June-Woo Park

Real-time PCR array to study effects of chemicals on the Hypothalamic-Pituitary-Gonadal axis of the Japanese medaka

This paper describes the development and validation of a PCR array for studying chemical-induced effects on gene expression of selected endocrine pathways along the hypothalamic-pituitary-gonadal (HPG) axis of the small, oviparous fish, the Japanese medaka (Oryzias latipes). The Japanese medaka HPG-PCR array combines the quantitative performance of SYBR Green-based real-time PCR with the multiple gene profiling capabilities of a microarray to examine expression profiles of 36 genes associated with endocrine pathways in brain, liver and gonad. The performance of the Japanese medaka HPG-PCR array was evaluated by examining effects of two model compounds, the synthetic estrogen, 17alpha-ethinylestradiol (EE2) and the anabolic androgen, 17beta-trenbolone (TRB) on the HPG axis of the Japanese medaka. Four-month-old medaka was exposed to three concentrations of EE2 (5, 50, 500 ng/L) or TRB (50, 500, 5000 ng/L) for 7d in a static renewal exposure system. A pathway-based approach was implemented to analyze and visualize concentration-dependent mRNA expression in the HPG axis of Japanese medaka. The compensatory response to EE2 exposure included the down-regulation of male brain GnRH RI and testicular CYP17. The down-regulation of AR-alpha expression in brain of EE2-exposed males was associated with suppression of male sexual behavior. Compensatory responses to TRB in the female HPG axis included up-regulation of brain GnRH RII and ovary steroidogenic CYP19A. Overall, the results suggested that the Japanese medaka HPG-PCR array has potential not only as a screening tool of potential endocrine-disrupting chemicals but also in elucidating mechanisms of action.

Time‐Dependent transcriptional profiles of genes of the hypothalamic‐pituitary‐gonadal axis in medaka (Oryzias latipes) exposed to fadrozole and 17β‐trenbolone

Both the anabolic androgen 17beta-trenbolone (TRB) and the aromatase inhibitor fadrozole (FAD) can cause decreased plasma concentrations of estrogen (E2) and reduce fecundity of fish. However, the underlying mechanisms and the molecular pathways involved are largely unknown. The present study was designed to assess time-dependent effects of FAD and TRB on the transcriptional responses of the hypothalamic-pituitary-gonadal (HPG) axis of Japanese medaka (Oryzias latipes). Fourteen-week-old Japanese medaka were exposed to 50 microg FAD/L or 2 microg TRB/L in a 7-d static renewal test, and the expression profiles of 36 HPG axis genes were measured by means of a medaka HPG real-time reverse-transcription polymerase chain reaction array after 8 h, 32 h, or 7 d of exposure. Exposure to TRB or FAD caused lesser fecundity of Japanese medaka and down-regulated transcription of vitellogenin and choriogenin (CHG) gene expression in the liver of females. Exposure to FAD for 8 h resulted in an 8-fold and 71-fold down-regulation of expression of estrogen receptor alpha and choriogenin L (CHG L), respectively, in female liver. 17beta-Trenbolone caused similar down-regulation of these genes, but the effects were not observed until 32 h of exposure. These results support the hypothesis that FAD reduces plasma E2 more quickly by inhibiting aromatase enzyme activity than does TRB, which inhibits the production of the E2 precursor testosterone. Exposure to FAD and TRB resulted in rapid (after 8 h) down-regulation of luteinizing hormone receptor and low-density-lipoprotein receptor in the testis to compensate for excessive androgen levels. Overall, the molecular responses observed in the present study differentiate the mechanisms of the reduced fecundity by TRB and FAD.

