三丁基锡对褐菖鮋精巢性激素受体表达的影响

三丁基锡(Tributyltin,TBT)能够影响鱼类生殖功能,而有关TBT对鱼类生殖细胞发育至关重要的雄激素和雌激素受体的影响还未见报道。本实验以海洋经济鱼类褐菖鲉(Sebastiscus marmoratus)为对象,研究环境水平TBT(1,10和100ng/L)对精巢雄激素和雌激素受体表达的影响。结果表明,TBT暴露能够抑制褐菖鲉精巢发育和精巢雌激素受体蛋白的水平,并且发现雌激素受体主要定位于精巢精小叶腔间隙。小叶腔间隙的雌激素受体蛋白水平降低可能影响支持细胞和间隙细胞的正常功能,从而影响精巢的正常发育。TBT暴露对雄激素受体的表达并没有显著的影响。该结果为全面了解TBT产生鱼类生殖毒性效应、途径和机制提供了新的重要的资料。     

17β-Estradiol induces changes in cytokine levels in head kidney and blood of juvenile sea bass (Dicentrarchus labrax,L., 1758)

The cytokine network is involved in the immune system communication. As estrogens influence the cytokine expression in mammals, this study investigated the impact of exogenous estrogenic pollutants on selected cytokines in Dicentrarchus labrax. The gene expression of Interleukin 6, Tumour Necrosis Factor α, Transforming Growth Factor β1 and Interleukin 1β was assessed and accomplished with protein measurements in the blood for the last two. Impacts through 17β-estradiol mainly occurred at the beginning of organ regionalisation, thus falling together with a developmentally induced increase of Interleukin 1β and Tumour Necrosis Factor α gene expression in 102 dph fish. 17β-estradiol depressed this modification after 35 days of exposure and the cytokine gene expression tended to be generally down-regulated independently of the 17β-estradiol concentrations after 56 days of exposure. This impact was confirmed at the protein level, showing that 17β-estradiol affects the fine control of the cytokine network in sea bass.     

The effect of tributyltin upon steroid titres in the female dogwhelk, Nucella lapillus, and the development of imposex

Exposure of female Nucella lapillus to tributyltin (TBT) in seawater at a concentration of 40 ng Sn/litre led to accumulation of TBT in the tissues, and an increase in penis length compared to control animals. Investigation of the steroid concentrations by radioimmunoassay revealed a significant increase in testosterone after 28 days exposure to TBT. No significant effect was noted upon the amounts of progesterone or estradiol-17β. When testosterone was injected into female N. lapillus in the absence of TBT, penis length was found to increase significantly. These observation suggest that there may be an association between the change in testosterone titre in response to exposure to TBT and the development of imposex in the dogwhelk.     

Effects of tributyltin on salmon interrenal CYP11β, steroidogenic factor-1 and glucocorticoid receptor transcripts in the presence and absence of second messenger activator,forskolin

The mechanisms by which TBT produces modulations of the endocrine systems are not fully described. In this study, juvenile salmon were force-fed diet containing TBT (0: solvent control, 0.1, 1 and 10 mg/kg fish) for 72 h. Subsequently, fish exposed to solvent control and 10 mg/kg TBT were exposed to waterborne concentration of the adenyl cyclase stimulator forskolin (200 μg/L) for 2 and 4 h. Tissue and blood were sampled from individual fish (n = 6). Gene expression patterns of CYP11β, steroidogenic factor-1 (SF-1), and glucocorticoid receptor (GlucR) were determined by qPCR. TBT generally decreased mRNA levels of CYP11β, GlucR and SF-1, compared to the solvent control and these effects were differentially modulated by the presence of forskolin. This study suggests that TBT may exert broader endocrine disrupting effects through possible modulation of cAMP/PKA second messenger systems.     

