17α‐ethynylestradiol‐induced vitellogenin gene transcription quantified in livers of adult males, larvae, and gills of fathead minnows (Pimephales promelas)

Abstract

We have applied a method for quantifying relative levels of messenger RNA (mRNA) transcription to assess chemically induced gene expression in fathead minnows (Pimephales promelas). Synthetic oligonucleotides designed for the fathead minnow vitellogenin gene transcription product were used in a reverse transcription polymerase chain reaction (RT-PCR) protocol. This sensitive and rapid strategy detected vitellogenin gene transcription in livers of male fathead minnows exposed to concentrations as low as 2 ng/L of the endocrine-disrupting compound 17alpha-ethynylestradiol for 24 h. Surprisingly, vitellogenin transcription products also were detected in gill tissue and in 48-h-old posthatch fathead minnow larvae. Relative levels of vitellogenin gene induction among individuals were quantified in a single-step reaction (PCR multiplex) with 18S rRNA universal primers and Competimers concurrently with fathead minnow vitellogenin oligonucleotides. This quantitative approach will markedly enhance detection of the first cellular event of estrogenic exposure to aquatic ecosystems in both field and laboratory systems. Use of the model provides sensitivity of detection at a concentration below those that cause mortality or visible signs of stress in fish or other aquatic organisms. The model may also provide an in vivo screening method for estrogenlike endocrine-disrupting compounds.

Comparison of relative binding affinities of endocrine active compounds to fathead minnow and rainbow trout estrogen receptors

“…The binding affinity of E2 to fhmER and rbtER essentially was the same, with mean IC50s of 6.7 Ϯ 1.3 nM (n ϭ 8) and 6.6 Ϯ 2.4 nM (n ϭ 31), respectively. In addition, numerous studies have shown that EE2 induces vitellogenin in both fathead minnows [23,[25][26][27][28] and rainbow trout [29][30][31]. The RBAs for DES, EE2, and E1 determined in this study were within fivefold of one another and are comparable with the values reported by Tollefsen et al (Table 1 [15]).…”

Effects of progesterone on reproduction and embryonic development in the fathead minnow (Pimephales promelas)

“…Vitellogenin mRNA analysis was performed using the method of Lattier et al [20], with modifications. The quality of the RNA was assessed and quantified spectrophotometrically with absorbance measurements at 260 nm with a Molecular Devices Spectramax M5e reader.…”

Effects of 17α-ethynylestradiol on early-life development, sex differentiation and vitellogenin induction in mummichog (Fundulus heteroclitus)

“…This makes comparison to other studies that had exposure concentrations <10 or >10 ng/L possible. Fathead minnow have failed development of secondary sex characteristics, altered mating behaviour, intersex and induced VTG in males at 1-5 ng/L EE 2 (Länge et al, 2001;Lattier et al, 2002;Parrott and Blunt, 2005); Japanese medaka display intersex and reduced copulations at 2-10 ng/L EE 2 (Balch et al, 2004); and zebrafish have reduced spawning, fertilization, intersex and GSI at 5-10 ng/L EE 2 (Van den Belt et al, 2001Belt et al, , 2002Nash et al, 2004). In contrast to freshwater species, in which effects are observed at concentrations 610 ng/L EE 2 , sheepshead minnow have reduced reproductive success at EE 2 between 20 and 200 ng/L (Zillioux et al, 2001) while mummichog have altered steroid synthesis, VTG induction, reproductive success and sex differentiation at >10 ng/L EE 2 (MacLatchy et al, 2003;Peters et al, 2007;this study).…”