VITELLOGENIN AS BIOMARKER INDICATOR ON SEX-REVERSAL OF GIANT GROUPER (EPINEPHELUS LANCEOLATUS)

Presented by: Ahmad Daud bin Om FRI Tg. Demong, 22200, Besut, Terengganu

Introductions
Giant grouper is Protogynous hermaphrodites

This kind of sexuality raises several problems for brood-stocks management, concerning the prediction of natural sex inversion (age and/or

size) and the artificial control of sex. Steroid treatments have been used successfully in several grouper species in order to control sex in fish culture.

 Vitellogenin (Vtg) is the major source

of nutrients for the developing embryo and elevated levels in female fish plasma signal gonadal development preceding spawning In oviparous fish where the developing embryo is dependent on the store food in the yolk, Vtg levels in the eggs could indicate its capability to support embryonic growth.

 Vtg plays an important role in oocyte

growth (from the previtellogenic oocytes to vitellogenic oocyte, up to the tertiary yolk globule stage) After their secretion into blood in maturing females, Vtg are transported to the ovary where they are selectively sequestered proteolyticall processed into the lipovitellin and phosvitin, the prodominent yolk protein.

 Vitellogenesis is defined as the estradiol-

induced hepatic synthesis of Vtg, its secretion and transport in the blood to the ovary, and its uptake into maturing oocytes.

 Present in adult vitellogenic females, absent

in males and immature females Both males and females have the gene for Vtg, but normally is produced in highest concentration in mature females undergoing oogenesis. Synthesis of Vtg may also be induced by oestogen treatment ( Estradiol ) of immature females and males Female have low levels of plasma Vtg, their eggs likely will be of lower quality

Vitellogenin as biomarkers
NRC (1987) defined biomarkers as indicators of

events in biological systems or samples Biomarker is: "a characteristic that is objectively measured and evaluated as an indicator of normal biologic processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention. Classified into Biomarkers of exposure, which is the identification of an exogenous substances (Vtg) within the biological system, the interactive between a compound substances and the endogenous components, or other events in the biological system related to exposure.

Molecule weight of Vitellogenin substance

Species Sea bass Atlantic Halibut Winter Flounder California Halibut Senegalese sole MW of Vtg Authors (kDa) 145 160 135 168 172 Mannos et al, 1994 Norberg, 1995 Harling, et al., 1997 Palumbo et al., 2007 Cuzman et al., 2008

Molecular Weight of vtg in different species

Vtg levels useful indicator for female

maturing condition

MAIN OBJECTIVES OF THE EXPERIMENTS

To characterize the Vtg To evaluate the effectiveness of

vitellogenin as biomarkers on sex reversal of Giant Grouper

METHODOLOGY

Series of experiments
1. Induction of Vitellogenin by used Estradiol

2. Determine the reproductive status of 56 pcs of Giant Grouper brood stock

3. Effects of Estradiol in sex reversal of Giant Grouper 4. Effects of HCG and LHRH on Vitellogenin development 5. Comparison of results on Vtg in Giant Grouper with Tiger Grouper

 Plasma will be collect from the fish at 3

categories Categorize: 1. Induced males by Estradiol 2. Female naturally(+ve control) 3. Male naturally (-ve control)

Brood stock management

BLOOD SAMPLING PROCEDURE

COLECT THE BLOOD FROM CAPILLARY GILLS

Aliqout plasma and protease inhibitor (2:1)

Store at -300C until used (Watts et al. 2003)

PUT THE BLOOD INTO MICROCENTRIFUGE TUBE

Collect the supernatant (plasma)

Acid Heparin / EDTA

Centrifuge (3000 rpm, 4oC , 15 min Matsubara, et al., 1994

Blood Sampling

SDS page

Exp 1 : Male treated by Estradiol to produce vitellogenin

Peanut oil + Estradiol

Inject on pectoral fin Blood collection

Protein characterization

Hormone preparation

Hormone injection

Protein separation by SDS-PAGE

Principle : separate the proteins (expected Vtg) according to their size

SDS PAGE method

Prepare 7% resolving gel & 5% stacking gell Destain using destaining solution 3 hours Determine the MW of purified plasma by comparing with MW of markers

