2006 Progress Report: Chemical Induced Changes in Gene Expression Patterns Along the HPG-axis at Different Organizational Levels Using a Small Animal Model (Japanese medaka)EPA Grant Number: R831846
Title: Chemical Induced Changes in Gene Expression Patterns Along the HPG-axis at Different Organizational Levels Using a Small Animal Model (Japanese medaka)
Investigators: Giesy, John P. , Hecker, Markus , Jones, Paul D. , Newsted, John L.
Institution: Michigan State University
EPA Project Officer: Hahn, Intaek
Project Period: September 1, 2004 through August 31, 2007
Project Period Covered by this Report: September 1, 2005 through August 31, 2006
Project Amount: $749,904
RFA: Computational Toxicology and Endocrine Disruptors: Use of Systems Biology in Hazard Identification and Risk Assessment (2004) RFA Text | Recipients Lists
Research Category: Health , Safer Chemicals , Endocrine Disruptors , Computational Toxicology , Health Effects
Endocrine-disrupting compounds have been defined as exogenous agents that interfere with the “synthesis, secretion, transport, binding, action, or elimination of natural hormones in the body that are responsible for the maintenance of homeostasis, reproduction, development, and/or behavior.” Much of the recent concern and energy has been focused on compounds that are hormone-direct agonists or antagonists, especially those that interact with the estrogen receptor (ER). Effects consistent with exposure to ER agonists have been observed in fish exposed to natural hormones and some synthetic chemicals such as nonylphenol, nonylphenol polyethoxylates, octylphenol. Because chemicals can cause both direct (receptor-mediated) and indirect effects through changes in signal transduction pathways, methods are needed that permit the screening of multiple effects. Furthermore, methods are needed that can screen for these effects simultaneously in a number of tissues, including during critical windows of development, when tissues may be small and the amount of material available for testing is small and difficult to remove from the organism. The objective of this research project is to develop a screening method to use molecular techniques such as in situ hybridization (ISH), in situ reverse transcriptase-polymerase chain reaction (RT-PCR), and immunohistochemical (ICS) staining to screen for effects of chemicals on the hypothalamic-pituitary-gonadal (HPG) axis with a special emphasis on steroidogenic pathways and hormonal control mechanisms along the HPG-axis in the Japanese medaka. The proposed method will allow for screening of multiple effects in multiple tissues, even at points in development when the tissues are too small to be accurately dissected for use in more traditional molecular techniques. The proposed methods will be applied to a set of model and test compounds for a set of target genes. Once the methods have been developed and validated, they can be adapted for use with other genes and/or species of interest and used to efficiently and completely screen for endocrine disruptor effects beyond simple receptor binding.
Tissue Sectioning and In Situ Hybridization Techniques
Paraffin Embedded Sections. The procedures for fixation and paraffin embedding of whole fish samples were established at the time of submission of the first progress report (December 2005); very few minor changes have been made to the protocols since that time. The techniques have been shown to be reproducible in all sizes of fish tested and result in tissue sections suitable for the applications of general histological staining, IHC, and ISH (Figure 1). These protocols are routine histological procedures that have been optimized to facilitate large samples with complex textural qualities. Briefly, whole fish are fixed in an 80 percent Histochoice/2 percent paraformaldehyde/0.05 percent glutaraldehyde solution overnight. Samples are then dehydrated, cleared, and embedded in paraffin. Sections are cut at 7 microns thickness, stretched out on a prewarmed ethanol covered slide, floated out onto a 40°C water bath, and mounted on Superfrost Plus slides.
Figure 1. Hematoxylin- and Eosin-Stained Section of an Adult Male Medaka
Development of Probes. Three genes were initially selected to develop and optimize ISH techniques. Beta actin was selected as the housekeeping gene, and two isoforms of the aromatase gene, CYP19a and b, were selected as initial target genes because their expression is highly tissue specific. Furthermore, aromatase is currently discussed as one of the key endpoints in endocrine disruptor research, and both the CYP19a and b genes have been shown to be responsive to series of environmental chemicals (e.g., Sanderson, et al., 2000; Ankley, et al., 2005; Villeneuve, et al., 2006).
