Characterization of zebrafish vitellogenin gene family for potential development of receptor mediated gene transfer method 4

If this gene delivery approach is feasible in fish, foreign genes could be specifically introduced into the oocytes after injection of the Vtg-DNA complexes into blood circulation of fem

Abstract

The potential of receptor-mediated gene transfer using vitellogenin protein as DNA carrier was explored In these preliminary experiments, tilapia Vtgs were induced, purified and labeled with 125I After injection, purified Vtgs could be preferably taken up by ovaries

By modification with N-succinimidyl 3-(2-pyridyldithio)-propionate (SPDP), three types

of Vtg-poly-L-lysine conjugates were constructed and used in complexes preparation However, the efficiency of mediating DNA uptake from the Vtg-poly-L-lysine conjugates

by oocytes was not significantly higher than those by other tissues Possible reasons for this were discussed Furthermore, recombinant Vtg fragments covering essentially the full

Vtg sequence were produced in E coli and attempts of determining receptor-binding domains were also made by in vivo binding assay, though inconclusive results were

observed

4.1 Introduction

Transgenic fish are not only an important experimental tool for developmental analyses of gene expression and function, but also have enormous potential in aquaculture In 1985,

Zhu et al successfully made transgenic gold fish (Carassius auratus) through gene

transfer by microinjection Since then, the production of transgenic fish has been reported

in many fish species and a variety of gene delivery methods have been developed and successfully employed (Fletcher and Davies, 1991; Maclean and Rahman, 1994; Gong and Hew, 1995; Chen et al., 1996) Common gene delivery methods used in transgenic fish research include microinjection, electroporation, sperm carrier and particle bombardment Each method has its advantages and drawbacks For example, microinjection is a popular gene delivery approach used by many researchers but requires special equipment and skilled personnel Moreover, only a limited number of eggs can be injected at a time for most fish species In contrast, other gene delivery methods such as electroporation and particle bombardment are more efficient in dealing with a large number of eggs but the germ line integration rate of foreign genes is usually very low and special equipment is also required Thus, new gene delivery methods need to be developed that overcome these deficiencies

It is well known that receptor-mediated endocytosis (RME) provides a major pathway for trafficking of extracellular molecules or ligands into animal cells Based on the RME process, a novel gene transfer method designated “receptor-mediated gene transfer” was proposed (Wu and Wu, 1987) In their experiment, foreign DNA was transported into hepatocytes by an asialoglycoprotein receptor-mediated pathway after administration of

conjugates (Wu and Wu, 1987) The main advantage of the receptor-mediated gene

transfer is that it can be used to target specific cells in vivo after intravenous injection

However, no attempts have been made to apply this gene delivery approach in fish

In fish, Vtgs are synthesized in the liver under the control of E2 and incorporated into oocytes via receptor-mediated endocytosis (Selman and Wallace, 1982; Tyler et al., 1987) Thus, Vtg is a candidate protein for a DNA carrier to use in transforming fish oocytes through receptor-mediated gene delivery approach If this gene delivery approach is feasible in fish, foreign genes could be specifically introduced into the oocytes after injection of the Vtg-DNA complexes into blood circulation of female fish and high percentage of transgenic offspring would be expected This gene delivery method is effective, and does not depend on experienced personnel or special equipment

In this study, receptor-mediated gene transfer will be tested in red tilapia (Oreochromis mossambica) The reason for shifting the experimental model from zebrafish to tilapia is

that it is much easier to inject experimental materials through caudal artery of the tilapia

than of the zebrafish Furthermore, the full-length cDNA sequence of tilapia (Oreochromis aureus) vtg1 became available from GenBank during the project and it encodes a Vtg with the homologous subdomains I-V (see Table 2-4 in Chapter 2) Thus, the tilapia vtg1 is a potential orthologue of the zebrafish vtg2 and it also encodes a Vtg that is complete in

primary structure

4.2 Materials and Methods

4.2.1 E 2 induction and blood serum isolation

Female red tilapia (Oreochromis mossambica) (body weight 400 – 500g) were purchased

from a local fish farm and acclimated in a tank for two weeks They were fed daily with commercial fish food E2 stock solution was prepared as described in Chapter 3 (Section 3.2.1) and was injected intraperitoneally on days 1, 5, 9 and 14 at 4 µg E2/g body weight according to Ding et al (1989) Blood samples were collected through the caudal artery from control and treated fish on days 4, 16 and 20, respectively, using pre-chilled syringes For serum isolation, blood was clotted on ice for 10-15 min prior to centrifugation at 6000g for 10 min at 4 °C Aprotinin (Sigma) was added to the serum at a final concentration of 40 µg/ml and the fish serum was stored at – 70 °C prior to purification

