Regulation of cholesterogenic gene expression in Japanese medaka (Oryzias latipes) exposed to 17β-trenbolone
ABSTRACT: The anabolic androgen 17β-trenbolone (TB) can cause masculinization and reduce fecundity of fish. However, the underlying mechanisms of various biological pathways including metabolism, biosynthesis etc. are largely unknown. Here, we evaluated the effects of TB using the medaka DNA microarray representing 36,398 genes. Larval medaka, Oryzias latipes, (within 24 hrs posthatch) were exposed to TB at various concentrations (2, 6, 20, 60, 100, 200 ng/L) for up to 7 days. Dose-response relationships in gene expression levels of the categorized genes were analyzed using the cumulative chisquared method. Microarray analyses of the TB-exposed larvae showed that 117 and 32 genes were determined as up and down-regulated genes, respectively. The most significant GO term for biological process identified within this gene list was lipid metabolic process, which contained 26 genes in up-regulated genes. In this category, “cholesterol biosynthetic process” was highlighted as an important subcategory with 15 genes, including hydroxymethylglutaryl-CoA synthase cytoplasmic, squalene monooxygenase, lanosterol synthase etc. RT-PCR measurements in these genes were consistent with the microarray results in the direction and magnitude of these changes in gene expression. On the other hands, in the category of “sexual differentiation and development”, genes related to hypothalamic-pituitary-gonadal (HPG) axis were not affected by TB treatment except for one gene encoded to cytochrome P450 19A1. Genes related to oogenesis, such as choriogenins and vitellogenins were weakly down regulated at 2-200 ng/L of TB. Our findings demonstrate that genes encoding cholesterol synthesis pathway via the mevalonate pathway were controlled by TB in larval medaka. TB concentrations used were 0 (control), 2, 6, 20, 60, 100 and 200 ng/L for 7 days of exposure. Each TB treatment had 90 larval medaka for each chamber. At day 7 of the exposures, triplicate samples (30 larvae/sample) from each chamber respectively were collected, flash-frozen in liquid nitrogen, and stored in liquid nitrogen until RNA extraction.
Project description:We assess gene expression patterns upon 17beta-estradiol (E2) exposure to Japanese medaka (Oryzias latipes) in order to appere the E2 effects using DNA microarray analysis. Larval medaka were exposed to 0, 3, 30 and 100 ng/L of E2 and concentration-dependent changes in gene expressions were examined using Agilent medaka DNA microarrays. At 7 day, fish were sacrificed and mRNA was extracted for gene expression analysis. In an effort to link gene expression changes to effects on higher levels of biological organization, sex characteristics, gonadal histology, GSI, and egg production and fertility were examined. In microarray experiments, the correlation factors between the controls were from 0.91 to 1.00 among control samples. We observed highly induced O. latipes Gene Indices (OLGI) related to egg-yolk protein such as vitellogenin and L-SF precursor etc., which were significantly affected in a concentration-dependent manner by E2 exposure. To clarify the function of expressed genes by E2 treatments, we selected statistically expressed genes from the microarray experiments. We found 190 genes and 72 genes which were statistically expressed in E2 treatment as induced and repressed genes, respectively. In the induced gene list, there were characteristic induced-genes in the categories of lipid metabolism, stress (oxidative stress, DNA and protein damage), and apoptosis with MAPK pathway. On the other hands, there were characteristic repressed genes in the categories of heat shock protein. Our result may suggest that gene expressions in yolk medaka is able to be used for detection of E2 effect by DNA microarray analysis. Larval medaka were exposed to 0, 3, 30 and 100 ng/L of E2 and concentration and time-dependent changes in gene expressions were examined using Agilent medaka DNA microarrays. At 7 day, three independent samples (one sample contained thirty whole medakas) were sacrificed and mRNA was extracted for gene expression analysis.
