E. David Thompson, Per-Erik Olsson, Gregory D. Mayer, Carl Haux, Patrick J.

Walsh, Erin Burge and Christer Hogstrand

Am J Physiol Regulatory Integrative Comp Physiol 280:527-535, 2001.

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on the following topics:
Biochemistry .. Complementary DNA
Biochemistry .. Metallothionein
Biophysics .. Vitellogenin
Physiology .. Estradiol
Physiology .. Estrogen
Physiology .. Beryciformes

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 Am J Physiol Regulatory Integrative Comp Physiol
280: R527–R535, 2001.

Effects of 17␤-estradiol on levels and distribution

of metallothionein and zinc in squirrelfish
E. DAVID THOMPSON,1 PER-ERIK OLSSON,2 GREGORY D. MAYER,1 CARL HAUX,3
PATRICK J. WALSH,4 ERIN BURGE,1 AND CHRISTER HOGSTRAND1,4
1
T.H. Morgan School of Biological Sciences, University of Kentucky, Lexington, Kentucky
40506-0225; 2Division of Physiology, Department of Cellular and Molecular Biology, Umeå
University, SE-901 87 Umeå, Sweden; 3European Commission, Joint Research Centre, Environment
Institute, TP 460, I-21020 Ispra, Italy; and 4Division of Marine Biology and Fisheries and National
Institute of Environmental Health Sciences Marine and Freshwater Biomedical Science Center,
Rosenstiel School of Marine and Atmospheric Sciences, University of Miami, Miami, Florida 33149
Received 8 November 1999; accepted in final form 15 September 2000

Thompson, E. David, Per-Erik Olsson, Gregory D. sis. One such protein believed to regulate intercellular
Mayer, Carl Haux, Patrick J. Walsh, Erin Burge, and zinc concentrations is metallothionein (MT), a low-

Downloaded from ajpregu.physiology.org on October 25, 2009

Christer Hogstrand. Effects of 17␤-estradiol on levels and weight (⬃6 kDa) protein characterized by high cysteine
distribution of metallothionein and zinc in squirrelfish. Am J content (30–35%) and a lack of aromatic amino acids
Physiol Regulatory Integrative Comp Physiol 280: R527– and histidine (21). MT binds elements of groups IB and
R535, 2001.—Females of the squirrelfish family (Holocentri-
IIB of the Periodic Table of Elements with a typical
dae) accumulate higher levels of zinc in the liver than any
other known animal. This zinc accumulation is made possible
stoichiometry of 7 metal atoms of zinc or cadmium per
by high expression of the zinc-binding protein, metallothio- MT molecule or 10–12 atoms of copper, mercury, or
nein (MT). In the present study, the squirrelfish (Holocentrus silver per MT molecule (22). Although the exact mech-
ascensionis) MT cDNA was cloned and sequenced. The de- anism is uncertain, these metals also induce transcrip-
duced amino acid sequence was very similar to other teleost tion of the MT gene (28) mediated by metal regulatory
MT. The role of estrogens on zinc metabolism was investi- elements of the MT promoter (35). MT has been shown
gated by injecting male and immature female squirrelfish to bind zinc with a dissociation constant of 3.2 ⫻ 10⫺13
with 17␤-estradiol (E2). E2 treatment triggered transient M⫺1 (31). Despite the strong affinity of MT for zinc,
increases in plasma zinc and vitellogenin (VTG) levels, and recent studies have shown that MT can donate and
both of these variables showed very similar time courses. accept zinc from zinc proteins (18, 25). These charac-
These results suggest that E2 is responsible for the large teristics suggest that MT would be well suited for
hepatoovarian translocation of zinc observed in female squir- regulating zinc balance within the cell, but this func-
relfish and that VTG might be a vehicle for zinc. E2 did not tion remains to be demonstrated.
directly alter the levels of zinc or MT mRNA in the liver.
The squirrelfish family (Holocentridae) presents an
However, the hepatic MT protein concentration increased
differentially in the nuclear fraction. Thus E2 is probably
interesting system for studying zinc homeostasis and
responsible for the association of MT with the nuclear frac- elucidating the function of MT. Females of this family
tion previously observed in untreated mature female squir- have been shown to maintain high levels of hepatic
relfish. zinc, up to 70 ␮mol/g wet wt, which is unseen in any
other studied organism (12, 14, 15). In the liver, fe-
zinc; metallothionein complimentary deoxyribonucleic acid; males also have unusually high MT levels that are
reproduction; endocrinology; teleost closely correlated to liver zinc concentrations (0.89 ⬍
r ⬍ 0.99) and not to other metals that can bind MT (15).
Zinc is believed to be the native metal of squirrelfish
ZINC IS AN ESSENTIALmicronutrient that plays a crucial MT. Moreover, the only organs displaying conspicu-
role in many cellular processes involving transcription, ously high zinc levels are the liver and the ovaries (12).
enzyme structure and activity, protein interactions, There are very high zinc concentrations in the retina as
and even cell signaling (45). Zinc is also involved in in other vertebrates (47), but this does not seem to be
antioxidant defense (5) and in maintaining membrane sex specific. Squirrelfish inhabit coral reefs throughout
integrity (1). As such, minimal levels must be main- the world, and females in all six species studied contain
tained for normal cellular function. However, zinc may high zinc and MT levels, suggesting that zinc accumu-
become toxic if accumulated at high levels (16), and lation is independent of geographical location (12, 14,
many proteins are therefore involved in zinc homeosta- 15). Also, analysis of intestinal content in wild male

