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Seven yolk proteins (YPs), four large lipoproteins (YPs1-4) and three minor yolk components (YPs5-7) including one phosphoprotein (YP7), were purified from extracts of vitellogenic ovaries of grey mullet (Mugil cephalus) by combinations of hydroxylapatite, ion exchange, immunoadsorbent, and gel filtration chromatography. The molecular masses of native YP1, YP2, YP3, and YP4 were estimated to be 330, 325, 335, and 570 kDa, respectively. The tertiary structures of YP1, YP2, and YP3 revealed by sodium dodecyl sulfate polyacrylamide gel electrophoresis were typical of teleost lipovitellins (Lvs), consisting of a heavy chain ( approximately 110, approximately 99, and approximately 97 kDa, respectively) and a light chain ( approximately 30, approximately 29, and approximately 21.5 kDa, respectively), while YP4 exhibited a heavy chain ( approximately 110 kDa) and two more polypeptide bands ( approximately 70 and approximately 54 kDa). Mapping of N-terminal peptide sequences of the purified YPs to the primary structure of multiple mullet vitellogenins (Vgs) deduced from their respective complete cDNAs, which were cloned and sequenced, conclusively identified YP1, YP2, and YP3 as Lvs derived from mullet VgA, VgB, and VgC, respectively. The fourth YP (YP4) appeared to be a proteolytic variant consisting of Lv and phosvitin components of VgA. Two other YPs (YP5 and YP6) were identified as beta'-components derived from VgA and VgB based on their structures and common, but not identical, antigenicity to salmonid beta'-component, while purified YP7, a phosphoprotein with a high content of serine residues, was identified as a phosvitin derived from VgB. This is the first report, of which we are aware, on purification and molecular classification of three distinct forms of Lv from any oviparous vertebrate.

cDNAs encoding the glycoprotein hormone alpha- and gonadotropin (GTH) II beta-subunits of Japanese eel (Anguilla japonica) pituitary were cloned using the polymerase chain reaction. The nucleotide sequence of the glycoprotein hormone alpha-subunit cDNA was 364 base pairs (bp) long, encoding 117 amino acids, and that of the GTH II beta-subunit cDNA was 433 bp long, encoding 140 amino acids. The deduced amino acid sequence of each mature subunit showed high homology with those of other teleosts, indicating that the structure of GTH subunits has been conserved during the evolution of teleosts. Changes in the expression of these subunit genes during ovarian development induced artificially by the injection of chum salmon pituitary homogenate were examined using Northern blot analysis. Glycoprotein hormone alpha-subunit mRNA increased almost linearly during ovarian development, whereas GTH II beta-subunit mRNA was detected only at the late vitellogenic and migratory nucleus stages. These data indicate that eel GTH II is synthesized mainly at the late vitellogenic and migratory nucleus stages, and suggest that GTH II plays an important role in final oocyte maturation of Japanese eel. Changes in the expression of glycoprotein hormone alpha- and GTH II beta-subunits mRNA correlate with the serum estradiol-17 beta (E(2)) and testosterone profile during ovarian development. The increase in mRNA of both subunits is probably due to positive feedback of E(2) and testosterone produced by ovarian follicles in response to the GTH contained in chum salmon pituitary homogenate.

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We monitored the contamination by environmental estrogens (EEs) of coastal areas in Korea and Japan using the wild grey mullet. The grey mullet were collected from Ansan, Jeju, Yeosu, Tongyeong, and Busan in Korea and Nagasaki, Omuta, and Fukuoka in Japan.Contamination by EEs was determined by measuring vitellogenin (VTG) levels in serum and identifying gonadal abnormalities histologically (i.e., testis-ova). In four sites in Korea (Ansan, Yeosu, Tongyeong, and Busan) and two sites in Japan (Nagasaki and Fukuoka), serum VTG in immature and male grey mullet was detected at levels greater than 1.0 g/ml, which is considered to be an abnormal level. Although, testis-ova were observed in some individuals collected in Ansan, Tongyeong, and Busan in Korea and Omuta in Japan, there was no correlation between individuals with testis-ova and individuals with abnormal levels of VTG. Furthermore, in Japan, serum VTG levels of fish collected from Nagasaki and Fukuoka was also greater than 1.0 g/ml. Although individuals with testis-ova were found in Omuta, these fish expressed normal levels of serum VTG. Our results suggest that the grey mullet living in these coastal areas are influenced by EEs in the environment. Furthermore, it appears that the production of VTG and the occurrence of testis-ova are caused by different mechanisms.

Immunoglobulin M (IgM) is known as a main factor in the humoral immune system of teleosts. In the present study, the effect of cortisol on plasma IgM concentrations was investigated using a specific antibody to IgM in masu salmon (Oncorhynchus masou). Cortisol was orally administered each day for 2 weeks at a dose of 1 mg g(-1) in the diet, and for the following week the fish were fed a non-treated diet. Blood plasma samples were collected at 0, 1, 2 and 3 weeks after the initiation of treatment. Oral administration of cortisol elevated plasma cortisol concentrations to about 40 ng/ml for 2 weeks after administration and slightly reduced plasma IgM concentration; the suppression was statistically significant one week after the period of hormone administration. However, treatment with cortisol did not affect plasma concentrations of total protein or α1-protein, one of the major serum proteins, during the experimental period. These results indicate that cortisol specifically suppresses plasma IgM concentrations.