Characterization of zebrafish vitellogenin gene family for potential development of receptor mediated gene transfer method 3

Chapter 3. Expression of vtgs

Chapter 3.
Expression of zebrafish vtg genes

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Chapter 3. Expression of vtgs
Abstract

All seven zebrafish vitellogenin genes (vtg1-7) are predominantly expressed in female
liver and can be induced in male liver by 17β-estradiol (E2). The level of vtg1 mRNA was
about 100X and 1000X higher than those of vtg2 and vtg3 mRNAs, respectively, in the
liver of female fish. vtg mRNAs were also detected in several non-liver tissues, but the
expression level is generally <10% of that in the liver. In-situ hybridization confirmed that
the extrahepatic expression was actually in adipocytes associated with several tissues such
as the intestine, ovary and E2-induced testis. Finally, the relative levels of estrogen
receptor α (ERα) mRNA in different tissues with or without E2 treatment were also
determined and good correlation between ERα and vtg mRNA levels was found in
different tissues, indicating the role of ERα in vtg induction.

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Chapter 3. Expression of vtgs
3.1. Introduction

The major sites for Vtg synthesis in egg-laying animals (oviparous) include the fat body
(in insects), intestine (in nematodes and echinoderms) and liver (in vertebrates) (Byrne et
al., 1989 and references within). In oviparous invertebrates, the presence of multiple Vtg

synthesis sites has been reported by several investigators. For example, in sea urchins, Vtg
is synthesized in both intestine and gonads of male and female individuals (Shyu et al.,
1986). In a marine shrimp (Penaeus semisulcatus), a ~7.8-kb vtg transcript was detected in
both hepatopancreas and ovary by Northern blot analysis, indicating these two tissues are
involved in Vtg synthesis (Avarre et al., 2003). Tsang et al. (2003) further demonstrated
that MeVg1 is expressed in both hepatopancreas and ovary, while MeVg2 is expressed
only in the hepatopancreas of the shrimp (Metapenaeus ensis). Similarly, in oviparous
vertebrates, multiple vtg expression (by liver and extrahepatic tissues) was observed in
white sturgeon (Acipenser transmontanus). Bidwell and Carlson (1995) reported that in
addition to the liver, white sturgeon vtg mRNAs were also detected in undifferentiated
gonads of estrogen treated fish and in the testis of both control and estrogen treated males
by Northern blot hybridization. In spotted ray (Torpedo marmorata), strong evidence
indicated that the ovarian follicle cells are involved in Vtg synthesis (Prisco et al., 2004).

The transcription of vtgs is under hormonal regulation which is stage-, sex- and tissuespecific. In insects, such as Drosophila and locust, the synthesis of Vtg in the fat body is
controlled mainly by juvenile hormone (Jowett and Postlethwait, 1980; Wyatt, 1988). In
oviparous vertebrates, Vtg is produced in the liver under the influence of E2, which is
synthesized in the ovarian follicle cells in response to pituitary gonadotropins (Ng and
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Chapter 3. Expression of vtgs
Idler, 1983; Wallace, 1985). It is believed that E2 binds to estrogen receptor (ER) in the
liver and subsequently interacts with estrogen response elements (EREs), resulting in
activation of E2 responsive genes including vtgs (Wahli, 1988; Lazier and Mackay, 1993).

Since seven vtgs have been identified in the zebrafish genome, it will be interesting to
examine the sites and levels of expression for different vtgs in order to verify potential
extrahepatic expression of vtgs in teleost fish. Furthermore, since the zebrafish Vtg2
contains homology subdomains I-V and is most complete in primary structure compared

with other Vtg members, it is an ideal candidate protein for receptor-mediated gene
transfer and receptor-binding domain identification studies (see Chapter 2). Thus, it is
relevant to examine the E2 inducibility of different vtgs and their expression levels as the
native candidate Vtgs will be purified from fish serum (preferably from E2 treated
individuals for better yielding) for preparation of DNA carrier. Therefore, Northern blot
and in-situ hybridizations were carried out to examine the tissue distribution pattern of the
seven vtg transcripts and their subcellular localization in various vtg expressing tissues.
Meanwhile, the expression levels of vtg1-3 mRNAs in control and E2 treated fish were
quantified by real-time PCR and the effect of E2 treatment on the expression of vtgs was
investigated.

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Chapter 3. Expression of vtgs
3.2. Materials and Methods
3.2.1. E2 treatment
E2 stock solution (1 mg/ml) was prepared by dissolving E2 (C18H24O2, Merck) in 100%
ethanol, followed by adjusting to final concentrations of 1% (v/v) for ethanol and 0.8%
(w/v) for NaCl (Chan et al., 1991). The solution was then sonicated for 30 min on ice and
stored at 4 ºC. Both male and female zebrafish were induced for 3 days at a concentration
of 5 µg E2 per liter water and male and female fish were kept in different tanks. The water
was changed daily and E2 stock solution was added afterwards to the same concentration.

