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.     

Development of an enzyme-linked immunosorbent assay (ELISA) for vitellogenin measurement in greenback flounder Rhombosolea tapirina

Vitellogenin (Vtg) was purified from the plasma of 17-estradiol (E₂)-injected male greenback flounder,Rhombosolea tapirina. The molecular weight of the native Vtg was estimated by gel filtration as 540 kD. SDS-PAGE and Western blotting analyses indicated that this protein consisted of three bands with molecular weights of 155, 104, 79 kD, respectively. A polyclonal antibody against the highest molecular weight band of putative Vtg was generated in sheep and an indirect antibody-capture competitive enzyme-linked immunosorbent assay (ELISA) was developed. The assay was validatedfor plasma Vtg measurement in greenback flounder. Serial dilutions of plasma from vitellogenic females parallelled the standard Vtg curve, whereas no cross-reaction was observed with the plasma of males in the ELISA. The Vtg ELISA was used to assess the induction of Vtg by E₂ in vivo in males. The induction of Vtg in greenback flounder showed a time- and dose-dependent response as in other species. In E₂-treated fish, detectable levels of Vtg were first found at 48 h, and reached a peak at 96 h post-injection. Plasma levels of Vtg increased as the E2 dose increased with a threshold of 0.1 mg kg^–1.     

Application MALDI TOF on protein identification of vitellogenin in giant grouper (Epinephelus lanceolatus)

A new proteomics technology has been implemented to study the protein repertoires of developing oocytes of giant grouper (Epinephelus lanceolatus). Knowledge of the chemical composition and physiochemical properties of vitellogenin (Vtg) is necessary to interpret the functional and biological properties attributed during ovulation. Vtg, as a biomarker indicator in sex determination, has been analyzed to determine the sex and maturational status of fish in the absence of the gonad tissue. A male giant grouper was induced by 2 mg/kg of 17ß-estradiol (E2), and blood was sampled at days 0, 1, 3, 5, and 10. SDS-PAGE 1D electrophoresis was used to analyze Vtg protein, and Vtg identification was done with 4800 Plus MALDI TOF/TOF? mass spectrophotometer (Applied Biosystems/MDS SCIEX, USA). Meanwhile, MS/MS de novo sequencing identified the proteins by matching sequences of tryptic peptides to the known sequences of other species. Vtg was confirmed by MASCOT at 95 % significant level, and molecular mass was 187 kDa. Protein resolved on SDS-PAGE as a double band of approximately the same mass as determined with MALDI-TOF. The N-terminal sequences and identification of Vtg were also determined. The potential of using MS methods to understand the structure and function of Vtg is discussed.     

Identification,partial characterization,and use of grey mullet ( Mugil cephalus ) vitellogenins for the development of ELISA and biosensor immunoassays

Vitellogenin (Vtg) has proven to be a sensitive and simple biomarker in determining sex, sexual maturity, and xenoestrogenic effects in fish. Thus, our investigation has been focused on identification, partial characterization, and quantification of grey mullet (Mugil cephalus) Vtg through the use of a variety of biochemical and immunological analytical techniques. Mullet is considered both a promising aquaculture candidate and an important species for improving sediment quality in polyculture systems. In the first part of this work, grey mullet Vtg was purified from plasma of 17β-estradiol (E2)-induced male fish by a one-step chromatographic protocol, and partially characterized. Specific polyclonal antibodies were then raised against the mullet Vtg, and both an indirect ELISA and an optical immunosensor were set up and validated to quantify plasma Vtg. The indirect ELISA and the optical immunosensor assay developed showed linear measuring in the range 56.8–1047.1 ng mL−1 and 70–739 ng mL−1 Vtg concentrations in standard solutions, respectively. The results obtained suggest that the indirect ELISA allows Vtg detection over a wide dynamic range, thus resulting more suitable for rapid and sensitive sample screening. Therefore, we suggest that the direct immunosensor is a promising tool which needs more investigation to improve the sensitivity.     

