004, Pellegrini et al., 2005). However, there was a striking difference in that the cyp19a1b transcripts appear to be exported into the extensions of the radial glial cells in zebrafish but not in trout (Menuet et al., 2003). Significance of such within organ, between species and or sex specific differences, as well as mechanisms of their regulation remains poorly understood. In this context, the inherent diversity of teleost species combined with functional specialization of the two isoforms provides unique opportunities to dissect the cellular and molecular basis of the diverse roles of estrogen in vertebrates at large.
In the present study, aromatase gene expression in the Murray River rainbowfish was investigated. This species has been utilized in laboratory experiments for various reasons including its ease of maintenance and short life cycle enabling the study of all life stages quickly (Pollino et al., 2007). The purpose of this study was to isolate the cDNA encoding the aromatase isoforms and to characterize their expression during ontogeny and adulthood.
Section snippets
Animals
Murray River rainbowfish were purchased from a commercial aquarium (Aquarium Industries, Melbourne, Australia) and reared at 25 ± 1 °C in 16:8 h light:dark regime in flow-through aquaria with carbon filtered aerated water. Throughout the maintenance, water quality parameters including temperature, dissolved oxygen, pH and conductivity were monitored and the fish were fed commercial fish pellets (Tetra color™, Blacksburg, VA, USA) twice daily. Reproductively active (spawning) male and female fish
Sequences and phylogenetic analysis
Two distinctly different isoforms of aromatase genes were isolated from brain and ovary. The ovarian aromatase gene (GenBank accession no. GU723457) was 1670 bp in length, with a GC content of 46.7% and having an ORF of 492 amino acids. In contrast, the brain isoform (GenBank accession no. GU723458) was longer (2056 bp) with an overall GC content of 44.5%, with the 5′ end (5′ of 5′ RACE primer) higher (47.4%) than the 3′ end (40.2% — 3′ of 3′ RACE primer) and an ORF of 499 amino acids. Although
Discussion
In this study we cloned full-length cyp19 isoforms encoding aromatase in M. fluviatilis along with their quantitative expression in key tissues for the first time.
The ovarian derived cyp19a1a comprises 1670 bp containing 1479 bp ORF and 492 amino acids; meanwhile the brain derived cyp19a1b comprises 2056 bp containing 1500 bp of ORF and 499 amino acid residues from the deduced protein. This is in agreement with what has been observed previously in other teleosts where generally the brain derived
Acknowledgments
We are grateful to RMIT University for International Postgraduate Scholarship to Shanthanagouda AH and we thank James Oliver for help with animal husbandry. Authors also thank two anonymous reviewers for providing useful comments and suggestions on an earlier draft of the manuscript.
References (64)
- et al.
Distinct cytochrome P450 aromatase isoforms in the common carp (Cyprinus carpio): sexual dimorphism and onset of ontogenic expression
Gen. Comp. Endocrinol.
(2008) - et al.
Cloning, sequencing and analysis, tissue distribution, and sex-specific expression of the neural form of P450 aromatase in juvenile sea bass (Dicentrarchus labrax)
Mol. Cell. Endocrinol.
(2004) - et al.
Molecular and cellular physiology of aromatase in the brain and retina
J. Steroid Biochem. Mol. Biol.
(1993) - et al.
Differential tissue distribution, developmental programming, estrogen regulation and promoter characteristics of cyp19 genes in teleost fish
J. Steroid Biochem. Mol. Biol.
(2001) - et al.
Two types of aromatase with different encoding genes, tissue distribution and developmental expression in Nile tilapia (Oreochromis niloticus)
Gen. Comp. Endocrinol.
(2005) - et al.
Molecular cloning of cytochrome P450 aromatases in the protogynous wrasse, Halichoeres tenuispinis
Comp. Biochem. Physiol. B
(2005) - et al.
European sea bass (Dicentrarchus labrax L.) cytochrome P450arom: cDNA cloning, expression and genomic organization
J. Steroid Biochem. Mol. Biol.
(2002) - et al.