Preparation of Probes. Two types of probes, cDNA and riboprobes, have been developed to identify target genes in medaka by ISH. cDNA probes are generated using PCR but have several drawbacks, including the fact that they must be denatured prior to hybridization and that the complementary strands compete with the target sequence, which can decrease the sensitivity of the hybridization. The advantage of riboprobes over cDNA probes is that they bind target molecules more strongly. The disadvantage is that riboprobes are more susceptible to degradation by RNases. One aim of this study was to identify the optimum probe type for use with ISH in whole mount sections of Japanese medaka.
Synthesis of cDNA Probes. Total RNA was extracted from Japanese medaka using the SV Total RNA Isolation System (Promega, WI). Because of tissue-specific expression of the genes of interest, gonads were used for the extraction of CYP19a, head for CYP19b, and whole body for beta actin. A sample containing 500 ng of total RNA was used to synthesize cDNA. Primers were designed in our laboratory based on consideration of guanine-cytosine (GC) content, length, secondary structure, and melting temperature of the primers using the program Beacon Designer 2 (PREMIER Biosoft Intl., CA) (Table 2). Synthesized cDNA was used as a template in the PCR reaction to produce PCR products.
Table 2. Primer Sequences for Synthesizing Probes
Primer specificities and optimal PCR conditions were verified by single PCR products after agarose gel electrophoresis and by DNA sequencing of the PCR products. No primer-dimer formation occurred. High homologies between PCR-product and target sequence (NCBI GeneBank) were observed with 90 percent, 98 percent, and 100 percent sequence identities for beta actin (488 bp), CYP19a (496 bp), and CYP19b (513 bp), respectively.
Amplified PCR products were purified and denatured and then labeled with fluorescent dye (Alexa Fluor 568, ULYSIS Nucleic Acid Labeling Kits, Molecular Probes, OR). Labeled cDNA probes were hybridized with total RNA extracted from ovary at 95°C for 10 minutes and were verified by the gel electrophoresis without ethidium bromide staining (Figure 2).
Figure 2. Electrophoresis of cDNA Probes Hybridized With Total RNA. 1: Hybridization of beta actin cDNA probe (1.5 μg) with Total RNA (4 μg); 2: Hybridization of CYP19a Probe (1.5 μg) with total RNA (4 μg); 3: Hybridization of CYP19b probe (1.5 μg) with total RNA (4 μg); 4: CYP19b cDNA probe (0.7 μg); 5: beta actin cDNA probe (0.7 μg).
Synthesis of Riboprobes (RNA probe). Riboprobes were generated by in vitro transcription of a cloned DNA sequence by RNA polymerase. Amplified PCR products were inserted into a vector (pGEM-T Easy Vector System, Promega, WI) using standard methods. These ligated vectors were transformed into the Escherichia coli (JM 109 High Efficiency Competent Cells, Promega, WI) through the heat-shock method followed by immediate cooling down. The cloned plasmid was extracted from successfully transfected E. coli colonies. After purification of the plasmid, homology of the cloned amplicon to the target gene was confirmed by automatic DNA sequencing and subsequent BLAST2 analysis (NCBI, MD). Cloned plasmid DNA was digested using Sal I (Invitrogen, CA) for sense probe and Nco I (Invitrogen, CA) for antisense probe. Size of sense and antisense sequences was confirmation by gel electrophoresis. Linearized plasmid DNA was then used as a template for in vitro transcription reaction. T7 RNA polymerase (Invitrogen, CA) was used for synthesizing sense probe, and SP6 RNA polymerase (Roche Applied Science, IN) was used for synthesizing antisense probe in the mixture of transcription (10X digoxigenin [DIG]) labeling mix; transcription buffer, 0.1M DTT; RNAseOut). Synthesized riboprobes were purified using a spin column (CENTRI-SEP, Princeton Separation Inc., NJ) to remove unincorporated DIG labeling mix. Quality of the riboprobes was tested using morpholine propanesulfonic acid (MOPS)-formaldehyde gel electrophoresis (Figure 3).
