Sexual maturation and reproductive zinc physiology in the female squirrelfish
1 T. H. Morgan School of Biological Sciences, 101 Morgan Building,
University of Kentucky, Lexington, KY 40506, USA
2 Division of Marine Biology and Fisheries, and NIEHS Marine and Freshwater
Biomedical Science Center, Rosenstiel School of Marine and Atmospheric Science
(RSMAS), University of Miami, 4600 Rickenbacker Causeway, Miami, FL 33149,
USA
3 School of Health and Life Sciences, King's College London,
FranklinWilkins Building, 150 Stamford Street, London SE1 9NN,
UK
* Author for correspondence (e-mail: christer.hogstrand{at}kcl.ac.uk)
Accepted 5 August 2002
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Summary |
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Key words: zinc, metallothionein, vitellogenin, estradiol, sexual maturity, reproductive physiology, female, squirrelfish
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Introduction |
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Zinc is an essential micronutrient that is required for normal cellular
function (for a general review, see Vallee
and Falchuk, 1993). The absorption of zinc in marine fish occurs
by two major pathways. Aqueous Zn2+ is taken in through the gills
while the majority of zinc uptake occurs via intestinal absorption of dietary
zinc and waterborne zinc (Hogstrand and
Wood, 1996
). Upon entering the bloodstream, zinc is bound to
proteins such as albumin, which act in its transport
(Fletcher and Fletcher, 1980
;
Dyke et al., 1987
). Although
the exact mechanism remains unclear, the recent discovery of the ZIP family of
membrane-associated proteins seems to indicate a means for zinc to pass into
the cell (Guerinot, 2000
). The
cellular accumulation of zinc is likely to be the result of interplay between
the relative activities of this ZIP-mediated zinc import, and export by other
membrane proteins such as ZnT-1 (Palmiter
and Findley, 1995
; Cousins and
McMahon, 2000
). Once within the cell, zinc is almost
instantaneously chelated to a variety of zinc-binding proteins
(Vallee and Falchuk, 1993
).
One such zinc-binding protein is metallothionein (MT). MT is capable of
binding not only zinc but also other elements of groups IB and IIB of the
periodic table (Kagi and Schaffer,
1988
). One function of MT is to sequester potentially toxic metal
species within the cell, which is probably an extension of its role in the
regulation of labile intracellular zinc. It has been shown previously that
zinc can be toxic if accumulated in large amounts
(Hogstrand and Wood, 1996
).
Thus, it is remarkable that the concentrations of zinc accumulated in the
liver of female squirrelfish (up to 70 µmol g-1 wet weight) are
higher than any other studied organism (Hogstrand and Haux,
1991
,
1996
;
Hogstrand et al., 1996
). In
addition, it has also been discovered that female squirrelfish have high
hepatic concentrations of MT, and MT levels are closely correlated to zinc
concentrations (0.89<r<0.99) but not other metals that can bind
MT (Hogstrand and Haux, 1996
),
further suggesting a role for MT in protecting female squirrelfish against
potentially toxic zinc hyperaccumulation.
A seasonal shift in the subcellular localization of MT in female
squirrelfish liver also exists, with MT located primarily in the liver cell
cytosol in late spring and co-precipitating with the nuclear fraction in
winter months (Thompson et al.,
1999). Conversely, MT in male squirrelfish liver cells is
maintained in the cytosol throughout the year
(Hogstrand et al., 1996
;
Thompson et al., 1999
).
Overall, this gender specificity suggests that the dynamics of zinc and MT
accumulation and distribution observed in female squirrelfish are in some way
related to the reproductive cycle. However, the point of development at which
females become segregated from males in terms of zinc metabolism remains
unclear. In addition, the mechanism by which this occurs has not been
identified. Because squirrelfish are not bred in captivity and are collected
from their reef environment, it is logistically difficult to characterize
their reproductive biology, physiology and endocrinology. Thus, we are still
faced with the questions of when, as well as how, this unique phenomenon is
initiated.