Gonadal development in Japanese medaka (Oryzias latipes) exposed to 17 β-estradiol

The purpose of this research was to evaluate the Japanese medaka (Oryzias latipes) as an ecotoxicological model for the rapid evaluation of environmental estrogens. A novel short-term (48-h) exposure to 17 β-estradiol is proposed in development of a positive control for disruption of gonadal development. Recently hatched medaka fry (30 fry per dose group) with undifferentiated gonads were exposed to 4.0, 29.4, and 115.6 μg/liter of 17 β-estradiol (acetone carrier) for 48 h in a water bath at 25 °C. The fry were then grown-out in spring water for 2 weeks, killed, and processed for histological evaluation. High lethality was encountered during the grow-out period in the 115.6 μg/liter dose group. Fry in the spring water and acetone (carrier) control groups developed into females or males. Fry exposed to 17 β-estradiol developed primarily into females or had testis-ova.     

Influence of exposure history on vitellogenin induction in medaka: a case of ‘estrogen memory’?

In this study, we exposed female medaka to aqueous solutions of o,p′-DDT until tissues residues reached approximately 100 g/g. Male offspring of exposed females and unexposed males that were raised to 6 months of age differed from untreated controls in their response to subsequent 4-day static exposure to 17-β-estradiol (E2; 12 μg/l as determined by steroid EIA). Homogenized liver samples from males from DDT treatments, females from DDT treatments, control males and control females produced two vitellogenin bands on a Western blot with no qualitative differences in band size and location between the groups. To quantify the relative level of vitellogenin, 5 μg of protein from each sample was analyzed by slot blot. A statistical comparison revealed a significant difference in optical density of the slots between control males and DDT exposed males (analysis of variance, n=18, P=0.021; Tukey test, n=9, P=0.047) indicating that previous transgenerational exposure to DDT enhanced the vitellogenic response to E2 exposure in males. These results indicate that exposure to estrogenic chemicals may produce an ‘estrogen memory’ which potentiates the response to a subsequent exposure event.     

许氏平鮋( Sebastes schlegeli) CYP19B 基因cDNA克隆及在生殖周期中的表达分析

从许氏平鲉脑组织中克隆到细胞色素P450芳香化酶(P450aromB)(CYP19B)基因核心功能区cDNA。结果表明,许氏平鲉CYP19B主要功能区片段编码369个氨基酸,包括I-螺旋区、Ozol’s肽区及部分芳香化酶特异性保守区。RT-PCR分析表明,CYP19B在雌鱼和雄鱼各组织中表达情况不同,在雌鱼中表达较丰富,但是两者都在性腺和脑中有表达,且脑中的表达量高于性腺。生殖季节表达结果表明,CYP19B在繁殖前期表达较为丰富,退化期未见有表达。CYP19B在精巢处于Ⅲ期的许氏平鲉脑组织中表达最为丰富,在精巢发育Ⅳ期的脑组织表达量最低。上述研究结果说明,CYP19B在雌性和雄性许氏平鲉繁殖周期中均发挥重要生理作用。     

中华乌塘鳢雌雄异体和雌雄同体性类固醇激素含量的季节变化与差异

采用酶联免疫吸附法测定了野生中华鸟塘鳢（Bostrichthyssinensis）不同季节血清E2和1l—KT含量的变化,并比较了生殖季节养殖雌雄同体与野生雌雄异体之间E,、11一KT和17仪,2013一P三种性类固醇激素含量的差异．结果表明,野生雌鱼和雄鱼血清E：和11一KT的含量均具有明显的季节变化,其高低均依次为5〉8〉2〉11月．在相同月份,雌鱼的E,含量显著高于雄鱼,而雄鱼的ll—KT含量显著高于雌鱼．生殖高峰期,雌雄同体E：含量高于雌雄异体雄鱼,但低于雌雄异体雌鱼;雌雄同体l1一KT和17仅,20]3一P含量均高于雌雄异体雌鱼和雄鱼;野生雌鱼和雄鱼血清170L,2013一P含量没有显著差异．性类固醇激素在中华鸟塘鳢的生殖过程中起着重要作用,E：是野生中华乌塘鳢雌鱼的主要雌激素,而11一KT则是野生雄鱼的主要雄激素．     

