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Title: Development of quantitative real-time PCR assays for fathead minnow (Pimephales promelas) gonadotropin ß subunit mRNAs to support endocrine disruptor research.

Abstract

Fathead minnows (Pimephales promelas) are one of the most widely-used small fish models for regulatory ecotoxicology testing and research related to endocrine disrupting chemicals (EDCs). In this study, we isolated and sequenced cDNAs for fathead minnow follicle-stimulating hormone-like and luteinizing hormone-like β (FSHβ and LHβ) and glycoprotein α (GPα) subunits. Quantitative real-time PCR assays for measuring gonadotropin (GtH) β subunit transcripts were developed and used to examine “baseline” transcript levels over a range of age classes and reproductive states encompassed in EDC testing. In females, FSHβ and LHβ transcripts were greater in 4-5 month old than in younger fish and were significantly correlated with one another across all age classes examined. In males, FSHβ transcripts were greatest in 2-3 month old fish and were inversely correlated with various measures of testis development including, gonadal-somatic index (GSI), and histological stage. Overall, the pattern of GtHβ expression over age classes associated with gonad development was similar to that reported for other asynchronous-spawning fish. Despite significant changes in female GSI, gonad stage, and plasma vitellogenin within 24 h of spawning, GtHβ transcript levels in fish that had spawned within the preceding 24 h were not significantly different from those in fish that weremore » 2-3 days post-spawn and expected to spawn within the next 24 h based on spawning history. Results of this study provide insights related to the role of GtHs in fathead minnow reproductive development and function. Additionally they provide useful “baseline” data needed to design and interpret effective experiments for studying direct and indirect effects of EDCs on GtH subunit mRNA expression, which will facilitate a greater understanding of integrated system-wide responses of the fathead minnow brain-pituitary-gonadal axis to stressors including EDCs.« less

Authors:
; ; ; ; ; ; ; ; ;
Publication Date:
Research Org.:
Pacific Northwest National Lab. (PNNL), Richland, WA (United States)
Sponsoring Org.:
USDOE
OSTI Identifier:
985075
Report Number(s):
PNNL-SA-47802
Journal ID: ISSN 1532-0456; TRN: US201016%%1760
DOE Contract Number:
AC05-76RL01830
Resource Type:
Journal Article
Resource Relation:
Journal Name: Comparative Biochemistry and Physiology Part C: Toxicology and Pharmacology, 145(2):171-183; Journal Volume: 145; Journal Issue: 2
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; 99 GENERAL AND MISCELLANEOUS//MATHEMATICS, COMPUTING, AND INFORMATION SCIENCE; DESIGN; FATHEAD MINNOW; FEMALES; GLYCOPROTEINS; GONADOTROPINS; GONADS; MALES; PLASMA; TESTING; fathead minnow; endocrine disruptors; PCR

