PhD.

Research Proposal

2010

Purification and characterization of vitellogenin on

Giant Grouper (Epinephelus lanceolatus) and
Tiger Grouper (Epinephelus fuscoguttatus)

By

Ahmad Daud Om
GSK 0298

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A. LITERATURE REVIEW SUMMARY

Vitellogenin (Vtg) is precursor of egg yolk in female oviparous vertebrates. During

the process of vitellogenesis, Vtg is syntesized in the liver in response to circulating
estrogen, released into the bloodstream, and taken up by growing oocytes through
receptor-mediated endocytosis (Ng and Idler, 1983; Mommesen and Walsh 1988). In
the eggs, Vtg is cleaved to lipovitellin, phosvitin, and β-component which are used
as nutritive material by the developing embryos and larvae (Wallace, 1985; Specker
and Sullivan, 1994).

Males and immature fish do not normally produced significant amounts of Vtg,
yet it its synthesis can be induced by injection of, or exposure to estrogen or
estrogenic compounds. Thus the presence of eleveted levels of plasma Vtg, in male
fish particularly, is considered a biomaker for exposure to endocrine disrupting
contiminants (EDCs) (Tyler et al. 1996). Vtg, could be an easily identifiable marker
for the onset of maturation in female fish (Idler et al. 1981).

Vitelogenins have been purified and characterized from a number of fish species.
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,
1987). Vtg is specific to maturing females. This protein is easily purified, circulates
at high levels in the plasma of maturing fish, and is very antigenic, which has
facilatated development of immunoassays for Vtg detection in numerous fish species
(Specker and Sullivan, 1994).

Vtg is ussually measured by immunoassay and detected in Western blot using

specific antibodies raised againts purified Vtg. In general, antibodies recognize Vtg
cross-reactive epitopes between related species (Nilsen et al. 1998) yet several
studies have shown Vtg cross-reactivity between unrelated species, even with

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monoclonal antibodies (Covens et al. 1987). In contrast, there can be a lack of cross-
reactivity even between closely related species (Watts et al. 2003).

The aquaculture of high-value species (e.g. groupers, snappers, cobia, pompano

and tuna) is increasingly becoming popular in many parts of the world, and
continues to develop rapidly in the Southeast Asian region (Rimmer, 2004).
Groupers are one of the improtant species for aquaculture in both marine and
brackishwater pond and off-shore cages. They are particularly popular for the live
fish market, mainly in Hongkong, China,Singapore and Taiwan. Most of the grouper
aquaculture production came from Asia, with China and Taiwan topping the list,
followed by Indonesia and Thailand. In 2005, Asia produced a total 62,088 MT
representing 24% of the total world fisheries production for groupers (FAO, 2007).
The most common species for grouper culture are Orange-spotted grouper
(Epinephelus coioides), Malabar grouper (Epinephelus. malabaricus), and
Humpback grouper (Cromileptes altivelus).

Giant grouper (Epinephelus lanceolatus) and Brown-marbled grouper or Tiger

grouper (Epinephelus fuscoguttatus), is two of the grouper species which is has
bright prospect in the market. Fast growth and high economic value are the
advantages of this species compare with other grouper species. It is much sought
after for the live reef fish trade with Hong Kong import statistics revealing import of
around 2.4 tonnes of Giant grouper in 2004 (Lau, P.F. and Parry-Jones, R. 1999).
Although Taiwan has had some success in breeding, and sells Giant grouper
fingerlings in South East Asia, the amount of hatchery reared fish available is
thought to be small, and proportion of traded individuals from wild versus hatchery
production is unknown. Indonesia and Thailand are known to be conducting research
on the breeding of this species.

The success of seed production activity depends on the gonad maturity of

broodstock, which is naturally depend on the age, size, feed, environment and
season. To manage the technology culture of these species properly, its life history,

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including spawning and larval development, must be understood. Furthermore, since
this species suitable for culture, the development of hatchery techniques is essential
to produce large supplies of fries, either for culture too.