Specific accumulation of organotin compounds in tissues of the rock shell,Thais clavigera

Concentrations of organotin compounds (butyltins and phenyltins) were determined in gonad, accessory sex organs, penis, digestive gland, kidney, radula with sac, oesophagus with crop, stomach, hypobranchial gland, rectum, mantle, osphradium, ctenidium, heart, salivary gland, head ganglia and muscle of imposex-exhibiting female and male rock shells (Thais clavigera), by gas chromatography with flame photometric detection (GC-FPD). Different tissue distributions were observed between butyltin and phenyltin compounds. More than 1000 ng TBT/g wet wt. were observed in ovary, digestive gland, kidney, heart, ctenidium, osphradium, stomach, head ganglia and penis, of both imposex-exhibiting females and males. More than 1000 ng TPT/g wet wt. were found in almost all tissues of both sexes. Approximately one-third or more of total TBT was accumulated in the digestive glands of both females and males, respectively. Meanwhile, approximately 40–50% and one-half of total TPT accumulated in the digestive glands of females and males, respectively.     

Dumpton syndrome reduces the tributyltin (TBT) sterilising effect on Nucella lapillus (L.) by limiting the development of the imposed vas deferens

TBT induces imposex and sterilisation in female Nucella lapillus. Interestingly, there are populations where individuals (both males and imposexed females) may suffer from a genetic aberration [Dumpton Syndrome (DS)] which results in the development of no penis (i.e. aphally). This anomaly confers a so far unexplained protection against TBT. Six populations with contrasting DS frequency were sampled; the imposex stage in females and the mean calibre of every vas deferens (VDC) were assessed. While the sex ratio in the 600 specimens examined was close to 1, aphally was more frequent in females than in males (31 and 7%, respectively). VDC was significantly higher in normal than in aphallic (DS) specimens, independently of sex and/or site. It is concluded that the vas deferens imposed by TBT onto DS females is less prone to develop thick enough so as to interfere with normal egg laying.     

Reproductive endocrine function in female atlantic croaker exposed to pollutants

To examine whether xenobiotics impair teleost reproduction by altering reproductive endocrine function, steroid hormone secretion and ovarian growth were investigated in female Atlantic croaker (Micropogonias undulatus) after chronic oral administration with sublethal doses of three classes of reproductive toxins (lead, benzo[a]pyrene and Aroclor 1254). All the sublethal treatments significantly impaired ovarian growth as assessed by the gonadosomatic index. Reduced ovarian growth was accompanied by a significant decline in circulating 17β-estradiol levels in fish exposed to lead and benzo[a]pyrene. All three toxicants significantly decreased plasma testosterone levels. However, the steroidogenic capacity of ovarian tissue in vitro was not altered by xenobiotic exposure. The data suggest that the decreased ovarian growth in croaker after pollutant exposure may be a consequence of the decline in plasma 17β-estradiol levels. Further, this decrease in circulating levels of gonadal steroids does not appear to be caused by a direct effect of the chemicals on ovarian steroid synthesis.     