Figure 3. Assessment of Quality of RNA Probes Using MOPS-Formaldehyde Gel Electrophoresis. AP = antisense probe; SP = sense probe.
To estimate DIG labeling efficiency and the yield of riboprobes, a spot test with a DIG-labeled control was conducted (Figure 4). Direct comparison of the intensities of sample and control allows the estimation of labeling yield.
Figure 4. Estimating the Yield of DIG-Labeled DNA. AP =antisense probe; SP = sense probe.
Specificity of the antisense probe versus sense probe was tested using a slot-blot test (Bio-Dot SF Microfiltration Apparatus; BioRad, CA). Total RNA was allowed to hybridize with CYP19 sense and antisense probe. Although relatively high background staining occurred, a clear dose-dependent increase of the intensity of hybridization with increasing dose of total RNA was observed (Figure 5). Also, The staining was greater in samples incubated with the antisense probe when compared to samples incubated with the sense probe. This indicates that there is specific hybridization of the antisense probe with the target sequence. Currently work is underway to optimize hybridization condition to reduce background staining.
Figure 5. Dot Blotting of Different Concentrations of Total RNA (Head) vs. DIG-Labeled Riboprobe
Synthesis of Biotin-Labeled Riboprobe. The development of a multiplex ISH technique that can be used to simultaneously detect two genes per fish section is currently underway. In this technique two differentially labeled probes, biotin and DIG, will be hybridized parallel on the same section. Thus, in addition to the DIG-labeled probes, a biotinylated CYP19b probe was synthesized. Synthesis steps were similar to those required in the synthesis of DIG-labeled riboprobe (Figure 6). Labeling was conducted as described in the directions of the manufacturer (Roche Applied Science, IN). Currently, ISH techniques are adapted for the simultaneous hybridization of medaka sections with riboprobes of CYP19a labeled with DIG and CYP19b labeled with biotin.
Figure 6. Assessment of Quality of Biotinylated CYP19b RNA Probes Using MOPS-Formaldehyde Gel Electrophoresis. AP = antisense probe; SP = sense probe.
cDNA probes for beta actin, CYP19a, and CYP19b were successfully synthesized and fluorescently labeled. The probes showed good accordance to target sequences and were electrophoretically shown to recognize RNA of the appropriate size. However, because of potential complications inherent in using cDNA probes, including the problem of denaturation of the probe during hybridization, effort was put into development of riboprobes. DIG-labeled CYP19b riboprobes showed good accordance to target sequences with high labeling efficiencies. However, hybridization conditions still need to be optimized to remove background staining. As an additional check of hybridization specificity, we are purifying mRNA from total RNA (Oligo DT columns, Amersham Biosciences, NJ) to better evaluate the background staining in the slot blots. In addition, Northern blotting currently is used to further verify the hybridization of the riboprobes with the target gene. Once conditions and procedures have been optimized and validated, the other riboprobes will be tested with both the dot blot test and northern blotting.
In situ Hybridization. The ISH procedures are almost completely optimized at this point. The methods have demonstrated specific staining for all probes tested thus far–CYP19a, CYP19b, and beta actin. All probes are -labeled. They are visualized using standard protocols. In brief, the probes are synthesized with nucleotides tagged with a DIG molecule. This molecule is detected with an anti-DIG antibody that is conjugated to the alkaline phosphatase enzyme. Alkaline phosphatase is detected by using a substrate system (NBT/BCIP) that leaves a purple/blue product after reaction (Figures 7-10).
The sense and antisense probes did exhibit the expected binding patterns in all cases: no or background staining of the sense probe and clear tissue, and cell-type specific signal of the antisense probe (Figures 7-10). Hybridization with the CYP19b probe resulted in staining in the brain only, with the greatest response in the hypothalamus (Figure 7). In contrast, CYP19a only stained certain cell types in gonads. These cell types were early oocytes and supporting cells in the ovary of female fish (Figure 8), and early male germ cells (spermatogonia) in the testis of juvenile male medaka (Figure 9). A clear positive staining signal throughout all tissue-types was observed for the beta actin probe (Figure 10). The intensity of staining, however, depended on the tissue type. Overall, specificity of staining of the different probes was in accordance with tissues that have been reported to express these genes.