Mix the sample with sample buffer (2:1) & boil 5 min

Remove the gels and stain with coomasive brilliant blue overnight

Load the sample

Run at 80V, 100 mA for 2 hours

Visualization & Isolation of a protein

proteins migrate in an electrical field at rates

that depend upon their net charge, size, and shape Gel Electrophoresis; separation by charge (separation of in a media as gels) in a media as porous gel (polyacrylamide) : gels & staining SDS-PAGE: Sodium Dodecyl Sulfate polyacrylamide gel electrophoresis; proteins treated with ionic detergent that separates according to size SDS binds to protein @ 1 SDS/2 amino acid thus proportional to a protein's Molecular Weight

Protein Mapping by MALDI-TOF

BML UM, Kuala Lumpur

Results

Exp 1 : INDUCTION OF Vtg & BLOOD SAMPLING

To clarified the characterize (dis)similarity of

protein identification results MALDI-TOF Interpret results based on the MALDI-TOF ionization preferences Compare performance (quantification and identification) in a protein differential expression study

Exp 1 : INDUCTION OF Vtg & BLOOD SAMPLING

Males-injected with 17-estradiol (E2) to

enhance the production of vtg Amount of hormone used 2 mg E2/kg fish body weight Estradiol dissolved in ratio of 1:9 (Estradiol : peanut oil) (Mananos et al, 1994)
Dispersed in ultrasonic bath (Birgitta Norberg and Carl

Haux, 1985) Before inject anaesthetized with Ms 222 solution.

250 kDa

Vtg
130 kDa

95 kDa

Marker

Initial

Day 1

Day 3

Day 5

Day 10

Vitellogenin (Vtg) in Male Giant Grouper after induced by Estradiol

185 kDa

Vtg in Male plasma induced by Estradiol

Exp. 2 Reproductive status of Giant Grouper

Objective

To verified the status of reproductive

Vitellogenin as biomarker reproductive

Reproductive status of Giant Grouper

56 pcs were reared in Indoor and outdoor 150 ton tank Located at FRI research Institute Grouper were hermaphrodite protogrynous ; which is female for a certain years / size than

turn to male after several years / size Difficult to maintain at female : male ratio due to sex-reversal phenomena
Number MALE 12 FEMALE 7 UNKNOWN 34 Percents 22.6 13.2 64.2

Genital structure of Giant Grouper

FEMALE &UNKNOWN

MALE

Vtg

F1

F2

M1

M2

M3

M4 Maker

SDS-PAGE results on female and male naturally Giant Grouper

Female Giant Grouper from natural

Male Giant Grouper plasma from natural

Exp 3 : Effects of estradiol in unknown sex of grouper to female

At first , 10 fish/ treatment from unknown sex

will choose for this experiment. Giant grouper (body weight 35.045 kg) used in this study were reared separately in 150 ton indoor tank. At first , dose of Estradiol was 10 mg/kg for High Dose and 1 mg/kg for Low dose . Results shows, High Dose ; 100% mortality and Low dose 75% mortality. Finally; High dose = 0.5 mg/kg and Low dose 0.05 mg/kg was use in this experiment (3 pcs/ treatment)

 Marking all the fish with microchip and making

hormone pellet with using cholesterol powder + Estradiol (Source Syndell). 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. All sample are subjected for same analysis above.