Address for reprint requests and other correspondence: E. D. The costs of publication of this article were defrayed in part by the
Thompson, T. H. Morgan School of Biological Sciences, 101 Morgan payment of page charges. The article must therefore be hereby
Bldg., Univ. of Kentucky, Lexington, KY 40506-0225 (E-mail: marked ‘‘advertisement’’ in accordance with 18 U.S.C. Section 1734
[email protected]). solely to indicate this fact.

http://www.ajpregu.org 0363-6119/01 $5.00 Copyright © 2001 the American Physiological Society R527
R528 ESTROGEN CONTROL OF ZN AND MT IN SQUIRRELFISH

and female squirrelfish indicated that female-specific ferred to ⫺80°C, where they were stored until used for total
zinc accumulation is not a result of dietary differences RNA extraction as described below.
between the sexes (12). It has been observed previously Tissue zinc content. Liver and gonad samples were sub-
in rainbow trout (Oncorhynchus mykiss) that hepatic jected to acid digestion in 2.0 ml of 70% HNO3 (trace-metal
grade, Fisher). The tubes were heated in a sand bath for 3 h
MT and zinc levels increase during female sexual mat- at 120°C and then cooled to room temperature before 0.50 ml
uration (29, 30). These findings suggest that some of H2O2 was added. The temperature was then gradually
female-specific factor plays a role in zinc accumulation increased to 120°C until all liquid had evaporated. The dried
and that increased zinc and MT levels could be in- residues were reconstituted with 4.0 ml of 0.50% HNO3.
volved in female squirrelfish reproduction. One female- These samples were then analyzed for zinc content by air/
specific factor involved in the reproductive cycle is acetylene flame atomic absorption spectroscopy (model 2380;
17␤-estradiol (E2). E2 is the endocrine signal for the Perkin-Elmer). Plasma samples taken from these fish were
hepatic production of vitellogenin (VTG), the precursor also analyzed for zinc content in the same manner.
VTG and E2 analysis. Plasma samples from each fish were
to yolk proteins in all oviparous vertebrates, and the
measured for VTG and E2 content by ELISA (10, 37). The
vitelline envelope (zona radiata) proteins (17, 46). The VTG ELISAs were performed according to the procedure of
purpose of the present study was to evaluate if E2 plays Palmer et al. (32). Each plasma sample was diluted 1:100 in
a role in zinc accumulation and distribution in female PBS (0.15 M NaCl, 0.0015 M KH2PO4, 0.015 M Na2HPO4,
squirrelfish and also in the production and distribution and 0.002 M KCl, pH 7.4), and 100 ␮l of this dilution were
of MT. This aim can be relatively easily investigated in added per well in 96-well microtitre plates (Fisher). Squir-
fish because immature females and males can express relfish VTG was isolated by ion-exchange chromatography