3.2.2. Generation and tests of vtg gene-specific probes
Eight cDNA probes were used in the present study. Five of them were derived from the 3’
end sequences of the following clones by restriction enzyme digestion. Briefly, a 218-bp
fragment between 3’ end Xmn I and Kpn I from clone A248 was used as vtg1 probe.
Fragments between 3’ end Ase I and Kpn I from clones A391 (182-bp) and A220 (201-bp)
were used as probes for vtg4 and vtg6, respectively. Fragments between 3’ end BamH I

and Kpn I from clones A227 (185-bp) and A349 (170-bp) were used as probes for vtg5
and vtg7, respectively. All the Kpn I sites mentioned above were located in the pBluescrip
SK vector, 20-bp downstream of each insert’s poly-A tail (see Figs. 2-1, 2-4 to 2-7 in
Chapter 2; vector map not shown). Probes for vtg2, vtg3 and β-actin were generated by
PCR. Briefly, a gene specific primer vtg2MF4 and a vector primer T7 were used in PCR
amplification of a 769-bp fragment from clone A183 as vtg2 probe (Fig. 2-2 in Chapter 2).
Probes for vtg3 (2.3 kb) and β-actin (1.4 kb) were generated by PCR amplification using
vector primers SK and T7 from clones A376 and E398, respectively. After gel

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Chapter 3. Expression of vtgs
electrophoresis, each DNA fragment was recovered by using QIAquick Gel Extraction Kit
(Qiagen) and labeled by [α-32P]-dCTP using Random Primers DNA Labeling System
(Invitrogen, Life Technologies). The 32P-labeled probe was purification by NICK Column
(Pharmacia Biotech) and stored at 4 ºC for further use.

Since vtg1 and vtg4-7 have similar sequences, the specificity of their cDNA probes was
determined by Southern blot hybridization. Briefly, cDNA inserts of vtg1 and vtg7 were
prepared by restriction enzyme digestion using Xho I and Sma I on clones A248 and
A349, respectively. cDNA inserts of vtg4, vtg5 and vtg6 were generated by restriction
enzyme digestion using Xho I and EcoR I on clones A391, A227 and A220, respectively.
The cDNA fragments (~ 25 ng each) were resolved by gel electrophoresis, denatured insitu by incubation in 1.5 M NaCl, 0.5 N NaOH for 45 min and neutralized in 1 M Tris, pH
7.4, 1.5 M NaCl for 30 min. Finally, the denatured cDNA fragments were blotted onto a
nylon membrane (GeneScreen Plus, NEN Life Science Products) in 20 x SSC overnight.

Hybridization was carried out as described by Gong and Brandhorst (1987). Briefly, the
blot was pre-hybridized in hybridization buffer [50% formamide, 5 x Denhardt's Solution,
4 x SET (1 x = 0.15 M NaCl; 1 mM EDTA; and 20 mM Tris, pH 7.8), 0.2% NaPPi,

25mM phosphate buffer, 250 µg/ml tRNA and 0.5% SDS] at 42 ºC for 2 hours. The
hybridization process was continued for another 16 hr after addition of [α-32P] dCTPlabeled cDNA probe (106 cpm per ml of hybridization buffer). After that, a series of
washes with normal stringency were performed. First, the blots were washed in washing
solution (2 x SET, 0.5% SDS and 0.2% NaPPi) for 15 minutes with two changes at room

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Chapter 3. Expression of vtgs
temperature. Then, the blots were washed in the same washing solution at 65 ºC for 20
minutes with two changes. The final wash was conducted with a low salt washing solution
(0.2 x SET, 0.5% SDS) at 65 ºC for 30 min. If a high stringent wash was necessary, an
additional wash will be carried out in 0.2 x SET at 70 ºC for 30 min after the final wash.
The blots were then autoradiographed using Kodak's BioMax MS film and BioMax MS
Intensifying Screen at -80 ºC and films were developed by an automatic developer.

