Summary
Injection of male bullheads (Ameiurus nebulosus) with estradiol induced the production of a major serum phosphoprotein of molecular weight 145 000. This protein was immunoprecipitable by antisera raised against lipovitellin from bullhead eggs and was absent from the serum of control males. Production of this serum protein coincided with changes in the liver mRNA population, which suggested that estradiol had induced the synthesis of additional mRNA sequences in the high-frequency class. Agarose gel electrophoresis in the presence of methyl mercury hydroxide showed that this mRNA population contained at least one species which was not present in the liver of uninjected males. This new RNA was the major polyadenylated species present in the total cellular RNA and its size relative to ribosomal RNAs and locust vitellogenin mRNA was estimated as 5800 nucleotides. When the liver total RNAs were translated in the mRNA-dependent rabbit reticulocyte lysate system the major translation product from the induced fish had the same molecular weight (145 000) as the serum phosphoprotein and was immunoprecipitable by antilipovitellin antisera. This translation product was not coded for by RNA from control fish. These observations are consistent with the induction of vitellogenesis by estradiol as reported in other egg-laying vertebrates and they show that bullhead vitellogenin and its mRNA are significantly smaller than their avian and amphibian counterparts.
Methodology
Materials. All biochemicals were obtained from Sigma Chemical Co. with the following exceptions. Oligo(dT)-cellulose type T3 was purchased from Collaborative Research Inc., methyl mercury hydroxide from Ventron Corporation, phage DNAs and restriction endonucleases from Bethesda Research Labs., and protein molecular weight markers from Bio-Rad Laboratories. Escherichia coli ribosomal RNAs were from Boehringer Mannheim Corporation. Proteinase K was retailed by British Drug Houses and labelled compounds were purchased from New.England Nuclear. Total cellular RNA from locust fat body was a gift from Dr. G.R. Wyatt and co-workers, Department of Biology, Queen's University. Rat liver ribosomal RNAs were a gift from Dr. R. Kisilevsky, Department of Pathology, Queen's University, and AMV reverse transcriptase was kindly supplied through Dr. Chirigos of the National Cancer Institute, Bethesda, MD by Dr. Joseph Beard.
Collection of tissue. Brown bullheads (A. nebulosus) of average weight 200--300 g were obtained from the Lake.Opinicon region, Ontario, in the spring, and were kept in aquaria supplied with running tap water. Fish in the experimental groups were given two injections of estradiol, the second 7 days after the first. During each of these injections 17~-estradiol (4 mg) was administered intramuscularly in propylene glycol (0.2 ml). Livers were removed from these fish 3 days after the second injection and were immediately cut into several pieces and frozen in liquid nitrogen prior to storage at --60°C. Livers from control fish which had not received estradiol injection were similarly treated and, for both sets, livers from male and female fish were kept separately. The female fish were naturally vitellogenic at the time they were sampled and therefore the presence of roe was used as an indicator of the sex of the fish. Samples of plasma or serum were collected from male and female fish from both the control and injected groups at the time livers were removed and were stored at --20°C. Serum samples were prepared in the presence of PMSF (0.1 mM) and plasma samples in heparin (500 pg/ml). Roes were also retained from female bullheads and were stored at --60°C.
Raising antibodies to bullhead lipovitellin. Bullhead lipoviteUin was prepared from roe by adapting the method of Bernardi and Cook [13]. The final step in the purification of lipovitellin involved its precipitation from solution in 1 M NaC1 by the addition of 20 vol. water. The precipitate was collected by centrifugation, dried by lyophilization and then weighed. Rabbits (5 kg) were initially injected subcutaneously with 1 ml of a homogenate of equal volumes of crude bullhead egg protein (4 mg/ml) in rabbit saline (130 mM NaC1/7.5 mM MgC12/5 mM KC1) and Freund's Complete Adjuvant. These injections were followed by two intravenous injections of purified lipovitellin (2 mg in 1 ml of rabbit saline) 5 and 8 weeks afterwards. After 10 weeks serum was obtained from the rabbits and tested for reaction with purified lipovitellin and various bullhead sera on Ouchterlony plates.