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estrogen receptors and are responsive to injections of according to the protocol of Silversand and Haux (36), with
E2. Changes in treated individuals can then be com- minor modifications, and this purified VTG was used as a
standard. The samples were incubated in duplicate for 2 h at
pared with control individuals. Such experiments have
room temperature to allow for optimal adsorption of antigen
been used to study E2 induction of VTG and vitelline to the plates and were then washed four times with 300 ␮l
envelope proteins (17, 46) as well as xenoestrogens in PBS per well. After the washes, the plates were treated with
the environment (40). In addition, the present study 150 ␮l of blocking buffer (5% dried milk in PBS) for 30 min at
involves the cloning of the squirrelfish MT gene and room temperature. Blocking buffer was then removed, and
the examination of the primary amino acid sequence the wells were subjected to 100 ␮l of primary antibody and
along with comparisons with other known MT se- incubated overnight at 4°C. The primary antibody was a
quences to determine if the ability of female squir- rabbit anti-turtle VTG IgG (gift from Dr. Brent Palmer,
relfish to utilize massive amounts of zinc is a result of University of Kentucky) diluted 1:5,000 in blocking buffer.
The cross-reactivity of this antibody with squirrelfish VTG
some unique property of squirrelfish MT.
was confirmed by Western blot (data not shown). After the
primary antibody incubation, the plates were washed six
MATERIALS AND METHODS times with PBS, subjected to 100 ␮l of secondary antibody,
and incubated for 2 h at room temperature. The secondary
Animal care and E2 injections. Squirrelfish (Holocentrus antibody was a horseradish peroxidase-linked goat anti-rab-
ascensionis) were collected by scuba divers off Tavernier, FL, bit IgG (Bio-Rad) diluted 1:1,000 in blocking buffer. The
in May and early June. Fish were transported to the Rosen- plates were then washed four times with PBS, and 100 ␮l of
stiel School of Marine and Atmospheric Sciences, University a tetramethylbenzidine peroxidase enzyme immunoassay
of Miami, where they were housed in two tanks (4,000 liters) substrate (Bio-Rad) were added according to the manufactur-
supplied with a continuous flow of aerated seawater (28°C) er’s specifications. After a 15-min incubation, 50 ␮l of 0.5 M
from Biscayne Bay. Fish were fed daily to satiation with live H2SO4 were added to the substrate to stop the reaction, and
shrimp and were allowed to acclimate to laboratory condi- the plates were read on a microplate reader (model 450;
tions for a total of 4 days before experimentation. Bio-Rad) at 450 nm. Because of limitations in sample volume,
The squirrelfish in one tank were given interperitoneal these samples were not rerun. Although obtained values
injections of 5 mg E2/kg body wt (1 ml/kg in peanut oil) on from these samples may underestimate the true VTG con-
days 0, 2, and 4. A control group housed in the other tank was centrations, the data were not censored (27). The potential
injected with peanut oil only. Control and treated fish were error introduced would not affect any conclusions made. E2
killed on either day 5, 6, or 10. was analyzed using estradiol ELISA kits (Oxford Biomedical
Sampling. Fish were killed by an overdose of MS-222 (0.5 Research) according to the manufacturer’s specifications.
g/l) and weighed. One milliliter of blood was withdrawn from Hepatic subcellular fractionation. Subcellular fractions of
the caudal vessel with a heparinized syringe, and plasma liver were obtained by differential centrifugation of liver
was separated from blood cells by centrifugation (14,000 g) homogenates via the procedure for rainbow trout liver as
for 3 min. The plasma was divided into 50-␮l aliquots and described by Hogstrand et al. (12). Liver samples of known
was stored at ⫺80°C for subsequent analysis of E2, VTG, mass (⬃0.5 g) were homogenized individually in an ice-cold
zinc, and MT as described below. Livers and gonads were isotonic homogenization buffer (35 mM Tris 䡠 HCl, 0.20 M
dissected out, weighed, and immediately divided into ali- KCl, and 0.25 M sucrose, pH 7.4) using a glass-Teflon homog-
quots. A sample of tissue of weighted mass (⬃0.5 g) was enizer. The homogenate was centrifuged (370 g) for 5 min at
taken from each liver and subjected to subcellular fraction- 4°C, and the supernatant was removed, whereas the pellet
ation as described below. Samples of liver and gonad (⬃0.5 g) (nuclear fraction) was immediately placed on ice. The super-
were placed into individual 16 ⫻ 150-mm borosilicate glass natant was centrifuged (9,200 g) for 5 min at 4°C, and the
tubes for acid digestion and subsequent zinc analysis. The pellet (mitochondrial/lysosomal fraction) was saved and
remaining liver and gonads were wrapped in aluminum foil placed on ice. This supernatant was then centrifuged
and frozen in liquid nitrogen. The frozen samples were trans- (130,000 g) for 60 min at 4°C, resulting in a small pellet
 ESTROGEN CONTROL OF ZN AND MT IN SQUIRRELFISH R529

(microsomal fraction) that was saved and immediately placed mixture was shaken and incubated for 10 min before centrif-
on ice. The final supernatant (cytosolic fraction) was divided ugation (12,000 g) to pellet the RNA. The supernatant was
into 0.50-ml aliquots and immediately placed on ice. All removed, and 1.0 ml of 75% ethanol was added to the pellet.
pellets were resuspended in 0.50 ml of fresh homogenization The sample was vortexed and centrifuged (7,500 g) for 5 min
buffer, and subcellular fractions were divided into aliquots at 4°C to pellet the RNA. The pellet was allowed to partially
and transferred to liquid nitrogen. These samples were then air dry before being redissolved in RNase-free water. The
stored at ⫺80°C until used in Western analysis for MT. extracted RNA was then stored at ⫺80°C until used as
Western analysis. Each subcellular fraction was subjected described in Northern analysis.
to SDS-PAGE with a discontinuous buffer system according Squirrelfish MT cloning and sequencing. Mature female
to the protocol of Laemmli (23). The protein concentration for squirrelfish liver was used as a source of poly(A)⫹ RNA for
each subcellular fraction was determined by Bradford (4) construction of a ␭ ZAP cDNA library, using the ZAP Express
assay, and samples were diluted with distilled water as cDNA library kit (Stratagene). After construction, the li-
necessary. These samples were then mixed 1:4 with sample brary, which contained ⬃500,000 individual clones with an
buffer [62 mM Tris 䡠 HCl, pH 6.8, 10% glycerol, and 5.0% average size of 500 base pairs (squirrelfish MT cDNA is ⬃200
2-mercaptoethanol (added just before dilution), 2.0% SDS, base pairs), was amplified and used to screen for MT cDNA.
and 0.0012% bromphenol blue] and were heated at 100°C for The library was plated out at a density of 20,000 plaques/
5 min. A total of 25 ␮g of protein was loaded into each well. plate and was screened for MT cDNA using a digoxigenin
Perch MT was used as a standard (13). Electrophoresis was (Dig)-labeled perch MT cRNA probe. Several positive clones
carried out on a 4% stacking gel and a 12.5% separating gel were obtained, and their identity was confirmed after DNA
at 100 V for 2 h in a Mini-Protean II electrophoresis system sequencing. A full-length clone of squirrelfish MT cDNA was
(Bio-Rad). thereby obtained.

