Sex- and tissue-specific effects of waterborne estrogen on estrogen receptor subtypes and E2-mediated gene expression in the reproductive axis of goldfish.

This research examined the gene expression profile of three goldfish estrogen receptor (ER) subtypes in multiple tissues in relation to mRNA levels of aromatase B and vitellogenin (VTG) following waterborne estrogen exposures. The protocol consisted of: i) adult male goldfish in late gonadal recrudescence exposed to 1 nM 17beta-estradiol (E2); ii) adult male and female goldfish in early sexual regression exposed to 1 nM E2 for 3, 6, 12 and 24h; and, iii) sexually mature, adult male goldfish exposed to 0.3 nM 17alpha-ethynylestradiol (EE2) for 24h. Liver produced the most consistent response with up-regulation of ERalpha in sexually regressed, mature and recrudescing males and in sexually regressed females. The dose and length of exposure, reproductive state and sex affected the auto-regulation of ERbeta1 by E2. ERbeta2 was not affected in any experiments suggesting it may not be auto-regulated by E2. Aromatase B and VTG gene expression were affected by E2, but also by other experimental conditions. EE2 induced liver ERalpha and VTG mRNA levels indicating that high environmental EE2 levels induce E2-mediated gene expression in a model teleost. These studies reveal a more complicated action of estrogenic compounds that has important implications on estrogenic endocrine disruptors in teleosts.

Defining global neuroendocrine gene expression patterns associated with reproductive seasonality in fish.

BACKGROUND: Many vertebrates, including the goldfish, exhibit seasonal reproductive rhythms, which are a result of interactions between external environmental stimuli and internal endocrine systems in the hypothalamo-pituitary-gonadal axis. While it is long believed that differential expression of neuroendocrine genes contributes to establishing seasonal reproductive rhythms, no systems-level investigation has yet been conducted. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, by analyzing multiple female goldfish brain microarray datasets, we have characterized global gene expression patterns for a seasonal cycle. A core set of genes (873 genes) in the hypothalamus were identified to be differentially expressed between May, August and December, which correspond to physiologically distinct stages that are sexually mature (prespawning), sexual regression, and early gonadal redevelopment, respectively. Expression changes of these genes are also shared by another brain region, the telencephalon, as revealed by multivariate analysis. More importantly, by examining one dataset obtained from fish in October who were kept under long-daylength photoperiod (16 h) typical of the springtime breeding season (May), we observed that the expression of identified genes appears regulated by photoperiod, a major factor controlling vertebrate reproductive cyclicity. Gene ontology analysis revealed that hormone genes and genes functionally involved in G-protein coupled receptor signaling pathway and transmission of nerve impulses are significantly enriched in an expression pattern, whose transition is located between prespawning and sexually regressed stages. The existence of seasonal expression patterns was verified for several genes including isotocin, ependymin II, GABA(A) gamma2 receptor, calmodulin, and aromatase b by independent samplings of goldfish brains from six seasonal time points and real-time PCR assays. CONCLUSIONS/SIGNIFICANCE: Using both theoretical and experimental strategies, we report for the first time global gene expression patterns throughout a breeding season which may account for dynamic neuroendocrine regulation of seasonal reproductive development.

Mercury-induced reproductive impairment in fish.

Mercury is a potent neurotoxin, and increasing levels have led to concern for human and wildlife health in many regions of the world. During the past three decades, studies in fish have examined the effects of sublethal mercury exposure on a range of endpoints within the reproductive axis. Mercury studies have varied from highly concentrated aqueous exposures to ecologically relevant dietary exposures using levels comparable to those currently found in the environment. This review summarizes data from both laboratory and field studies supporting the hypothesis that mercury in the aquatic environment impacts the reproductive health of fish. The evidence presented suggests that the inhibitory effects of mercury on reproduction occur at multiple sites within the reproductive axis, including the hypothalamus, pituitary, and gonads. Accumulation of mercury in the fish brain has resulted in reduced neurosecretory material, hypothalamic neuron degeneration, and alterations in parameters of monoaminergic neurotransmission. At the level of the pituitary, mercury exposure has reduced and/or inactivated gonadotropin-secreting cells. Finally, studies have examined the effects of mercury on the reproductive organs and demonstrated a range of effects, including reductions in gonad size, circulating reproductive steroids, gamete production, and spawning success. Despite some variation between studies, there appears to be sufficient evidence from laboratory studies to link exposure to mercury with reproductive impairment in many fish species. Currently, the mechanisms underlying these effects are unknown; however, several physiological and cellular mechanisms are proposed within this review.