水温对文昌鱼生殖活动的影响及其内分泌机制

采用慢性实验方法,研究在不同水温条件下饲养性腺发生前的文昌鱼(Branchiostoma belcheri)对其性腺分化和发育的影响。另外,在繁殖季节研究在低温16℃、中温22℃和高温28℃下饲养性成熟的文昌鱼对其产卵和排精的影响。同时,采用免疫组织化学方法对几种生殖相关激素在不同发育期文昌鱼的生殖调控轴(脑-哈氏窝-性腺)中的分布进行免疫识别,并用磁酶免定量测定方法检测睾酮和17 β-雌二醇在文昌鱼性腺中的含量,以分析和探讨水温影响文昌鱼生殖活动的内分泌机制。结果表明:低温有利于文昌鱼性腺向雌性分化,雌雄比例为4.2:1,中温和高温组则不受影响,雌雄数量大致相等。高水温则有利于文昌鱼的精巢发育和生精活动。28℃海水适于文昌鱼的产卵和排精。根据免疫组织化学和磁酶免定量测定的结果分析提示,温度影响文昌鱼性别分化和发育以及繁殖活动的内分泌机制是:首先水温经皮肤或哈氏窝化学感受器,刺激神经系统(脑泡)释放GnRH。然后GnRH促进哈氏窝分泌LH,从而可能刺激性腺产生和分泌性类固醇激素,始动文昌鱼性腺的发育至成熟及其生殖活动。此外,脑中芳香化酶可能介导文昌鱼性别分化。     

牙鲆芳香化酶Cyp19a多克隆抗体制备及其应用

芳香化酶Cyp19a在鱼类性别决定和性别分化中起关键作用,通过调控体内雌、雄激素的转化来影响鱼类性别表型形成。为深入研究Cyp19a在牙鲆(Paralichthys olivaceus)性腺分化和发育过程中的表达规律及作用机制,本文从牙鲆cDNA中克隆获得1557bp的cyp19a基因编码序列,并成功构建了原核重组表达质粒。经体外重组与纯化获得较高纯度的重组Cyp19a蛋白;以此作为抗原免疫兔子,制备多克隆抗体。用间接酶联免疫吸附剂测定(ELISA)技术检测抗血清效价,评估其免疫原性。结果表明免疫结束后得到的抗体血清效价超过了1∶50000,且纯化后抗体具有较好活性。Western Blot(WB)检测结果表明Cyp19a兔多抗可以特异性识别牙鲆重组Cyp19a蛋白和内源性Cyp19a蛋白。蛋白水平的雌雄性腺差异表达分析显示,牙鲆Cyp19a蛋白在卵巢中高表达,在精巢中微量表达。利用Foxl2和Dmrt1重组蛋白处理牙鲆性腺分化期幼鱼可分别显著上调和下调Cyp19a的表达(P<0.05)。综上所述,本研究成功制备了牙鲆Cyp19a多克隆抗体并进行了应用,为深入研究牙鲆等鱼类性别分化机制提供有力工具。     

Plasma steroid levels in female flounder (Platichthys flesus) after chronic dietary exposure to single polycyclic aromatic hydrocarbons

The chronic effects of polycyclic aromatic hydrocarbons (PAHs) on ovary development, total hepatic lipids and plasma sex- and corticosteroid levels in female flounder (Platichthys flesus) were examined. Sexually mature feral female flounder were exposed via the diet to phenanthrene (0.5, 2.5 or 12.5 nmol/g food) or chrysene (0.4 nmol/g food) for 12 weeks, during the previtellogenic phase of the annual reproductive cycle. PAH exposure did not directly affect germ cell development since no structural and/or developmental differences were observed between control and exposed fish. On the contrary, all treatments resulted in altered plasma steroid levels. The most pronounced effect was the significant decrease in plasma 17β-estradiol to 19±11%, 27±7%, 63±20% and 61±12% in relation to control fish, respectively, in flounders exposed to 12.5, 2.5 or 0.5 nmol phenanthrene/g food and 0.4 nmol chrysene/g food. Impaired ovarian growth was not observed, most likely because experiments were ended before the period of vitellogenesis, even though a non-significant general decline in total hepatic lipids could be observed. Moreover, all exposed flounders, except fish fed with the highest amount of phenanthrene, showed a negative correlation between plasma 17β-estradiol and 17α-hydroxyprogesterone levels (r=−0.46). One possible explanation is that PAH action may be mediated by a specific inhibition of steroidogenic enzymes. These findings provide evidence that selected PAHs are antiestrogenic xenobiotics with the capability to impair female teleost reproductive function.     