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After electrophoresis, proteins were transferred from poly- Northern analysis. Total RNA extracted from liver sam-
acrylamide gels to nitrocellulose membranes (Schleicher & ples (⬃10 ␮g) was subjected to electrophoresis on a 1.5%
Schuell) by electroblotting, as described by Towbin et al. (43), agarose gel with formaldehyde as denaturant (34). To stan-
in a SemiPhor TE 70 semidry transfer unit (Hoefer Scientific) dardize results, a reference sample with a constant amount
at 0.8 mA/cm2 constant current for 60 min at room temper- of MT mRNA was loaded onto each gel. In addition, some
ature. Once proteins were transferred, the membranes were experimental samples overlapped between gels as a control
blocked with 5.0% dried milk in TBS-T (20 mM Tris 䡠 HCl, pH for interassay variability. After complete separation, RNA
7.4, 137 mM NaCl, and 0.10% Tween 20) for 60 min to was transferred to a 170-cm2 Hybond N nylon membrane
prohibit further nonspecific protein binding. All incubations (Amersham) by capillary/gravity blotting using the Turbo
were carried out at room temperature. The membranes were Blotter (Schleicher & Schuell). The RNA was ultravioletly
then subjected to a series of washes in fresh TBS-T (one 15 cross-linked to the membrane followed by incubation in 18 ml
min and two 5 min) followed by a 1-h incubation in primary of the prehybridization buffer [50% formamide, 5⫻ saline-
antibody. The primary antibody was a rabbit antiperch se- sodium citrate (SSC), 2.0% blocking reagent (Boehringer
rum (13) diluted 1:8,000 in TBS-T. After another series of the Mannheim), 0.10% N-lauroylsarcosine, and 0.02% SDS] at
same washes, the membranes were incubated 1 h in second- 68°C for 4 h. The prehybridization buffer was discarded, and
ary antibody. The secondary antibody was a horseradish the membrane was hybridized for 18 h at 68°C in 18 ml of the
peroxidase-conjugated donkey anti-rabbit IgG (Amersham) same buffer, with 0.30 ␮l probe/ml added, using a Dig-labeled
diluted 1:40,000 in TBS-T. After a final series of washes, antisense squirrelfish MT-cRNA probe. The probe was syn-
immunodetection was performed with an enhanced chemilu- thesized by in vitro transcription of squirrelfish MT-cDNA
minescence system (ECL; Amersham) according to the man- cloned downstream of the T7 promoter in the pBK-CMV
ufacturer’s specifications. Chemiluminescence was captured plasmid (Stratagene). The plasmid was linearized, and the
on photographic film (Kodak), and the optical density (OD) of Dig-labeled RNA transcript was made from the DNA tem-
each band was quantified using Sigma Gel software (Jandel plate by T7 polymerase in the presence of a nucleotide mix
Scientific). Each Western blot was exposed for 15, 30, and containing Dig-labeled dUTP (Boehringer Mannheim). After
60 s to ensure the OD linearity of the film. The OD of each hybridization, the membrane was subjected to stringency
unknown was compared with the OD of the internal standard washes (2 washes in 2⫻ SSC, 0.10% SDS for 5 min at room
of each gel. Although a known amount of the perch MT temperature; 2 washes in 0.1⫻ SSC, 0.1% SDS for 5 min at
standard was added to each gel, the results are presented as 68°C), followed by immunochemiluminescent detection of
arbitrary units because it has not been determined if the Dig-labeled probe using a Fab fragment of sheep anti-Dig-AP
immunoreactivity of perch and squirrelfish MT is the same. conjugate (Boehringer Mannheim) and CSPD (Boehringer
The relative amount of MT in each fraction was thus calcu- Mannheim) as chemiluminescent substrate. Chemilumines-
lated as the ratio of sample OD to standard OD. cence was captured on X-ray film, and MT mRNA bands were
Total RNA extraction. Liver and gonad tissues previously quantified using Sigma Gel software (Jandel Scientific). Each
stored at ⫺80°C were thawed, and 50–100 mg of each sample Northern blot was exposed for at least four exposure times to
were homogenized in 1.0 ml of TRIzol Reagent (GIBCO-BRL) ensure OD linearity of the film. Arbitrary units were derived
using a glass-Teflon homogenizer. After homogenization, by the ratio of sample OD to reference OD.
samples were centrifuged (12,000 g) to remove insoluble Statistics. Because gonadal examination is the only known
material. The RNA-containing supernatant was removed and way to distinguish gender and sexual maturity in squir-
treated with 0.20 ml chloroform. This mixture was shaken for relfish, injections were administered to all fish, and males
15 s, incubated for 2–3 min, centrifuged (12,000 g) for 15 min, and immature females (operationally defined as gonadoso-
and allowed to separate into aqueous and organic phases. matic index ⬍0.25) were sorted out after death. Statistical
The RNA-containing aqueous phase was transferred to fresh analysis, employing the Mann-Whitney U-test, showed that
tubes and treated with 0.25 ml isopropanol followed by 0.25 immature females were not significantly different from males
ml of a high-salt precipitation solution (1.20 M sodium citrate in all criteria studied in this experiment (Table 1). As such,
and 0.80 M NaCl) to remove glycogen, which otherwise made immature females and males are collectively analyzed
the final RNA pellet difficult to redissolve. The resulting throughout the remainder of the text.
R530 ESTROGEN CONTROL OF ZN AND MT IN SQUIRRELFISH

Table 1. Comparison of sham injection in males and immature females

Liver Zinc, Plasma Zinc, E 2, VTG, MT mRNA, MT C, MT N, MT M/L,
LSI ␮mol/g ␮mol/ml ng/ml ␮g/ml arbitrary units arbitrary units arbitrary units arbitrary units