3.2.3. Northern blot hybridization analysis of vtg mRNA expression
Whole fish and various tissues including brain, intestine, eye, gonad, skin, gill, liver and
muscle from E2 treated and untreated adult fish of both sexes were used for RNA
extraction. Fifteen-30 fish were sampled from each treatment and sex group. Tissues were
isolated, pooled and frozen immediately on dry ice before transferring to a - 80 ºC freezer.
Total RNAs were prepared using TRIzol Reagent (Invitrogen, Life Technologies)
according to the manufacturer's protocol. Ten µg of total RNA from each pooled tissue
was loaded on a 1.2% (w/v) denaturing agarose gel containing 6% (v/v) formaldehyde.
Following electrophoresis, RNAs were blotted onto a piece of nylon filter (GeneScreen
Plus, NEN Life Science Products). Hybridization was carried out as described in Section
3.2.2. Serial washes with normal stringency were employed in this experiment. After
autoradiography, some blots were stripped in a low salt solution (0.05 x SET, 0.1% SDS)
at 80 ºC for 30-40 minutes and then hybridized with another cDNA probe. The cDNA
probes used were generated from cDNA clones as described in Section 3.2.2.

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Chapter 3. Expression of vtgs
3.2.4. Synthesis of DIG-labeled RNA probes for in-situ hybridization
Prior to the synthesis of antisense riboprobes, cDNA clones were linearized by restriction
enzyme digestion at 5’ ends of inserts as following: A248 (vtg1) and A183 (vtg2) by Sma
I; A376 (vtg3) and E398 (β-actin) by EcoR I. The cDNA clone of liver fatty acid binding
protein (LFABP) (cloned into pGEM-T vector, isolated by Shizhen Zhu from our lab) was
linearized by Sac II. All linearized cDNA fragments were recovered from agarose gel
using a QIAquick Gel Extraction Kit (Qiagen). Antisense riboprobes were synthesized
using DIG RNA labeling kit (SP6/T7) based on the manufacturer’s protocol (Roche).
Briefly, 1 µg of DNA template was used in a 20 µl reaction, which contained 2 µl of 10 x
transcription buffer, 2 µl of 10 x NTP labeling mixture, 2 µl of T7 RNA polymerase (20
U/µl) and 1 µl of RNase inhibitor (20 U/µl). The mixture was incubated for 2 hr at 37 ºC
for probe synthesis. Subsequently, 2 µl of RNase free DNase I (10 U/µl) was added into
the reaction mixture and incubated for another 15 min at 37 ºC to digest the DNA
templates. The reaction was stopped by adding 2 µl of 0.2 M EDTA (pH 8.0) and the
product was precipitated by adding 2.5 µl of 4 M LiCl2 and 75 µl of 100% EtOH (cold),
followed by incubation on ice for 30 min. The precipitates were spun down at 14000 rpm
for 30 min at 4 ºC and washed with 250 µl of 70% EtOH. The RNA pellet was air-dried
before re- suspension in 100 µl of DEPC water. An aliquot was run on 1% (w/v) agarose
gel to check the quality of the riboprobes and the remaining was stored at -70 ºC.

3.2.5. In-situ hybridization on paraffin sections
3.2.5.1. Preparation of tissue sections
The following procedures for in-situ hybridization on paraffin sections were modified
from Jowett (1997). Briefly, tissues from both E2 treated and control adult zebrafish (male
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Chapter 3. Expression of vtgs
and female) were pre-fixed in freshly prepared 4% (w/v) PFA-PBS (pH 7.4) for 10 min at
4 ºC followed by fixation in fresh 4% (w/v) PFA-PBS at 4 ºC overnight. After washing in
1x DEPC treated PBS (DEPC-PBS) for 5-10 min at 4 ºC, the tissues were dehydrated at
room temperature in increasing gradients of ethanol (50%, 70% and 95%) for 1 hr each
and in two changes of 100% ethanol for 30 min each. The tissues were cleared in HistoClear II (National Diagnostics) and imbedded in molten paraffin (TissuePrep, Fisher
Scientific) at 60 ºC. Sections with 6-10 µm thickness were made using a microtome
(Reichert-Jung, 2030). Paraffin ribbons were floated on drops of DEPC-H2O on 3aminopropyltriethoxy silane (TESPA; Sigma) coated slides (Fisher Scientific), which
were kept on a 45-50 ºC heat block for several minutes to spread the ribbons. After that,
the water was blotted off and the slides were transferred to a 37 ºC heat block for complete
drying of the sections (5-6 hours). Slides were stored in a dry place at 4 ºC for later use.

3.2.5.2. Tissue preparation before hybridization
Tissue sections were de-waxed in two changes of Histo-Clear II for 10 min each and
rinsed in 100% ethanol for 2 min before re-hydration in decreasing gradients of ethanol
(100%, 95%, 70%, 50% and 30%) for 30 sec each and finally in DEPC-H2O for two
changes of 5 min each. The sections were then post-fixed in 4% PFA-PBS for 30 min at
room temperature, followed by washing in DEPC-PBS for 2 x 10 min. In order to reduce
non-specific binding, sections were acetylated in 0.25 % (v/v) acetic anhydride (Merck)
and 1.33 % (v/v) triethanolamine (Merck) (pH 7.5) for 10 min followed by washing in
DEPC-PBS for 2 x 5 min. Finally, sections were equilibrated in 5 x DEPC-SSC for 15 min
before pre-hybridization.