4.2.2 Vtg purification by anion-exchange chromatography

Vtg was purified from tilapia serum by anion-exchange chromatography according to Chan et al (1991) with modifications Briefly, to adjust the pH and ionic strength, buffer

A (50 mM Tris-HCl, pH 8.0) was added to the fish serum at a ratio of 3 : 1 (v/v) After filtration, 1 ml of diluted serum sample was injected into an UNO Q-1 column (Bio-Rad) integrated in a HPLC system (Pharmacia) Unbound proteins were washed away with 2.5

ml of buffer A (~ 2 times of bed volume) at a constant flow rate of 1 ml/min For elution,

a gradient of 0 – 35% of buffer B (50 mM Tris-HCl, pH 8.0, 1 M NaCl) was applied over

15 ml, followed by holding at 35% of buffer B for 4 ml (see Fig 4-3B) Elutes were collected in 1 ml fraction and stored at – 80 ºC

4.2.3 Iodination of Vtg

Purified tilapia Vtg was labeled with 125I using a solid phase oxidizing agent, tetrachloro-3α, 6α-diphenyl glycouril (Iodogen, Sigma) mediated method (Salacinski et al., 1981) Briefly, an Iodogen tube was prepared by dispensing 100 µl of Iodogen solution (0.1 mg/ml in chroloform) onto the bottom of a 12x75 mm glass tube, followed by vacuum evaporation A time course of radioiodination was determined based on the method of Rudick (1998) Briefly, the Iodogen tube was rinsed with 1 ml of 50 mM Tris-HCl (pH 8) to remove any loose flakes of Iodogen Then, the following components were added with gentle swirling: 90 µl of 50 mM Tris-HCl (pH 8.0), 10 µl of purified tilapia Vtg (20 µg) and 0.5 µl of Na125I (50 µCi) Immediately, 4 µl of solution was removed and spotted onto a nitrocellulose sheet This process was repeated at 3 min intervals for a total

1,3,4,6-of 21 min Finally, all nitrocellulose sheets were washed in 50 ml 1,3,4,6-of washing buffer (25

mM Tris-HCl, 192 mM glycine, 12.5 mM NaI, 20% methanol, pH 8.3) and countered by a Gammer counter (1470 Wizard, Wallac) Final labeling reaction was performed according

to the optimal duration determined and 20 µg of purified Vtg was radioiodinated at room temperature The 125I-labeled Vtg was purification on a Sephadex G-25 column (PD-10, Pharmacia Biotech) and stored at 4 °C prior to use

4.2.4 Synthesis of Vtg-pLys conjugates

Purified tilapia Vtg was coupled to poly-L-lysine (pLys) through a disulfide bond after modification by a heterobifunctional reagent, N-succinimidyl 3-(2-pyridyldithio)-propionate (SPDP, Pierce) (Jung et al., 1981; Wagner et al., 1990) In this experiment,

Vtg

Vtg

HN-C-CH2-CH2O

-S-S-poly-L-lysine

H2N

poly-L-lysineHS-CH2-CH2-C-NH

O

HN-C-CH2-CH2-S

O

poly-L-lysineS-CH2-CH2-C-NH

(pyridin -2-thione)

Fig 4-1 Flow chart depicting the formation of Vtg-poly-L-lysine conjugates Three steps are included in the process, which is described in detail in Materials and Methods (Section 4.2.4) SPDP, N-succinimidyl 3-(2-pyridyldithio)-propionate; DTT, dithiothreitol (adapted from Jung et al., 1981; Wagner et al., 1990)

two types of pLys with an average chain length of 36 (pLys36, MW 7500 Da, Sigma) and