We are currently in the final stages of fine-tuning the protocols to assure that the data generated are both precise and consistent.
Figure 7. Hybridization With CYP19b Riboprobe, Adult Female Medaka: Sense Probe (left) Shows No Staining; Antisense Probe (Right) Shows Specific Staining in the Hypothalamus of the Brain. No counterstain.
Figure 8. Hybridization With CYP19a Riboprobe, Juvenile Male Medaka: Sense Probe (left) Shows No Staining; Antisense Probe (Right) Shows Specific Staining (Purple) in the Testis. Counterstain: nuclear fast red (pink).
Figure 9. Hybridization With CYP19a Probe, Juvenile Female Medaka: Sense Probe (left) Shows No Staining; Antisense Probe (Right) Shows Specific Stain (Purple) in Early Stage Oocytes and Supporting Cells
Figure 10. Hybridization With Beta Actin Probe, Juvenile Female Medaka: Sense Probe (Left) Shows No Staining; Antisense Probe Shows Specific Stain (Purple) in Muscle Tissue
Analysis of Slide Image
After ISH has been performed on slides, digital images of whole fish are captured as well as magnified organs of interest using an Olympus digital camera and a compound microscope. All images are taken in manual mode with all variables (shutter speed, aperture, etc.) held constant for each picture. The pictures are saved as TIFF files to avoid the loss of resolution associated with JPG files. The pictures are then downloaded to ImageJ software (free software available from the National Institutes of Health) for analysis. We are currently working on a method to quantify the product of the alkaline phosphatase/NBT/BCIP reaction.
June 2006 Exposure of Juvenile Medaka to Fadrozole
An initial medaka exposure experiment was conducted in June of 2006 to verify the developed ISH methods for the CYP19a and b probes. In this experiment, fish were exposed to a known aromatase inhibitor, fadrozole, which has been previously shown to inhibit aromatase gene expression in teleost fish (Ankley, et al., 2005). Medaka were reared at our laboratory until 14 weeks of age, and then acclimated to exposure conditions for an additional 2 weeks until start of the experiment. Exposure concentrations were 0, 1, 10, and 100 μg/L fadrozole; each exposure concentration was run in two replicate tanks with 10 fish (5 male and 5 female) per tank, resulting in a total number of 80 fish that were used in the experiment. Fish were exposed under static renewal conditions for 7 days with a one-half volume water and chemical change daily. Water quality parameters (pH, hardness, dissolved oxygen, ammonia, nitrate, and temperature) were monitored daily; all stayed within acceptable ranges for the duration of the exposure. Water samples were also taken daily for fadrozole concentration analysis. At exposure termination, fish were euthanized in MS-222 and randomly assigned to be in one of two groups, ISH or RT-PCR. The ISH fish were processed to paraffin blocks and await analysis for gene expression by ISH. The RT-PCR fish had organs of interest (brain, liver, and gonad) removed. The organs were flash frozen in liquid nitrogen. We are in the process of extracting RNA and analyzing the expression of target genes in these samples by RT-PCR at present.
Whole fish ISH methods have been developed for an initial set of three genes, CYP19a, CYP19b, and beta actin. CYP19a and b were chosen based on their tissue specificity, their key role in the endocrine system, and responsiveness to endocrine disruptors. Beta actin was selected as the housekeeping gene (internal reference). Riboprobes for these genes, whole mount fish sectioning techniques, and ISH methods have been successfully developed and are currently optimized and validated. At this point, the probes in use are DIG-labeled and biotinylated because of their increased sensitivity when compared to fluorescent probes. Methods to quantify the product of the alkaline phosphatase/NBT/BCIP reaction using ImageJ software are underway. In addition to the initial set of genes, a second series of probes is currently being developed, including multiple key genes along the hypothalamus-pituitary-gonadal axis. These genes include ER, androgen receptor (AR), gonadotropin releasing hormone (GnRH), gonadotropins (GtH), and gonadotropin receptors (GtHR).