Project description:Using medaka fish embryo model, toxic effects of silver nanocolloids (SNC, 3.8±1.0nm diameter) on developmental morphology and gene expression profile were investigated. SNC caused morphological changes in embryos including cardiovascular malformations, ischemia, underdeveloped central nervous system and eyes, and kyphosis at exposures of 0.5mg/L and 1.0mg/L.. To determine in vivo distribution of SNC, medaka embryos were exposed to 0.5mg/L for 6 days and subjected to ICP-OES analyses. Silver was detected in medaka embryos and chorion at levels of 16.6±9.3pg and 720±29pg, respectively. TEM analyses showed SNC adhered to the chorion surface and inside the chorion. On investigation of oxidative mechanism, NAC (0.05mM) rescued all embryos by 96-hr post treatment, while 0.5mM GSH did not. NAC blocked lipid peroxidation. Furthermore, medaka oligo DNA microarray and qRT-PCR were used for gene expression profiling in embryos exposed for 48-hr to 0.05mg/L SNC. Six genes relative to embryogenesis and morphogenesis such as ctsL, Tpm1, RBP, mt, atp2a1 and hox6b6 turned out to be affected and their involvement to the above malformations was implied. In conclusion, SNC causes gross malformations in the cardiovascular and central nervous systems in developing medaka embryos through potentially SNC-affected differential expression of a series of genes related to oxidative stress, embryonic cellular proliferation, and morphological development. Three of the stage-21 medaka embryos per sample were exposed in triplicate to 0mg/L (control) and 0.05mg/L SNC for 48 hours. Therefore, for each exposure condition nine of the stage-21 embryos were prepared and used in the SNC exposure test.
Project description:Mycobacteria of the Mycobacterium tuberculosis complex (MTBC) greatly impact on human and animal health worldwide. Mycobacterial life cycle is complex and the mechanisms resulting in pathogen infection and survival in host cells are not fully understood. Eurasian wild boar (Sus scrofa) are natural reservoir hosts for MTBC and a model for mycobacterial infection and tuberculosis (TB). In the wild boar TB model, mycobacterial infection affects the expression of innate and adaptive immune response genes in mandibular lymph nodes and oropharyngeal tonsils and biomarkers have been proposed as correlates with resistance to natural infection. However, the mechanisms used by mycobacteria to manipulate host immune response are not fully characterized. Our hypothesis is that the immune system proteins under-represented in infected animals when compared to uninfected controls are used by mycobacteria to guarantee pathogen infection and transmission. To address this hypothesis, a comparative proteomics approach was used to compare host response between uninfected (TB-) and M. bovis-infected young (TB+) and adult animals with different infection status [TB lesions localized in the head (TB+) or affecting multiple organs (TB++)]. The results identified host immune system proteins that play an important role in host response to mycobacteria. Calcium binding protein A9, Heme peroxidase, Lactotransferrin, Cathelicidin and Peptidoglycan-recognition protein were under-represented in TB+ animals when compared to uninfected TB- controls but protein levels increased as infection progressed in TB++ animals when compared to TB- and/or TB+ adult wild boar. MHCI was the only protein over-represented in TB+ adult wild boar when compared to uninfected TB- controls. The results reported here suggested that M. bovis manipulates host immune response by reducing the production of immune system proteins. However, as infection progresses, wild boar immune response recover to limit pathogen multiplication and promote survival that also facilitates pathogen transmission.
