Atlantic halibut (Hippoglossus hippoglossus) vitellogenin: induction, isolation and partial characterization

Abstract

Vitellogenin (VTG) synthesis was induced by repeated injections of estradiol-17β in juvenile Atlantic halibut (Hippoglossus hippoglossus). VTG eluted as a large, phosphoprotein containing peak on DEAE-Sephacel chromatography of plasma from estradiol-17β treated juvenile and mature female, but not mature male halibut. A purification procedure for Atlantic halibut VTG was developed, where VTG was precipitated with MgCl2, EDTA and distilled water, and the precipitated protein submitted to anion exchange chromatography on DEAE-Sephacel. Precipitated VTG eluted as a broad, partly dissociated peak on DEAE-Sephacel, when chromatography was run at 4°C, but the protein appeared intact when analysed both by SDS PAGE and native PAGE. DEAE-Sephacel chromatography at room temperature resulted in an irregular elution pattern and a dissociated protein fraction, as analysed by SDS PAGE. Biochemical characterization of VTG showed that the molecular mass of the monomer was ca 160 kDa, as estimated by SDS-PAGE. The total lipid content was 19.8% w/w, with 64%, or 12.7% of the total weight, as phospholipid. Protein bound phosphorus constituted 0.62% w/w of halibut VTG. Plasma dilution curves from mature and maturing female halibut were parallel with a dilution curve from halibut egg yolk homogenate in an homologous RIA. Plasma from mature male, but not juvenile halibut crossreacted with the VTG antiserum.

Composition, accumulation and utilization of yolk lipids in teleost fish

“…Phospholipids constituted 61-73% of total VTG lipids in these two species, with TAGs and cholesterol making up most of the balance (Norberg and Haux, 1985;Riazi and Fr6mont, 1988). Similar lipid contents have been found in the VTGs of goldfish (Hori et al, 1979) and Atlantic halibut (Norberg, 1995). Phospholipid content of the goldfish VTG was 87% of total lipid and that of the halibut was 64%.…”

Development of quantitative vitellogenin-ELISAs for fish test species used in endocrine disruptor screening

“…From carp, large plasma volumes for isolation of Vtg are easily obtained. Using a modified selective precipitation procedure [8], we obtained a highly purified fraction of carp Vtg in a single step. This preparation was used for producing the standard in the fathead minnow Vtg assay.…”

“…Carp Vtg used as standard in the quantitative carp Vtg ELISA was purified from plasma of E2-exposed common carp by selective precipitation. This procedure is a modified version of the method described by Norberg [8]. Plasma (1 mL) was mixed with 4 mL 0.02 M EDTA, pH 7.7 and 0.3 mL 0.5 M MgCl 2 .…”

Fish Zona Radiata (Eggshell) Protein: A Sensitive Biomarker for Environmental Estrogens

“…The zona pellucida plays a significant role during fertilization and in the prevention of polyspermy (19), as does the teleostean zona radiata proteins, thus showing dear functional similarities. Very little Vtg or Zrp, if any, can be detected in male and in juvenile fish, presumably because of low estrogen concentrations, but it is known that these proteins are synthesized by the liver cells (in vivo and in vitro) of male and juvenile fish treated with 17,B-estradiol (12,20,21).…”