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Purification and characterization of vitellogenin on Giant Grouper (Epinephelus lanceolatus) and Tiger Grouper (Epinephelus fuscoguttatus)
Ahmad Daud Om GSK 0298
Specker and Sullivan. 1983. Vtg is specific to maturing females. in male fish particularly.A. Vtg. phosvitin. In general. and is very antigenic. and taken up by growing oocytes through receptor-mediated endocytosis (Ng and Idler. 1994). In the eggs. or exposure to estrogen or estrogenic compounds. Teleost Vtgs are phospholipoglycoproteins with high molecular mass of 300-640 kDa and are thought to be a dimer of two polypetide chains of 150 ± 250 kDa (Hara. yet it its synthesis can be induced by injection of. released into the bloodstream. LITERATURE REVIEW SUMMARY Vitellogenin (Vtg) is precursor of egg yolk in female oviparous vertebrates. During the process of vitellogenesis. Vtg is cleaved to lipovitellin. circulates at high levels in the plasma of maturing fish. is considered a biomaker for exposure to endocrine disrupting contiminants (EDCs) (Tyler et al. and -component which are used as nutritive material by the developing embryos and larvae (Wallace. 1998) yet several studies have shown Vtg cross-reactivity between unrelated species. Thus the presence of eleveted levels of plasma Vtg. 1994). 1987). could be an easily identifiable marker for the onset of maturation in female fish (Idler et al. antibodies recognize Vtg cross-reactive epitopes between related species (Nilsen et al. 1981). 1996). even with 2 . 1985. This protein is easily purified. Males and immature fish do not normally produced significant amounts of Vtg. Vtg is ussually measured by immunoassay and detected in Western blot using specific antibodies raised againts purified Vtg. Vtg is syntesized in the liver in response to circulating estrogen. which has facilatated development of immunoassays for Vtg detection in numerous fish species (Specker and Sullivan. Mommesen and Walsh 1988). Vitelogenins have been purified and characterized from a number of fish species.
which is naturally depend on the age. They are particularly popular for the live fish market. malabaricus). To manage the technology culture of these species properly. cobia. In 2005. 1987). P. environment and season. and Humpback grouper (Cromileptes altivelus). Indonesia and Thailand are known to be conducting research on the breeding of this species. pompano and tuna) is increasingly becoming popular in many parts of the world. and sells Giant grouper fingerlings in South East Asia. the amount of hatchery reared fish available is thought to be small. In contrast. feed. R. groupers. Fast growth and high economic value are the advantages of this species compare with other grouper species. The aquaculture of high-value species (e. snappers.monoclonal antibodies (Covens et al.g. mainly in Hongkong. its life history. and Parry-Jones. 1999). Most of the grouper aquaculture production came from Asia. followed by Indonesia and Thailand. The success of seed production activity depends on the gonad maturity of broodstock. size. The most common species for grouper culture are Orange-spotted grouper (Epinephelus coioides). there can be a lack of crossreactivity even between closely related species (Watts et al.4 tonnes of Giant grouper in 2004 (Lau. 3 . Groupers are one of the improtant species for aquaculture in both marine and brackishwater pond and off-shore cages.F. and continues to develop rapidly in the Southeast Asian region (Rimmer. is two of the grouper species which is has bright prospect in the market. with China and Taiwan topping the list. Although Taiwan has had some success in breeding. It is much sought after for the live reef fish trade with Hong Kong import statistics revealing import of around 2. 2004). Giant grouper (Epinephelus lanceolatus) and Brown-marbled grouper or Tiger grouper (Epinephelus fuscoguttatus). 2003).Singapore and Taiwan. Asia produced a total 62. China. Malabar grouper (Epinephelus.088 MT representing 24% of the total world fisheries production for groupers (FAO. 2007). and proportion of traded individuals from wild versus hatchery production is unknown.