144 lysine monomers (pLys144, MW 30,100 Da, Sigma) were used Stock solutions of 3.33 nmol/µl for pLys36 and 2 nmol/µl for pLys144 were prepared in sodium phosphate buffer (0.1 M sodium phosphate, pH 7.8, 0.1 M NaCl) The construction process included three steps shown in Fig 4-1 Results are summarized in Table 4-3

Step 1 Modification of Vtg with 3-(2-pyridyldithio)-propionate by SPDP First, a buffer exchange with sodium phosphate was performed on a PD-10 column for the HPLC purified Vtgs and the Vtg solution was concentrated using KwikSpin Micro Ultrafiltration Units (30 kDa MWCO, Pierce) For modification of Vtgs used for type II conjugates (see Table 4-3), 8 µl of SPDP stock solution (2 nmol/µl in 100% ethanol) was gradually added

to 430 µl of Vtg solution (0.86 nmol) The reaction mixture was vigorously mixed and kept at 4 °C for 5 hr Modified Vtgs were purified on PD-10 column with sodium phosphate buffer After an aliquot of modified Vtg was reduced by dithiothreitol (DTT), the amount of dithiopyridine linkers per Vtg molecule was determined based on a molar absorbance coefficient of 8.08 x 103 M-1• cm-1 at 343 nm for the released product pyridin-2-thione (Carlsson et al., 1978) For modification of Vtgs used for conjugates of types I and III, the molar ratios between Vtg and SPDP were adjusted accordingly (see Table 4-3) and the similar process was followed SPDP modified Vtgs were stored at 4 °C before use

Step 2 Modification of pLys with 3-mercaptopropionate by SPDP and DTT treatment First, for modification of pLys36 by SPDP at a molar ratio of pLys36 to SPDP of 1:1, 15 µl

of SPDP (20 nmol/µl) was mixed with 90 µl of pLys36 (3.33 nmol/µl) and 83 µl of sodium

phosphate buffer The solution was vigorously mixed and kept at room temperature for 2.5

hr, followed by gel filtration on PD-10 column with sodium acetate buffer (20 mM sodium acetate, pH 5.2, 0.1 M NaCl) For detection of the modified pLys, absorption at 211 nm was measured for each elute For modification of pLys144 by SPDP at a molar ratio of pLys144 to SPDP of 1:2, 30 µl of SPDP (20 nmol/µl) was mixed with 150 µl of pLys144 (2 nmol/µl) and the products were purified accordingly afterwards Two standard curves were prepared for quantification of pLys: 1) Y = 0.1035X-0.0061 for pLys36 and 2) Y = 0.3633X + 0.0139 for pLys144 (X: concentration of pLys in pmol/µl, Y: absorption at 211 nm) The amount of dithiopyridine linkers in modified pLys was determined as described

in step 1 Second, SPDP modified pLys was reduced by DTT to form mercaptopropionate modified pLys Briefly, 500 µl of SPDP modified pLys36 (8.21 nmol)

3-or pLys144 (3.01 nmol) was mixed with 12.5 µl of 1 M DTT and the solution was kept under N2 for 2 hr at room temperature After gel filtration on PD-10 column with sodium acetate buffer, the 3-mercaptopropionate modified pLys was stored at – 20 °C until use

Step 3 Synthesis of Vtg-pLys conjugates Three types of Vtg-pLys conjugates were synthesized, Vtg-pLys36 (type I), Vtg-pLys144 (H) (type II) and Vtg-pLys144 (L) (type III) (see Table 4-3) Briefly, for making type I conjugates, 1 ml (1.95 nmol) of 3-(2-pyridylditho)-propionate modified Vtg (with 4.9 linkers per molecule) was mixed with

100 µl (1.93 nmol) of 3-mercapto-propionate modified pLys36 and the reaction was kept under N2 at 4 °C for ~20 hr For making type II conjugates, the reaction was performed by mixing 351 µl (0.8 nmol) of modified Vtg (with 10.1 linkers per molecule) with 175 µl (0.832 nmol) of 3-mercaptopropionate modified pLys144 Similarly, for making type III

conjugates, 467 µl (0.214 nmol) of modified Vtg (with 1.1 linker per molecule) was mixed with 50 µl (0.238 nmol) of 3-mercapto-propionate modified pLys144 The Vtg-pLys conjugates were separated from uncoupled 3-mercaptopropionate modified pLys by gel filtration on a Bio-Gel P-100 column (with exclusion limit of 100 kDa) and eluted using