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Chapter 3. Expression of vtgs
3.2.5.3. Hybridization and wash

Tissue sections were pre-hybridized in 1 ml of hybridization buffer (50% formamide, 5 x
DEPC-SSC and 50 µg/ml salmon sperm DNA) per slide for 2-3 hr at 58 ºC in a humidified
chamber containing 5 x SSC. Then, the hybridization buffer was blotted off and 100 µl of
fresh hybridization buffer containing 0.05-0.1 µg denatured (80 ºC for 5 min) DIG-labeled
RNA probe was added onto the tissue sections. The slides were placed in a humidified
chamber containing 5 x SSC and 50 % formamide and hybridization was performed at 58
º

C overnight. After hybridization, sections were first washed in 2 x SSC at room

temperature for 30 min, followed by washing at 65 ºC in 2 x SSC for 1 hr and then in 0.1 x
SSC for another 1 hr.

3.2.5.4. Antibody staining
After washing, tissue sections were equilibrated in buffer 1 (100 mM Tris HCl, 150 mM
NaCl, pH 7.5) for 5 min and then in blocking buffer [10% (v/v) FCS (fetal calf serum) in
buffer 1] for 2 hr at room temperature. Then, 200 µl of 1 : 5000 diluted Anti-DIG-AP, Fab
fragments (Roche) in 10% FCS/buffer 1 was added to each slide, followed by incubation
in a humidified chamber overnight at 4 ºC. After that, the slides were washed in buffer 1
twice for 15 min each and equilibrated in buffer 2 (100 mM Tris HCl, 100 mM NaCl, 50
mM MgCl2, pH 9.5) for 5 min before adding 200 µl of staining solution [4.5 µl of NBT
(50 mg/ml in dimethyl formamide) and 3.5 µl of BCIP (50 mg/ml in dimethyl formamide)
per ml of buffer 2] per slide for color development. The slides were covered with
coverslips and incubated at room temperature in dark for several hours till color appeared.
The reaction was stopped by washing in TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0)
for 10 min, and background staining was removed by washing in 95% ethanol for a few
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Chapter 3. Expression of vtgs

minutes. Sections were rehydrated in dH2O for 15 min to remove Tris precipitates
followed by dehydration in increasing gradients of ethanol (70%, 95% and 100%) for 30
sec each. After cleared in Histo-Clear II for 15 min, tissue sections were mounted with
DePeX mounting medium (BDH) and observed using a microscope Axiovert 200M
(Zeiss).

3.2.6. Whole mount in-situ hybridization
Whole mount in-situ hybridization was performed as described by Korzh et al. (1998).
Briefly, isolated zebrafish ovaries were fixed in 4% PFA-PBS at 4 ºC overnight. Then, the
ovaries were washed in PBT (PBS, 0.1% Tween 20) for 4 x 20 min on a shaker at room
temperature. Prehybridization was carried out at 65 ºC overnight in hybridization buffer
(see Section 3.2.5.3 for composition). After adding denatured DIG-labeled RNA probe
(0.5-1 µg/ml buffer), the hybridization was proceeded at 68 ºC overnight. Upon finishing,
tissues were washed in 5 x SSC at 68 ºC for 1 hr, followed by washing in 0.2 x SSC at 68
º

C for 1 hr and in 1 x PBS at room temperature for 2 x 5 min. Antibody staining was

carried out as described in Section 3.2.5.4. Pictures were taken using a stereomicroscope
LEICA MZ12 (Leica).

3.2.7. Haematoxylin and eosin staining
After dewax and rehydration, tissue sections were first stained by Haematoxylin for 8-20
min. Excessive staining was removed by washing in tap water for 0.5-1 min. Then, slides
were dipped in 1% acid alcohol (1 ml HCl or H2SO4 in 100 ml 100% ethanol) for 10 sec
and washed in tap water for 0.5-1 min. After dipping in Scott’s Tap water (3.5 g NaHCO3
and 20 g MgSO4 in 1 L distilled water) for 3-4 min, sections were washed in tap water for
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Chapter 3. Expression of vtgs
1-2 min. Next, sections were counter-stained by eosin [1% (w/v) water solution] for 1-5
min and rinsed with tap water for 0.5-1 min. Finally, after dehydration in an increasing
gradient of ethanol, sections were cleared in Histo-clear II for 2 x 5 min and mounted with
DePeX.

