Vitellogenin in rare minnow (Gobiocypris rarus): identification and induction by waterborne diethylstilbestrol

Rare minnow (Gobiocypris rarus) is a tiny Chinese carp that has a short life cycle and is easily cultured in the laboratory. In this study, juvenile rare minnows were exposed to waterborne diethylstilbestrol (DES) at 0.05, 0.5 and 5 mug/l in laboratory aquaria. After exposure for 4, 8, 13 and 21 days, juvenile fish were collected and vitellogenin (Vtg) was measured in whole body homogenates. Native and SDS electrophoresis followed by Western blotting were performed for Vtg identification, and a non-competitive ELISA was developed. In the DES exposure groups (0.5 and 5 mug/l DES), Vtg appeared after 4 days, increased significantly after 8 days and reached a maximum on day 13. Further, a significant increase in the hepatosomatic index (HSI) was found in the 5 mug/l DES exposure group after 21 days. These results indicate that rare minnow provides a good model for assessing endocrine disruption by environmental estrogens. (C) 2004 Elsevier Inc. All rights reserved.

Differential expression of thyroid-stimulating hormone beta subunit in gonads during sex reversal of orange-spotted and red-spotted groupers

We have cloned and characterized the full-length cDNA encoding thyroid-stimulating hormone beta-subunit (TSHbeta) from orange-spotted grouper Epinephelus coioides. It contains 913 nucleotides with an open reading frame encoding 146 amino acids with a 20 amino acid signal peptide. The grouper mature TSHbeta has 75, 70, 61, 59, 41, 42 and 40% identities to that of rainbow trout, Atlantic salmon, zebrafish, European eel, chicken. mouse and human, respectively. RT-PCR analysis indicated that the TSHbeta mRNA was expressed abundantly not only in pituitary but also in gonads. A more interesting finding is to reveal the differential TSHbeta expressions between the ovaries and the transitional gonads or testes in natural individuals of orange-spotted grouper and red-spotted grouper Epinephelus akaara, and in artificial sex reversal individuals of red-spotted grouper induced by MT feeding. In situ hybridization localization provided direct evidence that the TSHbeta was transcribed in the germ cells. In the growing oocytes, the TSHbeta transcripts were concentrated on the ooplasm periphery. In testicular tissues, the intensively expressed TSHbeta cells were found to be spermatogonia and spermatocytes in the spermatogenic cysts. This is the first report of a TSHbeta expressed in the gonads of any vertebrates in addition to the expected expression in the pituitary, and it expresses more transcripts in the gonads during sex reversal or testis than in the ovaries both in E. coioides and E. akaara. Importantly, the TSHbeta identification in germ cells allows us to further investigate the functional roles and the molecular mechanisms in gametogenesis of groupers, especially in sex reversal and in spermatogenesis. (C) 2004 Elsevier Ireland Ltd. All rights reserved.

Microcalorimetric investigation of the effect of manganese(II) on the growth of Tetrahymena shanghaiensis S(1)99

The heat output of and the effect of manganese (II) on Tetrahymena shanghaiensis S(1)99 growth metabolism has been determined by means of a LKB-2277 BioActivity monitor. Different concentrations of manganese(II) ions have different effects on the growth of T. shanghaiensis. At low concentrations (0-40 mug/mL) culture growth is promoted, whereas high concentrations (60-800 mug/mL) slow growth. Furthermore, concentrations of 1200 mug/mL or greater stop the growth of this protozooa completely.

