Ontogenic and sexually dimorphic expression of cyp19 isoforms in the rainbowfish, Melanotaenia fluviatilis (Castelnau 1878)

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Abstract

To investigate the role of cytochrome P450 aromatase, we isolated cyp19 isoforms in the Murray River rainbowfish, M. fluviatilis. The cloned cDNA for cyp19a1a and cyp19a1b had an open reading frame (ORF) of 492 and 499 amino acid residues, with shared identity of up to 83% and 87% with the corresponding homologues of other teleosts respectively. In contrast, the cyp19a1a and cyp19a1b of the Murray River rainbowfish had a shared identity of only 61%. Not surprisingly, the phylogenetic analysis clustered the M. fluviatilis cyp19 isoforms with the corresponding isoforms of other teleosts, suggesting a shared evolutionary ancestry of the respective isoforms. We also studied the expression of cyp19 isoforms during ontogeny and in adult fish using quantitative Real-Time PCR (qPCR). Results suggest that uniquely only cyp19a1b transcripts are maternally inherited, suggesting its role in early development and growth in the species. In contrast to reports in many teleosts, the cyp19a1a was exclusively expressed in the ovarian tissue and completely absent in other tissues examined, including testis. The cyp19a1b like in most teleosts was predominantly expressed in the brain of both males and females with low level of expression in other tissues including gonads of both sexes.

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.

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