Genomic cloning and expression of vitellogenin gene from the self-fertilizing fish Rivulus marmoratus (Cyprinodontiformes, Rivulidae)

We cloned the vitellogenin gene from the self-fertilizing fish Rivulus marmoratus, and sequenced 12,326 bp. The number of exons of R. marmoratus and rainbow trout vitellogenin genes were different, and also the splicing junctions are different throughout most of the exons and introns but the amino acid similarity of R. marmoratus vitellogenin gene to other species was rather high. In promoter region of R. marmoratus vitellogenin gene, there were several E2 binding sites and the estrogen response element (ERE). We discuss here the gene structure and expression of R. marmoratus vitellogenin gene.     

Cloning and sequence analysis of the self-fertilizing fish Rivulus marmoratus immediate early gene c-fos

We have cloned the proto-oncogene c-fos from a self-fertilizing fish Rivulus marmoratus (Cyprinodontiformes, Rivulidae) after screening of R. marmoratus lambdaGEM-11 genomic DNA library, and sequenced over 12 kb including all exons, introns and the promoter region. The R. marmoratus c-fos gene consisted of one noncoding exon and four exons with high similarity to those of fugu and mammals. We sequenced approximately 7 kb of the R. marmoratus c-fos gene promoter region to gain a better understanding of the molecular anatomy of the immediate response of this gene upon cellular damage. In the promoter region, R. marmoratus c-fos gene has seven xenobiotic response elements (XREs) and eight metal response elements (MREs) as well as two estradiol (E2), 4 NFkappaB, 2 CarG, 2 prolactin (PRL) motifs and one pit1 site, while the 3'-UTR of this gene contains the estrogen response element (ERE). The seven XRE and eight MRE motifs raise the possibility of its regulation by exposure to environmental pollutants. In this paper, we discuss the gene structure of R. marmoratus c-fos gene and compare its promoter region with those of other organisms' c-fos genes. We propose its potential use in ecotoxicology.     

Cloning of cytochrome P450 1A (CYP1A) genes from the hermaphrodite fish Rivulus marmoratus and the Japanese medaka Oryzias latipes

To use two small fish Rivulus marmoratus (Cyprinodontiformes, Rivulidae) and the Japanese medaka Oryzias latipes (Belloniformes) as testing models in molecular ecotoxicology, we have cloned the cytochrome P450 1A (CYP1A) gene after screening of both genomic DNA libraries, and sequenced 11,863 and 7,243 bp including all the exons and introns with promoter regions, respectively. The Rivulus and the medaka CYP1A gene consisted of seven exons (including non-coding exons) with high homology to mammals. In the promoter region, Rivulus CYP1A gene has seven xenobiotic response elements (XREs) and two metal response elements (MREs), while the Japanese medaka CYP1A gene has six XREs and four MREs. Interestingly, medaka CYP1A gene has a number of MREs at the promoter, which may affect its response on metal exposure. We describe here the gene structure of both fish CYP1A genes.     

Cloning and sequence analysis of the self-fertilizing fish Rivulus marmoratus immediate early gene c-fos

We have cloned the proto-oncogene c-fos from a self-fertilizing fish Rivulus marmoratus (Cyprinodontiformes, Rivulidae) after screening of R. marmoratus λGEM-11 genomic DNA library, and sequenced over 12 kb including all exons, introns and the promoter region. The R. marmoratus c-fos gene consisted of one noncoding exon and four exons with high similarity to those of fugu and mammals. We sequenced ≈7 kb of the R. marmoratus c-fos gene promoter region to gain a better understanding of the molecular anatomy of the immediate response of this gene upon cellular damage. In the promoter region, R. marmoratus c-fos gene has seven xenobiotic response elements (XREs) and eight metal response elements (MREs) as well as two estradiol (E2), 4 NFκB, 2 CarG, 2 prolactin (PRL) motifs and one pit1 site, while the 3^′-UTR of this gene contains the estrogen response element (ERE). The seven XRE and eight MRE motifs raise the possibility of its regulation by exposure to environmental pollutants. In this paper, we discuss the gene structure of R. marmoratus c-fos gene and compare its promoter region with those of other organisms' c-fos genes. We propose its potential use in ecotoxicology.     