Males 1.21 ⫾ 0.10 0.41 ⫾ 0.03 0.036 ⫾ 0.004 2.61 ⫾ 0.46 0.65 ⫾ 0.45 0.45 ⫾ 0.13 0.46 ⫾ 0.09 0.15 ⫾ 0.04 0.17 ⫾ 0.03
Females 1.44 ⫾ 0.11 1.18 ⫾ 0.45 0.041 ⫾ 0.004 1.25 ⫾ 0.87 2.01 ⫾ 1.37 0.41 ⫾ 0.10 0.46 ⫾ 0.10 0.15 ⫾ 0.05 0.10 ⫾ 0.03
P value 0.138 0.382 0.368 0.057 0.254 0.923 0.804 0.999 0.088
Values are means ⫾ SE; no. of observations was 4–9 for immature females and 5–11 for males. Comparison of sham-injected males and
immature females in terms of liver somatic index (LSI), plasma zinc, 17␤-estradiol (E2), vitellogenin (VTG) levels, concentrations of
metallothionein (MT) mRNA in liver, and MT protein in cytosolic (C), nuclear (N), and mitochondrial/lysosomal (M/L) hepatic subcellular
fractions. Immature females were operationally defined as females with a gonadosomatic index of ⬍0.25. Males were not different from the
immature females in any of the variables listed. Statistical comparisons were calculated by the Mann-Whitney U-Test (P ⬍ 0.05).

Pairwise differences between variables in E2-treated fish zinc distribution as well as production and intracellu-
and the control at each sampling occasion were examined by lar distribution of hepatic MT. Quantification of
the Mann-Whitney U-test. Groups were considered signifi- plasma E2 indicated that injections were successful in
cantly different at P ⬍ 0.05. Because squirrelfish samples producing increased levels of E2 in treated fish (Fig.
were divided between the different laboratories involved in
the study and further separated into aliquots for different
1A). E2-injected squirrelfish sampled 1 day after the
analyses, the number of observations for each treatment final E2 injection (day 5) had E2 levels [10.71 ⫾ 3.44
group and sampling occasion was not the same for all vari- (SE) ng/ml, n ⫽ 3] fourfold higher than control fish
(2.60 ⫾ 1.15 ng/ml, n ⫽ 6). E2 levels in the treated

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ables.
The derived primary sequence of squirrelfish MT was group were still markedly elevated on day 6 (E2 treat-
submitted to a BLAST search in the SWISS-PROT protein ed: 6.64 ⫾ 1.24 ng/ml, n ⫽ 6; control: 1.84 ⫾ 1.25 ng/ml,
sequence database (41). The squirrelfish MT sequence was n ⫽ 3) but were not different from that of the sham-
compared with existing MT sequences, and similarities were injected control at day 10.
scored. The response to E2 in treated squirrelfish was eval-
RESULTS uated by measuring liver somatic index (LSI ⫽ liver
body mass⫺1 ⫻ 100) and plasma VTG levels. There was
Squirrelfish in the experimental group were injected a significant increase in LSI upon increases in E2
with E2 to determine any effect this hormone has on concentrations (Fig. 1B). By day 5, the LSI of E2-

Fig. 1. Comparisons of plasma 17␤-estradiol (E2) levels (A), liver

somatic index (LSI; B), and plasma vitellogenin (VTG) concen-
trations (C) in sham-injected (filled bars) and E2-treated (open
bars) squirrelfish. Fish were injected on days 0, 2, and 4 and were
sampled on days 5, 6, and 10 of the experiment. Bars show the
arithmetic means, with error bars indicating SE (n ⫽ 3- 8 fish).
* Values significantly different from the sham-treated control at
the same sampling point (P ⬍ 0.05; Mann-Whitney U-test).
 ESTROGEN CONTROL OF ZN AND MT IN SQUIRRELFISH R531

treated fish (3.00 ⫾ 0.26, n ⫽ 5) was 2.3 times higher The effect of increased E2 levels was also investi-
than the LSI of control fish (1.33 ⫾ 0.13, n ⫽ 7). At day gated in terms of MT induction and distribution. There
6, E2-treated animals still had a significantly higher was no statistically significant change in MT mRNA
LSI (2.43 ⫾ 0.14, n ⫽ 8) than controls (0.98 ⫾ 0.09, n ⫽ levels as a result of increased E2 concentrations at days
5), but this difference was no longer present at day 10. 5 and 6 of the sampling period (Fig. 3). There was an
Likewise, elevated E2 levels increased plasma VTG apparent increase in MT mRNA at day 10, but this was
concentrations in the experimental group (Fig. 1C), but found to be statistically insignificant (P ⫽ 0.059). How-
the time course was slightly delayed in relation to ever, increased levels of E2 did have an effect on the
plasma E2 concentrations and LSI. Although E2 levels MT protein levels and subcellular distribution of MT.
and LSI both peaked on day 5, the highest plasma VTG Subcellular fractions of control and E2-treated squir-
concentrations were measured in samples from day 6 relfish liver cells were subjected to Western analysis
(78.1 ⫾ 10.2 ␮g/ml, n ⫽ 8). Furthermore, plasma VTG for MT content (Table 2). There were no significant
was still markedly elevated on day 10 (E2 treated: changes in the absolute cytosolic or mitachondrial/
51.1 ⫾ 17.7 ␮g/ml, n ⫽ 5; sham-treated control: 1.37 ⫾ lysosomal MT content at any day of the sample period.
0.64 ␮g/ml, n ⫽ 8), although both E2 and LSI had MT was not detectable in microsomal fractions. There
returned to control levels at this point. was, however, an effect on nuclear MT (Fig. 4A). Nu-
The plasma zinc concentrations increased signifi- clear MT in E2-treated fish at day 5 (0.41 ⫾ 0.10, n ⫽
cantly after E2 injections compared with control fish 3) was significantly higher than that in sham-treated
and peaked on day 6 (E2 treated: 0.085 ⫾ 0.013 ␮mol control fish (0.15 ⫾ 0.06, n ⫽ 6). Still at day 6, nuclear
zinc/ml, n ⫽ 8; sham-treated control: 0.041 ⫾ 0.003 MT content in treated fish (0.58 ⫾ 0.10, n ⫽ 8) was