20 mM HEPES, pH 7.4, 0.15 M NaCl The coupling degree was estimated based on the increased OD343 value as described above

4.2.5 Formation of complexes between Vtg-pLys conjugates and DNA

Preparation of complexes between Vtg-pLys conjugates and DNA and gel retardation assay were carried out according to Wagner et al (1990, 1991) In order to form the complexes, Vtg-pLys and DNA were directly mixed at a certain ratio in a buffer containing 20 mM HEPES (pH 7.4) and 0.15 M NaCl, followed by incubation for 1 hr at room temperature The optimal ratio for neutralization between Vtg-pLys conjugates and DNA was determined by gel retardation assay Briefly, 1 µg of DNA (100-bp long, cut from a plasmid by restriction enzymes) was labeled by [α-32P]dCTP using Nick Translation Reagent Kit (BRL) according to the manufacturer’s protocol and purified by a NICK Column (Pharmacia Biotech) A series of complexes were prepared between 0.45 µl

of 32P-labeled DNA (~ 0.02 pmol) and increasing amount of Vtg-pLys conjugates.After that, the complex mixture was loaded into a 1% agarose gel and resolved by gel electrophoresis in 1x TAE buffer at 30 V for 2 hr Finally, the agarose gel was dried and autoradiography was performed at – 70 ˚C with Kodak's BioMax MS film

Table 4-1 Summary of primers used in amplification of eight tilapia vtg cDNA fragments

†Overlapping sequence with tilapia (Oreochromis aureus) vtg1 cDNA is in italic letters

For primer locations, see Fig 4-7

‡Tilapia (Oreochromis aureus) vtg1 cDNA sequence is from GenBank (accession No

AF017250) CDS, coding sequence

4.2.6 Amplification of eight tilapia vtg cDNA fragments by RT-PCR

Red tilapia (Oreochromis mossambica) liver total RNA was extracted using TRIzol

Reagent (GIBCO BRL) according to the manufacturer’s instructions Eight pairs of primer

with BamH I (or Bgl II) and EcoR I linkers were designed based on tilapia (Oreochromis aureus) vtg1 cDNA sequence (GenBank accession No AF017250) and used in amplification of eight tilapia vtg cDNA fragments by RT-PCR (Table 4-1) RT-PCR was performed using Access RT-PCR System (Promega) Briefly, first strand vtg cDNAs were

synthesized using AMV reverse transcriptase at 48°C for 45 min Inactivation of the reverse transcriptase and denaturation of mRNA/cDNA were performed at 94°C for 2 min, followed by 35 to 40 cycles of PCR for amplification of target sequences using the following conditions: 94 °C for 30 sec, 55~67 °C for 1 min, 72 °C for 2 min, final extension at 72 °C for 8 min Amplified products were resolved in 1% agarose gel and

bands with anticipated sizes were cut under UV illumination vtg cDNA fragments were

recovered from the gel using QIAquick Gel Extraction Kit (QIAGEN)

4.2.7 Construction of GST-Vtg fusion protein expression vectors

Glutathione S-transferase (GST)-Vtg fusion protein expression vectors were constructed based on an expression vector pGEX-2TK (Pharmacia Biotech, Fig 4-2A) Briefly, the expression vector pGEX-2TK was linearized by restriction enzyme digestion with BamH I

and EcoR I, and subsequently ligated with each of the following five vtg cDNA fragments, LVIa, LVIb, LVIc, PV and LVII, which were also digested by BamH I and EcoR I Ligation

was performed at 14 °C overnight in the presence of T4 DNA ligase (GIBCO) The

A pRSET

Fig 4-2 Maps of original and modified expression vectors used in expression of

recombinant Vtg fragments A: Vector map of pGEX-2TK Arrows indicate cloning sites

for vtg cDNA insert B: Vector map of pRSET-A (original) Arrows indicate cloning sites

for a new cDNA fragment LVIa’, which was amplified by PCR using a 48 mer forward

primer (for sequence, see Materials and Methods, Section 4.2.8) and the reverse primer 1R