Gender determination in the Paiche or Pirarucu (<Emphasis Type="Italic">Arapaima gigas</Emphasis>) using plasma vitellogenin, 17β-estradiol,and 11-ketotestosterone levels

Arapaima gigas is an air-breathing giant fish of Amazonian rivers. Given its great economic and cultural importance, the aquaculture development of this species represents an evident solution to face the decline of wild populations. In captivity, reproduction occurs generally in large earthen ponds where stocks of a few tens of brooders are maintained together at the beginning of the rainy season (December–March in the Peruvian Amazon). Fry production relies on the spontaneous formation of male and female pairs, which build a nest, delimit a territory and guard the offspring for at least 20 days from other congeners and predators. However, as sex determination of A. gigas is not possible by morphological criteria, it is very difficult to optimize reproduction conditions and fry production in each pond, which seriously hampers the culture of this species. This situation prompted us to develop sexing methodologies based on (1) the detection of female specific plasma Vitellogenin (Vtg) using an enzyme immuno assay (EIA), and (2) the determination of plasma 17β-estradiol and 11-ketotestosterone levels for immature specimens. The Vtg purification was performed by electro-elution after polyacrilamide gel electrophoresis (PAGE) from plasma of 17β-estradiol treated A. gigas juveniles. Two different Vtg molecules were isolated, (Vtg₁ and Vtg₂) with 184 and 112 kDa apparent molecular masses, respectively, and two antibodies were raised in rabbits for each Vtg molecule. Adult fish were 100% accurately sexed by Vtg EIA, while 100% of immature fish and 95% of adults were accurately sexed by 17β-Estradiol and 11-Ketestosterone ratios. We also observed different color pattern development in male and female adult fish (6-year-olds) around the reproductive period.     

Carp (Cyprinus carpio) lipovitellin is a highly stable phospholipoglycoprotein with the same immunogenicity as vitellogenin

Vitellogenin (Vtg) induction in carp (Cyprinus carpio) is a commonly used biomarker for oestrogenic contamination. However, the accurate quantification of Vtg was challenged because the easy degradation of Vtg standard would change the standard curves of the immunoassays. In this study, three yolk proteins were purified from carp ovarian extracts by a two‐step chromatographic method. The purified proteins were characterized as phospholipoglycoproteins with molecular weights of approximately 416, 398 and 383 kDa. In SDS‐PAGE, the purified proteins appeared as a major band of approximately 110 kDa and several faint bands. Based on these characteristics, purified proteins were identified as carp lipovitellin (Lv). Immunological analysis showed that anti‐Vtg antiserum reacted with the purified Lv. The results of stability analysis revealed that even heated at 60°C for 60 min, the electrophoretic patterns of carp Lv in native‐PAGE and SDS‐PAGE did not have a significant difference compared with the Lv solution stored at 4°C. Therefore, the purified carp Lv was confirmed to have similar immunogenicity with Vtg and possess exceptionally high stability, which would be helpful for the development of robust immunoassays for accurate quantification of carp Vtg.     

Purification of vitellogenin from the air breathing catfish, Clarias gariepinus

Vitellogenin (Vtg) is a female specific glycophospholipoprotein which can be induced both in male and female with estradiol and xeno-estrogens. The basic theme behind the purification of vitellogenin from the fish is to understand the evolutionary relationship and for the purification and characterization of the Vtg receptor. The male catfish, Clarias gariepinus was administrated with estradiol over a period of time for the synthesis of Vtg and the serum was collected. The Vtg was purified from the serum using a two step chromatographic technique. The serum was passed on to DEAE-ion exchange column and the protein was eluted using a salt gradient. The fractions containing the Vtg were pooled and passed onto a gel permeation chromatography column and the pure protein was obtained. The molecular weight is around 200 kDa on the SDS-PAGE and around 520 kDa on the native gel electrophoresis.     

Liver melanomacrophage centres as indicators of Atlantic bluefin tuna,Thunnus thynnus L. well‐being

Melanomacrophage centres (MMCs), located in different organs of non‐mammalian vertebrates, play a role in the destruction, detoxification or recycling of endogenous and exogenous materials. Cytochrome P450 monoxygenase 1A (CYP1A) is involved in xenobiotics biotransformation, and its liver expression is considered as a biomarker for detecting exposure to environmental pollutants. Atlantic bluefin tuna (ABFT), Thunnus thynnus L., liver samples were collected from: wild animals caught in the eastern Atlantic; juveniles reared in the central Adriatic; juveniles reared in the northern Adriatic; adults reared in the western Mediterranean. The samples were processed for basic histology, histochemistry and for CYP1A immunodetection. An unexpected high density of MMCs, containing ferric iron and lipofuscin–ceroids, was detected in the juveniles sampled in the northern Adriatic Sea. These individuals showed also a strong anti‐CYP1A immunopositivity in hepatocytes and in the epithelium of bile ducts. This study supports the utility of MMCs as biomarkers of fish ‘health status’ and gives concern for a potential contaminant accumulation in ABFT.     