Downloaded from ajpregu.physiology.org on October 25, 2009

␮mol zinc/ml, n ⫽ 3; Fig. 2A). There was no apparent significantly higher than that in control fish (0.12 ⫾
change in the hepatic zinc concentration, measured as 0.07, n ⫽ 5). This difference was no longer present at
micromoles zinc per gram liver, as a result of E2 injec- day 10. Total MT appeared to increase somewhat dur-
tions (Fig. 2B). In contrast, the hepatic zinc content, as ing the sampling period (Fig. 4B). Although these in-
measured by multiplying micromole zinc per gram creases were not significant compared with the simul-
liver by LSI, increased and followed the pattern of taneous controls at any single sampling point, the
the LSI, thus reflecting the vast increase in liver size overall effect of E2 on total MT was significant, as
(Fig. 2C). assessed by two-way ANOVA (P ⬍ 0.05). Thus E2

Fig. 2. Effects of E2 administration on zinc concentrations in the

blood plasma (A) and zinc concentration (B) and zinc content (C)
in liver of squirrelfish at days 5, 6, and 10 of the experiment (n ⫽
3–8 fish). Other details as in Fig. 1.
R532 ESTROGEN CONTROL OF ZN AND MT IN SQUIRRELFISH

Fig. 3. Effect of E2 administration on metallothionein (MT) mRNA

levels in the liver of squirrelfish at days 5, 6, and 10 of the experi-
ment (n ⫽ 3–8 fish). Other details as in Fig. 1.

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treatment seems to result in an increase in the concen-
tration of MT in the liver of squirrelfish without a
concomitant increase in MT mRNA levels (Fig. 3).
Squirrelfish MT cDNA was cloned and sequenced
(Fig. 5). The deduced amino acid sequence was com-
pared with other MT amino acid sequences by BLAST
search in the metallo.txt file of the SWISS-PROT pro-
tein sequence database (41). The homology of squir-
relfish MT to listed MTs from other vertebrates was
highly significant (P ⬍ 3.8e⫺25), with the closest resem-
blance found to other fish (Teleostei) species (⬎78%
identities; P ⬍ 3.5e⫺38). The highest score of sequence
homology was obtained for viviparous eelpout (Zoarces Fig. 4. Effect of E2 administration on nuclear MT (A) and total MT
viviparus) MT, with 95% identities to squirrelfish MT (B) in subcellular liver fractions obtained by differential centrifuga-
and only one “out-of-group” amino acid substitution. tion at days 5, 6, and 10 of the experiment (n ⫽ 2–8 fish). Other
details as in Fig. 1.
Figure 5 shows a comparison of squirrelfish MT with
MT from viviparous eelpout (41) and several other
species, including mouse (9), human (39), and domestic
pigeon (24), and is representative of four other teleost groups, the metal-chelating cysteinyl residues, were
orders (3, 6, 41). All sequenced teleost MTs, including perfectly conserved among all sequenced fish MTs,
squirrelfish MT, have one less residue than mouse or both in terms of numbers and positions.
human MT and two less residues than pigeon MT. In DISCUSSION
position 13 of the squirrelfish MT sequence, a serine (S)
has substituted the aspargine (N) found in almost all E2 administration did not change the zinc concentra-
other fish MTs. Squirrelfish MT differed from its tion in the liver of treated squirrelfish. In a previous
eelpout counterpart in two additional positions (19 and study in rainbow trout, E2 was found to mediate the
41). The uncharged polar threonine (T) at position 19 is accumulation of zinc in the liver when measured as a
the more noteworthy because many fish species have a function of total liver weight (30). This measurement
basic lysine (K) residue located here. The functional was obtained by multiplying zinc per gram liver by LSI