3.2.8. Reverse transcription (RT) for real-time PCR
First-strand cDNAs were synthesized with random hexamers using SuperScript FirstStrand Synthesis System for RT-PCR (Invitrogen, life technologies). The same batch of
RNA samples was used for both Northern blot hybridization and real-time RT-PCR. To
ensure the uniformity of RT efficiency, a master mix was prepared and reactions were
performed for all samples at the same time. Briefly, 5 µg of total RNA was used in each
20 µl reaction, which contained 1 µl of random hexamers (50 ng/µl), 1 µl of 10 mM dNTP
mix, 2 µl of 10 x RT buffer, 4 µl of 25 mM MgCl2, 2 µl of 0.1 M DTT, 1 µl of RNaseOUT
Recombinant Ribonuclease Inhibitor and 1 µl of SuperScript II RT (50 units). The reaction
was incubated at 42 ºC for 50 min and terminated at 70 ºC for 15 min. One µl of RNase H
was added and the reaction was incubated at 37 ºC for 20 min to degrade RNA templates
prior to storage at - 20 ºC.

3.2.9. Real-time PCR using LightCycler instrument
3.2.9.1. Determination of absolute number of vtg transcripts
To determine the transcript number of vtg1-3 in various tissues, first-strand cDNAs
(Section 3.2.8) were analyzed by real-time PCR using LightCycler FastStart DNA Master
SYBR Green I kit (Roche Applied Science) based on respective DNA standard curves. To
avoid amplification from contaminated genomic DNA, three pairs of primers
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Chapter 3. Expression of vtgs
(vtg1RTF1/vtg1RTR1, vtg2RTF3/vtg2RTR3 and vtg3RTF1/vtg3RTR1) were designed
crossing intron/exon boundaries (see Figs. 2-1 to 2-3 in Chapter 2 for primer sequences).

The partial 5’ end genomic sequence of vtg1 was from Shan (2002) and the 3’ end
genomic sequences of vtg2 and vtg3 were obtained by PCR amplification of genomic
DNA. One µl each of the first-strand RT reactions (Section 3.2.8) was used in 10 µl of
real-time PCR reaction, which contained 5.8 µl of dH2O, 1.2 µl of 25 mM MgCl2, 0.5 µl
each of forward and reverse primers (10 mM each) and 1 µl of LightCycler FastStart DNA
Master SYBR Green I reaction mixture. After pre-incubation at 95 ºC for 10 min, PCR
was performed for 45 cycles with the following conditions: denaturation at 95 ºC for 10 s,
annealing at 58 ºC for 5 s and extension at 72 ºC for 8 s. Melting curve analysis was
carried out according to manufacturer’s protocol. All samples were analyzed in duplicate.
Crossing point (CP) was calculated by the LightCycler software using the Second
Derivative Maximum Method and baseline adjustment was performed using the
Arithmetic Method.

Standard curves were generated by amplification of 10-fold diluted DNA standards
(linearized plasmid DNA or RT-PCR fragment). Briefly, for vtg2 and vtg3, the linearized
plasmids of A183 and A376 used for synthesizing riboprobes (see Section 3.2.4) were
used as DNA standards, respectively. For vtg1, a 173-bp vtg1 fragment was amplified
from liver total RNA by RT-PCR using primers vtg1RTF1/vtg1RTR1 and subsequently
used as DNA standards. The concentrations of all DNA standards were measured
spectrophotometrically. The ranges of concentration in the serially diluted DNA standards
were 1.168 x 104 - 1.168 x 109 copies/µl for vtg1, 3.904 x 102 - 3.904 x 107 copies/µl for
vtg2 and 3.857 x 103 - 3.857 x 107 copies/µl for vtg3. In quantification of unknown
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Chapter 3. Expression of vtgs
samples, two dilutions from a DNA standard series were included in each run so as to
make sure that constant CP values were obtained from those standard samples before
importing the existing standard curve into these runs (Kühne, 2003).

3.2.9.2. Determination of relative levels of estrogen receptor α transcript
Real-time one-step RT-PCR was performed for measuring the relative levels of estrogen
receptor α (ERα) transcript in various tissues using a LightCycler-RNA Amplification kit
SYBR Green I (Roche Applied Science). A forward primer, 5’-AGGATCTGTCTCTGCATGAC-3’ (nucleotides 1201-1220) and a reverse primer, 5’-GACACACAAATTCCTCCAGC-3’ (nucleotides 1421-1440) were designed in the less conserved ligand binding
E domain based on the zebrafish ERα mRNA sequence (GenBank Accessory No.
AB037185). The reaction composition and RT-PCR program were based on the
manufacturer’s instructions with adjustment. Briefly, each 10 µl of reaction mixture
contains 0.8 µl of 25 mM MgCl2, 0.5 µl of each primer (10 µM), 0.2 µl of RT-PCR
Enzyme Mix, 2 µl of RT-PCR Reaction Mix SYBR Green I, 1 µl of resolution solution
and 1 µl of RNA (250 ng/µl) and 4 µl of PCR-grade H2O. Reverse transcription was
performed at 55ºC for 10 min, followed by denaturation at 95ºC for 30 s. Subsequent PCR
were performed for 45 cycles with the parameters for each cycle as following:
denaturation at 95 ºC for 1 s, annealing at 60 ºC for 10 s and extension at 72 ºC for 13 s.
Melting curve analysis was carried out according to manufacturer’s protocol. All samples
were analyzed in duplicate.