彭泽鲫F_1、F_2代雌雄鱼VtgB和ZP2表达及卵黄蛋白原含量差异研究

雌核发育彭泽鲫后代理论上应为全雌群体,前期研究发现实验室养殖彭泽鲫F1代(相比池塘养殖)和实验室高密度养殖F2代(1.28尾/L,相比低密度0.64尾/L)组中出现了高比例的雄鱼。之前研究认为Vtg B和ZP2基因是雌性特异性表达的基因,本试验对F1、F2代雌雄鱼Vtg B和ZP2表达及卵黄蛋白原含量差异进行研究,结果发现,实验室养殖F1代雄鱼性腺中Vtg B和ZP2的表达分别极显著和显著高于雌鱼(P0.01,P0.05);池塘养殖F1代雄鱼性腺中Vtg B和ZP2的表达分别极显著高于和低于雌鱼(P0.01);F2代低密度养殖组中雄鱼性腺中ZP2的表达极显著高于雌鱼(P0.01)。实验室养殖F1代雄鱼肝胰脏中Vtg B和ZP2的表达分别极显著高于和低于雌鱼(P0.01);池塘养殖F1代雄鱼肝胰脏Vtg B和ZP2的表达均极显著低于雌鱼(P0.01);F2代雄鱼肝胰脏中Vtg B和ZP2的表达极显著高于雌鱼(P0.01)。F1代实验室养殖雄鱼全鱼卵黄蛋白原含量极显著低于雌鱼(P0.01);F1代池塘养殖雄鱼性腺、肝胰脏中卵黄蛋白原含量分别极显著高于和低于雌鱼(P0.01);F2代雄鱼性腺、肝胰脏中卵黄蛋白原含量极显著高于雌鱼(P0.01)。本研究表明,彭泽鲫Vtg B和ZP2基因并非雌性特异性表达,且不同的养殖方式、密度影响雌雄鱼Vtg B和ZP2表达及卵黄蛋白原含量。     

Development of an efficient bioreactor system for delivering foreign proteins secreted from liver into eggs with a vitellogenin signal in medaka Oryzias latipes

In this study, we developed a novel bioreactor system to deliver and accumulate foreign proteins in eggs using medaka fish Oryzias latipes with the aid of a partial sequence of vitellogenin (Vtg). In teleost fish, Vtg, the hepatically generated precursor of egg yolk proteins, is secreted into the bloodstream and then taken up into eggs. We predicted in silico a probable region (Vtg signal) of Vtg that mediates transportation of proteins from the liver into eggs. Then, we established two transgenic lines expressing the fused proteins including the Vtg signal and each reporter gene, enhanced green fluorescent protein (EGFP) or firefly luciferase (LUC)-fused EGFP, in the liver driven by a liver-specific choriogeninH (chgH) promoter. Each reporter signal was detected from the fertilized eggs spawned by the transgenic females, showing successful transportation of the proteins into the eggs with the Vtg signal. This is the first report demonstrating that the Vtg signal has capability to deliver exogenous proteins into eggs. Because Vtg is a highly conserved protein among most of oviparous organisms, our findings hold promise for establishing bioreactor systems viable in a wide range of organisms.

     

Indexing serum biochemical attributes of Lutjanus argentimaculatus (Forsskal, 1775) to instrument in health assessment

The present study was aimed to establish reference data for nine serum biochemical attributes (glucose, protein, albumin, globulin, albumin/globulin ratio, cholesterol, triglycerides, serum glutamate oxaloacetate transaminase [SGOT] and serum glutamate pyruvate transaminase [SGPT]) of a supreme aquaculture candidate, namely Lutjanus argentimaculatus. Further, the influence of ecotype (cage–wild), condition index and gender on biochemical attributes was evaluated. The results have shown that gender and condition index had no significant effect in the studied age group, whereas ecotype had a significant role, except on serum glucose levels, suggesting the prime importance of estimating ecotype‐specific reference intervals for interpreting fish health. Intra‐ecotype variability of serum biochemical attributes, namely serum protein, globulin, cholesterol and triglycerides, was less, suggesting the possibility of using these parameters as health indices within a specific ecotype. Consistency in terms of the least intra‐ecotype variability coupled with PCA results points serum protein as the best ecotype‐specific health index among the different parameters studied. Altogether, the results of the present study provide thought‐provoking insights on serum biochemistry of L. argentimaculatus as a platform for the health management of this supreme aquaculture candidate.     