Molecular cloning, characterization and expression analysis of gonadal P450aromatase in the half-smooth tongue-sole, Cynoglossus semilaevis
Aquaculture
(2009) - et al.
Sex determination and sex differentiation in fish: an overview of genetic, physiological, and environmental influences
Aquaculture
(2002) - et al.
Aromatase in the brain of teleost fish: expression, regulation and putative functions
Front. Neuroendocrinol.
(2010)
Neuroprotection by estradiol
Prog. Neurobiol.
Isolation of a goldfish brain cytochrome P450 aromatase cDNA: mRNA expression during the seasonal cycle and after steroid treatment
Mol. Cell. Endocrinol.
Aromatase activity in the European sea bass (Dicentrarchus labrax L.) brain. Distribution and changes in relation to age, sex, and the annual reproductive cycle
Gen. Comp. Endocrinol.
Localization and expression of aromatase mRNA in adult zebrafish
Gen. Comp. Endocrinol.
Isolation and characterization of two cytochrome P450 aromatase forms in killifish (Fundulus heteroclitus): differential expression in fish from polluted and unpolluted environments
Aquat. Toxicol.
Characterization of gonadal and extra forms of the cDNA encoding the Atlantic stingray (Dasyatis sabina) cytochrome P450 aromatase (CYP19)
Mol. Cell. Endocrinol.
Sex- and tissue-specific expression of P450 aromatase (cyp19a1a) in the yellowtail clownfish, Amphiprion clarkia
Comp. Biochem. Physiol. A
Molecular characterization and sex-specific tissue expression of estrogen receptor α (esr1), estrogen receptor βa (esr2a) and ovarian aromatase (cyp19a1a) in yellow perch (Perca flavescens)
Comp. Biochem. Physiol. B
Real-Time PCR analysis of ovary and brain-type aromatase gene expression during Atlantic halibut (Hippoglossus hippoglossus) development
Comp. Biochem. Physiol. B.
Tissue-and sex-specific regulation of CYP19A1 expression in the Atlantic croaker (Micropogonias undulatus)
Gen. Comp. Endocrinol.
Aromatase and 5α-reductase in the teleost brain, spinal cord, and pituitary gland
Gen. Comp. Endocrinol.
Tissue and sexually dimorphic expression of ovarian and brain aromatase mRNA in the Japanese medaka (Oryzias latipes): implications for their preferential roles in ovarian and neural differentiation and development
Gen. Comp. Endocrinol.
Relationships between aromatase and estrogen receptors in the brain of teleost fish
Gen. Comp. Endocrinol.
Real-time PCR analysis of cytochrome P450 aromatase expression in zebrafish: gene specific tissue distribution, sex differences, developmental programming and estrogen regulation
Gen. Comp. Endocrinol.
Brain aromatase from pejerrey fish (Odontesthes bonariensis): cDNA cloning, tissue expression, and immunohistochemical localization
Gen. Comp. Endocrinol.
Cloning and developmental expression of the cytochrome P450 aromatase gene (CYP19) in the European eel (Anguilla anguilla)
Gen. Comp. Endocrinol.
Relationship between brain and ovary aromatase activity and isoform-specific aromatase mRNA expression in the fathead minnow (Pimephales promelas)
Aquat. Toxicol.
Expression of two cytochrome P450 aromatase genes is regulated by endocrine disrupting chemicals in rare minnow Gobiocypris rarus juveniles
Comp. Biochem. Physiol. C.
Two distinct cytochrome P450 aromatases in the orange-spotted grouper (Epinephelus coioides): cDNA cloning and differential mRNA expression
J. Steroid Biochem. Mol. Biol.
Relationships between liver testosterone receptor isoforms and aromatase activity in female green frog Rana esculenta
Life Sci.
Isolation and characterization of the cDNA encoding the tilapia (Oreochromis niloticus) cytochrome P450 aromatase (P450arom): changes in P450arom mRNA, protein and enzyme activity in ovarian follicles during oogenesis
J. Mol. Endocrinol.
Two cyp19 (P450 aromatase) genes on duplicated zebrafish chromosomes are expressed in ovary or brain
Mol. Biol. Evol.
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