缢蛏多巴胺受体基因及其在损伤修复中的作用

多巴胺受体(dopamine receptor)作为生物体中重要的神经递质受体, 在生物体的生长、发育、代谢等多个生理过程中都发挥着重要的功能。本研究从缢蛏Sinonovacula constricta转录组文库中筛选获得多巴胺受体基因的部分片段, 结合RACE(rapid-amplification of cDNA ends)技术和降落 PCR(polymerase chain reaction)技术, 克隆得到缢蛏两个多巴胺受体的选择性多聚腺苷酸化(alternative polyadenylation, APA)的变异体, 命名为ScDopR2a和 ScDopR2b, 长度分别为1824bp和2758bp。两个变异体均包含相同的5′非翻译区(untranslated regions, UTR)(24bp)和开放阅读框(open reading frame, ORF)(1440bp), 共编码479个氨基酸残基。但是两个变异体的3′UTR 长度不同, 分别为360bp和1294bp, 其中ScDopR2b在polyA尾前插入936bp。在同源的ORF区设计引物, 采用实时定量PCR分析多巴胺受体基因在不同组织中的表达特征, 结果表明多巴胺受体基因在水管、鳃、斧足的表达量显著高于其他组织。在该基因序列3′UTR插入片段区域设计特异引物检测ScDopR2b的组织表达情况, 结果表明在水管、鳃、斧足的表达量仍高于其他组织, 表达趋势与ORF区大致相同。进一步设计缢蛏组织损伤实验, 对进水管前端进行损伤处理, 在处理后的第4h、8h、12h、24h、48h、60h和72h取样, 荧光定量检测结果显示, 同源区表达量在12h和48h呈上调, 在8h、24h和72h下调, 且ScDopR2b表达趋势与同源区表达模式大致相同。研究结果表明, 缢蛏多巴胺受体参与了损伤修复过程, 其表达特征可能与多巴胺作为补偿性神经递质的作用有关。     

联苯菊酯暴露对雄性褐菖鲉精巢发育的干扰

Bifenthrin(BF) is a pyrethroid insecticide that is widely used in agriculture, horticulture, and for residential purposes. However, few studies addressing the reproductive toxicity of BF on fishes are available. The present study was conducted to investigate the effects of BF on testicular development in Sebastiscus marmoratus and to gain insight into its mechanism. After exposure to 1, 10 and 100 ng/L BF for 50 days, there was a reduced number of mature sperm and an abundance of the late stages of spermatocysts in the testes. The levels of 17β-estradiol and testosterone were decreased significantly after BF exposure. The activity of caspase-3 was increased in a dosedependent manner after BF exposure, TUNEL assay indicated that BF exposure resulted in the occurrence of apoptosis in the testes, which might be main reason for the inhibition of spermatogenesis.     

Estrogenic effect of dioxin-like aryl hydrocarbon receptor (AhR) agonist (PCB congener 126) in salmon hepatocytes

Previous studies have shown that aryl hydrocarbon receptor (AhR) agonists possess anti-estrogenic activities and several mechanisms have been proposed to explain the interactions between AhR and estrogen receptor (ER) signalling pathways. In the present study, we show that 3,3'4,4'5-pentachlorobiphenyl (PCB126 - a dioxin-like AhR agonist) produced estrogenic responses in the absence of ER agonist, in fish in vitro system. We exposed salmon primary hepatocytes to PCB126 (1, 10 and 50 pM) and the ER agonist nonylphenol (NP; 5 and 10 microM) singly and also in combination. Vitellogenin (Vtg) and zona radiata proteins (Zr-proteins) levels were analysed by semi-quantitative ELISA. We observed that the protein levels of Vtg and Zr-proteins were significantly induced in a concentration-specific manner in cells treated with PCB126 and NP, singly or in combination. In general, these results show a novel aspect of dioxin-like PCB effect not previously demonstrated in fish system.     

Evidence for estrogenic endocrine disruption in an offshore flatfish, the dab (Limanda limanda L.)

