脊尾白虾翻译控制肿瘤蛋白基因TCTP克隆及自噬调控相关基因在卵巢发育期的表达

为深入了解脊尾白虾(Exopalaemon carinicauda)卵巢发育和卵黄蛋白原发生的分子机制,本研究克隆得到脊尾白虾翻译控制肿瘤蛋白基因TCTP,并结合自噬调控基因TCTP、Hif-1α、Beclin1和Bcl-2在卵巢发育期的表达水平,阐释脊尾白虾卵黄蛋白原合成过程中的分子调控特征。研究显示,脊尾白虾TCTP基因c DNA全长为732 bp,编码168个氨基酸,具有典型的TCTP1和TCTP2功能域以及PKC和TKⅡ等mTOR信号通路相关的磷酸化位点。同时发现,甲壳动物TCTP普遍缺乏在其他动植物中高度保守的C末端的cys残基。进化分析显示,脊尾白虾TCTP与中华绒螯蟹(Eriocheir sinensis)亲缘关系最近。4种自噬调控基因在脊尾白虾卵巢发育期的表达结果显示,肝胰腺TCTP基因从增殖期到产后恢复期呈递减趋势;肝胰腺Hif-1α、Beclin1和Bcl-2基因表达趋势相似,即从增殖期到小生长期高表达,大生长期极显著下降,表达量最低(P0.01),这与外源性卵黄蛋白原的合成趋势大致相反。这些自噬调控基因可能通过自噬作用共同调节外源性卵黄蛋白原的合成。卵巢TCTP基因在小生长期表达量最高;卵巢Hif-1α基因从增殖期到产后恢复期持续升高,这与内源性卵黄蛋白原表达趋势相似;卵巢Beclin1基因在大生长期表达量最高,与脊尾白虾卵巢Ec R表达趋势相似,与卵巢Bcl-2基因表达趋势相反,这些自噬调控基因可能通过自噬作用共同促进内源性卵黄蛋白的合成。本研究表明,自噬调控基因TCTP、Hif-1α、Beclin1和Bcl-2在脊尾白虾卵巢发育时期相互协调共同作用,可能通过自噬作用调节脊尾白虾卵黄蛋白原的合成和卵巢发育。     

Effects of the herbicide LASSO MTX (alachlor 42% W/V) on biometric parameters and liver biomarkers in the common carp (Cyprinus carpio)

The aim of the study was to evaluate the effect of subchronic exposure to the herbicide LASSO MTX (alachlor 42% W/V) on biometric parameters and important liver biomarkers in the common carp (Cyprinus carpio). One year old fish were exposed for 28 days to LASSO MTX added to the tank water at concentrations of 240 and 2400 μg L⁻¹. The exposure did not affect fish biometric parameters. Glutathione-S-tranferase (GST) activity in liver (hepatopancreas) remained unchanged in exposed fish when compared to controls. However, significant induction of total cytochrome P 450 (CYP 450), ethoxyresorufin-O-deethylase (EROD) activity and elevated glutathione (GSH) in liver of exposed fish were detected.     

不同倍性虹鳟卵黄蛋白原的组织定位

采用分步沉淀法提纯虹鳟卵黄蛋白,以同发育期二倍体虹鳟为对照,用免疫组化法对不同发育阶段的三倍体虹鳟性腺、肝脏、血液和肠道进行卵黄蛋白原(Vg)的细胞化学定位研究.试验结果表明:所纯化蛋白为与卵黄蛋白原具有相似免疫原性的卵黄脂磷蛋白,呈雌性特异性.性腺发育Ⅲ～Ⅴ期的二倍体雌性虹鳟,血液和肝细胞呈卵黄蛋白原阳性,Ⅳ～Ⅴ期卵巢呈卵黄蛋白原阳性,各发育期肠组织呈卵黄蛋白原阴性;性腺发育至Ⅰ～Ⅴ期的三倍体雌雄虹鳟及雄性二倍体虹鳟,其性腺、肝脏、血液和肠道组织呈卵黄蛋白原阴性.二倍体虹鳟外源性卵黄蛋白原在肝细胞内合成,经血液运送至卵巢,最终在卵母细胞中形成卵黄颗粒.由于三倍体雌性虹鳟卵巢发育受阻,无滤泡细胞分化,生殖细胞与体细胞的互作缺失,不能分泌足量的17β-E2以诱导Vg的合成,因而肝脏的Vg阴性并不能说明肝脏不具备合成Vg的能力,从卵黄发生的角度分析,卵黄蛋白原的缺乏不是导致三倍体虹鳟雌性不育的原因,而是其卵泡败育的结果.     

