Isolation, immunochemical detection, and observations of the instability of vitellogenin from four teleosts

Abstract

Vitellogenin was purified from plasma of estradiol-17P-treated cod (Gadus morhua) rainbow trout (Oncorhynchus mykiss), turbot (Scophthalmus maximus), and wolffish (Anarhichas lupus) by precipitation with EDTA:Mg2+, distilled water, and high-performance ion-exchange chromatography. Vitellogenin of high purity was obtained by precipitation followed by chromatography, as evaluated by an homologous antiserum developed for each species. The instability of vitellogenin demanded consistent low temperature and the use of protease inhibitor before blood sampling. When the necessary precautions were taken, vitellogenin from rainbow trout, turbot, and wolffish eluted as one regular peak during chromatography. Cod vitellogenin eluted as two peaks and these demonstrated identical migration patterns on SDS-PAGE. The observed differences in stability between the four species suggest that isolation procedures should be modified according to the requirements for each species. Electrophoresis of plasma from treated fish revealed the presence of several smaller proteins, with a molecular mass around 50 kDa, that were considered to be vitelline envelope proteins. Other minor plasma proteins were immunoreactive to antisera, directed against vitellogenin and therefore judged to be fragments of degraded vitellogenin. o 1993 Wiley-Liss, Inc.

Vitellogenin isolation, purification and antigenic cross-reactivity in three teleost species

“…In most species vtg is composed of monomers or dimers (Matsubara et al,1999;Specker and Sullivan, 1994), but vtg of goldfish (Carassius aurata) and Japanese eel (Anguilla japonica) also appear to be composed of from three to four subunits (reviewed in Utarabhand and Bunlipatanon, 1996). Only one dominant subunit was found in rainbow trout, in agreement with a previous description of vtg for this species (Silversand et al, 1993), but several minor bands were present which may also be attributable to vtg.…”

“…However, the value we obtained for rainbow trout vtg (340 kD) is low compared with the literature value (550 kD) (Specker and Sullivan, 1994). This may be explained either by the potential lack of precision of the molecular weight estimation method or by the extremely labile nature of the native vtg molecule, which has been shown to be susceptible to proteolysis during isolation (Silversand et al, 1993). It is possible that some degradation could have occurred during the isolation procedure.…”

Effects of Dietary Arachidonic Acid on the Reproductive Physiology of Female Atlantic Cod (Gadus morhua L.).

“…Briefly, VTG was isolated from plasma from E2-treated juvenile cod by anion exchange chromatography (Silversand et al, 1993). The concentration of the purified VTG was determined by UV absorbance spectrophotometry, using a coefficient of 0.66 corresponding to 1 mg/ml (Norberg and Haux, 1985).…”

“…Standards in triplicate, and diluted plasma samples in duplicate were added to 96-well Maxisorp immunoplates (NUNC), coated overnight with 50 ng VTG in 0.02 M NaHCO3, pH 9.6. Polyclonal anti-cod VTG (Silversand et al, 1993) was added to the wells at a dilution of 1:100,000. The immunoplates were incubated at 4°C overnight.…”

Effect of Photoperiod Advancement of Atlantic Cod Spawning on Egg Size and Biochemistry

“…The maturational cycle-beginning with endocrine-controlled secretion of estradiol (Wallace 1985), the synthesis of vitellogenin, the uptake and incorporation of lipids and protein from vitellogenin into the developing oocytes (Tyler 1993;Hyllner et al 1994), ovulation, and spawning-normally encompasses several months in most teleosts from northern latitudes. In Atlantic cod, vitellogenesis normally is initiated during September-November (Kjesbu 1994), commencing with vitellogenin synthesis induced by estradiol-17b (Silversand et al 1993). In Atlantic cod stocks adjacent to Newfoundland, spawning subsequently occurs during April-June and the major spawning period is in May (Brander 1993).…”