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

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.. Meanwhile, the expression levels of vtg1-3 mRNAs in

Chapter 3

Expression of zebrafish vtg genes

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

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 specific In insects, such as Drosophila and locust, the synthesis of Vtg in the fat body is

tissue-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

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

3.2 Materials and Methods

3.2.1 E 2 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

electrophoresis, each DNA fragment was recovered by using QIAquick Gel Extraction Kit

(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 in-situ 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,

hybridization process was continued for another 16 hr after addition of [α-32P] labeled 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

dCTP-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

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

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 Histo-Clear 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 3-aminopropyltriethoxy 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

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

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

ºC 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

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

ºC 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

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

strand cDNAs were synthesized with random hexamers using SuperScript Strand 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

First-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

(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

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, CATGAC-3’ (nucleotides 1201-1220) and a reverse primer, 5’-GACACACAAATTCC-TCCAGC-3’ (nucleotides 1421-1440) were designed in the less conserved ligand binding

5’-AGGATCTGTCTCTG-E domain based on the zebrafish 5’-AGGATCTGTCTCTG-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)

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

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), cross-

hybridizations 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

Normal stringency High stringency

uthern blot analysis A: Gel

electrophoresis of five vtg inserts, which are designated as fragments 1 (vtg1), 4 (vtg4), 5

4 4

5 5

6 6

7 7

1

Probe:

Fig 3-1 Specificity of vtg cDNA probes examined by So

(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

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

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

vtg1 vtg2

vtg3 vtg4 vtg5 vtg6 vtg7

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

32P-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

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 1 to

2-3 in Chapter 2) Primers of vtg2 and vtg2-3 were designed in the extended 2-3’ 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

500

100 200

400 300

dH2O gDNA

cDNA Plasmid DNA

E

dH2O gDNA Plasmid DNA cDNA

vtg1

vtg2

vtg3

D C

F

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

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

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

vtg1,

female (C)

vtg1,

female (E2) 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

Table 3-1 vtg mRNA concentrations (copy number per microgram of total RNA) in six

selected tissues from control (C) and E2 treated (E2) female fish and E2 treated male fish

† Percentage amount was obtained by dividing vtg mRNA concentrations in non-liver

tissues by that in the liver, assuming vtg mRNA concentration in the liver was 100% for

all three vtg transcripts

* Dashes represent extremely low concentrations of vtg mRNAs, which cannot be

estimated accurately using the current standard curves

Note: Total RNAs were extracted from 6 types of tissues, which were pooled from 15-30 fish, respectively