Toxicity of the brominated flame retardant tris-（2,3-dibromopropyl） isocyanurate in zebrafish （Danio rerio）

Tris-(2,3-dibromopropyl) isocyanurate (TBC) is a heterocyclic brominated flame retardant that was recently detected in the environment in China. TBC is semi-volatile and can accumulate in the lipid of some species, but little is known about its effect on aquatic organisms. We exposed adult zebrafish to 0, 0.25, 1 and 4 mg/L TBC for 28 d and measured the effect on survival, growth, histopathology, hormone levels, enzyme activity, and gene expression. TBC exposure had no effect on survival or growth. We observed significant damage to the liver and gill, including hepatocellular swelling and fatty degeneration in the liver as well as proliferation and edema of epithelial cells in the gills. In addition, exposure to 4 mg/L TBC induced proliferation of goblet cells in the intestine of both sexes, acellular areas in the testis, and thinly scattered vitellogenic granules in vitellogenic oocytes. TBC exposure had no effect on the levels of thyroid hormones, testosterone, estradiol, liver superoxide dismutase activity, malondialdehyde content, and brain cholinesterase activity. By contrast, hepatic vitellogenin and cytochrome P4501A gene expression was significantly down-regulated in both male and female zebrafish in response to TBC exposure. Our results suggest that exposure to TBC causes a variety of potential reproductive and endocrine toxic effects.     

Dynamic expression pattern of zebrafish bfabp （fabp7） during early embryogenesis

The expression of bfabp is correlated with the development of nervous system. Recently, a bfabp homologue (fabp7) was identified in zebrafish and its genomic structure, mRNA expression by RT-PCR and linkage mapping data were obtained. However, these studies did not provide the information of spatial and temporal expression pattern of fabp7 during zebrafish embryogenesis.     

Cloning and Expression of BTB/POZ-Domain from Zebrafish gcl （Germ Cell-Less）, and Appraisement of Its Polyclonal-Antibody

In this study, a recombinant pET28c-gBTB/POZ was constructed by cloning the sequence of the BTB/POZ domain of the zebrafish gcl （germ cell-less） into the expression vector pET28c, and pET28c-gBTB/POZ was transformed into BL21（DE3） pLysS strain to express the fusion protein for the preparation of antibody. Polyclonal-antibody against the GCL-BTB/POZ domain was prepared by immunizing rabbit with the fusion protein, and the Western Blot and immuno-histochemical analysis were performed to detect the quantity of the polyclonal-antibody. The result indicates that the polyclonal-antibodies were of good quantity and specification. Further studies will be performed to demonstrate the function and expression pattern of the GCL protein during the development process of zebrafish with the polyclonal-antibody.     

The expression of vasa gene during zebrafish ( Danio rerio ) oogenesis

vasa gene expression pattern during oogenesis of zebrafish was examined usingin situ hybridization and fluorescent quantitative RT-PCR. During zebrafish oogensis,vasa mRNA is expressed strongly and uniformly distributed in the cytoplasm in stage II oocytes, followed by a distribution among vacuome in stage III. Later in stage IV and V,vasa mRNA is enriched at the cortex and finally localized at the cortex. The fluorescent quantitative RT-PCR shows that the quantity ofvasa mRNA decreases from stage II to stage III, but remains relatively invariable from stage III to stage V. The observed differences invasa mRNA expression in the different stages of zebrafish oogenesis suggest thatvasa gene plays an important role during oogenesis. Foundation item: Supported by the National Natural Science Foundation of China (30370744, 30150005) Biography: XIANG Fang (1979-), male, Master candidate, research direction: molecular development of animals.     