The relative level of ERα mRNA was determined based on a RNA standard curve, which
was generated by RT-PCR amplification of serially diluted total RNAs (50 pg-500ng)
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Chapter 3. Expression of vtgs
from E2 treated female liver using the ERα primers. For relative quantification of unknown
samples, two dilutions from the liver total RNA standard were included in each run to
make sure that constant CP values were obtained from those dilutions before importing the
existing RNA standard curve into these runs. 100 units of ERα mRNAs were arbitrarily set
for 1 ng of total RNA from E2 treated female liver.

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Chapter 3. Expression of vtgs
3.3. Results
3.3.1. Multiple tissue expression of vtg mRNAs revealed by Northern blot analysis
3.3.1.1. Specificities of vtg cDNA probes
As the nucleotide sequences of vtg3 cDNA and the 3’ end region of vtg2 cDNA were
highly divergent from the other vtg sequences (only 40-53% sequence identities) and from
each other, no cross hybridization was expected when the probes of vtg2 and vtg3 were
used. However, based on the results of the multiple sequence alignment, the remaining
five vtg cDNA sequences are very similar. To examine whether the remaining five vtg
cDNA probes cross-hybridize with each other, Southern blots of five cDNA inserts (vtg1
and vtg4-7) were hybridized with 32P-labeled vtg cDNA probes. As shown in Fig. 3-1 (left
column), weak cross-hybridized signals were observed under normal stringency washing
conditions (final wash at 30 mM NaCl, 65 °C for 30 min). After washing with high
stringency (an additional wash applied at 30 mM NaCl, 70 °C for 30 min), crosshybridizations for probes of vtg4, vtg5 and vtg6 were effectively eliminated, while trace
amounts of cross-hybridization remained for vtg1 and vtg7 (Fig. 3-1, right column). For
the vtg7 probe, a general reduction of hybridization signal including a specific signal was
observed after a high stringency wash. In all cases, vector DNA was cross-hybridized by
all five probes probably due to the presence of a large quantity of vector DNAs (Fig. 3-1).

The above results suggested that in our Southern blot hybridization, the non-specific
signal due to cross-hybridization for probes of vtg4, vtg5 and vtg6, can be negligible under
the normal stringency wash and completely eliminated under the high stringency wash.
For vtg1 and vtg7, weak cross-hybridization existed under the normal stringent washing

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Chapter 3. Expression of vtgs

A

bp

M

3500

1

3000
2500
2000
1500

6

4

1000

B

5

Probe:

7

G

1

1

vtg1

H

C
4

vtg4

4

I

D

vtg5
5

5

J

E
6

vtg6

6

F

K
vtg7
7

Normal stringency

7

High stringency

Fig. 3-1. Specificity of vtg cDNA probes examined by Southern blot analysis. A: Gel
electrophoresis of five vtg inserts, which are designated as fragments 1 (vtg1), 4 (vtg4), 5
(vtg5), 6 (vtg6) and 7 (vtg7). B-F: After hybridized with [α-32P]-dCTP labeled vtg cDNA
probes, the blots were subjected to a normal stringent wash (30 mM NaCl, 65 °C for 30
min), followed by 1st exposure. G-K: The same set of blots were washed to high
stringency (30 mM NaCl, 70 °C for 30 min) followed by 2nd exposure. The vtg probes
used are indicated on the right. The position of linearized pBluescript SK vector is
indicated by arrowheads.

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Chapter 3. Expression of vtgs
condition and can not be completely eliminated by the high stringency wash. Thus, this
weak cross-hybridization may affect the investigation of vtg7 expression, since the

expression level of vtg7 was much lower that that of vtg1. In the subsequent Northern blot
analysis, normal stringency wash was performed for all seven vtg probes.