Table 2. Subcellular distribution of MT

C N M/L

Control E2 Control E2 Control E2

Day 5 0.40 ⫾ 0.15 0.84 ⫾ 0.41 0.15 ⫾ 0.06 0.41 ⫾ 0.10* 0.13 ⫾ 0.04 0.29 ⫾ 0.09
Day 6 0.54 ⫾ 0.17 0.78 ⫾ 0.11 0.12 ⫾ 0.07 0.58 ⫾ 0.10* 0.15 ⫾ 0.06 0.30 ⫾ 0.07
Day 10 0.45 ⫾ 0.09 0.74 ⫾ 0.16 0.16 ⫾ 0.05 0.33 ⫾ 0.07 0.14 ⫾ 0.03 0.31 ⫾ 0.18
Values are means ⫾ SE; no. of observations varied from 2 to 8 fish. Subcellular distribution of MT (arbitrary units) in the cytosolic, nuclear,
and mitochondrial/lysosomal fractions of control and E2-treated squirrelfish livers sampled at days 5, 6, and 10 of the experiment. Microsomal
fractions are not shown since no MT was detected. * Significantly different from the sham-treated control at the same sampling point
(P ⬍ 0.05; Mann-Whitney U-test).
 ESTROGEN CONTROL OF ZN AND MT IN SQUIRRELFISH R533

Fig. 5. Deduced amino acid sequence of squir-

relfish MT (sq) compared with the sequences
of viviparous eelpout (zoa), winter flounder
(wf), rainbow trout (rbt), Atlantic cod (cod),
zebra fish (zeb), human (hu), mouse (mou),
and domestic pigeon (pi). Differences in amino
acid residues relative to squirrelfish MT are
indicated by substitution of the appropriate
one-letter code. 1Data from Kille and Olsson
(41); 2Chan et al. (6); 3Bonham et al. (3);
4
Stennard et al. (39); 5Durnam et al. (9); 6Lin
et al. (24).

to compensate for the increase in liver size that accom- transcription would have been a secondary effect of E2
panies VTG synthesis. When the same calculation is administration. Evidence for such indirect transcrip-
made using the data from this study, zinc per total tional activation has been presented for rainbow trout

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liver is indeed significantly increased (P ⬍ 0.05) on day (28). Such an increase in MT mRNA levels could be the
6 in E2-treated individuals (1.28 ⫾ 0.47, n ⫽ 8) com- result of alterations in hepatic zinc pools after VTG
pared with controls (0.45 ⫾ 0.06, n ⫽ 5). There were no production.
significant changes at any other sampling point. How- E2 increased the hepatic MT concentration and also
ever, previous studies on squirrelfish show that hepatic had an effect on the intracellular localization of MT in
zinc concentrations in mature female squirrelfish are squirrelfish liver cells. Specifically, most of the in-
much higher than in males and fluctuate throughout crease in MT level occurred in the nuclear fraction
the year (42). Therefore, the evidence suggests that E2 where, on day 6 (peak of MT accumulation), the MT
is not the signal for the massive hepatic zinc accumu- concentration was 380% higher than the simultaneous
lation observed in female squirrelfish. control. This should be seen against the smaller (and
In accordance with the previously indicated E2-in- not statistically significant) increase in cytosol and
duced zinc redistribution from the liver to the ovaries mitochondria/lysosome of 44 and 100%, respectively.
(12), E2 injections resulted in an increased zinc concen- These results suggest that MT is preferentially accu-
tration of the plasma. This effect of E2 on plasma zinc mulated in the nuclear fraction of E2-treated individ-
concentration has previously been noted in male red uals. The fact that MT mRNA did not increase as a
drum (Sciaenops ocellatus), and in this species almost direct response to E2 suggests that MT in the nuclear
all of the plasma zinc eluted together with VTG upon fraction was not the result of increased MT production
size-exclusion chromatography (11). Isolated VTG from but possibly a differential decrease in the degradation
red drum and Xenopus have been demonstrated to of MT. Previous studies have demonstrated that squir-
contain zinc, with binding stoichiometry of three atoms relfish liver cells have a sex-specific subcellular MT
of zinc per VTG monomer for red drum (11) and one distribution, with MT being a cytosolic protein in males
atom zinc per VTG monomer for Xenopus (26). In the and primarily associated with the nuclear fraction in
present study, plasma VTG concentrations in E2- females (12). The present study indicates that E2 may
treated squirrelfish were elevated highly and reached a be responsible for this sex-specific MT distribution.
maximum on day 6, 2 days after the third and final E2 Interestingly, this shift in MT distribution occurred in
injection. The same pattern was found for plasma zinc. parallel to the presumed VTG exocytosis (based on
These peaks in plasma VTG and zinc occurred just VTG levels in plasma) from the liver into the blood.
when liver size, measured as LSI, started to decline, Whether these events were coordinated or even related
suggesting that the liver was releasing newly synthe- remains to be investigated.
sized VTG into the plasma. If this VTG was carrying Questions arise as to the nature and significance of
zinc, as in red drum and Xenopus, it could possibly the redistribution of MT from the cytosolic to the nu-
explain the concomitant increase in plasma zinc and clear fraction. Immunohistochemical studies have sug-
also the E2-dependant transfer of zinc from liver to gested that MT accumulates in the nucleus of a variety
ovaries observed in female squirrelfish (12). of fetal and tumor cells (7) and also in regenerating rat
E2 did not elicit a significant increase in MT mRNA hepatocytes under cytokine stimulation (44). Thus the
levels, although there was a possible increase at day 10 function of nuclear MT may be related to the replica-
(P ⫽ 0.059). If this increase is real, it occurred at a time tion of DNA associated with the rapid proliferation of
when all other E2-mediated changes had already sub- regenerating and cancerous tissues. This explanation
sided and the circulating E2 levels had in fact returned is probably unlikely in our study because the hepato-
to normal. This suggests that E2 itself does not induce cytes were not undergoing periods of rapid prolifera-
transcription of the MT gene but rather any increase in tion. A possible scenario could be that nuclear MT in
R534 ESTROGEN CONTROL OF ZN AND MT IN SQUIRRELFISH