Therfore in order to develop grouper culture technology the seed production

technology must be prepared. The success of seed production activity depends on the
gonad maturity of broodstock, which is naturally depend on the age, size, feed,
environment and season. To manage the technology culture of these species
properly, its life history, including spawning and larval development, must be
understood. Therefore in order to fulfill seed demand, this study is proposed
sequentially to develop culture technology of this species.

References

Covens M, Covens L, Ollevier F., Loof AD. (1987) A compartive study of some
properties of Vitellogenin (Vg) and yolk proteins in a number of freshwater
and marine teleost fishes. Comp Biochem Physiol 88B:75-80.

FAO, (2007). Fisheries statistic database. Food and Agriculture Organization of the
United

Hara, (1987). Studies on female-specific serum proteins (vitellogenin) and yolk

proteins in teleost : immunochemical, physiocochemical and structural studies.
Mem. Fac. Fish. Hokkaido University 34: 1-59.

Idler,D.R., Hwang, S.J., Crim, L.W. and Reddin, D. (1981). Determination of sexual
maturation stages of Atlantic Salmon (Salmo salar) capture at sea. Can J. Fish.
Aquat. Sci. 38: 405-413.

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Ng TB and Idler D R.(1983). Yolk formation and differantion in teleost fishes. In :
Hoar WS, Randall DJ, Donaldson EM (eds). Reproduction. Endocrine tissue
and hormons. Fish physiology, vol 9A. Academic Press, New York pp 373
-404.

Nilsen BM, Berg K, Arukwe A. (1998). Monoclonal and polyclonal antibodies

against fish vitellogenin for used in endocrine disruptor screening. Anal
Bioanal Chem 378:621-633.

Mommesen TP, Walsh PJ. (1988). Vitellogenesis and oocyte assembly. In: Hoar
WS, Randall DJ, Donaldson EM (eds). Reproduction. Endocrine tissue and
hormons. Fish physiology, vol 9A. Academic Press, New York pp 347 -406.

Rimmer, M.A., 2004. Section 1 – Introduction. In Advances in Grouper Aquaculture

(M.A. Rimmer, S.McBride and K.C. Williams, editors). Australian Center for
International Agricultural Research, Canberra, Australia. pp. 1-5.

Specker JL, Sullivan CV (1994). Vitellogenesis in fishes: status and prospective in

comparative endocrinplogy. National Research Council Canada, Ottawa, pp
304-315.

Tyler CR, Van der Eerden B, Jobling S et al (1996). Measurement of vitellogenin, a

biomaker for exposure to oestrogenic chemicals, in a wide variety of cyprinid
fish. J. Comp Physiol 166:418-426.

Wallace, (1985). Vitellogenesis and oocyte growth in non-mammalian vertebrates .

In: Browder LW (ed). Developmental biology: a comprehensive synthesis, vol
1. Oogenesis. Plenum Press, London New York, pp 127-177.

Watts M, Pankhurst NW, Pryce A, Sun B (2003). Vitellogenin isolation, purification

and antigenic cross-reactivity in three teleost species. Comp Biochem Physiol
134B: 467-476.

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 B. SPECIFIC OBJECTIVE

1) To isolate and purify the Vtg - Gel Filtration Chromatography

2) To characterize the purified Vtg - SDS-PAGE
3) To developed and Enzyme Linked Immunosorbant Assay (ELISA) to quantify
females gaint grouper and tiger grouper Vtg.
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.

c. METHODOLOGY

Experiment 1: Purification of vitellogenin on Tiger and Giant Grouper

1) Experimental fish and administration of estradiol.

 10 of Tiger Grouper (1 kg) and Giant Grouper (15 kg) will induce to produce
Vtg by intraperitoneal or intramuscular injection of 17 β estradiol, 20 mg/kg
body weight, twice a week for a period of three weeks.
 Use Estradiol : peanut oil (1:9)
 Individual fish were anesthetized by immersion in MS 222, 75 mg/liter.
 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.
 Sample will centrifuge at 3000 rpm / 4oC / 15 min and keep in -30 oC prior to
analysis.