中国对虾卵黄蛋白原合成部位的研究

十足目甲壳类卵巢中卵黄的来源,在过去的几十年研究中一直存在争议,内源性合成和外源性合成都有报道。本文以雌性中国对虾为实验材料,根据组织学观察确定其发育阶段,试验虾可以分为:卵巢未发育期;卵黄发生前期;初级卵黄发生期和次级卵黄发生期4个时期。从处于不同卵巢发育期对虾的卵巢和肝胰腺中提取总RNA,用RT-PCR的方法初步探讨不同发育期的中国对虾卵巢和肝胰腺中卵黄蛋白原mRNA的表达,确定是否存在卵黄蛋白原的合成功能。结果发现,从卵巢未发育期到次级卵黄发生期的卵巢和肝胰腺中都有卵黄蛋白原mRNA的表达,说明二者为卵黄蛋白的合成部位。     

Pesticide—induced reproductive dysfunction in Indian fishes

Numerous studies are available reporting the effects of pesticides on reproductive activity in Indian fishes. The majority of these reports deals with histopathological changes in gonads and endocrine glands involved in the regulation of reproduction following treatment with different pesticides. Pesticides are reported to cause degenerative changes in gonads and arrest gametogenic processes either by acting directly on the gonads or by interfering with the secretory activity of the hypothalamo-hypophyseal-gonadal/thyroid axis that regulates various reproductive events. Secretion of hormones such as gonadotropin-releasing hormone (GnRH), gonadotropin, growth hormone, adrenocorticotropic hormone (ACTH), testosterone, estrogens, 17,20β-dihydroxyprogesterone and thyroid hormones are in general lowered, leading to cessation of gametogenesis, vitellogenesis, oocyte maturation, ovulation, spermiation, etc. Adverse effects of pesticides have also been demonstrated on fecundity, fertilization, hatching, and postembryonic development. The effects are highly variable and depend on the nature, dose, and mode of application of the pesticides.     

Disrupted sexual cycles in female grass carp (Ctenopharyngodon idella) raised in tropical conditions

In the Ivory Coast, grass carp (Ctenopharyngodon idella) have been integrated in the tilapia-based polyculture in order to increase pond productivity. In the tropical conditions prevailing there, female cycles seemed disrupted. We describe oogenesis in these conditions using histological observations, monitoring of individual cycles with intraovarian biopsies, endocrinal monitoring (development of a specific enzyme-linked immunosorbent assay (ELISA) for vitellogenin (Vg), measurement of plasma oestradiol-17β (E2) and testosterone (T)) and comparison with grass carp raised in Poland. We noted that oogenesis was blocked in all females at the migrating germinal vesicle stage, precluding ovulation or spawning without artificial induction. High rates of atypical post-vitellogenic oocytes (translucent, not filled with yolk granules) were observed in many females. Female individual cycles also displayed atypical features: cycles were sometimes (10% of females) blocked at the beginning of vitellogenesis (BV), for females displaying abnormally low E2 (0.5 ng/ml) and Vg (30 μg/ml) levels compared to “normal” females (1.4 ng/ml and 223 μg/ml, respectively). The duration of the cycles was highly variable among females (a few days to several weeks). Sexual cycles were unsynchronised (all the ovarian stages could be found for all seasons) and the number of females at the end of vitellogenesis (EV) was low (<40% most of the year). These characteristics raise problems for artificial induction of spawning in small-scale hatcheries: a large stock of broodfish is required as is regular checking of female broodstock with intraovarian biopsies to select responsive females.     

Endocrine disruption and metabolic changes following exposure of Cyprinus carpio to diethyl phthalate

Diethyl phthalate (DEP) enter into aquatic environment from industries manufacturing cosmetics, plastic and many commercial products and can pose potential fish and human health hazard. This experiment evaluated effects of DEP in adult male (89 g) common carp (Cyprinus carpio) by exposing them to fractions of LC₅₀ (1/500-1/2.5) doses with every change of water for 28 days. Vitellogenin induction metabolic enzymes, somatic indices and bioaccumulation were studied on 7th, 14th, 21st and 28th day. The 96th hour LC₅₀ of DEP in fingerlings was found to be 48 mg/L. Compared to control, except increase (P < 0.01) in alkaline phosphatase activity (EC 3.1.3.1) and liver size, there was decrease (P < 0.01) in activity of acid phosphatase (EC 3.1.3.2), aspartate aminotransferase (EC 2.6.1.1), alanine aminotransferase (EC 2.6.1.2) and testiculosomatic index following exposure to 1, 5 and 20 ppm DEP. Significant (P < 0.01) dose dependant vitellogenin induction was observed with exposure of fish to 0.1, 1 and 5 ppm DEP. The bioaccumulation of DEP in testis, liver, brain, gills and more importantly in muscle tissues of fish increased significantly (P < 0.01) with increase of dose from 1 to 5 ppm. Significant interaction (P < 0.01) of dose and duration of exposure indicated that exposure period of a week to two was sufficient to bring about changes in quantifiable parameters studied. Fish exposed to 20 ppm DEP became lethargic and discolored during onset of the 4th week. This is the first report describing metabolic changes and vitellogenin induction following exposure of C. carpio to DEP dose that is as low as 1/500th fraction of LC₅₀.     

