No 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.

The induction of metallothionein and vitellogenin synthesis in rainbow trout liver was studied after injection of oestradiol-17 beta alone or in combination with cadmium or zinc. Intraperitoneal injection of oestradiol-17 beta increased the liver somatic index, with subsequent induction of vitellogenin synthesis. Oestradiol-17 beta did not induce metallothionein synthesis. Injection of cadmium induced the synthesis of metallothionein mRNA and metallothionein. Injection of oestradiol-17 beta in combination with cadmium resulted in inhibition of transcription and translation of both vitellogenin and metallothionein. Chromatography of liver cytosols revealed that cadmium, when co-injected with oestradiol-17 beta, did not bind to metallothionein but would initially bind to high-molecular-mass (HMr) cytosolic proteins. In fish injected with cadmium in combination with oestradiol-17 beta, cadmium was gradually redistributed from HMr proteins to metallothionein. This resulted in induction of metallothionein synthesis and in binding of most of the cadmium to metallothionein. Induction of vitellogenin mRNA was observed 15 days after injection, as cadmium was being redistributed to newly synthesized metallothionein. These findings indicate that cadmium inhibits the transcription of vitellogenin. The binding of cadmium to these non-metallothionein proteins represses the induction of metallothionein and results in increased toxicity of the metal. Preinduction of metallothionein by zinc injections resulted in decreased cadmium sensitivity of the fish and a decrease in the repression of vitellogenin mRNA. Furthermore, a role for metallothionein in the detoxification of cadmium is indicated by the induction of vitellogenin synthesis that occurs once metallothionein has begun sequestering cadmium.

No abstract

In order to study the initial formation of the vitelline envelope and the appearance of vitellogenin in oocytes of rainbow trout, females were sampled monthly from 19 to 5 mo before ovulation. Immunohistochemistry revealed that the formation of the vitelline envelope starts when the oocytes reach a diameter of about 450 microns. Oocytes of this size were first found in females sampled a year before ovulation at the time when plasma levels of estradiol-17 beta increased from 0.2 to 0.6 ng/ml. An antiserum directed against vitellogenin crossreacted with small vesicles (around 2 microns) present just inside the oolemma, when the oocytes reached a diameter of 600 microns. This was interpreted as an active uptake of vitellogenin. Oocytes of this size were first found in females sampled 9 mo before ovulation at the time when estradiol-17 beta levels increased from 0.6 to 1.0 ng/ml and the gonadal somatic index was doubled. Oocytes with a diameter of 600 microns had an immunoreactive vitelline envelope with a thickness of about 3 microns. It is apparent that the initial formation of the vitelline envelope starts before the active uptake of vitellogenin and that the low previtellogenic plasma levels of estradiol-17 beta observed in females are of physiological significance.