The purpose of the present study was therefore twofold. The first phase was
to investigate the role of sexual maturation in female squirrelfish as
pertaining to the accumulation and localization of zinc and MT. To do so, we
have measured zinc, MT, MT mRNA and sex steroids in female squirrelfish as a
function of ovary size. In a previous study involving mature females,
administration of 17ß-estradiol (E2) elicited a decrease in
hepatic zinc and at the same time moderated an increase in ovarian zinc
(Hogstrand et al., 1996).
Therefore, to further characterize the effects of this female sex hormone, the
second aspect of this study was to inject immature and mature female
squirrelfish with E2, followed by analyses of the same variables as
described above, to determine if E2 is responsible for any
previously observed physiological differences in zinc regulation brought on by
the onset of sexual maturity.
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Materials and methods |
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The squirrelfish in one tank were given interperitoneal injections of 5 mg E2 kg body weight-1 (1 ml kg-1 in peanut oil) on days 0, 2 and 4, to maintain circulating E2 levels. A control group housed in the other tank was injected with peanut oil only. Control and treated fish were sacrificed on either day 5, 6 or 10.
Sampling
Fish were euthanized by overdose of MS222 (0.5 g l-1) and
weighed. Approximately 2 ml of blood was withdrawn from the caudal vessel with
a heparinized syringe, and plasma was separated from blood cells by
centrifugation (14000g) for 3 min. The plasma was divided into
50 µl samples and stored at -80°C for subsequent analysis of
E2, testosterone (T), progesterone (P), vitellogenin (VTG), zinc
and MT as described below. Livers and gonads were removed, weighed and
immediately divided into aliquot samples. A tissue sample (approximately 0.5
g) was taken from each liver and subjected to subcellular fractionation as
described below. Samples of liver (approximately 0.5 g) were placed into
individual 16 mmx150 mm borosilicate glass tubes for acid digestion and
subsequent zinc analysis as described below. The remaining liver samples were
wrapped in aluminum foil and frozen in liquid nitrogen. The frozen samples
were transferred to -80°C, where they were stored until used for total RNA
extraction as described below.
Tissue zinc content
Liver samples were subjected to acid digestion in 2.0 ml of 70%
HNO3 (trace-metal grade, Fisher, Pittsburgh, PA, USA). The tubes
were heated in a sand bath for 3 h at 120°C and then cooled to room
temperature before 0.5 ml of H2O2 was added. The
temperature was then gradually increased to 120°C until all liquid had
evaporated. The dried residues were reconstituted with 5.0 ml of 0.5%
HNO3. These samples were then analyzed for zinc content by
air/acetylene flame atomic absorption spectroscopy (Perkin Elmer, model 2380,
Shelton, CT, USA). Plasma samples (0.05 ml) were analyzed for zinc content in
the same manner.
VTG and steroid analysis
Plasma samples from each fish were measured for VTG and steroid content by
enzyme-linked immunosorbent assay (ELISA)
(Specker and Anderson, 1994).
The VTG ELISAs were performed according to the procedure of Thompson et al.
(2001
). Each plasma sample was
diluted 1:100 in phosphatebuffered saline [PBS (0.15 mol l-1 NaCl,
0.0015 mol l-1 KH2PO4, 0.015 mol
l-1 Na2HPO4, 0.002 mol l-1 KCl, pH
7.4)] and 100µl of this dilution was added per well in 96-well
Nunc-ImmunoTM MaxiSorpTM microtiter plates (Nalge Nunc
International, Rochester, NY, USA). Squirrelfish VTG was isolated by
ion-exchange chromatography according to the protocol of Silversand and Haux
(1989
), with minor
modifications. This purified squirrelfish VTG was used to produce standard
curves. The samples were incubated in duplicate for 1 h at 4°C to allow
for optimal adsorption of antigen to the plates and were then washed four
times with 300 µl PBS per well. Following the washes, the plates were
treated with 150 µl of blocking buffer (5% dehydrated nonfat milk in PBS)
for 30 min at room temperature. Blocking buffer was then removed and the wells
were subjected to 100 µl of primary antibody and incubated for 1 h at room
temperature on an orbital shaker. The primary antibody (gift from Dr Ram
Abuknesha, King's College London) was a sheep immunoglobulin G (IgG) reared
against a conserved VTG peptide sequence diluted 1:30000 in PBS. The
crossreactivity and specificity of this antibody with squirrelfish VTG was
confirmed by western blot (data not shown). After the primary antibody
incubation, the plates were washed four times with PBS, subjected to 100 µl
of secondary antibody, and incubated for 1 h at 37°C. The secondary
antibody was a horseradish peroxidase-linked donkey anti-sheep IgG (BioRad,
Hercules, CA, USA) diluted 1:2000 in PBS. The plates were then washed four
times with PBS, and 100 µl of a tetramethylbenzidine (TMB) peroxidase
enzyme immunoassay substrate (BioRad) was added according to manufacturer's
specifications. After a 15 min incubation, 50 µl of 0.5 mol l-1
H2SO4 was added to the substrate to stop the reaction,
and the plates were read on a microplate reader (BioRad, model 450) at 450 nm.