斑马鱼（Branchydaniorerio）视网膜正常结构的定量研究

本文采用半薄切片甲苯胺蓝染色方法，对斑马鱼视网膜正常结构进行了形态学观察，并对视网膜各层厚度，特别是神经节细胞层中细胞核的大小和密度进行了定量分析。结果表明：斑马鱼视网膜正常结构象大多数脊椎动物的一样分为１０层，其总厚度为１６７．５±１２．５μｍ、除外、内界膜外、其余各层由外向内分别为：色素上皮层约２９．６±５．９μｍ，视杆视锥层２５．４±３．４μｍ，外核层８．９±ｌ．４μｍ，外网状层８．９±２．０μｍ，内核层２９．５±４．８μｍ，内网状层３７．８±５．６μｍ，神经节细胞层与视神经纤维层一起共２５．９±３．７μｍ，其中神经节细胞核长轴平均约８．０±１．２μｍ，，短轴约７．３±１．１μｍ，每ｍｍ长度中约含１２５．８±４０．０个神经节细胞核。这些数据可为以后更深入的科研工作提供参考价值。同时讨论了斑马鱼视网膜具有典型的光照条件好，白昼活动型的结构特点。     

内分泌扰乱化学物质对斑马鱼生长发育影响研究进展

随着内分泌扰乱化学物质研究的逐渐深入,人们越来越关心其对脊椎动物生长发育的影响。斑马鱼具有胚胎透明、性成熟周期短、繁殖力强等优点,是研究脊椎动物发育生物学的重要模式物种之一,被广泛地应用于内分泌扰乱化学物质的研究。本文综述了内分泌扰乱化学物质对斑马鱼胚胎发育、生长、性别分化、繁殖的影响及其机制,展望了该领域的发展方向。     

斑马鱼3种胰蛋白酶原基因的克隆、序列分析及组织表达

 胰蛋白酶是丝氨酸蛋白酶类超家族成员之一,在动物蛋白消化中起着重要作用。为深入研究胰蛋白酶在鱼类中的蛋白结构和生理功能,利用RT-PCR和RACE方法,成功获得了斑马鱼3种胰蛋白酶原cDNA序列(zftry1a、zftry1b和zftry2)。结果表明,zftry1a和zftry1b均有242个氨基酸残基组成,其中包括15个氨基酸的信号肽和5个氨基酸(LDDDK)的激活肽。zftry2由247个氨基酸残基组成,其中包括15个氨基酸的信号肽和9个氨基酸(APLGDDDDK)的激活肽。氨基酸序列比对结果显示,三者具备胰蛋白酶原的保守结构特征,如含有催化三联体氨基酸(His-57、Asp-102和Ser-195),12个半胱氨酸,位于底物结合口袋底部Asp-189和口袋开口处的Gly-216、Gly-226等。进化树结果显示,斑马鱼zftry1a和zftry1b属于group I,为阴离子胰蛋白酶原;斑马鱼zftry2属于group II,为阳离子型胰蛋白酶原。RT-PCR结果显示,三者组织分布模式类似,且在肠中有最高表达量。这些结果为研究鱼类胰蛋白酶原的基因进化和功能以及进一步探讨鱼类消化生理的分子机制奠定了基础。     

椭圆背角无齿蚌卵子发生的研究

作者通过石蜡切片技术与苏木精－伊红染色的组织学方法及超薄切片技术和透射电镜观察，较系统地研究了椭圆背角无齿蚌卵细胞的发生、发育与成熟过程的细胞学特征：原始生殖细胞在滤泡壁上分裂增殖，成为卵原细胞、卵原细胞发育、膨大成为卵母细胞而排入滤泡腔。在滤泡腔中卵母细胞不断地进行卵黄的合成与积累，体积继续增大。最后成熟的卵母细胞，在未完成减数分裂的情况下直接排入外鳃叶腔，与随水流入的精子相遇而受精，在观察的基础上，作者比较分析了双壳纲淡水贝类生殖过程中所具有的一般特征，着重讨论了卵子发生过程中的双核仁现象与卵黄发生，成熟卵母细胞减数分裂的阻断与重新起始。     

