脊尾白虾VgR基因克隆及其在卵巢发育过程中的表达分析

为了解卵黄蛋白原受体(vitellogenin receptor,Vg R)在脊尾白虾卵巢发育中的作用,采用同源克隆和RACE技术,克隆了脊尾白虾Vg R基因全长c DNA序列,用实时荧光定量PCR方法分析了Vg R基因在雌虾不同组织、卵巢发育不同时期的表达特征。结果显示,脊尾白虾Vg R基因全长5892 bp,开放阅读框5661 bp,编码1886个氨基酸。脊尾白虾Vg R具有低密度脂蛋白受体(LDLR)家族典型结构特征,属于LDLR家族,进化上与日本沼虾等甲壳动物Vg R亲缘关系最近,然后与昆虫Vg R分支聚为一支,而与LDLR家族中其他成员亲缘关系较远。脊尾白虾Vg R在各组织中均有表达,但主要在卵巢内表达。随着卵巢的发育,卵巢Vg R表达量逐渐升高,在III期达到最大,与肝胰腺Vg表达量、血液Vg浓度变化趋势相一致;而在卵巢成熟时,卵巢Vg R表达量降到了最低,与肝胰腺Vg表达情况截然相反;排卵后的恢复期,血液中Vg浓度仍维持在较高水平,卵巢Vg R表达量又升至III期水平,同时卵巢Vg表达量也升至最高。由此可见,甲壳动物与昆虫Vg R起源于同一祖先,但在进化上已形成独立一支;脊尾白虾卵巢成熟前,卵巢主要通过Vg R介导作用摄取血液中Vg,以供卵巢快速成熟;卵巢成熟期,肝胰腺呈现补偿性合成Vg,以尽快恢复其营养储备功能;卵巢恢复期,卵巢通过Vg R介导摄入外源Vg和内源合成Vg两种途径,为卵巢二次发育提供营养物质。     

In vitro effect of pituitary,interrenal and gonadal hormones on vitellogenin synthesis in primary hepatocyte cultures of Chalcalburnus tarichi

Vitellogenin (Vtg) is an important precursor yolk protein in egg‐laying vertebrates, including fish. The 17β‐oestradiol (E2) plays a crucial role in the Vtg synthesis; moreover, certain hormones can stimulate Vtg synthesis. We investigated the possible role of E2, carp recombinant growth hormone (crGH), insulin (Ins), progesterone (P4) and 11‐deoxycortisol (11‐DOC) hormones in Vtg synthesis on primary juvenile Chalcalburnus tarichi hepatocyte culture. The amount of Vtg in the medium was measured at 2‐day intervals using enzyme‐linked immunosorbent assay (ELISA). The hepatocytes were maintained in culture for more than 2 weeks without the addition of serum components. Vitellogenin localization was visualized with the immunofluorescence method in E2‐supplemented hepatocytes. Among hormones applied to the culture, only E2 had an influence on Vtg synthesis in a time‐dependent manner, while crGH, Ins, P4 and 11‐DOC had no effect. However, in hepatocytes stimulated with E2 in combination with P4, a lower Vtg production was seen compared with Vtg produced when hepatocytes were stimulated with E2 alone. P4 proved to have potentiating effects on co‐treatment with E2‐induced Vtg production. As a result, E2 and P4 are the most important hormones for Vtg synthesis in juvenile C. tarichi hepatocyte culture.     

水生动物卵黄蛋白原研究新进展

卵黄蛋白原（vitellogenin,VTG）是卵生动物卵黄中主要成分的前体。文章简述了水产动物如鱼类、两栖类及甲壳类动物中卵黄蛋白原基因的鉴定及其基因家族的分类及进化研究,介绍了VTG分子的生物学新功能,以及VTG作为标记分子在环境雌激素监测、反映雌性动物性腺成熟度、区分雌雄性别等方面的应用,以期为VTG在水产动物中的研究以及繁殖中的应用提供参考。     

实时荧光定量PCR检测凡纳滨对虾和罗氏沼虾卵黄蛋白原mRNA在卵巢和肝胰腺中的表达

卵黄磷蛋白作为卵黄蛋白的主要成分, 可为甲壳动物胚胎和早期幼体发育提供能量, 为研究其来源及合成规律, 实验应用SYBR GreenⅠ荧光定量PCR法检测了凡纳滨对虾和罗氏沼虾性腺不同发育时期卵巢和肝胰腺两种组织中卵黄蛋白原mRNA的表达水平。结果发现,凡纳滨对虾和罗氏沼虾的卵巢和肝胰腺中都有卵黄蛋白原mRNA的表达。随着卵巢的发育, 凡纳滨对虾卵巢中卵黄蛋白原mRNA的相对表达量在前5个阶段不断增加, 分别为1.1, 5.9, 10.4, 26.9, 85.0, 恢复期急剧减少, 为1.6。肝胰腺中的相对表达量也不断增加, 分别为1.3, 3.3, 7.1, 37.3, 51.6, 恢复期急剧减少, 为1.0。罗氏沼虾肝胰腺中卵黄蛋白原mRNA的相对表达量在前四个阶段不断增加, 分别为3.4, 12.6, 15.2, 38.9, 抱卵期急剧减少, 为2.9;而卵巢在整个发育过程中对卵黄蛋白原合成的贡献比较小, 分别为1.0, 1.3, 1.7, 4.8, 1.5。研究表明, 两种虾类的肝胰腺和卵巢均具合成卵黄蛋白的功能, 而且在不同的卵巢发育阶段呈现明显的规律性。     