Dab (Limanda limanda) caught in UK offshore waters show evidence of being exposed to estrogenic endocrine disrupters at a relatively low level. Two of 449 males caught between June and July 2005 had markedly elevated levels of vitellogenin (VTG; 21 and 750 microg/ml) and the remainder ranged from <0.01 to 8.6 microg/ml. Omitting the two outliers, there was a very significant positive relationship with the mass of individual males (a feature noted in previous studies on cod). Mean VTG concentrations in males differed significantly between sites. The site with the highest mean (1.6 microg/ml) was North East of the Dogger Bank and the site with the lowest (0.04 microg/ml) was in Cardigan Bay. Mean VTG concentrations in all North Sea fish were significantly higher than English Channel and Irish Sea fish, but this difference disappeared when fish mass was taken into account. VTG concentrations showed no relationship to water depth, stage of sexual maturity or age of the males. Sixty selected male plasmas were assayed for 17beta-estradiol but only two had measurable amounts (assay limit 0.04 ng/ml). Despite being the start of summer, the gonads of many of the males and females (especially those caught in the North Sea) showed signs of sexual maturity (presence of sperm in males and vitellogenic eggs in females). Many females had high VTG concentrations (up to 14 mg/ml) and 78 out of 80 had measurable concentrations of 17beta-estradiol. The cause of elevated VTG levels in male dab is unknown. As seen in cod, the presence of affected males does not appear to be linked to proximity to land or to known point sources of endocrine disrupters. However, our data, showing that larger fish are more likely to have elevated VTG concentrations, suggests a gradual accumulation by marine fish, probably through feeding, of persistent (probably relatively weak) estrogenic compounds.     

方斑东风螺性畸变现象的解剖学和组织学研究

以解剖学和组织学方法研究了方斑东风螺未成熟和成熟的正常雌、雄个体以及性畸变个体的生殖系统结构.性畸变个体除具有正常的雌性器官外,还有输精管或阴茎.在正常雄性输精管的个体发育过程中再现了系统发育的过程,而且在性畸变个体输精管的形成过程和发育过程中组织学结构特征的变化与正常雄性输精管的个体发育过程的相似.性畸变雌体的性腺发育受阻.目前,性畸变现象仅存在于成熟个体,但这并不表明未成熟个体中不会发生性畸变现象.具有较高食用价值的方斑东风螺因有机锡污染可能会对人类健康不利,应引起人们的关注.     

Gonadal alterations in male eelpout (Zoarces viviparus) exposed to ethinylestradiol and trenbolone separately or in combination

To evaluate the interaction between 17β-trenbolone (TB) and 17α-ethinylestradiol (EE2) in relevant environmental concentrations, male eelpout Zoarces viviparus were exposed in a flow-through seawater-system for 21 days to 5 ng l⁻¹ EE2, 5 ng l⁻¹ or 20 ng l⁻¹ TB or to combinations of both compounds. The effects on hepatosomatic index (HSI), gonadosomatic index (GSI) and gonadal histology were studied. No significant effects on HSI were observed in any treatment; in contrast, decreased GSI was observed in males exposed to EE2 alone or in combination with TB compared to controls (p < 0.05). The histology revealed that the males were in the beginning of spermatogenesis. Males from the control group and some from the TB groups showed tubules with cysts containing spermatogonia, spermatocytes and spermatids; however, some testes of males exposed to TB showed slight to moderate interstitial fibrosis. Nevertheless, the most severely affected were males exposed to EE2 showing marked interstitial fibrosis, necrosis of germinal cells and reduced number of spermatocytes and spermatogonia in the cyst. Likewise, increased tubule number and proportionally decreased tubule diameter were observed in the testis of all EE2 exposed groups (p < 0.05). Finally, a similar tubule number was observed in males exposed to EE2 + 20 ng l⁻¹ TB compared to control (p > 0.05). This study shows that EE2 dramatically disrupts the spermatogenesis and low doses of 17β-trenbolone are unable to effectively counteract the morphological effects of EE2.