Use of vitellogenin mRNA as a biomarker for endocrine disruption in feral and cultured fish

Abstract

The presence of the female-specific yolk protein precursor vitellogenin in blood and liver from male fish is widely used as an indicator of endocrine disruption. We studied the induction of vitellogenin mRNA in liver from several species of fish, both maintained in fish tanks or captured in the wild. Our procedure requires minute amounts of liver samples (down to 50 mg), and can be applied to field samples if the appropriate RNA-stabilisation agent is used. We used reverse-transcriptase PCR and quantitative real-time PCR for detection and precise quantitation of vitellogenin mRNA levels. Male mummichog (Fundulus heteroclitus) exposed to 17 beta-estradiol contained levels of vitellogenin mRNA up to 30 times higher than in untreated females and treatment with nonylphenol resulted in a weak but consistent induction of this transcript. We also studied levels of vitellogenin mRNA in a population of common carp (Cyprinus carpio) from the Anoia river, a river known for its high levels of estrogenic alkylphenols. The results were consistent with recorded data for fish from this sampling site. Finally, we also detected vitellogenin mRNA in barbs (Barbus graellsi), a species for which no vitellogenin sequence was available. The use of mRNA quantitation techniques for analysis of feral and cultured fish of different species opens the possibility of more precise detection and further control of the noxious effects of contaminants on the local fauna exposed to them.

Macroscopic gonadal deviations and intersex in developing whitefish Coregonus lavaretus

“…As a reference, mRNA of the 18s gene was measured using a TaqMan Riboso-mal RNA Control Reagents kit (Applied Biosystems). Transcript abundance of VTG was normalized to the abundance of 18s and reported as number of VTG copies 1000 -1 copies of 18s according to Pfaffl (2001) and Garcia-Reyero et al (2004).…”

Estrogen receptor-related receptors in the killifish Fundulus heteroclitus: diversity, expression, and estrogen responsiveness

“…Primers for ERRa, ERRba, ERRbb, ERRgb, and b-actin (Table 1) were designed with one primer spanning a predicted exon-exon junction to avoid amplification of genomic DNA. Primers for EF-1 (Bears et al 2006) and vitellogenin (Garcio-Reyero et al 2004) were taken from published studies. cDNA was synthesized from 2 mg total RNA using random hexamers and the Omniscript cDNA Synthesis Kit (Qiagen).…”

Evaluation of environmental impact on natural populations of the Mediterranean killifish Aphanius fasciatus by quantitative RNA biomarkers

“…In this context, markers based on RNA quantification methods, such qRT-PCR, provide a suitable alternative, as they only require minute amounts of sample Rotchell and Ostrander, 2003;Valasek and Repa, 2005). These techniques have proved their usefulness for monitoring of different types of environmental pollutants, like estrogens (Barucca et al, 2006;Garcia-Reyero et al, 2004;George et al, 2004), metals (Cheung et al, 2004;Navarro et al, 2009;Tom et al, 2004), and organic substances (Eljarrat et al, 2008;Quirós et al, 2007a;Quirós et al, 2007b). It is clear that biomarkers based on mRNA levels only reflect the initial steps of a cascade of events linking environmental insults to ecological impact (van der Oost et al, 2003).…”

“…In this work, we develop mRNA markers for monitoring A. fasciatus exposure to different kinds of pollutants, using known DNA sequences from evolutionarily related species (mainly Cyprinodonts and Percomorphs). Genes used in this analysis included the main metal-responsive metallothionein (MT) gene (Cheung et al, 2004;Navarro et al, 2009;Schlenk et al, 1995;Tom et al, 2004;Van Cleef-Toedt et al, 2001), the egg-forming, liver-expressed vitellogenin (Vtg), a known marker for estrogens (Barucca et al, 2006;Denslow et al, 2001;Garcia-Reyero et al, 2004), the dioxin-like responsive Cytochrome P4501A (CYP1A, Chung-Davidson et al, 2004;Craft et al, 2001;Quirós et al, 2007a;Quirós et al, 2007b), the oxidative stress-related gene Superoxide dismutase (SOD, Cho et al, 2006;Gonzalez et al, 2006;Pandey et al, 2003), and the protein chaperone HSP70, which are involved in the processing of missfolded proteins due to different kinds of stress (Carnevali and Maradonna, 2003;Maradonna and Carnevali, 2007;Schroder et al, 2000;Varo et al, 2002). In addition, probes for two housekeeping genes (ß actin and the elongation translation factor EF1)…”