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2) Purification of Vtg
 Plasma sample will prepare and clarified by centrifugal ultrafiltration
 Dilute in seven volume of 50 mM Tris-HCL buffer (pH 8.0). Place into an
ultrafiltration tube and centrifuged at 3000 g.
 Isolate by anion exchange and gel filtration chromatography.
 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 8.0
 Fraction around the peak will pooled, concentrated by centrifugal
ultrafiltration, and further purified by gel filtration chromatography
 Molecular weight of Grouper will determine by gel filtration chromatography
calibrated with molecular weight markers (158 – 669 kDa).

Experiment 2 : Characterization of vitellogenin on Tiger and Giant Grouper

1) SDS-PAGE and staining

 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).
 The molecular weight standards from 67 to 669 kDa will use.
 Gel will run in Bio-Rad Mini – Cell apparatus at 100 V for 240 min.
 After electrophoresis, gel will washed and incubate in 50 mM Tris-HCL
buffer, pH 9, for 30 min at 5oC.
 Thereafter, gel will wash, and fix with Coomassie brilliant blue in a
methanol-acetic solution (50:20:50)
 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.

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2) Western Blot
 Protein were electroblotted from the gel onto a polyvinylidene fluoride
(PVDF) membrane using a Blot module.
 The transfer will carried out at 15 V overnight at 4oC using NuPAGE
transfer buffer with 10% methanol and 0.001% NuPAGE antioxidant.
 Detection with a protein detector Western blot kit.

3) Amino Acid analysis

 Purified Vtg solution will successively wash with 4oC Ultrapure water,
pass through Ultra centrifugal filters devices (10 kDa molecular weight,
cut-off, Millipore) ti remove buffer
 Vtg will hydrolyzed in 6 N HCL, 0.1% phenol, 0.1% thioglycolic acid for
20 hours at 110oC.
 Amino Acid composition of Vtg, will crried out on a amino acid analyzer
using ion-exchange chromatography, post-column derivatization with
ninhydrin.
 Detection will perform at two wave length, 570 nm and 440 nm.

4) Vtg N-terminal sequencing

 N-termonal sequencing analyses of Vtg will repurified by two-
dimensional PAGE to remove products of Vtg degradation during
storage, electroblotted onto Problot membranes.
 Stained with Coommassie blue, excised from the membranes, and
inserted into the sequencing cartridge of an Applied Biosystems
automated protein sequencer

5) Enzyme-linked immunosorbent assay (ELISA) of Vtg

 Antibody and antigen dilution curves will initially performed to optimize
reagent concentrations in the assay.

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  Standard will make by serially diluting a stock solution of purified Vtg
grouper in PBST (0.01 M NaPO4, 0.15 M NaCl, and 0.05% Tween-20,
pH 7.4)
 Vtg standard concentration ranged from 9.75 to 5000 ng/ml
 Plasma dilution will also make in PBST-NGS.
 96 microtiter plates will coat with 200 µl/ well of either a 500 ng Vtg /ml
solution in carbonate buffer (0.05 M Sodium carbonate, pH 9.6) or an
equal concentration of bovine serum albumin
 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.

Experiments 3. The Effects of estradiol in undetected sex of grouper to female

 10 fish from undetected sex will choose for this experiment.

 Giant grouper (body weight 35.0–45 kg) and Tiger grouper (8 – 10 kg)
used in this study were reared separately in 150 ton indoor tank at FRI
Tg. Demong, Besut, Terengganu.
 Treatment: 3 unknown fish as control, 3 unknown fish as low
concentration implant with E2 Estradiol (0.5 mg/kg), another 3 unknown
as high concentration implant with E2 Estradiol (0.05 mg/kg), were
culture together in one tank.
 Marking all the fish with microchip and making hormone pellet with
using cholesterol powder + Estradiol.
 9 fish reared in normal condition will implant with hormone E2 Estradiol
 Every month; Implant with pellet hormone and blood will sampling
 Keep in -20oC prior analysis.
 Implant will follow in every month until the sixth month.

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 Sex reversal of Giant Grouper (9 pcs) / Tiger grouper

Control Low dose High dose

(3 pcs) Estradiol (0.05 mg/kg) Estradiol (0.5 mg/kg)
3 pcs 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. Statistical significance was inferred at
P < 0.05.

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