Quantification of rainbow trout ( Oncorhynchus mykiss) zona radiata and vitellogenin mRNA levels using real-time PCR after in vivo treatment with estradiol-17β or α-zearalenol

Abstract

Estrogen receptor-mediated induction of zona radiata (ZR) and vitellogenin (VTG) mRNA and protein in rainbow trout ( Oncorhynchus mykiss) was compared to assess their utility as biomarkers for exposure to estrogenic compounds. Partial sequences of rainbow trout ZR and β-actin were cloned by reverse transcriptase polymerase chain reaction (RT-PCR) using degenerate primers based on conserved regions across a number of species. A 549 bp fragment of the rainbow trout ZR-gene showed a high degree of amino acid sequence identity to that of salmon (77%), winter flounder (64%), carp ZP2 (63%) and medaka (61%) ZR-proteins. The 1020 bp β-actin fragment was approximately 100% identical to sequences from several species. Real-time PCR was used to quantify the induction of ZR-gene and VTG in rainbow trout liver after in vivo exposure to estradiol-17β (E 2) (0.01, 0.1, 1.0 or 10 mg/kg body weight (bw) fish) or α-zearalenol (α-ZEA) (0.1, 1.0 or 10 mg/kg bw). Real-time PCR and indirect enzyme-linked immunosorbent assay (ELISA) showed that ZR and VTG were induced in both the liver and the plasma after a single injection of E 2 or α-ZEA. ZR was more responsive to low levels of E 2 and α-ZEA than VTG, and real-time PCR was shown to be more sensitive than the ELISA. Rainbow trout ZR-gene and proteins provide a sensitive biomarker for assessing estrogenic activity.