Waterborne exposure to fluorotelomer alcohol 6:2 FTOH alters plasma sex hormone and gene transcription in the hypothalamic-pituitary-gonadal (HPG) axis of zebrafish

Fluorotelomer alcohols (FTOHs) have shown estrogenic activity in vitro and in vivo, but the mechanism of this activity is not known. In this study, 18-week-old zebrafish (Danio rerio) were exposed to 0, 0.03, 0.3 and 3.0 mg/l 1H, 1H, 2H, 2H-perfluorooctan-1-ol (6:2 ETCH) for 7 days, and the effects on plasma sex hormone levels were measured followed by use of real-time PCR to examine selected gene expression in hypothalamic-pituitary-gonadal (HPG) axis and liver. Exposure to 6:2 FTOH significantly increased plasma estradiol (E2) and testosterone (T) levels in both males and females. Furthermore, the ratio of T/E2 was reduced in females while increased in males. In females, the increase of E2 was accompanied by up-regulated hepatic estrogenic receptor alpha (ER alpha) and vitellogenin (VTG1 and VTG3) expression. In males, the elevation of the T level is consistent with the up-regulation of cytochrome P450 c17 alpha-hydroxylase, 17, 20-lase (CYP17) and the down-regulation of cytochrome P450 aromatase A (CYP19A). The present study demonstrated that waterborne exposure to 6:2 FTOH alter plasma sex hormone levels and the ratio of T/E2, as well as the transcriptional profiles of some genes in the HPG axis and liver. The results suggested that FTOHs may disturb fish reproduction through endocrine disrupted activity. (C) 2009 Elsevier B.V. All rights reserved.

8～12岁男孩的性激素与智力、人格的关系

Recently there emerged more and more researhes concerning about sex hormone-related cognitive ability and behaviours. Few were carried out in pre- to early adolescent children., and the objective of the current study is to investigate whether there are covariance between sex hormones, intelligence and personality in 232 pre- to early adolescent boys, including 62 gifted boys. Indexes of sex hormone levels were salivary testosterone and estradiol concentrations, the 2D:4D digit ratio ( a reliable pointer of prenatal sex hormone concentraions). The Cattell Culture Fair Intelligence Test and a Chinese version of Children’s Personality Questionaire was applied in the current study. The main findings are: 1) salivary sex hormone concentrations significantly positively correlated with intelligence performance in 10-year-old boys; 2) salivary testosterone negatively related to intelligence performance in 12-year-old boys; 3) gifted boys bears lower testosterone concentrations in both prenatal period and pre- to early adolescence; 4) for personality, higher salivary estradiol was related to extraversion and digit ratios correlated with several personality factors in 8-year-old boys. In conclusion, results in the current study suggested that for male early maturers, intelligence may be negatively influenced by early coming androgen surge. In contrast, male late matures may benefit from their lately and moderately increasing hormone. Besides, the results suggested that the relationship between 2D:4D digit ratio and personality may also be paid attention to.

Enzyme-linked immunosorbent assay of changes in serum levels of growth hormone (cGH) in common carps (Cyprinus carpio)

The aim of the present study was to purify the common native carp growth hormone (ncGH), produce monoclonal antibodies (mAbs) to common native carp growth hormone (ncGH), and further enhance the sensitivity of enzyme-linked immunosorbent assays (ELISA) for ncGH. Additionally, we investigated changes in serum ncGH levels in carps raised in different environmental conditions. The recombinant grass carp (Ctenopharyngodon idella) growth hormone was purified and used as antigen to immunize the rabbit. The natural ncGH was isolated from the pituitaries of common carp. SDS-PAGE and Western blot utilizing the polyclonal anti-rgcGH antibody confirmed the purification of ncGH from pituitaries. Purified ncGH was then used as an immunogen in the B lymphocyte hybridoma technique. A total of 14 hybridoma cell lines (FMU-cGH 1-14) were established that were able to stably secrete mAbs against ncGH. Among them, eight clones (FMU-cGH1-6, 12 and 13) were successfully used for Western blot while nine clones (FMU-cGH 1-7, 9 and 10) were used in fluorescent staining and immunohistochemistry. Epitope mapping by competitive ELISA demonstrated that these mAbs recognized five different epitopes. A sensitive sandwich ELISA for detection of ncGH was developed using FMU-cGH12 as the coating mAb and FMU-cGH6 as the enzyme labeled mAb. This detection system was found to be highly stable and sensitive, with detection levels of 70 pg/mL. Additionally, we found that serum ncGH levels in restricted food group and in the net cage group increased 6.9-and 5.8-fold, respectively, when compared to controls, demonstrating differences in the GH stress response in common carp under different living conditions.