Fluorescence in situ hybridization techniques (FISH) to detect changes in CYP19a gene expression of Japanese medaka (Oryzias latipes)

The aim of this study was to develop a sensitive in situ hybridization methodology using fluorescence-labeled riboprobes (FISH) that allows for the evaluation of gene expression profiles simultaneously in multiple target tissues of whole fish sections of Japanese medaka (Oryzias latipes). To date FISH methods have been limited in their application due to autofluorescence of tissues, fixatives or other components of the hybridization procedure. An optimized FISH method, based on confocal fluorescence microscopy was developed to reduce the autofluorescence signal. Because of its tissue- and gender-specific expression and relevance in studies of endocrine disruption, gonadal aromatase (CYP19a) was used as a model gene. The in situ hybridization (ISH) system was validated in a test exposure with the aromatase inhibitor fadrozole. The optimized FISH method revealed tissue-specific expression of the CYP19a gene. Furthermore, the assay could differentiate the abundance of CYP19a mRNA among cell types. Expression of CYP19a was primarily associated with early stage oocytes, and expression gradually decreased with increasing maturation. No expression of CYP19a mRNA was observed in other tissues such as brain, liver, or testes. Fadrozole (100 microg/L) caused up-regulation of CYP19a expression, a trend that was confirmed by RT-PCR analysis on excised tissues. In a combination approach with gonad histology, it could be shown that the increase in CYP19a expression as measured by RT-PCR on a whole tissue basis was due to a combination of both increases in numbers of CYP19a-containing cells and an increase in the amount of CYP19a mRNA present in the cells.

Advanced fluorescence in situ hybridization to localize and quantify gene expression in Japanese medaka (Oryzias latipes) exposed to endocrine‐disrupting compounds

In an earlier study, we described the development of fluorescence in situ hybridization (FISH) using confocal microscopy to localize and quantify gene expression in fish. Here, we report the results of FISH application to investigate effects of model endocrine-disrupting chemicals (EDCs), 17alpha-ethinylestradiol (EE2) and 17beta-trenbolone (TB), on expressions of EDC-responsive genes in Japanese medaka (Oryzias latipes) at the cellular/tissue level paired with histological observation. Gene expressions of vitellogenin-II (Vit-II), androgen receptor (AR), and cytochrome P450 gonadal aromatase (CYP19a) were determined after exposure to 5, 50, or 500 ng/L of EE2 or 50, 500, or 5,000 ng/L of TB for 7 d. Exposure to the greatest concentration of EE2 or TB significantly reduced fecundity and caused histological alterations in gonads. 17alpha-Ethinylestradiol induced Vit-II expression in both male gonads and liver relative to controls and resulted in greater intensity of hematoxylin staining in hepatocytes, which was significantly correlated with Vit-II induction in liver. When exposed to EE2 at less than 50 ng/L, CYP19a expression associated with early stage oocytes was greater than that in controls. However, at 500 ng/L, this trend was reversed. The greater Vit-II expression in testis from all EE2 groups, and the lesser expression of CYP19a in ovaries from the 500 ng/L group, likely is related to changes in the number of cells in which these genes are predominantly expressed rather than to an increase in expression per cell. 17beta-Trenbolone significantly induced AR expression in ovaries but did not alter AR expression in female liver. It was concluded that FISH combined with histology enables advanced elucidation of molecular effects of chemicals by associating changes in gene expression with certain tissues and/or cell types and allows these changes to be related to histological effects.

In situ hybridization to detect spatial gene expression in medaka

A whole-animal tissue section in situ hybridization (ISH) system with radio-labeled probes was developed to detect differential gene expression among tissues of the small, oviparous teleost fish, Japanese medaka (Oryzias latipes). Because of its tissue- and gender-specific expression, gonadal aromatase (CYP19a) was selected as a model gene to demonstrate the potential of the system. The ISH system was validated with a 7d exposure to the model aromatase inhibitor, fadrozole. Fadrozole did not affect the magnitude of gene expression in testes, but significantly up-regulated CYP19a gene expression in ovaries. These results were confirmed with quantitative real-time-polymerase chain reaction (RT-PCR). Histological evaluation revealed that females exposed to 100microg/L fadrozole lacked mature oocytes. Male gonadal morphology was normal in all treatments. The ISH method developed in this study allowed tissue-specific resolution of gene expression in a whole animal model, as well as the ability to analyze cellular morphological detail in the same organism.