3种鲈形目鱼类精子发生早期的超微结构研究

采用透射电镜技术,研究了3种鲈形目鱼类褐菖鲉Sebastiscus marmoratus、黑鲷Sparus macrocephalus及平鲷Rhabdosargus sarba(Forskal)精子发生早期精原细胞、初级精母细胞及次级精母细胞的超显微结构变化。结果表明,3种鱼生精细胞形态结构及代谢活动变化主要体现在核的变化、线粒体数目与结构的变化及高尔基体和溶酶体的行为变化。3种鱼核的变化基本一致:细胞核由近圆形或椭圆形转变为多角形;核质逐渐浓缩;核仁从明显结构至完全消失。褐菖鲉精原细胞时期线粒体分布较多,并出现拟染色质与线粒体相粘附;而平鲷与黑鲷精原细胞时期的线粒体分布较少,无拟染色质。3种鱼高尔基体与溶酶体的变化也有相似性。黑鲷的初级精母细胞中还观察到典型的环层状的髓样小体的结构。     

褐菖鲉仔、稚鱼生长特性及其关键变态期的研究

根据1991～1994年间褐菖YouSebastiscus marmoratus人工苗种培育中仔、稚鱼生物学测定资料及其关键变态期出现时间、特征等资料，研究了日龄与褐菖You仔、稚鱼生长参数间的相互关系，分析了褐菖You仔、稚鱼阶段的关键变态期的出现规律及其生长参数间的关系，探讨了褐褐菖You仔、稚鱼关键变态期的发生机理及其应对措施。     

Development of hepatic CYP1A and blood vitellogenin in eel (Anguilla anguilla) for use as biomarkers in the Thames Estuary, UK.

The potential of eel (Anguilla anguilla) as a monitoring species for the Thames Estuary, UK, was examined. Hepatic cytochrome P4501A [7-ethoxyresorufin O-deethylase (EROD) activity] and blood vitellogenin (Western analysis) were investigated as biomarkers of exposure to, respectively, organic contaminants and to contaminants showing estrogenic activity. Hepatic microsomal EROD activities in A. anguilla from seven sites in the Thames Estuary in May 1998 varied three-fold (111 +/- 24 to 355 +/- 42 pmol min-1 mg protein-1) (mean +/- S.E.M.) and showed correlation with salinity; however, the latter relationship was not maintained at other times of the year. The range of EROD activities was two- to eight-fold higher than the 37 +/- 8 pmol min-1 mg-1 for A. anguilla from the relatively clean Tamar Estuary. beta-Naphthoflavone treatment (5 mg kg-1 wet wt.; 2 days) of Thames A. anguilla produced a two-fold increase in hepatic microsomal EROD activity. Comparing the Thames EROD data with those for A. anguilla from well-characterised contaminated sites in the Netherlands (Van der Oost, R., Goks?yr, A., Celander, M., Heida, H., & Vermeulen, N. P. E. 1996. Aquatic Toxicology, 36, 189-222), the Thames is suggested to be moderately impacted by polycyclic aromatic hydrocarbons and related contaminants. 17-beta-Estradiol treatment produced the appearance of a plasma protein of 211 Kd app. mol. wt. (recognised by antibodies to vitellogenin of Morone saxatilis), but putative vitellogenin could not be detected in A. anguilla from selected sites in the Thames Estuary.     

Array technology as a tool to monitor exposure of fish to xenoestrogens

A variety of anthropogenic chemicals are capable of binding to the estrogen receptor of vertebrate species. Binding of these compounds can interfere with homeostasis by disrupting normal gene expression patterns. The purpose of this study was to investigate the feasibility of applying array technology as a monitoring tool for detecting the presence and distribution of estrogenic compounds in coastal habitats using sheepshead minnows as our model. cDNA clones that were isolated from differential display, including vitellogenin alpha and beta, vitelline envelope protein (ZP2), and transferrin, among others, were spotted on the macroarray. The results of these experiments demonstrate a characteristic expression pattern of estrogen responsive genes in sheepshead minnows exposed to 17 beta-estradiol (E2).     

