Identification and Characterization of Androgen-Responsive Genes in Zebrafish Embryos.

Responsive genes for fish embryos have been identified so far for some endocrine pathways but not for androgens. Using transcriptome analysis and multiple concentration-response modeling, we identified putative androgen-responsive genes in zebrafish embryos exposed to 0.05-5000 nM 11-ketotestosterone for 24 h. Four selected genes with sigmoidal concentration-dependent expression profiles (EC50 = 6.5-30.0 nM) were characterized in detail. The expression of cyp2k22 and slco1f4 was demonstrated in the pronephros; lipca was detected in the liver, and sult2st3 was found in the olfactory organs and choroid plexus. Their expression domains, the function of human orthologs, and a pathway analysis suggested a role of these genes in the metabolism of hormones. Hence, it was hypothesized that they were induced to compensate for elevated hormone levels. The induction of sult2st3 and cyp2k22 by 11-ketotestosterone was repressed by co-exposure to the androgen receptor antagonist nilutamide supporting a potential androgen receptor mediated regulation. Sensitivity (expressed as EC50 values) of sult2st3 and cyp2k22 gene expression induction after exposure to other steroidal hormones (11-ketotestosterone ∼ testosterone > progesterone > cortisol > ethinylestradiol) correlated with their known binding affinities to zebrafish androgen receptor. Hence, these genes might represent potential markers for screening of androgenic compounds in the zebrafish embryo.

[1]  J. Bogerd,et al.  Functional characterization and expression analysis of the androgen receptor in zebrafish (Danio rerio) testis. , 2008, Reproduction.

[2]  Xiaowei Zhang,et al.  Zebrafish embryos/larvae for rapid determination of effects on hypothalamic-pituitary-thyroid (HPT) and hypothalamic-pituitary-interrenal (HPI) axis: mRNA expression. , 2013, Chemosphere.

[3]  I. Katsiadaki,et al.  Development of a stickleback kidney cell culture assay for the screening of androgenic and anti-androgenic endocrine disrupters. , 2006, Aquatic toxicology.

[4]  C. Haux,et al.  An 11-ketotestosterone induced kidney-secreted protein: the nest building glue from male three-spined stickleback, Gasterosteus aculeatus , 2004, Fish Physiology and Biochemistry.

[5]  M. Pfaffl,et al.  A new mathematical model for relative quantification in real-time RT-PCR. , 2001, Nucleic acids research.

[6]  R. Altenburger,et al.  The internal concentration of organic substances in fish embryos—A toxicokinetic approach , 2013, Environmental toxicology and chemistry.

[7]  M. S. Hossain,et al.  Zebrafish Androgen Receptor: Isolation, Molecular, and Biochemical Characterization1 , 2008, Biology of reproduction.

[8]  É. Fetter,et al.  Comparative analysis of goitrogenic effects of phenylthiourea and methimazole in zebrafish embryos. , 2015, Reproductive toxicology.

[9]  Chau‐Ching Liu,et al.  Sulphonation of dehydroepiandrosterone and neurosteroids: molecular cloning, expression, and functional characterization of a novel zebrafish SULT2 cytosolic sulphotransferase. , 2003, The Biochemical journal.

[10]  D. Posada,et al.  Model selection and model averaging in phylogenetics: advantages of akaike information criterion and bayesian approaches over likelihood ratio tests. , 2004, Systematic biology.

[11]  Bart De Moor,et al.  BioMart and Bioconductor: a powerful link between biological databases and microarray data analysis , 2005, Bioinform..

[12]  L Earl Gray,et al.  Competitive binding comparison of endocrine‐disrupting compounds to recombinant androgen receptor from fathead minnow, rainbow trout, and human , 2007, Environmental toxicology and chemistry.

[13]  S. Scholz.  Zebrafish embryos as an alternative model for screening of drug-induced organ toxicity , 2013, Archives of Toxicology.

[14]  Aldert H Piersma,et al.  Relative embryotoxicity of two classes of chemicals in a modified zebrafish embryotoxicity test and comparison with their in vivo potencies. , 2011, Toxicology in vitro : an international journal published in association with BIBRA.

[15]  W. J. Kent,et al.  BLAT--the BLAST-like alignment tool. , 2002, Genome research.

[16]  David R. Anderson,et al.  AIC model selection and multimodel inference in behavioral ecology: some background, observations, and comparisons , 2011, Behavioral Ecology and Sociobiology.

[17]  O. Kah,et al.  Early regulation of brain aromatase (cyp19a1b) by estrogen receptors during zebrafish development , 2009, Developmental dynamics : an official publication of the American Association of Anatomists.

[18]  A I Saeed,et al.  TM4: a free, open-source system for microarray data management and analysis. , 2003, BioTechniques.

[19]  M. Halpern,et al.  Androgen receptor gene expression in the developing and adult zebrafish brain , 2008, Developmental dynamics : an official publication of the American Association of Anatomists.

[20]  M. Hammerschmidt,et al.  Zebrafish in endocrine systems: recent advances and implications for human disease. , 2011, Annual review of physiology.

[21]  S. Reibe,et al.  Analysis of origin and growth of the thyroid gland in zebrafish , 2006, Developmental dynamics : an official publication of the American Association of Anatomists.

[22]  Taisen Iguchi,et al.  Rapid fluorescent detection of (anti)androgens with spiggin-gfp medaka. , 2014, Environmental science & technology.