睾酮与TBTC抑制斑马鱼卵巢发育的分子机制

为探讨雄激素与三丁基锡对鱼类卵巢发育和配子发生的影响及其分子差异,分别用1μg·L-1睾酮(T)、1μg·L-1三丁基氯化锡(TBTC)单独处理以及T、TBTC(两者含量均为1μg·L-1)联合处理对斑马鱼(Daniorerio)雌鱼进行了90d暴露。组织学结果表明:与对照相比,经T、TBTC单独处理的斑马鱼成熟系数(GSI)下降,卵巢发育受阻,卵子发生抑制,且TBTC单独处理的斑马鱼发育后期的卵母细胞大量凋亡;经T、TBTC联合处理的斑马鱼卵母细胞尽管在发育后期发生凋亡,但仍有少量卵母细胞持续发育,GSI值明显升高。定量PCR结果表明:T和TBTC单独处理都能显著下调雌激素(E2)合成酶基因cyp19a及其上游调控基因foxl2的表达(p<0.05),而两者联合处理则显著上调cyp19a和foxl2基因表达(p<0.05);T单独处理的斑马鱼雄激素合成酶基因P450 11β的表达显著升高(p<0.05),而TBTC单独处理使该基因表达显著下降(p<0.05),T、TBTC联合处理对该基因表达无显著影响;T、TBTC单独处理均不影响孕激素合成酶基因20βHSD的表达,但T、TBTC联合处理却显著上调该基因的表达(p<0.05)。研究认为T、TBTC抑制斑马鱼卵巢发育和卵子发生的机制可能既相似又有所差异。
     

多种环境雌激素低剂量联合处理诱导斑马鱼精子发生障碍

用E2、EE2、DES、4-t-OP、4-NP、BPA等6种环境雌激素暴露雄性斑马鱼(Danio rerio)成鱼120 d。对暴露后斑马鱼精巢的组织学观察发现,其中精子的发生被明显抑制,精子大量丢失,出现空腔;早期生殖细胞大量增生。采用半定量RT-PCR对暴露后斑马鱼精巢发育和精子发生相关基因的表达进行研究,结果表明联合处理后,斑马鱼精巢中雄激素合成酶基因P450 11β、cyp17a1的表达显著下调(p<0.05),而孕激素合成酶基因20β〖KG0.01mm〗-hsd和雌激素合成酶基因cyp19a1a的表达量没有显著变化,但雌激素受体基因esr1的表达则显著上调(p<0.05);减数分裂标记基因dmc1没有显著变化,而scp3基因的表达则显著下调(p<0.05)。另外,视黄酸(Retinoic acid,RA)合成酶基因aldh1a2的表达显著上调(p<0.05),而RA降解酶基因cyp26b1的表达则无显著变化。研究推测多种环境雌激素的联合处理可能通过下调雄激素合成酶的表达、上调雌激素效应和增强减数分裂起始并抑制减数分裂后期过程诱导斑马鱼成鱼精子发生障碍;因此多种环境雌激素低剂量联合暴露对渔业资源造成的生殖风险值得警惕。
     

斑马鱼视网膜神经节细胞数量及分布特点

研究了斑马鱼（ Brachydanio rerio） 视网膜整体装片的组织结构,着重观察和分 析了视网膜神经节细胞的数目及密度分布情况. 结果表明,其视网膜组织结构符合脊椎动物 的基本模式. 视网膜面积为3.04±0.29 mm2,视神经乳头位于视网膜的偏腹颞侧. 视网膜 神经节细胞总数约为40　000～56 000个,分成6级密度水平. 在视神经乳头偏颞侧部位有一 高密度区即中央区,沿鼻颞轴分布. 神经节细胞核大小均匀,呈规则的圆形.     

用RT-PCR方法检测达乌尔黄鼠(Spermophilus dauricus)白色脂肪组织中瘦素的基因表达

达乌尔黄鼠（Spermophilus dauricus）作为研究冬眠的优良模式动物,已经在医学、生理学以及生态学领域得到广泛的应用。但是目前尚未见使用分子生物学手段对达乌尔黄鼠（Spermophilus dauricus）进行冬眠机理方面的研究。瘦素在调节摄食、能量平衡和入眠方面都有非常重要的作用。尝试使用RT-PCR这一分子生物学技术对达乌尔黄鼠的瘦素和β-actin进行特异性扩增,并对其进行克隆测序,得到了达乌尔黄鼠的瘦素和β-actin基因片段的序列,为研究达乌尔黄鼠冬眠的分子机制提供基础数据。同时总结了用RT-PCR方法检测未知基因序列物种有关基因表达时需要注意的一些问题。