E2, T and P were analyzed using the respective steroid ELISA kits
(Oxford Biomedical Research, Oxford, MI, USA) according to manufacturer's
specifications.
Hepatic subcellular fractionation
Subcellular fractions of liver were obtained by differential centrifugation
of liver homogenates immediately following dissection via the procedure for
rainbow trout liver described by Julshamn et al.
(1988), modified for
squirrelfish by Hogstrand et al.
(1996
). Liver samples
(approximately 0.5 g) were individually homogenized in an ice-cold
homogenization buffer (35 mmol l-1 Tris-HCl, 0.20 mol
l-1 KCl, 0.25 mol l-1 sucrose, pH 7.4) using a
glass-Teflon homogenizer. The homogenate was centrifuged (370 g) for
5 min at 4°C and the supernatant was removed, while the pellet (nuclear
fraction) was immediately placed on ice. The supernatant was centrifuged (9200
g) for 5 min at 4°C, and the pellet (mitochondrial/lysosomal
fraction) was saved and placed on ice. This supernatant was then centrifuged
(13,0000 g) for 60 min at 4°C, resulting in a small pellet
(microsomal fraction), which was saved and immediately placed on ice. The
final supernatant (cytosolic fraction) was divided into 0.5 ml aliquot samples
and immediately placed on ice. All pellets were resuspended in 0.5 ml of fresh
homogenization buffer and subcellular fractions were divided into aliquots and
transferred into liquid nitrogen. These samples were then stored at -80°C
until used in western analysis for MT.
Western analysis
Each subcellular fraction was subjected to SDS-PAGE with a discontinuous
buffer system according to the protocol of Laemmli
(1970). The protein
concentration for each subcellular fraction was determined by Bradford assay
(Bradford, 1976
), and samples
were diluted with distilled water as necessary. These samples were then
diluted 1:4 with sample buffer [62 mmol l-1 Tris-HCl, pH 6.8, 10%
glycerol, 5.0% 2-mercaptoethanol (added just before dilution), 2.0% SDS,
0.0012% Bromophenol Blue] and heated at 100°C for 5 min. A total of 25
µg of protein was loaded into each well. Perch (Perca fluviatilis)
MT was used as a standard (Hogstrand and
Haux, 1990
). Electrophoresis was carried out on a 4% stacking gel
and a 12.5% separating gel at 100 V for 2 h in a Mini-Protean II
electrophoresis system (BioRad).
After electrophoresis, proteins were transferred from polyacrylamide gels
onto 54 cm2 nitrocellulose membranes (Schleicher & Schuell, 0.2
µm pore diameter) by electroblotting, as described by Towbin et al.
(1979), in a SemiPhor TE 70
semi-dry transfer unit (Hoefer Scientific, Moorestown, NJ, USA) at 0.8 mA
cm-2 constant current for 60 min at room temperature. Once proteins
were transferred, the membranes were blocked with 5% dehydrated nonfat milk in
TBS-T (20 mmol l-1 Tris-HCl, pH 7.4, 137 mmol l-1 NaCl,
0.10% Tween-20) for 60 min to prohibit further nonspecific protein binding.