Alligator gar (Atractosteus spatula,Lacépède 1803) vitellogenin: purification,characterization and establishment of an enzyme‐linked immunosorbent assay

Alligator gar (Atractosteus spatula) is a non‐teleost bony fish distributed in North America. Gar populations have drastically declined as a consequence of habitat deterioration and the lack of regulation for their capture. Control of reproduction is critical for recovering their natural populations. The impossibility to distinguish genders and the determination of sexual maturity have hindered their successful reproduction. This research was aimed at developing an enzyme‐linked immunosorbent assay (ELISA) for the quantitative estimation of vitellogenin (VTG), a female‐specific protein. Plasmatic VTG from 17β‐estradiol (E2)‐induced juveniles and ovary vitellin from adult females were purified and characterized. Polyclonal antibodies against both proteins were produced to develop an ELISA. The immunoassay was validated by quality tests such as sensitivity, parallelism, recovery, reproducibility and specificity. Vitellogenin was determined in different tissues (plasma, mucus, liver and gills) of alligator gar. Vitellogenin and E2 concentrations in female breeders were found to be higher in November, before the spring spawning season. This approach represents a quick, reliable and non‐invasive practical alternative to distinguish genders and evaluate gonad maturation.     

蜜蜂卵黄原蛋白的研究进展

卵黄原蛋白是当前蜜蜂生理生化研究中最活跃的领域之一。主要介绍了卵黄原蛋白的国内外研究进展。蜜蜂作为一种进化的社会性昆虫,有其特殊的调控机制,卵黄原蛋白是其调控机制的主要因子。蜜蜂卵黄原蛋白不仅与保幼激素相互调节,而且与长寿存在一定关系。卵黄原蛋白调控着使执行巢内任务的蜜蜂转变为采集蜂,而且血淋巴中卵黄原蛋白的效价高低影响蜜蜂抗氧化能力,蜂王比工蜂的抗氧化能力强,从而蜂王寿命比一般工蜂长;越冬蜂却是例外,由于不执行哺育任务和采集行为,并且减少了外界环境的影响,越冬蜂的寿命增加;此外,卵黄原蛋白影响蜜蜂繁殖力,产卵蜂王卵黄原蛋白的效价很高,而无生殖力的工蜂卵黄原蛋白效价很低,相反产卵工蜂的卵黄原蛋白效价与老蜂王相似。     

In vitro study of a putative role of gonad‐inhibiting hormone in oocyte growth stimulation in Penaeus monodon

Ovarian development in crustacean is controlled by several factors, among which a neuropeptide gonad‐inhibiting hormone (GIH) is known to inhibit vitellogenin (Vg) synthesis in the ovary. It has been postulated that GIH may control Vg synthesis by inhibiting the release of gonad‐stimulating factor (GSF) from brain and thoracic ganglia. To prove this hypothesis, this study was primarily aimed to investigate the influence of GIH on the release of GSF from thoracic ganglia of Penaeus monodon. Our result showed that GIH did not suppress the release of putative GSF from thoracic ganglia by calcium ionophore A23187 as the induction of oocyte growth in the ovary explants that were cocultured with thoracic ganglia in the presence of A23187 was not affected by the addition of recombinant GIH protein. In addition and interestingly, when the ovary explants were incubated with the recombinant GIH alone, the oocyte growth was increased at the rate comparable to that induced by A23187 in the presence of thoracic ganglia. Hence, our in vitro study demonstrated that the stimulation of GSF released from thoracic ganglia is independent of GIH, and that the GIH has a dual function in oocyte growth stimulation and inhibition of Vg synthesis in the early stage of ovarian development. This expands our knowledge on the regulation of ovarian development in shrimp by GIH. Further in vivo studies in this novel aspect of GIH function will be useful for the improvement of shrimp ovarian maturation in the future.     

Ovarian maturation and haemolymphatic vitellogenin concentration of Pacific white shrimp Litopenaeus vannamei (Boone) fed increasing levels of total dietary lipids and HUFA

Two experiments were carried out to determine the quantitative lipid requirements for ovarian maturation of Litopenaeus vannamei (Boone) broodstock. The first experiment tested the effect of total dietary lipid (TDL) levels of 8.1%, 8.8%, 9.8% and 11.2% on the gonadosomatic index (GSI), hepatosomatic index (HSI), survival and body weight. The second experiment tested the effect of total dietary levels of highly unsaturated fatty acids (TDH) 0.6%, 1.3%, 2.0% and 2.7% on the same parameters mentioned above and on the haemolymphatic vitellogenin (vg) concentration. The GSI was higher in females fed 8.1% TDL as compared with those fed 8.8% TDL (P ≤ 0.05), and higher in females fed 8.1% and 8.8% TDL than in those fed 9.8% and 11.2% TDL. Increasing TDH levels led to increasing levels of highly unsaturated fatty acids (HUFA) in the ovaries (r² = 0.999). However, the GSI was not affected by dietary treatments (P > 0.05). The vg concentration was higher in animals receiving 2.0% TDH as compared with those receiving 2.7% TDH. In conclusion, high TDL levels affected ovarian maturation in a negative way, why TDH levels had no pronounced effect on ovarian maturation within the range tested in this study.