Estradiol stimulates growth hormone production in female goldfish

The effects of estradiol (E(2)) on growth hormone (GH) production was investigated in gonad-intact female goldfish. It was first necessary to generate a specific antibody for use in immunocytochemistry, Western, and dot-blot analyses of GH production. To accomplish this, grass carp GH (gcGH) cDNA was cloned by the reverse transcription polymerase chain reaction (RT-PCR) and expressed in Echerichia coli and a specific polyclonal antibody to recombinant gcGH was generated in the rabbit. In Western blot, the anti-gcGH antibody specifically immunoreacted with recombinant gcGH, purified natural common carp GH, and with a single 21.5-kDa GH form from pituitary extracts of grass carp, common carp, goldfish, and zebrafish but not salmon, trout, or tilapia. Intraperitoneal injection of the recombinant gcGH enhanced the growth rates of juvenile common carp demonstrating biological activity of this GH preparation. Electron microscopic studies showed that the anti-gcGH-I antibody specifically reacted with GH localized in the secretory granules of the goldfish somatotroph. Using anti-gcGH-I in a dot-blot assay, it was found that in vivo implantation of solid silastic pellets containing E(2) (100 mu g/g body weight for 5 days) increased pituitary GH content by 150% in female goldfish. In a second, independent study employing a previously characterized anticommon carp GH antibody for radioimmunoassay, it was found that E(2) increased pituitary GH content by 170% and serum GH levels by approximately 350%. The E(2)-induced hypersecretion of GH and increase in pituitary GH levels was not associated with changes in steady-state pituitary GH mRNA levels, suggesting that this sex steroid may enhance GH synthesis at the posttranscriptional or translational level. Previous observations indicate that GH can stimulate ovarian E(2) production. The present results show that E(2) can in turn stimulate GH production, indicating the existence of a novel pituitary GH-ovarian feedback system in goldfish. (C) 1997 Academic Press.

Alteration of metallothionein mRNA in bay scallop Argopecten irradians under cadmium exposure and bacteria challenge

Metallothionein (MT) is a superfamily of cysteine-rich proteins contributing to metal metabolism, detoxification of heavy metals, and immune response such as protecting against ionizing radiation and antioxidant defense. A metallothionein (designated AiMT2) gene was identified and cloned from bay scallop, Argopecten irradians. The full length cDNA of AiMT2 consisted of an open reading frame (ORF) of 333 bp encoding a protein of 110 amino acids. with nine characteristic Cys-X-Cys, five Cys-X-X-Cys, five Cys-X-X-X-Cys and two Cys-Cys motif arrangements and a conserved structural pattern Cys-x-Cys-x(3)-Cys-Tyr-x(3)Cys-x-Cys-x(3)-Cys-x-Cys-Arg at the C-terminus. The cloned ANT showed about 50% identity in the deduced amino acid sequence with previously published MT sequences of mussels and oysters. The conserved structural pattern and the close phylogenetic relationship of AiMT2 shared with MTs from other mollusc especially bivalves indicated that AiMT2 was a new member of molluscan MT family. The mRNA transcripts in hemolymph of AiMT2 under cadmium (Cd) exposure and bacteria challenge were examined by real-time RT-PCR. The mRNA expression of AiMT2 was up-regulated to 3.99-fold at 2 h after Listonella anguillarum challenge, and increased drastically to 66.12-fold and 126.96-fold at 16 and 32 h post-challenge respectively. Cadmium ion exposure could induce the expression of AiMT2, and the expression level increased 2.56-fold and 6.91-fold in hemolymph respectively after a 10-day exposure of 100 mu g L-1 and 200 mu g L-1 CdCl2. The sensitivity of AiMT2 to bacteria challenge and cadmium stress indicated it was a new Cd-dependent MT in bay scallop and also regulated by an immune challenge. The changes in the expression of AiMT2 could be used as an indicator of exposure to metals in pollution monitoring programs and oxidative stress, and bay scallop as a potential sentinel organism for the cadmium contamination in aquatic environment. (C) 2008 Elsevier Inc. All rights reserved.

Ergosterimide, a new natural Diels-Alder adduct of a steroid and maleimide in the fungus Aspergillus niger

Ergosterimide (1), a natural Diels-Alder adduct of ergosteroid and maleimide, was characterized from the culture extract of Aspergillus niger EN-13, an endophytic fungus isolated from the marine brown alga Colpomenia sinuosa. In addition, four known steroids including (22E,24R)-ergosta-5,7,22-trien-3 beta-ol (2), (22E,24R)-ergosta-4,6,8(14),22-tetraen-3one (3), (22E,24R)-5 alpha,8 alpha-epidioxyergosta-6,22-dien-3 beta-ol (4), and (22E,24R)-ergosta-7,22dien-3 beta,5 alpha,6 beta-triol. (5) were also isolated and identified. The structures of these compounds were elucidated by extensive analysis of 1D and 2D NMR and IR spectra and MS data. The plausible biosynthetic pathway of 1 was also discussed. To the best of our knowledge, 1 is the first natural Diels-Alder adduct of steroid and maleimide reported so far. (C) 2007 Elsevier Inc. All rights reserved.