All incubations were carried out at room temperature. The membranes were then
subjected to a series of washes in fresh TBS-T (one 15 min and two 5 min)
followed by a 1 h incubation in primary antibody. The primary antibody was
rabbit anti-perch MT (Hogstrand and Haux,
1990
) diluted 1:8000 in TBS-T. After another series of the same
washes, the membranes were incubated for 1 h in secondary antibody. The
secondary antibody was a horseradish peroxidase-conjugated donkey anti-rabbit
IgG (Amersham, Piscataway, NJ, USA) diluted 1:40000 in TBS-T. Following a
final series of washes, immunodetection was performed with an enhanced
chemiluminescence system (ECL, Amersham) according to manufacturer's
specifications. Chemiluminescence was captured on photographic film (Kodak),
and the optical density (OD) of each band was quantified using Sigma Gel
software (Jandel Scientific, Chicago, IL, USA). Each western blot was exposed
for 15 s, 30 s and 60 s to ensure the OD linearity of the film. The OD of each
unknown was compared with the OD of the internal standard of each gel.
Although a known amount of the perch MT standard was added to each gel, the
results are presented as arbitrary units because it has not been determined if
the immunoreactivity of perch and squirrelfish MT are the same. The relative
amount of MT in each fraction was thus calculated as the ratio of sample OD to
standard OD.
Total RNA extraction
Liver and gonad tissues previously stored at -80°C were thawed, and
50-100 mg of each sample was homogenized in 1 ml of TRIzol Reagent (Gibco BRL,
Rockville, MD, USA) using a glass-Teflon homogenizer. Total RNA was then
extracted from the homogenates according to manufacturer's specifications,
with minor modifications as suggested by the manufacturer. Specifically, the
homogenates were centrifuged (12,000 g) to remove insoluble material,
and treated with a high salt precipitant solution (1.20 mol l-1
sodium citrate, 0.80 mol l-1 NaCl) to remove glycogen.
Northern analysis
Total RNA extracted from liver samples (approximately 10 µg) was
subjected to electrophoresis on a 1.5% agarose gel with formaldehyde as
denaturant (Sambrook et al.,
1989). To standardize results, a reference sample with a constant
amount of MT mRNA was loaded onto each gel. In addition, some experimental
samples overlapped between gels as a control for interassay variability. After
completed separation, RNA was transferred onto a 170 cm2 Hybond N
nylon membrane (Amersham) by capillary/gravity blotting using the Turbo
Blotter (Schleicher & Schuell, Moorestown, NJ, USA). The RNA was UV
crosslinked to the membrane followed by incubation in 18 ml of the
prehybridization buffer [50% formamide, 5 x SSC, 2.0% Blocking reagent
(Boehringer Mannheim, Indianapolis, IN, USA), 0.10% N-laurosarcosine, 0.02%
SDS] at 68°C for 4 h. The prehybridization buffer was discarded and the
membrane was hybridized for 18 h at 68°C in 18 ml of the same buffer, with
0.30 µl probe ml-1 added, using a digoxigenin (DIG)-labeled
antisense squirrelfish MT-cRNA probe
(Thompson et al., 2001
). The
plasmid was linearized and the DIG-labeled RNA riboprobe was made from the DNA
template by T7 RNA polymerase in the presence of a nucleotide mix containing
DIG-labeled dUTP (Boehringer Mannheim). After hybridization, the membrane was
subjected to stringency washes (2 washes in 2 x SSC, 0.10% SDS for 5 min
at room temperature; two washes in 0.1 x SSC, 0.1% SDS for 5 min at
68°C), followed by immunochemiluminescent detection of DIG-labeled probe
using a Fab fragment of sheep anti-DIG-AP conjugate (Boehringer Mannheim) and
CSPD (Boehringer Mannheim) as chemiluminescent substrate. Chemiluminescence
was captured on X-ray film (Kodak), and MT mRNA bands were quantified using
Sigma Gel software (Jandel Scientific). Each northern blot was exposed for at
least four exposure times to ensure OD linearity of the film. Arbitrary units
were derived by the ratio of sample OD to reference OD.
