Fish Zona radiata (eggshell) proteins: evaluation and validation as a biomarker for xenoestrogen monitoring

New cellular and molecular tools are enabling researchers to study the basic mechanisms of action of natural reproductive hormones, and of endocrine disrupters, including xenoestrogens. Zonagenesis is defined by the hepatic synthesis of the eggshell Zona radiata proteins (Zrp), their secretion and transport in the blood and their uptake by maturing oocytes. Zrps are comprised of three-to-four protein monomers in the 50–60 kDa MW range and show structural similarity to proteins of the Zona pellucida; an extracellular envelope that lies immediately outside the plasma membrane in the eggs of placental mammals. We studied the xenoestrogen-induced elevation in hepatic mRNA and plasma levels of Zrp and vitellogenin (Vtg) in several fish species. Eggshell Zrp protein and mRNA was induced in juvenile salmon by exposure to nonylphenol (NP: given singly or in combination with other xenoestrogens/xenobiotics). In English sole (Pleuronectes vetulus), Zrp was induced in the plasma of mature female, E₂- and NP-treated male fish. We are currently using salmon cDNA probes to study estrogen-receptor, Zrp and Vtg mRNA expression in English sole treated with E₂- and NP. Validation in a semi-field experiment showed that effluent from an oil refinery treatment plant caused a dose-dependent induction of Zrp and Vtg in juvenile salmon. However, a component corresponding to Zrp-β was more responsive than Zrp-α and Zrp-γ and also more than Vtg. Zrp seem to be a sensitive complement to Vtg analysis, and appear to be a more ecologically significant biomarker of estrogenicity.