3.3.1.2. Tissue distribution of seven vtg mRNAs
Northern blot hybridization showed that transcripts of all seven vtgs were expressed
predominantly in the liver of control female fish with the sizes estimated as ~ 4.3 kb for
vtg1 and vtg3-7 mRNAs, and ~ 5.5 kb for vtg2 mRNA (Fig. 3-2, left column).
Interestingly, transcripts of all seven vtgs were also detected in the intestine samples of
female though the levels of expression were much lower than those in the liver. Also
noteworthy is the fact that very weak vtg hybridization signals were detected in the ovary
samples of both control and E2 treated female fish when vtg1 or vtg2 probe was used (Fig.
3-2, arrowheads). Other vtg mRNAs were not detectable in the ovary samples probably
because the expression levels were too low. After E2 treatment, the expression levels for
all seven vtgs were enhanced in the liver and intestine of female fish as indicated by the
increased intensity of vtg hybridization signals in these tissues. However, the induction of
ovary expression was not obvious following E2 treatment.

No vtg hybridization signal was observed in total RNA samples prepared from various
tissues of control male fish (data not shown). In E2 treated male fish, transcripts of the
seven vtgs were induced predominantly in the liver (Fig. 3-2, right column). Interestingly,
vtg hybridization signals were also detected in the intestine, testis and very weakly in the
muscle of E2 treated male.
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Chapter 3. Expression of vtgs

Control female
B I E O SG LM F
28S

E2 treated female
B I EO SG L M F

E2 treated male
B I E T SG LM F

Probe:

vtg1

18S
28S

vtg2

18S
28S

vtg3

18S
28S

vtg4

18S
28S

vtg5

18S
28S

vtg6

18S
28S

vtg7

18S
β-actin

Fig. 3-2. Northern blot analysis of tissue distribution of vtg mRNAs in both female and
male fish with or without E2 treatment. 10 µg of total RNA was loaded to a 1.2%
formaldehyde agarose gel and resolved by electrophoresis. Blots were hybridized with
32
P-labeled vtg cDNA probes and washed under normal stringent condition (30 mM NaCl,
65 °C for 30 min). All blots were stripped and re-probed with β-actin cDNA probes and
the results are representatively shown at the bottom. No vtg hybridization signal was
detected in tissues of control male fish (data not shown). The positions of 28S and 18S
RNAs are marked on the left and names of vtg transcripts on the right. Weak vtg signals in
ovary samples are marked by arrowheads. B, brain; I, intestine; E, eye; O, ovary; T, testis;
S, skin; G, gill; L, liver; M, muscle; F, whole fish.

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Chapter 3. Expression of vtgs

Thus, in addition to the liver, vtg mRNAs were also detected in two extrahepatic tissues in
female fish, the intestine and ovary. In E2 treated male fish, the expressions of vtg mRNAs
were observed in the liver and three non-liver tissues including intestine, testis and
muscle.

3.3.2. Quantitative analysis of vtg and ERα mRNAs
3.3.2.1. Quantification of vtg mRNA copy number
3.3.2.1.1. Specificities of vtg primers
To quantitatively evaluate the relative levels of vtg mRNAs in liver and extrahepatic
tissues of control and E2 treated fish, the expression levels of vtg1, vtg2 and vtg3 were
determined by real-time PCR as the three vtg genes represent the three vtg groups
identified in the zebrafish.

To avoid amplification from potentially contaminated genomic DNA in the total RNA
preparations, all three pairs of primers were designed crossing intron/exon junctions based
on partial vtg genomic sequences and corresponding cDNA sequences (see Figs. 2-1 to 23 in Chapter 2). Primers of vtg2 and vtg3 were designed in the extended 3’ end coding
region and in a divergent region near the 3’ end of cDNA sequence, respectively, for
specificity of amplification. As shown in Fig. 3-3A,C,E, by melting curve analysis,
specific and overlapping peaks were produced in samples containing first-strand vtg
cDNAs and vtg DNA standards, but not in samples containing genomic DNA or dH2O for
all three pairs of vtg primers used. The specificity was further confirmed by gel
electrophoresis, as only one band was amplified from each experimental sample and the

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Chapter 3. Expression of vtgs

A

C

cDNA
RT-PCR fragment
gDNA
dH2O

cDNA
Plasmid DNA
gDNA
dH2O

B bp

M

1

2

3

4

500
400
300
200
100

D

bp

vtg1

M

1

2

3

4

500
400
300
200

vtg2

100

E

cDNA
Plasmid DNA
gDNA

dH2O

F
vtg3

1

2

3

4

M

bp
500
400
300
200
100

Fig. 3-3. Demonstration of specificities of vtg primers used in real-time PCR by melting
curve analysis and gel electrophoresis. Specific products were amplified by real-time PCR
from samples containing first-strand vtg cDNAs and vtg DNA standards when primers for
vtg1 (A), vtg2 (C) or vtg3 (E) were used. The amount of templates in each 10 µl of PCR
reaction was 1 µl of first-strand vtg cDNAs, 1 µl of 173 bp vtg1 RT-PCR fragment (1.168
x 107 copies), 1 µl of linearized vtg2 cDNA clone A183 (3.904 x 104 copies), 1 µl of
linearized vtg3 cDNA clone A376 (3.857 x 104 copies) or 50 ng of zebrafish genomic
DNA. PCR products from A,C,E were resolved on agarose gel shown in B,D,F,

respectively. Lanes 1, 2, 3 and 4 in B,D,F contain PCR products from first-strand cDNA,
DNA standards, genomic DNA and dH2O, respectively.