Statistics
Because gonadal examination is the only known way to distinguish gender and
sexual maturity in squirrelfish, immature females [operationally defined as
gonadosomatic index (GSI) <0.25] and mature females were sorted into
separate groups after sacrifice. To further characterize that selected females
were indeed immature and not merely adults in a regressed ovarian stage, the
mean body mass of immature and mature females was compared (immature,
157.5±5.54 g, N=9; mature, 245.8±10.8 g,
N=14). E2-injected fish were also sorted into groups of
immature and mature females following sacrifice (immature, 153.9±11.6
g, N=9; mature, 240.8±11.5 g, N=13).
Squirrelfish samples were divided between the different laboratories involved in the study and further divided for different analyses. Therefore, the number of observations for each treatment group and sampling occasion was not the same for all variables, and the resulting distributions were not normal and did not exhibit equal variances. As a result, nonparametric statistical analyses have been used. Pair-wise comparisons between immature and mature female squirrelfish were determined by the Mann-Whitney U-test. For the E2-injection experiment, trend analyses were performed by Kruskal-Wallis one-way analysis of variance (ANOVA), and any significant findings were further subjected to post-hoc pair-wise comparisons performed by Dunn's Test. Data are presented as means±S.E.M. Results were considered significant at P<0.05.
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Results |
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Hepatic MT mRNA levels (Table 1) observed in mature female squirrelfish were nearly four times that of their immature counterparts. Furthermore, the levels of hepatic MT mRNA were significantly correlated with increasing GSI (Fig. 3, P<0.001, N=21). On a subcellular basis, MT protein in the hepatic nuclear fraction (Table 1) was significantly greater in mature females than in immature females. There was no significant difference in the amount of MT protein observed in the cytosolic or mitochondrial/lysosomal fractions, and MT in the microsomal fraction was undetectable.
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The effects of E2 treatment were determined for both immature and mature female squirrelfish. E2 treatment resulted in a significant increase in LSI of immature females during the sampling period as compared with control individuals (Fig. 4A), with the highest measurements occurring at day 5 and remaining elevated in E2-treated fish at day 6 before returning to control levels by day 10 (KruskalWallis ANOVA; P=0.009, N=18). The trend was similar for mature female squirrelfish (Fig. 4B), with a peak in the LSI of E2-treated individuals at day 5 before falling back to levels similar to control individuals by day 10 (P=0.008, N=13). E2 treatment had no effect on the GSI of immature female squirrelfish (Fig. 5A). The overall effect of E2 on GSI of mature females was not found to be statistically significant (P=0.101, N=13). Interestingly, pair-wise comparison of means indicates that GSI of E2-treated individuals was significantly greater than control measurements both at days 5 and 6 of the sampling period, while significantly less than control GSI measurements at day 10 (Fig. 5B). E2 treatment had no effect on the hepatic accumulation of zinc in either immature or mature females (Tables 2, 3). However, there was an effect on zinc accumulation in the blood plasma of immature females (Fig. 6A) with plasma zinc remaining significantly elevated throughout the sampling period (P=0.002, N=6). The same effect was observed in mature female squirrelfish (Fig. 6B; P<0.001, N=12), although levels at day 10 were approximately 50% less than plasma zinc concentrations at days 5 and 6 in E2-treated individuals. Likewise, plasma VTG concentrations were significantly increased in E2-treated fish compared with simultaneous controls in both immature (Table 2, P=0.006, N=8) and mature (Table 3, P=0.001, N=11) female squirrelfish, with VTG concentrations in both E2-treated groups reaching the highest levels at day 5 of the sampling period.
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E2 treatment of mature female squirrelfish did result in increased plasma E2 levels during the sampling period when compared with controls (Table 3; P= 0.024, N=10). Conversely, the overall effect of E2 concentration in plasma of immature females was not found to be statistically significant (Table 2; P=0.142, N=7), even though there was a pair-wise significance at day 5 of the sampling period. This probably resulted from low sample numbers plus the lower-than-control average E2 level of treated fish on day 10. Plasma E2 levels peaked in mature females at day 6 of the sampling period before dropping to levels at day 10 that were approximately 10 times less than the levels of simultaneous controls (Table 3). E2 treatment resulted in a significant decrease (up to 30 times less) in plasma T levels of immature (Table 2; P=0.002, N=7) and mature (Table 3; P=0.001, N=10) females throughout the sampling period when compared with control female squirrelfish. Likewise, plasma P levels were significantly decreased (approximately 10 times less) in E2-treated immature females (Table 2; P=0.002, N=7) and mature females (Table 3; P=0.002, N=9) when compared with controls. These drastic reductions in the levels of T and P in both immature and mature female squirrelfish are likely to be in response to increased concentrations of E2. Thus, the addition of large doses of exogenous E2 to female squirrelfish seems to indicate a negative feedback loop mechanism within the hypophysealgonadal sex steroid production axis.