(Table 4-1) from the plasmid pGST-LVIa C: Partial map of the modified pRSET-A

vector (pRSET’), showing an extra protein kinase site (arrow) located between the six

histidine residues and vtg cDNA insert Restriction sites flanking the vtg insert are shown

resulting expression vectors were named as pGST-LVIa, pGST-LVIb, pGST-LVIc, pGST-PV and pGST-LVII

4.2.8 Construction of 6xHis-tagged Vtg expression vectors

The original 6xHis expression vector pRSET A (Invitrogen, Fig 4-2B) was first modified with an insertion of a protein kinase recognition site coding sequence to facilitate the labeling of 6xHis-tagged Vtgs by [γ-32P]ATP Briefly, a 48-mer forward primer (5'-CCAGATCTCGTCGTGCATCTGTT-GGATCCGACCAGTCCAACTTGGCCC-3') was designed which contains (5’ to 3’) a Bgl II cutting site (AGATCT), a sequence (CGTCGTGCATCTGTT) encoding a protein kinase recognition motif (RRASV) and the sequence of primer 1F (excluding the two protection nucleotides at 5’ end), which contains a BamH I site PCR was performed using this 48-mer primer and the reverse

primer 1R in order to amplify a fragment (LVIa’) from the plasmid pGST-LVIa After

restriction enzyme digestion with Bgl II and EcoR I, the amplified fragment was ligated with the pRSET A vector (linearized by BamH I and EcoR I), resulting in a modified vector, named as pRSET'-LVIa, which encodes a 6xHis-tagged LVIa with a protein kinase recognition motif (Fig 4-2C) For construction of other 6xHis-Vtg expression vectors, the

pRSET'-LVIa was cut by BamH I and EcoR I, followed by ligation with a respective vtg

cDNA fragment, which was obtained either by restriction enzyme digestion of construct pGST-LVIb, pGST-LVIc or pGST-LVII (by BamH I and EcoR I), or by RT-PCR

amplification for cDNA fragment LVIb1, LVIb2 or LVIb3 Thus, seven 6xHis-Vtg

expression vectors were constructed and named as LVIa, LVIb, LVIc, pRSET'-LVII, pRSET'-LVIb1, pRSET'-LVIb2 and pRSET'-LVIb3

pRSET'-4.2.9 Expression and purification of recombinant vtg fragments

Recombinant proteins of GST-Vtgs and 6xHis-Vtgs were expressed in E coli BL21 cells

at 28 ˚C after induction with 0.1-1.0 mM (final concentration) of galactopyranoside (IPTG, Sigma) Bacteria were lysed with lysozyme and Triton X-100 according to Gong and Hew (1994) Briefly, after centrifugation, the bacterial pellet was re-suspended in lysis buffer at a ratio of bacteria to lysis buffer of 1 : 2 (w: v) followed by addition of lysozyme to a final concentration of 2.5 mg/ml The lysis buffer contained 15% (w/v) sucrose, 2 mM EDTA, 5 µg/ml aprotinin, 1 mM PMSF, 1 mM DTT and 50

isopropyl-1-thio-β-D-mM Tris-HCl (pH 8) (GST-Vtgs) or 15% (w/v) sucrose, 5 µg/ml aprotinin, 1 isopropyl-1-thio-β-D-mM PMSF,

500 mM NaCl and 50 mM sodium phosphate buffer (pH 7) (6xHis-Vtgs) After mixing, Triton X-100 was added to a final concentration of 1% (v/v) and the suspension was mixed vigorously Then, an equal volume of lysis buffer was added and the solution was supplemented with MgCl2 and Dnase I (final concentrations of 5 mM and 20 – 40 µg/ml, respectively) Finally, after centrifugation at 15000 rpm, 4 °C for 15 min, the supernatant containing soluble recombinant proteins was removed and stored at 4 °C