Kinetics of vitellogenin protein and mRNA induction and depuration in fish following laboratory and environmental exposure to oestrogens

Plaice, flounder and sand goby were exposed to ethynylestradiol (EE2) for 21 days and then followed for up to 31 days after removal of the oestrogen. Plasma vitellogenin (VTG) and hepatic VTG mRNA were determined in groups of fish sampled during the induction and post-exposure phases. VTG mRNA increased slightly earlier than plasma protein, but both reached maxima by 21 days. In contrast, VTG mRNA decayed much more rapidly than protein after EE2 exposure was terminated (typical values t(1/2) mRNA 3 days, protein 15-30 days). Vitellogenin and VTG mRNA thus measure different temporal events and this is illustrated by field data of male flounder in which both parameters have been determined. Few fish show co-ordinate increased VTG mRNA and vitellogenin but rather more fish have increased vitellogenin. Low level increases of VTG mRNA (5 X) is observed in some fish without increased vitellogenin and this may represent polymorphic differences between individual fish.     

The effect of estrogen on cadmium distribution in rainbow trout (Oncorhynchus mykiss)

The effects of injections of 17β-estradiol (E2) and Cd, on the distribution of Cd and the induction of metallothionein (MT) mRNA and vitellogenin mRNA was investigated. Bone and liver were the main organs accumulating Cd. However, E2 redirected the metal accumulation from the bone and liver to the gill, gut and muscle upon exposure to E2. Cd did not induce the hepatic MT mRNA levels in animals treated with E2. The VTG mRNA levels were also reduced following co-injection of E2 and Cd. However, the kidney responded to Cd exposure by upregulating MT mRNA even in the presence of E2 treatment. In the liver the reduced MT mRNA induction led to a redistribution of CD from MT to non-MT proteins. The toxicological significance of these alterations in Cd handling remains to be determined.     

Effects of an environmental estrogen, 17alpha-ethinyl-estradiol,on the maternal-fetal trophic relationship in the eelpout Zoarces viviparus (L)

In the present experiment pregnant eelpout Zoarces viviparus females were exposed to 0, 5, 10, 50, 500 ng/l of the synthetic estrogen ethinylestradiol (EE2) and to 500 ng/l estradiol-17beta (E2) under flow-through conditions during 3 weeks and the maternal-fetal trophic response was investigated. The circulating yolk-precursor protein vitellogenin, measured by ELISA, increased from a mean control value of 0.017-36 mg/ml in the plasma of the motherfish exposed to the highest concentration of EE2 This increase in vitellogenin was also depicted by a 288% increase in circulating calcium levels. During pregnancy the ovary represents a new route of calcium loss from the maternal blood for the growth of the embryos. However, a significant decrease (120% in the group exposed to the highest concentration of EE2) in the calcium level in the ambient medium of the embryos, the ovarian fluid, was observed concomitant with the increase in maternal plasma calcium in the EE2-exposed females. In contrast, the level of circulating amino acids decreased in the maternal blood, with a slight concomitant increase in the ovarian fluid of the exposed fish. These and other metabolic observations indicate that exposure to different doses of EE2 and to estradiol-17beta affects the maternal-fetal trophic relationship.     

线纹尖塘鳢的形态特征、染色体核型及分类学地位

以1996年由澳大利亚Fiyzroy河水系引进的野生线纹尖塘鳢幼鱼群体和经池塘驯养、混合选育、繁殖多代的幼成鱼为材料,进行形态生物学的观测、分类地位研究;采用PHA腹腔注射、肾细胞直接制片法,进行染色体核型分析。结果表明,线纹尖塘鳢(Oxyeleotris lineolatus)与云斑尖塘鳢(O. marmoratus)是尖塘鳢属中亲缘关系最接近的单系类群,完成了其系统分类学的定位,支持将尖塘鳢从塘鳢属中分出的传统分类处理,从细胞学角度,证明传统分类的正确性。提出两种尖塘鳢种间杂交可能性很大,与塘鳢属、乌塘鳢属和沙塘鳢属间杂交的可能性也存在。     