E2 treatment did not result in increased hepatic MT mRNA levels in either immature or mature female squirrelfish (Tables 2, 3) during the sampling period. However, E2 treatment did result in an increase in the levels of MT protein in the nuclear fraction of immature female squirrelfish liver cells (Fig. 7A) when compared with control individuals (P=0.002, N=8), with levels peaking at day 6 of the sampling period. There was no significant trend in the levels of MT in the nuclear fraction of E2-treated mature females (Fig. 7B). Furthermore, E2 treatment resulted in increased levels of cytosolic MT protein in immature females (Table 2; P=0.046, N=8), while, again, a significant effect did not occur in mature females (Table 3). In fact, there was a significant increase in the ratio of nuclear MT to cytosolic MT in E2-treated immature females (P=0.046, N=8). This trend did not occur in E2-injected mature female squirrelfish.
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Discussion |
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In addition to hepatic zinc, circulating levels of zinc in the bloodstream
also increased as a result of sexual maturity. This corroborates evidence from
previous studies in squirrelfish, which indicates that zinc is accumulated in
the liver and passed on to the ovaries via the bloodstream in mature females,
under the influence of E2
(Hogstrand et al., 1996;
Thompson et al., 2001
).
Furthermore, E2 administration in this study resulted in an
increase in plasma zinc levels. As stated previously, hepatic zinc
concentrations were not altered by increased E2 levels. If zinc in
the plasma originates from the liver, then both import and export of zinc in
the liver must be proportionately increased by E2. Interestingly,
the hepatocytes of female squirrelfish have indeed been shown to have higher
zinc efflux as well as higher zinc influx than the hepatocytes of males
(Hogstrand et al., 1996
). It
remains to be investigated if this upregulation of bilateral zinc fluxes is
under the control of E2 or some other endocrine factor.
Zinc transport from the liver followed the same time course as the hepatic
production and transport of VTG. However, the molar ratio of plasma zinc to
VTG was consistently high (approximately 11:1), suggesting that either
squirrelfish VTG has the capacity to bind much more zinc than do VTGs of other
species or that VTG is not the primary vehicle of hepatoovarian zinc
transport in squirrelfish. In the present study, immature and mature females
were not significantly different in terms of LSI, an indication of hepatic VTG
production. Furthermore, there was no difference in the plasma VTG levels
between immature and mature female squirrelfish from the respective control
groups. This was in spite of 35% higher plasma zinc levels in mature females
in comparison with immature females. E2 administration of immature
and mature females did result in increased plasma concentrations of VTG and
zinc in all females irrespective of sexual development stage. However, whereas
plasma VTG levels decrease by day 10 of the sampling period in both immature
and mature females, plasma zinc levels only decreased in mature females.
Furthermore, plasma zinc levels were twice as high in mature females than
immature females after E2 administration, while VTG concentrations
were similar in both mature and immature females. These findings seem to
suggest that plasma zinc is not primarily bound to VTG in the plasma of female
squirrelfish. In Xenopus, VTG-bound zinc appears to be the only form
of zinc taken up by the developing oocyte
(Falchuk et al., 1995). Thus,
it is unclear if the zinc in the plasma of squirrelfish that is not bound to
VTG is a direct indication of zinc that is available for uptake into the
ovaries. Certainly, the zinc concentration in squirrelfish ovaries is
unusually high, so zinc is likely to be taken up by the follicle in one form
or another.