(2007). (1987). To manage the technology culture of these species properly. size. Loof AD. Mem. Determination of sexual maturation stages of Atlantic Salmon (Salmo salar) capture at sea. L.J. Ollevier F. which is naturally depend on the age.. must be understood. Hwang.. including spawning and larval development. Crim. The success of seed production activity depends on the gonad maturity of broodstock. and Reddin. FAO. physiocochemical and structural studies. (1987) A compartive study of some properties of Vitellogenin (Vg) and yolk proteins in a number of freshwater and marine teleost fishes. Fish. 4 .including spawning and larval development. Therfore in order to develop grouper culture technology the seed production technology must be prepared. S. since this species suitable for culture.D. D. must be understood. this study is proposed sequentially to develop culture technology of this species. Can J. the development of hatchery techniques is essential to produce large supplies of fries. Aquat. Fisheries statistic database. Hokkaido University 34: 1-59. its life history. (1981). References Covens M. Furthermore. Sci. feed. Comp Biochem Physiol 88B:75-80. Food and Agriculture Organization of the United Hara. Studies on female-specific serum proteins (vitellogenin) and yolk proteins in teleost : immunochemical. Fac. Idler. Therefore in order to fulfill seed demand. Fish. environment and season.R. either for culture too.. 38: 405-413. Covens L.W.
Williams. Donaldson EM (eds). pp. Vitellogenesis and oocyte assembly. purification and antigenic cross-reactivity in three teleost species. (1988). In: Hoar WS.(1983). 2004. Academic Press. Donaldson EM (eds). Van der Eerden B. M. Measurement of vitellogenin. Comp Biochem Physiol 134B: 467-476. Developmental biology: a comprehensive synthesis. Sun B (2003). Mommesen TP. Arukwe A. Academic Press. vol 9A. Ottawa. Vitellogenin isolation. 5 . Randall DJ. Anal Bioanal Chem 378:621-633. Watts M. Monoclonal and polyclonal antibodies against fish vitellogenin for used in endocrine disruptor screening. Reproduction. Vitellogenesis and oocyte growth in non-mammalian vertebrates . Yolk formation and differantion in teleost fishes. in a wide variety of cyprinid fish. Plenum Press. Oogenesis. Section 1 ± Introduction. Berg K. Australian Center for International Agricultural Research.McBride and K. S.C. Randall DJ. Reproduction.. Tyler CR. Comp Physiol 166:418-426. Wallace. pp 304-315. Australia. Endocrine tissue and hormons. a biomaker for exposure to oestrogenic chemicals. 1-5. editors). Vitellogenesis in fishes: status and prospective in comparative endocrinplogy. (1998). (1985). In: Browder LW (ed). Fish physiology. Pryce A. pp 127-177. Canberra. Specker JL. New York pp 373 404.Ng TB and Idler D R. London New York. Jobling S et al (1996). Pankhurst NW. Nilsen BM. vol 9A. J.A. Walsh PJ. National Research Council Canada. vol 1. Sullivan CV (1994). Rimmer. New York pp 347 -406. In Advances in Grouper Aquaculture (M.A. Fish physiology. Rimmer. In : Hoar WS. Endocrine tissue and hormons.
estradiol.B.SDS-PAGE 3) To developed and Enzyme Linked Immunosorbant Assay (ELISA) to quantify females gaint grouper and tiger grouper Vtg. 75 mg/liter. y y y Use Estradiol : peanut oil (1:9) Individual fish were anesthetized by immersion in MS 222. c. 20 mg/kg 6 . y Sample will centrifuge at 3000 rpm / 4oC / 15 min and keep in -30oC prior to analysis. Sample of blood (500 µl) before induction and as much as possible when collected to the first estradiol injection and 2-4 days after last estradiol injection. SPECIFIC OBJECTIVE 1) To isolate and purify the Vtg . twice a week for a period of three weeks. 4) Test the effectiveness of Estradiol on sex reversal of Giant Grouper and Tiger Grouper 5) To developed a rapid and sensitive biochemical test for gender and maturity of adult female grouper. y 10 of Tiger Grouper (1 kg) and Giant Grouper (15 kg) will induce to produce Vtg by intraperitoneal or intramuscular injection of 17 body weight. METHODOLOGY Experiment 1: Purification of vitellogenin on Tiger and Giant Grouper 1) Experimental fish and administration of estradiol.Gel Filtration Chromatography 2) To characterize the purified Vtg .
