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Low-dose exposure to 17alpha-ethinylestradiol rapidly regulates expression networks of gonad differentiation in the adult fathead minnow testes

ABSTRACT: Environmental estrogens are ubiquitous in aquatic systems worldwide. In wild fish inhabiting wastewater effluent with estrogenic components, the presence of both oocytes and spermatogenic tissue in the testes has been documented. However, the molecular networks underlying the manifestation of the intersex condition are not completely characterized. In this study, we used an environmentally relevant concentration of 17alpha-ethinylestradiol (EE2), the active component of the birth control pill, to determine the response of the fathead minnow testis transcriptome after 96 hrs. The goal was to capture early molecular initiating events that may lead to the intersex condition following exposure to an estrogen that is present in wastewater effluent. There were no effects of EE2 on morphometric endpoints such as gonadosomatic index nor proportion of gametes within the testes. However, in vitro production of 11-ketotestosterone and testosterone in the testis was decreased relative to controls following EE2 exposure. Gene expression profiling revealed that there were 10 transcripts that were differentially expressed in the testis using a 8x60K fathead minnow microarray, the most dramatic change being that of coagulation factor XIII A chain (20-fold increase). Transcripts that included guanine nucleotide binding protein (Beta Polypeptide 2), peroxisome proliferator-activated receptor delta, and WNK lysine deficient protein kinase 1a, were down-regulated by EE2. Subnetwork enrichment analysis revealed that EE2 suppressed transcriptional networks associated with reproduction such as steroid metabolism, hormone biosynthesis, and sperm mobility. Most interesting was that gene networks associated with doublesex and mab-3 related transcription factor 1 (dmrt1) were suppressed in the adult testis, despite the fact that dnmt1 itself was not differentially expressed from controls. Moreover cell processes involving forkhead box L2 (foxL2) such as ovarian follicle development and granulosa cell development were increased in expression in the testis. Cell processes that included glucose homeostasis, response to heavy metal, amino acid catabolism, and the cyclooxygenase pathway were decreased with EE2 while lymphocyte chemotaxis, intermediate filament polymerization, glucocorticoid metabolism, carbohydrate utilization, and anterior/posterior axis specification were increased, shedding light on the transcriptional responses that may be perturbed prior to gonadal remodelling following exposure to estrogenic contaminants. These data demonstrate that low concentrations of EE2 rapidly suppresses male hormone secretion, suppresses gene networks related to male sex differentiation, and induces transcriptional networks related to female sexual differentiation in the adult testis. These responses are hypothesized to be the molecular initiating events that occur prior to the development of the intersex phenotype after prolonged exposures to estrogens.

ORGANISM(S): Pimephales promelas

PROVIDER: GSE81290 | GEO | 2016/05/11

SECONDARY ACCESSION(S): PRJNA321209

REPOSITORIES: GEO

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Project description:Three surface waters in Gainesville, Florida were used in a 48 hour whole effluents exposure to assess gene expression profiles of male fathead minnow liver. Microarray analysis was used to determine changes in gene expression of exposed fish to waters from a site downstream of a wastewater treatment plant (streamwater), a wastewater treatment plant (wastewater), and a lake (stormwater). Differences in gene expression between fish exposed to collected waters and controls were observed. Number of altered genes and biological processes were 1028 and 18 for stormwater; 787 and 19 for streamwater; and: 575 and 12 for wastewater. In general, the effects observed in all exposed fish were related with fatty acid metabolism, DNA repair, oxidation-reduction process, cell wall catabolic process and apoptosis. All exposed fish showed altered expression of genes related with DNA damage repair. In particular fish exposed to stormwater and streamwater showed downregulation of several key intermediates transcripts of cholesterol. The presence and environmental persistence of perfluorinated chemicals (PFCs) in these waters, the resemblance in known effects on transcripts with those found in this study, suggest that the set of genes differentially regulated in fathead minnows after 48 hours of exposure may be attributed to exposure to PFCs. Three surface water sites were chosen for effluent collection in Gainesville, Florida: A lake (stormwater), surface water downstream of a wastewater treatment plant (streamwater), and a wastewater treatment plant effluent used for landscaping irrigation (wastewater). Water from each site was collected two days prior to the fish exposure experiment using Chemfluor ® tubing and a 120 liters steel barrels coated with polyester resin (gel coat) to avoid cross-contamination. Three barrels for each effluent were collected during day 1. Water from the barrel was transported to the laboratory and pumped into four fiberglass cylinders in the aquatic toxicology facility. Water from each cylinder was then pumped into four replicate aquariums per treatment and kept for 1 day without fish (pre-treatment). On day 2, four male fathead minnows from a common tank were transferred to each replicate aquarium and kept for 48 hours, with one 75% water change after first 24 hours. The exposure system consisted of 40 L glass aquaria. Each exposure was conducted in quadruplicate and each aquarium contained the four male fish in 25 L of treatment water . The water used in the control treatment was carbon filtered, dechlorinated tap water. The positions of the treatment tanks were randomized and test initiation times were staggered to ensure an exposure/sampling interval of 48 h. The fish were not fed during the experiment. The temperature range of the water was 24-26 °C with a photoperiod of 16 h light: 8 h dark. Liver was isolate from 4 males indviduals for each treatment except for control group (3 individuals).

Project description:Rivers containing effluents from water treatment plants are complex soups of compounds, ranging from pharmaceuticals to natural hormones. Male fathead minnows (Pimephales promelas) were exposed for 3 weeks to effluent waters from the Metropolitan Wastewater Treatment Plant in St. Paul, MN. Fish were tested for their competitive nest holding behavior. Changes in vitellogenin were measured and these were correlated to changes in gene expression using a 22,000 gene microarray developed specifically for fathead minnows. Significant changes in gene expression were observed in both liver and gonad, which correlate to phenotypic changes of vitellogenin induction and reduced competitive behavior. We also compared by real-time PCR the expression changes in key genes related to steroid biosynthesis and metabolism in fish exposed to the effluent as well as in fish exposed to a model estrogen and a model androgen. While the gene expression signature from effluent-exposed fish shared some elements with estrogen and androgen signatures, overall it was different, underscoring the complexity of compounds present in sewage and their different modes of action. Fathead minnow 22,000 gene arrays were designed by EcoArray (Alachua, FL) and were purchased from Agilent. Array hybridizations were performed using a reference design, where each sample was compared to a reference sample. The reference consisted of equal amounts of RNA from control female and male tissues (liver, brain and gonad) and was prepared as a standard for several experiments. Four replicates consisting of four different individuals were analyzed for each of the tissues, liver and gonad for both the control and the effluent-exposed fish. cDNA synthesis, cRNA labeling, amplification and hybridization were performed following the manufacturerM-bM-^@M-^Ys kits and protocols (Agilent Low RNA Input Fluorescent Linear Amplification Kit and Agilent 60-mer oligo microarray processing protocol; Agilent, Palo Alto, CA). Ovarian and liver samples from the fish were labeled with Cy5 while the reference sample was labeled with Cy3.

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Low-dose exposure to 17alpha-ethinylestradiol rapidly regulates expression networks of gonad differentiation in the adult fathead minnow testes

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