Biochemical and Serological Characterization of Flavobacterium columnare from Freshwater Fishes of Eastern India

Flavobacterium columnare is an important pathogen of freshwater fishes often causing high mortality. Seven strains of F. columnare have been isolated from gill necrosis, skin lesions and internal organs of Catla catla, Labeo rohita, Cirrhinus mrigala, Carassius auratus, Anabas testudineus and Clarias batrachus and characterized by biochemical reactions and serological tests viz indirect enzyme‐linked immunosorbent assay (ELISA), Dot‐ELISA, agglutination test. All the strains showed binding to Congo red dye as well as hemolysis. All the seven strains were susceptible to amikacin, gentamycin and ofloxacin. In vivo LD₅₀ dose of virulent strain MS2 was found to be 6 × 10⁴ CFU/mL after experimental infection to L. rohita. Serologically all the seven strains showed a positive result to agglutination, Dot‐ELISA and indirect ELISA. The agglutination titer was found to be in the range of 256‐131,072. The lysozyme activity of hyperimmune sera was found to be 39.37 ± 0.80 units/mL. This is the first extensive study that report about the various strains of F. columnare from freshwater fishes of Eastern India and their differentiation by biochemical and serological methods.     

Correlation between oocyte development and plasma concentrations of steroids and vitellogenin in greenback flounder Rhombosolea tapirina

The development of oocytes in association with changes in plasma concentrations of vitellogenin (Vtg), 17β-estradiol (E₂) and testosterone (T) was investigated in maturing female greenback flounder Rhombosolea tapirina over the first part of a reproductive season. During vitellogenesis, increasing oocyte size was accompanied by the sustained elevation of plasma Vtg and E₂. There was a marked increase in T towards the end of vitellogenesis.     

Development of an ELISA to measure the humoral immune response of hybrid striped bass Morone chrysops×M. saxatilis to Streptococcus iniae

A previously developed monoclonal antibody (mAb) specific for the heavy chain of hybrid striped bass (HSB) Morone chrysops×M. saxatilis immunoglobulin was used in an assay to detect the humoral response to antigens of Streptococcus iniae. In order to validate this assay, an anti‐S. iniae antibody was produced in HSB by immunization with formalin‐killed cells of S. iniae mixed with Freund's complete adjuvant (FCA). After boosting with cells mixed with Freund's incomplete adjuvant (FIA), fish were challenged with live S. iniae by i.p. injection. This resulted in mortalities of 100%, 13% and 7% for non‐immunized, immunized and non‐challenged fish respectively. Live S. iniae were recovered only from moribund non‐immunized challenged fish, indicating that the immunization conferred protection against S. iniae. Sera were taken at 28 and 42 days post challenge and anti‐S. iniae antibody was measured by both agglutination and indirect ELISA. Titres of anti‐S. iniae antibody increased only in the immunized group as measured by agglutination and ELISA. Serum complement measured in the final bleeding was significantly higher in the immunized group. Western blotting using immune HSB serum indicated that the predominant antigen in this case was the high molecular weight polysaccharide of S. iniae.     

Lipid transport in female Fundulus heteroclitus during the reproductive season

Lipid transport during the reproductive cycle of seasonally spawning teleosts has been well described. However, there is little information on lipid transport in a multiple spawning teleost. We examined lipid transport during the reproductive cycle in Fundulus heteroclitus, a teleost that spawns in a semilunar fashion. Plasma levels of triglycerides more than double prior to spawning events and then drop off sharply around the time of spawning. Plasma levels of total cholesterol also changed throughout the spawning cycle, but those changes were not so pronounced. The fraction of lipid carried by very low density lipoprotein (VLDL) and low density lipoprotein (LDL) was greatest prior to spawning events, while high density lipoprotein (HDL) carried the greatest portion of lipid during the spawning period. Vitellogenin (Vtg) was detected prior to spawning events, but it never represented more than 5% of total plasma lipid, making it very unlikely that Vtg is responsible for the changes in plasma lipids that we observed. It is also unlikely that Vtg is solely responsible for transport of lipid to growing ovaries because it carries only a very small portion of plasma lipid. This is the first study we are aware of, that tracks changes in plasma lipids of a multiple spawning teleost with a great enough sampling frequency to resolve single spawning events. This revised version was published online in August 2006 with corrections to the Cover Date.     