for 30 min at 5oC. and fix with Coomassie brilliant blue in a methanol-acetic solution (50:20:50) y y Destaining carried out in methanol-acetic acid-water solution (35:10:55) A log-linear plot of the mobility of the MW standards will construct and use to develop a regression equation for calculating the relative mobility of grouper Vtg. 7 . y y y Isolate by anion exchange and gel filtration chromatography. and further purified by gel filtration chromatography y Molecular weight of Grouper will determine by gel filtration chromatography calibrated with molecular weight markers (158 ± 669 kDa). pH 8. y Thereafter. After electrophoresis. gel will washed and incubate in 50 mM Tris-HCL buffer. y y y The molecular weight standards from 67 to 669 kDa will use. gel will wash. concentrated by centrifugal ultrafiltration. 2 ml of ultrfiltered plasma will applied to DEAE column Vtg will elute with a linear gradient of 0 ± 550 mM NaCl in 50mM Tris HCl buffer. pH 9.0 y Fraction around the peak will pooled.0). Experiment 2 : Characterization of vitellogenin on Tiger and Giant Grouper 1) SDS-PAGE and staining y Sodium dodecyl sulfate 4 -15% polyacrylamide gradient gel electrophoresis (SDS-PAGE) of purified grouper Vtg and blood plasma from vitellogenic and non vitellogenic fish (control).2) Purification of Vtg y y Plasma sample will prepare and clarified by centrifugal ultrafiltration Dilute in seven volume of 50 mM Tris-HCL buffer (pH 8. Place into an ultrafiltration tube and centrifuged at 3000 g. Gel will run in Bio-Rad Mini ± Cell apparatus at 100 V for 240 min.
2) Western Blot y Protein were electroblotted from the gel onto a polyvinylidene fluoride (PVDF) membrane using a Blot module. excised from the membranes.1% phenol.001% NuPAGE antioxidant.1% thioglycolic acid for 20 hours at 110oC. 0. Millipore) ti remove buffer y Vtg will hydrolyzed in 6 N HCL. will crried out on a amino acid analyzer using ion-exchange chromatography. y The transfer will carried out at 15 V overnight at 4oC using NuPAGE transfer buffer with 10% methanol and 0. y Amino Acid composition of Vtg. 8 . y Detection will perform at two wave length. post-column derivatization with ninhydrin. pass through Ultra centrifugal filters devices (10 kDa molecular weight. and inserted into the sequencing cartridge of an Applied Biosystems automated protein sequencer 5) Enzyme-linked immunosorbent assay (ELISA) of Vtg y Antibody and antigen dilution curves will initially performed to optimize reagent concentrations in the assay. y Detection with a protein detector Western blot kit. 570 nm and 440 nm. electroblotted onto Problot membranes. y Stained with Coommassie blue. 4) Vtg N-terminal sequencing y N-termonal sequencing analyses of Vtg will repurified by two- dimensional PAGE to remove products of Vtg degradation during storage. 3) Amino Acid analysis y Purified Vtg solution will successively wash with 4oC Ultrapure water. 0. cut-off.
3 unknown fish as low concentration implant with E2 Estradiol (0. Terengganu.01 M NaPO4. and 0. Besut.15 M NaCl. 96 microtiter plates will coat with 200 µl/ well of either a 500 ng Vtg /ml solution in carbonate buffer (0.75 to 5000 ng/ml Plasma dilution will also make in PBST-NGS. Experiments 3. y Giant grouper (body weight 35. 9 .05 mg/kg).05% Tween-20. The Effects of estradiol in undetected sex of grouper to female y 10 fish from undetected sex will choose for this experiment.0±45 kg) and Tiger grouper (8 ± 10 kg) used in this study were reared separately in 150 ton indoor tank at FRI Tg. pH 7. y Treatment: 3 unknown fish as control.4) y y y Vtg standard concentration ranged from 9. Demong. 0. y Marking all the fish with microchip and making hormone pellet with using cholesterol powder + Estradiol. y 9 fish reared in normal condition will implant with hormone E2 Estradiol y Every month.y Standard will make by serially diluting a stock solution of purified Vtg grouper in PBST (0. y Implant will follow in every month until the sixth month. Implant with pellet hormone and blood will sampling y Keep in -20oC prior analysis.5 mg/kg). another 3 unknown as high concentration implant with E2 Estradiol (0.05 M Sodium carbonate. were culture together in one tank.6) or an equal concentration of bovine serum albumin y y Incubate for 2 hour at 37oC 0r overnight at 4oC Blocked the reaction by addition of 350 µl of 5% NGS in carbonate buffer followed by incubation for 1 h at 37oC. pH 9.
5 mg/kg) 3 pcs Statistics Statistical analysis of data consisted of analysis of variance (ANOVA) followed by Duncan¶s multiple range test or Student¶s t-test.05 mg/kg) 3 pcs High dose Estradiol (0.Sex reversal of Giant Grouper (9 pcs) / Tiger grouper Control (3 pcs) Low dose Estradiol (0.05. Statistical significance was inferred at P < 0. 10 .
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