FISH

Wt (KG)

DOSE 1st Months

HORMONE IMPLANT & BLOOD SMPLING 2nd Months 3rd Months 4th Months 5th Months 6th Months

CONTROL

0.0 g/kg

C1
C2 C3 LOW L1 L2

52
47.5 43.5

0
0 0 50 g/kg

/
/ /

/
/ /

/
/ /

/
/ /

/
/ /

/
/ /

53.5 43

2.675 2.15

/ /

/ /

/ /

/ +ve

L3 HIGH
H1 H2 H3

45.5

2.275 500 g/kg

31.5 31 40

15.75 15.5 20

/ / /

/ +ve /

+ve

Effects of estradiol to change Unknown sex to female in Low, high and control treatment

The effects of Estradiol overdose on Giant Grouper

Vtg

C1

C2

C3

C4

C5

H1

H2

H3

H4

H5

H1 H5 represent as months 1,..5 C1,C5 represent as months 1,.5

SDS-PAGE results on High dose and Control treatment of sex-reversal effects after 4 and 5 months implants by using 500 ug/kg of estradiol.

Vtg

LOW DOSE

HIGH MARKER DOSE

SDS-PAGE results on female Giant Grouper induced by female with low dose Estradiol and female with high dose of Estradiol

Female Giant Grouper effects by implant with Estradiol Low dose (0.05 mg/kg)

Female Giant Grouper effects by implant with Estradiol High dose (0.5 mg/kg)

Exp. 4 Effects of HCG and LHRH on Vitellogenin development

DOSE: 1ST INJECTION 1000 IU/KG 2ND INJECTION 500 IU/KG (After 24 hrs) 3RD INJECTION 50 Ul/KG of LHRH (After 24 hrs)

Hormone preparation & injection

EGGS DEVELOPMENT (IN GONAD)

0 hrs (Initial)

24 hrs

36 hrs

60 hrs

48 hrs

EGGS DEVELOPMENT AFTER FERTILE

0 hours

3:30 hours

6:00 hours

5:30 hours

Vtg

0 hrs

24 hrs 48 hrs

SDS-PAGE results on maturing female of Giant Grouper

Maturing female Giant Grouper shows the vitellogenin spectrum

5. Comparison of results on Vtg in Giant Grouper with Tiger Grouper

Two experiment were taken to analysis the

vtg of tiger grouper i) Induction of vtg by using Estradiol at same dose with Giant grouper(2 mg/kg) ii)Sex reversal experiment with using same dose (Low = 50 ug/kg and High = 500 ug/kg & control = 0 ug/kg)

b) Ikan Kerapu Harimau (tangki)

A1AE B7CD

250 130 95 72 55

M: Penanda
0: Permulaan 1: Suntikan Hari Pertama 3: Suntikan Hari Ketiga 5: Suntikan Hari Kelima

A5

A3

A1

A0

C5

C3

C1

C0

Vtg

Marker

Day 0

Day 1

Day 3

Day 5

Induction of Vitellogenin of Tiger Grouper by using estradiol and sampling plasma at day 0,1,3,and 5

Sex reversal experiment of Tiger Grouper

FISH Wt (KG) DOSE 1st Months HORMONE IMPLANT & BLOOD SMPLING 2nd Months 3rd Months 4th Months 5th Months 6th Months

CONTROL C1
C2 C3 LOW L1 L2 4.36 5.87

0.0 g/fish 3.78

5.37 5.23

0
0 0 50 g/fish 0.218 0.294

/
/ /

/
/ /

/
/ /

/
/ /

/ /

/ /

+ve / /

L3
HIGH H1 H2 H3

4.85

0.242
500 g/fish

6.03 5.07 8.16

3.015 2.54 4.08

/ / /

/ + ve /

+ve

Genital structure of tiger Grouper after 3 months experiments

Low Dose

High Dose

Control

Conclusion
Vtg has confirm found in female , not in male Vtg has marked at 187 molecular weight kDa Vtg has confirm found in induced unknown female by Estradiol AND slowly can produced at High dose of 0.05 mg/kg (4 months) and low dose of 0.5 mg/kg (2 months) Can be use as biomarker reproductive in Giant Grouper Tiger grouper also shows same pattern on vtg in response to testing on sex reversal and induction of vtg.

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