Effects of fluoxetine on the reproductive axis of female goldfish (Carassius auratus).

We investigated the effects of fluoxetine, a selective serotonin reuptake inhibitor, on neuroendocrine function and the reproductive axis in female goldfish. Fish were given intraperitoneal injections of fluoxetine twice a week for 14 days, resulting in five injections of 5 microg fluoxetine/g body wt. We measured the monoamine neurotransmitters serotonin, dopamine, and norepinephrine in addition to their metabolites with HPLC. Homovanillic acid, a metabolite in the dopaminergic pathway, increased significantly in the hypothalamus. Plasma estradiol levels were measured by radioimmunoassay and were significantly reduced approximately threefold after fluoxetine treatment. We found that fluoxetine also significantly reduced the expression of estrogen receptor (ER)beta1 mRNA by 4-fold in both the hypothalamus and the telencephalon and ERalpha mRNA by 1.7-fold in the telencephalon. Fluoxetine had no effect on the expression of ERbeta2 mRNA in the hypothalamus or telencephalon. Microarray analysis identified isotocin, a neuropeptide that stimulates reproductive behavior in fish, as a candidate gene affected by fluoxetine treatment. Real-time RT-PCR verified that isotocin mRNA was downregulated approximately sixfold in the hypothalamus and fivefold in the telencephalon. Intraperitoneal injection of isotocin (1 microg/g) increased plasma estradiol, providing a potential link between changes in isotocin gene expression and decreased circulating estrogen in fluoxetine-injected fish. Our results reveal targets of serotonergic modulation in the neuroendocrine brain and indicate that fluoxetine has the potential to affect sex hormones and modulate genes involved in reproductive function and behavior in the brain of female goldfish. We discuss these findings in the context of endocrine disruption because fluoxetine has been detected in the environment.

Hormone cross-regulation in the tadpole brain: developmental expression profiles and effect of T3 exposure on thyroid hormone- and estrogen-responsive genes in Rana pipiens.

During metamorphosis, the tadpole neuroendocrine brain is a major target for the organisational effects of hormones acting via both endocrine feedback mechanisms and local hormone production. While the receptor-mediated actions of thyroid hormones in brain development have been well described, there is evidence that thyroid hormones could also be an important modulator of estrogen action during metamorphosis. To better understand hormone action and potential cross-regulation between thyroid hormone and estrogen, we examined changes in thyroid hormone receptors (TRalpha and TRbeta) and the estrogen receptor (ERalpha) in the brain of Rana pipiens throughout metamorphosis and in response to 48 h waterborne triiodothyronine (T3) exposure (0.5, 5 and 50 nM). We also measured mRNA levels of iodothyronine deiodinase (D2 and D3) and aromatase, key enzymes responsible for local synthesis and availability of thyroid hormones and estrogen, respectively. A real-time PCR strategy targeting these genes was developed using either a fluorescent dual-labelled probe- or SYBR Green I-based method. TRbeta mRNA levels were increased during development and in response to T3 exposure. Deiodinase (D2 and D3) enzymes were differentially regulated during development, but mRNA levels of both were increased with 50 nM T3 exposure. ERalpha and aromatase mRNA levels significantly increased at metamorphic climax, but whereas estrogen receptor alpha mRNA levels were increased by 50 nM T3, aromatase mRNA levels were decreased. These results (1) demonstrate that the developing amphibian brain is an important site for stage-specific thyroid hormone regulation of nuclear receptors and hormone synthesis enzymes and (2) provide the basis for further studies exploring the physiological and functional significance of the cross-regulation between thyroid status and estrogen-sensitive genes in the brain during amphibian metamorphosis.

Characterization of Inhibitor of Growth 2 tumor suppressor in Alligator mississippiensis, its conservation in Archosauria, and response to thyroid stimulating hormone.

Inhibitor of growth 2 (ING2) belongs to a family of tumor suppressors that are important regulators of a wide range of cellular processes including proliferation, apoptosis, and DNA repair. ING family members are found in yeast, plants, invertebrates and many vertebrate species. However, to date, ING has not been characterized in reptiles. Herein we describe the isolation of expressed ING2 sequence in the American alligator, Alligator mississippiensis, and compare this sequence with that isolated in the chicken. We identify features that are unique to these two representatives of the Archosaurs including conservation of specific amino acid residues and the absence of an adenylate residue in the 5' end of the nucleotide sequence relative to frogs and mammals. The latter feature results in an alteration of the coding potential leading to distinctive N-termini. Injection of juvenile alligators with thyroid stimulating hormone (TSH), which increases endogenous thyroid hormones, results in the modulation of ING2 transcript levels. Quantitative real time polymerase chain reaction analyses revealed a reduction in the steady-state levels of ING2 mRNA in the phallus/cliterophallus, lung, and liver by 48 h after TSH injection. ING2 expression in the thyroid gland, gonad, and heart was unaffected by TSH treatment. These data indicate that control of ING2 expression by the thyroid axis may be conserved among species and is tissue-dependent.

