Anchoring ethinylestradiol induced gene expression changes with testicular morphology and reproductive function in the medaka
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ABSTRACT: This study assessed the implications of a 14 day sub-chronic exposure of ethinylestradiol (EE2; 1.0 or 10.0 M-BM-5g/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 M-BM-5g/L EE2 was 91.3% (M-BM-14.4), 62.8% (M-BM-18.3) and 28.8% (M-BM-15.8), 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 M-BM-5g/L EE2, and 10.0 M-BM-5g/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: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.
Project description:17alpha-ethinylestradiol (EE2) is one of the most potent estrogens that have the ability to interfere with the endocrine system of fish. The objective was to investigate the effects and mechanisms of action caused by 60 days of dietary exposure to 0.2 mg EE2/kg and 0.07 mg EE2/kg feed in female largemouth bass (LMB) during the reproductive season. Microarrays and pathway analyses were performed on hepatic tissues to identify genes and biological processes altered in female LMB by EE2 exposure. The hypothesis was that the two concentrations of EE2 would produce dose-response changes in sensitive genes. Body and ovary weights were measured and blood was collected for measurement of plasma steroid hormones (17beta-estradiol (E2), testosterone (T)) and vitellogenin (VTG) using ELISA. The 0.2 mg EE2/kg feed exposure reduced the gonadosomatic index (GSI) by 75%, and plasma levels of E2 and T were reduced by over 90%. Plasma VTG was increased by approximately 100% (from 4 to 8mg/ml) by the 0.2 mg/kg treatment. T levels, from the 0.07 mg EE2/kg feed, reduced GSI by approximately 30% and circulating E2 and T by ~80% but did not affect VTG concentrations. We found 1,594 and 1,165 genes were significantly affected (p<0.05) by the 0.07 mg EE2/kg feed and 0.2 mg EE2/kg feed, respectively. Gene ontology (GO) analysis revealed that there were different biological processes regulated by the two concentrations of EE2. Pathway analysis showed that the 0.07 mg EE2/kg feed exposure caused differential regulation of genes associated with fatty acid biosynthesis and glycolysis, indicating some metabolic effects. In contrast, the 0.2 mg EE2/kg feed exposure altered transcription of genes involved in immune response and apoptosis, suggesting a toxic response at this concentration. These results suggest that the two concentrations demonstrated distinct physiological responses, with the higher concentration inducing complete endocrine disruption in LMB. These findings demonstrate the usefulness of microarrays to identify possible biomarkers and modes of toxic action to dietary exposure in LMB. Two concentrations of EE2 would produce dose-response changes in sensitive genes. Female LMB were fed 5 days per week for 60 days with floating pellets that contained 0.07 or 0.2 mg/kg of EE2.
Project description:The objective of this study was to investigate pathway signatures altered in the livers of female largemouth bass (LMB) and their potential links with biological responses by dietary exposure to 0.2 mg EE2/Kg (1% of their body weight) over two months using a transcriptomics approach. A high concentration of dietary 17alpha-ethinylestradiol (EE2) can activate key signaling pathways in response to oxidative damage may occur regardless of tumorigenesis and cancer. Female LMB received about 1.2g EE2/day/fish (from EE2-laced feed containing 0.2 mg EE2/Kg) for 60 days.
Project description:17-ethinylestradiol (EE2) is a synthetic estrogen commonly used as an active substance in oral contraceptives. It is frequently found in waste water effluent and raise concern due to its persistent nature. EE2 binds to estrogen receptors with similar affinity to oestradiol and acts as one of the most potent hormone mimics found in the environment. Estrogen is involved in many aspects of the development of the neuroendocrine system influencing both brain structure and behavior. We and others have reported a significant effect on non-reproductive behaviors in adult fish and in recent studies we found that developmental exposure to EE2 resulted in an anxiogenic phenotype as adults even after a long remediation period. In this study we aim to study possible mechanisms behind the behavior alterations of zebrafish developmentally exposed to EE2 by sequencing the whole brain transcriptome. Zebrafish embryos were exposed to 0, 2.14 and 7.34 ng/L EE2 from 1 day to 80 days post fertilization. After the exposure period a remediation period of 120 days followed before the fish were sampled. 3 male brains from the control group (0 ng/L) and the 2.14 ng/L group were sampled and 3 female brains from the control group (0 ng/L) and 7.34 ng/L were sampled.
Project description:17α-Ethinylestradiol (EE2) is a ubiquitous aquatic contaminant shown to decrease fish fertility at low concentrations, especially in fish exposed during development. The mechanisms of the decreased fertility are not fully understood. In this study, we perform transcriptome analysis by RNA sequencing of testes from zebrafish with previously reported lowered fertility due to exposure to low concentrations of EE2 during development. Fish were exposed to 1.2 and 1.6 ng/L (measured concentration) of EE2 from fertilization to 80 days of age, followed by 82 days of remediation in clean water.
Project description:Adult male marine fish were exposed to 100ng/L of ethinyl estradiol (EE2) for 7 days in 1.8L vessels, dimethyl sulfoxide (DMSO) was used as carrier never reaching concentrations > 0.001% v/v. The same volume of DMSO was added to the solvent control vessels. Culture conditions, monitored daily, were as follows: temperature 18.8 ± 0.2°C, pH 7.94±0.03, dissolved oxygen saturation 99.3% ± 1.1 and 16:8 hours of light:dark photoperiod. 90% of the water was renewed daily and EE2 and DMSO concentrations were adjusted after that.
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:Sticklebacks were caught from the River Aire (Yorkshire, UK) and Siblyback reservoir (Cornwall, UK), depurated for 4 months at Exeter University, split into groups of 20 individuals before exposure. Fish were exposed to either control conditions, three concentrations of copper (3.2, 32, 128 µg/L; prepared using copper sulphate), three concentrations of ethinyl-estradiol (EE2) (1, 10, 32 ng/L) and three mixtures (3.2 µg Cu/L and 32 ng EE2/L; 128 µg Cu/L and 1 ng EE2/L; 128 µg Cu/L and 32 ng EE2/L) in duplicate tanks for 4 days. After sampling liver of male fish were stored at -80C for transcriptomics. Total RNA was prepared from fish livers, aliquots of all samples were pooled and labeled with Cy3-dCTP as a common reference, individual samples were labeled with Cy5-dCTP. Each hybridisation to the stickleback PGPS2 cDNA microarray consisited of one Cy5-labeled individual sample and one aliquot of the Cy3 labeled commmon reference sample. Data were captured with an Axon scanner using Genepix software.
Project description:The anabolic androgen 17M-NM-2-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, M-bM-^@M-^\cholesterol biosynthetic processM-bM-^@M-^] 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 M-bM-^@M-^\sexual differentiation and developmentM-bM-^@M-^], 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:During development, the inherited DNA methylation patterns from the parental gametes needs to be remodeled into a state compatible with embryonic pluripotency. In Zebrafish, this remodeling is achieved by the maternal methylome becoming hypomethylated to match the paternal methylome. However, how this is achieved in medaka (another teleost fish) is currently not known. Moreover, how DNA methylation remodeling is impacted in hybrid organisms, and the effects this may have on their development, is also not known. Here we address these questions by generation whole genome bisulfite sequencing data for zebrafish, medaka and zebrafish medaka embryos.