Profiling neuroendocrine gene expression changes following fadrozole-induced estrogen decline in the female goldfish

Abstract

Teleost fish represent unique models to study the role of neuroestrogens because of the extremely high activity of brain aromatase (AroB; the product of cyp19a1b). Aromatase respectively converts androstenedione and testosterone to estrone and 17␤-estradiol (E2). Specific inhibition of aromatase activity by fadrozole has been shown to impair estrogen production and influence neuroendocrine and reproductive functions in fish, amphibians, and rodents. However, very few studies have identified the global transcriptomic response to fadrozole-induced decline of estrogens in a physiological context. In our study, sexually mature prespawning female goldfish were exposed to fadrozole (50 g/l) in March and April when goldfish have the highest AroB activity and maximal gonadal size. Fadrozole treatment significantly decreased serum E2 levels (4.7 times lower; P ϭ 0.027) and depressed AroB mRNA expression threefold in both the telencephalon (P ϭ 0.021) and the hypothalamus (P ϭ 0.006). Microarray expression profiling of the telencephalon identified 98 differentially expressed genes after fadrozole treatment (q value Ͻ0.05). Some of these genes have shown previously to be estrogen responsive in either fish or other species, including rat, mouse, and human. Gene ontology analysis together with functional annotations revealed several regulatory themes for physiological estrogen action in fish brain that include the regulation of calcium signaling pathway and autoregulation of estrogen receptor action. Real-time PCR verified microarray data for decreased (activin-␤A) or increased (calmodulin, ornithine decarboxylase 1) mRNA expression. These data have implications for our understanding of estrogen actions in the adult vertebrate brain.aromatase; microarray; fish; brain MAIN ESTROGENS estradiol-17␤ (E2) and estrone (E1) play fundamental regulatory roles in neuroendocrine and reproductive systems. Through binding to its nuclear estrogen receptors (nER)-␣ and nER-␤, E2 regulates transcriptional process of target genes whose promoters contain estrogen-responsive elements (ERE) (57). In addition to this classical nuclear action, E2 also has membrane actions in which a unique membrane receptor is utilized to rapidly activate a series of signaling pathways (52,70). Since the signaling pathway activation will ultimately influence the transcriptional activities of downstream transcription factors including nERs, membrane actions of E2 provide another mode to regulate genomic gene expression (70). Fish are unique models in which to study E2 action because there is high activity of the B-subtype aromatase (AroB; the product of cyp19a1b), the enzyme responsible for synthesizing E2 from testosterone (T), in the neuroendocrine brain, telencephalon (Tel) and hypothalamus (Hyp) (42). Moreover, in contrast to mammals where aromatase and nER are commonly expressed in neurons, current studies in fish and birds strongly suggest that AroB is exclusively expressed in radial glial cells and nER is mostly expressed in neurons (16,43). Thus, the local...

Regeneration of breeding tubercles on zebrafish pectoral fins requires androgens and two waves of revascularization

“…Here we show that androgens (T and DHT) and an aromatase inhibitor (fadrozole) induce the formation of BTs in both intact and regenerating pectoral fins of female zebrafish. Fadrozole can effectively decrease the serum levels of 4331 RESEARCH ARTICLE Zebrafish breeding tubercle regeneration E2, leading to differentially expressed estrogen-responsive genes and impaired ovarian development in female goldfish (Zhang et al, 2009). Similarly, fadrozole reduces the serum levels of E2 in female fathead minnows, whereas androgen serum levels remain stable (Ankley et al, 2002).…”

“…Flutamide (Sigma-Aldrich), in our hands, is effective at 2 mg/l when dissolved in ethanol and added to system water. Fadrozole was dissolved in system water at 50 μg/l (Ankley et al, 2002;Zhang et al, 2009). PTK787 (Selleck Chemicals) was dissolved in system water at an effective final concentration of 500 nM (Bayliss et al, 2006).…”

Analysis of Sexually Dimorphic Expression of Genes at Early Gonadogenesis of Pejerrey <i>Odontesthes bonariensis</i> Using a Heterologous Microarray

“…For estrogen receptors, HSP90 has the dual role of maintaining receptor inactivity in the absence of estradiol as well as ensuring an efficient hormonal response in mammals [Lee et al, 2002]. In goldfish, hypothalamic hsp90 was upregulated after aromatase inhibition with fadrozole, implying an association with estrogen signaling [Zhang et al, 2009]. Therefore, the high expression of hsp90 in pejerrey may be involved in estrogen-dependent processes associated with ovarian differentiation at the FPT.…”

“…The methods used were similar to those validated previously by some of the authors [Martyniuk et al, 2006;Zhang et al, 2009]. For microarray hybridizations, total RNA was extracted using RNeasy Plus Micro Kit (Qiagen) following the manufacturer's protocol.…”

“…There is, however, a general lack of these tools for most non-model fish species as the analyses have typically centered on the study of well-characterized model organisms [Garcia-Reyero et al, 2008]. We [Martyniuk et al, 2006;Popesku et al, 2008;Zhang et al, 2009] and others [Bar-Or et al, 2007;von Schalburg et al, 2008;Williams et al, 2008] have shown that cross-species microarray hybridizations are amenable for the study of diverse biological processes in numerous fishes. In this approach, target RNA and microarray probes are from different, but closely related species.…”