Citation Formats

Villeneuve, Daniel L., Miracle, Ann L., Jensen, Kathleen M., Degitz, Sigmund J., Kahl, Michael D., Korte, Joseph J., Greene, Katie J., Blake, Lindsey S., Linnum, Ann, and Ankley, Gerald T.. Development of quantitative real-time PCR assays for fathead minnow (Pimephales promelas) gonadotropin ß subunit mRNAs to support endocrine disruptor research.. United States: N. p., 2007. Web. doi:10.1016/j.cbpc.2006.11.003.
Villeneuve, Daniel L., Miracle, Ann L., Jensen, Kathleen M., Degitz, Sigmund J., Kahl, Michael D., Korte, Joseph J., Greene, Katie J., Blake, Lindsey S., Linnum, Ann, & Ankley, Gerald T.. Development of quantitative real-time PCR assays for fathead minnow (Pimephales promelas) gonadotropin ß subunit mRNAs to support endocrine disruptor research.. United States. doi:10.1016/j.cbpc.2006.11.003.
Villeneuve, Daniel L., Miracle, Ann L., Jensen, Kathleen M., Degitz, Sigmund J., Kahl, Michael D., Korte, Joseph J., Greene, Katie J., Blake, Lindsey S., Linnum, Ann, and Ankley, Gerald T.. Thu . "Development of quantitative real-time PCR assays for fathead minnow (Pimephales promelas) gonadotropin ß subunit mRNAs to support endocrine disruptor research.". United States. doi:10.1016/j.cbpc.2006.11.003.
@article{osti_985075,
title = {Development of quantitative real-time PCR assays for fathead minnow (Pimephales promelas) gonadotropin ß subunit mRNAs to support endocrine disruptor research.},
author = {Villeneuve, Daniel L. and Miracle, Ann L. and Jensen, Kathleen M. and Degitz, Sigmund J. and Kahl, Michael D. and Korte, Joseph J. and Greene, Katie J. and Blake, Lindsey S. and Linnum, Ann and Ankley, Gerald T.},
abstractNote = {Fathead minnows (Pimephales promelas) are one of the most widely-used small fish models for regulatory ecotoxicology testing and research related to endocrine disrupting chemicals (EDCs). In this study, we isolated and sequenced cDNAs for fathead minnow follicle-stimulating hormone-like and luteinizing hormone-like β (FSHβ and LHβ) and glycoprotein α (GPα) subunits. Quantitative real-time PCR assays for measuring gonadotropin (GtH) β subunit transcripts were developed and used to examine “baseline” transcript levels over a range of age classes and reproductive states encompassed in EDC testing. In females, FSHβ and LHβ transcripts were greater in 4-5 month old than in younger fish and were significantly correlated with one another across all age classes examined. In males, FSHβ transcripts were greatest in 2-3 month old fish and were inversely correlated with various measures of testis development including, gonadal-somatic index (GSI), and histological stage. Overall, the pattern of GtHβ expression over age classes associated with gonad development was similar to that reported for other asynchronous-spawning fish. Despite significant changes in female GSI, gonad stage, and plasma vitellogenin within 24 h of spawning, GtHβ transcript levels in fish that had spawned within the preceding 24 h were not significantly different from those in fish that were 2-3 days post-spawn and expected to spawn within the next 24 h based on spawning history. Results of this study provide insights related to the role of GtHs in fathead minnow reproductive development and function. Additionally they provide useful “baseline” data needed to design and interpret effective experiments for studying direct and indirect effects of EDCs on GtH subunit mRNA expression, which will facilitate a greater understanding of integrated system-wide responses of the fathead minnow brain-pituitary-gonadal axis to stressors including EDCs.},
doi = {10.1016/j.cbpc.2006.11.003},
journal = {Comparative Biochemistry and Physiology Part C: Toxicology and Pharmacology, 145(2):171-183},
number = 2,
volume = 145,
place = {United States},
year = {Thu Mar 01 00:00:00 EST 2007},
month = {Thu Mar 01 00:00:00 EST 2007}
}
  • No abstract prepared.
  • Three concurrent studies were conducted to determine the chronic effect of prespawning exposure to various concentrations of copper on fathead minnow reproduction. Copper was introduced into the three exposure systems to give 6-, 3-, and 0-months exposure prior to spawning. Prespawning exposure time had no significant effect on reproduction. Number of eggs produced per female decreased, however, with increase in copper concentrations. Egg production at copper concentrations of 37 ..mu..g l/sup -1/ and higher was significantly lower (P = 0.05) than in the control, but at concentrations of 24 ..mu..g l/sup -1/ and lower it was not different. The maximummore » acceptable toxicant concentrations (MATC) was estimated to be 32 ..mu..g Cu l/sup -1/, which is 0.07 of the 96 h LC/sub 50/. This application factor for copper is similar to those found in other studies.« less
  • The rate at which radiation was delivered to fathead minnows (Pimephales promelas) was found to affect survival time. For a dose of 3000 rad, the time to 50% mortality at 2.62 rad/min was over 60 days, at 37.8 rad/min it was 28 days, and at 1730 rad/min it was 17 days. At a dose rate of 1730 rad/min the 50% endpoint for mortality at 12 days was independent of dose from 6000 to at least 24,000 rad. These results further substantiate that dose rate has a significant effect on the survival time in fish, if the environmental temperature is constant.
  • Gene microarrays provide the field of ecotoxicology new tools to identify mechanisms of action of chemicals and chemical mixtures. Herein we describe the development and application of a 2,000-gene oligonucleotide microarray for the fathead minnow Pimephales promelas, a species commonly used in ecological risk assessments in North America. The microarrays were developed from various cDNA and subtraction libraries that we constructed. Consistency and reproducibility of the microarrays were documented by examining multiple technical replicates. To test application of the fathead minnow microarrays, gene expression profiles of fish exposed to 17-estradiol, a well-characterized estrogen receptor (ER) agonist, were examined. For thesemore » experiments, adult male fathead minnows were exposed for 24 h to waterborne 17-estradiol (40 or 100 ng/L) in a flow-through system, and gene expression in liver samples was characterized. Seventy-one genes were identified as differentially regulated by estradiol exposure. Examination of the gene ontology designations of these genes revealed patterns consistent with estradiol’s expected mechanisms of action and also provided novel insights as to molecular effects of the estrogen. Our studies indicate the feasibility and utility of microarrays as a basis for understanding biological responses to chemical exposure in a model ecotoxicology test species.« less
  • The largest environmental release of coal ash in U.S. history occurred in December 2008 with the failure of a retention structure at the Tennessee Valley Authority (TVA) Kingston Fossil Plant in East Tennessee. A byproduct of coal-burning power plants, coal ash is enriched in metals and metalloids such as selenium and arsenic with known toxicity to fish including embryonic and larval stages. The effects of contact exposure to sediments containing up to 78 % coal ash from the Kingston spill on the early development of fish embryos and larvae were examined in 7-day laboratory tests with the fathead minnow (Pimephalesmore » promelas). No significant effects were observed on hatching success, incidences of gross developmental abnormalities, or embryo-larval survival. Results suggest that direct exposures to sediment containing residual coal ash from the Kingston ash release may not present significant risks to fish eggs and larvae in waterways affected by the spill.« less