Levels of Vitellogenin in Male Japanese Medaka (Oryzias latipes) Exposed to the Chemotherapeutics: Oxytetracycline,Romet-30®, and Copper Sulfate

ABSTRACT

Japanese medaka (Oryzias latipes) were exposed to oxytetracycline and Romet-30^® at daily doses via feed of 0.06 mg, 0.11 mg, and 0.28 mg per gram body weight for 11 days, and at daily doses of 0.05 mg, 0.10 mg, and 0.15 mg per gram body weight for 5 days, respectively. In addition, fish were exposed to copper sulfate in their aquaria water at 1.85 mg/L for 1 h each day (over a span of 1 day, 7 consecutive days, or 1 day in every 3 to 5 days for a total of seven doses). Vitellogenin levels were measured in fish using a sandwich enzyme-linked immunosorbent assay (ELISA). Vitellogenin levels were not induced from exposure to either oxytetracycline or Romet-30® (P > 0.05); however, copper sulfate for 1 h on 1 day showed a decrease in vitellogenin from the control fish and other treatment groups (P = 0.007). Under these test conditions, oxytetracycline, Romet-30, and copper sulfate were not found to act in medaka as estrogenic compounds by inducing vitellogenin.     

绿盲蝽卵黄原蛋白(Vg)基因片段克隆及不同生境表达量分析

为了探讨不同果园生境对绿盲蝽生殖的影响,从分子角度确定绿盲蝽适合的产卵生境,本研究首先采用绿盲蝽专用诱芯调查了枣园、葡萄园的绿盲蝽发生量,然后通过比较半翅目,直翅目,膜翅目,鳞翅目等昆虫cDNA同源序列,根据基因保守序列设计一对简并性引物,克隆绿盲蝽卵黄原蛋白(Vg)基因片段,采用RT-PCR的方法检测了2种生境中Vg基因相对表达量。结果显示,枣园绿盲蝽的发生量始终高于葡萄园,枣园有4个发生高峰,而葡萄园有2个。基因克隆获得一段大小为732 bp的基因片段,编码244个氨基酸;该基因序列与其他蝽类昆虫Vg基因氨基酸序列的同源性达80%以上;系统进化分析表明,绿盲蝽Vg基因与赤须盲蝽、烟盲蝽及黑肩绿盲蝽的亲缘关系最近,克隆的基因片段为绿盲蝽Vg基因片段,并命名为AL-Vg。不同生境Vg基因表达量比较可知,枣园的表达量低于葡萄园,结合发生量表明枣园生境更适合绿盲蝽生殖产卵。     

Comparative responses in rare minnow exposed to 17β-estradiol during different life stages

Present in the excrement of humans and animals, 17β-estradiol (E₂) has been detected in the aquatic environment in a range from several nanograms to several hundred nanograms per liter. In this study, the sensitivities of rare minnows during different life stages to E₂ at environmentally relevant (5, 25, and 100 ng l⁻¹) and high (1000 ng l⁻¹) concentrations were compared using vitellogenin (VTG) and gonad development as biomarkers under semistatic conditions. After 21 days of exposure, VTG concentrations in whole-body homogenates were analyzed; the results indicated that the lowest observed effective concentration for VTG induction was 25 ng l⁻¹ E₂ in the adult stage, but 100 ng l⁻¹ E₂ in the larval and juvenile stages. After exposure in the early life stage, the larval and juvenile fish were transferred to clean water until gonad maturation. No significant difference in VTG induction was found between the exposure and control groups in the adults. However, a markedly increased proportion of females and appearance of hermaphrodism were observed in the juvenile-stage group exposed to 25 ng l⁻¹ E₂. These results showed that VTG induction in the adult stage is more sensitive than in larval and juvenile stages following exposure to E₂. The juvenile stage may be the critical period of gonad development. Sex ratio could be a sensitive biomarker indicating exposure to xenoestrogens in early-life-stage subchronic exposure tests. The results of this study provide useful information for selecting sensitive biomarkers properly in aquatic toxicology testing.