Purification of recombinant Vtgs was carried out using either GST Sepharose 4B (Pharmacia Biotech) for GST-Vtgs or Talon Metal Affinity Resins (Clontech) for 6xHis-Vtgs according to the manufacturers’ protocols Briefly, 50% bead slurry was added to the clear supernatant of bacteria lysate at a ratio of 1 : 20 (v/v), followed by incubation at 4 °C for 30 – 60 min on a rocking platform Then, beads were spun down at 700 g, 4 °C for 5 min and washed with cold washing buffer (100 mM NaCl, 0.5% NP-40, 1 mM DTT in 20

mM Tris-HCl, pH 8 for washing GST fusions; 500 mM NaCl, 25 mM Imidazole, 0.5%

NP-40 in 50 mM sodium phosphate buffer, pH 7 for washing 6xHis-Vtgs) three times with

10 min each at 4 °C on a rocking platform Finally, elution buffer (containing 10 mM reduced GSH in 50 mM Tris-HCl, pH 8 for GST-Vtgs or 300 mM NaCl, 150 mM Imidazole in 50 mM sodium phosphate buffer, pH 7 for 6xHis-Vtgs) was added to the beads, followed by incubation at room temperature for 5 min After centrifugation, the supernatant containing purified recombinant Vtgs was collected and kept at 4 °C for further analysis

4.2.10 Labeling of 6xHis-Vtgs by [ γ- 32 P]ATP

6xHis-Vtgs were labeled by [γ- 32P]ATP on beads according to the manufacturer’s protocol (Pharmacia Biotech) Briefly, recombinant protein bound beads were washed in 1

x heart muscle kinase buffer (HMK) (20 mM Tris, pH 7.5, 0.1 M NaCl, 12 mM MgCl2), spun down and kept on ice To 20 µl of beads, 30 µl of protein kinase reaction mixture [3

µl of 10 x HMK, 3 µl of 10U/µl bovine heart kinase (dissolved in 1 x HMK buffer), 3 µl

of [γ- 32P]ATP (3000 Ci/mmol) and 21 µl of dH2O] was added Then, the solution was mixed by gentle agitation and incubated at 4 °C for 30 min After that, the beads were washed twice in ice-cold 1 x PBS and spun down Finally, radio-labeled recombinant Vtgs were eluted from the beads and stored at 4 °C until use GST was also labeled by [γ-

32P]ATP as a control

4.2.11 Polyacrylamide gel electrophoresis

One-dimensional discontinuous sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was carried out using a Bio-Rad minigel apparatus according to Celis and

Olsen (1998) The composition of the gels were 5% (w/v) acrylamide-bisacrylamide (Acr/Bis) mixture (C=3.3), 0.125 M Tris-HCl (pH 6.8) and 0.1% SDS (for stacking gel); 8-10% (w/v) Acr/Bis, 0.374 M Tris-HCl (pH 8.8) and 0.1% SDS (for separating gel) Running buffer contained 25 mM Tris-HCl (pH 8.3), 0.192 M glycine and 0.1% SDS, and the sample buffer contains 80 mM Tris-HCl (pH 6.8), 2% SDS, 7.5% (v/v) glycerol and 5% 2-mercaptoethanol (v/v) For resolving 125I-labeled native Vtgs, nondenaturing polyacrylamide gel electrophoresis (NPAGE) was performed according to Safer (1998) Briefly, the composition of the gels were 3% (w/v) Acr/Bis and 0.124 M Tris-HCl (pH 6.7) for the stacking gel; 5.5% (w/v) Acr/Bis and 0.374 M Tris-HCl (pH 8.9) for the separating gel Running buffer was composed of 50 mM Tris-HCl (pH 8.3) and 0.384 M glycine; sample buffer composed of 62 mM Tris-HCl (pH 6.8) and 10% glycerol (v/v) Samples were either denatured at 95 ºC for 5 min prior to loading (for SDS-PAGE) or directly loaded without denaturation (for NPAGE) Gel electrophoresis was performed with constant voltage at 50 V for 0.5 h, followed by 100 V for 2 h Proteins were stained with 0.1% (w/v) Coomassie Brilliant Blue R-250 (BDH) which was dissolved in 25% methanol and 10% acetic acid Protein quantification was performed based on Bradford colorimetric assay method using Bio-Rad Protein Assay Kit (standard or microassay procedure) with BSA (Sigma) as a standard