105


Chapter 3. Expression of vtgs
sizes of the amplified products were the same as estimated from vtg cDNA sequences, i.e.,
173 bp for vtg1, 190 bp for vtg2 and 130 bp for vtg3 (Fig. 3-3B,D,F).

3.3.2.1.2. Determination of absolute number of vtg mRNAs
To determine the copy number of vtg mRNAs by real-time PCR, a standard curve was
constructed by amplifying serially diluted vtg1, vtg2 or vtg3 DNA standards. As shown in
Fig. 3-4, these standard curves were linear over 4-5 orders of magnitude with a correlation
coefficient (R2) near 0.99 for each curve. Amplification curves showing crossing point
(CP) values are presented in Fig. 3-5. By using the standard curves, concentrations of vtg
mRNAs (copy number per microgram of total RNA) are calculated and the results are
summarized in Table 3-1.

The concentrations (copy number per µg of total RNA) of vtg1-3 mRNAs in the liver were
the highest compared with those in other tissues in the control female fish (Table 3-1). In
comparison, the mRNA levels of vtg2 (1.8 x 107 copies/µg) and vtg3 (2.1 x 106 copies/µg)
were about 100 and 1000 times lower than that of vtg1 mRNA (1.4 x 109 copies/µg) in the
control female liver. The concentrations of vtg mRNAs in the intestine of control female
fish were measured as 7.2 x 107, 1.8 x 106 and 1.0 x 105 copies/µg for vtg1, vtg2 and vtg3,
respectively. Thus, the concentrations of vtg1-3 mRNAs in the intestine were
approximately 10-20 times lower than those in the liver in the control female fish. In the
ovary of control females, the concentrations of vtg mRNAs were 2.6 x 106 copies/µg for
vtg1 and 1.2 x 105 copies/µg for vtg2 which are about 500 and 150 times lower than those
in the liver, respectively. The vtg3 mRNA level in the ovary of control female was too low

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Chapter 3. Expression of vtgs

A

y = -3.785x + 45.246

30

R2 = 0.9875

CP value

25
20

vtg1

15
10
5
0
0

1

2

3

4

5

6

7

8

9

10

Log concentration (copy number)

B

y = -3.99x + 42.62

35

2

R = 0.9989

30

CP value

25
20

vtg2

15
10
5
0
0

1

2

3

4

5

6

7

8

Log concentration (copy number)

C

y = -3.982x + 45.381

35

2

R = 0.9998

30
CP value

25
20

vtg3

15
10
5
0
0

1

2

3

4

5

6

7

8

Log concentration (copy number)

Fig. 3-4. Standard curves used for determination of absolute copy numbers of vtg1 (A),
vtg2 (B) and vtg3 mRNAs (C). Each standard curve was generated by plotting log
concentration (copy number) of DNA standard series against cycle number (CP). For each
dilution point, duplicated reactions were performed and the mean CP value was used. R2,
squared correlation coefficient.

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Chapter 3. Expression of vtgs

A

vtg1,
female (C)

C

D

CP

vtg2,
female (C, E2)

vtg3,
female (C, E2)

B

vtg1,
female (E2)

CP

CP

CP

CP

CP

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Chapter 3. Expression of vtgs

E

F

G

vtg1,
male (E2)

vtg2,
male (E2)

vtg3,
male (E2)

CP

CP

CP

Fig. 3-5. Quantification of vtg mRNA copy number in various tissues of control and E2
treated female fish and E2 treated male fish by real-time PCR. Amplification curves with
fluorescence values versus cycle number (CP) are shown. A,B,C,D: Amplification curves
for measuring the copy numbers of vtg1-3 mRNAs in samples of female fish. E,F,G:
Amplification curves for measuring the copy numbers of vtg1-3 mRNAs in samples of E2
treated male fish. All samples were analyzed in duplicate. G, gill; S, skin; M, muscle; O,
ovary; T, testis; I, intestine; L, liver; W, whole fish. Affixes C and E indicate control and
E2 treated samples, respectively.

109