云斑尖塘鳢人工育苗技术初步研究

对云斑尖塘鳢（Oxyeleotris marmoratus （Bleeker））人工育苗技术进行了初步的研究,利用燃油热水炉加温进行亲鱼越冬,采取人工辅助控温、保温的方法,配合其生态条件的调控和投喂优质饲料,培育亲鱼成熟并能自然产卵,使用人工饵料、代用饵料、生物饵料等混合饵料互相搭配组合,经过2个月左右的培育鱼苗全长达3cm左右。亲鱼具多次产卵习性,观察到一年当中最多产卵4次。利用外源激素催产率达到95．0％,自然产卵的受精率和孵化率均高于人工催产。     

Uptake of estradiol from sediment by hornyhead turbot (Pleuronichthys verticalis) and effects on oxidative DNA damage in male gonads

Male and female hornyhead turbot (Pleuronichthys verticalis) were exposed to four concentrations (0, 0.75, 14.7 and 46.5 ng/g dry weight) of E2-amended sediment for 7 days. Sediment-derived E2 was bioavailable to the flatfish, though the route of uptake was unclear. A concentration of 46.5 ng/g E2 in sediment led to a significant increase in vitellogenin concentrations in the plasma in both sexes after 7 days of exposure. Though plasma E2 concentrations increased significantly in males at sediment E2 concentrations of 0.75 ng/g dry weight and above, a dose-dependent increase was not observed. There was also no correlation between sediment E2 concentrations, plasma E2 concentrations, and oxidative DNA damage in male gonads. The results suggest that the DNA damage previously seen in the gonads of feral hornyhead turbot at a sewage outfall is likely not caused by acute exposure to exogenous E2 from sediments.     

瓦氏黄颡鱼(Pelteobagrus vachelli)卵黄蛋白原的纯化、性质鉴定及ELISA检测方法的建立

腹腔注射17β-雌二醇(E2),使瓦氏黄颡鱼雄鱼在7天内产生卵黄蛋白原(Vtg)。采用凝胶过滤和离子交换两种层析技术,从E2诱导的雄性瓦氏黄颡鱼血浆中分离、纯化出Vtg,采用糖、磷、脂蛋白染色技术证明分离、纯化的蛋白为Vtg,该Vtg在非变性条件下分子量约为240kDa,在SDS变性条件下分子量约为143kDa。纯化的瓦氏黄颡鱼Vtg经检测显示可能含有类胡萝卜素,但没有二硫键,对热相对稳定。利用纯化的瓦氏黄颡鱼Vtg,制备了兔抗瓦氏黄颡鱼Vtg多克隆抗血清。用双向免疫扩散法测得抗血清的纯度较高,效价为1︰32;Western blotting检测显示抗血清的特异性较好。以瓦氏黄颡鱼Vtg多克隆抗血清为抗体,以纯化的瓦氏黄颡鱼Vtg为抗原,建立了间接酶联免疫吸附反应(ELISA)方法检测瓦氏黄颡鱼体内Vtg的含量,标准曲线线性部分的线性方程为y=0.099x+0.4529(R2=0.9327),该方法检测的灵敏度为15.6ng/ml,工作范围为31.2—4000ng/ml,在此范围内,标准曲线具有良好的线性。     

Cloning of cytochrome P450 1A (CYP1A) genes from the hermaphrodite fish Rivulus marmoratus and the Japanese medaka Oryzias latipes

To use two small fish Rivulus marmoratus (Cyprinodontiformes, Rivulidae) and the Japanese medaka Oryzias latipes (Belloniformes) as testing models in molecular ecotoxicology, we have cloned the cytochrome P450 1A (CYP1A) gene after screening of both genomic DNA libraries, and sequenced 11,863 and 7,243 bp including all the exons and introns with promoter regions, respectively. The Rivulus and the medaka CYP1A gene consisted of seven exons (including non-coding exons) with high homology to mammals. In the promoter region, Rivulus CYP1A gene has seven xenobiotic response elements (XREs) and two metal response elements (MREs), while the Japanese medaka CYP1A gene has six XREs and four MREs. Interestingly, medaka CYP1A gene has a number of MREs at the promoter, which may affect its response on metal exposure. We describe here the gene structure of both fish CYP1A genes.     

Comparative vitellogenic responses in three teleost species: extrapolation to in situ field studies.