The onset of sexual maturation also resulted in increased levels of hepatic
MT mRNA. However, administration of E2 in immature and mature
female squirrelfish did not enhance MT mRNA levels. This result is similar to
results obtained previously with male squirrelfish
(Thompson et al., 2001).
Similarly, in Oncorhynchus mykiss, E2 treatment did not
directly result in the production of MT mRNA
(Olsson et al., 1989
).
Furthermore, promoter analyses of various fish MT loci have revealed no
elements that would be expected to respond to sex steroids
(Olsson, 1993
). Thus, there is
at present little experimental evidence to suggest that E2 directly
triggers MT gene transcription in fish in general. Likewise, E2
treatment alone does not seem to be sufficient to induce MT transcription in
squirrelfish. It seems more likely that accumulation of zinc itself, or in
combination with a sex hormone (e.g. E2), activates transcription
of MT. This would mean that zinc absorption and its uptake in liver are
upregulated during female maturation. Indeed, recent results suggest the
female squirrelfish have an unusually high intestinal zinc uptake rate and
that absorbed zinc is rapidly translocated to the liver
(Glover et al., 2002
).
Furthermore, a study on isolated squirrelfish hepatocytes demonstrated that
hepatocytes from females take up zinc more rapidly than those of males
(Hogstrand et al., 1996
).
Whatever the signal for MT synthesis, the fact remains that the very potent
production of MT allows zinc to be accumulated at very high levels in the
liver of mature female squirrelfish.
Not only are the levels of MT mRNA and MT protein increased during
maturation of the female squirrelfish, but the subcellular distribution of MT
protein is also altered. Mature female squirrelfish liver has a greater
proportion of MT in the cell nuclear fraction than immature females. This
distribution could be the result of increased levels of E2, as
immature females treated with E2 do indeed show increased
proportions of MT in the nuclear fraction of liver cells. However, this is not
the case in E2-treated mature females. This result was unexpected,
and is difficult to explain, but could mean that the female liver has to be in
a responsive state for the occurrence of MT movement into the nuclear
fraction. Interestingly, the increase of MT in the nuclear fraction of
immature females did not occur at the expense of MT in the cytosolic fractions
of these fish, indicating a possible preferential degradation or other
post-transcriptional mechanism by which E2 increases total liver
MT. Similar results have been presented for male squirrelfish
(Thompson et al., 2001).
E2 treatment also resulted in an increase in the GSI of mature
female squirrelfish, but this effect is not observed in E2-treated
immature females. The pre-spawning development of the female teleost oocyte
occurs in two phases, a period of bulk growth and a period of final maturation
(Wallace, 1985). Oocyte growth
is primarily the result of the uptake of VTG
(Wallace, 1985
). VTG is
produced in the liver in response to E2, and is transported via the
bloodstream to the ovary where it is incorporated into the developing oocyte
(Wallace, 1985
). Indeed,
plasma VTG concentrations were increased in E2-treated mature and
immature females. The uptake of VTG by the oocyte is a receptor-mediated
endocytotic process (Wallace,
1985
) via a membrane receptor specific for VTG
(Tyler and Lancaster, 1993
).
One possibility for the lack of gonadal growth stimulation by E2 in
immature female squirrelfish could be that sexually immature oocytes lack
functional VTG receptors and therefore do not respond to circulating VTG in
the same manner as in mature females.
We have now established the developmental stage for the female-specific
zinc and MT accumulation and distribution in squirrelfish to be concurrent
with sexual maturation. As in most vertebrates, E2 is a very
important regulator of sexual maturation in fish. Yet E2 does not
seem to be sufficient for enhancing hepatic uptake of zinc or transcription of
MT. Thus, the factors that initiate these physiological differences indicative
of sexual maturity in female squirrelfish remain unclear. Studies of sex
hormone profiles during the female squirrelfish reproductive cycle are
currently under way. E2 does seem to stimulate an increase in
plasma zinc concentration and we hypothesize that this is to supply zinc to
the oocytes. The purpose of such a hepatoovarian zinc transfer is not
known, but Thompson et al.
(2001) speculated that it
might be to supply the nocturnal squirrelfish larvae with sufficient zinc to
ensure proper night vision.
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Acknowledgments |
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