4.2.12 Western blot

Western blot was performed according to the manufacturer’s protocol (Pierce) Briefly, after SDS-PAGE, resolved proteins were transferred onto a piece of nitrocellulose paper using Bio-Rad Mini Trans-Blot Cell at 250 mA for 2.5 hr The transfer buffer contained

Tris-HCl, pH 8.0, 50 mM NaCl, 50 mg/ml BSA) at 4 ºC overnight, the nitrocellulose membrane was incubated in 10 ml of blocking buffer with 1 : 5000 diluted Anti-HisG Antibody (1 mg/ml, Invitrogen) for 1 hr at room temperature with shaking Then, the membrane was washed 3 times in washing buffer (25 mM Tris-HCl, pH 8.0, 50 mM NaCl,

5 mg/ml BSA) for 10 min each, followed by incubation in 1 : 2000 diluted horseradish peroxidase conjugated goat anti-mouse IgG secondary antibody (0.8 mg/ml, Pierce) for 1

hr at room temperature After final washes, SuperSignal West Pico Chemiluminescent Substrate (Pierce) was added to the membrane Signals were captured by exposure with a piece of Hyperfilm ECL (Amersham pharmacia biotech)

4.2.13 Administration method, sampling criteria and sample treatment

Red tilapia (Oreochromis mossambica) (~ 10 cm in body length) was administrated

radioisotope labeled proteins or DNAs by injection into the caudal artery The injected materials included 125I-labedled native tilapia Vtgs (~ 40,000 cpm/fish), 32P-labeled 100

bp DNA (~ 200,000 cpm/fish), complexes of 32P-labeled DNA and Vtg-pLys conjugates (~ 200,000 cpm/fish) and 32P-labeled recombinant Vtg fragments (~250,000 cpm/fish)

To evaluate the developmental stages of oocytes, ovaries from experimental fish were removed and weighed The diameter of randomly selected oocytes was measured prior to further processing of the ovary samples The gonadal index (GI) was calculated according

to the following formula GI = (weight of ovary/weight of fish body) x 100% Female fish bearing oocytes at developmental stages between vitellogenic (oocyte diameter = ~ 0.7 mm) and preovulatory stages (oocyte diameter = ~ 2 x 3 mm) (Kraft and Peter, 1963;

Quek, 1985) were used for further analysis Experimental fish bearing oocytes out of the above ranges were discarded accordingly

Seven tissues were isolated, including the ovary, liver, gut, gill, heart, spleen and kidney The radioactivities in those tissues were either measured directly by a gamma counter (1470 Wizard, Wallac) for 125I or measured after treatment by a liquid scintillation counter (1414 Guardian, Wallac) for 32P 32P samples were pre-treated with a tissue solubilizer NNCS502 (Amersham) at 10 ml/g tissue overnight before counting For 125I-injected fish, the collective radioactivity in the remaining parts of fish was calculated by summing the radioactivities from all remaining tissues and labeled as “remains” For 32P injected fish, the collective radioactivity in the remaining parts of fish (labeled as “others”) was obtained by measuring the radioactivities of portions of the remaining tissues and then multiplying accordingly based on the proportion of the sampled tissues in the remaining parts of fish (by weight) The “radioactivity recovery rate” was defined as the total radioactivity of whole fish divided by the injected radioactivity, and the “relative radioactivity” was defined as the radioactivity of each tissue divided by the total radioactivity of whole fish

4.3 Results and Discussion

4.3.1 Purification of tilapia vitellogenin proteins

To obtain a sufficient amount of native vitellogenin for construction of Vtg-polylysine conjugates, female red tilapia were injected with E2 After injection, serum proteins were examined by SDS gel electrophoresis We found that multiple Vtg proteins were induced

by E2 treatment, especially after the 4th injection, as revealed by SDS-PAGE analysis (Fig 4-3A) As shown in Fig 4-3A, two proteins with molecular weights of ~190 and ~130 kDa were greatly enhanced in female fish after the 4th E2 injection compared with those from the control females and females after the 1st E2 injection In serum of male fish, both bands were absent (Fig 4-3A, lane 1) It was reported that two Vtg subunits of 180 and