A first exposure experiment has been successfully conducted using fadrozole, a specific aromatase inhibitor, as the model compound. One-half of the fish from this experiment were embedded in paraffin using fixation protocols developed at our laboratory and are currently processed (sectioned and mounted) for subsequent ISH analyses. From the remaining 50 percent of these fish tissues (brain, gonads, liver) have been dissected. RNA has been extracted from these tissue samples and is currently subjected to quantitative RT-PCR analysis as a confirmation of the ISH experiments. Results for both ISH and RT-PCR analyses are expected to be available by the end of October 2006. In addition to this initial experiment, a second series of exposures is currently prepared. In these experiments male and female medaka will be exposed to four different model compounds (fadrozole, EE2, ZM 189,154, and trenbolone) for three different time periods (12, 48, and 144 hours) to identify changes in gene expression patterns along the HPG-axis as a function of dose and time. The chemicals were chosen based on the specific mechanisms by which they interact with the endocrine system.
We currently are finalizing the validation of the ISH methods for CYP19a, CYP19b, and beta actin. We will begin analyzing the samples from our June 2006 exposure gene expression by ISH within the next month. We are also in the process of developing a method for quantification of the ISH signal, using ImageJ software to convert digital images of the slides to a form that can be quantified and analyzed using conventional statistics. In addition, we are currently analyzing the RT-PCR subset of samples from our June 2006 exposure to gauge expression of the target genes in organs of interest along the HPG-axis. These analyses should be finished and results submitted for publication by January 2007. Also, we are currently in the process of writing detailed standard operating procedures (SOPs) for all methods developed. Separate SOPs are issued for developing and synthesizing probes, fixing and sectioning whole fish mounts for ISH, and ISH procedures.
Our team submitted three abstracts to present our findings at the 27th Annual Society of Environmental Toxicology and Chemistry meeting in Montreal, Canada, this November; all were accepted. We will be covering the results, both RT-PCR and ISH, from the June 2006 exposure as well as a new multiplexing method we are developing to visualize spatial expression of two genes in the same tissue section.
We are also planning a second chemical exposure for late November or early December involving a series of model compounds with different modes of action. Some of the chemicals selected for these experiments differ from those listed in the proposal. This was done because the compounds originally selected all interact with the endocrine system primarily through receptor-mediated mechanisms. Therefore, vinclozolin, a weak androgen receptor antagonist, will be replaced with fadrozole to capture effects at the level of estrogen synthesis, a mechanism deemed highly relevant in the current endocrine disruptor discussions. Thus, the chemicals to be tested are ethinylestradiol (ER agonist), ZM 189,154 (ER antagonist), trenbolone (AR agonist), and fadrozole (aromatase inhibitor). In addition to dose dependent alterations in gene expression patterns, we will also investigate the timing of effects by analyzing subsamples of fish after 12, 48, and 144 hours of exposure to identify critical time points at which certain genes are affected.
Also, efforts are currently underway to develop a second series of probes. Genes of interest are the ERs, ARs, GnRH, GtH, and GtHR. The reason for the selection of these genes is their key role in mediating gonad maturation and ovulation. Release of GnRH and subsequent secretion of GtHs from the hypothalamus and pituitary, respectively, are believed to be linked to local formation of estrogens and their binding to the ER in the brain of both male and female fish. The binding of GtH to its receptors in turn triggers the synthesis of sex steroids in the gonads that then exert their action by binding to their specific receptors (e.g., production of vitellogenin in response to binding of E2 to the ER in the liver) but also result in negative feedback to the brain. Thus, once this set of gene probes (including both CYP19 isoforms previously established) is established, we will be able to characterize and evaluate the above described key processes along the HPG axis, which is the major goal of this study.