Whole-body amino acid pattern of F-4 human growth hormone gene-transgenic red common carp (Cyprinus carpio) fed diets with different protein levels

F-4 generation of human growth hormone (hGH) gene-transgenic red common carp, and the non-transgenic controls were fed for 8 weeks on purified diets with 20%, 30% or 40% protein. Analysis of whole-body amino acids showed that the proportions of lysine, leucine, phenylalanine, valine and alanine, as percentages of body protein, increased significantly, while those of arginine, glutamic acid and tyrosine decreased, with increases in dietary protein level in at least one strain of fish. Proportions of the other amino acids were unaffected by the diets. The proportions of lysine and arginine were significantly higher, while those of leucine and alanine were lower in the transgenics than in the controls in at least one diet group. Proportions of the other amino acids were unaffected by strain. The results suggest that the whole-body amino acid profile of transgenic carp, when expressed as proportions of body protein, was in general, similar to that of the non-transgenic controls. (C) 2000 Elsevier Science B.V. All rights reserved.

Growth and feed utilization by F-4 human growth hormone transgenic carp fed diets with different protein levels

The F-4 generation of human growth hormone (hGH) transgenic red common carp Cyprinus carpio had significantly higher growth rates than the non-transgenic controls. Protein and energy intakes were significantly higher in the transgenic carp than in the controls fed the 20% protein diet, but were not different between the two strains fed diets with 30 and 40% protein. Faecal protein loss, as a proportion of protein intake, was significantly lower in the transgenics than in the controls fed diets with 20 and 30% protein, but was not different between the two strains Fed diet with 40% protein. Faecal energy loss, as a proportion of energy intake, was significantly lower in the transgenics than in the controls fed diet with 20% protein, but was not different between the two strains fed diets with 30 and 40% protein. Recovered protein, as a proportion of protein intake, was significantly higher in the transgenics than in the controls fed all diets, whereas recovered energy was significantly higher in the transgenic fish fed the 40% protein diet. For fish fed each diet, the transgenics had significantly higher body contents of dry matter and protein, but lower contents of lipid than the controls. It was concluded that transgenics were more efficient in utilizing dietary protein than the controls. it a lower dietary protein level; transgenics achieved higher growth rates mainly by increasing feed intake; at higher levels of dietary protein, transgenics achieved higher growth rates mainly through a higher energy conversion efficiency. (C) 1998 The Fisheries Society of the British Isles.

A one-year investigation of the relationship between serum GH levels and the growth of F-4 transgenic and non-transgenic common carp Cyprinus carpio

It has been demonstrated that growth hormone (GH) transgenic fish often posses a trait for fast growth. Here, we investigated the growth of F-4 'all-fish' GH transgenic carp Cyprinus carpio and their serum GH levels for a year. The results showed that F-4 all-fish GH transgenic carp were significantly larger in body mass (c. two-fold, P < 0 center dot 001) and body length (c. 1 center dot 3 fold, P < 0 center dot 001), compared with the non-transgenic group. The discrepancy of serum GH levels between the transgenic carp group and control group is 54 fold, when the water temperature was 12-34 degrees C. When the water temperature decreased to 3 center dot 5 degrees C in January, the discrepancy was 256 fold. The serum GH level of the transgenic group was relatively constant, while that of control varied greatly based on month and water temperature. The changes of growth rates between the transgenic group and the control group were similar for a year. Taken together, the results indicated that F-4 all-fish GH transgenic carp had not only higher and constant serum GH levels but also a significant fast-growing effect, compared with the control. To our knowledge, this is the first report on a one-year investigation of growth trait and serum growth hormone level in F-4 all-fish GH transgenic carp.

Molecular cloning of growth hormone receptor (GHR) from common carp (Cyprinus carpio L.) and identification of its two forms of mRNA transcripts

The cDNA of growth hormone receptor (GHR) was cloned from the liver of 2-year common carp (Cyprinus carpio L.) by reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA end (RACE). Its open reading frame (ORF) of 1806 nucleotides is translated into a putative peptide of 602 amino acids, including an extracellular ligand-binding domain of 244 amino acids (aa), a single transmembrane domain of 24 aa and an intracellular signal-transduction domain of 334 aa. Sequence analysis indicated that common carp GHR is highly homologous to goldfish (Carassius auratus) GHR at both gene and protein levels. Using a pair of gene-specific primers, a GHR fragment was amplified from the cDNA of 2-year common carp, a 224 bp product was identified in liver and a 321 bp product in other tissues. The sequencing of the products and the partial genomic DNA indicated that the difference in product size was the result of a 97 bp intron that alternatively spliced. In addition, the 321 bp fragment could be amplified from all the tissues of 4-month common carp including liver, demonstrating the occurrence of the alternative splicing of this intron during the development of common carp. Moreover, a semi-quantitative RT-PCR was performed to analyze the expression level of GHR in tissues of 2-year common carp and 4-month common carp. The result revealed that in the tissues of gill, thymus and brain, the expression level of GHR in 2-year common carp was significantly tower than that of 4-month common carp.