Cloning,expression, and induction by 17-β estradiol (E<Subscript>2</Subscript>) of a vitellogenin gene in the white cloud mountain minnow <Emphasis Type="Italic">Tanichthys albonubes</Emphasis>

Vitellogenins (Vtgs), the precursors for the yolk proteins, are very important for the embryonic development of teleosts, and have also been studied extensively as biomarkers for environmental estrogenic mimics. The cDNA for a Vtg was isolated from the liver of the female white cloud mountain minnow (Tanichthys albonubes) by 3′- and 5′-RACE methods. It is 4,171 bp in full length, and encodes a putative protein of 1,326 amino acids. This putative Vtg, designated as wcmmVtg, contains complete portions of LVI and PV, but lacks the C-terminal half of LVII and thus belongs to type I vitellogenin. In addition to the liver of the female fish, wcmmVtg was also shown to be expressed in the ovary. During ovarian development, the mRNA expression of wcmmVtg in both the liver and ovary was continuously increased from the previtellogenic to late vitellogenic stages, but then decreased significantly at post-spawning stage. In the male fish, expression of wcmmVtg mRNA was induced in the liver by treatment with E₂ (10 and 100 ng/l) for 14 days. These results suggest that the Vtg originated from the ovary of the white cloud mountain minnow may also contribute to the accumulation of yolk proteins during oocyte growth, and that the male white cloud mountain minnow is sensitive to the estrogenic treatment in terms of Vtg mRNA expression, which could also be applied to monitor the environmental estrogenic mimics.     

Correlation between dietary lipid:protein ratios and plasma growth and thyroid hormone levels in juvenile Arctic charr, Salvelinus alpinus (Linnaeus)

Sexually immature Arctic charr, Salvelinus alpinus (Linnaeus), were fed one of five isoenergetic practical diets of differing lipid:protein ratios (0.98, 0.67, 0.41, 0.26, 0.19) for an 84‐day period to examine the influence of diet composition on growth, and growth hormone (GH) and thyroid hormone physiology. All five diets supported growth at approximately the same rate, but the diet with a lipid:protein ratio of 0.98 had the lowest weight gain and highest food conversion ratios. A GH enzyme‐linked immunosorbent assay (ELISA), developed for use with oncorhynchid fishes, was validated for use with Arctic charr. Plasma GH concentrations were significantly higher in fish fed the diet with a lipid:protein ratio of 0.98, and there were significant direct and inverse correlations between plasma GH levels and dietary lipid and protein content respectively. There were no significant differences in pre‐ and post‐prandial plasma GH concentrations for any group. There were significant post‐prandial elevations of plasma triiodothyronine (T₃) and thyroxine (T₄) for fish fed the lower lipid:protein ratio diets, but there were no differences related to the diets. The results are discussed in terms of GH as a factor in the regulation of lipid and protein homeostasis in fishes.     

Sea bass Dicentrarchus labrax (L.) bacterial infection and confinement stress acts on F‐type lectin (DlFBL) serum modulation

The F‐lectin, a fucose‐binding protein found from invertebrates to ectothermic vertebrates, is the last lectin family to be discovered. Here, we describe effects of two different types of stressors, bacterial infection and confinement stress, on the modulation of European sea bass Dicentrarchus labrax (L.) F‐lectin (DlFBL), a well‐characterized serum opsonin, using a specific antibody. The infection of the Vibrio alginolyticus bacterial strain increased the total haemagglutinating activity during the 16‐day testing period. The DlFBL value showed an upward regulation on the first, second and last days and underwent a slight downward regulation 4 days post‐challenge. In contrast, the effect of confinement and density stress showed a decrease in the plasma concentration of lectin, ranging from 50% to 60% compared with the control. The modulation of DlFBL is in line with the hypothesis that humoral lectins could be involved and recruited in the initial recognition step of the inflammation, which leads to agglutination, and the activation of mechanisms responsible for killing of the pathogens.