Induction of vitellogenin (VTG) was compared among three teleostean species to determine their relative sensitivity of exposure to 17 beta-estradiol (E2). Japanese medaka (Oryzias latipes), sunshine bass (Morone saxatalis x Morone chrysops) and channel catfish (Ictalurus punctatus) were exposed to aqueous concentrations of E2 ranging from 10 to 100,000 ng/l for 21 days. Respective EC50 values for plasma VTG detected by western blot in medaka, catfish and bass were 200, 170 and 1560 ng E2/l. Since these EC50 values are based on VTG induction curves calculated relative to control values, they indicate differences in species' sensitivity to E2 exposure. Catfish and bass VTG responses obtained in laboratory exposures were compared to VTG responses previously observed with 21-day wastewater treatment plant effluent exposures. Plasma VTG induction in effluent-exposed fish ranged from 14 to 82% above reference values depending on species. Extrapolation of field responses with laboratory-exposed fish indicate catfish and bass were exposed to the equivalent of 27-240 ng E2/l in sewage effluent.     

Exposure to environmentally relevant concentrations of different nonylphenol formulations in Japanese medaka.

The time course of exposure to p-nonylphenol (NP) from two different sources was compared to equivalent exposures of 17-beta-estradiol (E2) and a solvent control (ethanol; EtOH). Japanese medaka were exposed for 4 days to a nominal concentration of 20 micrograms/l of either NP-I (Schenectady International, Inc.), NP-II (Aldrich), or E2, and were then placed in untreated water for 5 days. Tissue samples were taken at two time points during the 4-day exposure and two time points during the 5 days following exposure. Liver homogenates were analyzed using a western blot to detect vitellogenin (VTG) and quantified by measuring the optical density for each lane. Preliminary results indicate that E2 significantly increased VTG staining above the level observed in EtOH-treated controls for both males and females. A two-way analysis of variance (ANOVA) indicates that NP from both sources, as well as E2, significantly increased VTG staining in males (ANOVA, n = 48, P < 0.001; Tukey pairwise tests, all P < 0.008). A significant increase in VTG was observed in E2-treated males and females the first day following transfer into toxicant-free water (two-way ANOVAs, both n = 48, P < 0.003; Tukey pairwise tests, all P < 0.019). If confirmed, this extended response observed for low-level exposures may represent a significant factor for sampling scenarios following pulsitile exposure.     

Preliminary investigations on vitellogenin m-RNA induction in some bioindicator Mediterranean fish species

This paper describes the development of a RT-PCR method for assaying Vtg gene expression in different marine fish as a potentially valuable and sensitive biomarker of exposure to estrogenic chemicals. The levels of Vtg mRNA have been analyzed using primers specifically designed for the various species and the procedures have been standardized relative to actine mRNA expression levels. Different species were analyzed including organisms with a great potential as bioindicators in the Mediterranean (i.e. the red mullet Mullus barbatus, the striped mullet Mugil cephalus, the European eel Anguilla anguilla) or exposed to biomagnification of halogenated hydrocarbons and with elevated commercial value (the bluefin tuna Thunnus thynnus). The analysis of vitellogenin mRNA levels has been standardized in feral fish providing suitable indications for a future development of this approach.     

Influence of cadmium exposure on plasma calcium, vitellogenin and calcitonin in vitellogenic rainbow trout

Effects of cadmium exposure on plasma levels of calcitonin and free and protein-bound calcium were studied in vitellogenic female rainbow trout kept in brackish water (7%(.)). Fish were exposed to 100 μg cadmium litre⁻¹ for four weeks. Exposure of female rainbow trout in the stage of vitellogenesis, with increased total plasma levels of calcium, resulted in a complex hypocalcemic response. Thus, hypocalcemia was found to be due to three different processes: (1) a decrease in the free plasma calcium, and a reduction in protein-bound calcium; due both to (2) decreased plasma levels of vitellogenin; and (3) a reduced binding of calcium to vitellogenin. These findings support the concept of an interference of cadmium with ionregulating tissues as a mechanism for hypocalcemia in rainbow trout. A direct effect on the vitellogenin-binding of calcium was also observed and reproductive function in the females was affected by decreased plasma levels of vitellogenin. In spite of the marked changes of plasma calcium in exposed fish, no significant effects on plasma calcitonin were observed, indicating a lack of a direct relationship between plasma calcium and calcitonin levels in rainbow trout.