Project description:This study assessed the implications of a 14 day sub-chronic exposure of ethinylestradiol (EE2; 1.0 or 10.0 µg/L EE2) on male medaka fertility, testicular histology and testicular gene expression. The findings demonstrate that a 14 day exposure to EE2 induced impaired male reproductive capacity and time- and dose-dependent alterations in testicular morphology and gene expression. The average fertilization rate/day following the exposure for control, 1.0 and 10.0 µg/L EE2 was 91.3% (±4.4), 62.8% (±8.3) and 28.8% (±5.8), respectively. The testicular morphologic alterations observed include increased germ cell apoptosis, decreased germinal epithelium and thickening of the interstitium. The morphologic changes observed were highly associated with gene expression changes observed using a medaka-specific microarray. A pathway analysis of the differentially expressed genes emphasized genes and pathways associated with apoptosis, cell cycle and proliferation, collagen production/extracellular matrix organization, hormone signaling, male reproduction and protein ubiquitination among others. Six month old male medaka were exposed to ethinyl estradiol (EE2) for a 14 day time period. Treatment exposures were completed in triplicate including DMSO (vehicle control), 1.0 µg/L EE2, and 10.0 µg/L EE2. Fish were sampled for gene expression on days 1, 7 and 14 of exposure. Five male fish were placed in 2-liter beaker replicates for each treatment and sampling time point.
Project description:For further identify the differentiation between latent and clinical tuberculosis (TB), we employed whole genome microarray expression profiling to study genes with significant expression change in peripheral CD4+T cells between healthy control, latent tuberculosis (LTB) and clinical tuberculosis (TB). Our experiment included 4 groups: healthy donor (HD), latent TB1 (LTB1) with low IFN-gamma release level, latent TB2 (LTB2) with high IFN-gamma release level, and tuberculosis (TB) with high IFN-gamma release level. Human peripheral blood mononuclear cells were collected, from which CD4+T cells were isolated. Total RNA of each individuals of each group was extracted from peripheral CD4+T cells. One μg of RNA mixture, pooled equivalently by each individual total RNA of each group, was administrated microarray test. Compared with HD, through analyzing enriched-Gene Ontology (GO) terms and KEGG pathways of each group, we found peripheral CD4+T cells might had different ability for mycobacterium tuberculosis infection in LTB1, LTB2 and TB. Finally we detected that TNFSF13/APRIL and TNFSF13B/BAFF was significant up-regulation in both CD4+T cells and serum of TB by real time PCR and ELISA, respectively. Peripheral CD4+ T cells were purified by positive selection using magnetic beads (BD IMagTM anti-human CD4 Particles-DM, BD Biosciences). Total RNA was extracted from CD4+ T cells (CD4-RNA) of LTB1, LTB2 and TB patients and healthy controls by RNeasy Mini Kit (QIAGEN).Then, RNA quality was checked and mixed with an equal quantity of each individual. The number of included-individuals in each group was 11 for HD, 11 for LTB1, 12 for LTB2 and 11 for TB.
Project description:Infection with Mycobacterium tuberculosis (M. tb) is initiated when an aerosol droplet carrying a few bacilli is inhaled into an alveolus. Alveolar epithelial cells (AEC) (type II and type I) are among the first cells encountered by the infecting bacteria and greatly outnumber macrophages in the alveolus. M. tb replicates dramatically (>20,000 fold) in a “non-migrating” compartment in the lung prior to the development of the cell-mediated immune response in aerosol-infected mice (Wolf AJ, 2008). M. tb DNA has been found in type II AEC in autopsied lung tissue of latently infected individuals (Hernandez-Pando R et. al, 2000; Barrios-Payan J et. al 2012), and M. tb-infected AEC in broncho-alveolar lavage (BAL) and in sputum samples from TB patients indicates the infection of these cells in active as well as latent human TB (Eum SY et. al, 2010). M. tb has been shown to infect and replicate in the human type II AEC line, A549, and passaging of M. tb through A549 increases M. tb invasiveness (Bermudez LE et.al, 2002). In this work, we have used DNA microarray analysis to investigate the transcriptome of M. tb replicating in type II AEC (A549) compared to M. tb grown logarithmically in Middlebrook 7H9 broth media in order to identify M. tb adaptations to this intracellular environment as well as M. tb mechanisms/factors contributing to M. tb replication and increased invasiveness in primary infection. The global gene expression of M. tb H37Rv replicating in A549 cells at 72 hr was compared to that of M. tb grown to log phase in Middlebrook 7H9 media.