female squirrelfish interacts with zinc finger proteins large increase in the hepatic production and secretion
to regulate the hepatic transcription of the large of VTG. E2 was also found to markedly elevate plasma
amounts of RNA related to the synthesis of VTG (8). zinc levels. In fact, the peaks in plasma zinc and VTG
It must also be considered that MT may not actually occurred simultaneously, indicating that VTG could be
be present in the nucleus but rather associated with involved in the transport of zinc from the liver to the
the nuclear envelope. Another possibility is that the ovaries. It will be interesting to isolate and character-
MT found in the nuclear fraction is not associated with ize squirrelfish VTG to ascertain whether this protein
the nucleus at all but rather in other structures, such perhaps has a higher binding capacity for zinc than the
as dense vesicles, that would coprecipitate with nuclei VTGs of other species.
during subcellular fractionation via differential cen-
trifugation. The export of VTG from hepatocytes into Perspectives
the plasma involves large Golgi vesicles (46). Recent
It is fascinating to speculate why squirrelfish accu-
studies have shown that MT can donate zinc to the
mulate such high levels of zinc. A key to the biological
apoforms of zinc proteins (18), despite lower affinities
function may be that only liver and gonads of females
of these proteins for zinc than MT, and that this trans-
display higher concentrations of zinc than males or
fer is mediated by the redox activities of glutathione
females of other fish species (16). This clue, together
(19). Perhaps MT is localized in Golgi vesicles and is
with the finding that E2 treatment triggers redistribu-
acting to donate zinc to VTG, or other vehicle proteins,
tion of zinc from liver to ovaries in mature females,
for transfer to the ovaries. Such an explanation would
suggests that zinc accumulation is related to reproduc-
be consistent with the increase in plasma zinc concen-

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tion (12). Our results have prompted us to further
trations observed in E2-injected squirrelfish.
hypothesize that zinc is initially bound to MT in liver
With the use of the suggested criteria for defining the
and subsequently transferred to a vehicle, which trans-
phylogenetic relationships of the MT superfamily (2),
ports the zinc to the ovaries where it is incorporated
squirrelfish MT fall into family 1, vertebrate MTs, and
into the developing oocyte.
are further divided into subfamily t, teleostean MT.
Squirrelfish are nocturnal animals with extremely
Specifically, squirrelfish MT most closely resembles,
large eyes. There is a connection between eyes and
with 95% identity, that of the viviparous eelpout (Z.
zinc, in that eyes are the organs with the highest zinc
viviparus), which belongs to the superorder Acan-
concentration in the vertebrate body (16, 47). Further-
thopterygii and the order Perciformes. Squirrelfish
more, nocturnal animals tend to have more zinc in
sorts under the same superorder as the viviparous
their eyes than their day-active relatives (47). Indeed,
eelpout (Acanthopterygii) but to a different order,
in squirrelfish of both sexes, the zinc concentration of
namely Beryciformes. Of the teleostean superorders
the retina is almost as high as that of the female liver
entered into the SWISS-PROT database, the order of
(12). In the vertebrate retina, zinc is localized to glu-
peptide sequence homology scores to squirrelfish MT
tamatergic neurons in which it acts as a neuromodu-
was Acanthopterygii ⬎ Procanthopterygii ⬎ Paracan-
lator (38). The nocturnally adapted eyes of squirrelfish,
thopterygii ⬎ Ostariophysi. On the basis of the high
coupled to their physical size, may necessitate large
homology with other teleost MTs and the fact that all
amounts of zinc to be incorporated in the developing
cysteinyl residues are perfectly conserved between MT
retina. Thus the significance of high zinc content in
from squirrelfish and other teleosts, we suggest that
female squirrelfish would be to supply the embryo with
squirrelfish MT probably does not bind zinc any differ-
sufficient zinc for eye development. This hypothesis
ently than other teleostean MTs, nor would other func-
remains untested.
tional biochemical properties be different. Therefore,
the massive accumulation of zinc in female squirrelfish We thank Dr. Brent Palmer, University of Kentucky, for providing
is most likely not the result of any unusual features of antibodies to VTG, Henry Feddern at Tavernier, Florida, for supply-
the MT primary sequence. ing the fish, and Tom Capo, Rosenstiel School of Marine and Atmo-
spheric Sciences, for assistance in handling the fish.
E2 had no direct effect on the concentrations of zinc This study was supported by Grant IBN-9631441 from the Na-
or MT mRNA in the liver of treated squirrelfish. Be- tional Science Foundation (to C. Hogstrand).
cause zinc and MT accumulations observed in squir- Present addresses: E. Burge: Dept. of Environmental Sciences,
relfish are known to be specific to females and because Virginia Institute of Marine Science, College of William and Mary,
immature females from the present study were not Gloucester Point, VA 23062-1346; C. Hogstrand: Div. of Life Sci-
ences, King’s College London, Franklin-Wilkins Bldg., 150 Stamford
different from males in these respects, it seems likely St., London SE1 8WA, United Kingdom.
that some kind of endocrine regulation, linked to the
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