Gene expression profiling in the neuroendocrine brain of male goldfish (Carassius auratus) exposed to 17alpha-ethinylestradiol.

17-alpha ethinylestradiol (EE2), a pharmaceutical estrogen, is detectable in water systems worldwide. Although studies report on the effects of xenoestrogens in tissues such as liver and gonad, few studies to date have investigated the effects of EE2 in the vertebrate brain at a large scale. The purpose of this study was to develop a goldfish brain-enriched cDNA array and use this in conjunction with a mixed tissue carp microarray to study the genomic response to EE2 in the brain. Gonad-intact male goldfish were exposed to nominal concentrations of 0.1 nM (29.6 ng/l) and 1.0 nM (296 ng/l) EE2 for 15 days. Male goldfish treated with the higher dose of EE2 had significantly smaller gonads compared with controls. Males also had a significantly reduced level of circulating testosterone (T) and 17beta-estradiol (E2) in both treatment groups. Candidate genes identified by microarray analysis fall into functional categories that include neuropeptides, cell metabolism, and transcription/translation factors. Differentially expressed genes verified by real-time RT-PCR included brain aromatase, secretogranin-III, and interferon-related developmental regulator 1. Our results suggest that the expression of genes in the sexually mature adult brain appears to be resistant to low EE2 exposure but is affected significantly at higher doses of EE2. This study demonstrates that microarray technology is a useful tool to study the effects of endocrine disrupting chemicals on neuroendocrine function and suggest that exposure to EE2 may have significant effects on localized E2 synthesis in the brain by affecting transcription of brain aromatase.

Isolation of the alligator (Alligator mississippiensis) thyroid hormone receptor alpha and beta transcripts and their responsiveness to thyroid stimulating hormone.

Thyroid hormones (THs) play key regulatory roles in growth, development and metabolism in vertebrates. Modulation of the cellular hormonal response is largely through the activity of two nuclear TH receptors, TRalpha and TRbeta, which act as transcription factors and alter gene expression programs. Little information is available regarding their structure and regulation in reptiles. We have cloned the expressed sequences encoding these two receptors in the American alligator, Alligator mississippiensis. The encoded putative proteins share a high degree of amino acid sequence conservation with other vertebrates, however, both alligator TRs contain putative N-terminal truncations. This phenomenon is shared with the chicken for TRbeta, but not for TRalpha, making this the first demonstration of this type of TRalpha isoform. We measured the steady-state levels of TR transcripts in heart, lung, liver, thyroid, cliterophallus/phallus, and gonad of juvenile alligators 24 and 48 h after injection with thyroid stimulating hormone (TSH). TRalpha transcript levels were increased in the heart, decreased in the lung and cliterophallus/phallus, and unaffected in the liver, thyroid, and gonad. TRbeta transcript levels were increased in the heart, lung, and gonad whereas estrogen receptor alpha transcript levels were elevated by TSH treatment only in the gonad. Modulation of these transcripts in the gonad is consistent with TH playing an important role in this tissue's function since seasonal TH fluctuations coincide with reproductive events. These data demonstrate that alligator tissues are differentially responsive to TSH by regulation of TR expression and provide an important comparative framework among vertebrates.

Proteolytic processing and differential distribution of secretogranin-II in goldfish.

Secretoneurin (SN) is a 33-34 amino acid neuropeptide derived by endoproteolysis of secretogranin-II (SgII), a chromogranin. A multi-antigenic strategy was used to generate a rabbit polyclonal goldfish SN antiserum that was characterized for Western blot analysis. In the goldfish pituitary two intermediate proteins containing SN and likely processed from the 69.6-kDa SgII precursor were detected. No immunoreactive proteins were observed in the goldfish interrenal, ovary, cerebellum, and telencephalon whereas SgII mRNA was expressed in all these tissues. Immunoreactive levels of the approximately 57 kDa product were higher in the pars distalis (PD) than in the neurointermediate lobe (NIL). The abundance of the approximately 57 kDa protein indicates that this SgII-product containing the SN sequence is a major stored form in secretory granules of the goldfish pituitary. High expression and processing of SN in the hypothalamus and pituitary suggest important roles for SgII-derived peptides in neuroendocrine tissues.