Project description:We have improved the DamID method to make it applicable for vertebrate life specimens. We use iDamID-seq to profile the binding profile of the transcription factor Rx2 in medaka embryos at stage 22 of development. We compare two replicates of the fusion Dam-Rx2 against two replicates of the fusion Dam-GFP as control. Medaka embryos at 1-cell stage were injected with mRNA transcribed in vitro from one of the two Dam fusions. The embryos were allowed to grow in normal conditions up to stage 22. The genomic DNA was isolated and treated to extract only the DpnI fragments that had adenine methylation. These fragments were subjected to deep sequencing.
Project description:Mycobacterium tuberculosis (M. tb), the cause of tuberculosis (TB), utilizes the blood circulation to spread systemically and establish infection, and the risk of developing active TB (pulmonary and extrapulmonary) is significantly increased in individuals infected with human immunodeficiency virus (HIV). In this work, we have used DNA microarray analysis to investigate the transcriptome of M. tb replicating in human whole blood from both HIV-negative and HIV-positive donors compared to M. tb grown in Middlebrook 7H9 broth media in order to identify M. tb adaptations to this host environment as well as M. tb mechanisms/factors contributing to increased active and disseminated TB during M. tb/HIV co-infection. We compared the global gene expression of M. tb H37Rv replicating in whole blood from 6 HIV- and 6 HIV+ individulas at 96 hr to M. tb grown to log phase in Middlebrook 7H9 media.
Project description:Recently, omics techniques have been widely applied to the discovery of potential bio-markers and explore triggering mechanism. To get a more comprehensive diagnosis of HBCD impacts on marine medaka (Oryzias melastigma), the larvae (within 24 hours post-hatch) were exposed to gradient doses of HBCD. After exposure for 7 days, the profiles of genes expression were examined using a custom-commercial 26, 430-oligonucleotide arrays (4×44K) of Japanese medaka which is shared much genomic information with marine medaka.At the end of the treatment period, 30 larvae/sample were pooled for RNA extraction and labeled by One-Color. A total of twelve independent arrays: three control (DMSO), three low-concentration HBCD (0.2 nM) exposures, three medium-concentration HBCD (2 nM) exposures, and three high-concentration HBCD (20 nM) exposures. The larvae of marine medaka (within 24 hours post-hatch) were exposed to to 0 (control), 0.2nM, 2nM and 20nM of HBCD (dimethyl sulfoxide with a final concentration of 1:30000 v/v water) for 7 days. Each HBCD treatment had three replicates with 100 larvae for each Petri dish. At the end of the treatment period, 30 larvae/sample were pooled for RNA extraction. A total of twelve independent arrays: three control (DMSO), three low-concentration HBCD (0.2 nM) exposures, three medium-concentration HBCD (2 nM) exposures, and three high-concentration HBCD (20 nM) exposures.
Project description:Mammalian ATF6α/β are membrane-bound transcription factors which are activated by endoplasmic reticulum (ER) stress-induced proteolysis to upregulate various ER quality control proteins to maintain the homeostasis of the ER. ATF6α- and ATF6β-single knockout mice develop normally but ATF6α/β-double knockout causes embryonic lethality, the reason for which remains unknown. Here, we showed that medaka fish exhibits the same phenotype regarding the effects of deleting ATF6α, ATF6β, and both. Analyses revealed that ER stress occurred physiologically during early embryonic development, particularly in the brain, otic vesicle and notochord. The absence of transcriptional induction of ER chaperones in ATF6α/β-double knockout blocked notochord development, which was partially rescued by microinjection-mediated overexpression of the major ER chaperone BiP. Thus, ATF6α/β-mediated adjustment of chaperones to the increased demands in the ER is essential for development of the notochord, which synthesizes and secretes large amounts of extracellular matrix proteins to serve as the body axis. Gene expression profile at stage 24 of DKO fish (ATF6a -/-b-/-) and control fish (ATF6a+/- b+/-) was determined. Two independent experiments were performed.