Huiling Xiong

The goldfish (Carassius auratus) as a model for neuroendocrine signaling

Goldfish (Carassius auratus) are excellent model organisms for the neuroendocrine signaling and the regulation of reproduction in vertebrates. Goldfish also serve as useful model organisms in numerous other fields. In contrast to mammals, teleost fish do not have a median eminence; the anterior pituitary is innervated by numerous neuronal cell types and thus, pituitary hormone release is directly regulated. Here we briefly describe the neuroendocrine control of luteinizing hormone. Stimulation by gonadotropin-releasing hormone and a multitude of classical neurotransmitters and neuropeptides is opposed by the potent inhibitory actions of dopamine. The stimulatory actions of gamma-aminobutyric acid and serotonin are also discussed. We will focus on the development of a cDNA microarray composed of carp and goldfish sequences which has allowed us to examine neurotransmitter-regulated gene expression in the neuroendocrine brain and to investigate potential genomic interactions between these key neurotransmitter systems. We observed that isotocin (fish homologue of oxytocin) and activins are regulated by multiple neurotransmitters, which is discussed in light of their roles in reproduction in other species. We have also found that many novel and uncharacterized goldfish expressed sequence tags in the brain are also regulated by neurotransmitters. Their sites of production and whether they play a role in neuroendocrine signaling and control of reproduction remain to be determined. The transcriptomic tools developed to study reproduction could also be used to advance our understanding of neuroendocrine-immune interactions and the relationship between growth and food intake in fish.

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.

Auto-regulation of estrogen receptor subtypes and gene expression profiling of 17β-estradiol action in the neuroendocrine axis of male goldfish

Auto-regulation of the three goldfish estrogen receptor (ER) subtypes was examined simultaneously in multiple tissues, in relation to mRNA levels of liver vitellogenin (VTG) and brain transcripts. Male goldfish were implanted with a silastic implant containing either no steroid or 17beta-estradiol (E2) (100 microg/g body mass) for one and seven days. Liver transcript levels of ERalpha were the most highly up-regulated of the ERs, and a parallel induction of liver VTG was observed. In the testes (7d) and telencephalon (7d), E2 induced ERalpha. In the liver (1d) and hypothalamus (7d) ERbeta1 was down-regulated, while ERbeta2 remained unchanged under all conditions. Although aromatase B levels increased in the brain, the majority of candidate genes identified by microarray in the hypothalamus (1d) decreased. These results demonstrate that ER subtypes are differentially regulated by E2, and several brain transcripts decrease upon short-term elevation of circulating E2 levels.

Using Generalized Procrustes Analysis (GPA) for normalization of cDNA microarray data

BackgroundNormalization is essential in dual-labelled microarray data analysis to remove non-biological variations and systematic biases. Many normalization methods have been used to remove such biases within slides (Global, Lowess) and across slides (Scale, Quantile and VSN). However, all these popular approaches have critical assumptions about data distribution, which is often not valid in practice.ResultsIn this study, we propose a novel assumption-free normalization method based on the Generalized Procrustes Analysis (GPA) algorithm. Using experimental and simulated normal microarray data and boutique array data, we systemically evaluate the ability of the GPA method in normalization compared with six other popular normalization methods including Global, Lowess, Scale, Quantile, VSN, and one boutique array-specific housekeeping gene method. The assessment of these methods is based on three different empirical criteria: across-slide variability, the Kolmogorov-Smirnov (K-S) statistic and the mean square error (MSE). Compared with other methods, the GPA method performs effectively and consistently better in reducing across-slide variability and removing systematic bias.ConclusionThe GPA method is an effective normalization approach for microarray data analysis. In particular, it is free from the statistical and biological assumptions inherent in other normalization methods that are often difficult to validate. Therefore, the GPA method has a major advantage in that it can be applied to diverse types of array sets, especially to the boutique array where the majority of genes may be differentially expressed.

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, GABAA 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.

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

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 17beta-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 mcirog/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 = 0.027) and depressed AroB mRNA expression threefold in both the telencephalon (P = 0.021) and the hypothalamus (P = 0.006). Microarray expression profiling of the telencephalon identified 98 differentially expressed genes after fadrozole treatment (q value <0.05). 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-betaA) or increased (calmodulin, ornithine decarboxylase 1) mRNA expression. These data have implications for our understanding of estrogen actions in the adult vertebrate brain.

Mural granulosa cell gene expression associated with oocyte developmental competence

BackgroundOvarian follicle development is a complex process. Paracrine interactions between somatic and germ cells are critical for normal follicular development and oocyte maturation. Studies have suggested that the health and function of the granulosa and cumulus cells may be reflective of the health status of the enclosed oocyte. The objective of the present study is to assess, using an in vivo immature rat model, gene expression profile in granulosa cells, which may be linked to the developmental competence of the oocyte. We hypothesized that expression of specific genes in granulosa cells may be correlated with the developmental competence of the oocyte.MethodsImmature rats were injected with eCG and 24 h thereafter with anti-eCG antibody to induce follicular atresia or with pre-immune serum to stimulate follicle development. A high percentage (30-50%, normal developmental competence, NDC) of oocytes from eCG/pre-immune serum group developed to term after embryo transfer compared to those from eCG/anti-eCG (0%, poor developmental competence, PDC). Gene expression profiles of mural granulosa cells from the above oocyte-collected follicles were assessed by Affymetrix rat whole genome array.ResultsThe result showed that twelve genes were up-regulated, while one gene was down-regulated more than 1.5 folds in the NDC group compared with those in the PDC group. Gene ontology classification showed that the up-regulated genes included lysyl oxidase (Lox) and nerve growth factor receptor associated protein 1 (Ngfrap1), which are important in the regulation of protein-lysine 6-oxidase activity, and in apoptosis induction, respectively. The down-regulated genes included glycoprotein-4-beta galactosyltransferase 2 (Ggbt2), which is involved in the regulation of extracellular matrix organization and biogenesis.ConclusionsThe data in the present study demonstrate a close association between specific gene expression in mural granulosa cells and the developmental competence of oocytes. This finding suggests that the most differentially expressed gene, lysyl oxidase, may be a candidate biomarker of oocyte health and useful for the selection of good quality oocytes for assisted reproduction.

Bioinformatic Approach to Identify Penultimate Amino Acids Efficient for N-Terminal Methionine Excision

More than half of proteins in prokaryotes and eukaryotes undergo N-terminal methionine excision (NME). While it is known that the penultimate amino acid affects the efficiency of NME in several bacterial and eukaryotic species, it is experimentally difficult and tedious to verify which amino acid at the penultimate site (the site after initiator Met) is the most efficient for NME in different species. Here we present a new bioinformatic approach to identify penultimate amino acids that are efficient for NME. Amino acids most efficient for NME are alanine, serine and proline in human, and alanine, glycine, valine, proline and serine in the yeast Saccharomyces cerevisiae. This finding also helps resolve the two hypotheses that have been proposed to explain the presence of+4G site in the Kozak consensus for translation initiation.