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.

Osmoregulatory actions of growth hormone in juvenile tilapia (Oreochromis niloticus)

Growth hormone (GH) effectively promotes seawater (SW) adaptation in salmonids, but little is known of its effect in tilapias. Experiments were performed to investigate the effects of recombinant eel GH (reGH) on osmoregulatory actions and ultrastructural features of gill chloride cells in juvenile tilapia, Oreochromis niloticus. Tilapia showed a markedly improved SW survival, when directly transferred from freshwater (FW) to 62.5% SW 24h after a single reGH injection (0.25 or 2.5 mu g g(-1)) or 3 reGH injections (0.25 mu g g(-1) every other day). Plasma Na+ and Mg2+ levels were significantly reduced by reGH (0.25 and 2.5 mu g g(-1)) compared with saline injections; Ca2+ concentrations were reduced significantly by high dose of reGH (2.5 mu g g(-1)) after SW transfer. However, fish failed to survive more than 24h when directly transferred to 70 % SW, although the fish treated with reGH could survive longer than the controls. When examined by electron microscopy, the chloride cells were identified as mitochondrion-rich and an extensive tubular system was induced by GH treatment. The results of the present study suggest that, similar to its effect on salmonids, GH also exerts acute osmoregulatory actions and enhances SW adaptation in juvenile tilapia. GH also stimulates the differentiation of chloride cells toward SW adaptation.

Effect of parental stock size on F-1 genetic structure in the bay scallop, Argopecten irradians (Lamarck, 1819)

Three F-1 families of the bay scallop, Argopecten irradians, were produced from one, two and 10 individuals. The genetic changes in these populations, which suffered recent and different levels of bottleneck, were analysed using amplified fragment length polymorphism (AFLP) techniques. In the parental stock, a total of 330 bands were detected using seven AFLP primer pairs, and 70% of the loci were polymorphic. All F-1 groups had a significantly lower proportion of polymorphic loci when compared with the initial stock, and loss of the rare loci and reduction in heterozygosity both occurred. The progeny of the larger population (i.e., N=10) exhibited a lesser amount of genetic differentiation compared with the progeny from N=2, which showed lesser differentiation than progeny from N=1. The effective population sizes (N-e) in N=1, 2 and 10 were estimated as 1.50, 1.61 and 2.49. Based on regression analysis, we recommend that at least 340 individuals be used in hatchery populations to maintain genetic variation.

Statistical analysis of synthetic earthquake catalogs generated by models with various levels of fault zone disorder

The stress release model, a stochastic version of the elastic rebound theory, is applied to the large events from four synthetic earthquake catalogs generated by models with various levels of disorder in distribution of fault zone strength (Ben-Zion, 1996) They include models with uniform properties (U), a Parkfield-type asperity (A), fractal brittle properties (F), and multi-size-scale heterogeneities (M). The results show that the degree of regularity or predictability in the assumed fault properties, based on both the Akaike information criterion and simulations, follows the order U, F, A, and M, which is in good agreement with that obtained by pattern recognition techniques applied to the full set of synthetic data. Data simulated from the best fitting stress release models reproduce, both visually and in distributional terms, the main features of the original catalogs. The differences in character and the quality of prediction between the four cases are shown to be dependent on two main aspects: the parameter controlling the sensitivity to departures from the mean stress level and the frequency-magnitude distribution, which differs substantially between the four cases. In particular, it is shown that the predictability of the data is strongly affected by the form of frequency-magnitude distribution, being greatly reduced if a pure Gutenburg-Richter form is assumed to hold out to high magnitudes.

Fatigue crack-growth from a circular notch under high-levels of biaxial stress

A series of experiments have been conducted on cruciform specimens to investigate fatigue crack growth from circular notches under high levels of biaxial stress. Two stress levels (Δσ₁= 380 and 560 MPa) and five stress biaxialities (λ=+1.0, +0.5, 0, −0.5 and −1.0; where λ=σ₂/σ₁ were adopted in the fatigue tests in type 316 stainless steel having a monotonic yield strength of 243 MPa. The results reveal that fatigue crack growth rates are markedly influenced by both the stress amplitude and the stress biaxiality. A modified model has been developed to describe fatigue crack growth under high levels of biaxial stress.