130 kDa were induced in male tilapia (Oreochromis aureus) after E2 treatment (Ding et al., 1989) Similarly, in male tilapia (O mossambicus), the molecular weights of two E2

induced Vtg subunits were reported as 200 and 130 kDa, respectively (Kishida and Specker, 1993) Thus, the enhanced proteins of ~190 and ~130 kDa in this experiment were assumed to be two Vtg subunits

Based on a procedure used in purifying Vtgs from nile tilapia (O niloticus) (Chan et al., 1991), tilapia (Oreochromis mossambica) Vtgs were purified by HPLC from serum

proteins of E2 treated female fish As shown in Fig 4-3B, a potential Vtg peak was identified in the gel filtration profile when the concentration of NaCl reached 0.35 M in the elution buffer After SDS-PAGE analysis, it was found that the potential Vtg peak was mainly composed of the Vtg subunit of ~190 kDa (Fig 4-3A, lane 6)

23 24

~500 kDa

Fig 4-3 Gel electrophoresis and HPLC purification profile of tilapia serum proteins A:

SDS-PAGE analysis of tilapia serum proteins (lanes 1-4) and HPLC fractions (lanes 5-8) NPAGE analysis of 125I-labeled Vtg is shown in lane 9 Lanes 1, 2, 3 and 4 contain serum proteins from control male, control female, female after first injection with E2 and female after fourth injection with E2, respectively (0.5 µg protein/lane) Lanes 5, 6, 7 and 8 contain HPLC fractions 18, 19, 20 and 21 (in B), respectively (20 µl fraction/lane) Two Vtg subunits of ~190 and ~130 kDa are marked by arrowheads and asterisks, respectively

B: HPLC profile of serum proteins from female tilapia after first injection with E2 An arrow indicates a potential Vtg peak

4.3.2 Purified native vitellogenins were preferably taken up by ovaries

To test whether the HPLC purified tilapia Vtgs can be recognized by their receptors and subsequently taken up by fish oocytes, tilapia Vtgs were labeled with 125I, and injected into tilapia caudal artery Based on the radioiodination time course, Vtg proteins were labeled with Na125I by the Iodogen mediated method for 15 min at room temperature, resulting in a 82% incorporation rate of Na125I The integrity of 125I-labeled Vtgs was monitored by nondenaturing polyacrylamide gel electrophoresis (NPAGE) analysis As shown in Fig 4-3A (lane 9), no apparent degradation was observed after radioiodination

After injection of 125I-labeled Vtgs (~ 40000 cpm/fish) into female fish, various tissues were isolated at different time points and the radioactivities of certain tissues and the remains of fish were measured using a gamma counter The mean radioactivity recovery rates ranged from 43.4% to 58.0% at different time points, indicating there was no significant difference in the radioactivity recovery rates between different time points (Table 4-2) Essentially, nearly half of the injected radioactivity was lost at 12 hr after injection and probably part of it was lost during the injection Thus, for subsequent assays, relative radioactivity was used, which was defined as the radioactivity of each tissue divided by the total radioactivity of whole fish As shown in Table 4-2 and Fig 4-4, 12 hr after injection, more than half of the total radioactivity of whole fish (52.4%) was detected

in the remaining tissues of fish, while only 19.7% of the total radioactivity was accumulated in the ovary However, with the time lapsed, the relative radioactivity in the ovary increased to 45.5%, 52.0% and 68.1% at 24, 48 and 72 hr respectively after injection In contrast, the relative radioactivities in essentially all other tissues decreased

Table 4-2 Relative radioactivities in seven tissues and the remains of fish examined at 12,

24, 48 and 72 hr after injection with 125I-labeled Vtg

Time

Tissue

12 hr†(n = 3)

24 hr (n = 5)

48 hr (n = 4)

72 hr (n = 4) Ovary 19.7 ± 11.3‡ 45.5 ± 4.0 52.0 ± 28.6 68.1 ± 6.1

†The mean gonad indexes of fish in experimental groups of 12, 24, 48 and 72 hr were 4.67

± 0.16 %, 4.16 ± 2.67 %, 4.69 ± 1.74 % and 3.68 ± 1.92 % (mean ± SD%), respectively

‡Mean ± SD (%)