[23]  J. Takeo,et al.  Rainbow trout androgen receptor-alpha fails to distinguish between any of the natural androgens tested in transactivation assay, not just 11-ketotestosterone and testosterone. , 2000, General and comparative endocrinology.

[24]  Jens C. Streibig,et al.  Bioassay analysis using R , 2005 .

[25]  J S Denny,et al.  Alternatives to in vivo tests to detect endocrine disrupting chemicals (EDCs) in fish and amphibians – screening for estrogen, androgen and thyroid hormone disruption , 2013, Critical reviews in toxicology.

[26]  M. Popović,et al.  Organic anion transporting polypeptides (OATP) in zebrafish (Danio rerio): Phylogenetic analysis and tissue distribution. , 2010, Comparative biochemistry and physiology. Part A, Molecular & integrative physiology.

[27]  R. Fischer,et al.  Transcriptome alterations in zebrafish embryos after exposure to environmental estrogens and anti-androgens can reveal endocrine disruption. , 2013, Reproductive toxicology.

[28]  Christian Laggner,et al.  Rapid behavior—based identification of neuroactive small molecules in the zebrafish , 2009, Nature chemical biology.

[29]  Zhan Yin,et al.  Use of TSHβ:EGFP transgenic zebrafish as a rapid in vivo model for assessing thyroid-disrupting chemicals. , 2012, Toxicology and applied pharmacology.

[30]  B. Thisse,et al.  High-resolution in situ hybridization to whole-mount zebrafish embryos , 2007, Nature Protocols.

[31]  O. Kah,et al.  Expression and estrogen‐dependent regulation of the zebrafish brain aromatase gene , 2005, The Journal of comparative neurology.

[32]  Menghang Xia,et al.  Mechanism-based testing strategy using in vitro approaches for identification of thyroid hormone disrupting chemicals. , 2013, Toxicology in vitro : an international journal published in association with BIBRA.

[33]  Yuh-Shyong Yang,et al.  Identification of novel hydroxysteroid-sulfating cytosolic SULTs, SULT2 ST2 and SULT2 ST3, from zebrafish: cloning, expression, characterization, and developmental expression. , 2006, Archives of biochemistry and biophysics.

[34]  Demetrio Raldúa,et al.  Simple, rapid zebrafish larva bioassay for assessing the potential of chemical pollutants and drugs to disrupt thyroid gland function. , 2009, Environmental science & technology.

[35]  Rolf Altenburger,et al.  High‐throughput concentration–response analysis for omics datasets , 2015, Environmental toxicology and chemistry.

[36]  Olivier Kah,et al.  Screening Estrogenic Activities of Chemicals or Mixtures In Vivo Using Transgenic (cyp19a1b-GFP) Zebrafish Embryos , 2012, PloS one.

[37]  D. Versteeg,et al.  Hepatic gene expression profiling using GeneChips in zebrafish exposed to 17alpha-methyldihydrotestosterone. , 2008, Aquatic toxicology.

[38]  C. Martyniuk,et al.  Transcriptomics profiling and steroid production in mummichog (Fundulus heteroclitus) testes after treatment with 5α-dihydrotestosterone. , 2014, General and comparative endocrinology.

[39]  G. Ankley,et al.  Gene expression profiling of the androgen receptor antagonists flutamide and vinclozolin in zebrafish (Danio rerio) gonads. , 2011, Aquatic toxicology.

[40]  J. Yang,et al.  Generation of a fluorescent transgenic zebrafish for detection of environmental estrogens. , 2010, Aquatic toxicology.

[41]  T. Cajthaml,et al.  New in vitro reporter gene bioassays for screening of hormonal active compounds in the environment , 2010, Applied Microbiology and Biotechnology.

[42]  E. Birney,et al.  Mapping identifiers for the integration of genomic datasets with the R/Bioconductor package biomaRt , 2009, Nature Protocols.

[43]  J. Dalton,et al.  Pharmacokinetics and Pharmacodynamics of Nonsteroidal Androgen Receptor Ligands , 2006, Pharmaceutical Research.

[44]  M. H. Angelis,et al.  Zebrafish and steroids: What do we know and what do we need to know? , 2013, The Journal of Steroid Biochemistry and Molecular Biology.

[45]  Stefan Scholz,et al.  Predicting adult fish acute lethality with the zebrafish embryo: relevance of test duration, endpoints, compound properties, and exposure concentration analysis. , 2012, Environmental science & technology.

[46]  E. Nikkilä,et al.  Regulation of hepatic lipase and serum lipoproteins by sex steroids. , 1987, American heart journal.

[47]  J. Hackermüller,et al.  CEM-designer: design of custom expression microarrays in the post-ENCODE Era. , 2014, Journal of biotechnology.

[48]  G. Ying,et al.  Trace analysis of 28 steroids in surface water, wastewater and sludge samples by rapid resolution liquid chromatography-electrospray ionization tandem mass spectrometry. , 2011, Journal of chromatography. A.

[49]  D. Balciunas,et al.  Development and Notch Signaling Requirements of the Zebrafish Choroid Plexus , 2008, PloS one.

[50]  J. Muncke,et al.  Vitellogenin 1 mRNA as an early molecular biomarker for endocrine disruption in developing zebrafish (Danio rerio) , 2006, Environmental toxicology and chemistry.

[51]  Jeffrey M Peters,et al.  Xenobiotic metabolism, disposition, and regulation by receptors: from biochemical phenomenon to predictors of major toxicities. , 2011, Toxicological sciences : an official journal of the Society of Toxicology.