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

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 beta -actin were cloned by rever se transcriptase polymerase chain reaction (RT-PCR) using degenerate primer s 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 id entity to that of salmon (77%,), winter flounder (64%), carp ZP2 (63%) and medaka (61%) ZR-proteins. The 1020 bp beta -actin fragment was approximatel y 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 i n vivo exposure to estradiol-17 beta (E-2) (0.01, 0.1, 1.0 or 10 mg/kg body weight (bw) fish) or alpha -zearalenol (alpha -ZEA) (0.1. 1.0 or 10 mg/kg bw). Real-time PCR and indirect enzyme-linked immunosorbent assay (ELISA) s howed that ZR and VTG were induced in both the liver and the plasma after a single injection of E-2 or alpha -ZEA. ZR was more responsive to low level s of E-2 and alpha -ZEA than VTG, and real-time PCR was shown to be more se nsitive than the ELISA. Rainbow trout ZR-gene and proteins provide a sensit ive biomarker fur assessing estrogenic activity. (C) 2001 Elsevier Science Ltd. All rights reserved.